| MSV000084442 | Cancer | Melanoma | Prediction of HLA epitopes is important for the development of cancer immunotherapies and vaccines. However, current prediction algorithms have limited predictive power, in part because they were not trained on high-quality epitope datasets covering a broad range of HLA alleles. To enable prediction of endogenous HLA class I-associated peptides across a large fraction of the human population, we used mass spectrometry to profile >185,000 peptides eluted from 95 HLA-A, -B, -C and -G mono-allelic cell lines. We identified canonical peptide motifs per HLA allele, unique and shared binding submotifs across alleles and distinct motifs associated with different peptide lengths. By integrating these data with transcript abundance and peptide processing, we developed HLAthena, providing allele-and-length-specific and pan-allele-pan-length prediction models for endogenous peptide presentation. These models predicted endogenous HLA class I-associated ligands with 1.5-fold improvement in positive predictive value compared with existing tools and correctly identified >75% of HLA-bound peptides that were observed experimentally in 11 patient-derived tumor cell lines. | For primary tumors and patient cell lines, HLA-peptide complexes were immunoprecipitated from 0.1 to 0.2g tissue or up to 50 million cells. Solid tumor samples were dissociated using tissue homogenizer (Fisher Scientific 150) and HLA complexes were enriched as described above. 8-10mm skin punch biopsies were obtained from healthy human skin discarded during skin surgeries. Subcutaneous fat was removed and remaining skin was snap frozen prior to processing. For skin HLA immunoprecipitations, frozen samples were dissociated in lysis buffer using TissueRaptorTM before immunoprecipitation (Qiagen). | Peptides were resuspended in 3% ACN, 5% FA and loaded onto an analytical column (20-30 cm, 1.9 μm C18 Reprosil beads (Dr. Maisch HPLC GmbH), packed in-house PicoFrit 75 μM inner diameter, 10 μM emitter (New Objective)). Peptides were eluted with a linear gradient (EasyNanoLC 1000 or 1200, Thermo Scientific) ranging from 6-30% Buffer B (either 0.1% FA or 0.5% AcOH and 80% or 90% ACN) over 84 min, 30-90% B over 9 min and held at 90% Buffer B for 5 min at 200 nl/min. During data dependent acquisition, peptides were analyzed on a QExactive Plus (QE+), QExactive HF (QE-HF) or Fusion Lumos (Thermo Scientific). | Key eligibility criteria were clinically or radiographically evident, pathologically confirmed stage IIIB/C and IVM1a/b melanoma | Key eligibility criteria were clinically or radiographically evident, pathologically confirmed stage IIIB/C and IVM1a/b melanoma deemed amenable to complete surgical resection and an ECOG performance status of 0 or 1. | Fresh tumor cell suspensions or thawed cryopreserved cells were washed and cultured in tissue culture plates containing OptiMEM GlutaMax media (Gibco,Thermofisher) supplemented with fetal bovine serum (5%), sodium pyruvate (1mM), penicillin and streptomycin (100units/ml), gentamycin (50µg/ml), insulin (5µg/ml) and epidermal growth factor (5ng/ml; Sigma-Aldrich). Cell cultures were dissociated and passaged using versene (Gibco,Thermofisher). The expanding cell lines were tested mycoplasma free and verified as melanoma through immunohistochemical stains using antibodies against the melanoma markers HMB45, MITF, MART-1, Melan-A, and S100 that were performed in the Dana-Farber/Harvard Cancer Center Specialized Histopathology Core Laboratory. | A large peptidome dataset improves HLA class I epitope prediction across most of the human population;An immunogenic personal neoantigen vaccine for patients with melanoma |
| MSV000084442 | Cancer | Ovarian Cancer | Prediction of HLA epitopes is important for the development of cancer immunotherapies and vaccines. However, current prediction algorithms have limited predictive power, in part because they were not trained on high-quality epitope datasets covering a broad range of HLA alleles. To enable prediction of endogenous HLA class I-associated peptides across a large fraction of the human population, we used mass spectrometry to profile >185,000 peptides eluted from 95 HLA-A, -B, -C and -G mono-allelic cell lines. We identified canonical peptide motifs per HLA allele, unique and shared binding submotifs across alleles and distinct motifs associated with different peptide lengths. By integrating these data with transcript abundance and peptide processing, we developed HLAthena, providing allele-and-length-specific and pan-allele-pan-length prediction models for endogenous peptide presentation. These models predicted endogenous HLA class I-associated ligands with 1.5-fold improvement in positive predictive value compared with existing tools and correctly identified >75% of HLA-bound peptides that were observed experimentally in 11 patient-derived tumor cell lines. | For primary tumors and patient cell lines, HLA-peptide complexes were immunoprecipitated from 0.1 to 0.2g tissue or up to 50 million cells. Solid tumor samples were dissociated using tissue homogenizer (Fisher Scientific 150) and HLA complexes were enriched as described above. 8-10mm skin punch biopsies were obtained from healthy human skin discarded during skin surgeries. Subcutaneous fat was removed and remaining skin was snap frozen prior to processing. For skin HLA immunoprecipitations, frozen samples were dissociated in lysis buffer using TissueRaptorTM before immunoprecipitation (Qiagen). | Peptides were resuspended in 3% ACN, 5% FA and loaded onto an analytical column (20-30 cm, 1.9 μm C18 Reprosil beads (Dr. Maisch HPLC GmbH), packed in-house PicoFrit 75 μM inner diameter, 10 μM emitter (New Objective)). Peptides were eluted with a linear gradient (EasyNanoLC 1000 or 1200, Thermo Scientific) ranging from 6-30% Buffer B (either 0.1% FA or 0.5% AcOH and 80% or 90% ACN) over 84 min, 30-90% B over 9 min and held at 90% Buffer B for 5 min at 200 nl/min. During data dependent acquisition, peptides were analyzed on a QExactive Plus (QE+), QExactive HF (QE-HF) or Fusion Lumos (Thermo Scientific). | Ovarian cancer patient | surgical resection | Ovarian cancer patient-derived cells were propagated within a xenograft model, which was generated by serial passaging of tumor cells from a patient with advanced ovarian cancer. These cells originated from solid tumor or pleural effusion (3 million cells/mouse) that were injected orthotopically in the abdominal cavity in NOD-SCID mice (8-week old, Jackson labs). Tumor growth was monitored weekly by observing mice for signs of abdominal distension. Cells were harvested 4 months after initial injection and banked for future experiments. For interferon stimulation, cultured cells were stimulated with 2000Unit/ml of IFNγ (Peprotech) for 3 days and were used in IP/MS analysis. | A large peptidome dataset improves HLA class I epitope prediction across most of the human population |
| MSV000084442 | Cancer | Kidney Clear Cell Cancer | Prediction of HLA epitopes is important for the development of cancer immunotherapies and vaccines. However, current prediction algorithms have limited predictive power, in part because they were not trained on high-quality epitope datasets covering a broad range of HLA alleles. To enable prediction of endogenous HLA class I-associated peptides across a large fraction of the human population, we used mass spectrometry to profile >185,000 peptides eluted from 95 HLA-A, -B, -C and -G mono-allelic cell lines. We identified canonical peptide motifs per HLA allele, unique and shared binding submotifs across alleles and distinct motifs associated with different peptide lengths. By integrating these data with transcript abundance and peptide processing, we developed HLAthena, providing allele-and-length-specific and pan-allele-pan-length prediction models for endogenous peptide presentation. These models predicted endogenous HLA class I-associated ligands with 1.5-fold improvement in positive predictive value compared with existing tools and correctly identified >75% of HLA-bound peptides that were observed experimentally in 11 patient-derived tumor cell lines. | For primary tumors and patient cell lines, HLA-peptide complexes were immunoprecipitated from 0.1 to 0.2g tissue or up to 50 million cells. Solid tumor samples were dissociated using tissue homogenizer (Fisher Scientific 150) and HLA complexes were enriched as described above. 8-10mm skin punch biopsies were obtained from healthy human skin discarded during skin surgeries. Subcutaneous fat was removed and remaining skin was snap frozen prior to processing. For skin HLA immunoprecipitations, frozen samples were dissociated in lysis buffer using TissueRaptorTM before immunoprecipitation (Qiagen). | Peptides were resuspended in 3% ACN, 5% FA and loaded onto an analytical column (20-30 cm, 1.9 μm C18 Reprosil beads (Dr. Maisch HPLC GmbH), packed in-house PicoFrit 75 μM inner diameter, 10 μM emitter (New Objective)). Peptides were eluted with a linear gradient (EasyNanoLC 1000 or 1200, Thermo Scientific) ranging from 6-30% Buffer B (either 0.1% FA or 0.5% AcOH and 80% or 90% ACN) over 84 min, 30-90% B over 9 min and held at 90% Buffer B for 5 min at 200 nl/min. During data dependent acquisition, peptides were analyzed on a QExactive Plus (QE+), QExactive HF (QE-HF) or Fusion Lumos (Thermo Scientific). | patients with clear cell renal cell carcinoma (ccRCC) | Surgically resected ccRCC tumor tissue was mechanically dissociated with scalpels | Surgically resected ccRCC tumor tissue was mechanically dissociated with scalpels, and then enzymatically dissociated using a mixture of collagenase D (Roche), Dispase (STEMCELL Technologies), and DNase I (New England BioLabs) at room temperature, and filtered through a 100 micron cell strainer using the sterile plunger of a syringe. Red blood cells were lysed using ammonium-chloride-potassium buffer (Gibco). The cell suspension was stained for viability (Zombie Aqua; BioLegend), anti-CD45 (BV605; BD Biosciences), and anti-carbonic anhydrase IX (PE; R&D Systems). Viable, CD45-negative, CAIX-positive tumor cells were isolated by FACS (BD FACSAria II cell sorter;BD Biosciences). Cells were cultured in a specialized growth medium consisting of OptiMEM GlutaMax media (Gibco), 5% fetal bovine serum, 1mM sodium pyruvate (Gibco), 100 units/mL penicillin and streptomycin, 50 micrograms/mL gentamicin, 5 micrograms/mL insulin (Sigma), and 5 ng/mL epidermal growth factor (Sigma). Following successive passages, CAIX expression was confirmed by flow cytometry (anti-CAIX, PE-conjugated; R&D Systems) and by immunohistochemical analysis of a cell pellet. | A large peptidome dataset improves HLA class I epitope prediction across most of the human population |
| PXD004746 | Cancer | Lymphoma | Cancer somatic mutations can generate neoantigens that distinguish malignant from normal cells. Such neoantigens have been implicated in response to immunotherapies including immune checkpoint blockade, yet their identification and validation remains challenging. Here we discover neoantigens in human mantle cell lymphomas using an integrated strategy for genomic and proteomic tumor antigen discovery that interrogates peptides presented within the tumor major histocompatibility complex (MHC) class I and class II molecules. We applied this approach to systematically identify neoantigen peptides in diagnostic tumor specimens from 17 patients. Remarkably, the 52 discovered neoantigenic peptides were invariably derived from the lymphoma immunoglobulin (Ig) heavy or light chain variable regions. Although we could identify MHC presentation of private germline polymorphic alleles, no mutated peptides were recovered from non-Ig somatically mutated genes. The immunoglobulin variable region somatic mutations were almost exclusively presented by MHC-II. We found T-cells specific for an immunoglobulin-derived neoantigen in the blood of a patient using MHC-II tetramers, and these T-cell clones expanded in frequency following tumor vaccination. These results demonstrate that an integrative approach combining MHC isolation, peptide identification and exome sequencing is an effective platform to uncover tumor neoantigens. Application of this strategy to human lymphoma implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy. | MHC-class I and class II peptidomes were extracted from 17 MCL-patients and 2 cell lines. In brief, cells were lysed and the lysate was subjected to centrifugation and then precleared using rProtein A Sepharose fast-flow beads (GE Healthcare). For all MHC-1 captures the precleared lysate was incubated with the pan HLA-A-, B-, and C- antibody W6/32 coupled to rProtein A Sepharose fast-flow beads. For MHC class-2 captures the precleared lysate was incubated with the HLA-DR specific antibody L243 coupled to rProtein A Sepharose fast-flow beads. Following the immune-capture, the beads were washed with TBS and peptides were eluted from the purified MHC-molecules using 10% acetic acid. The peptides were further purified with a MWCO size filter, followed by a concentration step and then desalted on C18 based StageTips. The isolated peptides were analyzed on an LTQ Orbitrap Elite mass spectrometer (Thermo Fischer Scientific). | For all MHC-1 captures the precleared lysate was incubated with the pan HLA-A-, B-, and C- antibody W6/32 coupled to rProtein A Sepharose fast-flow beads.Following the immune-capture, the beads were washed with TBS and peptides were eluted from the purified MHC-molecules using 10% acetic acid. The peptides were further purified with a MWCO size filter, followed by a concentration step and then desalted on C18 based StageTips. The isolated peptides were analyzed on an LTQ Orbitrap Elite mass spectrometer (Thermo Fischer Scientific). | Samples were collected from patients with untreated MCL by excisional lymph node biopsy, or splenectomy, or peripheral blood leukapheresis, and cryopreserved. | Samples were collected from patients with untreated MCL by excisional lymph node biopsy, or splenectomy, or peripheral blood leukapheresis, and cryopreserved. | Cell lines were grown per ATCC recommendations. | Antigen presentation profiling reveals recognition of lymphoma immunoglobulin neoantigens |
| PXD028309 | Cancer | Colon Carcinoma | Colorectal cancer is the second leading cause of cancer death worldwide, and the incidence of this disease is expected to increase as global socioeconomic changes occur. Immune checkpoint inhibition therapy is effective in treating a minority of colorectal cancer tumors; however, microsatellite stable tumors do not respond well to this treatment. Emerging cancer immunotherapeutic strategies aim to activate a cytotoxic T cell response against tumor-specific antigens, presented exclusively at the cell surface of cancer cells. These antigens are rare and are most effectively identified with a mass spectrometry-based approach, which allows the direct sampling and sequencing of these peptides. While the few tumor-specific antigens identified to date derived from coding regions of the genome, recent findings indicate that a large proportion of tumor-specific antigens originate from allegedly noncoding regions. Here, we employed a novel proteogenomic approach to identify tumor antigens in a collection of colorectal cancer-derived cell lines and biopsy samples consisting of matched tumor and normal adjacent tissue. The generation of personalized cancer databases paired with mass spectrometry analyses permitted the identification of more than 30 000 unique MHC I-associated peptides. We identified 19 putative tumor-specific antigens in both microsatellite stable and unstable tumors, over two-thirds of which were derived from non-coding regions. Many of these peptides were derived from source genes known to be involved in colorectal cancer progression, suggesting that antigens from these genes could have therapeutic potential in a wide range of tumors. These findings could benefit the development of T cell-based vaccines, in which T cells are primed against these antigens to target and eradicate tumors. Such a vaccine could be used in tandem with existing immune checkpoint inhibition therapies, to bridge the gap in treatment efficacy across subtypes of colorectal cancer with varying prognoses. | CRC cell line pellet samples (2 × 108 cells per replicate, four replicates per cell line) were resuspended with PBS up to 2 ml and then solubilized by adding 2 ml of ice-cold 2× lysis buffer (1% w/v CHAPS). Tumor and NAT samples (average 568 mg) were cut into small pieces (cubes, ∼3 mm in size), and 5 ml of ice-cold PBS containing protein inhibitor cocktail (Sigma, cat#P8340-5 ml) was added. Tissues were first homogenized twice for 20 s using an Ultra Turrax T25 homogenizer (IKA-Labortechnik) set at a speed of 20,000 rpm and then 20 s using an Ultra Turrax T8 homogenizer (IKA-Labortechnik) set at speed 25,000 rpm. Then, 550 μl of ice-cold 10× lysis buffer (5% w/v CHAPS) was added to each sample. After 60-min incubation with tumbling at 4 °C, tissue samples and CRC cell line samples were spun at 10,000g for 30 min at 4 °C. Supernatants were transferred into tubes containing 1 mg of W6/32 antibody covalently cross-linked protein A magnetic beads, and MAPs were immunoprecipitated as described (30). MAP extracts were then dried using a Speed-Vac and kept frozen before MS analyses. | MHC class I surface density of the CRC cell lines was determined by Qifikit (Agilent) using the W6/32 anti-HLA class I antibody (BioXCell), according to the manufacturer’s instructions.CRC cell line pellet samples (2 × 108 cells per replicate, four replicates per cell line) were resuspended with PBS up to 2 ml and then solubilized by adding 2 ml of ice-cold 2× lysis buffer (1% w/v CHAPS). Tumor and NAT samples (average 568 mg) were cut into small pieces (cubes, ∼3 mm in size), and 5 ml of ice-cold PBS containing protein inhibitor cocktail (Sigma, cat#P8340-5 ml) was added. Tissues were first homogenized twice for 20 s using an Ultra Turrax T25 homogenizer (IKA-Labortechnik) set at a speed of 20,000 rpm and then 20 s using an Ultra Turrax T8 homogenizer (IKA-Labortechnik) set at speed 25,000 rpm. Then, 550 μl of ice-cold 10× lysis buffer (5% w/v CHAPS) was added to each sample. After 60-min incubation with tumbling at 4 °C, tissue samples and CRC cell line samples were spun at 10,000g for 30 min at 4 °C. Supernatants were transferred into tubes containing 1 mg of W6/32 antibody covalently cross-linked protein A magnetic beads, and MAPs were immunoprecipitated as described (30). MAP extracts were then dried using a Speed-Vac and kept frozen before MS analyses. | Four CRC cell lines (COLO 205 [ATCC CCL-222], HCT 116 [ATCC CCL-247], RKO [ATCC CRL-2577], SW620 [SW-620] [ATCC CCL-227]) and one normal fetal small intestine cell line (HIEC6 [ATCC CRL3266]) were obtained from the American Type Culture Collection (ATCC).Six pairs of primary human samples consisting of matched colon adenocarcinoma tumor and normal adjacent tissue (NAT) were purchased from Tissue Solutions. | Tissue samples were taken from patients receiving surgery as the first line of treatment and were flash frozen in liquid nitrogen. | COLO205, HCT116, and SW620 were grown in RPMI-1640 (Gibco) supplemented with 10% fetal bovine serum (FBS), RKO was grown in Eagle’s minimum essential medium (ATCC) supplemented with 10% FBS, and HIEC-6 was grown in OptiMEM 1 reduced serum medium (Gibco) supplemented with 20 mM Hepes (Gibco), 10 mM GlutaMAX (Gibco), 10 ng/ml epidermal growth factor (Gibco), and FBS to a final concentration of 4%. All cells were maintained at 37 °C with 5% CO2. | Immunopeptidomic analyses of colorectal cancers with and without microsatellite instability. |
| PXD004894 | Cancer | Melanoma | Although mutations may represent attractive targets for immunotherapy, direct identification of mutated peptide ligands isolated from human leukocyte antigens (HLA) on the surface of native tumor tissue has so far not been successful. Using advanced mass spectrometry (MS) analysis, we survey the melanoma-associated immunopeptidome to a depth of 95,500 patient-presented peptides. We thereby discover a large spectrum of attractive target antigen candidates including cancer testis antigens and phosphopeptides. Most importantly, we identify peptide ligands presented on native tumor tissue samples harboring somatic mutations. Four of eleven mutated ligands prove to be immunogenic by neoantigen-specific T-cell responses. Moreover, tumor-reactive T cells with specificity for selected neoantigens identified by MS are detected in the patient`s tumor and peripheral blood. We conclude that direct identification of mutated peptide ligands from primary tumor material by MS is possible and yields true neoepitopes with high relevance for immunotherapeutic strategies in cancer. | For the purification of HLA complexes, snap-frozen melanoma tissue samples were homogenized for 10 s on ice using ULTRA-TURRAX (IKA, Staufen, Germany) in a tube containing 5–10 ml of lysis buffer and incubated at 4 °C for 1 h. The lysis buffer contained 0.25% sodium deoxycholate, 0.2 mM iodoacetamide, 1 mM EDTA, 1:200 Protease Inhibitors Cocktail (Sigma-Aldrich, MO), 1 mM PMSF, 1% octyl-β-D glucopyranoside (Sigma-Aldrich, MO) in PBS. The lysates were cleared by 20 min centrifugation at 40,000g. Lysates were passed through a column containing Protein-A Sepharose beads (Invitrogen, CA) to deplete the endogenous antibodies. Subsequently, HLA-I molecules were immunoaffinity purified from cleared lysate with the W6/32 antibody covalently bound to Protein-A Sepharose beads (Invitrogen, Camarillo, CA). HLA-II molecules were then purified by transferring the flow through onto similar affinity columns containing the HB-145 antibody. Affinity columns were washed first with 10 column volumes of 150 mM NaCl, 20 mM Tris–HCl (buffer A), 10 column volumes of 400 mM NaCl, 20 mM Tris–HCl, 10 volumes of buffer A again, and finally with seven column volumes of 20 mM Tris–HCl, pH 8.0. HLA molecules were eluted at room temperature by adding 500 μl of 0.1 N acetic acid, in total seven elutions for each sample. Eluted HLA peptides and the subunits of the HLA complexes were loaded on Sep-Pak tC18 (Waters, MA) cartridges that were prewashed with 80% acetonitrile (ACN) in 0.1% trifluoracetic acid (TFA) and with 0.1% TFA. The peptides were separated from the much more hydrophobic HLA heavy chains and B2M on the C18 cartridges by eluting them with 30% CAN in 0.1% TFA. They were further purified using a Silica C-18 column tips (Harvard Apparatus, Holliston MA) and eluted again with 30% ACN in 0.1% TFA. The peptides were concentrated and the volume was reduced to 15 μl using vacuum centrifugation. Remaining immunoaffinity purified HLA heavy chains and the B2M molecules were eluted from the Sep-Pak tC18 cartridges with 80% ACN in 0.1%TFA. For western-blot detection, 1% of each of those protein containing samples were used. Anti human B2M antibody EP2978Y (1:5,000, Abcam, Cambridge, United Kingdom) was used and was detected with donkey anti-rabbit IgG HRP conjugate secondary antibody (1:5,000, Thermo Fisher Scientific) in a peroxidase assay using SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific). | For the purification of HLA complexes, snap-frozen melanoma tissue samples were homogenized for 10 s on ice using ULTRA-TURRAX (IKA, Staufen, Germany) in a tube containing 5–10 ml of lysis buffer and incubated at 4 °C for 1 h. The lysis buffer contained 0.25% sodium deoxycholate, 0.2 mM iodoacetamide, 1 mM EDTA, 1:200 Protease Inhibitors Cocktail (Sigma-Aldrich, MO), 1 mM PMSF, 1% octyl-β-D glucopyranoside (Sigma-Aldrich, MO) in PBS. The lysates were cleared by 20 min centrifugation at 40,000g. Lysates were passed through a column containing Protein-A Sepharose beads (Invitrogen, CA) to deplete the endogenous antibodies. Subsequently, HLA-I molecules were immunoaffinity purified from cleared lysate with the W6/32 antibody covalently bound to Protein-A Sepharose beads (Invitrogen, Camarillo, CA). HLA-II molecules were then purified by transferring the flow through onto similar affinity columns containing the HB-145 antibody. Affinity columns were washed first with 10 column volumes of 150 mM NaCl, 20 mM Tris–HCl (buffer A), 10 column volumes of 400 mM NaCl, 20 mM Tris–HCl, 10 volumes of buffer A again, and finally with seven column volumes of 20 mM Tris–HCl, pH 8.0. HLA molecules were eluted at room temperature by adding 500 μl of 0.1 N acetic acid, in total seven elutions for each sample. Eluted HLA peptides and the subunits of the HLA complexes were loaded on Sep-Pak tC18 (Waters, MA) cartridges that were prewashed with 80% acetonitrile (ACN) in 0.1% trifluoracetic acid (TFA) and with 0.1% TFA. The peptides were separated from the much more hydrophobic HLA heavy chains and B2M on the C18 cartridges by eluting them with 30% CAN in 0.1% TFA. They were further purified using a Silica C-18 column tips (Harvard Apparatus, Holliston MA) and eluted again with 30% ACN in 0.1% TFA. The peptides were concentrated and the volume was reduced to 15 μl using vacuum centrifugation. Remaining immunoaffinity purified HLA heavy chains and the B2M molecules were eluted from the Sep-Pak tC18 cartridges with 80% ACN in 0.1%TFA. For western-blot detection, 1% of each of those protein containing samples were used. Anti human B2M antibody EP2978Y (1:5,000, Abcam, Cambridge, United Kingdom) was used and was detected with donkey anti-rabbit IgG HRP conjugate secondary antibody (1:5,000, Thermo Fisher Scientific) in a peroxidase assay using SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific). | Tumor tissue samples from 25 melanoma patients. | Tumour tissue samples were collected from patients, who underwent tumour resection at the Department of Surgery, Klinikum rechts der Isar of the TU München. Immediately after resection (within 30 min), tumour tissue was macroscopically dissected by an experienced pathologist, snap frozen and stored in liquid nitrogen (−196 °C) at the MRI-TUM-Biobank (MTBIO) until usage. Additional tumour tissue was formalin-fixed and paraffin-embedded (FFPE). | | Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry |
| PXD007635 | Cancer | Ovarian Cancer | Immunopeptidome analysis of ovarian carcinoma tissues was performed to characterize the HLA class I and class II presented ligandome of this malignancy. | HLA class I and II molecules were isolated by standard immunoaffinity purification as described previously (PMID: 23329485). Pan-HLA class I-specific mAb W6/32 was employed for HLA class I isolation and pan-HLA class II mAb Tü39 as well as HLA-DR-specific mAb L243 were used for HLA class II isolation. Between 0.5 g and 3 g of tissue samples were employed for lysis and HLA ligand isolation. | HLA class I and II molecules were isolated by standard immunoaffinity purification as described previously (PMID: 23329485). Pan-HLA class I-specific mAb W6/32 was employed for HLA class I isolation and pan-HLA class II mAb Tü39 as well as HLA-DR-specific mAb L243 were used for HLA class II isolation. Between 0.5 g and 3 g of tissue samples were employed for lysis and HLA ligand isolation. | EOC, benign ovary, and fallopian tube tissues | EOC, benign ovary, and fallopian tube tissues were freshly collected from patients undergoing tumor resection/ debulking or salpingoophorectomy. | | The immunopeptidomic landscape of ovarian carcinomas. |
| PXD007935 | Cancer | B-Acute Lymphoblastic Leukemia | Significant technological advances in both affinity chromatography and mass spectrometry have facilitated the identification of peptides associated with the major histocompatibility complex class I (MHC I) molecules, and enabled a greater understanding of the dynamic nature of the immunopeptidome of normal and neoplastic cells. While the isolation of MHC I-associated peptides (MIPs) typically used mild acid elution (MAE) or immunoprecipitation (IP), limited information currently exists regarding their respective analytical merits. Here, we present a comparison of these approaches for the isolation of two different B-cell lymphoblasts cell models, and report on the recovery, reproducibility, scalability and complementarity of identification from each method. Both approaches yielded reproducible datasets for peptide extracts obtained from 2 to 100 million cells, with 2016 to 5093 MIPs, respectively. The IP typically provides up to 6.4 fold increase in MIPs compared to the MAE. We extended the comprehensiveness of these immunopeptidome analyses using personalized genomic database of B-cell lymphoblasts, and discovered that 0.4 % of their respective MIP repertoire harbored non-synonymous single nucleotide variations (also known as minor histocompatibility antigens, MiHAs). | The absolute membrane density of MHC I was evaluated by indirect labeling with a purified anti-human HLA-ABC (clone W6/32), using commercially available QIFIKIT® (Dako) according to the manufacturer’s instructions. Peptide isolation by immunoprecipitation The W6/32 antibodies were incubated in PBS for 60 minutes at room temperature with PureProteome protein A magnetic beads at a ratio of 1 mg of antibody per mL of slurry. Antibodies were covalently cross-linked to magnetic beads using dimethylpimelidate. Biological replicates of cell pellets were resuspended in 1 mL PBS pH 7.2 and solubilized by adding 1 mL of detergent buffer containing PBS pH 7.2, 1% (w/v) CHAPS supplemented with Protease inhibitor cocktail. After 60 minute incubation with tumbling at 4°C, samples were spun at 10000g for 30 minutes at 4°C. Post-nuclear supernatants were transferred into new tubes containing magnetic beads coupled to W6/32 antibodies at a ratio of 10 mg of W6/32 antibody per 1 × 106 cells. Samples were incubated with tumbling for 180 minutes at 4°C and placed on a magnet to recover bound MHC I complexes to magnetic beads. Magnetic beads were first washed with 4 × 1 mL PBS, then with 1 × 1 mL of 0.1X PBS and finally with 1 × 1 mL of water. MHC I complexes were eluted from the magnetic beads by acidic treatment using 0.2% trifluoroacetic acid (TFA). To remove any residual magnetic beads, eluates were transferred into 2.0 mL Costar mL Spin-X centrifuge tube filters (0.45 mm) and spun 2 minutes at 3000g. Filtrates containing peptides were separated from MHC I subunits (HLA molecules and b-2 macroglobulin) using home-made stage tips packed with twenty 1 mm diameter octadecyl (C-18) solid phase extraction disks. Stage tips were pre-washed first with methanol then with 80% acetonitrile (ACN) in 0.2% TFA and finally with 0.1% formic acid (FA). Samples were loaded onto the stage tips and the peptides were retained on the stage tips while the HLA molecules and b-2 macroglobulin were found in the flow through. Stage tips were washed with 0.1% FA and peptides were eluted with 30% ACN in 0.1%TFA. The peptides were dried using vacuum centrifugation and then stored at -20°C until MS analysis. Peptide isolation by mild acid elution Peptides were released by mild acid elution using citrate pH 3.3 buffer. Samples were desalted using an HLB cartridge and filtered with a 3,000 Da cut-off column. | The absolute membrane density of MHC I was evaluated by indirect labeling with a purified anti-human HLA-ABC (clone W6/32), using commercially available QIFIKIT® (Dako) according to the manufacturer’s instructions. Peptide isolation by immunoprecipitation The W6/32 antibodies were incubated in PBS for 60 minutes at room temperature with PureProteome protein A magnetic beads at a ratio of 1 mg of antibody per mL of slurry. Antibodies were covalently cross-linked to magnetic beads using dimethylpimelidate. | B-ALL specimen used in this study was from an adult male B-ALL patient. | B-ALL specimen used in this study was from an adult male B-ALL patient. | | Comparison of the MHC I Immunopeptidome Repertoire of B-Cell Lymphoblasts using Two Isolation Methods |
| PXD008127 | Cancer | Glioblastoma | Glioblastoma multiforme (GBM) is the most aggressive brain tumor with poor prognosis to most patients. Immunotherapy of GBM is a potentially beneficial treatment option, whose optimal implementation may depend on familiarity with tumor specific antigens, presented as HLA peptides by the GBM cells. Furthermore, early detection of GBM, such as by a routine blood test, may improve survival, even with the current treatment modalities. This study includes large-scale analyses of the HLA peptidome (immunopeptidome) of the plasma-soluble HLA molecules (sHLA) of 142 plasma samples and the membranal HLA of GBM tumors of 10 tumor samples of the same patients. Tumor samples were fresh-frozen immediately after surgery and plasma samples were collected before, and at multiple visits after surgery. In total, this HLA peptidome analysis involved 52 different HLA allotypes and resulted in the identification of more than 35,000 different HLA peptides. Strong correlations were observed in the signal intensities and in the repertoires of identified peptides between the tumors and plasma-soluble HLA peptidomes of the same patients; yet, low correlations were observed between these HLA peptidomes and the tumors’ proteomes. HLA peptides derived from Cancer/Testis Antigens (CTAs) were selected based on their presence among the HLA peptidomes of the patients and absence of expression of their source genes from any healthy and essential human tissues, except from immune-privileged sites. Additionally, peptides were selected as potential biomarkers if their levels in the plasma-sHLA peptidome were significantly reduced after the removal of tumor mass. The CTAs identified among the analyzed HLA peptidomes provide new opportunities for personalized immunotherapy and for early diagnosis of GBM. | HLA class I molecules from the cleared lysate or from the fresh human plasma were immunoaffinity purified using the W6/32 mAb bound to Amino-Link beads (Thermo-Fisher Scientific) as in (28, 46). The HLA molecules with their bound peptides were eluted from the affinity column with five column volumes of 1% TFA. The eluted HLA class I proteins, and the released peptides were loaded on disposable C18 micro-columns (Harvard Apparatus, Holliston, MA), and the peptides fraction was recovered with 30% acetonitrile in 0.1% TFA, whereas the protein fraction was recovered with 80% acetonitrile in 0.1% TFA, as in (46). The peptide fractions were dried using vacuum centrifugation, reconstituted in 100 μl of 0.1% TFA, reloaded on C18 Stage-Tips (47), eluted with 80% acetonitrile, dried, and reconstituted with 0.1% formic acid for LC-MS/MS analysis. | HLA class I molecules from the cleared lysate or from the fresh human plasma were immunoaffinity purified using the W6/32 mAb bound to Amino-Link beads (Thermo-Fisher Scientific) as in (28, 46). The HLA molecules with their bound peptides were eluted from the affinity column with five column volumes of 1% TFA. The eluted HLA class I proteins, and the released peptides were loaded on disposable C18 micro-columns (Harvard Apparatus, Holliston, MA), and the peptides fraction was recovered with 30% acetonitrile in 0.1% TFA, whereas the protein fraction was recovered with 80% acetonitrile in 0.1% TFA, as in (46). The peptide fractions were dried using vacuum centrifugation, reconstituted in 100 μl of 0.1% TFA, reloaded on C18 Stage-Tips (47), eluted with 80% acetonitrile, dried, and reconstituted with 0.1% formic acid for LC-MS/MS analysis. | tumor samples were obtained from 10 of the 52 GBM patients for mHLA peptidome analysis, as well as for tumor proteome analysis. | Tumor samples were frozen in liquid nitrogen immediately after dissection and stored at −80 °C until use, for both mHLA peptidome and proteome analyses of the same tissue extracts. | | Identification of Tumor Antigens Among the HLA Peptidomes of Glioblastoma Tumors and Plasma. |
| PXD009738 | Normal | Breast | Here we analysed the immunopeptidomes of 6 HLA-A2-positive triple negative breast cancer (TNBC) samples by nano-ultra performance liquid chromatography tandem mass spectrometry (nUPLC-MS2). | | One mg per sample of human anti-HLA class I antibody (W6/32, ATCC HB-95) was bound and cross-linked to 1 ml Protein A beads (GE healthcare) and used for immunoprecipitation of HLA complexes. In brief, lysates were incubated with the antibody beads over night at 4ºC and washed subsequently with 50 mM Tris, pH8.0 containing either 150 mM, 450 mM and finally no salt. Peptides were eluted with 5 ml of 10% acetic acid. Dried peptides were resuspended and injected onto a 4.6 x 50 mm ProSwift RP-1S column (Thermo Fisher Scientific). Peptides were separated from larger complex components by elution using a 500 μl/min flow rate over 10 min from 2-25% ACN in 0.1% TFA. Alternate fractions were pooled and two final fractions were analysed by nUPLC-MS2. HLA peptides eluted from tissues were separated on an Ultimate 3000 RSLCnano system (Thermo Scientific) using a PepMap C18 column, 2 μm particle size, 75 μm x 50 cm (Thermo Scienific) with a 30 min (two technical replicates) and 1h (single run) linear gradient of 3-25% buffer B (0.1% formic acid, 5% DMSO in acetonitrile) in buffer A (0.1% formic acid, 5% DMSO in water) at a flowrate of 250 µl/min. Peptides were introduced using an Easy-Spray source at 2000V and to a Fusion Lumos (Thermo Scientific). | Six pairs of TNBC and matching normal tissues | TNBC biopsies | | Immunopeptidomic Profiling of HLA-A2-Positive Triple Negative Breast Cancer Identifies Potential Immunotherapy Target Antigens. Proteomics. |
| PXD009738 | Cancer | Breast Cancer | Here we analysed the immunopeptidomes of 6 HLA-A2-positive triple negative breast cancer (TNBC) samples by nano-ultra performance liquid chromatography tandem mass spectrometry (nUPLC-MS2). | Approximately 0.5 cm3 of breast cancer and matching adjacent normal tissue biopsy material was homogenized in lysis buffer (1% Igepal, 300 mM sodium chloride, 100 mM Tris, pH 8.0) supplemented with protease inhibitor cocktail (Roche) using a bead beater (Precellys 24 bead-beater, Bertin Technologies) five times for 10 s at 6500 rpm. Lysates were cleared by subsequent centrifugation steps at 300 × g for 10 min and then 20 000 × g for 60 min. One milligram per sample of human anti-HLA class I antibody (W6/32, ATCC HB-95) was bound and cross-linked to 1 mL Protein A beads (GE Healthcare) and used for immunoprecipitation of HLA complexes as described previously.16 In brief, lysates were incubated with the antibody beads overnight at 4 °C and washed subsequently with 50 mM Tris, pH 8.0 containing first 150 mM, then 450 mM and finally 0 mM NaCl. Peptides were eluted with 5 mL of 10% acetic acid. Dried peptides were resuspended and injected onto a 4.6 × 50 mm ProSwift RP-1S column (Thermo Fisher Scientific). Peptides were separated from larger complex components by elution using a 500 μL min−1 flow rate over 10 min from 2 to 25% acetonitrile in 0.1% trifluoroacetic acid. Alternate fractions were pooled and two final fractions were analyzed by nano-ultra performance liquid chromatography tandem mass spectrometry (nUPLC-MS2). | One mg per sample of human anti-HLA class I antibody (W6/32, ATCC HB-95) was bound and cross-linked to 1 ml Protein A beads (GE healthcare) and used for immunoprecipitation of HLA complexes. In brief, lysates were incubated with the antibody beads over night at 4ºC and washed subsequently with 50 mM Tris, pH8.0 containing either 150 mM, 450 mM and finally no salt. Peptides were eluted with 5 ml of 10% acetic acid. Dried peptides were resuspended and injected onto a 4.6 x 50 mm ProSwift RP-1S column (Thermo Fisher Scientific). Peptides were separated from larger complex components by elution using a 500 μl/min flow rate over 10 min from 2-25% ACN in 0.1% TFA. Alternate fractions were pooled and two final fractions were analysed by nUPLC-MS2. HLA peptides eluted from tissues were separated on an Ultimate 3000 RSLCnano system (Thermo Scientific) using a PepMap C18 column, 2 μm particle size, 75 μm x 50 cm (Thermo Scienific) with a 30 min (two technical replicates) and 1h (single run) linear gradient of 3-25% buffer B (0.1% formic acid, 5% DMSO in acetonitrile) in buffer A (0.1% formic acid, 5% DMSO in water) at a flowrate of 250 µl/min. Peptides were introduced using an Easy-Spray source at 2000V and to a Fusion Lumos (Thermo Scientific). | Six pairs of TNBC and matching normal tissues | TNBC biopsies | | Immunopeptidomic Profiling of HLA-A2-Positive Triple Negative Breast Cancer Identifies Potential Immunotherapy Target Antigens. Proteomics. |
| PXD015957 | Cancer | Melanoma | Predicting the outcome of immunotherapy treatment in melanoma patients is challenging. Alterations in genes involved in antigen presentation and the interferon gamma (IFNγ) pathway play an important role in the immune response to tumors. We describe here that the overexpression of PSMB8 and PSMB9, two components of the immunoproteasome, is predictive of better survival and improved response to immune-checkpoint inhibitors of melanoma patients. We study the mechanism that underlies this connection by analyzing the antigenic peptide repertoire of cells that overexpress these subunits using HLA peptidomics. We find a higher response of patient-matched tumor infiltrating lymphocytes against antigens deferentially presented after immunoproteasome overexpression. which may explain the higher immune infiltration observed in patients with high immunoproteasome expression levels. Importantly, we find that PSMB8 and PSMB9 expression levels are much stronger predictors of melanoma patients immune response to checkpoint inhibitors than the tumors mutational burden. Taken together, these results suggest that their expression levels can serve as important biomarkers for stratifying melanoma patients for immune-checkpoint treatment. | For the HLA peptidomics analysis we used three experimental replicates per each cell line and each condition (empty vector/ overexpression and treated/ non-treated cells). Samples were processed as described previously23,25,36. Briefly, cell pellets were lysed with lysis buffer containing 0.25% sodium deoxycholate, 0.2 mM iodoacetamide, 1 mM EDTA, 1:200 protease inhibitors cocktail (Sigma-Aldrich), 1 mM PMSF and 1%octyl-b-D glucopyranoside in PBS, and then incubated at 4 °C for 1 h. The lysates were cleared by centrifugation at 4 °C and 48,000g for 60 min, and then passed through a pre-clearing column containing Protein-A Sepharose beads.HLA-I molecules were immunoaffinity purified from cleared lysate with the pan-HLA-I antibody (W6/32 antibody purified from HB95 hybridoma cells) covalently bound to Protein-A Sepharose beads (Thermo Fisher Scientific, as in ref. 23). Affinity column was washed first with 10 column volumes of 400 mM NaCl, 20 mM Tris–HCl, pH 8.0 and then with 10 volumes of 20 mM Tris-HCl, pH 8.0. The HLA peptides and HLA molecules were eluted with 1% TFA followed by separation of the peptides from the proteins by binding the eluted fraction to disposable reversed-phase C18 columns (Harvard Apparatus) as in ref. 37. Elution of the peptides was done with 30% acetonitrile (ACN) in 0.1% trifluoracetic acid (TFA). The eluted peptides were cleaned also by C18 stage tip38. | For the HLA peptidomics analysis we used three experimental replicates per each cell line and each condition (empty vector/ overexpression and treated/ non-treated cells). Samples were processed as described previously23,25,36. Briefly, cell pellets were lysed with lysis buffer containing 0.25% sodium deoxycholate, 0.2 mM iodoacetamide, 1 mM EDTA, 1:200 protease inhibitors cocktail (Sigma-Aldrich), 1 mM PMSF and 1%octyl-b-D glucopyranoside in PBS, and then incubated at 4 °C for 1 h. The lysates were cleared by centrifugation at 4 °C and 48,000g for 60 min, and then passed through a pre-clearing column containing Protein-A Sepharose beads.HLA-I molecules were immunoaffinity purified from cleared lysate with the pan-HLA-I antibody (W6/32 antibody purified from HB95 hybridoma cells) covalently bound to Protein-A Sepharose beads (Thermo Fisher Scientific, as in ref. 23). Affinity column was washed first with 10 column volumes of 400 mM NaCl, 20 mM Tris–HCl, pH 8.0 and then with 10 volumes of 20 mM Tris-HCl, pH 8.0. The HLA peptides and HLA molecules were eluted with 1% TFA followed by separation of the peptides from the proteins by binding the eluted fraction to disposable reversed-phase C18 columns (Harvard Apparatus) as in ref. 37. Elution of the peptides was done with 30% acetonitrile (ACN) in 0.1% trifluoracetic acid (TFA). The eluted peptides were cleaned also by C18 stage tip38. | Cell lines 12T and 108T and their TILs were derived from pathologyconfirmed metastatic melanoma tumor resections collected from patients enrolled in institutional review board (IRB)-approved clinical trials at the Surgery Branch of the National Cancer Institute | Cell lines 12T and 108T and their TILs were derived from pathologyconfirmed metastatic melanoma tumor resections collected from patients enrolled in institutional review board (IRB)-approved clinical trials at the Surgery Branch of the National Cancer Institute | | Immunoproteasome expression is associated with better prognosis and response to checkpoint therapies in melanoma. |
| PXD017149 | Cancer | Kidney Clear Cell Cancer | Clear cell renal cell carcinoma (ccRCC) is the dominant subtype of renal cancer. With currently available therapies, cure of advanced and metastatic ccRCC is achieved only in rare cases. Here, we developed a workflow integrating different -omics technologies to identify ccRCC-specific HLA-presented peptides as potential drug targets for ccRCC immunotherapy. We analyzed frequent ccRCC-specific peptides by MS-based HLA ligandomics of 55 ccRCC tumors (cohort 1), paired non-tumor renal tissues and 158 benign tissues from other organs. Pathways enriched in ccRCC compared to its cell type of origin were identified by transcriptome and gene set enrichment analyses in 51 tumor tissues of the same cohort. To retrieve a list of candidate target genes with involvement in ccRCC pathogenesis, ccRCC-specific pathway genes were intersected with the source genes of tumor-exclusive peptides. The candidates were validated in an independent cohort from the Cancer Genome Atlas (TCGA KIRC, n=452), yielding 113 candidate genes. DNA methylation (TCGA KIRC, n=273), and somatic mutations (TCGA KIRC, n=392), as well as correlations with tumor metabolites (cohort 1, n=30) and immune-oncological markers (cohort 1, n=37) were analyzed to refine regulatory and functional involvements of candidates. Immunogenicity analysis identified candidate epitopes able to activate native CD8+ T cells. Functional analysis of EGLN3, a candidate with frequent ccRCC-specific immunogenic peptides, revealed possible tumor-promoting functions. Integration of HLA ligandomics, transcriptomics, genetic and epigenetic data leads to the identification of novel functionally relevant therapeutic targets for ccRCC immunotherapy. Validation of the identified targets is now mandatory to expand the treatment landscape of ccRCC. | HLA ligandomics was performed by reversed phase liquid chromatography coupled mass spectrometry as previously described (Löffler et al. Genome Med 2019, Kowalewski and Stevanović Methods Mol Biol 2013, Nelde et al. Methods Mol Biol 2019). The monoclonal antibodies W6/32, Tü39 and L243 (in-house production at the Department of Immunology, University of Tübingen, Tübingen, Germany) were used for immunoaffinity purification of HLA class I and II-peptide complexes. Five technical replicate LC-MS runs were acquired per sample. | HLA ligandomics was performed by reversed phase liquid chromatography coupled mass spectrometry as previously described (Löffler et al. Genome Med 2019, Kowalewski and Stevanović Methods Mol Biol 2013, Nelde et al. Methods Mol Biol 2019). The monoclonal antibodies W6/32, Tü39 and L243 (in-house production at the Department of Immunology, University of Tübingen, Tübingen, Germany) were used for immunoaffinity purification of HLA class I and II-peptide complexes. Five technical replicate LC-MS runs were acquired per sample. | Primary ccRCC tumors and paired non-tumor renal tissues (n = 55) were collected | surgical resection | | Integrative -omics and HLA-ligandomics analysis to identify novel drug targets for ccRCC immunotherapy. |
| PXD017149 | Normal | Kidney | Clear cell renal cell carcinoma (ccRCC) is the dominant subtype of renal cancer. With currently available therapies, cure of advanced and metastatic ccRCC is achieved only in rare cases. Here, we developed a workflow integrating different -omics technologies to identify ccRCC-specific HLA-presented peptides as potential drug targets for ccRCC immunotherapy. We analyzed frequent ccRCC-specific peptides by MS-based HLA ligandomics of 55 ccRCC tumors (cohort 1), paired non-tumor renal tissues and 158 benign tissues from other organs. Pathways enriched in ccRCC compared to its cell type of origin were identified by transcriptome and gene set enrichment analyses in 51 tumor tissues of the same cohort. To retrieve a list of candidate target genes with involvement in ccRCC pathogenesis, ccRCC-specific pathway genes were intersected with the source genes of tumor-exclusive peptides. The candidates were validated in an independent cohort from the Cancer Genome Atlas (TCGA KIRC, n=452), yielding 113 candidate genes. DNA methylation (TCGA KIRC, n=273), and somatic mutations (TCGA KIRC, n=392), as well as correlations with tumor metabolites (cohort 1, n=30) and immune-oncological markers (cohort 1, n=37) were analyzed to refine regulatory and functional involvements of candidates. Immunogenicity analysis identified candidate epitopes able to activate native CD8+ T cells. Functional analysis of EGLN4, a candidate with frequent ccRCC-specific immunogenic peptides, revealed possible tumor-promoting functions. Integration of HLA ligandomics, transcriptomics, genetic and epigenetic data leads to the identification of novel functionally relevant therapeutic targets for ccRCC immunotherapy. Validation of the identified targets is now mandatory to expand the treatment landscape of ccRCC. | HLA ligandomics was performed by reversed phase liquid chromatography coupled mass spectrometry as previously described (Löffler et al. Genome Med 2019, Kowalewski and Stevanović Methods Mol Biol 2013, Nelde et al. Methods Mol Biol 2019). The monoclonal antibodies W6/32, Tü39 and L243 (in-house production at the Department of Immunology, University of Tübingen, Tübingen, Germany) were used for immunoaffinity purification of HLA class I and II-peptide complexes. Five technical replicate LC-MS runs were acquired per sample. | HLA ligandomics was performed by reversed phase liquid chromatography coupled mass spectrometry as previously described (Löffler et al. Genome Med 2019, Kowalewski and Stevanović Methods Mol Biol 2013, Nelde et al. Methods Mol Biol 2019). The monoclonal antibodies W6/32, Tü39 and L243 (in-house production at the Department of Immunology, University of Tübingen, Tübingen, Germany) were used for immunoaffinity purification of HLA class I and II-peptide complexes. Five technical replicate LC-MS runs were acquired per sample. | Primary ccRCC tumors and paired non-tumor renal tissues (n = 55) were collected | surgical resection | | Integrative -omics and HLA-ligandomics analysis to identify novel drug targets for ccRCC immunotherapy. |
| MSV000084442 | Cancer | Chronic Lymphocytic Leukemia | Prediction of HLA epitopes is important for the development of cancer immunotherapies and vaccines. However, current prediction algorithms have limited predictive power, in part because they were not trained on high-quality epitope datasets covering a broad range of HLA alleles. To enable prediction of endogenous HLA class I-associated peptides across a large fraction of the human population, we used mass spectrometry to profile >185,000 peptides eluted from 95 HLA-A, -B, -C and -G mono-allelic cell lines. We identified canonical peptide motifs per HLA allele, unique and shared binding submotifs across alleles and distinct motifs associated with different peptide lengths. By integrating these data with transcript abundance and peptide processing, we developed HLAthena, providing allele-and-length-specific and pan-allele-pan-length prediction models for endogenous peptide presentation. These models predicted endogenous HLA class I-associated ligands with 1.5-fold improvement in positive predictive value compared with existing tools and correctly identified >75% of HLA-bound peptides that were observed experimentally in 11 patient-derived tumor cell lines. | For primary tumors and patient cell lines, HLA-peptide complexes were immunoprecipitated from 0.1 to 0.2g tissue or up to 50 million cells. Solid tumor samples were dissociated using tissue homogenizer (Fisher Scientific 150) and HLA complexes were enriched as described above. 8-10mm skin punch biopsies were obtained from healthy human skin discarded during skin surgeries. Subcutaneous fat was removed and remaining skin was snap frozen prior to processing. For skin HLA immunoprecipitations, frozen samples were dissociated in lysis buffer using TissueRaptorTM before immunoprecipitation (Qiagen). | Peptides were resuspended in 3% ACN, 5% FA and loaded onto an analytical column (20-30 cm, 1.9 μm C18 Reprosil beads (Dr. Maisch HPLC GmbH), packed in-house PicoFrit 75 μM inner diameter, 10 μM emitter (New Objective)). Peptides were eluted with a linear gradient (EasyNanoLC 1000 or 1200, Thermo Scientific) ranging from 6-30% Buffer B (either 0.1% FA or 0.5% AcOH and 80% or 90% ACN) over 84 min, 30-90% B over 9 min and held at 90% Buffer B for 5 min at 200 nl/min. During data dependent acquisition, peptides were analyzed on a QExactive Plus (QE+), QExactive HF (QE-HF) or Fusion Lumos (Thermo Scientific). | PBMC from patients with chronic lymphocytic leukemia (CLL) were enriched for CD19 positive CLL tumor cells and were used in IP/MS analysis. | | Cells were cultured in a specialized growth medium consisting of OptiMEM GlutaMax media (Gibco), 5% fetal bovine serum, 1mM sodium pyruvate (Gibco), 100 units/mL penicillin and streptomycin, 50 micrograms/mL gentamicin, 5 micrograms/mL insulin (Sigma), and 5 ng/mL epidermal growth factor (Sigma). | A large peptidome dataset improves HLA class I epitope prediction across most of the human population |
| MSV000084442 | Cancer | Glioblastoma | Prediction of HLA epitopes is important for the development of cancer immunotherapies and vaccines. However, current prediction algorithms have limited predictive power, in part because they were not trained on high-quality epitope datasets covering a broad range of HLA alleles. To enable prediction of endogenous HLA class I-associated peptides across a large fraction of the human population, we used mass spectrometry to profile >185,000 peptides eluted from 95 HLA-A, -B, -C and -G mono-allelic cell lines. We identified canonical peptide motifs per HLA allele, unique and shared binding submotifs across alleles and distinct motifs associated with different peptide lengths. By integrating these data with transcript abundance and peptide processing, we developed HLAthena, providing allele-and-length-specific and pan-allele-pan-length prediction models for endogenous peptide presentation. These models predicted endogenous HLA class I-associated ligands with 1.5-fold improvement in positive predictive value compared with existing tools and correctly identified >75% of HLA-bound peptides that were observed experimentally in 11 patient-derived tumor cell lines. | For primary tumors and patient cell lines, HLA-peptide complexes were immunoprecipitated from 0.1 to 0.2g tissue or up to 50 million cells. Solid tumor samples were dissociated using tissue homogenizer (Fisher Scientific 150) and HLA complexes were enriched as described above. 8-10mm skin punch biopsies were obtained from healthy human skin discarded during skin surgeries. Subcutaneous fat was removed and remaining skin was snap frozen prior to processing. For skin HLA immunoprecipitations, frozen samples were dissociated in lysis buffer using TissueRaptorTM before immunoprecipitation (Qiagen). | Peptides were resuspended in 3% ACN, 5% FA and loaded onto an analytical column (20-30 cm, 1.9 μm C18 Reprosil beads (Dr. Maisch HPLC GmbH), packed in-house PicoFrit 75 μM inner diameter, 10 μM emitter (New Objective)). Peptides were eluted with a linear gradient (EasyNanoLC 1000 or 1200, Thermo Scientific) ranging from 6-30% Buffer B (either 0.1% FA or 0.5% AcOH and 80% or 90% ACN) over 84 min, 30-90% B over 9 min and held at 90% Buffer B for 5 min at 200 nl/min. During data dependent acquisition, peptides were analyzed on a QExactive Plus (QE+), QExactive HF (QE-HF) or Fusion Lumos (Thermo Scientific). | Snap frozen meningioma tissues from patients (3830-NJF, 3849-BR, 3912-BAM, 3865-DM) were obtained from the University Hospital of Lausanne (CHUV, Lausanne, Switzerland). | | | A large peptidome dataset improves HLA class I epitope prediction across most of the human population;High-throughput and Sensitive Immunopeptidomics Platform Reveals Profound Interferonγ-Mediated Remodeling of the Human Leukocyte Antigen (HLA) Ligandome |
| MSV000084787 | Cancer | Melanoma | Tumor epitopes, peptides that are presented on surface-bound MHC I proteins, provide targets for cancer immunotherapy and have been identified extensively in the annotated protein-coding regions of the genome. Motivated by the recent discovery of translated novel unannotated open reading frames (nuORFs) using ribosome profiling (Ribo-seq), we hypothesized that cancer-associated processes could generate nuORFs that can serve as a new source of tumor antigens that harbor somatic mutations or show tumor-specific expression. To identify cancer-specific nuORFs, we generated Ribo-seq profiles for 29 malignant and healthy samples, developed a sensitive analytic approach for hierarchical ORF prediction, and constructed a high-confidence database of translated nuORFs across tissues. Peptides from 3,555 unique translated nuORFs were presented on MHC I, based on analysis of an extensive dataset of MHC I-bound peptides detected by mass spectrometry, with >20-fold more nuORF peptides detected in the MHC I immunopeptidomes compared to whole proteomes. We further detected somatic mutations in nuORFs of cancer samples and identified nuORFs with tumor-specific translation in melanoma, chronic lymphocytic leukemia and glioblastoma. NuORFs thus expand the pool of MHC I-presented, tumor-specific peptides, targetable by immunotherapies. | Soluble lysates from up to 50 million HLA expressing B721.221 cells or 0.1 to 0.2g cancer cells were immunoprecipitated with W6/32 antibody (sc-32235, Santa Cruz) as described previously1,2. 10 mM iodoacetamide was added to the lysis buffer to alkylate cysteines during the lysis and incubation step (3h, 4C) (Supplementary Note 2) for 71 alleles and 10 tumor samples (Supplementary Table 7). | Peptides were resuspended in 3% ACN, 5% FA and loaded onto an analytical column (20-30 cm, 1.9 µm C18 Reprosil beads (Dr. Maisch HPLC GmbH), packed in-house PicoFrit 75 µm inner diameter, 10 µm emitter (New Objective)). Peptides were eluted with a linear gradient (EasyNanoLC 1000 or 1200, ThermoFisher Scientific) ranging from 6-30% Buffer B (either 0.1% FA or 0.5% AcOH and 80% or 90% ACN) over 84 min, 30-90% B over 9 min and held at 90% Buffer B for 5 min at 200 nl/min. | A375 cells were cultured in DMEM media (Gibco), supplemented with 5% fetal bovine serum (FBS). | | A375 cells were cultured in DMEM media (Gibco), supplemented with 5% fetal bovine serum (FBS). | Unannotated proteins expand the MHC-I-restricted immunopeptidome in cancer |
| MSV000087743 | Cancer | Melanoma | Mass spectrometry is the most effective method to directly identify peptides presented on HLA molecules. However, current standard approaches often use 500 million or more cells as input to achieve high coverage of the immunopeptidome and therefore these methods are not compatible with the often limited amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples off-line followed by ion mobility coupled to LC-MS/MS for analysis. The combination of these two separation methods enabled identification of 20% to 50% more peptides compared to samples analyzed without either prior fractionation or use of ion mobility alone. We demonstrate coverage of HLA immunopeptidomes with up to 8,107 distinct peptides starting with as few as 100 million cells. The increased sensitivity obtained using our methods can provide data useful to improve HLA binding prediction algorithms as well as to enable detection of clinically relevant epitopes such as neoantigens. | Soluble lysates from up to 50 million cells and up to 0.2 g from tumor tissue or tumor-derived cell lines were immunoprecipitated with W6/32 antibody (sc-32235; Santa Cruz) as described previously (8) (supplemental Table S1). Iodoacetamide (10 mM) was added to the lysis buffer to alkylate cysteines. For each experiment, two IPs were pooled (equivalent to 100 × 106 cells), HLA-I bound peptides were acid eluted on 1 cc 50 mg tC18 SepPak Cartridges (WAT054960; Waters) on a vacuum manifold 1 to 2 days prior to analysis on the instrument. SepPaks are equilibrated with 200 μl MeOH 2×, 100 μl 50% acetonitrile (ACN)/1% formic acid (FA), and 500 μl 1% FA 4×, respectively. Beads with HLA-I bound peptides were resuspended in 3% ACN/5% FA and transferred to the equilibrated cartridge. Peptides were eluted from HLA-I proteins with 10% acetic acid for 5 min twice and then desalted with 4× 500 μl 1% FA. Finally, peptides were eluted into the same tube from the desalt matrix using 250 μl 15% ACN/1% FA followed by 250 μl 50% ACN/1% FA and dried down. This step was performed for every sample described here. Patient samples were typically immunoprecipitated from the same lysate pool but eluted on different days to prevent/reduce peptide loss because of peptide degradation and adsorption to plastic while waiting for analysis. An overview of cell input and processing details for each sample is provided in supplemental Table S1. | For primary tumors and patient-derived cell lines, HLA–peptide complexes were immunoprecipitated from 0.1 to 0.2 g tissue or up to 50 million cells. Solid tumor samples were dissociated using a tissue homogenizer (Fisher Scientific 150 Homogenizer package; Fisher Scientific), and HLA complexes were enriched as described later. | melanoma (MEL) tumor cell lines | | | Optimized Liquid and Gas Phase Fractionation Increases HLA-Peptidome Coverage for Primary Cell and Tissue Samples |
| MSV000087927 | Cancer | Colon Carcinoma | The HCT116 cell line was obtained from ATCC (American Type Culture Collection, Manassas,VA). Cells were grown in T75 flasks to a density of 1e9 cells before purification of HLA-I peptides for MS experiments. | In brief, 1 × 109 cells were dissociated using 40 mL of lysis buffer with 0.25% Sodium deoxycholate, 1% n-octyl glucoside, 100 mM PMSF and protease inhibitor cocktails in PBS at 4 °C for 60 min. Lysate were further cleared by 30 min centrifugation at 14,000× g. Cleared lysate were immunoaffinity purified with pan-HLA class I complexes antibody covalently bound to Protein-A Sepharose CL-4B beads. Beads were first washed with 10 column volumes of 150 mM NaCl, 20 mM Tris HCl (buffer A), then 10 column volumes of 400 mM NaCl, 20 mM Tris HCl, then 10 volumes of buffer A again, and finally with 10 column volumes of 20 mM Tris HCl, pH 8.0. The HLA-I molecules were eluted at room temperature using 0.1 N acetic acid. Eluate were then loaded on Sep-Pak tC18 cartridges (Waters, 50 mg) and washed with 0.1% TFA. The peptides were separated from HLA-I complexes on the C18 cartridges by eluting with 30% ACN in 0.1% TFA and concentrated to 20 µL using vacuum centrifugation. Finally, a 5 µL sample was used for MS analysis. | In brief, 1 × 109 cells were dissociated using 40 mL of lysis buffer with 0.25% Sodium deoxycholate, 1% n-octyl glucoside, 100 mM PMSF and protease inhibitor cocktails in PBS at 4 °C for 60 min. Lysate were further cleared by 30 min centrifugation at 14,000× g. Cleared lysate were immunoaffinity purified with pan-HLA class I complexes antibody covalently bound to Protein-A Sepharose CL-4B beads. Beads were first washed with 10 column volumes of 150 mM NaCl, 20 mM Tris HCl (buffer A), then 10 column volumes of 400 mM NaCl, 20 mM Tris HCl, then 10 volumes of buffer A again, and finally with 10 column volumes of 20 mM Tris HCl, pH 8.0. The HLA-I molecules were eluted at room temperature using 0.1 N acetic acid. Eluate were then loaded on Sep-Pak tC18 cartridges (Waters, 50 mg) and washed with 0.1% TFA. The peptides were separated from HLA-I complexes on the C18 cartridges by eluting with 30% ACN in 0.1% TFA and concentrated to 20 µL using vacuum centrifugation. Finally, a 5 µL sample was used for MS analysis. | HCT116 cell line | | Cells were grown in T75 flasks to a density of 1 × 109 cells before harvesting for experiments. | IntroSpect: Motif-Guided Immunopeptidome Database Building Tool to Improve the Sensitivity of HLA I Binding Peptide Identification by Mass Spectrometry |
| PXD000394 | Cancer | Breast Cancer | HLA class I molecules reflect the health state of cells to cytotoxic T-cells by presenting a repertoire of endogenously derived peptides. However, the extent to which the proteome shapes the peptidome is still largely unknown. Here we present a high-throughput mass-spectrometry-based workflow that allows stringent and accurate identification of thousands of such peptides and direct determination of binding motifs. Applying the workflow to seven cancer cell lines and primary cells, yielded more than 22,000 unique HLA peptides across different allelic binding specificities. By computing a score representing the HLA-I sampling density, we show a strong link between protein abundance and HLA-presentation (P<0.0001). When analyzing over-presented proteins - those with at least five-fold higher density score than expected for their abundance – we noticed that they are degraded almost 3 hours faster than similar but non-presented proteins (top 20% abundance class; median half-life 20.8h vs. 23.6h, p<0.0001). This validates protein degradation as an important factor for HLA presentation. Ribosomal, mitochondrial respiratory chain and nucleosomal proteins as particularly well presented. Taking a set of proteins associated with cancer, we compared the predicted immunogenicity of previously validated T-cell epitopes with other peptides from these proteins in our dataset. The validated epitopes indeed tend to have higher immunogenic scores than the other detected HLA peptides, suggesting the usefulness of combining MS-analysis with immunogenesis prediction for ranking and selection of epitopes for therapeutic use. | HLA-I peptidomes were obtained from 3-4 biological replicates per cell line. HLA-I complexes were purified from about 5x108 cell pellets after lysis with 0.25% sodium deoxycholate, 0.2 mM iodoacetamide, 1 mM EDTA, 1:200 Protease Inhibitors Cocktail (Sigma, MO), 1 mM PMSF, 1% octyl-β-D glucopyranoside (Sigma, MO) in PBS at 4 °C for 1 h. The lysates were cleared by 30 min centrifugation at 40,000 × g. We immunoaffinity purified HLA-I molecules from cleared lysate with the W6/32 antibody covalently bound to Protein-A Sepharose beads (Invitrogen, CA), because covalent binding of W6/32 antibody to the beads improves the purity of the eluted HLA-I complexes, diminishes co-elution of the antibodies which otherwise overload the C-18 cartridges and enables the reuse of the column. This affinity column was washed first with 10 column volumes of 150 mM NaCl, 20 mM Tris•HCl (buffer A), 10 column volumes of 400 mM NaCl, 20 mM Tris•HCl, 10 volumes of buffer A again, and finally with seven column volumes of 20 mM Tris•HCl, pH 8.0. The HLA-I molecules were eluted at room temperature by adding 500 µl of 0.1 N acetic acid, in total 7 elutions for each sample. Small aliquots of each elution fraction were analyzed by 12% SDS-PAGE to evaluate the yield and purity of the eluted HLA-I. Purification and concentration of HLA-I peptides. Eluted HLA-I peptides and the subunits of the HLA complex were loaded on Sep-Pak tC18 (Waters, MA) cartridges that were pre-washed with 80% acetonitrile (ACN) in 0.1% trifluoroacetic acid (TFA) and with 0.1% TFA only. After loading, the cartridges were washed with 0.1% TFA. The peptides were separated from the much more hydrophobic HLA-I heavy chains on the C18 cartridges by eluting them with 30% ACN in 0.1% TFA. They were further purified using a Silica C-18 column tips (Harvard Apparatus, MA) and eluted again with 30% ACN in 0.1% TFA. The peptides were concentrated and the volume was reduced to 15 µl using vacuum centrifugation. For MS analysis, we used 5 µl of this highly enriched HLA peptides. | Eluted HLA-I peptides and the subunits of the HLA complex were loaded on Sep-Pak tC18 (Waters, Milford, MA) cartridges that were prewashed with 80% acetonitrile (ACN) in 0.1% trifluoroacetic acid (TFA) and with 0.1% TFA only. After loading, the cartridges were washed with 0.1% TFA. The peptides were separated from the much more hydrophobic HLA-I heavy chains on the C18 cartridges by eluting them with 30% ACN in 0.1% TFA. They were further purified using a Silica C-18 column tips (Harvard Apparatus, Holliston, MA) and eluted again with 30% ACN in 0.1% TFA. The peptides were concentrated and the volume was reduced to 15 μl using vacuum centrifugation. For MS analysis, we used 5 μl of this highly enriched HLA peptides. | HCC1143 and HCC1937 cell lines | | HCC1143 and HCC1937 cells were maintained in RPMI 1640 medium and HCT116 cells in DMEM medium. | Mass spectrometry of human leukocyte antigen class I peptidomes reveals strong effects of protein abundance and turnover on antigen presentation. |
| PXD000394 | Cancer | Colon Carcinoma | HLA class I molecules reflect the health state of cells to cytotoxic T-cells by presenting a repertoire of endogenously derived peptides. However, the extent to which the proteome shapes the peptidome is still largely unknown. Here we present a high-throughput mass-spectrometry-based workflow that allows stringent and accurate identification of thousands of such peptides and direct determination of binding motifs. Applying the workflow to seven cancer cell lines and primary cells, yielded more than 22,000 unique HLA peptides across different allelic binding specificities. By computing a score representing the HLA-I sampling density, we show a strong link between protein abundance and HLA-presentation (P<0.0001). When analyzing over-presented proteins - those with at least five-fold higher density score than expected for their abundance – we noticed that they are degraded almost 3 hours faster than similar but non-presented proteins (top 20% abundance class; median half-life 20.8h vs. 23.6h, p<0.0001). This validates protein degradation as an important factor for HLA presentation. Ribosomal, mitochondrial respiratory chain and nucleosomal proteins as particularly well presented. Taking a set of proteins associated with cancer, we compared the predicted immunogenicity of previously validated T-cell epitopes with other peptides from these proteins in our dataset. The validated epitopes indeed tend to have higher immunogenic scores than the other detected HLA peptides, suggesting the usefulness of combining MS-analysis with immunogenesis prediction for ranking and selection of epitopes for therapeutic use. | HLA-I peptidomes were obtained from 3-4 biological replicates per cell line. HLA-I complexes were purified from about 5x108 cell pellets after lysis with 0.25% sodium deoxycholate, 0.2 mM iodoacetamide, 1 mM EDTA, 1:200 Protease Inhibitors Cocktail (Sigma, MO), 1 mM PMSF, 1% octyl-β-D glucopyranoside (Sigma, MO) in PBS at 4 °C for 1 h. The lysates were cleared by 30 min centrifugation at 40,000 × g. We immunoaffinity purified HLA-I molecules from cleared lysate with the W6/32 antibody covalently bound to Protein-A Sepharose beads (Invitrogen, CA), because covalent binding of W6/32 antibody to the beads improves the purity of the eluted HLA-I complexes, diminishes co-elution of the antibodies which otherwise overload the C-18 cartridges and enables the reuse of the column. This affinity column was washed first with 10 column volumes of 150 mM NaCl, 20 mM Tris•HCl (buffer A), 10 column volumes of 400 mM NaCl, 20 mM Tris•HCl, 10 volumes of buffer A again, and finally with seven column volumes of 20 mM Tris•HCl, pH 8.0. The HLA-I molecules were eluted at room temperature by adding 500 µl of 0.1 N acetic acid, in total 7 elutions for each sample. Small aliquots of each elution fraction were analyzed by 12% SDS-PAGE to evaluate the yield and purity of the eluted HLA-I. Purification and concentration of HLA-I peptides. Eluted HLA-I peptides and the subunits of the HLA complex were loaded on Sep-Pak tC18 (Waters, MA) cartridges that were pre-washed with 80% acetonitrile (ACN) in 0.1% trifluoroacetic acid (TFA) and with 0.1% TFA only. After loading, the cartridges were washed with 0.1% TFA. The peptides were separated from the much more hydrophobic HLA-I heavy chains on the C18 cartridges by eluting them with 30% ACN in 0.1% TFA. They were further purified using a Silica C-18 column tips (Harvard Apparatus, MA) and eluted again with 30% ACN in 0.1% TFA. The peptides were concentrated and the volume was reduced to 15 µl using vacuum centrifugation. For MS analysis, we used 5 µl of this highly enriched HLA peptides. | Eluted HLA-I peptides and the subunits of the HLA complex were loaded on Sep-Pak tC18 (Waters, Milford, MA) cartridges that were prewashed with 80% acetonitrile (ACN) in 0.1% trifluoroacetic acid (TFA) and with 0.1% TFA only. After loading, the cartridges were washed with 0.1% TFA. The peptides were separated from the much more hydrophobic HLA-I heavy chains on the C18 cartridges by eluting them with 30% ACN in 0.1% TFA. They were further purified using a Silica C-18 column tips (Harvard Apparatus, Holliston, MA) and eluted again with 30% ACN in 0.1% TFA. The peptides were concentrated and the volume was reduced to 15 μl using vacuum centrifugation. For MS analysis, we used 5 μl of this highly enriched HLA peptides. | HCT116 cell line | | HCC1143 and HCC1937 cells were maintained in RPMI 1640 medium and HCT116 cells in DMEM medium. | Mass spectrometry of human leukocyte antigen class I peptidomes reveals strong effects of protein abundance and turnover on antigen presentation. |
| PXD000394 | Cancer | B-Acute Lymphoblastic Leukemia | HLA class I molecules reflect the health state of cells to cytotoxic T-cells by presenting a repertoire of endogenously derived peptides. However, the extent to which the proteome shapes the peptidome is still largely unknown. Here we present a high-throughput mass-spectrometry-based workflow that allows stringent and accurate identification of thousands of such peptides and direct determination of binding motifs. Applying the workflow to seven cancer cell lines and primary cells, yielded more than 22,000 unique HLA peptides across different allelic binding specificities. By computing a score representing the HLA-I sampling density, we show a strong link between protein abundance and HLA-presentation (P<0.0001). When analyzing over-presented proteins - those with at least five-fold higher density score than expected for their abundance – we noticed that they are degraded almost 3 hours faster than similar but non-presented proteins (top 20% abundance class; median half-life 20.8h vs. 23.6h, p<0.0001). This validates protein degradation as an important factor for HLA presentation. Ribosomal, mitochondrial respiratory chain and nucleosomal proteins as particularly well presented. Taking a set of proteins associated with cancer, we compared the predicted immunogenicity of previously validated T-cell epitopes with other peptides from these proteins in our dataset. The validated epitopes indeed tend to have higher immunogenic scores than the other detected HLA peptides, suggesting the usefulness of combining MS-analysis with immunogenesis prediction for ranking and selection of epitopes for therapeutic use. | HLA-I peptidomes were obtained from 3-4 biological replicates per cell line. HLA-I complexes were purified from about 5x108 cell pellets after lysis with 0.25% sodium deoxycholate, 0.2 mM iodoacetamide, 1 mM EDTA, 1:200 Protease Inhibitors Cocktail (Sigma, MO), 1 mM PMSF, 1% octyl-β-D glucopyranoside (Sigma, MO) in PBS at 4 °C for 1 h. The lysates were cleared by 30 min centrifugation at 40,000 × g. We immunoaffinity purified HLA-I molecules from cleared lysate with the W6/32 antibody covalently bound to Protein-A Sepharose beads (Invitrogen, CA), because covalent binding of W6/32 antibody to the beads improves the purity of the eluted HLA-I complexes, diminishes co-elution of the antibodies which otherwise overload the C-18 cartridges and enables the reuse of the column. This affinity column was washed first with 10 column volumes of 150 mM NaCl, 20 mM Tris•HCl (buffer A), 10 column volumes of 400 mM NaCl, 20 mM Tris•HCl, 10 volumes of buffer A again, and finally with seven column volumes of 20 mM Tris•HCl, pH 8.0. The HLA-I molecules were eluted at room temperature by adding 500 µl of 0.1 N acetic acid, in total 7 elutions for each sample. Small aliquots of each elution fraction were analyzed by 12% SDS-PAGE to evaluate the yield and purity of the eluted HLA-I. Purification and concentration of HLA-I peptides. Eluted HLA-I peptides and the subunits of the HLA complex were loaded on Sep-Pak tC18 (Waters, MA) cartridges that were pre-washed with 80% acetonitrile (ACN) in 0.1% trifluoroacetic acid (TFA) and with 0.1% TFA only. After loading, the cartridges were washed with 0.1% TFA. The peptides were separated from the much more hydrophobic HLA-I heavy chains on the C18 cartridges by eluting them with 30% ACN in 0.1% TFA. They were further purified using a Silica C-18 column tips (Harvard Apparatus, MA) and eluted again with 30% ACN in 0.1% TFA. The peptides were concentrated and the volume was reduced to 15 µl using vacuum centrifugation. For MS analysis, we used 5 µl of this highly enriched HLA peptides. | Eluted HLA-I peptides and the subunits of the HLA complex were loaded on Sep-Pak tC18 (Waters, Milford, MA) cartridges that were prewashed with 80% acetonitrile (ACN) in 0.1% trifluoroacetic acid (TFA) and with 0.1% TFA only. After loading, the cartridges were washed with 0.1% TFA. The peptides were separated from the much more hydrophobic HLA-I heavy chains on the C18 cartridges by eluting them with 30% ACN in 0.1% TFA. They were further purified using a Silica C-18 column tips (Harvard Apparatus, Holliston, MA) and eluted again with 30% ACN in 0.1% TFA. The peptides were concentrated and the volume was reduced to 15 μl using vacuum centrifugation. For MS analysis, we used 5 μl of this highly enriched HLA peptides. | SupB15RT cell line | | SupB15RT cells were grown with the addition of 1 μm imatinib to the medium (Cayman Chemical, Ann Arbor, MI). | Mass spectrometry of human leukocyte antigen class I peptidomes reveals strong effects of protein abundance and turnover on antigen presentation. |
| PXD003790 | Cancer | Glioblastoma | Treatment of cancer cells with anti-cancer drugs often fails to achieve complete remission. Yet, such drug treatments may induce alteration in the tumor’s gene expression patterns, including those of Cancer/Testis Antigens (CTA). The degradation products of such antigens can be presented as HLA peptides on the surface of the tumor cells and be developed into anticancer immunotherapeutics. For example, the DNA methyl transferase inhibitor, 5-aza-2'deoxycytidine (Decitabine) has limited anti-tumor efficacy, yet it induces the expression of many genes, including CTAs that are normally silenced in the healthy adult tissues. In this study, the presentation of many new HLA peptides derived from CTAs and induced by Decitabine was demonstrated in three human Glioblastoma cell lines. Such presentation of CTA-derived HLA peptides can be exploited for development of new treatment modalities, combining drug treatment with anti-CTA targeted immunotherapy. The Decitabine-induced HLA peptidomes include many CTAs that are not normally detected in healthy tissues or in cancer cells, unless treated with the drug. In addition, the study included large-scale analyses of the simultaneous effects of Decitabine on the transcriptomes, proteomes and HLA peptidomes of the human Glioblastoma cells. It demonstrates the poor correlations between these three levels of gene expression, both in their total levels and in their response to the drug. The proteomics and HLA peptidomics data are available via ProteomeXchange with identifier PXD003790 and the transcriptomics data are available via GEO with identifier GSE80137. | Affinity purification of HLA molecules: HLA class I molecules were purified from three biological replicates for each Glioblastoma cell line. Each replica with about 5*108 cells was lysed with 0.25% sodium deoxycholate, 0.2 mM iodoacetamide, 1 mM EDTA, 1:200 Protease Inhibitors Cocktail (Sigma), 1 mM PMSF and 1% octyl-β-D glucopyranoside (Sigma) in PBS at 4°C for 1 hour. The cell extracts were cleared by centrifugation for 45 minutes at 18,000 rpm, 4°C. The recovered HLA class I molecules were immunoaffinity purified using the W6/32 mAb bound to Amino-Link beads (Thermo Scientific) . The HLA molecules with their bound peptides were eluted from the affinity column with five column volumes of 0.1 N acetic acid. The eluted HLA class I proteins and the released peptides were loaded on disposable C18 columns (Harvard Apparatus) and the peptides fraction was recovered with 30% acetonitrile in 0.1% TFA. The peptide fractions were dried using vacuum centrifugation, reconstituted in 100 μl of 0.1% TFA, reloaded on Stage-Tips , eluted with 80% ACN, dried and reconstituted with 0.1% formic acid for μLC-MS-MS analysis. | HLA class I molecules were purified from three biological replicates for each Glioblastoma cell line. Each replica with about 5 × 108 cells was lysed with 0.25% sodium deoxycholate, 0.2 mm iodoacetamide, 1 mm EDTA, 1:200 Protease Inhibitors Mixture (Sigma), 1 mm PMSF, and 1% octyl-β-D glucopyranoside (Sigma) in PBS at 4 °C for 1 h. The cell extracts were cleared by centrifugation for 45 min at 18,000 rpm, 4 °C. The recovered HLA class I molecules were immunoaffinity purified using the W6/32 mAb bound to Amino-Link beads (Thermo Scientific, Waltham, MA) as in (49). The HLA molecules with their bound peptides were eluted from the affinity column with five column volumes of 0.1 N acetic acid. The eluted HLA class I proteins and the released peptides were loaded on disposable C18 columns (Harvard Apparatus, Holliston, MA) and the peptides fraction was recovered with 30% acetonitrile in 0.1% TFA. The peptide fractions were dried using vacuum centrifugation, reconstituted in 100 μl of 0.1% TFA, reloaded on Stage-Tips (50), eluted with 80% ACN, dried, and reconstituted with 0.1% formic acid for μLC-MS-MS analysis. | Three human Glioblastoma cell lines, U-87, T98G, and LNT-229 | | Three human Glioblastoma cell lines, U-87, T98G, and LNT-229 (obtained from the ATCC, Manassas, VA) were maintained in DMEM supplemented with 10% FBS, 1% l-glutamine, 1% Na-pyruvate, 0.1 mg/ml streptomycin, and 100 μ/ml penicillin and 0.1% HEPES. The cells were kept in 5% CO2 humidified incubator at 37 °C. The monoclonal antibodies W6/32 (anti-HLA class I) were affinity purified from growth medium of the HB95 hybridoma (obtained from the ATCC). | HLA peptides derived from tumor antigens induced by inhibition of DNA methylation for development of drug-facilitated immunotherapy. Mol Cell Proteomics |
| PXD005704 | Cancer | Lymphoma | Cancer somatic mutations can generate neoantigens that distinguish malignant from normal cells. Such neoantigens have been implicated in response to immunotherapies including immune checkpoint blockade, yet their identification and validation remains challenging. Here we discover neoantigens in human mantle cell lymphomas using an integrated strategy for genomic and proteomic tumor antigen discovery that interrogates peptides presented within the tumor major histocompatibility complex (MHC) class I and class II molecules. We applied this approach to systematically identify neoantigen peptides in diagnostic tumor specimens from 17 patients and several cell lines. Remarkably, the discovered neoantigenic peptides were invariably derived from the lymphoma immunoglobulin (Ig) heavy or light chain variable regions. Although we could identify MHC presentation of private germline polymorphic alleles, no mutated peptides were recovered from non-Ig somatically mutated genes. The immunoglobulin variable region somatic mutations were almost exclusively presented by MHC-II. We found T-cells specific for an immunoglobulin-derived neoantigen in the blood of a patient using MHC-II tetramers, and these T-cell clones expanded in frequency following tumor vaccination. These results demonstrate that an integrative approach combining MHC isolation, peptide identification and exome sequencing is an effective platform to uncover tumor neoantigens. Application of this strategy to human lymphoma implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy. | MHC-class I peptidomes were extracted from both a Raji wildtype and a Raji transduced cell line with the HLA B*35:01 allele. In brief, cells were lysed and the lysate was subjected to centrifugation and then precleared using rProtein A Sepharose fast-flow beads (GE Healthcare). The precleared lysate was incubated with the pan HLA-A-, B-, and C- antibody W6/32 coupled to rProtein A Sepharose fast-flow beads. Following the immune-capture, the beads were washed with TBS and peptides were eluted from the purified MHC-molecules using 10% acetic acid. The peptides were further purified with a MWCO size filter, followed by a concentration step and then desalted on C18 based StageTips. The isolated peptides were analyzed on an LTQ Orbitrap Elite mass spectrometer (Thermo Fischer Scientific). | MHC-class I peptidomes were extracted from both a Raji wildtype and a Raji transduced cell line with the HLA B*35:01 allele. | Both a Raji wildtype and a Raji transduced cell line with the HLA B*35:01 allele | | Cell lines were grown per ATCC recommendations. | Antigen presentation profiling reveals recognition of lymphoma immunoglobulin neoantigens. |
| PXD006939 | Cancer | Meningioma | Comprehensive knowledge of the HLA class-I and class-II peptides presented to T-cells is crucial for designing innovative therapeutics against cancer and other diseases. However methodologies for their extraction for mass-spectrometry analysis have been a major limitation. We designed a novel high-throughput, reproducible and sensitive method for sequential extraction of HLA-I and -II peptides from up to 96 samples in a plate format from cell lines or tissues. Our methodology drastically reduces sample-handling and can be completed within six hours. We report identification and quantification of thousands of peptides with excellent reproducibility (Pearson correlations reaching 0.98 and 0.97, class I and II, respectively). Due to improved sensitivity, as many as 1,846 HLA-I and 2,633 HLA-II peptides were accurately identified from only 107 B-cells. We demonstrate the feasibility of performing drug-screening by using ovarian cancer cells treated with interferon gamma. This straightforward method is applicable for basic and clinical applications. | For high-throughput HLA-I and HLA-II purification, we employ the Waters Positive Pressure-96 Processor (Waters, 186006961) and customized it by adding extended self-made metal rods to accommodate the stacking of up to five 96-well plates. We used for immuno-affinity purifications the 96-well micro-plate with 3µm glass fiber and 10µm polypropylene membranes which are compatible with the processor and are commercially available (Seahorse Bioscience, 360063).The positive pressure processor was used in each step of the procedure to generate homogenous flow of liquid through the plates. Preparation of lysates: In the first experimental set-up we purified the HLA-I and II peptidome from JY, CD165, PD42, CM467, RA957, TIL1 and TIL3. Cell lysis was performed with PBS containing 0.25% sodium deoxycholate (Sigma-Aldrich, 30970), 0.2 mM iodoacetamide (Sigma-Aldrich, I6125), 1 mM EDTA, 1:200 Protease Inhibitors Cocktail (Sigma-Aldrich, 04693132001), 1 mM Phenylmethylsulfonylfluoride (Roche, 329-98-6), 1% octyl-beta-D glucopyranoside (Sigma-Alrich, 08001) at 4°C for 1 hour. In general, lysis buffer was added to the cells at a concentration of 1 x 108 cells/mL. Lysates were cleared by centrifugation with a table-top centrifuge (Eppendorf Centrifuge, 5430R) at 4°C at 14200 rpm for 50 min. For each cell line, lysate from a total of 3 x 108 cells were pooled and evenly distributed as 1 x 108 triplicates into designated wells. In a second experimental set-up, snap-frozen meningioma tissue samples were placed in tubes containing ice cold lysis buffer (mentioned above) and homogenized on ice in 3-5 short intervals of 5 seconds each using an Ultra Turrax homogenizer (IKA, T10 standard) with maximum speed. For one gram of tissue, 10 mL of lysis buffer was required. Lysates were cleared by centrifugation at 25,000 rpm in a high speed centrifuge (Beckman Coulter, JSS15314) at 4°C for 50 minutes. To test the limits of sensitivity with our method, we extracted HLA-I and -II peptides from 10, 30, 50 and 70 million cells as described above and we split the lysate of 1.3 x 108 CD165 B-cells proportionally to the desired cell amount; this was performed in triplicates. Last, four replicates of 1.5 x 108 UWB.1 289 cells untreated and four replicates treated with IFNy were processed separately but in parallel for HLAp extraction. Preparation of plates: First, empty plates’ wells were washed and equilibrated with 1 ml of 100% acetonitrile (ACN; Sigma-Aldrich, 34967), followed by 1 ml of 0.1% trifluoroacetic acid wash (TFA; Merck Millipore, 1082620100) and last with 2 ml of 0.1 M Tris-HCl pH 8 (Thermo Fisher Scientific, 15568025). Anti-pan HLA-I and HLA-II antibodies cross-linked to beads were loaded on their respective plates (named “HLA I†and “HLA IIâ€, see Figure 1 and Supplementary Video 1) at a final bead volume of 75 uL in 0.1 M Tris-HCl. For tissue samples, a depletion step of endogenous antibodies was required. Therefore, an additional plate (named “Pre-clear†plate) with wells containing 100 uL Pro-A beads was prepared. The beads alone or antibodies cross-linked to beads were conditioned with lysis buffer before lysate loading. Purification of HLA-I and HLA-II peptides: Two Sep-Pak tC18 96-well plates (Waters, 186002321) were required for the purification and concentration of HLA-I and HLA-II peptides. Each Sep-Pak plate was handled separately. Firstly, we conditioned the plates with 1 mL of 80% ACN in 0.1 % TFA and then with 2 mL of 0.1% TFA. The affinity plate was stacked on top of the C18 plate to achieve direct elution of the HLA complexes and the bound peptides with 500 uL 1% TFA. This is followed by washing the Sep-Pak wells with 2 mL of 0.1 % TFA. Thereafter we eluted the HLA-I peptides with 500 uL of 28% ACN in 0.1% TFA. HLA-II peptides were eluted from the class II Sep-Pak plate with 500 uL of 32% ACN in 0.1% TFA. Both HLA-I and -II peptides elutions were transferred into eppendorf tubes. Recovered HLA-I and -II peptides were dried using vacuum centrifugation (Thermo Fisher Scientific, N09Y-442492-NY) and stored at -20C. Dry peptides were resuspended in a designated volume prior to injection into the mass spectrometry. | Two Sep-Pak tC18 100 mg Sorbent 96-well plates (named “C18 solid phase extraction” plate) (ref number: 186002321, Waters) were required for the purification and concentration of HLA-I and HLA-II peptides. Each C18 plate was handled separately. Firstly, we conditioned the plates with 1 ml of 80% ACN in 0.1% trifluoroacetic acid (TFA) and then with 2 ml of 0.1% TFA. The affinity plate was stacked on top of the C18 plate to achieve direct elution of the HLA complexes and the bound peptides with 500 μl 1% TFA. The use of TFA leads to complete denaturation of antibodies and results in a high recovery of HLAp. This is followed by washing the C18 wells with 2 ml of 0.1% TFA. Thereafter, we eluted the HLA-I peptides with 500 μl of 28% ACN in 0.1% TFA. HLA-II peptides were eluted from the class II C18 plate with 500 μl of 32% ACN in 0.1% TFA. Both HLA-I and -II peptides elutions were transferred into eppendorf tubes. Recovered HLA-I and -II peptides were dried using vacuum centrifugation (Concentrator plus Eppendorf) and stored at −20 °C. The overall time required for sample drying may vary according to the specification of the vacuum centrifuge, the user settings and amount of samples. | Snap frozen meningioma tissues from patients (3830-NJF, 3849-BR, 3912-BAM, 3865-DM) were obtained from the University Hospital of Lausanne (CHUV, Lausanne, Switzerland). | | | High-throughput and Sensitive Immunopeptidomics Platform Reveals Profound Interferonγ-Mediated Remodeling of the Human Leukocyte Antigen (HLA) Ligandome. |
| PXD006939 | Cancer | Ovarian Cancer | Comprehensive knowledge of the HLA class-I and class-II peptides presented to T-cells is crucial for designing innovative therapeutics against cancer and other diseases. However methodologies for their extraction for mass-spectrometry analysis have been a major limitation. We designed a novel high-throughput, reproducible and sensitive method for sequential extraction of HLA-I and -II peptides from up to 96 samples in a plate format from cell lines or tissues. Our methodology drastically reduces sample-handling and can be completed within six hours. We report identification and quantification of thousands of peptides with excellent reproducibility (Pearson correlations reaching 0.98 and 0.97, class I and II, respectively). Due to improved sensitivity, as many as 1,846 HLA-I and 2,633 HLA-II peptides were accurately identified from only 107 B-cells. We demonstrate the feasibility of performing drug-screening by using ovarian cancer cells treated with interferon gamma. This straightforward method is applicable for basic and clinical applications. | For high-throughput HLA-I and HLA-II purification, we employ the Waters Positive Pressure-96 Processor (Waters, 186006961) and customized it by adding extended self-made metal rods to accommodate the stacking of up to five 96-well plates. We used for immuno-affinity purifications the 96-well micro-plate with 3µm glass fiber and 10µm polypropylene membranes which are compatible with the processor and are commercially available (Seahorse Bioscience, 360063).The positive pressure processor was used in each step of the procedure to generate homogenous flow of liquid through the plates. Preparation of lysates: In the first experimental set-up we purified the HLA-I and II peptidome from JY, CD165, PD42, CM467, RA957, TIL1 and TIL3. Cell lysis was performed with PBS containing 0.25% sodium deoxycholate (Sigma-Aldrich, 30970), 0.2 mM iodoacetamide (Sigma-Aldrich, I6125), 1 mM EDTA, 1:200 Protease Inhibitors Cocktail (Sigma-Aldrich, 04693132001), 1 mM Phenylmethylsulfonylfluoride (Roche, 329-98-6), 1% octyl-beta-D glucopyranoside (Sigma-Alrich, 08001) at 4°C for 1 hour. In general, lysis buffer was added to the cells at a concentration of 1 x 108 cells/mL. Lysates were cleared by centrifugation with a table-top centrifuge (Eppendorf Centrifuge, 5430R) at 4°C at 14200 rpm for 50 min. For each cell line, lysate from a total of 3 x 108 cells were pooled and evenly distributed as 1 x 108 triplicates into designated wells. In a second experimental set-up, snap-frozen meningioma tissue samples were placed in tubes containing ice cold lysis buffer (mentioned above) and homogenized on ice in 3-5 short intervals of 5 seconds each using an Ultra Turrax homogenizer (IKA, T10 standard) with maximum speed. For one gram of tissue, 10 mL of lysis buffer was required. Lysates were cleared by centrifugation at 25,000 rpm in a high speed centrifuge (Beckman Coulter, JSS15314) at 4°C for 50 minutes. To test the limits of sensitivity with our method, we extracted HLA-I and -II peptides from 10, 30, 50 and 70 million cells as described above and we split the lysate of 1.3 x 108 CD165 B-cells proportionally to the desired cell amount; this was performed in triplicates. Last, four replicates of 1.5 x 108 UWB.1 289 cells untreated and four replicates treated with IFNy were processed separately but in parallel for HLAp extraction. Preparation of plates: First, empty plates’ wells were washed and equilibrated with 1 ml of 100% acetonitrile (ACN; Sigma-Aldrich, 34967), followed by 1 ml of 0.1% trifluoroacetic acid wash (TFA; Merck Millipore, 1082620100) and last with 2 ml of 0.1 M Tris-HCl pH 8 (Thermo Fisher Scientific, 15568025). Anti-pan HLA-I and HLA-II antibodies cross-linked to beads were loaded on their respective plates (named “HLA I†and “HLA IIâ€, see Figure 1 and Supplementary Video 1) at a final bead volume of 75 uL in 0.1 M Tris-HCl. For tissue samples, a depletion step of endogenous antibodies was required. Therefore, an additional plate (named “Pre-clear†plate) with wells containing 100 uL Pro-A beads was prepared. The beads alone or antibodies cross-linked to beads were conditioned with lysis buffer before lysate loading. Purification of HLA-I and HLA-II peptides: Two Sep-Pak tC18 96-well plates (Waters, 186002321) were required for the purification and concentration of HLA-I and HLA-II peptides. Each Sep-Pak plate was handled separately. Firstly, we conditioned the plates with 1 mL of 80% ACN in 0.1 % TFA and then with 2 mL of 0.1% TFA. The affinity plate was stacked on top of the C18 plate to achieve direct elution of the HLA complexes and the bound peptides with 500 uL 1% TFA. This is followed by washing the Sep-Pak wells with 2 mL of 0.1 % TFA. Thereafter we eluted the HLA-I peptides with 500 uL of 28% ACN in 0.1% TFA. HLA-II peptides were eluted from the class II Sep-Pak plate with 500 uL of 32% ACN in 0.1% TFA. Both HLA-I and -II peptides elutions were transferred into eppendorf tubes. Recovered HLA-I and -II peptides were dried using vacuum centrifugation (Thermo Fisher Scientific, N09Y-442492-NY) and stored at -20C. Dry peptides were resuspended in a designated volume prior to injection into the mass spectrometry. | Two Sep-Pak tC18 100 mg Sorbent 96-well plates (named “C18 solid phase extraction” plate) (ref number: 186002321, Waters) were required for the purification and concentration of HLA-I and HLA-II peptides. Each C18 plate was handled separately. Firstly, we conditioned the plates with 1 ml of 80% ACN in 0.1% trifluoroacetic acid (TFA) and then with 2 ml of 0.1% TFA. The affinity plate was stacked on top of the C18 plate to achieve direct elution of the HLA complexes and the bound peptides with 500 μl 1% TFA. The use of TFA leads to complete denaturation of antibodies and results in a high recovery of HLAp. This is followed by washing the C18 wells with 2 ml of 0.1% TFA. Thereafter, we eluted the HLA-I peptides with 500 μl of 28% ACN in 0.1% TFA. HLA-II peptides were eluted from the class II C18 plate with 500 μl of 32% ACN in 0.1% TFA. Both HLA-I and -II peptides elutions were transferred into eppendorf tubes. Recovered HLA-I and -II peptides were dried using vacuum centrifugation (Concentrator plus Eppendorf) and stored at −20 °C. The overall time required for sample drying may vary according to the specification of the vacuum centrifuge, the user settings and amount of samples. | UWB.1 289 ovarian carcinoma cells | | UWB.1 289 ovarian carcinoma cells (ATCC® CRL-2945™) were maintained in a 1:1 mix of HuMEC Ready medium (Thermo Fisher Scientific, Waltham, MA) supplemented with HuMEC Supplement Kit (Thermo Fisher Scientific) and RPMI 1640 + GlutaMAX medium, with addition of 1% Penicillin/Streptomycin Solution and 3% heat-inactivated FBS. | High-throughput and Sensitive Immunopeptidomics Platform Reveals Profound Interferonγ-Mediated Remodeling of the Human Leukocyte Antigen (HLA) Ligandome. |
| PXD008937 | Cancer | Melanoma | The influence of CD74 expression on the HLA peptidome of the H1 melanoma cell line was analyzed by label-free quantitation mass spectrometry of HLA ligand extracts obtained from CD74 siRNA & control siRNA treated cells. | HLA class I and class II molecules were isolated from 50Mio CD74siRNA treated and mock treated H1 cells using standard immunoaffinity purification as described previously,(PMID 23329485) . The pan-HLA class I-specific mAb W6/32 was utilized for isolation of Class I molecules. A 1:1 mixture of the pan-HLA class II-specific mAb Tü-39 and the HLA-DR- specific mAB L243 was utilized for the isolation of HLA class II. Label-free relative quantitation (LFQ) of the HLA peptidome composition was performed by LC-MS analysis of HLA class II ligand extracts from treated and control cells in five technical replicates. Peptide samples were separated by nanoflow high- performance liquid chromatography (RSLCnano, Thermo Fisher Scientific) using a 50 μm x 25 cm PepMap C18 column (Thermo Fisher Scientific) and a linear gradient ranging from 2.4% to 32.0% acetonitrile over the course of 90 min. Eluting peptides were analyzed in an an online-coupled Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) in data dependent acquisition mode using collision- induced dissociation fragmentation. MS2 spectra for 2+ and 3+ precursors of 400- 650 m/z were acquired at 30k resolution with AGC target values of 70,000 and maximum injection times of 150ms. Normalized collision energy was set to 35%, dynamic exclusion was set to 7s. On column peptide amounts for the 5 LFQ replicates were adjusted based on initial dose-finding LC-MS analysis. | HLA class I and class II molecules were isolated from 50Mio CD74siRNA treated and mock treated H1 cells using standard immunoaffinity purification as described previously,(PMID 23329485) . The pan-HLA class I-specific mAb W6/32 was utilized for isolation of Class I molecules. A 1:1 mixture of the pan-HLA class II-specific mAb Tü-39 and the HLA-DR- specific mAB L243 was utilized for the isolation of HLA class II. Label-free relative quantitation (LFQ) of the HLA peptidome composition was performed by LC-MS analysis of HLA class II ligand extracts from treated and control cells in five technical replicates. Peptide samples were separated by nanoflow high- performance liquid chromatography (RSLCnano, Thermo Fisher Scientific) using a 50 μm x 25 cm PepMap C18 column (Thermo Fisher Scientific) and a linear gradient ranging from 2.4% to 32.0% acetonitrile over the course of 90 min. Eluting peptides were analyzed in an an online-coupled Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) in data dependent acquisition mode using collision- induced dissociation fragmentation. MS2 spectra for 2+ and 3+ precursors of 400- 650 m/z were acquired at 30k resolution with AGC target values of 70,000 and maximum injection times of 150ms. Normalized collision energy was set to 35%, dynamic exclusion was set to 7s. On column peptide amounts for the 5 LFQ replicates were adjusted based on initial dose-finding LC-MS analysis. | In total, we investigated 236 embedded tissue samples including BM of: melanoma (n = 96), non small cell lung cancer (NSCLC, n = 56), breast carcinoma (n = 31), renal cell carcinoma (RCC, n = 18), small cell lung cancer (SCLC n = 8), colon carcinoma (n = 10), carcinomas which were not otherwise specified (carcinoma NOS n = 8) and specimens of rare tumors summarized as others (n = 9). | | | CD74 regulates complexity of tumor cell HLA class II peptidome in brain metastasis and is a positive prognostic marker for patient survival |
| PXD008984 | Cancer | Glioblastoma | Glioblastoma is an aggressive incurable primary malignant brain tumor. Measles virus (MeV) therapy is a promising upcoming treatment strategy with proven preclinical efficacy and clinical safety. Using RNA Sequencing and immunopeptidome analyses, we aimed at identifying a synergistic combination regimen of MeV with conventional therapeutic strategies for glioblastoma patients, i.e. radiotherapy and temozolomide or lomustine, and to understand the underlying molecular mechanism. | Isolation of HLA ligands Cells subjected to treatment regimens as mentioned above were harvested at 96 h, washed twice with cold phosphate buffered saline (PBS; Lonza), frozen at -80°C with subsequent iosolation of HLA class I molecules using standard immunoaffinity purification. In brief, cell pellets were lysed in 10 mM 3-[(3-Cholamidopropyl)-Dimethylammonio]-1-Propanesulfonate (CHAPS; Applichem, Gatersleben, Germany)/PBS (Lonza) containing protease inhibitor (Roche, Basel, Switzerland). HLA molecules were purified using the pan-HLA class I–specific monoclonal W6/32 Ab, which is covalently linked to CNBr-activated Sepharose (GE Healthcare, Little Chalfont, U.K.). Repeated addition of 0.2% trifluoroacetic acid (Merck Millipore) eluted HLA molecules and peptides. The peptides were isolated employing ultrafiltration with centrifugal filter units (Merck Millipore), extracted and desalted using ZipTip C18 pipette tips (Merck Millipore). Peptides were then eluted in 35 µL acetonitrile (Merck Millipore)/0.1% trifluoroacetic acid, vacuum centrifuged to 5 µL, and resuspended in 25 µL of 1% acetonitrile/0.05% trifluoroacetic acid. | In brief, cell pellets were lysed in 10 mM 3-[(3-Cholamidopropyl)-Dimethylammonio]-1-Propanesulfonate (CHAPS; Applichem, Gatersleben, Germany)/PBS (Lonza) containing protease inhibitor (Roche, Basel, Switzerland). HLA molecules were purified using the pan-HLA class I–specific monoclonal W6/32 Ab, which is covalently linked to CNBr-activated Sepharose (GE Healthcare, Little Chalfont, U.K.). Repeated addition of 0.2% trifluoroacetic acid (Merck Millipore) eluted HLA molecules and peptides. The peptides were isolated employing ultrafiltration with centrifugal filter units (Merck Millipore), extracted and desalted using ZipTip C18 pipette tips (Merck Millipore). Peptides were then eluted in 35 µL acetonitrile (Merck Millipore)/0.1% trifluoroacetic acid, vacuum centrifuged to 5 µL, and resuspended in 25 µL of 1% acetonitrile/0.05% trifluoroacetic acid. | The human long-term glioma cell lines LN229 and LNZ308 | | they were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum and 50 μg/mL gentamycin (Thermo Fisher Scientific). | Measles Virus-Based Treatments Trigger a Pro-inflammatory Cascade and a Distinctive Immunopeptidome in Glioblastoma |
| PXD009752 | Cancer | Lung Cancer | MHC I-associated peptides (MAPs) presented at the surface of nucleated cells play a central role in CD8 T-cell immunosurveillance. MAPs presented by mature (i.e. MHC IIhi) medullary thymic epithelial cells (mTEChi) are essential to eliminate self-reactive CD8 T cells in a process called central tolerance. On tumor cells, MAPs that do not induce tolerance (i.e. non-tolerogenic MAPs), because absent from mTEChi or any other normal cells, are referred to as tumor-specific antigens (TSAs). Despite their clinical relevance, very few have been identified, even in highly mutated tumor types. Thus, we developed a novel proteogenomic workflow able to characterize the full TSA landscape of any tumor. Briefly, using RNA-seq data, we subtracted the mTEChi from the tumor signal to generate tumor-specific protein databases enriched in non-tolerogenic sequences. Using these databases to analyze the MAP repertoire of two murine cell lines (CT26 and EL4) sequenced by mass spectrometry, we identified a total of 21 TSAs, 90% of which derived from allegedly non-coding regions. Interestingly, our results highlighted that 70% of those TSAs derived from non-mutated yet tumor-restricted sequences, e.g. endogenous retroelements. Moreover, we showed that our approach is easily amenable to analyze human primary samples as we were able to identify TSAs in three lung tumor biopsies and four B-ALL specimens. Focusing on 5 TSAs, we demonstrated that both TSA expression and TSA-specific T-cell frequency in the pre-immune repertoire influenced the overall survival of pre-immunized tumor-bearing mice. In conclusion, this proof-of-concept study demonstrates that non-coding-derived TSAs are frequent and protective in vivo, while they could be shared by several individuals. Altogether, our findings will help expand the repertoire of human TSAs and facilitate their prioritization in the clinic. | Lung tumor biopsies (wet weight ranging from 771 mgs to 1825 mgs), were cut in small pieces (cubes, ~3 mm in size) and 5 ml of ice-cold PBS containing protein inhibitor cocktail was added to each tissue sample. Tissues were first homogenized twice for 20 seconds using an Ultra Turrax T25 homogenizer (IKA-Labortechnik) set at speed 20000 rpm and then once for 20 seconds using an Ultra Turrax T8 homogenizer (IKA-Labortechnik) set at speed 25000 rpm. Then, 550 µl of ice-cold 10X lysis buffer (10% w/v CHAPS) was added to each sample. After 60 minute incubation with tumbling at 4°C, samples were spun at 10000g for 30 minutes at 4°C. Supernatants were transferred into new tubes containing magnetic beads coupled to W6/32 antibodies. Samples were incubated with tumbling for 180 minutes at 4°C and placed on a magnet to recover bound MHC I complexes to magnetic beads. Magnetic beads were first washed with 8 × 1 mL PBS, then with 1 × 1 mL of 0.1X PBS and finally with 1 × 1 mL of water. MHC I complexes were eluted from the magnetic beads by acidic treatment using 0.2% trifluoroacetic acid (TFA). Eluates were transferred into 2.0 mL Costar mL Spin-X centrifuge tube filters and spun 2 minutes at 3000g. Filtrates containing peptides were separated from MHC I subunits using home-made stage tips packed with twenty 1 mm diameter octadecyl (C-18) solid phase extraction disks (EMPORE). Stage tips were pre-washed first with methanol then with 80% acetonitrile (ACN) in 0.2% TFA and finally with 0.1% formic acid (FA). Samples were loaded and then washed with 0.1% FA. Peptides were eluted with 30% ACN in 0.1%TFA, dried using vacuum centrifugation and then stored at -20°C. MAP extracts were loaded on a home-made C18 analytical column (15 cm x 150 𝜇m i.d. packed with C18 Jupiter Phenomenex) with a 100-min gradient from 5–28 % acetonitrile (0.2 % formic acid) and a 600 nl.min-1 flow rate on a nEasy-LC II system. | Lung tumor biopsies (wet weight ranging from 771 mgs to 1825 mgs), were cut in small pieces (cubes, ~3 mm in size) and 5 ml of ice-cold PBS containing protein inhibitor cocktail was added to each tissue sample. Tissues were first homogenized twice for 20 seconds using an Ultra Turrax T25 homogenizer (IKA-Labortechnik) set at speed 20000 rpm and then once for 20 seconds using an Ultra Turrax T8 homogenizer (IKA-Labortechnik) set at speed 25000 rpm. Then, 550 µl of ice-cold 10X lysis buffer (10% w/v CHAPS) was added to each sample. After 60 minute incubation with tumbling at 4°C, samples were spun at 10000g for 30 minutes at 4°C. Supernatants were transferred into new tubes containing magnetic beads coupled to W6/32 antibodies. Samples were incubated with tumbling for 180 minutes at 4°C and placed on a magnet to recover bound MHC I complexes to magnetic beads. Magnetic beads were first washed with 8 × 1 mL PBS, then with 1 × 1 mL of 0.1X PBS and finally with 1 × 1 mL of water. MHC I complexes were eluted from the magnetic beads by acidic treatment using 0.2% trifluoroacetic acid (TFA). Eluates were transferred into 2.0 mL Costar mL Spin-X centrifuge tube filters and spun 2 minutes at 3000g. Filtrates containing peptides were separated from MHC I subunits using home-made stage tips packed with twenty 1 mm diameter octadecyl (C-18) solid phase extraction disks (EMPORE). Stage tips were pre-washed first with methanol then with 80% acetonitrile (ACN) in 0.2% TFA and finally with 0.1% formic acid (FA). Samples were loaded and then washed with 0.1% FA. Peptides were eluted with 30% ACN in 0.1%TFA, dried using vacuum centrifugation and then stored at -20°C. MAP extracts were loaded on a home-made C18 analytical column (15 cm x 150 𝜇m i.d. packed with C18 Jupiter Phenomenex) with a 100-min gradient from 5–28 % acetonitrile (0.2 % formic acid) and a 600 nl.min-1 flow rate on a nEasy-LC II system. | Lung tumor biopsies (wet weight ranging from 771 mgs to 1825 mgs), were cut in small pieces (cubes, ~3 mm in size) and 5 ml of ice-cold PBS containing protein inhibitor cocktail was added to each tissue sample. | | | Noncoding regions are the main source of targetable tumor-specific antigens |
| PXD009754 | Cancer | Lung Cancer | MHC I-associated peptides (MAPs) presented at the surface of nucleated cells play a central role in CD8 T-cell immunosurveillance. MAPs presented by mature (i.e. MHC IIhi) medullary thymic epithelial cells (mTEChi) are essential to eliminate self-reactive CD8 T cells in a process called central tolerance. On tumor cells, MAPs that do not induce tolerance (i.e. non-tolerogenic MAPs), because absent from mTEChi or any other normal cells, are referred to as tumor-specific antigens (TSAs). Despite their clinical relevance, very few have been identified, even in highly mutated tumor types. Thus, we developed a novel proteogenomic workflow able to characterize the full TSA landscape of any tumor. Briefly, using RNA-seq data, we subtracted the mTEChi from the tumor signal to generate tumor-specific protein databases enriched in non-tolerogenic sequences. Using these databases to analyze the MAP repertoire of two murine cell lines (CT26 and EL4) sequenced by mass spectrometry, we identified a total of 21 TSAs, 90% of which derived from allegedly non-coding regions. Interestingly, our results highlighted that 70% of those TSAs derived from non-mutated yet tumor-restricted sequences, e.g. endogenous retroelements. Moreover, we showed that our approach is easily amenable to analyze human primary samples as we were able to identify TSAs in three lung tumor biopsies and four B-ALL specimens. Focusing on 5 TSAs, we demonstrated that both TSA expression and TSA-specific T-cell frequency in the pre-immune repertoire influenced the overall survival of pre-immunized tumor-bearing mice. In conclusion, this proof-of-concept study demonstrates that non-coding-derived TSAs are frequent and protective in vivo, while they could be shared by several individuals. Altogether, our findings will help expand the repertoire of human TSAs and facilitate their prioritization in the clinic. | Lung tumor biopsies (wet weight ranging from 771 mgs to 1825 mgs), were cut in small pieces (cubes, ~3 mm in size) and 5 ml of ice-cold PBS containing protein inhibitor cocktail was added to each tissue sample. Tissues were first homogenized twice for 20 seconds using an Ultra Turrax T25 homogenizer (IKA-Labortechnik) set at speed 20000 rpm and then once for 20 seconds using an Ultra Turrax T8 homogenizer (IKA-Labortechnik) set at speed 25000 rpm. Then, 550 µl of ice-cold 10X lysis buffer (10% w/v CHAPS) was added to each sample. After 60 minute incubation with tumbling at 4°C, samples were spun at 10000g for 30 minutes at 4°C. Supernatants were transferred into new tubes containing magnetic beads coupled to W6/32 antibodies. Samples were incubated with tumbling for 180 minutes at 4°C and placed on a magnet to recover bound MHC I complexes to magnetic beads. Magnetic beads were first washed with 8 × 1 mL PBS, then with 1 × 1 mL of 0.1X PBS and finally with 1 × 1 mL of water. MHC I complexes were eluted from the magnetic beads by acidic treatment using 0.2% trifluoroacetic acid (TFA). Eluates were transferred into 2.0 mL Costar mL Spin-X centrifuge tube filters and spun 2 minutes at 3000g. Filtrates containing peptides were separated from MHC I subunits using home-made stage tips packed with twenty 1 mm diameter octadecyl (C-18) solid phase extraction disks (EMPORE). Stage tips were pre-washed first with methanol then with 80% acetonitrile (ACN) in 0.2% TFA and finally with 0.1% formic acid (FA). Samples were loaded and then washed with 0.1% FA. Peptides were eluted with 30% ACN in 0.1%TFA, dried using vacuum centrifugation and then stored at -20°C. MAP extracts were loaded on a home-made C18 analytical column (15 cm x 150 𝜇m i.d. packed with C18 Jupiter Phenomenex) with a 100-min gradient from 5–28 % acetonitrile (0.2 % formic acid) and a 600 nl.min-1 flow rate on a nEasy-LC II system. | Lung tumor biopsies (wet weight ranging from 771 mgs to 1825 mgs), were cut in small pieces (cubes, ~3 mm in size) and 5 ml of ice-cold PBS containing protein inhibitor cocktail was added to each tissue sample. Tissues were first homogenized twice for 20 seconds using an Ultra Turrax T25 homogenizer (IKA-Labortechnik) set at speed 20000 rpm and then once for 20 seconds using an Ultra Turrax T8 homogenizer (IKA-Labortechnik) set at speed 25000 rpm. Then, 550 µl of ice-cold 10X lysis buffer (10% w/v CHAPS) was added to each sample. After 60 minute incubation with tumbling at 4°C, samples were spun at 10000g for 30 minutes at 4°C. Supernatants were transferred into new tubes containing magnetic beads coupled to W6/32 antibodies. Samples were incubated with tumbling for 180 minutes at 4°C and placed on a magnet to recover bound MHC I complexes to magnetic beads. Magnetic beads were first washed with 8 × 1 mL PBS, then with 1 × 1 mL of 0.1X PBS and finally with 1 × 1 mL of water. MHC I complexes were eluted from the magnetic beads by acidic treatment using 0.2% trifluoroacetic acid (TFA). Eluates were transferred into 2.0 mL Costar mL Spin-X centrifuge tube filters and spun 2 minutes at 3000g. Filtrates containing peptides were separated from MHC I subunits using home-made stage tips packed with twenty 1 mm diameter octadecyl (C-18) solid phase extraction disks (EMPORE). Stage tips were pre-washed first with methanol then with 80% acetonitrile (ACN) in 0.2% TFA and finally with 0.1% formic acid (FA). Samples were loaded and then washed with 0.1% FA. Peptides were eluted with 30% ACN in 0.1%TFA, dried using vacuum centrifugation and then stored at -20°C. MAP extracts were loaded on a home-made C18 analytical column (15 cm x 150 𝜇m i.d. packed with C18 Jupiter Phenomenex) with a 100-min gradient from 5–28 % acetonitrile (0.2 % formic acid) and a 600 nl.min-1 flow rate on a nEasy-LC II system. | Lung tumor biopsies (wet weight ranging from 771 mgs to 1825 mgs), were cut in small pieces (cubes, ~3 mm in size) and 5 ml of ice-cold PBS containing protein inhibitor cocktail was added to each tissue sample. | | | Noncoding regions are the main source of targetable tumor-specific antigens |
| PXD009755 | Cancer | Lung Cancer | MHC I-associated peptides (MAPs) presented at the surface of nucleated cells play a central role in CD8 T-cell immunosurveillance. MAPs presented by mature (i.e. MHC IIhi) medullary thymic epithelial cells (mTEChi) are essential to eliminate self-reactive CD8 T cells in a process called central tolerance. On tumor cells, MAPs that do not induce tolerance (i.e. non-tolerogenic MAPs), because absent from mTEChi or any other normal cells, are referred to as tumor-specific antigens (TSAs). Despite their clinical relevance, very few have been identified, even in highly mutated tumor types. Thus, we developed a novel proteogenomic workflow able to characterize the full TSA landscape of any tumor. Briefly, using RNA-seq data, we subtracted the mTEChi from the tumor signal to generate tumor-specific protein databases enriched in non-tolerogenic sequences. Using these databases to analyze the MAP repertoire of two murine cell lines (CT26 and EL4) sequenced by mass spectrometry, we identified a total of 21 TSAs, 90% of which derived from allegedly non-coding regions. Interestingly, our results highlighted that 70% of those TSAs derived from non-mutated yet tumor-restricted sequences, e.g. endogenous retroelements. Moreover, we showed that our approach is easily amenable to analyze human primary samples as we were able to identify TSAs in three lung tumor biopsies and four B-ALL specimens. Focusing on 5 TSAs, we demonstrated that both TSA expression and TSA-specific T-cell frequency in the pre-immune repertoire influenced the overall survival of pre-immunized tumor-bearing mice. In conclusion, this proof-of-concept study demonstrates that non-coding-derived TSAs are frequent and protective in vivo, while they could be shared by several individuals. Altogether, our findings will help expand the repertoire of human TSAs and facilitate their prioritization in the clinic. | Lung tumor biopsies (wet weight ranging from 771 mgs to 1825 mgs), were cut in small pieces (cubes, ~3 mm in size) and 5 ml of ice-cold PBS containing protein inhibitor cocktail was added to each tissue sample. Tissues were first homogenized twice for 20 seconds using an Ultra Turrax T25 homogenizer (IKA-Labortechnik) set at speed 20000 rpm and then once for 20 seconds using an Ultra Turrax T8 homogenizer (IKA-Labortechnik) set at speed 25000 rpm. Then, 550 µl of ice-cold 10X lysis buffer (10% w/v CHAPS) was added to each sample. After 60 minute incubation with tumbling at 4°C, samples were spun at 10000g for 30 minutes at 4°C. Supernatants were transferred into new tubes containing magnetic beads coupled to W6/32 antibodies. Samples were incubated with tumbling for 180 minutes at 4°C and placed on a magnet to recover bound MHC I complexes to magnetic beads. Magnetic beads were first washed with 8 × 1 mL PBS, then with 1 × 1 mL of 0.1X PBS and finally with 1 × 1 mL of water. MHC I complexes were eluted from the magnetic beads by acidic treatment using 0.2% trifluoroacetic acid (TFA). Eluates were transferred into 2.0 mL Costar mL Spin-X centrifuge tube filters and spun 2 minutes at 3000g. Filtrates containing peptides were separated from MHC I subunits using home-made stage tips packed with twenty 1 mm diameter octadecyl (C-18) solid phase extraction disks (EMPORE). Stage tips were pre-washed first with methanol then with 80% acetonitrile (ACN) in 0.2% TFA and finally with 0.1% formic acid (FA). Samples were loaded and then washed with 0.1% FA. Peptides were eluted with 30% ACN in 0.1%TFA, dried using vacuum centrifugation and then stored at -20°C. MAP extracts were loaded on a home-made C18 analytical column (15 cm x 150 𝜇m i.d. packed with C18 Jupiter Phenomenex) with a 100-min gradient from 5–28 % acetonitrile (0.2 % formic acid) and a 600 nl.min-1 flow rate on a nEasy-LC II system. | Lung tumor biopsies (wet weight ranging from 771 mgs to 1825 mgs), were cut in small pieces (cubes, ~3 mm in size) and 5 ml of ice-cold PBS containing protein inhibitor cocktail was added to each tissue sample. Tissues were first homogenized twice for 20 seconds using an Ultra Turrax T25 homogenizer (IKA-Labortechnik) set at speed 20000 rpm and then once for 20 seconds using an Ultra Turrax T8 homogenizer (IKA-Labortechnik) set at speed 25000 rpm. Then, 550 µl of ice-cold 10X lysis buffer (10% w/v CHAPS) was added to each sample. After 60 minute incubation with tumbling at 4°C, samples were spun at 10000g for 30 minutes at 4°C. Supernatants were transferred into new tubes containing magnetic beads coupled to W6/32 antibodies. Samples were incubated with tumbling for 180 minutes at 4°C and placed on a magnet to recover bound MHC I complexes to magnetic beads. Magnetic beads were first washed with 8 × 1 mL PBS, then with 1 × 1 mL of 0.1X PBS and finally with 1 × 1 mL of water. MHC I complexes were eluted from the magnetic beads by acidic treatment using 0.2% trifluoroacetic acid (TFA). Eluates were transferred into 2.0 mL Costar mL Spin-X centrifuge tube filters and spun 2 minutes at 3000g. Filtrates containing peptides were separated from MHC I subunits using home-made stage tips packed with twenty 1 mm diameter octadecyl (C-18) solid phase extraction disks (EMPORE). Stage tips were pre-washed first with methanol then with 80% acetonitrile (ACN) in 0.2% TFA and finally with 0.1% formic acid (FA). Samples were loaded and then washed with 0.1% FA. Peptides were eluted with 30% ACN in 0.1%TFA, dried using vacuum centrifugation and then stored at -20°C. MAP extracts were loaded on a home-made C18 analytical column (15 cm x 150 𝜇m i.d. packed with C18 Jupiter Phenomenex) with a 100-min gradient from 5–28 % acetonitrile (0.2 % formic acid) and a 600 nl.min-1 flow rate on a nEasy-LC II system. | Lung tumor biopsies (wet weight ranging from 771 mgs to 1825 mgs), were cut in small pieces (cubes, ~3 mm in size) and 5 ml of ice-cold PBS containing protein inhibitor cocktail was added to each tissue sample. | | | Noncoding regions are the main source of targetable tumor-specific antigens |
| PXD009749 | Cancer | B-Acute Lymphoblastic Leukemia | MHC I-associated peptides (MAPs) presented at the surface of nucleated cells play a central role in CD8 T-cell immunosurveillance. MAPs presented by mature (i.e. MHC IIhi) medullary thymic epithelial cells (mTEChi) are essential to eliminate self-reactive CD8 T cells in a process called central tolerance. On tumor cells, MAPs that do not induce tolerance (i.e. non-tolerogenic MAPs), because absent from mTEChi or any other normal cells, are referred to as tumor-specific antigens (TSAs). Despite their clinical relevance, very few have been identified, even in highly mutated tumor types. Thus, we developed a novel proteogenomic workflow able to characterize the full TSA landscape of any tumor. Briefly, using RNA-seq data, we subtracted the mTEChi from the tumor signal to generate tumor-specific protein databases enriched in non-tolerogenic sequences. Using these databases to analyze the MAP repertoire of two murine cell lines (CT26 and EL4) sequenced by mass spectrometry, we identified a total of 21 TSAs, 90% of which derived from allegedly non-coding regions. Interestingly, our results highlighted that 70% of those TSAs derived from non-mutated yet tumor-restricted sequences, e.g. endogenous retroelements. Moreover, we showed that our approach is easily amenable to analyze human primary samples as we were able to identify TSAs in three lung tumor biopsies and four B-ALL specimens. Focusing on 5 TSAs, we demonstrated that both TSA expression and TSA-specific T-cell frequency in the pre-immune repertoire influenced the overall survival of pre-immunized tumor-bearing mice. In conclusion, this proof-of-concept study demonstrates that non-coding-derived TSAs are frequent and protective in vivo, while they could be shared by several individuals. Altogether, our findings will help expand the repertoire of human TSAs and facilitate their prioritization in the clinic. | MHC I-associated peptides from three replicates of 650 x 10^6 cells were obtained by mild acid elution using, per replicate, 2.5ml of citrate buffer at pH 3.3 (0.131M citric acid / 0.066M Na2HPO4, 150mM NaCl) containing aprotinin and iodoacetamide (1:100). Peptides were then desalted on an Oasis HLB cartridge (30 mg, Waters) and filtered on a 3 kDa molecular weight cut-off (Amicon Ultra-4, Millipore) to remove 𝛽2m proteins. After being dried using a Speed-Vac, peptides were re-suspended in 0.2 % formic acid, loaded on a home-made C18 analytical column (15 cm x 150 𝜇m i.d. packed with C18 Jupiter Phenomenex) with a 56-min gradient from 0–40 % acetonitrile (0.2 % formic acid) and a 600 nl.min-1 flow rate on a nEasy-LC II system. | MHC I-associated peptides from three replicates of 650 x 10^6 cells were obtained by mild acid elution using, per replicate, 2.5ml of citrate buffer at pH 3.3 (0.131M citric acid / 0.066M Na2HPO4, 150mM NaCl) containing aprotinin and iodoacetamide (1:100). Peptides were then desalted on an Oasis HLB cartridge (30 mg, Waters) and filtered on a 3 kDa molecular weight cut-off (Amicon Ultra-4, Millipore) to remove 𝛽2m proteins. After being dried using a Speed-Vac, peptides were re-suspended in 0.2 % formic acid, loaded on a home-made C18 analytical column (15 cm x 150 𝜇m i.d. packed with C18 Jupiter Phenomenex) with a 56-min gradient from 0–40 % acetonitrile (0.2 % formic acid) and a 600 nl.min-1 flow rate on a nEasy-LC II system. | 1-2 x 106 B-ALL cells were thawed and transplanted via i.v. injection into 8–12-week-old sub-lethally irradiated (250 cGy, 137 Cs-gamma source) NSG mice. Mice were sacrificed at signs of disease and cell suspensions were prepared from a mix of splenocytes, bone marrow and peritoneal ascites. From there, Ficoll gradients were used to enrich for B-ALL cells prior to MAP isolation. | | 1-2 x 106 B-ALL cells were thawed and transplanted via i.v. injection into 8–12-week-old sub-lethally irradiated (250 cGy, 137 Cs-gamma source) NSG mice. Mice were sacrificed at signs of disease and cell suspensions were prepared from a mix of splenocytes, bone marrow and peritoneal ascites. From there, Ficoll gradients were used to enrich for B-ALL cells prior to MAP isolation. | Noncoding regions are the main source of targetable tumor-specific antigens |
| PXD009750 | Cancer | B-Acute Lymphoblastic Leukemia | MHC I-associated peptides (MAPs) presented at the surface of nucleated cells play a central role in CD8 T-cell immunosurveillance. MAPs presented by mature (i.e. MHC IIhi) medullary thymic epithelial cells (mTEChi) are essential to eliminate self-reactive CD8 T cells in a process called central tolerance. On tumor cells, MAPs that do not induce tolerance (i.e. non-tolerogenic MAPs), because absent from mTEChi or any other normal cells, are referred to as tumor-specific antigens (TSAs). Despite their clinical relevance, very few have been identified, even in highly mutated tumor types. Thus, we developed a novel proteogenomic workflow able to characterize the full TSA landscape of any tumor. Briefly, using RNA-seq data, we subtracted the mTEChi from the tumor signal to generate tumor-specific protein databases enriched in non-tolerogenic sequences. Using these databases to analyze the MAP repertoire of two murine cell lines (CT26 and EL4) sequenced by mass spectrometry, we identified a total of 21 TSAs, 90% of which derived from allegedly non-coding regions. Interestingly, our results highlighted that 70% of those TSAs derived from non-mutated yet tumor-restricted sequences, e.g. endogenous retroelements. Moreover, we showed that our approach is easily amenable to analyze human primary samples as we were able to identify TSAs in three lung tumor biopsies and four B-ALL specimens. Focusing on 5 TSAs, we demonstrated that both TSA expression and TSA-specific T-cell frequency in the pre-immune repertoire influenced the overall survival of pre-immunized tumor-bearing mice. In conclusion, this proof-of-concept study demonstrates that non-coding-derived TSAs are frequent and protective in vivo, while they could be shared by several individuals. Altogether, our findings will help expand the repertoire of human TSAs and facilitate their prioritization in the clinic. | MHC I-associated peptides from three replicates of 650 x 10^6 cells were obtained by mild acid elution using, per replicate, 2.5ml of citrate buffer at pH 3.3 (0.131M citric acid / 0.066M Na2HPO4, 150mM NaCl) containing aprotinin and iodoacetamide (1:100). Peptides were then desalted on an Oasis HLB cartridge (30 mg, Waters) and filtered on a 3 kDa molecular weight cut-off (Amicon Ultra-4, Millipore) to remove 𝛽2m proteins. After being dried using a Speed-Vac, peptides were re-suspended in 0.2 % formic acid, loaded on a home-made C18 analytical column (15 cm x 150 𝜇m i.d. packed with C18 Jupiter Phenomenex) with a 56-min gradient from 0–40 % acetonitrile (0.2 % formic acid) and a 600 nl.min-1 flow rate on a nEasy-LC II system. | MHC I-associated peptides from three replicates of 650 x 10^6 cells were obtained by mild acid elution using, per replicate, 2.5ml of citrate buffer at pH 3.3 (0.131M citric acid / 0.066M Na2HPO4, 150mM NaCl) containing aprotinin and iodoacetamide (1:100). Peptides were then desalted on an Oasis HLB cartridge (30 mg, Waters) and filtered on a 3 kDa molecular weight cut-off (Amicon Ultra-4, Millipore) to remove 𝛽2m proteins. After being dried using a Speed-Vac, peptides were re-suspended in 0.2 % formic acid, loaded on a home-made C18 analytical column (15 cm x 150 𝜇m i.d. packed with C18 Jupiter Phenomenex) with a 56-min gradient from 0–40 % acetonitrile (0.2 % formic acid) and a 600 nl.min-1 flow rate on a nEasy-LC II system. | 1-2 x 106 B-ALL cells were thawed and transplanted via i.v. injection into 8–12-week-old sub-lethally irradiated (250 cGy, 137 Cs-gamma source) NSG mice. Mice were sacrificed at signs of disease and cell suspensions were prepared from a mix of splenocytes, bone marrow and peritoneal ascites. From there, Ficoll gradients were used to enrich for B-ALL cells prior to MAP isolation. | | 1-2 x 106 B-ALL cells were thawed and transplanted via i.v. injection into 8–12-week-old sub-lethally irradiated (250 cGy, 137 Cs-gamma source) NSG mice. Mice were sacrificed at signs of disease and cell suspensions were prepared from a mix of splenocytes, bone marrow and peritoneal ascites. From there, Ficoll gradients were used to enrich for B-ALL cells prior to MAP isolation. | Noncoding regions are the main source of targetable tumor-specific antigens |
| PXD009751 | Cancer | B-Acute Lymphoblastic Leukemia | MHC I-associated peptides (MAPs) presented at the surface of nucleated cells play a central role in CD8 T-cell immunosurveillance. MAPs presented by mature (i.e. MHC IIhi) medullary thymic epithelial cells (mTEChi) are essential to eliminate self-reactive CD8 T cells in a process called central tolerance. On tumor cells, MAPs that do not induce tolerance (i.e. non-tolerogenic MAPs), because absent from mTEChi or any other normal cells, are referred to as tumor-specific antigens (TSAs). Despite their clinical relevance, very few have been identified, even in highly mutated tumor types. Thus, we developed a novel proteogenomic workflow able to characterize the full TSA landscape of any tumor. Briefly, using RNA-seq data, we subtracted the mTEChi from the tumor signal to generate tumor-specific protein databases enriched in non-tolerogenic sequences. Using these databases to analyze the MAP repertoire of two murine cell lines (CT26 and EL4) sequenced by mass spectrometry, we identified a total of 21 TSAs, 90% of which derived from allegedly non-coding regions. Interestingly, our results highlighted that 70% of those TSAs derived from non-mutated yet tumor-restricted sequences, e.g. endogenous retroelements. Moreover, we showed that our approach is easily amenable to analyze human primary samples as we were able to identify TSAs in three lung tumor biopsies and four B-ALL specimens. Focusing on 5 TSAs, we demonstrated that both TSA expression and TSA-specific T-cell frequency in the pre-immune repertoire influenced the overall survival of pre-immunized tumor-bearing mice. In conclusion, this proof-of-concept study demonstrates that non-coding-derived TSAs are frequent and protective in vivo, while they could be shared by several individuals. Altogether, our findings will help expand the repertoire of human TSAs and facilitate their prioritization in the clinic. | MHC I-associated peptides from three replicates of 650 x 10^6 cells were obtained by mild acid elution using, per replicate, 2.5ml of citrate buffer at pH 3.3 (0.131M citric acid / 0.066M Na2HPO4, 150mM NaCl) containing aprotinin and iodoacetamide (1:100). Peptides were then desalted on an Oasis HLB cartridge (30 mg, Waters) and filtered on a 3 kDa molecular weight cut-off (Amicon Ultra-4, Millipore) to remove 𝛽2m proteins. After being dried using a Speed-Vac, peptides were re-suspended in 0.2 % formic acid, loaded on a home-made C18 analytical column (15 cm x 150 𝜇m i.d. packed with C18 Jupiter Phenomenex) with a 56-min gradient from 0–40 % acetonitrile (0.2 % formic acid) and a 600 nl.min-1 flow rate on a nEasy-LC II system. | MHC I-associated peptides from three replicates of 650 x 10^6 cells were obtained by mild acid elution using, per replicate, 2.5ml of citrate buffer at pH 3.3 (0.131M citric acid / 0.066M Na2HPO4, 150mM NaCl) containing aprotinin and iodoacetamide (1:100). Peptides were then desalted on an Oasis HLB cartridge (30 mg, Waters) and filtered on a 3 kDa molecular weight cut-off (Amicon Ultra-4, Millipore) to remove 𝛽2m proteins. After being dried using a Speed-Vac, peptides were re-suspended in 0.2 % formic acid, loaded on a home-made C18 analytical column (15 cm x 150 𝜇m i.d. packed with C18 Jupiter Phenomenex) with a 56-min gradient from 0–40 % acetonitrile (0.2 % formic acid) and a 600 nl.min-1 flow rate on a nEasy-LC II system. | 1-2 x 106 B-ALL cells were thawed and transplanted via i.v. injection into 8–12-week-old sub-lethally irradiated (250 cGy, 137 Cs-gamma source) NSG mice. Mice were sacrificed at signs of disease and cell suspensions were prepared from a mix of splenocytes, bone marrow and peritoneal ascites. From there, Ficoll gradients were used to enrich for B-ALL cells prior to MAP isolation. | | 1-2 x 106 B-ALL cells were thawed and transplanted via i.v. injection into 8–12-week-old sub-lethally irradiated (250 cGy, 137 Cs-gamma source) NSG mice. Mice were sacrificed at signs of disease and cell suspensions were prepared from a mix of splenocytes, bone marrow and peritoneal ascites. From there, Ficoll gradients were used to enrich for B-ALL cells prior to MAP isolation. | Noncoding regions are the main source of targetable tumor-specific antigens |
| PXD009753 | Cancer | B-Acute Lymphoblastic Leukemia | MHC I-associated peptides (MAPs) presented at the surface of nucleated cells play a central role in CD8 T-cell immunosurveillance. MAPs presented by mature (i.e. MHC IIhi) medullary thymic epithelial cells (mTEChi) are essential to eliminate self-reactive CD8 T cells in a process called central tolerance. On tumor cells, MAPs that do not induce tolerance (i.e. non-tolerogenic MAPs), because absent from mTEChi or any other normal cells, are referred to as tumor-specific antigens (TSAs). Despite their clinical relevance, very few have been identified, even in highly mutated tumor types. Thus, we developed a novel proteogenomic workflow able to characterize the full TSA landscape of any tumor. Briefly, using RNA-seq data, we subtracted the mTEChi from the tumor signal to generate tumor-specific protein databases enriched in non-tolerogenic sequences. Using these databases to analyze the MAP repertoire of two murine cell lines (CT26 and EL4) sequenced by mass spectrometry, we identified a total of 21 TSAs, 90% of which derived from allegedly non-coding regions. Interestingly, our results highlighted that 70% of those TSAs derived from non-mutated yet tumor-restricted sequences, e.g. endogenous retroelements. Moreover, we showed that our approach is easily amenable to analyze human primary samples as we were able to identify TSAs in three lung tumor biopsies and four B-ALL specimens. Focusing on 5 TSAs, we demonstrated that both TSA expression and TSA-specific T-cell frequency in the pre-immune repertoire influenced the overall survival of pre-immunized tumor-bearing mice. In conclusion, this proof-of-concept study demonstrates that non-coding-derived TSAs are frequent and protective in vivo, while they could be shared by several individuals. Altogether, our findings will help expand the repertoire of human TSAs and facilitate their prioritization in the clinic. | MHC I-associated peptides from three replicates of 650 x 10^6 cells were obtained by mild acid elution using, per replicate, 2.5ml of citrate buffer at pH 3.3 (0.131M citric acid / 0.066M Na2HPO4, 150mM NaCl) containing aprotinin and iodoacetamide (1:100). Peptides were then desalted on an Oasis HLB cartridge (30 mg, Waters) and filtered on a 3 kDa molecular weight cut-off (Amicon Ultra-4, Millipore) to remove 𝛽2m proteins. After being dried using a Speed-Vac, peptides were re-suspended in 0.2 % formic acid, loaded on a home-made C18 analytical column (15 cm x 150 𝜇m i.d. packed with C18 Jupiter Phenomenex) with a 56-min gradient from 0–40 % acetonitrile (0.2 % formic acid) and a 600 nl.min-1 flow rate on a nEasy-LC II system. | MHC I-associated peptides from three replicates of 650 x 10^6 cells were obtained by mild acid elution using, per replicate, 2.5ml of citrate buffer at pH 3.3 (0.131M citric acid / 0.066M Na2HPO4, 150mM NaCl) containing aprotinin and iodoacetamide (1:100). Peptides were then desalted on an Oasis HLB cartridge (30 mg, Waters) and filtered on a 3 kDa molecular weight cut-off (Amicon Ultra-4, Millipore) to remove 𝛽2m proteins. After being dried using a Speed-Vac, peptides were re-suspended in 0.2 % formic acid, loaded on a home-made C18 analytical column (15 cm x 150 𝜇m i.d. packed with C18 Jupiter Phenomenex) with a 56-min gradient from 0–40 % acetonitrile (0.2 % formic acid) and a 600 nl.min-1 flow rate on a nEasy-LC II system. | 1-2 x 106 B-ALL cells were thawed and transplanted via i.v. injection into 8–12-week-old sub-lethally irradiated (250 cGy, 137 Cs-gamma source) NSG mice. Mice were sacrificed at signs of disease and cell suspensions were prepared from a mix of splenocytes, bone marrow and peritoneal ascites. From there, Ficoll gradients were used to enrich for B-ALL cells prior to MAP isolation. | | 1-2 x 106 B-ALL cells were thawed and transplanted via i.v. injection into 8–12-week-old sub-lethally irradiated (250 cGy, 137 Cs-gamma source) NSG mice. Mice were sacrificed at signs of disease and cell suspensions were prepared from a mix of splenocytes, bone marrow and peritoneal ascites. From there, Ficoll gradients were used to enrich for B-ALL cells prior to MAP isolation. | Noncoding regions are the main source of targetable tumor-specific antigens |
| PXD009925 | Cancer | Meningioma | HLA-I molecules bind short peptides and present them to CD8+ T cells for TCR recognition. The length of HLA-I ligands typically ranges from 8 to 12 amino acids, but high variability is observed between different alleles. Here we used recent HLA peptidomics data to analyze in an unbiased way peptide length distributions over 85 different HLA-I alleles. Our results revealed clear clustering of HLA-I alleles with distinct peptide length distributions, which enabled us to unravel some of the molecular basis of peptide length distributions and predict peptide length distributions based on HLA-I sequences only. We further took advantage of our collection of curated HLA peptidomics studies to investigate multiple specificity in HLA-I molecules and validated these observations with binding assays. Explicitly modeling peptide length distributions and multiple specificity significantly improved predictions of naturally presented HLA-I ligands, as demonstrated in an independent benchmarking based on ten newly generated HLA peptidomics datasets from meningioma samples. | We extracted HLA-I peptides from the meningioma samples as previously described (ref MCP). Briefly, snap-frozen meningioma tissue samples were placed in tubes containing ice cold PBS lysis buffer comprising of 0.25% sodium deoxycholate (Sigma-Aldrich), 0.2 mM iodoacetamide (Sigma-Aldrich), 1 mM EDTA, 1:200 Protease Inhibitors Cocktail (Sigma-Aldrich), 1 mM Phenylmethylsulfonylfluoride (Roche, Basel, Switzerland), 1% octyl-beta-D glucopyranoside (Sigma-Alrich) and homogenized on ice in 3-5 short intervals of 5 seconds each using an Ultra Turrax homogenizer (IKA, Staufen, Germany) at maximum speed. Cell lysis was performed at 4°C for 1 hour. Lysates were cleared by centrifugation at 20,000 rpm in a high speed centrifuge (Beckman Coulter, JSS15314, Nyon, Switzerland) at 4°C for 50 minutes. The lysates were loaded on stacked 96-well single-use micro-plates (3µm glass fiber, 10µm polypropylene membranes; ref number 360063, Seahorse Bioscience, North Billerica, Massachusetts). The first plate contained protein-A sepharose 4B (Pro-A) beads (Invitrogen, Carlsbad, California) for depletion of antibodies, and the second plate contained same beads cross-linked to W6/32 anti HLA monoclonal antibodies as previously described (ref MCP). We then employed the Waters Positive Pressure-96 Processor (Waters, Milford, Massachusetts). We washed the second plate with 4 times 2 mL of 150 mM sodium chloride (NaCl) (Carlo-Erba, Val de Reuil, France) in 20 mM Tris-HCl pH 8, 4 times 2 mL of 400 mM NaCl in 20 mM Tris-HCl pH 8 and again with 4 times 2 mL of 150 mM NaCl in 20 mM Tris-HCl pH 8. Finally, we washed the beads twice with 2 mL of 20 mM Tris-HCl pH 8. We stacked the affinity plate on top of a Sep-Pak tC18 100 mg Sorbent 96-well plate (ref number: 186002321, Waters) already equilibrated with 1 mL of 80% ACN in 0.1 % TFA and with 2 mL of 0.1% TFA. We eluted the HLA and peptides with 500 µL 1% TFA into the Sep-Pak plate and then we washed this plate with 2 mL of 0.1 % TFA. Thereafter, we eluted the HLA-I peptides with 500 µL of 28% ACN in 0.1% TFA into a collection plate. Recovered HLA-I peptides were dried using vacuum centrifugation (Concentrator plus Eppendorf) and stored at -20°C. Prior to MS analysis HLA-I peptide samples were re-suspended in 10 µL of 2% ACN in 0.1 % FA and aliquots of 3 ul for each MS run were placed in the Ultra HPLC autosampler. | We extracted HLA-I peptides from the meningioma samples as previously described (ref MCP). Briefly, snap-frozen meningioma tissue samples were placed in tubes containing ice cold PBS lysis buffer comprising of 0.25% sodium deoxycholate (Sigma-Aldrich), 0.2 mM iodoacetamide (Sigma-Aldrich), 1 mM EDTA, 1:200 Protease Inhibitors Cocktail (Sigma-Aldrich), 1 mM Phenylmethylsulfonylfluoride (Roche, Basel, Switzerland), 1% octyl-beta-D glucopyranoside (Sigma-Alrich) and homogenized on ice in 3-5 short intervals of 5 seconds each using an Ultra Turrax homogenizer (IKA, Staufen, Germany) at maximum speed. Cell lysis was performed at 4°C for 1 hour. Lysates were cleared by centrifugation at 20,000 rpm in a high speed centrifuge (Beckman Coulter, JSS15314, Nyon, Switzerland) at 4°C for 50 minutes. The lysates were loaded on stacked 96-well single-use micro-plates (3µm glass fiber, 10µm polypropylene membranes; ref number 360063, Seahorse Bioscience, North Billerica, Massachusetts). The first plate contained protein-A sepharose 4B (Pro-A) beads (Invitrogen, Carlsbad, California) for depletion of antibodies, and the second plate contained same beads cross-linked to W6/32 anti HLA monoclonal antibodies as previously described (ref MCP). We then employed the Waters Positive Pressure-96 Processor (Waters, Milford, Massachusetts). We washed the second plate with 4 times 2 mL of 150 mM sodium chloride (NaCl) (Carlo-Erba, Val de Reuil, France) in 20 mM Tris-HCl pH 8, 4 times 2 mL of 400 mM NaCl in 20 mM Tris-HCl pH 8 and again with 4 times 2 mL of 150 mM NaCl in 20 mM Tris-HCl pH 8. Finally, we washed the beads twice with 2 mL of 20 mM Tris-HCl pH 8. We stacked the affinity plate on top of a Sep-Pak tC18 100 mg Sorbent 96-well plate (ref number: 186002321, Waters) already equilibrated with 1 mL of 80% ACN in 0.1 % TFA and with 2 mL of 0.1% TFA. We eluted the HLA and peptides with 500 µL 1% TFA into the Sep-Pak plate and then we washed this plate with 2 mL of 0.1 % TFA. Thereafter, we eluted the HLA-I peptides with 500 µL of 28% ACN in 0.1% TFA into a collection plate. Recovered HLA-I peptides were dried using vacuum centrifugation (Concentrator plus Eppendorf) and stored at -20°C. Prior to MS analysis HLA-I peptide samples were re-suspended in 10 µL of 2% ACN in 0.1 % FA and aliquots of 3 ul for each MS run were placed in the Ultra HPLC autosampler. | Snap frozen meningioma tissues from patients were obtained from the Centre hospitalier universitaire vaudois (CHUV, Lausanne, Switzerland). | | | Sensitive Immunopeptidomics by Leveraging Available Large-Scale Multi-HLA Spectral Libraries, Data-Independent Acquisition, and MS/MS Prediction |
| PXD009935 | Cancer | Lung Cancer | Mass spectrometry analysis of the immunopeptidome after TNF/IFN stimulation | The LC-MS/MS analyses of the HLA and the tryptic peptides were performed with a Q-Exactive-Plus mass spectrometer fitted with either with Easy nLC 1000 capillary HPLC (Thermo-Fisher Scientific) or with Ultimate 3000 RSLC nano-capillary UHPLC (Thermo-Fisher Scientific). The reversed phase chromatographies were performed with home-made 30 cm long, 75 μm inner diameter, packed with 3.5 μm silica ReproSil-Pur C18-AQ resin (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany), as in Ishihama et al. (87). The HLA peptides were eluted using a linear gradient of 5–28% of acetonitrile in 0.1% formic acid, at a flow rate of 0.15 μl/min for 2 h. Data was acquired using a data-dependent “top 10” method, fragmenting the peptides by higher-energy collisional dissociation (HCD). Full scan MS spectra were acquired at a resolution of 70,000 at 200 m/z with a target value of 3 × 106 ions. Fragmented masses were accumulated to AGC (automatic gain control) target value of 105 with a maximum injection time of 100 ms. No fragmentation was attempted for HLA peptides with unassigned precursor charge states. The peptide match option was set to Preferred. The normalized collision energy was set to 25% and MS/MS resolution was 17,500 at 200 m/z. Fragmented m/z values were dynamically excluded from further selection for 20 s. | Membranal HLA (mHLA) class I molecules were purified similarly to Hunt et al. (83) with modifications as described in Milner et al. (84) with minor modifications, from about 5 × 108 cells. The cells were lysed with 0.25% sodium deoxycholate, 0.2 mM iodoacetamide, 1 mM EDTA, 1:200 Protease Inhibitors Cocktail (Sigma Aldrich, St. Louis, MO), 1 mM PMSF and 1% octyl-β-D glucopyranoside (Sigma Aldrich) in PBS at 4°C for 1 h. Cell extracts were cleared by centrifugation for 45 min, at 48,000 g and at 4°C. The HLA class I molecules were immunoaffinity purified using the W6/32 mAb bound to protein A-resin beads (Genscript, Piscataway, NJ) as in Barnea et al. (85). The HLA molecules with their bound peptides were eluted from the affinity column with five column volumes of 1% TFA. The eluted HLA class I proteins and the released peptides were loaded on disposable C18 columns (Harvard Apparatus, Holliston, MA) and the peptides fraction was recovered with 30% acetonitrile in 0.1% TFA, as in Milner et al. (84). The peptides were dried using vacuum centrifugation, reconstituted with 100 μl of 0.1% TFA, reloaded on C18 StageTips, prepared as in Rappsilber et al. (86), eluted with 80% acetonitrile, dried and reconstituted with 0.1% formic acid for the LC-MS/MS analysis. | A549 cells | | A549 cells were grown in DMEM supplemented with 10% fetal bovine serum, 1% Penicillin/streptomycin and L-glutamine (2 mM) (Biological industries) at 37°C with 5% CO2. Cells were treated with 400 U*mL-1 TNFα and/or 200 U*mL-1 IFNγ (peprotech) for the indicated amount of time. | Pro-inflammatory Cytokines Alter the Immunopeptidome Landscape by Modulation of HLA-B Expression |
| PXD010808 | Cancer | Lymphoma | Immunoglobulin gene rearrangement and somatic hypermutation have the potential to create neoantigens in non-Hodgkin B cell lymphoma. However, the presentation of these putative immunoglobulin neoantigens by B cell lymphomas has not been proven. We used MHC immunoprecipitation followed by liquid chromatography and tandem mass spectrometry (LC-MS/MS) to define antigens presented by follicular lymphomas (FL), chronic lymphocytic leukemias (CLL), diffuse large B cell lymphoma (DLBCL) and mantle cell lymphomas (MCL). We found presentation of the clonal immunoglobulin molecule, including neoantigens by both class I and class II MHC, though more commonly in class II MHC. To determine whether B cell activation could promote presentation of immunoglobulin neoantigens, we used a toll-like receptor 9 (TLR9) agonists to upregulate expression of MHC-II. This resulted in enhanced class II MHC presentation of the immunoglobulin variable region including neoantigens. These findings demonstrate that immunoglobulin neoantigens are presented across most subtypes of B cell lymphomas. Activation of lymphoma cells to upregulate antigen presentation boosts presentation of immunoglobulin neoantigens and represents a strategy for augmenting lymphoma immunotherapies. | In brief, cells were lysed and the lysate was subjected to centrifugation and then precleared using rProtein A Sepharose fast-flow beads (GE Healthcare). For all MHC-1 captures the precleared lysate was incubated with the pan HLA-A-, B-, and C- antibody W6/32 coupled to rProtein A Sepharose fast-flow beads. For MHC class II captures the precleared lysate was incubated with the HLA-DR specific antibody L243 coupled to rProtein A Sepharose fast-flow beads. Following the immune-capture, the beads were washed with TBS and peptides were eluted from the purified MHC-molecules using 10% acetic acid. The peptides were further purified with a MWCO size filter, followed by a concentration step and then desalted on C18 based StageTips. The isolated peptides were analyzed on an LTQ Orbitrap Elite mass spectrometer (Thermo Fischer Scientific) or Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fischer Scientific). | In brief, cells were lysed and the lysate was subjected to centrifugation and then precleared using rProtein A Sepharose fast-flow beads (GE Healthcare). For all MHC-1 captures the precleared lysate was incubated with the pan HLA-A-, B-, and C- antibody W6/32 coupled to rProtein A Sepharose fast-flow beads. For MHC class II captures the precleared lysate was incubated with the HLA-DR specific antibody L243 coupled to rProtein A Sepharose fast-flow beads. Following the immune-capture, the beads were washed with TBS and peptides were eluted from the purified MHC-molecules using 10% acetic acid. The peptides were further purified with a MWCO size filter, followed by a concentration step and then desalted on C18 based StageTips. The isolated peptides were analyzed on an LTQ Orbitrap Elite mass spectrometer (Thermo Fischer Scientific) or Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fischer Scientific). | follicular lymphomas (FL), chronic lymphocytic leukemias (CLL), diffuse large B cell lymphoma (DLBCL) and mantle cell lymphomas (MCL). | | | B cell lymphomas present immunoglobulin neoantigens. |
| PXD011723 | Cancer | T-Acute Lymphoblastic Leukemia | We propose a new pipeline for the refinement of spectrum-peptide assignment, designed specifically for MHC ligand identification. By modeling the peptidome as a collection of a limited number of specificities, corresponding to the MHC alleles of the cell line, our method achieves increased sequencing depth, while at the same time removing potential experimental outliers and contaminants. | Jurkat cells were resuspended in 1ml 2x lysis buffer per 108 cells. Lysates were cleared by subsequent centrifugation steps at 500 g for 10 min and then 20,000 g for 60 min. 1 mg per sample of anti-HLA class I antibody (W6/32, ATCC HB-95) was bound and cross-linked to 1 ml Protein A beads (GE healthcare) and used for immunoprecipitation of HLA complexes. In brief, lysates were incubated with the antibody beads over night at 4ºC in 1x lysis buffer and washed subsequently with 10 bed volumes of 50 mM Tris, pH 8.0 containing either 150 mM, 450 mM and finally no salt. Peptides were eluted with 5 ml of 10% acetic acid. Dried peptides were suspended and injected onto a 4.6 x 50 mm ProSwift RP-1S column (Thermo Fisher Scientific). Peptides were separated from larger complex components by elution using a 500 μl/min flow rate over 10 min from 2-34% ACN in 0.1% TFA. Alternate fractions that did not contain the beta-2-microglobulin were pooled and two final fractions were analyzed by nUPLC-MS2. | Jurkat cells were resuspended in 1ml 2x lysis buffer per 108 cells. Lysates were cleared by subsequent centrifugation steps at 500 g for 10 min and then 20,000 g for 60 min. 1 mg per sample of anti-HLA class I antibody (W6/32, ATCC HB-95) was bound and cross-linked to 1 ml Protein A beads (GE healthcare) and used for immunoprecipitation of HLA complexes. In brief, lysates were incubated with the antibody beads over night at 4ºC in 1x lysis buffer and washed subsequently with 10 bed volumes of 50 mM Tris, pH 8.0 containing either 150 mM, 450 mM and finally no salt. Peptides were eluted with 5 ml of 10% acetic acid. Dried peptides were suspended and injected onto a 4.6 x 50 mm ProSwift RP-1S column (Thermo Fisher Scientific). Peptides were separated from larger complex components by elution using a 500 μl/min flow rate over 10 min from 2-34% ACN in 0.1% TFA. Alternate fractions that did not contain the beta-2-microglobulin were pooled and two final fractions were analyzed by nUPLC-MS2. | Jurkat cells | | Jurkat cells were cultured in RPMI 1640 (Sigma) supplemented with 10% heatinactivated fetal calf serum, 2 mM L-glutamine, 1 mM pyruvate, 0.1mM Non-Essential Amino Acids Solution and 100U penicillin/ml, and was incubated at 37˚C in 5% CO2 (9). | MS-Rescue: A Computational Pipeline to Increase the Quality and Yield of Immunopeptidomics Experiments |
| PXD011766 | Cancer | Melanoma | The efficacy of cancer immunotherapy, including treatment with immune-checkpoint inhibitors, is often limited by ineffective presentation of antigenic peptides that can elicit T-cell mediated anti-tumor cytotoxic responses. Therefore, manipulating antigen presentation is an emerging approach for enhancing the immunogenicity of tumors in immunotherapy settings. ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that trims peptides that can bind onto MHC class I molecules (MHC-I). We hypothesized that pharmacologically inhibiting ERAP1 in cells can regulate the global cellular immunopeptidome. To test this hypothesis, we treated the A375 melanoma cell line with a recently developed potent ERAP1 inhibitor and analyzed the presented MHC-I peptide repertoire by isolating MHC-I, eluting the bound peptides and identifying them using capillary chromatography and tandem mass spectrometry. Although the inhibitor did not negatively affect overall MHC-I presence on the cell surface, it induced significant changes on the presented peptidomes, both at the qualitative and quantitative levels. Specifically, inhibitor treatment altered about half of the total 3204 identified peptides and about one third of the peptides predicted to be good ligands for MHC-I, affected length and sequence without however interfering with basic binding motifs. Strikingly, the inhibitor enhanced overall MHC-I binding affinity by reducing presentation of sub-optimal long peptides and generating many high-affinity 9-12mers, suggesting that baseline ERAP1 activity in this cell line is destructive for many potential epitopes. Our results suggest that chemical inhibition of ERAP1 is a valid approach for manipulating the immunopeptidome of cancer and autoimmunity. | Preparation of immunoaffinity columns W6/32 antibody (2mg per column) was dialyzed in coupling buffer (NaHCO3 0.1M, NaCl 0.5M pH 8.3) overnight. To generate one 1ml bed volume of CNBr-activated Sepharose 4B (GE Healthcare 17-0430-01) 0.285g of dry beads were weighted. The Sepharose was moisturized with 1mM HCl for 30min and then washed thoroughly with coupling buffer. The solution of the antibody was added to the beads and left for coupling overnight at 4°C. After coupling, the beads were washed with coupling buffer and then with blocking buffer (Tris-HCl 0.1M, pH 8.0). After the washes the beads were transferred to a 50ml tube with blocking buffer and were mixed for 3h at room temperature. Finally, the beads were washed with 3 cycles of acidic (CH3COONa 0.1M, NaCl 0.5M, pH 4.0) and basic (Tris-HCl 0.1M, NaCl 0.5M, pH 8.0) buffer and then with 20mM Tris-HCl pH 7.5, 150mM NaCl. For the pre-columns the exact same procedure was followed with the exception of the W6/32 coupling step. The columns and the pre-columns were stored at 4°C until needed. Isolation of MHCI immunopeptidome For the isolation of the immunopeptidome 5x108 cells per sample were used. Cells were lysed with 20ml lysis buffer (Tris-HCL pH 7.5, 150mM NaCl, 0.5% Igepal CA-630, 0.25% sodium deoxycholate, 1mM EDTA pH 8.0, 1x complete EDTA free protease inhibitor cocktail tablets) for 1h at 4°C. The cell lysate was cleared with ultracentrifugation at 100000g for 1h at 4°C and then loaded onto a CN-Br activated Sepharose pre-column, blocked as described above. The flow through from the pre-column was passed through W6/32 coupled beads three times and then washed with 20 bed volumes 20mM Tris-HCl pH 8.0, 150mM NaCl, 20 bed volumes 20mM Tris-HCl pH 8.0, 400mM NaCl, 20 bed volumes 20mM Tris-HCl pH 8.0, 150mM NaCl and finally with 40 bed volumes 20mM Tris-HCl pH 8.0. The MHCI-peptide complexes were eluted from the immunoaffinity column with 1% TFA. The peptides were separated from the MHCI molecules using reversed phase C18 disposable spin columns (Thermo Scientific). The fraction containing the peptides was dried prior to LC-MS/MS analysis. Mass spectrometry analysis Each sample was analyzed in a Q-Exactive-Plus mass spectrometer (Thermo Fisher Scientific) as described previously 67. The peptides were resolved using 7– 40% acetonitrile gradients with 0.1% formic acid for 180 min and 0.15 l/min on a 0.075 mm x 30 cm capillary column pressure-packed with Reprosil C18-Aqua (Dr. Maisch, GmbH, Ammerbuch-Entringen, Germany) as described previously 68. The dynamic exclusion was set to 20 s; the selected masses were fragmented from the survey scan of mass to charge ratio (m/z) 300 –1,800 atomic mass units at resolution of 70,000. MS/MS spectra were acquired starting at m/z 200 with a resolution of 17,500. The target value was set to 1x105, and the isolation window was set to 1.8 Da. | W6/32 antibody (2 mg per column) was dialyzed in coupling buffer (NaHCO3 0.1 M, NaCl 0.5 M, pH 8.3) overnight. To generate one 1 ml bed volume of cyanogen bromide-activated Sepharose 4B (GE Healthcare 17-0430-01), 0.285 g of dry beads was used. Sepharose was rehydrated with 1 mM HCl for 30 min and then washed thoroughly with coupling buffer. The solution of the antibody was added to the beads and left for coupling overnight at 4 °C. After coupling, the beads were washed with coupling buffer and then with blocking buffer (Tris–HCl 0.1 M, pH 8.0). After the washes the beads were transferred to a 50-ml tube with blocking buffer and were mixed for 3 h at room temperature. Finally, the beads were washed with three cycles of acidic buffer (CH3COONa 0.1 M, NaCl 0.5 M, pH 4.0) and then basic buffer (Tris–HCl 0.1 M, NaCl 0.5 M, pH 8.0) solutions and then with 20 mM Tris–HCl, pH 7.5, 150 mM NaCl. For the pre-columns, the exact same procedure was followed with the exception of the W6/32 coupling step. The columns and the pre-columns were stored at 4 °C until needed.For the isolation of the immunopeptidome, 5 × 108 cells per sample were used. Cells were lysed with 20 ml lysis buffer (Tris–HCl, pH 7.5, 150 mM NaCl, 0.5% IGEPAL CA-630, 0.25% sodium deoxycholate, 1 mM EDTA, pH 8.0, 1 × complete EDTA-free protease inhibitor cocktail tablets) for 1 h at 4 °C. The cell lysate was cleared with ultracentrifugation at 100000 g for 1 h at 4 °C and then loaded onto a cyanogen bromide-activated Sepharose pre-column, blocked as described above. The flow through from the pre-column was passed through W6/32-coupled beads three times and then washed with 20 bed volumes 20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 20 bed volumes 20 mM Tris–HCl, pH 8.0, 400 mM NaCl, 20 bed volumes 20 mM Tris–HCl, pH 8.0, 150 mM NaCl and finally with 40 bed volumes 20 mM Tris–HCl, pH 8.0. The MHC-I–peptide complexes were eluted from the immunoaffinity column with 1% TFA. The peptides were separated from the MHC-I molecules using reversed-phase C18 disposable spin columns (Thermo Scientific). The fraction containing peptides was dried prior to LC-MS/MS analysis. | A375 cells | | Cells were cultured in DMEM containing stable glutamine, supplemented with 10% heat-inactivated FBS (Gibco), penicillin and streptomycin and incubated at 37 °C, 5% CO2. | Editing the immunopeptidome of melanoma cells using a potent inhibitor of endoplasmic reticulum aminopeptidase 1 (ERAP1). |
| PXD012083 | Cancer | Acute Myeloid Leukemia | Somatic mutations in cancer are a potential source of cancer specific neoantigens. Acute myeloid leukemia (AML) has common recurrent mutations shared between patients in addition to private mutations specific to individuals. We hypothesized that neoantigens derived from recurrent shared mutations would be attractive targets for future immunotherapy and sought to study the Class I and II HLA ligandomes of thirteen primary AML tumor samples and two AML cell lines (OCI-AML3 and MV4-11) using mass spectrometry. We identified two endogenous, mutation-bearing HLA Class I ligands from NPM1, which are predicted to bind the common HLA haplotypes, HLA-A*03:01 and HLA-A*02:01 respectively. We further derived CD8+ T cells from healthy donor peripheral blood samples which bound mutant-peptide loaded A*03:01 and A*02:01 tetramers, suggesting a new source of NPM1 mutation-specific T cell receptors (TCRs) for future evaluation. Since NPM1 is mutated in approximately one-third of patients with AML, the finding of endogenous NPM1 neoantigens supports future studies evaluating immunotherapeutic approaches against this target, for this subset of patients with AML. | MHC-class I and class II peptidomes were extracted from primary AML tumor samples and two AML cell lines. In brief, cells were lysed and the lysate was subjected to centrifugation and then precleared using rProtein A Sepharose fast-flow beads (GE Healthcare). For all MHC-1 captures the precleared lysate was incubated with the pan HLA-A-, B-, and C- antibody W6/32 coupled to rProtein A Sepharose fast-flow beads. For MHC class II captures the precleared lysate was incubated with the HLA-DR specific antibody L243 coupled to rProtein A Sepharose fast-flow beads. Following the immune-capture, the beads were washed with TBS and peptides were eluted from the purified MHC-molecules using 10% acetic acid. The peptides were further purified with a MWCO size filter, followed by a concentration step and then desalted on C18 based StageTips. The isolated peptides were analyzed on an LTQ Orbitrap Elite mass spectrometer (Thermo Fischer Scientific) or Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fischer Scientific). | For all MHC-1 captures the precleared lysate was incubated with the pan HLA-A-, B-, and C- antibody W6/32 coupled to rProtein A Sepharose fast-flow beads. For MHC class II captures the precleared lysate was incubated with the HLA-DR specific antibody L243 coupled to rProtein A Sepharose fast-flow beads. Following the immune-capture, the beads were washed with TBS and peptides were eluted from the purified MHC-molecules using 10% acetic acid. The peptides were further purified with a MWCO size filter, followed by a concentration step and then desalted on C18 based StageTips. | primary AML tumor samples.The OCI-AML3 cell lineThe MV4-11 cell line | | Peripheral blood mononuclear cells (PBMCs) were isolated from patient tumor samples using Ficoll-Paque density gradient centrifugation and placed in 20% fetal calf serum with 10% DMSO, with storage in either -80°C or vapor phase of liquid nitrogen until use.The OCI-AML3 cell line, which has NPM1 mutation A (p.W88fs*12) [18], was a kind gift of Dr. Beverly Mitchell. The MV4-11 cell line, which has mutated FLT3-ITD [19], was obtained from ATCC. The two cell lines were grown to 2 X 109 in complete RPM1 (10% FBS) and complete IMDM (10% FBS) respectively. Cells were washed twice in PBS, flash frozen in liquid nitrogen, and stored in -80°C until use. | Acute myeloid leukemia immunopeptidome reveals HLA presentation of mutated nucleophosmin |
| PXD013649 | Cancer | Melanoma | Efforts to precisely identify tumor human leukocyte antigen presented peptides (HLAp) capable of mediating T cell based tumor rejection still face important challenges. Recent reports suggest that non-canonical cancer HLAp could be immunogenic but their identification requires highly sensitive and accurate mass-spectrometry (MS)-based proteogenomics approaches. Here, we present a novel MS-based analytical pipeline that can precisely characterize the non-canonical HLAp repertoire, incorporating whole exome sequencing, bulk and single cell transcriptomics, ribosome profiling, and a combination of two MS/MS search tools. This approach results in the accurate identification of hundreds of shared and tumor-specific non-canonical HLAp. Albeit often at low levels and in distinct subpopulations of cells, numerous non-canonical HLAp are shared across tumors. This analytical platform holds great promise for the discovery of novel cancer antigens for cancer immunotherapy. | We performed HLA immunoaffinity purification according to our previously established protocols39,72. W6/32 and HB145 monoclonal antibodies were purified from the supernatants of HB95 (ATCC® HB-95™) and HB145 cells (ATCC® HB-145™) using protein-A sepharose 4B (Pro-A) beads (Invitrogen), and antibodies were then cross-linked to Pro-A beads. Cells were lysed with PBS containing 0.25% sodium deoxycholate (Sigma Aldrich), 0.2 mM iodoacetamide (Sigma Aldrich), 1 mM EDTA, a 1:200 protease inhibitors cocktail (Sigma Aldrich), 1 mM phenylmethylsulfonylfluoride (Roche), and 1% octyl-beta-d glucopyranoside (Sigma Alrich) at 4 °C for 1 h. The lysates were cleared by centrifugation in a table-top centrifuge (Eppendorf) at 4 °C for 50 min at 21,191 x g. Snap-frozen tissue samples were homogenized on ice in 3–5 short intervals of 5 s each using an Ultra Turrax homogenizer (IKA) at maximum speed. The lysates were then cleared by centrifugation at 75,600 x g in a high-speed centrifuge (Beckman Coulter, Avanti JXN-26 Series, JA-25.50 rotor) at 4 °C for 50 min. For HLA immunopurification, we employed the Waters Positive Pressure-96 Processor (Waters) and 96-well single-use micro-plates with 3 µm glass fibers and 10 µm polypropylene membranes (Seahorse Bioscience, ref no: 360063). Anti pan HLA-I and HLA-II antibodies cross-linked to beads were loaded onto separate plates, respectively. For tissue samples, depletion of endogenous antibodies was required with Pro-A beads. The lysates were passed sequentially through the first plate containing pan HLA-I antibody-cross-linked beads, then through the second plate with pan HLA-II antibody-cross-linked beads, at 4 °C. The beads in the plates were then washed separately with varying concentrations of salts using the processor. Finally, the beads were washed twice with 2 mL of 20 mM Tris-HCl pH 8.Sep-Pak tC18 100 mg Sorbent 96-well plates (Waters, ref no: 186002321) were used for the purification and concentration of HLA-I and HLA-II peptides. The C18 sorbents were conditioned, and the HLA complexes and bound peptides were directly eluted from the affinity plate with 1% trifluoroacetic acid (TFA; Sigma Aldrich). After washing the C18 sorbents with 2 mL of 0.1% TFA, HLA-I peptides were eluted with 28% acetonitrile (ACN; Sigma Aldrich) in 0.1% TFA, and HLA-II peptides were eluted with 32% ACN in 0.1% TFA. Recovered HLA-I and -II peptides were dried using vacuum centrifugation (Concentrator plus, Eppendorf) and stored at −20 °C. | We performed HLA immunoaffinity purification according to our previously established protocols39,72. W6/32 and HB145 monoclonal antibodies were purified from the supernatants of HB95 (ATCC® HB-95™) and HB145 cells (ATCC® HB-145™) using protein-A sepharose 4B (Pro-A) beads (Invitrogen), and antibodies were then cross-linked to Pro-A beads. Cells were lysed with PBS containing 0.25% sodium deoxycholate (Sigma Aldrich), 0.2 mM iodoacetamide (Sigma Aldrich), 1 mM EDTA, a 1:200 protease inhibitors cocktail (Sigma Aldrich), 1 mM phenylmethylsulfonylfluoride (Roche), and 1% octyl-beta-d glucopyranoside (Sigma Alrich) at 4 °C for 1 h. The lysates were cleared by centrifugation in a table-top centrifuge (Eppendorf) at 4 °C for 50 min at 21,191 x g. Snap-frozen tissue samples were homogenized on ice in 3–5 short intervals of 5 s each using an Ultra Turrax homogenizer (IKA) at maximum speed. The lysates were then cleared by centrifugation at 75,600 x g in a high-speed centrifuge (Beckman Coulter, Avanti JXN-26 Series, JA-25.50 rotor) at 4 °C for 50 min. For HLA immunopurification, we employed the Waters Positive Pressure-96 Processor (Waters) and 96-well single-use micro-plates with 3 µm glass fibers and 10 µm polypropylene membranes (Seahorse Bioscience, ref no: 360063). Anti pan HLA-I and HLA-II antibodies cross-linked to beads were loaded onto separate plates, respectively. For tissue samples, depletion of endogenous antibodies was required with Pro-A beads. The lysates were passed sequentially through the first plate containing pan HLA-I antibody-cross-linked beads, then through the second plate with pan HLA-II antibody-cross-linked beads, at 4 °C. The beads in the plates were then washed separately with varying concentrations of salts using the processor. Finally, the beads were washed twice with 2 mL of 20 mM Tris-HCl pH 8.Sep-Pak tC18 100 mg Sorbent 96-well plates (Waters, ref no: 186002321) were used for the purification and concentration of HLA-I and HLA-II peptides. The C18 sorbents were conditioned, and the HLA complexes and bound peptides were directly eluted from the affinity plate with 1% trifluoroacetic acid (TFA; Sigma Aldrich). After washing the C18 sorbents with 2 mL of 0.1% TFA, HLA-I peptides were eluted with 28% acetonitrile (ACN; Sigma Aldrich) in 0.1% TFA, and HLA-II peptides were eluted with 32% ACN in 0.1% TFA. Recovered HLA-I and -II peptides were dried using vacuum centrifugation (Concentrator plus, Eppendorf) and stored at −20 °C. | Melanoma cell lines (0D5P, 0MM745, 0NVC) were generated as follows: patient-derived tumors were cut into small pieces before being transferred into a digestion buffer containing collagenase type I (Sigma Aldrich) and DNase I (Roche) for at least 1 h.The primary melanoma cell lines T1185B, T1015A, Me290, and Me275 were generated at the Ludwig Institute for Cancer Research, Department of Oncology, University of Lausanne70,71. | | Melanoma cell lines (0D5P, 0MM745, 0NVC) were generated as follows: patient-derived tumors were cut into small pieces before being transferred into a digestion buffer containing collagenase type I (Sigma Aldrich) and DNase I (Roche) for at least 1 h. Dissociated cells were washed and maintained in RPMI 1640 + lutaMAX medium (Life Technologies) supplemented with 10% heat-inactivated FBS (Dominique Dutscher) and 1% Penicillin/Streptomycin Solution (BioConcept). If fibroblasts appeared, they were selectively eliminated with G418 (Geneticin; Gibco) treatment. The primary melanoma cell lines T1185B, T1015A, Me290, and Me275 were generated at the Ludwig Institute for Cancer Research, Department of Oncology, University of Lausanne70,71. All established melanoma cells were subsequently grown to 1 × 108 cells, collected by centrifugation at 151 x g for 5 min, washed twice with ice-cold PBS and stored as dry cell pellets at −20 °C until use. For the in vitro 72 h treatment with IFNγ (100 IU/mL, Miltenyi Biotec), T1185B cells were grown to 2 × 108 in triplicate. For the treatment with DAC (Sigma Aldrich), 2 × 108 melanoma cells were grown for 8 days in medium containing 0.5 µM DAC, and the drug was readministered on the fourth day. | Integrated proteogenomic deep sequencing and analytics accurately identify non-canonical peptides in tumor immunopeptidomes. |
| PXD013649 | Cancer | Lung Cancer | Efforts to precisely identify tumor human leukocyte antigen presented peptides (HLAp) capable of mediating T cell based tumor rejection still face important challenges. Recent reports suggest that non-canonical cancer HLAp could be immunogenic but their identification requires highly sensitive and accurate mass-spectrometry (MS)-based proteogenomics approaches. Here, we present a novel MS-based analytical pipeline that can precisely characterize the non-canonical HLAp repertoire, incorporating whole exome sequencing, bulk and single cell transcriptomics, ribosome profiling, and a combination of two MS/MS search tools. This approach results in the accurate identification of hundreds of shared and tumor-specific non-canonical HLAp. Albeit often at low levels and in distinct subpopulations of cells, numerous non-canonical HLAp are shared across tumors. This analytical platform holds great promise for the discovery of novel cancer antigens for cancer immunotherapy. | We performed HLA immunoaffinity purification according to our previously established protocols39,72. W6/32 and HB145 monoclonal antibodies were purified from the supernatants of HB95 (ATCC® HB-95™) and HB145 cells (ATCC® HB-145™) using protein-A sepharose 4B (Pro-A) beads (Invitrogen), and antibodies were then cross-linked to Pro-A beads. Cells were lysed with PBS containing 0.25% sodium deoxycholate (Sigma Aldrich), 0.2 mM iodoacetamide (Sigma Aldrich), 1 mM EDTA, a 1:200 protease inhibitors cocktail (Sigma Aldrich), 1 mM phenylmethylsulfonylfluoride (Roche), and 1% octyl-beta-d glucopyranoside (Sigma Alrich) at 4 °C for 1 h. The lysates were cleared by centrifugation in a table-top centrifuge (Eppendorf) at 4 °C for 50 min at 21,191 x g. Snap-frozen tissue samples were homogenized on ice in 3–5 short intervals of 5 s each using an Ultra Turrax homogenizer (IKA) at maximum speed. The lysates were then cleared by centrifugation at 75,600 x g in a high-speed centrifuge (Beckman Coulter, Avanti JXN-26 Series, JA-25.50 rotor) at 4 °C for 50 min. For HLA immunopurification, we employed the Waters Positive Pressure-96 Processor (Waters) and 96-well single-use micro-plates with 3 µm glass fibers and 10 µm polypropylene membranes (Seahorse Bioscience, ref no: 360063). Anti pan HLA-I and HLA-II antibodies cross-linked to beads were loaded onto separate plates, respectively. For tissue samples, depletion of endogenous antibodies was required with Pro-A beads. The lysates were passed sequentially through the first plate containing pan HLA-I antibody-cross-linked beads, then through the second plate with pan HLA-II antibody-cross-linked beads, at 4 °C. The beads in the plates were then washed separately with varying concentrations of salts using the processor. Finally, the beads were washed twice with 2 mL of 20 mM Tris-HCl pH 8.Sep-Pak tC18 100 mg Sorbent 96-well plates (Waters, ref no: 186002321) were used for the purification and concentration of HLA-I and HLA-II peptides. The C18 sorbents were conditioned, and the HLA complexes and bound peptides were directly eluted from the affinity plate with 1% trifluoroacetic acid (TFA; Sigma Aldrich). After washing the C18 sorbents with 2 mL of 0.1% TFA, HLA-I peptides were eluted with 28% acetonitrile (ACN; Sigma Aldrich) in 0.1% TFA, and HLA-II peptides were eluted with 32% ACN in 0.1% TFA. Recovered HLA-I and -II peptides were dried using vacuum centrifugation (Concentrator plus, Eppendorf) and stored at −20 °C. | We performed HLA immunoaffinity purification according to our previously established protocols39,72. W6/32 and HB145 monoclonal antibodies were purified from the supernatants of HB95 (ATCC® HB-95™) and HB145 cells (ATCC® HB-145™) using protein-A sepharose 4B (Pro-A) beads (Invitrogen), and antibodies were then cross-linked to Pro-A beads. Cells were lysed with PBS containing 0.25% sodium deoxycholate (Sigma Aldrich), 0.2 mM iodoacetamide (Sigma Aldrich), 1 mM EDTA, a 1:200 protease inhibitors cocktail (Sigma Aldrich), 1 mM phenylmethylsulfonylfluoride (Roche), and 1% octyl-beta-d glucopyranoside (Sigma Alrich) at 4 °C for 1 h. The lysates were cleared by centrifugation in a table-top centrifuge (Eppendorf) at 4 °C for 50 min at 21,191 x g. Snap-frozen tissue samples were homogenized on ice in 3–5 short intervals of 5 s each using an Ultra Turrax homogenizer (IKA) at maximum speed. The lysates were then cleared by centrifugation at 75,600 x g in a high-speed centrifuge (Beckman Coulter, Avanti JXN-26 Series, JA-25.50 rotor) at 4 °C for 50 min. For HLA immunopurification, we employed the Waters Positive Pressure-96 Processor (Waters) and 96-well single-use micro-plates with 3 µm glass fibers and 10 µm polypropylene membranes (Seahorse Bioscience, ref no: 360063). Anti pan HLA-I and HLA-II antibodies cross-linked to beads were loaded onto separate plates, respectively. For tissue samples, depletion of endogenous antibodies was required with Pro-A beads. The lysates were passed sequentially through the first plate containing pan HLA-I antibody-cross-linked beads, then through the second plate with pan HLA-II antibody-cross-linked beads, at 4 °C. The beads in the plates were then washed separately with varying concentrations of salts using the processor. Finally, the beads were washed twice with 2 mL of 20 mM Tris-HCl pH 8.Sep-Pak tC18 100 mg Sorbent 96-well plates (Waters, ref no: 186002321) were used for the purification and concentration of HLA-I and HLA-II peptides. The C18 sorbents were conditioned, and the HLA complexes and bound peptides were directly eluted from the affinity plate with 1% trifluoroacetic acid (TFA; Sigma Aldrich). After washing the C18 sorbents with 2 mL of 0.1% TFA, HLA-I peptides were eluted with 28% acetonitrile (ACN; Sigma Aldrich) in 0.1% TFA, and HLA-II peptides were eluted with 32% ACN in 0.1% TFA. Recovered HLA-I and -II peptides were dried using vacuum centrifugation (Concentrator plus, Eppendorf) and stored at −20 °C. | Snap-frozen normal and lung tumor tissue materials from the C3N-02289 (Lung squamous cell carcinoma, grade 2) and C3N-02671 (lung adenocarcinoma, G2) samples were kindly provided by the International Institute of Molecular Oncology. | | | Integrated proteogenomic deep sequencing and analytics accurately identify non-canonical peptides in tumor immunopeptidomes. |
| PXD013649 | Normal | Lung | Efforts to precisely identify tumor human leukocyte antigen presented peptides (HLAp) capable of mediating T cell based tumor rejection still face important challenges. Recent reports suggest that non-canonical cancer HLAp could be immunogenic but their identification requires highly sensitive and accurate mass-spectrometry (MS)-based proteogenomics approaches. Here, we present a novel MS-based analytical pipeline that can precisely characterize the non-canonical HLAp repertoire, incorporating whole exome sequencing, bulk and single cell transcriptomics, ribosome profiling, and a combination of two MS/MS search tools. This approach results in the accurate identification of hundreds of shared and tumor-specific non-canonical HLAp. Albeit often at low levels and in distinct subpopulations of cells, numerous non-canonical HLAp are shared across tumors. This analytical platform holds great promise for the discovery of novel cancer antigens for cancer immunotherapy. | We performed HLA immunoaffinity purification according to our previously established protocols39,72. W6/32 and HB145 monoclonal antibodies were purified from the supernatants of HB95 (ATCC® HB-95™) and HB145 cells (ATCC® HB-145™) using protein-A sepharose 4B (Pro-A) beads (Invitrogen), and antibodies were then cross-linked to Pro-A beads. Cells were lysed with PBS containing 0.25% sodium deoxycholate (Sigma Aldrich), 0.2 mM iodoacetamide (Sigma Aldrich), 1 mM EDTA, a 1:200 protease inhibitors cocktail (Sigma Aldrich), 1 mM phenylmethylsulfonylfluoride (Roche), and 1% octyl-beta-d glucopyranoside (Sigma Alrich) at 4 °C for 1 h. The lysates were cleared by centrifugation in a table-top centrifuge (Eppendorf) at 4 °C for 50 min at 21,191 x g. Snap-frozen tissue samples were homogenized on ice in 3–5 short intervals of 5 s each using an Ultra Turrax homogenizer (IKA) at maximum speed. The lysates were then cleared by centrifugation at 75,600 x g in a high-speed centrifuge (Beckman Coulter, Avanti JXN-26 Series, JA-25.50 rotor) at 4 °C for 50 min. For HLA immunopurification, we employed the Waters Positive Pressure-96 Processor (Waters) and 96-well single-use micro-plates with 3 µm glass fibers and 10 µm polypropylene membranes (Seahorse Bioscience, ref no: 360063). Anti pan HLA-I and HLA-II antibodies cross-linked to beads were loaded onto separate plates, respectively. For tissue samples, depletion of endogenous antibodies was required with Pro-A beads. The lysates were passed sequentially through the first plate containing pan HLA-I antibody-cross-linked beads, then through the second plate with pan HLA-II antibody-cross-linked beads, at 4 °C. The beads in the plates were then washed separately with varying concentrations of salts using the processor. Finally, the beads were washed twice with 2 mL of 20 mM Tris-HCl pH 8.Sep-Pak tC18 100 mg Sorbent 96-well plates (Waters, ref no: 186002321) were used for the purification and concentration of HLA-I and HLA-II peptides. The C18 sorbents were conditioned, and the HLA complexes and bound peptides were directly eluted from the affinity plate with 1% trifluoroacetic acid (TFA; Sigma Aldrich). After washing the C18 sorbents with 2 mL of 0.1% TFA, HLA-I peptides were eluted with 28% acetonitrile (ACN; Sigma Aldrich) in 0.1% TFA, and HLA-II peptides were eluted with 32% ACN in 0.1% TFA. Recovered HLA-I and -II peptides were dried using vacuum centrifugation (Concentrator plus, Eppendorf) and stored at −20 °C. | We performed HLA immunoaffinity purification according to our previously established protocols39,72. W6/32 and HB145 monoclonal antibodies were purified from the supernatants of HB95 (ATCC® HB-95™) and HB145 cells (ATCC® HB-145™) using protein-A sepharose 4B (Pro-A) beads (Invitrogen), and antibodies were then cross-linked to Pro-A beads. Cells were lysed with PBS containing 0.25% sodium deoxycholate (Sigma Aldrich), 0.2 mM iodoacetamide (Sigma Aldrich), 1 mM EDTA, a 1:200 protease inhibitors cocktail (Sigma Aldrich), 1 mM phenylmethylsulfonylfluoride (Roche), and 1% octyl-beta-d glucopyranoside (Sigma Alrich) at 4 °C for 1 h. The lysates were cleared by centrifugation in a table-top centrifuge (Eppendorf) at 4 °C for 50 min at 21,191 x g. Snap-frozen tissue samples were homogenized on ice in 3–5 short intervals of 5 s each using an Ultra Turrax homogenizer (IKA) at maximum speed. The lysates were then cleared by centrifugation at 75,600 x g in a high-speed centrifuge (Beckman Coulter, Avanti JXN-26 Series, JA-25.50 rotor) at 4 °C for 50 min. For HLA immunopurification, we employed the Waters Positive Pressure-96 Processor (Waters) and 96-well single-use micro-plates with 3 µm glass fibers and 10 µm polypropylene membranes (Seahorse Bioscience, ref no: 360063). Anti pan HLA-I and HLA-II antibodies cross-linked to beads were loaded onto separate plates, respectively. For tissue samples, depletion of endogenous antibodies was required with Pro-A beads. The lysates were passed sequentially through the first plate containing pan HLA-I antibody-cross-linked beads, then through the second plate with pan HLA-II antibody-cross-linked beads, at 4 °C. The beads in the plates were then washed separately with varying concentrations of salts using the processor. Finally, the beads were washed twice with 2 mL of 20 mM Tris-HCl pH 8.Sep-Pak tC18 100 mg Sorbent 96-well plates (Waters, ref no: 186002321) were used for the purification and concentration of HLA-I and HLA-II peptides. The C18 sorbents were conditioned, and the HLA complexes and bound peptides were directly eluted from the affinity plate with 1% trifluoroacetic acid (TFA; Sigma Aldrich). After washing the C18 sorbents with 2 mL of 0.1% TFA, HLA-I peptides were eluted with 28% acetonitrile (ACN; Sigma Aldrich) in 0.1% TFA, and HLA-II peptides were eluted with 32% ACN in 0.1% TFA. Recovered HLA-I and -II peptides were dried using vacuum centrifugation (Concentrator plus, Eppendorf) and stored at −20 °C. | Snap-frozen normal and lung tumor tissue materials from the C3N-02289 (Lung squamous cell carcinoma, grade 2) and C3N-02671 (lung adenocarcinoma, G2) samples were kindly provided by the International Institute of Molecular Oncology. | | | Integrated proteogenomic deep sequencing and analytics accurately identify non-canonical peptides in tumor immunopeptidomes. |
| PXD014062 | Cancer | Ovarian Cancer | High-grade serous ovarian cancer (HGSC), the principal cause of death from gynecological malignancies in the world, has not significantly benefited from recent progress in cancer immunotherapy. While HGSC infiltration by lymphocytes correlates with superior survival, the nature of antigens that can elicit anti-HGSC immune responses is unknown. The goal of this study was to establish the global landscape of HGSC tumor-specific antigens (TSAs) using a mass spectrometry pipeline that interrogates all reading frames of all genomic regions. In 23 HGSC tumors, we identified 113 TSAs. Classic TSA discovery approaches focusing only on mutated exonic sequences would have uncovered only seven of these TSAs. Other mutated TSAs resulted from out-of-frame exonic translation or from non-exonic sequences. The most interesting group of TSAs (n = 94) derived from aberrantly expressed unmutated genomic sequences which are not expressed in normal tissues. These aberrantly expressed TSAs (aeTSAs) derived primarily from non-exonic sequences, in particular intronic (31%) and intergenic (22%). Their expression was regulated at the transcriptional level by variations in gene copy number and DNA methylation. While mutated TSAs were unique to individual tumors, aeTSAs were shared by a large proportion of HGSCs. We conclude that, in view of their number and the fact that they are shared by many tumors, aeTSAs may be the most attractive targets for HGSC immunotherapy. | Tumor fragments of HGSC1-6 and OV606 were cut into small pieces (cubes, ∼3 mm in size) and 5 mL of ice-cold PBS containing protein inhibitor cocktail (Sigma, cat. #P8340-5mL) was added. Tissues were first homogenized twice for 20 seconds using an Ultra Turrax T25 homogenizer (IKA-Labortechnik) set at speed 20,000 rpm and then 20 seconds using an Ultra Turrax T8 homogenizer (IKA-Labortechnik) set at speed 25,000 rpm. Then, 550 μL of ice-cold 10× lysis buffer (5% w/v CHAPS, Sigma, cat. # C9426-5G) was added to each sample. After a 60-minute incubation with tumbling at 4°C, samples were spun at 10,000 × g for 30 minutes at 4°C. Supernatants were transferred into new tubes containing 1 mg of W6/32 antibody (Bio X Cell, cat. #BE0079) covalently cross-linked to 1 mL of protein A magnetic beads (Pure Proteome, cat. #LSKMAGA10) using dimethylpimelidate (26), and MAPs were immunoprecipitated as previously described (27). MAP extracts were then dried using a Speed-Vac and kept frozen at −20°C until MS analyses. | Tumor fragments of HGSC1-6 and OV606 were cut into small pieces (cubes, ∼3 mm in size) and 5 mL of ice-cold PBS containing protein inhibitor cocktail (Sigma, cat. #P8340-5mL) was added. Tissues were first homogenized twice for 20 seconds using an Ultra Turrax T25 homogenizer (IKA-Labortechnik) set at speed 20,000 rpm and then 20 seconds using an Ultra Turrax T8 homogenizer (IKA-Labortechnik) set at speed 25,000 rpm. Then, 550 μL of ice-cold 10× lysis buffer (5% w/v CHAPS, Sigma, cat. # C9426-5G) was added to each sample. After a 60-minute incubation with tumbling at 4°C, samples were spun at 10,000 × g for 30 minutes at 4°C. Supernatants were transferred into new tubes containing 1 mg of W6/32 antibody (Bio X Cell, cat. #BE0079) covalently cross-linked to 1 mL of protein A magnetic beads (Pure Proteome, cat. #LSKMAGA10) using dimethylpimelidate (26), and MAPs were immunoprecipitated as previously described (27). MAP extracts were then dried using a Speed-Vac and kept frozen at −20°C until MS analyses. | Tumor fragments of HGSC1-6 and matched normal adjacent tissues of HGSC1-3 were obtained from Tissue Solutions. | | | Proteogenomics uncovers a vast repertoire of shared tumor-specific antigens in ovarian cancer. |
| PXD015039 | Cancer | Acute Myeloid Leukemia | We report the use of complementary peptide antigen enrichment and comprehensive mass spectrometric acquisition strategies to provide in-depth immunopeptidome data for AML cell line THP1. | Briefly, frozen cell pellets (1 × 109 ) were pulverised using a cryogenic mill (Retsch Mixer Mill MM 400), reconstituted in Lysis Buffer [0.5% IGEPAL (Sigma-Aldrich, USA), 50 mM Tris pH 8, 150 mM NaCl and protease inhibitors (Complete Protease Inhibitor Cocktail Tablet, 1 tablet per 50 mL solution; Roche Molecular Biochemicals, Switzerland)] and incubated for 1 h at 4 °C with rotation. The supernatant was passed through a PAS pre-column (500 μL) to remove non-specific binding material, followed by serial affinity capture of HLA-A*02:01 peptides (BB7.2 column) and HLA-B*15:11 and -C*03:03 peptides (W6/32 column). Bound complexes were eluted from the column by using 5 column volumes of 10% acetic acid (Supplementary Fig. 1). The eluate was mixed and split into two equal volumes representing peptides isolated from 5 × 108 cells, which were then processed differently. One part of the eluate was fractionated by RP-HPLC on a 4.6 mm internal diameter 100 mm long RP monolithic C18 HPLC column (Chromolith Speed Rod, Merck-Millipore, Germany) using an ÄKTA micro HPLC system (GE Healthcare) running a mobile phase consisting of buffer A (0.1% trifluoroacetic acid [TFA; Thermo Fisher Scientific, USA]) and buffer B (80% acetonitrile [ACN; Thermo Fisher Scientific]/0.1% TFA). A flowrate of 2 mL/min was used to separate the peptides into 1 mL fractions across a changing gradient; 0–15% buffer B over 0.25 min., 15–30% buffer B over 4 min., 30–40% buffer B over 8 min., 40–45% buffer B over 10 min., and 45–99% buffer B over 2 min. Peptide-containing fractions were collected, pooled using a concatenation strategy into 10 fractions, vacuum concentrated, and reconstituted in 0.1% formic acid (FA; Thermo Fisher Scientific). The second half of the eluate was passed through a 5 kDa MWCO filter (Amicon), which had been pre-washed with optima water (Thermo Fisher Scientific) and then equilibrated with 10% acetic acid by centrifugation at 16,060g for 30 min. at room temperature (RT). The sample flow through containing HLA peptides was collected. Finally, the filter was rinsed with buffer A and centrifuged at 16,060g for 30 min. at RT. The filtered sample was centrifugally evaporated and desalted by reverse phase C18 stage tips (Omix, Agilent). The C18 tips were washed with 0.1% TFA and peptides were eluted with 40% ACN in 0.1% TFA. | Briefly, frozen cell pellets (1 × 109 ) were pulverised using a cryogenic mill (Retsch Mixer Mill MM 400), reconstituted in Lysis Buffer [0.5% IGEPAL (Sigma-Aldrich, USA), 50 mM Tris pH 8, 150 mM NaCl and protease inhibitors (Complete Protease Inhibitor Cocktail Tablet, 1 tablet per 50 mL solution; Roche Molecular Biochemicals, Switzerland)] and incubated for 1 h at 4 °C with rotation. The supernatant was passed through a PAS pre-column (500 μL) to remove non-specific binding material, followed by serial affinity capture of HLA-A*02:01 peptides (BB7.2 column) and HLA-B*15:11 and -C*03:03 peptides (W6/32 column). Bound complexes were eluted from the column by using 5 column volumes of 10% acetic acid (Supplementary Fig. 1). The eluate was mixed and split into two equal volumes representing peptides isolated from 5 × 108 cells, which were then processed differently. One part of the eluate was fractionated by RP-HPLC on a 4.6 mm internal diameter 100 mm long RP monolithic C18 HPLC column (Chromolith Speed Rod, Merck-Millipore, Germany) using an ÄKTA micro HPLC system (GE Healthcare) running a mobile phase consisting of buffer A (0.1% trifluoroacetic acid [TFA; Thermo Fisher Scientific, USA]) and buffer B (80% acetonitrile [ACN; Thermo Fisher Scientific]/0.1% TFA). A flowrate of 2 mL/min was used to separate the peptides into 1 mL fractions across a changing gradient; 0–15% buffer B over 0.25 min., 15–30% buffer B over 4 min., 30–40% buffer B over 8 min., 40–45% buffer B over 10 min., and 45–99% buffer B over 2 min. Peptide-containing fractions were collected, pooled using a concatenation strategy into 10 fractions, vacuum concentrated, and reconstituted in 0.1% formic acid (FA; Thermo Fisher Scientific). The second half of the eluate was passed through a 5 kDa MWCO filter (Amicon), which had been pre-washed with optima water (Thermo Fisher Scientific) and then equilibrated with 10% acetic acid by centrifugation at 16,060g for 30 min. at room temperature (RT). The sample flow through containing HLA peptides was collected. Finally, the filter was rinsed with buffer A and centrifuged at 16,060g for 30 min. at RT. The filtered sample was centrifugally evaporated and desalted by reverse phase C18 stage tips (Omix, Agilent). The C18 tips were washed with 0.1% TFA and peptides were eluted with 40% ACN in 0.1% TFA. | THP1 cells | | All the cell lines used in the study i.e. THP-1 parental and THP1 parental transfected with HLA B*27:05 (Battle et al., 2013; Tsuchiya et al., 1980) along with HMy2.C1R cells (Zemmour et al., 1992) transfected with HLA-A*02:01 (hereafter named C1R.A*02:01; see Supplementary Table 1for details) were maintained in RF10 (RPMI 1640 (Gibco) supplemented with 2 mM MEM nonessential amino acid solution (Gibco), 100 mM HEPES (Gibco), 2 mM L-glutamine (Gibco), Penicillin/Streptomycin (Gibco), 50 μM 2-mercaptoethanol (Sigma-Al-drich) and 10% heat inactivated FCS (Sigma-Aldrich). | In-depth mining of the immunopeptidome of an acute myeloid leukemia cell line using complementary ligand enrichment and data acquisition strategies. |
| PXD018542 | Cancer | Acute Myeloid Leukemia | The development of therapeutic anticancer vaccines calls for the identification of tumor-specific antigens (TSAs). Though a combination of four cutting-edge proteogenomic approaches, we performed a deep exploration of the MHC-I presented peptides (MAPs) of 19 acute myeloid leukemia (AML) patients and identified various TSAs that could serve for the design of an anti-AML vaccine. | The W6/32 antibodies (BioXcell) were incubated in PBS for 60 min at room temperature with PureProteome protein A magnetic beads (Millipore) at a ratio of 1 mg of antibody per mL of slurry. Antibodies were covalently cross-linked to magnetic beads using dimethylpimelidate as described (Lamoliatte et al., 2017). The beads were stored at 4°C in PBS pH 7.2 and 0.02% NaN3. For frozen cell pellet samples (98 million cells/pellet), cells were thawed and resuspended in 1 mL PBS pH 7.2 and solubilized by adding 1 mL of detergent buffer containing PBS pH 7.2, 1% (w/v) CHAPS (Sigma) supplemented with Protease inhibitor cocktail (Sigma). For solid tumor samples from NSG mice, the tissue (480mg) was cut into small pieces (cubes, ∼3 mm in size) and 5 mL of ice-cold PBS containing Protease inhibitor cocktail was added. Tissue pieces were first homogenized twice for 20 s using an Ultra Turrax T25 homogenizer (IKA-Labortechnik) set at speed 20000 rpm and then 20 s using an Ultra Turrax T8 homogenizer (IKA-Labortechnik) set at speed 25000 rpm. Then, 550 μL of ice-cold 10X lysis buffer (5% w/v CHAPS) was added to the sample. Solubilized cell pellets and solid tumor samples were incubated 60 min with tumbling at 4°C and then spun at 10000 g for 20 min at 4°C. Supernatants were transferred into new tubes containing 1 mg of W6/32 antibody covalently-cross-linked protein A magnetic beads per sample and incubated with tumbling for 180 min at 4°C. Samples were placed on a magnet to recover bound MHC I complexes to magnetic beads. Magnetic beads were first washed with 8 × 1 mL PBS, then with 1 × 1 mL of 0.1X PBS and finally with 1 × 1 mL of water. MHC I complexes were eluted from the magnetic beads by acidic treatment using 0.2% formic acid (FA). To remove any residual magnetic beads, eluates were transferred into 2.0 mL Costar mL Spin-X centrifuge tube filters (0.45 μm, Corning) and spun 2 min at 855 g. Filtrates containing peptides were separated from MHC I subunits (HLA molecules and β-2 microglobulin) using home-made stage tips packed with twenty 1 mm diameter octadecyl (C-18) solid phase extraction disks (EMPORE). Stage tips were pre-washed first with methanol then with 80% acetonitrile (ACN) in 0.2% trifluoroacetic acid (TFA) and finally with 0.2% FA. Samples were loaded onto the stage tips and washed with 0.2% FA. Peptides were eluted with 30% ACN in 0.1%TFA, dried using vacuum centrifugation and then stored at −20°C until MS analysis. | The W6/32 antibodies (BioXcell) were incubated in PBS for 60 min at room temperature with PureProteome protein A magnetic beads (Millipore) at a ratio of 1 mg of antibody per mL of slurry. Antibodies were covalently cross-linked to magnetic beads using dimethylpimelidate as described (Lamoliatte et al., 2017). The beads were stored at 4°C in PBS pH 7.2 and 0.02% NaN3. For frozen cell pellet samples (98 million cells/pellet), cells were thawed and resuspended in 1 mL PBS pH 7.2 and solubilized by adding 1 mL of detergent buffer containing PBS pH 7.2, 1% (w/v) CHAPS (Sigma) supplemented with Protease inhibitor cocktail (Sigma). For solid tumor samples from NSG mice, the tissue (480mg) was cut into small pieces (cubes, ∼3 mm in size) and 5 mL of ice-cold PBS containing Protease inhibitor cocktail was added. Tissue pieces were first homogenized twice for 20 s using an Ultra Turrax T25 homogenizer (IKA-Labortechnik) set at speed 20000 rpm and then 20 s using an Ultra Turrax T8 homogenizer (IKA-Labortechnik) set at speed 25000 rpm. Then, 550 μL of ice-cold 10X lysis buffer (5% w/v CHAPS) was added to the sample. Solubilized cell pellets and solid tumor samples were incubated 60 min with tumbling at 4°C and then spun at 10000 g for 20 min at 4°C. Supernatants were transferred into new tubes containing 1 mg of W6/32 antibody covalently-cross-linked protein A magnetic beads per sample and incubated with tumbling for 180 min at 4°C. Samples were placed on a magnet to recover bound MHC I complexes to magnetic beads. Magnetic beads were first washed with 8 × 1 mL PBS, then with 1 × 1 mL of 0.1X PBS and finally with 1 × 1 mL of water. MHC I complexes were eluted from the magnetic beads by acidic treatment using 0.2% formic acid (FA). To remove any residual magnetic beads, eluates were transferred into 2.0 mL Costar mL Spin-X centrifuge tube filters (0.45 μm, Corning) and spun 2 min at 855 g. Filtrates containing peptides were separated from MHC I subunits (HLA molecules and β-2 microglobulin) using home-made stage tips packed with twenty 1 mm diameter octadecyl (C-18) solid phase extraction disks (EMPORE). Stage tips were pre-washed first with methanol then with 80% acetonitrile (ACN) in 0.2% trifluoroacetic acid (TFA) and finally with 0.2% FA. Samples were loaded onto the stage tips and washed with 0.2% FA. Peptides were eluted with 30% ACN in 0.1%TFA, dried using vacuum centrifugation and then stored at −20°C until MS analysis. | Diagnostic AML samples (cryovials of DMSO-frozen leukemic blasts) were obtained from the Banque de cellules leucémiques du Québec program (BCLQ, bclq.org). | | Diagnostic AML samples (cryovials of DMSO-frozen leukemic blasts) were obtained from the Banque de cellules leucémiques du Québec program (BCLQ, bclq.org). One hundred million cells of each AML sample were thawed (1 min in 37°C water bath) and resuspended in 48 ml of 4°C PBS. | Atypical acute myeloid leukemia-specific transcripts generate shared and immunogenic MHC class-I-associated epitopes |
| PXD019643 | Normal | Adrenal Gland | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 50% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Aorta | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 51% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Bladder | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 52% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Bone Marrow | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 53% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Brain | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 54% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Breast | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 55% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Cerebellum | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 56% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Colon | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 57% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Esophagus | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 58% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Gallbladder | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 59% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Heart | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 60% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Kidney | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 61% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Liver | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 62% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Lung | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 63% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Lymph Node | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 64% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Muscle | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 65% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Nerve | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 66% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Ovary | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 67% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Pancreas | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 68% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Prostate | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 69% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Skin | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 70% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Small Intestine | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 71% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Spleen | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 72% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Stomach | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 73% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Testis | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 74% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Thymus | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 75% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Thyroid | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 76% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Tongue | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 77% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Trachea | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 78% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD019643 | Normal | Uterus | The human leukocyte antigen (HLA) complex regulates the adaptive immune response by showcasing the intracellular and extracellular protein content to the immune system. T cells recognize these HLA-presented peptides as self or foreign and can elicit an immune response. In this work, we describe the HLA-Ligand-Atlas, a comprehensive map of HLA-I and HLA-II-presented peptides from 30 benign tissues, 51 HLA-I alleles, and 86 HLA-II alleles. Nearly 79% of HLA ligands have not been previously described. Due to the scarcity of benign human samples, the tissue was extracted from different organs at autopsy from human subjects without any diagnosed malignancy. Furthermore, we were able to identify non-canonical HLA-I peptides on benign tissues, showing that their processing is not unique to cancer. This dataset holds great promise in answering both basic and translational questions in fields such as autoimmunity, organ/tissue transplantation, and cancer immunotherapy. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | Human tissue samples were obtained post-mortem during autopsy performed for medical reasons at the University Hospital Zürich. None of the subjects included in this study was diagnosed with any malignant disease. Tissue samples were collected during autopsy, which was performed within 72 hours after death. Tissue annotation was performed by a board-certified pathologist. Tissue samples were immediately snap-frozen in liquid nitrogen.Thymus samples were obtained from the University Children's Hospital Zürich/ Switzerland. Thymus tissue was removed during heart surgery for other medical reasons.Furthermore, two benign ovarian tissue samples were collected for the time series experiments (OVA-DN278 and OVA-DN281). Both patients were post-menopausal and had a bilateral ovarectomy for cystadenofibromas, which were diagnosed by histopathological examination of the specimen. The samples were obtained from a normal part of the ovary. | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD020186 | Cancer | Glioblastoma | A use case of the benign HLA-Ligand-Atlas is the prioritization of tumor-associated targets for e.g. peptide vaccination. Based on three glioblastoma samples, we illustrated how many of these peptides are covered in the benign dataset. The experimental and computational workflow for the isolation and identification of the glioblastoma immunopeptidomes is the same, as for all samples included in the HLA Ligand Atlas. As the three glioblastoma samples are not included in the HLA Ligand Atlas dataset, they have been deposited under a separate submission. | HLA-I and HLA-II molecules were isolated from snap-frozen tissue using standard immunoaffinity chromatography. The antibodies employed were the pan-HLA-I-specific antibody W6/32,45 and the HLA-DR-specific antibody L243,46 produced in house (University of Tübingen, Department of Immunology) from HB-95, and HB-55 cells (ATCC, Manassas, Virginia, USA), respectively. Furthermore, the pan-HLA-II-specific antibody Tü39 was employed and produced in house from a hybridoma clone as previously described.47 The antibodies were cross-linked to CNBr-activated sepharose (Sigma-Aldrich, St. Louis, Missouri, USA) at a ratio of 40 mg sepharose to 1 mg antibody for 1 g tissue with 0.5 M NaCl, 0.1 M NaHCO3 at pH 8.3. Free activated CNBr reaction sites were blocked with 0.2 M glycine.For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | For the purification of HLA-peptide complexes, tissue was minced with a scalpel and further homogenized with the Potter-Elvehjem instrument (VWR, Darmstadt, Germany). The amount of tissue employed for each purification is documented in online supplemental table S1. This information is not available for seven tissues, annotated as n.d. in said table. Tissue homogenization was performed in lysis buffer consisting of CHAPS (Panreac AppliChem, Darmstadt, Germany) and one cOmplete protease inhibitor cocktail tablet (Roche) in PBS. Thereafter, the lysate was sonicated and cleared by centrifugation for 45 min at 4000 rpm, interspaced by 1-hour incubation periods on a shaker at 4°C. Lysates were further cleared by sterile filtration employing a 5 µm filter unit (Merck Millipore, Darmstadt, Germany). The first column contained 1 mg of W6/32 antibody coupled to sepharose, whereas the second column contained equal amounts of Tü39 and L243 antibody coupled to sepharose. Finally, the lysates were passed through two columns cyclically overnight at 4°C. Affinity columns were then washed for 30 min with PBS and for 1 hour with water. Elution of peptides was achieved by incubating four times successively with 100–200 µL 0.2% trifluoroacetic acid (TFA) on a shaker. All eluted fractions were subsequently pooled. Peptides were separated from the HLA molecule remnants by ultrafiltration employing 3 kDa and 10 kDa Amicon filter units (Merck Millipore) for HLA-I and HLA-II, respectively. The eluate volume was then reduced to approximately 50 µL by lyophilization or vacuum centrifugation. Finally, the reduced peptide solution was purified five times using ZipTip pipette tips with C18 resin and 0.6 µL bed volume (Merck,) and eluted in 32.5% acetonitrile (ACN)/0.2% TFA. Each peptide eluate was purified by loading it five times onto the same ZipTip pipette tip. The tip was passed sequentially by pipetting ten times up and down through 32.5% ACN/ 0.2% TFA for purification, 0.1% TFA for equilibration, the sample for binding the peptides, 0.1% TFA for desalting and 32.5% ACN/0.2% TFA for elution. This entire sequence was repeated five times using the same ZipTip pipette tip. The purified peptide solution was concentrated by vacuum centrifugation and supplemented with 1% ACN/0.05% TFA and stored at −80°C until LC-MS/MS analysis. | three primary glioblastoma tumor samples | | | HLA Ligand Atlas: a benign reference of HLA-presented peptides to improve T-cell-based cancer immunotherapy |
| PXD020224 | Cancer | Melanoma | Many tumors escape by activating multiple cellular pathways that induce immunosuppression. One pivotal immune-suppressive mechanism is the production of tryptophan metabolites along the kynurenine pathway by IFNγ-induced IDO1 enzyme production 4-8. Phase III clinical trials using chemical inhibition of IDO1 in combination with PD1 pathway blockade, however, failed to improve melanoma treatment 9-12. This points at an incomplete understanding of the role of IDO1 and the consequent tryptophan degradation on mRNA translation and cancer progression. Here, we investigated the effects of prolonged IFNγ treatment on mRNA translation in melanoma cells by ribosome profiling. Surprisingly, we observed a massive accumulation of ribosomes ~20 amino acids downstream of tryptophan codons (termed here as W-Bumps) along with their expected stalling at the tryptophan codon itself. This indicated ribosomal bypass of the tryptophan codons in the absence of tryptophan. Detailed examination of W-Bumps position and its corresponding peptide sequences pinpointed towards ribosomal frameshifting events and their effects in the ribosome exit tunnel. In particular, W-Bumps strength was associated with the disorderedness level of potential out-of-frame peptides predicted downstream of tryptophan codons. Indeed, reporter assays demonstrated the induction of ribosomal frameshifting, and the generation of trans-frame proteins and their presentation at the cell surface after IFNγ treatment. Proteomics and immunopeptidomics analyses verified the production of IFNγ-induced trans-frame and out-of-frame aberrant peptides and their presentation on HLA class I molecules. Priming of naïve T cells from healthy donors with aberrant peptides resulted in identification of reactive, peptide-specific T cells. Altogether, our results suggest that IFNγ-induced IDO1-mediated tryptophan depletion plays a role in the immune recognition of melanoma cells by contributing to the diversity of the peptidome landscape, and by inducing the presentation of aberrant peptides. | In brief, cell pellets were lysed with lysis buffer containing 0.25% sodium deoxycholate, 0.2 mM iodoacetamide, 1 mM EDTA, 1:200 protease inhibitors cocktail (Sigma-Aldrich), 1 mM PMSF and 1% octyl-β-d-glucopyranoside in phosphate-buffered saline (PBS), and then incubated at 4 °C for 1 h. The lysates were cleared by centrifugation at 4 °C and 48,000g for 60 min, and then passed through a pre-clearing column containing Protein-A Sepharose beads (GenScript). HLA-I molecules were immunoaffinity-purified from cleared lysate with the pan-HLA-I antibody (W6/32 antibody purified from HB95 hybridoma cells) covalently bound to Protein-A sepharose beads). The affinity column was washed first with 10 column volumes of 400 mM NaCl, 20 mM Tris–HCl, pH 8.0 and then with 10 volumes of 20 mM Tris–HCl, pH 8.0. The HLA peptides and HLA molecules were eluted with 1% TFA followed by separation of the peptides from the proteins by binding the eluted fraction to disposable reversed-phase Sep-Pak tC18 (Waters). Elution of the peptides was done with 30% acetonitrile (ACN) in 0.1% TFA. The HLA peptides were dried by vacuum centrifugation, resolubilized with 0.1% formic acid and separated using reversed-phase chromatography using the nanoAquity system (Waters), with a Symmetry trap column (180 × 20 mm) and HSS T3 analytical column, 0.75 × 250 mm (Waters). The chromatography system was coupled by electrospray to tandem mass spectrometry to Q-Exactive-Plus (Thermo Fisher Scientific). The HLA peptides were eluted with a linear gradient over 2 h from 5 to 28% acetonitrile with 0.1% formic acid at a flow rate of 0.35μl min−1. Data were acquired using a data-dependent ‘top 10’ method, fragmenting the peptides by higher-energy collisional dissociation (HCD). Full-scan MS spectra were acquired at a resolution of 70,000 at 200 m/z with a target value of 3 × 106 ions. Ions were accumulated to an AGC target value of 105 with a maximum injection time of 100 ms in general. The peptide match option was set to preferred. Normalized collision energy was set to 25% and MS/MS resolution was 17,500 at 200 m/z. Fragmented m/z values were dynamically excluded from further selection for 20 s. | In brief, cell pellets were lysed with lysis buffer containing 0.25% sodium deoxycholate, 0.2 mM iodoacetamide, 1 mM EDTA, 1:200 protease inhibitors cocktail (Sigma-Aldrich), 1 mM PMSF and 1% octyl-β-d-glucopyranoside in phosphate-buffered saline (PBS), and then incubated at 4 °C for 1 h. The lysates were cleared by centrifugation at 4 °C and 48,000g for 60 min, and then passed through a pre-clearing column containing Protein-A Sepharose beads (GenScript). HLA-I molecules were immunoaffinity-purified from cleared lysate with the pan-HLA-I antibody (W6/32 antibody purified from HB95 hybridoma cells) covalently bound to Protein-A sepharose beads). The affinity column was washed first with 10 column volumes of 400 mM NaCl, 20 mM Tris–HCl, pH 8.0 and then with 10 volumes of 20 mM Tris–HCl, pH 8.0. The HLA peptides and HLA molecules were eluted with 1% TFA followed by separation of the peptides from the proteins by binding the eluted fraction to disposable reversed-phase Sep-Pak tC18 (Waters). Elution of the peptides was done with 30% acetonitrile (ACN) in 0.1% TFA. | Untreated (n = 4), IFNγ-treated (n = 4), and Trp-depleted (n = 4) MD55A3 cell | | | Anti-tumour immunity induces aberrant peptide presentation in melanoma. |
| PXD023038 | Cancer | Breast Cancer | Peptide vaccination remains a viable approach to induce T-cell mediated killing of tumours. To identify potential T-cell targets for Triple-Negative Breast Cancer (TNBC) vaccination, we examined the effect of the pro-inflammatory cytokine interferon-γ (IFNγ) on the transcriptome, proteome and immunopeptidome of the TNBC cell line MDA-MB-231. Using high resolution mass spectrometry, we identified a total of 84,131 peptides from 9,647 source proteins presented by human leukocyte antigen (HLA)-I and HLA-II alleles. Treatment with IFNγ resulted in a remarkable remoulding of the immunopeptidome, with only a 34% overlap between untreated and treated cells across the HLA-I immunopeptidome, and expression of HLA-II only on treated cells. IFNγ increased the overall number, diversity and abundance of the immunopeptidome, as well as the proportion of coverage of source antigens. The suite of peptides displayed under conditions of IFNγ treatment included many known tumour associated antigens, with the HLA-II repertoire sampling 265 breast cancer associated antigens absent from those sampled by HLA-I. Quantitative analysis of the transcriptome (10,248 transcripts) and proteome (6783 proteins) of these cells revealed 229 proteins and transcripts were commonly differentially expressed, most of which involved in downstream targets of IFNγ signalling including components of the antigen processing machinery such as tapasin and HLA. However, these changes in protein expression did not explain the dramatic modulation of the immunopeptidome following IFNγ treatment. These results demonstrate the high degree of plasticity in the immunopeptidome TNBC cells following cytokine stimulation and provide evidence that under pro-inflammatory conditions a greater variety of HLA-I and HLA-II vaccine targets are unveiled to the immune system. This has important implications for the development of personalised cancer vaccination strategies. | HLA-I and -II peptides were eluted from three biological replicates of 5 × 108 MDA-MB-231 cells prior to or after treatment with IFNγ (i.e. n = 6) as described (36). Briefly, cells were lysed in 0.5% IGEPAL, 50 mM Tris-HCl pH 8.0, 150 mM NaCl supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail Tablet; Roche Molecular Biochemicals) for 45 min at 4°C. Lysates underwent ultracentrifugation at 40,000 g and HLA-I and -II complexes were purified using a pan HLA-I antibody (w6/32) whilst HLA-II complexes were purified with the allele-specific antibodies of LB3.1 (HLA-DR), B721 (HLA-DP), and SPV-L3 (HLA DQ). The HLA peptide bound molecules were then eluted from the affinity column with five column volumes of 10% acetic acid. The eluted HLA-I or HLA-II peptide complexes were loaded onto a 4.6 mm internal diameter × 50 mm monolithic C18 RP-HPLC column (Chromolith Speed Rod; Merck) at a flow rate of 1 ml/min using an EttanLC HPLC system (GE Healthcare) with buffer A [0.1% trifluoroacetic acid (TFA)] and peptides separated by increasing concentrations of buffer B (80% ACN/0.1% TFA) as described in (36). Fractionated peptides were then concatenated into 10 pools and analyzed with an Orbitrap Fusion™ Tribrid™ mass spectrometer (ThermoFisher Scientific). | HLA-I and -II peptides were eluted from three biological replicates of 5 × 108 MDA-MB-231 cells prior to or after treatment with IFNγ (i.e. n = 6) as described (36). Briefly, cells were lysed in 0.5% IGEPAL, 50 mM Tris-HCl pH 8.0, 150 mM NaCl supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail Tablet; Roche Molecular Biochemicals) for 45 min at 4°C. Lysates underwent ultracentrifugation at 40,000 g and HLA-I and -II complexes were purified using a pan HLA-I antibody (w6/32) whilst HLA-II complexes were purified with the allele-specific antibodies of LB3.1 (HLA-DR), B721 (HLA-DP), and SPV-L3 (HLA DQ). The HLA peptide bound molecules were then eluted from the affinity column with five column volumes of 10% acetic acid. The eluted HLA-I or HLA-II peptide complexes were loaded onto a 4.6 mm internal diameter × 50 mm monolithic C18 RP-HPLC column (Chromolith Speed Rod; Merck) at a flow rate of 1 ml/min using an EttanLC HPLC system (GE Healthcare) with buffer A [0.1% trifluoroacetic acid (TFA)] and peptides separated by increasing concentrations of buffer B (80% ACN/0.1% TFA) as described in (36). Fractionated peptides were then concatenated into 10 pools and analyzed with an Orbitrap Fusion™ Tribrid™ mass spectrometer (ThermoFisher Scientific). | MDA-MB-231 cells | | MDA-MB-231 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 1% Penicillin/streptomycin and L-glutamine (2 mM) (Gibco) at 37°C with 5% CO2. Cells were titrated at 50,100 and 200 IU of lyophilized human IFNγ (Miltenyi Biotec #130-096-484) for 48 h before being examined at a time course across 16, 24, 48-h intervals. IFNγ treated cells were then treated with 50 IU of lyophilized human IFNγ (Miltenyi Biotec #130-096-484) for 48 h for all subsequent experiments with all cells being cultured from a single seed flask for comparative analysis. | IFNγ Modulates the Immunopeptidome of Triple Negative Breast Cancer Cells by Enhancing and Diversifying Antigen Processing and Presentation. |
| PXD024562 | Cancer | Melanoma | Peptides displayed by MHC molecules on a cell’s surface, referred to as its immunopeptidome, play an important role in the adaptive the immune response. Antigen processing for MHC class I presentation is a ubiquitous pathway present in all nucleated cells which generate and present peptides of both self and non-self origin. Peptides with post-translational modifications (PTMs) are one of the classes of peptides presented by MHC class I molecules. However, due to the high background of self-peptides presented by the cells, the diversity of peptides with post-translational modifications is not well reported. In this study, we have carried out MHC Class I immunopeptidomics analysis on Jurkat and A375 cell lines to characterize the diversity of post-translational modifications among MHC class I peptides. Using high resolution mass spectrometry, we identified 25,761 MHC-bound peptides across both the cell lines using Bolt and Sequest search engines. High specificity of the enrichment method is demonstrated by identifying ~90% of the peptides with typical length distribution of 8-12 aa and enriched motifs within those peptides similar to the binding motifs of MHC alleles. Among the MHC-bound peptides, we identified phosphorylation as a major post-translational modification followed by deamidation. We observed site-specific localization of these post-translational modifications, at position P4 for phosphorylated peptides and position P3 for deamidated peptides. We identified a smaller number of peptides with acetylated and methylated lysine, possibly due to very low stoichiometric levels of these post-translational modifications compared to phosphorylation and deamidation. Using PEAKS de novo sequencing algorithm, we identified spliced peptides that account for ~5-7% of MHC-bound peptides across the two cell lines. These peptides share similar features with respect to normal MHC-bound peptides such as peptide length distribution and binding motifs. We validated the identification of several post-translationally modified peptides and spliced peptides using synthetic peptide sequences. In conclusion, our study demonstrates unbiased identification of these low stoichiometric PTMs and unusual spliced peptides using high resolution mass spectrometry. | Pan-specific MHC Class I antibody (W6/32 clone) was ordered from BioXCell. 5 mg of antibody was loaded on 1 mL of Protein A-Sepharose 4B beads (Invitrogen) packed in a polypropylene column (BioRad) and incubated for 30 min. Antibody-bound beads was washed with borate buffer (pH 9) and incubated with 20 mM dimethyl pimelimidate (DMP) linker for 45 min. Crosslinking reaction was stopped by incubating the column with ethanolamine for 2 h. Then the column was washed with phosphate buffer saline and stored in PBS with 0.02% sodium azide at 4 °C. On the day of experiment, column was washed with 0.1 N acetic acid and equilibrated with 100 mM Tris–HCl, pH 8.0.MHC–peptide complexes were enriched using previously described protocol (Bassani-Sternberg et al. 2016). Loucy and A375 cell lines were lysed in buffer containing 0.25% sodium deoxycholate, 0.2 mM IAA, 1 mM EDTA, 1 mM PMSF, 1% Octyl-B-glucopyranoside, 1:200 protease inhibitor cocktail for 1 h on ice. Cell debris was separated by centrifugation of the lysate at 25,000×g, 4 °C for 50 min. The supernatant was loaded onto an MHC class I crosslinked affinity column and incubated for 1 h with gentle rotation. The column was washed with 150 mM NaCl in 20 mM Tris HCl pH 8.0, 400 mM NaCl in 20 mM Tris HCl pH 8.0 followed by 150 mM NaCl in 20 mM Tris HCl pH 8.0 and 20 mM Tris HCl pH 8.0. MHC-bound peptide complexes were eluted using 1% TFA and the eluate was subjected to C18 cleanup to purify the eluted peptides. The peptides were dried using a speedvac concentrator prior to LC–MS/MS analysis. | MHC–peptide complexes were enriched using previously described protocol (Bassani-Sternberg et al. 2016). Loucy and A375 cell lines were lysed in buffer containing 0.25% sodium deoxycholate, 0.2 mM IAA, 1 mM EDTA, 1 mM PMSF, 1% Octyl-B-glucopyranoside, 1:200 protease inhibitor cocktail for 1 h on ice. Cell debris was separated by centrifugation of the lysate at 25,000×g, 4 °C for 50 min. The supernatant was loaded onto an MHC class I crosslinked affinity column and incubated for 1 h with gentle rotation. The column was washed with 150 mM NaCl in 20 mM Tris HCl pH 8.0, 400 mM NaCl in 20 mM Tris HCl pH 8.0 followed by 150 mM NaCl in 20 mM Tris HCl pH 8.0 and 20 mM Tris HCl pH 8.0. MHC-bound peptide complexes were eluted using 1% TFA and the eluate was subjected to C18 cleanup to purify the eluted peptides. The peptides were dried using a speedvac concentrator prior to LC–MS/MS analysis. | A375 cell lines | | A375 cell line was cultured in DMEM medium. The cell lines were maintained in CO2 incubator with 5% CO2 level. Upon reaching confluence, cell lines were washed with phosphate buffered saline (PBS) three times, flash frozen in liquid nitrogen and stored at – 80 °C. | Digging deeper into the immunopeptidome: characterization of post-translationally modified peptides presented by MHC I |
| PXD024562 | Cancer | T-Acute Lymphoblastic Leukemia | Peptides displayed by MHC molecules on a cell’s surface, referred to as its immunopeptidome, play an important role in the adaptive the immune response. Antigen processing for MHC class I presentation is a ubiquitous pathway present in all nucleated cells which generate and present peptides of both self and non-self origin. Peptides with post-translational modifications (PTMs) are one of the classes of peptides presented by MHC class I molecules. However, due to the high background of self-peptides presented by the cells, the diversity of peptides with post-translational modifications is not well reported. In this study, we have carried out MHC Class I immunopeptidomics analysis on Jurkat and A375 cell lines to characterize the diversity of post-translational modifications among MHC class I peptides. Using high resolution mass spectrometry, we identified 25,761 MHC-bound peptides across both the cell lines using Bolt and Sequest search engines. High specificity of the enrichment method is demonstrated by identifying ~90% of the peptides with typical length distribution of 8-12 aa and enriched motifs within those peptides similar to the binding motifs of MHC alleles. Among the MHC-bound peptides, we identified phosphorylation as a major post-translational modification followed by deamidation. We observed site-specific localization of these post-translational modifications, at position P4 for phosphorylated peptides and position P3 for deamidated peptides. We identified a smaller number of peptides with acetylated and methylated lysine, possibly due to very low stoichiometric levels of these post-translational modifications compared to phosphorylation and deamidation. Using PEAKS de novo sequencing algorithm, we identified spliced peptides that account for ~5-8% of MHC-bound peptides across the two cell lines. These peptides share similar features with respect to normal MHC-bound peptides such as peptide length distribution and binding motifs. We validated the identification of several post-translationally modified peptides and spliced peptides using synthetic peptide sequences. In conclusion, our study demonstrates unbiased identification of these low stoichiometric PTMs and unusual spliced peptides using high resolution mass spectrometry. | Pan-specific MHC Class I antibody (W6/32 clone) was ordered from BioXCell. 5 mg of antibody was loaded on 1 mL of Protein A-Sepharose 4B beads (Invitrogen) packed in a polypropylene column (BioRad) and incubated for 30 min. Antibody-bound beads was washed with borate buffer (pH 9) and incubated with 20 mM dimethyl pimelimidate (DMP) linker for 45 min. Crosslinking reaction was stopped by incubating the column with ethanolamine for 2 h. Then the column was washed with phosphate buffer saline and stored in PBS with 0.02% sodium azide at 4 °C. On the day of experiment, column was washed with 0.1 N acetic acid and equilibrated with 100 mM Tris–HCl, pH 8.0.MHC–peptide complexes were enriched using previously described protocol (Bassani-Sternberg et al. 2016). Loucy and A375 cell lines were lysed in buffer containing 0.25% sodium deoxycholate, 0.2 mM IAA, 1 mM EDTA, 1 mM PMSF, 1% Octyl-B-glucopyranoside, 1:200 protease inhibitor cocktail for 1 h on ice. Cell debris was separated by centrifugation of the lysate at 25,000×g, 4 °C for 50 min. The supernatant was loaded onto an MHC class I crosslinked affinity column and incubated for 1 h with gentle rotation. The column was washed with 150 mM NaCl in 20 mM Tris HCl pH 8.0, 400 mM NaCl in 20 mM Tris HCl pH 8.0 followed by 150 mM NaCl in 20 mM Tris HCl pH 8.0 and 20 mM Tris HCl pH 8.0. MHC-bound peptide complexes were eluted using 1% TFA and the eluate was subjected to C18 cleanup to purify the eluted peptides. The peptides were dried using a speedvac concentrator prior to LC–MS/MS analysis. | MHC–peptide complexes were enriched using previously described protocol (Bassani-Sternberg et al. 2016). Loucy and A375 cell lines were lysed in buffer containing 0.25% sodium deoxycholate, 0.2 mM IAA, 1 mM EDTA, 1 mM PMSF, 1% Octyl-B-glucopyranoside, 1:200 protease inhibitor cocktail for 1 h on ice. Cell debris was separated by centrifugation of the lysate at 25,000×g, 4 °C for 50 min. The supernatant was loaded onto an MHC class I crosslinked affinity column and incubated for 1 h with gentle rotation. The column was washed with 150 mM NaCl in 20 mM Tris HCl pH 8.0, 400 mM NaCl in 20 mM Tris HCl pH 8.0 followed by 150 mM NaCl in 20 mM Tris HCl pH 8.0 and 20 mM Tris HCl pH 8.0. MHC-bound peptide complexes were eluted using 1% TFA and the eluate was subjected to C18 cleanup to purify the eluted peptides. The peptides were dried using a speedvac concentrator prior to LC–MS/MS analysis. | Loucy cell lines | | Loucy cell line was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The cell lines were maintained in CO2 incubator with 5% CO2 level. Upon reaching confluence, cell lines were washed with phosphate buffered saline (PBS) three times, flash frozen in liquid nitrogen and stored at – 80 °C. | Digging deeper into the immunopeptidome: characterization of post-translationally modified peptides presented by MHC I |
| PXD024871 | Cancer | Chronic Lymphocytic Leukemia | In recent years the clinical success of T cell-based immunotherapy approaches has revolutionized treatment of solid tumors and hematological malignancies. However, still some patients do not respond to available therapies at all, others for limited time only. A promising low side-effect approach is peptide-based immunotherapy, which relies on specific immune recognition of tumor-associated human leucocyte antigen (HLA)-presented peptides. In this study, we developed a workflow for the immunopeptidome-guided design of off-the shelf warehouses for personalized peptide vaccines using the example of chronic lymphocyte leukemia (CLL). The so defined warehouses could provide the basis for different T cell-based immunotherapy approaches such as TCR-engineered T cell transfer or multi-peptide vaccinations. The warehouse approach enables a fast and cost-effective way to provide a personalized T cell-based immunotherapeutic approach. The here defined peptide warehouse is already utilized for a personalized multi-peptide vaccine trial (iVAC-XS15-CLL01, NCT04688385). | HLA class I and HLA class II molecules were isolated by standard immunoaffinity purification (28) using the pan-HLA class I-specific monoclonal antibody W6/32, the pan-HLA class II-specific monoclonal antibody Tü-39, and the HLA-DR-specific monoclonal antibody L243 (all produced in-house) to extract HLA ligands. | HLA class I and HLA class II molecules were isolated by standard immunoaffinity purification (28) using the pan-HLA class I-specific monoclonal antibody W6/32, the pan-HLA class II-specific monoclonal antibody Tü-39, and the HLA-DR-specific monoclonal antibody L243 (all produced in-house) to extract HLA ligands. | Peripheral blood mononuclear cells (PBMCs) from CLL patients as well as PBMCs from healthy volunteers (HVs) were isolated by density gradient centrifugation and stored at -80°C until further use for subsequent HLA immunoprecipitation or T cell-based assays. | | Peripheral blood mononuclear cells (PBMCs) from CLL patients as well as PBMCs from healthy volunteers (HVs) were isolated by density gradient centrifugation and stored at -80°C until further use for subsequent HLA immunoprecipitation or T cell-based assays. | Immunopeptidomics-Guided Warehouse Design for Peptide-Based Immunotherapy in Chronic Lymphocytic Leukemia. |
| PXD027182 | Cancer | Neuroblastoma | We surveyed the immunopeptidome of neuroblastoma tumors to identify tumor-specific targets derived from non-mutated self proteins originating from core-regulatory circuit proteins. | HLA class I molecules were isolated using standard immunoaffinity purification as described previously,53,54. In brief, cell pellets were lysed in 10 mM CHAPS/PBS (AppliChem/Lonza) containing 1× protease inhibitor (Complete; Roche). Mouse MHC molecules were removed using a 1 h immunoaffinity purification with H-2K-specific monoclonal antibody 20-8-4S, covalently linked to CNBr-activated sepharose (GE Healthcare). Remaining HLA molecules were purified overnight using the pan-HLA class I-specific monoclonal antibody W6/32 or a mix of the pan-HLA class II-specific monoclonal antibody Tü39 and the HLA-DR-specific monoclonal antibody L243, covalently linked to CNBr-activated Sepharose. MHC–peptide complexes were eluted by repeated addition of 0.2% trifluoroacetic acid (Merck). Elution fractions E1–E4 were pooled, and free MHC ligands were isolated by ultrafiltration using centrifugal filter units (Amicon; Merck Millipore). MHC ligands were extracted and desalted from the filtrate using ZipTip C18 pipette tips (Merck Millipore). Extracted peptides were eluted in 35 µl of acetonitrile (Merck)/0.1% trifluoroacetic acid, centrifuged to complete dryness and resuspended in 25 µl of 1% acetonitrile/0.05% trifluoroacetic acid. Samples were stored at −20 °C until analysis by LC–MS/MS. | HLA class I molecules were isolated using standard immunoaffinity purification as described previously,53,54. In brief, cell pellets were lysed in 10 mM CHAPS/PBS (AppliChem/Lonza) containing 1× protease inhibitor (Complete; Roche). Mouse MHC molecules were removed using a 1 h immunoaffinity purification with H-2K-specific monoclonal antibody 20-8-4S, covalently linked to CNBr-activated sepharose (GE Healthcare). Remaining HLA molecules were purified overnight using the pan-HLA class I-specific monoclonal antibody W6/32 or a mix of the pan-HLA class II-specific monoclonal antibody Tü39 and the HLA-DR-specific monoclonal antibody L243, covalently linked to CNBr-activated Sepharose. MHC–peptide complexes were eluted by repeated addition of 0.2% trifluoroacetic acid (Merck). Elution fractions E1–E4 were pooled, and free MHC ligands were isolated by ultrafiltration using centrifugal filter units (Amicon; Merck Millipore). MHC ligands were extracted and desalted from the filtrate using ZipTip C18 pipette tips (Merck Millipore). Extracted peptides were eluted in 35 µl of acetonitrile (Merck)/0.1% trifluoroacetic acid, centrifuged to complete dryness and resuspended in 25 µl of 1% acetonitrile/0.05% trifluoroacetic acid. Samples were stored at −20 °C until analysis by LC–MS/MS. | Five neuroblastoma cell line xenografts and three patients derived xenografts | | Human-derived neuroblastoma cell lines, including SK-N-AS, SK-N-FI and NB-SD were obtained from the Maris Lab cell line bank. Neuroblastoma cell lines were cultured in RPMI supplemented with 10% fetal bovine serum (FBS), 100 U ml−1 penicillin, 100 µg ml−1 streptomycin, and 2 mM l-glutamine. Other human cancer cell lines, including Jurkat,SW620, HEPG2 and KATO III were obtained from American Type Culture Collection (ATCC). Jurkat cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% FBS, 100 U ml−1 penicillin, 100 µg ml−1 streptomycin and 2 mM l-glutamine. SW620 cells were cultured in RPMI supplemented with 10% FBS, 100 U ml−1 penicillin, 100 µg ml−1 streptomycin, and 2 mM l-glutamine. HEPG2 cells were cultured in Eagle’s minimum essential medium (EMEM) supplemented with 10% FBS, 100 U ml−1 penicillin, 100 µg ml−1 streptomycin and 2 mM l-glutamine. KATO III cells were cultured in IMDM supplemented with 20% FBS, 100 U ml−1 penicillin, 100 µg ml−1 streptomycin and 2 mM l-glutamine. Packaging cell lines including Platinum-A cells and HEK 293T cells were obtained from Cell BioLabs and ATCC, respectively. Both packaging cell lines were cultured in DMEM supplemented with 10% FBS, 100 U ml−1 penicillin, 100 µg ml−1 streptomycin and 2 mM l-glutamine. All cell lines were grown under humified conditions in 5% CO2 at 37 °C, and samples were regularly tested for mycoplasma contamination. | Cross-HLA targeting of intracellular oncoproteins with peptide-centric CARs. |