Tertiary lymphoid structures (TLSs) are associated with improved responses to immune checkpoint blockade across solid tumours1,2, but how they impact the phenotypic properties of tumour-specific T cells remains unclear. Here we found, across 24 treatment-naive renal cell carcinoma (RCC) tumours, that TLS-containing tumours are more heavily infiltrated by exhausted CD8+ T cells and have a reduced terminal exhaustion transcriptional program compared with TLS- tumours. Specificity screening of 554 T cell clonotypes expanded within the microenvironment of 6 RCC tumours revealed 82 TCRs that were reactive against tumour cells and/or RCC antigens. A subset of tumour-specific T cell clonotypes (12%) was enriched within TLSs, and these expressed an increased program of stem-like progenitor exhaustion, associated with favourable anti-tumour immunity. However, in 60 independent RCC tumours, macrophages within tumour margins of TLS-containing tumours had an inferred immunosuppressive phenotype and were colocalized with exhausted putative tumour-reactive T cells in a subgroup that was further analysed, therefore supporting this mode of immune evasion as a counterbalance to T cell immune pressure. Our data reveal that TLSs are reservoirs of tumour-specific T cells with stem-like progenitor features that could be leveraged by T cell immunotherapies.

Despite widespread vaccination, Bordetella pertussis (Bp) cases are resurging globally. Although CD4+ T cells are known to be essential for sustained protection, the antigens they recognize are not fully characterized, hindering vaccine refinement. Using immunopeptidomics, bioinformatics, and functional T cell assays, we identified high-affinity epitopes from reference and clinical Bp strains presented on MHC-II I-Ab. A subset of these epitopes stimulated systemic and mucosal CD4+ T cells of mice immunized with heat-killed Bp, and peripheral blood T cells from humans vaccinated with the whole-cell pertussis vaccine. Mice immunized with a subunit vaccine comprising two recombinant proteins identified in our screen were subsequently challenged with Bp. Bacterial burden was nearly eliminated from the lower respiratory tract and significantly reduced in the upper respiratory tract. Th1/Th17-polarized CD4+ tissue-resident memory T cells (Trms) were induced in nasal and pulmonary tissues. Depleting memory CD4+ T cells before challenge abolished protection, confirming that antigen-specific CD4+ T cells are critical for clearing Bp from the respiratory tract. Our integrated antigen identification and T cell assay approach revealed previously untested Bp antigens that elicit protective CD4+ T cell-mediated immunity, suggesting that incorporating them into new vaccines may help curb the resurgence of pertussis.

Abstract CD4 + T cells recognize peptides presented by human leukocyte antigen (HLA) II, implementing a fundamental mediation mechanism of the adaptive immune system. Although post-translational modifications (PTMs) alter immune responses, PTM-peptide-HLA interaction prediction remains challenging due to data scarcity resulting from substoichiometric levels of PTMs. To overcome this, we developed PepChem, a deep learning model utilizing novel, molecular-level peptide representations that enable predictions for sidechain modifications. Using monoallelic datasets that we reanalyze for PTMs of interest, we show accurate predictions on PTMs that were unseen during training. Furthermore, we introduce a novel training protocol that improves PTM-peptide generalization compared to conventional methods. We predict and experimentally validate citrullination-induced binding increase of rheumatoid arthritis (RA)-linked peptides to HLA II risk allele DRB1*04:01. This framework bridges the critical gap in PTM-aware immune recognition prediction, with immediate applications in autoimmunity, cancer, and infectious disease.

T cell-mediated immune surveillance is critical for cancer control, yet its role in bone marrow malignancies remains poorly understood. Here, we integrate TCR profiling, HLA immunopeptidomics, and functional screening to characterize tumor-reactive T cells in the bone marrow of patients with multiple myeloma (MM) and acute myeloid leukemia (AML). These cells are transcriptionally defined by a conserved effector program distinct from the exhausted phenotype of tumor-reactive T cells in solid cancers. Immunopeptidomic profiling reveals a partially shared antigen landscape enriched for noncanonical peptides driving convergent TCR responses. We develop TFiT (tumor-reactive features in T cells), a transcriptional classifier that identifies these cells and stratifies immunotherapy, but not chemotherapy, response across independent MM and AML cohorts, supporting its specificity for T cell-mediated tumor control. These findings reveal a latent but activatable anti-tumor T cell compartment in bone marrow malignancies and provide a framework for engaging endogenous immunity in MM and AML.

A major scientific drive is to characterize the protein-coding genome, which is a primary basis for studying human health. But the fundamental question remains of what has been missed in previous analyses. Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states1–3, with major implications for biomedical science. However, a key gap in knowledge has been which ncORFs produce small microproteins or alternative protein molecules that contribute to the human proteome. Here we report the collaborative efforts of the TransCODE Consortium4 to produce a consensus landscape of protein-level evidence for ncORFs. We show that about 25% of a set of 7,264 ncORFs gives rise to detectable peptides in a large-scale analysis of 95,520 proteomics experiments. We develop an annotation framework for ncORF-encoded microproteins as human proteins and codify the new conceptual model of ‘peptideins’ as microproteins that have indeterminate potential as functional proteins. To probe the biological implications of peptideins, we create an evolutionary analysis approach, termed ORF relative branch length (ORBL), and determine that evolutionary constraint is common and associates with observation of ncORF-derived peptides. We then characterize a pan-essential cellular phenotype for one peptidein from the OLMALINC long non-coding RNA. Overall, we generate public research tools supported by GENCODE and PeptideAtlas and advance biomedical discovery for understudied components of the human proteome. A large-scale proteomics analysis of the dark proteome by the TransCODE Consortium reveals many translated non-canonical open reading frames to encode microproteins and peptideins.

Human leukocyte antigen (HLA)-bound tumor peptides can be routinely isolated from cancer samples and identified using mass spectrometry (MS). However, MS approaches can be stochastic or rely on spectral libraries, which are not customarily available for individual-specific peptides, thus limiting the ability to discover novel peptides. Here, we introduce Pepyrus, which generates user-defined, individual-specific or disease-specific peptide libraries in Escherichia coli to improve the sensitivity and confidence of MS peptide identification, including lowly abundant neoantigens. Using Pepyrus-generated peptide libraries paired with an HLA-specific data-independent acquisition strategy, we recover >75% of the expected sequences per single injection for libraries of >10,000 peptides and identify 0.1 fmol of spiked-in peptides in a complex background. We apply Pepyrus to create personalized libraries, facilitating identification of clinically relevant HLA peptides, including several novel peptides from cell lines derived from persons with melanoma and renal cell carcinoma. Pepyrus enables identification of rare HLA-bound peptides and provides the ability to generate large training datasets to improve spectra, retention time and ion mobility prediction tools.

Clear cell renal cell carcinoma (ccRCC), despite having a low mutational burden, is considered immunogenic because it occasionally undergoes spontaneous regressions and often responds to immunotherapies. The signature lesion in ccRCC is inactivation of the VHL tumor suppressor gene and consequent upregulation of the HIF transcription factor. An earlier case report described a ccRCC patient who was cured by an allogeneic stem cell transplant and later found to have donor-derived T cells that recognized a ccRCC-specific peptide encoded by a HIF-responsive endogenous retrovirus (ERV), ERVE-4. We report that ERVE-4 is one of many ERVs that are induced by HIF, translated into HLA-bound peptides in ccRCCs, and capable of generating antigen-specific T cell responses. Moreover, ERV expression can be induced in non-ccRCC tumors with clinical-grade HIF stabilizers. These findings have implications for leveraging ERVs for cancer immunotherapy.

Over the past 3 decades, the Hunt laboratory has developed advancements in mass spectrometry-based technologies to enable the identification of peptides bound to major histocompatibility complex (MHC) molecules. The MHC class I processing pathway is responsible for presenting these peptides to circulating cytotoxic T cells, allowing them to recognize and eliminate malignant cells, many of which have aberrant signaling. Professor Hunt hypothesized that due to the dysregulation in phosphorylation in cancer that abnormal phosphopeptides could be presented by this pathway, and went on to demonstrate that this was, in fact, the case. Thereafter, the laboratory continued to sequence MHC-associated phosphopeptides and contributed several improved methods for their enrichment, detection, and sequencing. This article summarizes the most recent advancements in identification of modified MHC-associated peptides and includes the cumulative list of phosphopeptides sequenced by the Hunt lab. Further, many other post-translational modifications (PTMs) were found to modify MHC peptides, including O-GlcNAcylation, methylation, and kynurenine; in total, we present here a list of 2450 MHC-associated PTM peptides. Many of these were disease-specific and found across several patients, thus highlighting their potential as cancer immunotherapy targets. We are sharing this list with the field in hopes that it might be used in investigating this potential. Overall, the Hunt lab's contributions have significantly advanced our understanding of antigen presentation and dysregulation of PTMs, supporting modern immunotherapy and vaccine development efforts.

Defining viral proteomes is crucial to understanding viral life cycles and immune recognition but the landscape of translated regions remains unknown for most viruses. We have developed massively parallel ribosome profiling (MPRP) to determine open reading frames (ORFs) across tens of thousands of designed oligonucleotides. MPRP identified 4208 unannotated ORFs in 679 human-associated viral genomes. We found viral peptides originating from detected noncanonical ORFs presented on class-I human leukocyte antigen in infected cells and hundreds of upstream ORFs that likely modulate translation initiation of viral proteins. The discovery of viral ORFs across a wide range of viral families-including highly pathogenic viruses-expands the repertoire of vaccine targets and reveals potential cis-regulatory sequences.

A major scientific drive is to characterize the protein-coding genome as it provides the primary basis for the study of human health. But the fundamental question remains: what has been missed in prior genomic analyses? Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states, with major implications for proteomics, genomics, and clinical science. However, the impact of ncORFs has been limited by the absence of a large-scale understanding of their contribution to the human proteome. Here, we report the collaborative efforts of stakeholders in proteomics, immunopeptidomics, Ribo-seq ORF discovery, and gene annotation, to produce a consensus landscape of protein-level evidence for ncORFs. We show that at least 25% of a set of 7,264 ncORFs give rise to translated gene products, yielding over 3,000 peptides in a pan-proteome analysis encompassing 3.8 billion mass spectra from 95,520 experiments. With these data, we developed an annotation framework for ncORFs and created public tools for researchers through GENCODE and PeptideAtlas. This work will provide a platform to advance ncORF-derived proteins in biomedical discovery and, beyond humans, diverse animals and plants where ncORFs are similarly observed.

T cell-mediated immune surveillance is critical for cancer control, yet its endogenous effectiveness in hematological malignancies remains limited and poorly understood. Here, we integrate single-cell T cell receptor (TCR) profiling, HLA immunopeptidomics and functional antigen mapping to dissect the specificity landscape of bone marrow lymphocytes (BMLs) in multiple myeloma (MM) and acute myeloid leukemia (AML). We identify a rare subset of tumor-reactive T cells that exhibit a stereotyped transcriptional state distinct from bystander and virus-specific populations. Across both malignancies, immunopeptidomic profiling uncovers a partially conserved antigen repertoire enriched for noncanonical peptides, including products of novel or unannotated open reading frames (nuORFs), pseudogenes, and clonotypic immunoglobulin sequences. Several of these epitopes are recurrently presented and associated with convergent TCR responses across individuals. Based on this immune architecture, we develop a TCR-intrinsic fitness model that infers BML tumor specificity from transcriptional cues and stratifies immunotherapy response across three independent patient cohorts. Together, these findings map the latent potential of endogenous anti-tumor immunity in two biologically distinct diseases and provide a framework for decoding and restoring productive immune surveillance of hematological malignancies. Highlights ### Competing Interest Statement M.J.F reports speaker honoraria from Pfizer, Roche and Kerna Ventures and is a consultant for Moonwalk Biosciences. M.P. and E.W.G. are founders of Tcelltech. S.A.C. is a member of the scientific advisory boards of Kymera, PTM BioLabs, Seer and PrognomIQ. S.F. reports consultancy fees from Illumina. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Deutsche José Carreras Leukämie-Stiftung, https://ror.org/00826gz80, 01ZI/2022 Dr. Rolf M. Schwiete Stiftung, https://ror.org/03v7rwj71, 2025-018 Else Kröner-Fresenius-Stiftung, 2025_EKMS.52 Heidelberg University, https://ror.org/038t36y30, ExU 6.1.12

Translation of the noncoding genome in cancer can generate cryptic (noncanonical) peptides capable of presentation by human leukocyte antigen class I (HLA-I); however, the cancer specificity and immunogenicity of noncanonical HLA-I-bound peptides (ncHLAp) are incompletely understood. Using high-resolution immunopeptidomics, we discovered that cryptic peptides are abundant in the pancreatic cancer immunopeptidome. Approximately 30% of ncHLAp exhibited cancer-restricted translation, and a substantial subset were shared among patients. Cancer-restricted ncHLAp displayed robust immunogenic potential in a sensitive ex vivo T cell priming platform. ncHLAp-reactive, T cell receptor-redirected T cells exhibited tumoricidal activity against patient-derived pancreatic cancer organoids. These findings demonstrate that pancreatic cancer harbors cancer-restricted ncHLAp that can be recognized by cytotoxic T cells. Future therapeutic strategies for pancreatic cancer, and potentially other solid tumors, may include targeting cryptic antigens.

The aminopeptidase, endoplasmic reticulum aminopeptidase 1 (ERAP1), trims peptides for loading into major histocompatibility complex class I (MHC class I), and loss of this activity has broad effects on the MHC class I peptidome. Here, we investigated the impact of targeting ERAP1 in immune checkpoint blockade (ICB), as MHC class I interactions mediate both activating and inhibitory functions in antitumor immunity. Loss of ERAP sensitized mouse tumor models to ICB, and this sensitivity depended on CD8+ T cells and natural killer (NK) cells. In vivo suppression screens revealed that Erap1 deletion inactivated the inhibitory NKG2A-HLA-E checkpoint, which requires presentation of a restricted set of invariant epitopes (VL9) on HLA-E. Loss of ERAP altered the HLA-E peptidome, preventing NKG2A engagement. In humans, ERAP1 and ERAP2 showed functional redundancy for the processing and presentation of VL9, and loss of both inactivated the NKG2A checkpoint in cancer cells. Thus, loss of ERAP phenocopies the inhibition of the NKG2A-HLA-E pathway and represents an attractive approach to inhibit this critical checkpoint.

Abstract Translation of the ostensibly non-coding genome in cancer can generate novel peptides capable of presentation by major histocompatibility complex class I (MHC-I, HLA-I in humans). These non-canonical peptide sources can broaden the landscape of potentially targetable antigens in low-to-intermediate mutational burden malignancies, typified by pancreatic ductal adenocarcinoma (PDA). However, non-canonical MHC-I-associated peptides (ncMAPs) have yet to be explored in pancreatic cancer. We purified the malignant compartment from low tumor cellularity PDA specimens using patient-derived organoids (PDOs) and developed a personalized proteogenomics pipeline coupled with high-depth immunopeptidomics to deeply characterize the repertoire of HLA:peptide targets presented specifically on malignant cells. We demonstrate that ncMAPs are abundant on PDA and predominate over mutation-derived neoepitopes in the detectable immunopeptidome. While emerging evidence in other tumor types has implicated dysregulated translation of non-canonical open reading frames (ncORFs), it is currently unknown to what extent these translation products are truly cancer-restricted and how effectively the resulting ncMAPs can elicit a cytolytic T lymphocyte (CTL) response. To investigate the cancer-specificity of non-canonical peptides, we developed a translation-centric analysis pipeline that examines ncORF expression across a range of healthy tissues, including healthy thymus. We provide evidence that ~30% of ncMAPs exhibit cancer-restricted translation patterns, and a substantial subset of these are shared among pancreatic cancer patients with the appropriate HLA. To investigate immunogenicity, we employed a highly sensitive ex vivo platform to prime and expand antigen-specific T cells. We demonstrate that PDA-restricted ncMAPs are highly immunogenic, on par with or even exceeding the immunogenicity observed with mutation-derived peptides or tumor-associated antigens assayed using the same platform. These findings uncover a critical role for dysregulated translation in pancreatic cancer as a potential source for recurrent cancer-restricted epitopes capable of recognition by cytotoxic T cells. We envision that this novel class of antigens will accelerate ongoing efforts to treat pancreatic cancer patients with vaccines and cell-based therapies. Citation Format: Zackery A. Ely, Zachary J. Kulstad, Eva K. Verzani, Jennifer G. Abelin, Sudarsana Addepalli, Karl R. Clauser, Marta Casarrubios, Kevin S. Kapner, Miles P. Agus, Connor J. Hennessey, Sine R. Hadrup, Susan Klaeger, Jennifer Su, Alex M. Jaeger, Brian M. Wolpin, Srivatsan Raghavan, Philip D. Greenberg, Andrew J. Aguirre, Steven A. Carr, Tyler Jacks, William A. Freed-Pastor. Non-canonical MHC class I-associated antigens in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A038.

T cell alloreactivity against minor histocompatibility antigens (mHAgs)-polymorphic peptides resulting from donor-recipient (D-R) disparity at sites of genetic polymorphisms-is at the core of the therapeutic effect of allogeneic hematopoietic cell transplantation (allo-HCT). Despite the crucial role of mHAgs in graft-versus-leukemia (GvL) and graft-versus-host disease (GvHD) reactions, it remains challenging to consistently link patient-specific mHAg repertoires to clinical outcomes. Here we devise an analytic framework to systematically identify mHAgs, including their detection on HLA class I ligandomes and functional verification of their immunogenicity. The method relies on the integration of polymorphism detection by whole-exome sequencing of germline DNA from D-R pairs with organ-specific transcriptional- and proteome-level expression. Application of this pipeline to 220 HLA-matched allo-HCT D-R pairs demonstrated that total and organ-specific mHAg load could independently predict the occurrence of acute GvHD and chronic pulmonary GvHD, respectively, and defined promising GvL targets, confirmed in a validation cohort of 58 D-R pairs, for the prevention or treatment of post-transplant disease recurrence.