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Deciphering the immunopeptidome in vivo reveals new tumour antigens


Immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex class I (MHC-I) molecules1,2,3,4,5. Current approaches to profiling of MHC-I-associated peptides, collectively known as the immunopeptidome, are limited to in vitro investigation or bulk tumour lysates, which limits our understanding of cancer-specific patterns of antigen presentation in vivo6. To overcome these limitations, we engineered an inducible affinity tag into the mouse MHC-I gene (H2-K1) and targeted this allele to the KrasLSL-G12D/+Trp53fl/fl mouse model (KP/KbStrep)7. This approach enabled us to precisely isolate MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma and from lung adenocarcinoma (LUAD) in vivo. In addition, we profiled the LUAD immunopeptidome from the alveolar type 2 cell of origin up to late-stage disease. Differential peptide presentation in LUAD was not predictable by mRNA expression or translation efficiency and is probably driven by post-translational mechanisms. Vaccination with peptides presented by LUAD in vivo induced CD8+ T cell responses in naive mice and tumour-bearing mice. Many peptides specific to LUAD, including immunogenic peptides, exhibited minimal expression of the cognate mRNA, which prompts the reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance8. Beyond cancer, the KbStrep allele is compatible with other Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease and autoimmunity.

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Fig. 1: Design and validation of the KP/KbStrep mouse model.
Fig. 2: The LUAD immunopeptidome is dynamic and heterogenous throughout tumour evolution.
Fig. 3: Transcription and translation of LUAD-unique peptides.
Fig. 4: Discovery of new tumour antigens in LUAD.

Data availability

All MS data have been deposited to the Proteomics Identifications Database (PRIDE) repository with the dataset identifier PXD033232. Raw RNA-seq and Ribo-seq data have been submitted to the Gene Expression Omnibus with the dataset identifier GEO178944Source data are provided with this paper.


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We thank members of the Jacks Lab for critical comments on the work presented here, especially Z. Ely and N. Pattada for conceptual and technical support; G. Eng for support with organoid work; T. Koller and R. Schiavoni of the Koch Institute Proteomics core for technical insight on proteomic workflows; and staff at the Koch Institute Swanson Biotechnology Center for technical support, specifically the Flow Cytometry, Histology, Preclinical Modeling, Imaging and Testing, and Integrative Genomics and Bioinformatics core facilities. Data accessibility from the Tabula Muris was essential for multiple analyses in this manuscript. This work was supported by the Damon Runyon Cancer Research Foundation (A.M.J.), an NCI K99 Pathway to Independence Award (A.M.J.), the Howard Hughes Medical Institute, the Johnson & Johnson Lung Cancer Initiative, NCI Cancer Center Support Grant P30-CA1405, the Lustgarten Foundation Pancreatic Cancer Research Laboratory at MIT, the Stand Up To Cancer-Lustgarten Foundation Pancreatic Cancer Interception Translational Cancer Research Grant (grant number SU2C-AACR-DT25-17, W.A.F.-P. and T.J.), the MIT Center for Precision Cancer Medicine (L.E.S., R.A. and F.M.W.), the Margaret A. Cunningham Immune Mechanisms of Cancer Research Fellowship (L.E.S.), the Melanoma Research Alliance (L.E.S.), NIH Training Grant (T32-ES007020), the Ludwig Center at MIT (R.A.), the Pew-Stewart Scholars Program for Cancer Research (A.K.S.), and the Cancer Research Institute (T.F. and S.S.).

Author information

Authors and Affiliations



A.M.J. and T.J. conceived, designed and directed the study. L.E.S., R.A. and F.M.W. directed all MS analyses. A.M.J., L.E.S., R.A., E.A.S., D.A.S. and R.E.K. performed all experiments. W.A.F.-P. conducted all orthotopic and autochthonous pancreatic surgeries. S.N. provided guidance and reagents for AT2 organoid culture. W.M.R. conducted mouse ESC targeting and chimera generation. A.M.J., L.E.S., R.A. and T.F. performed data analyses. P.M.K.W. assisted with custom pMHC tetramer generation. K.B.N. and S.S. provided assistance with vaccination and ELISPOT analysis. P.S.W. and A.K.S. provided guidance for analysis of the scRNA-seq data. J.S. and S.-L.S. provided technical and conceptual support of the study. A.M.J., L.E.S., R.A., F.M.W. and T.J. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Tyler Jacks.

Ethics declarations

Competing interests

T.J. is a member of the Board of Directors of Amgen and Thermo Fisher Scientific, and a co-Founder of Dragonfly Therapeutics and T2 Biosystems. T.J. serves on the Scientific Advisory Board of Dragonfly Therapeutics, SQZ Biotech and Skyhawk Therapeutics. T.J. is also President of Break Through Cancer. His laboratory currently receives funding from Johnson & Johnson and The Lustgarten Foundation, and funds from the Lustgarten Foundation supported the research described in this manuscript. S.S. is a co-founder of Danger Bio and serves on the Scientific Advisory Board of Related Sciences/DanderBio, Ankyra Therapeutics, Arcus Biosciences, Takeda and Ribon, and serves as an advisor for Dragonfly Therapeutics and Merck. The S.S. Lab currently receives funding from Leap Therapeutics. A.K.S. reports compensation for consulting and/or SAB membership from Merck, Honeycomb Biotechnologies, Cellarity, Repertoire Immune Medicines, Ochre Bio, Third Rock Ventures, Hovione, Relation Therapeutics, FL82, Empress Therapeutics and Dahlia Biosciences. None of these affiliations influenced the work conducted or analysis of data presented in this manuscript.

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Peer review statement: Nature thanks Lélia Delamarre and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 (Related to Fig. 1). In vitro validation of the KbStrep allele.

a) Structural model depicting the topology of the KbStrep protein during affinity purification. The StrepTagII engagement with Streptactin affinity resin does not interfere with peptide or B2m binding. b) Southern blot analysis of KbStrep targeted KP* ES cells. c) Representative genotyping for WT/WT, KbStrep/WT heterozygotes, and KbStrep/KbStrep homozygotes. d) Brightfield images of KP and KP/ KbStrep pancreatic organoids pre- and post- Ad-CMV-Cre mediated transformation ex vivo. e) RT-PCR analysis of KP or KP/KbStrep pancreatic organoids with or without Cre recombination and with or without IFN-γ treatment. Each row represents a distinct primer set showing no discernable alterations in mRNA splicing with or without StrepTagII activation. f) Representative flow cytometry plots detecting cell surface expression of PD-L1 and StrepTagII at baseline (red) and following Cre activation and IFN-γ treatment (blue). g) Quantification of the median fluorescence intensity (MFI) of StrepTagII staining in KP (orange) and KP/KbStrep (blue) organoids in control, IFN-γ treated, Cre transformed, and Cre+IFN-γ treated samples. Data are mean ± sem (n = 3). Two-sided Student’s t-test. h) Immunoblot analysis of whole cell lysate from KP or KP/KbStrep PDAC cells after adaptation to 2D following treatment with IFN-γ. i) Immunoblot depicting affinity purification of intact MHC-I with Streptactin resin as evidenced by the co-precipitation of B2m. j) Coomassie staining of samples taken from KP or KP/KbStrep lysates at various stages of purification. In this experiment, the elution was taken by incubating washed Streptactin resin with SDS-PAGE loading buffer.

Extended Data Fig. 2 (related to Fig. 1). Isolation of MHC-I complexes from PDAC in vivo.

a) Experimental illustration of samples used for immunopeptidome comparison in PDAC. b) Multiplexed immunofluorescence of a representative autochthonous PDAC tumor. White arrows indicate cancer cell nests. c) High magnification multiplexed immunofluorescence image depicting the specificity of StrepTagII staining on cancer cells. d) Schematic illustration of the traditional method of peptide extraction (top) and the method used for competitive elution of MHC-I complexes with biotin used in this study (bottom). e) Immunoblot demonstrating efficient precipitation of the MHC-I heavy and light chain with acid (1% TFA) prior to peptide clean-up with C18 ziptip. f) Representative immunoblot demonstrating purification of MHC-I specifically from KP/KbStrep tumor bearing mice. g) Number of unique peptides identified in each sample type after filtering for length (8-11 amino acids) and NetMHCPan predicted affinity (<1000 nM). h) Amino acid length distribution of all peptides identified from 2D, Ortho, Auto, or WT samples. i) Peptide motifs of 8- and 9-mers isolated from 2D, orthotopic, and autochthonous tumors. j) Venn diagram comparison of peptides found in 2D, Ortho, or Auto samples. Peptides only found in vivo are outlined in red. k) Venn diagram comparing MHC-I peptides derived from normal pancreas in Schuster et. al. (gray) versus orthotopic transplant (light blue) or autochthonous PDAC (dark blue) in this study. l) UMAP embedding of reanalyzed pancreatic scRNAseq data from the Tabula Muris. For clarity of presentation in Extended Data Fig. 2m, cells from a specific lineage were collapsed into a single cluster if they originally separated into multiple clusters (i.e. Alpha 1 and Alpha 2 → Alpha). m) Expression of gene signatures derived from genes encoding for normal pancreas peptides (gray), orthotopic PDAC peptides (light blue), and autochthonous PDAC peptides (dark blue) in all cell types of the normal pancreas as measured by scRNAseq from Tabula Muris.

Extended Data Fig. 3 (related to Fig. 1). In vivo validation of KbStrep allele in KP LUAD.

a) Representative H&E images demonstrating adenocarcinoma in KP and KP/KbStrep tumors. b) Multiplexed immunofluorescence of 16-week KP/KbStrep tumors demonstrating specific StrepTagII detection on tumor cells within the tumor microenvironment. c) Multiplexed immunofluorescence of a WT KP tumor demonstrating no detection of the StrepTagII in tumors outlined in white dotted lines. d) Gating strategy for isolating cells positive for an alveolar type 2 (AT2) phenotype from KP tumor-bearing lung tissue. e) Histograms depicting StrepTagII staining intensity across all CD45- cells (left) or after gating for AT2 cells (right) in KP (pink) or KP; KbStrep (purple) tumors. f) Quantification of StrepTagII MFI on AT2 or CD45 cells in the tumor microenvironment from KP or KP/KbStrep tumors. Data are mean ± sem. Two-sided Student’s t-test. g) Relative abundance of CD4+ T cells, CD8+ T cells, Macrophages, and CD45+MHCII+ immune cells in the tumor microenvironment of KP and KP/KbStrep tumors. Data are mean ± sem. Two-sided Student’s t-test. h) RT-PCR analysis of diverse tissues in KP/KbStrep mice with and without intratracheal Adeno-SPC-Cre administration. Expression of the Strep tagged Kb allele is only present in the lung after Cre induction. i) Immunoblot depicting isolation of intact MHC-I complexes specifically from KP/KbStrep tissue as evidenced by co-purification of B2m. j) Comparison of predicted peptide hydrophobicity (GRAVY) versus median peptide retention time in MS analysis for all peptides presented in Fig. 1i. Linear regression is shown for peptides identified in Normal-Ab, Tumor-Ab, and Tumor-Strep datasets indicating no detectable differences in the biochemical features of identified peptides across methods. k) (left) Non-metric multidimensional scaling (nmds) plots depicting clusters of 8- and 9-mer peptides identified from Normal-Ab, Tumor-Ab, and Tumor-Strep samples. (right) Histogram showing the distribution of unique peptides from antibody (Ab) and Streptactin affinity purification (Strep) methods across peptide clusters identified with nmds analysis (n.s. – not significant, Fisher’s exact test). (l) Distribution of predicted peptide affinity for MHC-I peptides identified in 6 KP/KbStrep replicates. m) Upset plot depicting the peptide identification overlap between 6 KP/KbStrep replicates after length and affinity filtering. >77% of all identified peptides were found in at least 2/6 replicates.

Source data

Extended Data Fig. 4 (related to Fig. 1). Biochemical comparison of WT and Strep tagged H2-Kb.

a) Isolation strategy for KP and KP/KbStrep cell lines. b) Histograms depicting fluorescence staining intensity of H2-Kb across KP and KP/KbStrep cell lines at baseline (gray) or following treatment with IFN-γ (red). c) Median fluorescence intensity quantification of H2-Kb staining across KP or KP/KbStrep cell lines at baseline (gray) or following treatment with IFN-γ (red). d) Relative H2-Kb staining intensity on KP (gray) or KP/KbStrep (red) cell lines following incubation with brefeldin A (BFA, left) or acute stripping with acid (300 mM glycine, pH 3.0, right) for the indicated times. e) Representative immunoblot depicting relative amounts of H2-Kb immunoprecipitation with antibody (Y3-Ab), Streptactin, or antibody following Streptactin (Y3-F.T.). f) Densitometric quantification of immunoprecipitated B2m intensity following antibody (Y3), streptactin, or antibody following streptactin (Y3 after Strep) purification schemes. g) Representative immunoblot depicting Strep-tagged H2-Kb expression in KP or KP/KbStrep cell lines following incubation with the aminopeptidase inhibitors ERAP1-in-1 or Bestatin. h) Experimental schematic for comparison of KP and KP/KbStrep immunopeptidomes from cultured cells using a quantitative, tandem mass tag (TMT) mass spectrometry strategy. i) Immunoblot depicting the abundance of immunoprecipitated MHC-I from samples described in h). j) Quantitative abundance comparison between all peptides identified in KP and KP/KbStrep samples. k) Illustration of the lentiviral constructs used for stable expression of SIINFEKL in KP and KP/KbStrep cell lines. l) Flow cytometric analysis of SIINKFEKL-H2-Kb complex surface expression using 25-D1.16 antibody. m) Experimental schematic used to evaluate specific T cell killing mediated by OT-I TCR transgenic T cells. n) Representative flow cytometry histograms depicting raw data used for calculating % specific lysis. o) Quantification of OT-I T cell killing in KP and KP/KbStrep cells.

Source data

Extended Data Fig. 5 (Related to Fig. 2). Analysis of the LUAD immunopeptidome throughout tumor evolution.

a) UMAP embedding of clusters used for signature expression analysis in Fig. 2a. b) Gene expression profiles of cell type marker genes indicating robust clustering of known cell types in the healthy lung. c) Representative H&E stains for healthy lung (AT2), Early-, Mid- and Late-stage tumor samples. d) Peptide motifs of 8- and 9-mer peptides identified from healthy AT2 cells. e) Length and affinity characteristics of peptides identified in AT2, Early-, Mid-, and Late-stage tumors. f) Venn diagram comparison of peptides identified in bulk, healthy lung versus those identified specifically presented by normal AT2 cells. Pathways enriched by gene ontology depicted on bottom. g) Comparative analysis of gene signatures derived from peptides detected on AT2 cells versus bulk lung applied to the Tabula Muris data. Volcano plot shows the strong enrichment for an AT2 phenotype in the AT2 immunopeptidome versus bulk lung immunopeptidome. h) Comparison of AT2 and Early-, Mid-, and Late-Stage immunopeptidomes. i) Quantification of percent overlap from h). j) UMAP embedding of clusters from reanalyzing scRNAseq data from Marjonovic et. al. and used for analysis of Fig. 2j. k) Loess regression analysis across all cells scored for AT2, Early, Mid, and Late peptide signatures versus the AT2, GI-Epi, and Mixed transcriptional modules. l) Signature distribution for AT2-, Early-, Mid-, and Late-Stage peptide signatures across all KP scRNAseq clusters. m) Comparison of the immunopeptidome in control tumors (gray), tumors chronically depleted of CD8 T-cells (light teal), and tumors acutely depleted of CD8 T-cells (dark teal). n) Comparison of the immunopeptidome from control tumors (gray) or tumors treated with agonistic-CD40 antibody and Flt3-L (orange). CD8 depletion experiments and CD40/Flt3L experiments were carried out independently and analyzed on different MS runs separated by ~2.5 months.

Extended Data Fig. 6 (Related to Fig. 3). Identification of differentially translated genes in LUAD versus AT2 cells.

a) Representative images of normal AT2 and tumor organoid cultures in 3D organotypic culture or after transient adaptation to 2D monolayer culture prior to RiboSeq and RNAseq processing. b) Denaturing PAGE gels of RNA purified from RiboLace purification. Excised bands used for RiboSeq are indicated on the right and were selected for RNA that was ~ 30 bp in length. c) Localization of RPF alignments within transcripts depicting enrichment for annotated coding sequences (CDS). d) Normalized metagene density profiles of reads from normal and tumor cells at translation initiation (left) and termination (right). Both normal and tumor metaprofiles exhibit 3 nucleotide periodicity, indicative of active translation. e) Volcano plot depicting differential mRNA expression in tumor versus normal AT2 cells. f) Volcano plot depicting differential RPF abundance in tumor versus normal AT2 cells. g) Volcano plot depicting differential translation efficiency in tumor versus normal AT2 cells.

Extended Data Fig. 7 (Related to Fig. 3). Transcriptomic and proteomic data associated with LUAD-unique peptides.

a) mRNA expression of genes encoding for Normal (bulk-lung), AT2, All-LUAD, or LUAD-unique peptides across normal AT2 cells, early-, mid-, and late-stage sorted tumor cells. (Adapted from Chuang et. al.). b) Subcellular compartment distribution of source proteins for peptides found in bulk Normal Lung, AT2 cells, all tumor peptides, and LUAD-unique peptides. c) Distribution of Protein length, thermal stability, or protein half-life for source proteins of peptides found in Normal Tissue, All Tumor peptides, or LUAD-unique peptides. d) StringDb analysis of source proteins for LUAD-unique peptides indicated in Fig. 3a. Clusters of enriched protein families are depicted. e) Gene ontology analysis of LUAD-unique peptides from KEGG and Reactome databases. f) Expression of the LUAD-unique signature across all cells in the KP scRNAseq dataset (Marjonovic et. al.) g) Expression of the LUAD-unique signature across tumor progression in KP scRNAseq. h) Expression of LUAD-unique peptide signature across clusters in KP scRNAseq. i) Expression of individual genes encoding LUAD-unique peptides across KP timepoints. j) Correlation of the LUAD-unique peptide signature to all genes detected in scRNAseq. Genes related to antigen presentation are highlighted in red and genes related to metastasis are highlighted in blue.

Source data

Extended Data Fig. 8 (Related to Fig. 3). Modulation of protein folding through Hsp90 inhibition alters the immunopeptidome in vivo.

a) Experimental schematic of KP/KbStrep tumor treatment with either vehicle control or 0.5 mg/kg/day NVP-HSP990 prior to tumor specific MHC-I isolation. b) Immunoblot analysis of purified MHC-I from Vehicle (Veh) and Hsp90 inhibitor (Hsp90i) treated tumor samples or KP control tumors. c) Length and affinity distribution of peptides found in Veh (grey) and Hsp90i (blue) treated samples. d) Venn diagram of peptides found in 12-week control tumors or Hsp90i treated samples. e) Number of Hsp90 clients giving rise to peptide in either Veh (grey) or Hsp90i (blue) samples. f) Distribution of peptides identified in Veh or Hsp90i treated samples across subcellular compartments. g) Gene ontology analysis of source proteins for peptides that were only found in Hsp90i treated samples ranked according to FDR enrichment significance. h) Comparing the rank ordered abundance of all common peptides between Hsp90i treatment and control. i) Density plots of raw peptide abundance for non-clients, synthesis clients or constitutive clients in Veh (top) or Hsp90i (bottom) treated samples. j) Rank ordered abundance of peptides derived from Non-clients (No, gray), synthesis clients (Synth, light purple) or constitutive clients (Const, dark purple) in Veh and Hsp90i treated samples. P calculated with the Kologorov-Smirnov Test. k) RNA abundance, translation rate, and melting temperatures across non-clients (No), synthesis clients (Synth) and constitutive clients (Const) that are source proteins for MHC-I presentation. P calculated with Mann-Whitney U Test. l) Comparison of peptides unique to Flt3L/aCD40 treatment and those unique to Hsp90i.

Source data

Extended Data Fig. 9 (Related to Fig. 4). Expression and presentation of putative tumor specific and tumor associated antigens.

a) Correlelogram and heatmap depicting transcript abundance (transcripts per million, TPM) of putative TSA genes across mouse tissues. b) Correlelogram and heatmap depicting TPM abundance of putative TAA genes across mouse tissues. c) Experimental schematic showing the derivation of samples for 2D immunopeptidomics. d) Venn diagram depicting the relationship between peptides identified by KP tumors in vivo and those identified in vitro. e) Boxplot showing the predicted affinity distributions of peptides isolated in vivo and in vitro. f) Distribution of source protein subcellular compartments for peptides identified in vivo (gray) and in vitro (green). P calculated with Fisher’s Exact test with Monte Carlo simulation. g) Volcano plot indicating differentially expressed genes between EPCAM+ cells from embryonic day 16.5 and post-natal day 28 mouse lung (Adapted from Lung Map Project). Data analyzed and P calculated with DEseq2. All genes detected are shown in grey and genes encoding for LUAD-unique peptides are indicated with black dots.h) Flow cytometry analysis of tumor-bearing lung tissue from naïve and vaccinated mice stained with control pMHC-I tetramer (SIINFEKL) or TAA tetramer (SVAHFINL). i) Peptides identified in A549 cells (Javitt et. al.) with and without treatment of IFN-γ/TNFα. Peptides derived from source proteins homologous to those using in the pooled vaccine are indicated in red. j) Heatmap depicting expression of the human homologs of putative TSAs and TAAs from this study and whether or not peptides derived from those genes were found to be presented on A549 cells from Javitt et. al. k) Heatmap depicting the RNA Expression of homologs of potential TSA and TAA genes as found in Fig. 4a across all individual human tissues and 33 cancer types within TCGA.

Source data

Extended Data Fig. 10 (Related to Fig. 4). Mass spectrometry validation of immunogenic epitopes with synthetic peptides.

a) Mass spectrometry comparison of spectra from endogenously identified VNVYFALL peptide (Slc26a4) and a synthetic standard. b) Mass spectrometry comparison of spectra from endogenously identified SVAHFINL peptide (Prdm15) and a synthetic standard. c) Mass spectrometry comparison of spectra from endogenously identified AVLLYEKL peptide (Ift74) and a synthetic standard. In the left panel, y-, b-, and a-ions are colored in bold. In the right panel, common peaks are drawn in darker colour.

Supplementary information

Supplementary Fig. 1

Raw images of western blots.

Reporting Summary

Supplementary Table 1

List of peptides identified in PDAC and normal pancreas samples.

Supplementary Table 2

List of peptides identified in LUAD, AT2 and normal lung samples.

Supplementary Table 3

Comparison of peptides Identified in LUAD with CD8a depletion.

Supplementary Table 4

Comparison of peptides identified in LUAD with CD40/FLT3L treatment.

Supplementary Table 5

RNA-seq and ribosome profiling data in AT2 and KP LUAD organoids.

Supplementary Table 6

List of peptides identified in HSP90i-treated samples.

Supplementary Table 7

Label-free quantification of peptides in HSP90i-treated samples.

Supplementary Table 8

Peptides identified in healthy heart, spleen, liver and lung.

Supplementary Table 9

List of peptides identified in cultured KP/KbStrep cell lines.

Supplementary Table 10

Inclusion list information for MS analysis of synthetic peptides.

Supplementary Table 11

List of antibodies used.

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Jaeger, A.M., Stopfer, L.E., Ahn, R. et al. Deciphering the immunopeptidome in vivo reveals new tumour antigens. Nature 607, 149–155 (2022).

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