Non-small cell lung cancer (NSCLC) has poor survival even with modern checkpoint inhibitor therapies. Personalised vaccines based on short peptide neoantigens containing tumour mutations are an attractive precision medicine strategy, but identifying therapeutically relevant neoantigens remains challenging, with existing methods yielding positive responses in only 6% of candidates tested. We developed an immunopeptidomics approach to improve neoantigen identification in 24 NSCLC patients (15 adenocarcinoma, 9 squamous cell carcinoma). We directly identified one neoantigen and using whole exome sequencing, transcriptomics and mass spectrometry-based immunopeptidomics, we filtered predicted neoantigens based on observed cohort HLA peptide presentation. This approach achieved positive functional responses in 5 of 6 patients tested (83% success rate) with 13% of putative neoantigens (9 out of 70) eliciting strong responses. Bayesian modelling of our initial rules-based neoantigen selection further revealed patient specific peptide presentation patterns and propensities. Our findings demonstrate that incorporating donor-specific HLA peptide presentation data substantially improves neoantigen identification success rates and immune response specificity, advancing personalised cancer vaccine development.

2082 Background: CD4⁺ T cell help is central to durable anti-tumour immunity and predicts response to neoantigen vaccination. Antigen presentation to CD4⁺ T cells is governed by HLA class II molecules, yet the class II landscape in glioma remains poorly defined. Germline HLA diversity can influence immunity only if tumours retain antigen-presentation capacity. We hypothesised that the prognostic impact of HLA class II heterozygosity in glioma is conditional on tumour MHC-II expression. Methods: We performed germline HLA class II typing (DRB1, DQB1, DPB1) in 893 TCGA gliomas (388 glioblastoma, 505 lower-grade glioma) using a weighted consensus of four algorithms (HLA-HD, hla-genotyper, SOAP-HLA, Kourami). Tumour MHC-II expression was quantified using a nine-gene antigen-presentation signature. The primary analysis tested the interaction between germline heterozygosity and tumour MHC-II status on overall survival using multivariable Cox regression. Allele-level survival analyses were exploratory. Results: Gliomas showed extensive class II polymorphism (54 DRB1, 28 DQB1, 47 DPB1 alleles), with 35.9% of patients homozygous at one or more loci. The most frequent alleles were DRB107:01 (12.7%), DQB103:01 (20.7%), and DPB1*04:01 (38.7%). Germline heterozygosity was not prognostic in unstratified analyses (HR 1.04, 95% CI 0.83–1.32, p = 0.72). However, a significant interaction with tumour MHC-II expression was observed (p-interaction = 0.044). In MHC-II–low tumours, germline heterozygosity independently predicted improved survival (HR 0.71, 95% CI 0.52–0.97, p = 0.031), whereas no effect was seen in MHC-II–high tumours (p = 0.89). Tumour MHC-II expression itself was independently prognostic (p = 0.004). Exploratory allele-level analysis identified DRB1*03:01 (10.2%) as nominally associated with worse survival (median 23.7 vs 34.9 months, uncorrected p = 0.041). Conclusions: Germline HLA class II diversity influences survival in glioma only when tumour antigen presentation is impaired. Combined tumour MHC-II loss and limited germline diversity identify a biologically and clinically high-risk group with compounded antigen-presentation deficits. These findings have direct implications for neoantigen vaccine design and patient stratification, suggesting that restoration of antigen presentation may be required prior to immunotherapy in selected patients. The adverse association with DRB1*03:01 merits mechanistic investigation. Independent validation in 322 glioblastoma patients is ongoing.

Improving responses to cancer immunotherapies requires deeper insight into the cellular mechanisms governing T cell-mediated anti-tumor immunity. TMEM33 is an endoplasmic reticulum-resident transmembrane protein enriched across multiple tumor types, with reported functions in anti-viral immunity as well as calcium and lipid homeostasis, yet its role in tumor immunosurveillance remains unknown. Using murine genetic models, we demonstrate that host TMEM33 constrains anti-tumor CD8+ T cell responses. Constitutive Tmem33 -/- mice exhibited delayed melanoma tumor growth and increased CD8+ T cell infiltration. Antigen-specific CD8+ compartments in tumors of Tmem33 -/- mice showed TCF-1+PD-1+ progenitor-exhausted cell (Tpex) enrichment, elevated effector function and reduced exhaustion, alongside improved effector memory expansion and T-bet expression in draining lymph nodes. We highlight that TMEM33 functions intrinsically within the T cell compartment, as TMEM33 deletion (1) enhanced polyclonal activation of naive CD8+ T cells ex vivo, (2) promoted preferential Tpex accumulation among adoptively transferred naive OT-I cells in B16F10-OVA tumors and draining lymph nodes, and (3) improved the potency of ex vivo-expanded OT-I cells in controlling tumor growth during adoptive cell therapy. Finally, in a large, prospectively recruited metastatic melanoma cohort, lower TMEM33 expression in patient CD8+ T cells significantly correlated with improved survival and elevated TCF-7 (encoding TCF-1). Collectively, our findings define TMEM33 as a formerly unrecognized intrinsic determinant of tumor-directed CD8+ T cell fate that limits Tpex maintenance, and restrains cell therapy responses, suggesting that its modulation may strengthen immunotherapeutic efficacy.

The killer-cell immunoglobulin-like receptors (KIR) are a family of activating and inhibitory HLA class I (HLA-I) binding receptors expressed on natural killer (NK) cells and subsets of T cells. The KIR detect HLA-I molecules in a peptide-dependent manner, with some KIR displaying exquisite peptide-specificity. Studying peptide recognition by KIR often uses TAP-deficient cell lines expressing single HLA-I alleles, which are heterogenous and time consuming to generate. Here, we established an alternative approach using peptide-exchange technologies hitherto developed for studying T cell recognition of HLA-I. We tested two methods; dipeptide-mediated peptide exchange and ‘open-HLA-I’, HLA-I molecules consisting of heavy chain-β 2 m disulphide bonded dimers. We combined peptide-exchange technologies with SpyTag-SpyCatcher chemistry to allow rapid detection of KIR binding via HLA-I displayed on plates or cells. We demonstrated the fidelity of this system with peptides of known KIR specificity bound to HLA-C*05:01. We then screened a peptide library to identify novel strong KIR2DS4 binding peptides presented by HLA-C*04:01. Peptide-exchanged HLA-C was functionally competent, promoting activation of KIR2DS4+ NK cells and inhibiting activation of KIR2DL1+ NK cells. Together, we show that peptide-exchangeable HLA-I molecules are ligands for KIR, presenting a flexible, efficient system for examining the peptide-sequence dependent recognition of HLA-I by KIR.

2654 Background: Personalised neoantigen vaccines induce detectable CD8 + T cell responses for fewer than one-third of selected peptides. Current pipelines prioritise candidates by predicted immunogenicity and select peptides independently, overlooking two constraints: efficacy depends on the peptide set as a whole, and tumours adapt antigen processing under immune pressure. We developed a framework that optimises peptide combinations for resilience to tumour escape and tested whether escape-resilience predicts clinical immunogenicity. Methods: We modelled peptide susceptibility to five antigen-processing escape mechanisms: TAP downregulation, immunoproteasome-to-constitutive proteasome switching, aminopeptidase upregulation, tapasin loss, and HLA loss of heterozygosity. Selection was formulated as a minimax optimisation, maximising predicted efficacy under worst-case tumour adaptation. We analysed five neoantigen vaccine trials with per-epitope CD8 + T cell response data, consisting of 571 neoantigens, 3,806 peptides, and 174 patients. Mixed-effects models tested associations between escape-resilience and immunogenicity, adjusting for binding affinity (NetMHCpan-4.1 %rank), pMHC stability (NetMHCstabpan), mutation type, and clonality. Six hypotheses were pre-registered with Bonferroni correction. Results: Escape-resilience predicted immunogenicity independently of established features. After adjustment, vulnerability to TAP loss (OR 0.42 per SD, 95% CI 0.24–0.71, p=0.0018) and proteasome switching (OR 0.54 per SD, 95% CI 0.34–0.86, p=0.0089) were associated with failure to elicit CD8 + responses. Among peptides with comparable predicted binding affinity, escape-resilient peptides were significantly more likely to be immunogenic. Composite escape-resilience scores discriminated immunogenic from non-immunogenic peptides (AUC 0.71, 95% CI 0.66–0.76), outperforming binding affinity alone (AUC 0.58) and an affinity-stability model (AUC 0.64). Adding escape-resilience improved discrimination (ΔAUC 0.07, p=0.003). Retrospective re-ranking altered 38% (95% CI 31–45%) of vaccine compositions, replacing high-affinity but escape-vulnerable peptides with lower-affinity, processing-robust alternatives. Associations were stronger for truncal mutations (OR 2.8, 95% CI 1.6–4.9) than subclonal mutations (OR 1.4, 95% CI 0.8–2.4; interaction p=0.041), indicating that processing robustness is most consequential for clonally dominant neoantigens. Conclusions: Optimising neoantigen selection against tumour escape identifies peptides more likely to elicit CD8 + T cell responses, independent of binding affinity and stability. The stronger effects in truncal mutations suggest escape-aware ranking may be particularly valuable for durable, clone-targeted vaccination strategies. Prospective trials are needed to assess clinical impact.

Introduction:The killer-cell immunoglobulin-like receptors (KIR) are a family of activating and inhibitory Class I human leukocyte antigen (HLA-I) binding receptors expressed on natural killer (NK) cells and subsets of T cells. The KIR detect HLA-I molecules in a peptide-dependent manner, with some KIR displaying exquisite peptide specificity. Studying peptide recognition by KIR often uses TAP-deficient cell lines expressing single HLA-I alleles, which are heterogenous and time consuming to generate. Here, we established an alternative approach using peptide-exchange technologies hitherto developed for studying T cell recognition of HLA-I. Methods:We tested two methods; dipeptide-mediated peptide exchange and "open-HLA-I", HLA-I molecules consisting of heavy chain-β2m disulphide bonded dimers. We combined peptide-exchange technologies with SpyTag-SpyCatcher chemistry to allow rapid detection of KIR binding via HLA-I displayed on plates or cells. Results:We demonstrated the fidelity of this system with peptides of known KIR specificity bound to HLA-C*05:01. We then screened a peptide library to identify novel strong KIR2DS4 binding peptides presented by HLA-C*04:01. Peptide-exchanged HLA-C was functionally competent, promoting activation of KIR2DS4+ NK cells and inhibiting activation of KIR2DL1+ NK cells. Conclusion:Together, we show that peptide-exchangeable HLA-I molecules are ligands for KIR, presenting a flexible, efficient system for examining the peptide sequence dependent recognition of HLA-I by KIR.

2653 Background: Personalized neoantigen vaccines aim to match epitopes to a patient's HLA genotype, yet many peptides bind promiscuously across multiple HLA alleles. Promiscuous binding may reflect intrinsic peptide properties, including enhanced processing efficiency and structural stability, that drive immunogenicity independent of any single HLA match. We hypothesized that peptides with broader HLA binding profiles would show higher clinical immunogenicity, even when restricted to the patient's own HLA alleles. Methods: We curated 17 neoantigen vaccine trials (174 patients) and selected five with per-epitope CD8⁺ T cell immunogenicity data for primary analysis. Across 571 neoantigen sequences (3,806 derived peptides), we predicted binding and presentation using NetMHCpan-4.2, MHCflurry 2.0, and PRIME-2.0, and estimated peptide–MHC stability with NetMHCstabpan. A reference panel of 62 common HLA class I alleles (>95% global coverage) was used to quantify incidental coverage, defined as predicted binding (IC50 <500 nM) to non-patient HLA alleles. Mixed-effects models adjusted for patient-specific binding affinity, predicted stability, mutation type, neoantigen length, and trial structure, with Bonferroni correction for 5 pre-specified hypotheses. Results: Off-target HLA binding independently predicted clinical immunogenicity. Among peptides selected for vaccination, 35.2% bound at least one non-patient HLA allele, with the most promiscuous peptides binding up to 37 alleles. After adjusting for patient-specific binding affinity and stability, peptides from immunogenic neoantigens showed greater incidental coverage than non-immunogenic peptides (mean 2.46 vs 1.80 additional HLAs; p=0.0034). This association was consistent across tumour types and prediction methods. Among immunogenic peptides, the breadth of off-target binding correlated with response magnitude (Spearman ρ=0.46, p=0.034), with the strongest effect observed in glioblastoma. High-affinity off-target matches (IC50 <50 nM) and high-stability interactions showed the most robust associations with immunogenicity. Conclusions: Promiscuous HLA binding independently predicts neoantigen immunogenicity in clinical trials, beyond binding affinity and stability to the patient’s own HLA alleles. These results point to peptide-intrinsic properties linked to MHC stability and processing that are overlooked by current selection pipelines. Explicit modelling of HLA promiscuity and stability may improve neoantigen prioritisation, particularly in low–mutation burden tumours and patients with rare HLA genotypes.

Non-small cell lung cancer (NSCLC) is frequently diagnosed late and has poor survival. The two predominant subtypes of NSCLC, adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC), are currently differentially diagnosed using immunohistochemical markers; however, they are increasingly recognized as very different cancer types suggestive of potential for new, more targeted therapies. There are extensive efforts to find more precise and noninvasive differential diagnostic tools. Here, we examined these two NSCLC subtypes for differences that may inform treatment and identify potential novel therapeutic pathways. We presented a comparative analysis of transcriptomic and proteomic expression in tumors from a cohort of 22 NSCLC patients: 8 LUSC and 14 LUAD. Comparing NSCLC subtypes, we found differential gene expression related to cell differentiation for LUSC and cellular structure and immune response regulation for LUAD. Differential protein expression between NSCLC subtypes was related to extracellular structure for LUSC and metabolic processes, including glucose metabolism for LUAD. This direct comparison was more informative about subtype-specific pathways than between each subtype and control (nontumor) tissues. Many of our observations between NSCLC subtypes support and inform existing observations and reveal differences that may aid research seeking to identify and validate novel subtype biomarkers or druggable targets.

Tumor promoting inflammation and the ability to evade immune destruction are two of the hallmarks of cancer, but there is limited prospective evidence for the role of specific inflammation and immune-related pathways and protein markers in the development of prostate cancer. This study used data on the circulating levels of 368 inflammation and immune-related proteins (Olink Explore Inflammation I panel) from a nested case-control design within the European Prospective Investigation into Cancer and Nutrition (EPIC), including 1, 434 men who developed prostate cancer and 1, 434 matched controls - 488 of the cases also had clinically aggressive disease. Conditional logistic regression was used to estimate protein associations with cancer risk in EPIC, per standard deviation and the effective number of tests (ENT) was used to control for multiple testing. We additionally performed a fixed-effect meta-analysis combining associations for these proteins estimated in EPIC with those in 21, 481 men from UK Biobank (UKBB), of whom 1, 147 developed prostate cancer. Logistic regression was used to estimate exome protein score association with cancer risk in UKBB and replication analyses were performed in the Multiethnic Cohort (MEC). Mendelian randomisation and colocalisation analyses using data on 79, 148 overall prostate cancer cases from the PRACTICAL consortium were also conducted to aid triangulation. In EPIC, no inflammatory proteins were associated with prostate cancer risk overall or with more clinically aggressive subtypes after correction for multiple testing. In the EPIC+UKBB meta-analyses, FLT3LG and CNTNAP2 were significantly associated with overall prostate cancer risk (RRMeta: 0.88, 95% CI: 0.84-0.92 and RRMeta: 1.10, 95% CI: 1.05-1.16, respectively), and in the analyses stratified by time to diagnosis, IL15 was associated with risk of prostate cancer diagnosed more than seven years after blood draw (RRMeta: 0.86, 95% CI: 0.81-0.93), along with five other proteins (FLT3LG, BCL2L11, PGF, CKAP4, and TNFRSF11A). Exome protein scores in UKBB identified PARP1 as associated with an increased risk of prostate cancer (OR: 1.05, 95% CI: 1.02-1.07), which replicated in MEC (OR: 1.04, 95% CI: 1.01-1.08). MR and colocalisation analyses also identified SPINT2 (OR: 2.11, 95% CI: 1.70-2.63) and NME3 (OR: 1.25, 95% CI: 1.11-1.41) as being associated with an increased risk of prostate cancer overall. Our findings suggest higher levels of proteins involved in immunosurveillance pathways, including the recruitment and activation of natural killer and T cells, may be linked to a lower risk of developing prostate cancer, while PARP1 inhibition may be relevant for prostate cancer prevention. Mahboubeh Parsaeian, Wing Ching Chan, Joshua Atkins, Keren Papier, Trishna Desai, Zhe Huang, David Conti, David Bogumil, Jiayi Shen, Malcolm Sim, Konstantinos Tsilidis, James Yarmolinsky, Sabina Rinaldi, Rudolf Kaaks, Verena Katzke, Matthias Schulze, Catarina Schiborn, Saverio Caini, Lorenzo Milani, Raul Zamora-Ros, Marcela Guevara, Maria-Jose Sánchez, María-Dolores Chirlaque, Pilar Amiano, The PRACTICAL Consortium, Timothy Elliott, Ian Mills, Elio Riboli, Loïc Le Marchand, Christopher Haiman, Tim Key, Karl Smith-Byrne, Ruth Travis. Prospective and genetic analyses implicate lower immunosurveillance in the aetiology of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2268.

Peptide binding to major histocompatibility complex class I molecules (MHC-I) and their presentation to cytotoxic immune cells is a keystone of the adaptive immune system. The selection of MHC-I bound peptides is facilitated by the chaperone tapasin, which allows MHC-I to iteratively sample peptides until they are loaded with optimal binding peptides, known as peptide editing. However, some MHC-I allotypes can select high affinity binding peptides independently of tapasin, and the molecular mechanism(s) for such peptide editing are unknown. Here, we used enhanced sampling molecular dynamics simulations of peptide-deficient MHC-I to investigate tapasin-independent peptide editing. Our simulations revealed transient disruption of hydrogen bonds between MHC-I and the peptide backbone could allow for peptide editing, a process we term "active displacement". Destabilisation of interactions with the peptide backbone, necessitates sequence-specific sidechain interactions to maintain peptide binding. Our active displacement model predicts surface expression levels for multiple MHC-I allotypes and accounts for the presentation of an immunogenic mutant KRAS-G12D neoepitope by HLA-C*08:02, but not by closely related HLA-C*05:01. Together our data provide a molecular mechanism for tapasin-independent MHC-I peptide editing, influencing the surface immunopeptidome and anti-tumour immunity. Significance Statement Major histocompatibility complex class I molecules (MHC-I) bind and present peptides to specialised killer cells of the immune system. These immune cells can unleash their cytotoxic effector functions if they recognise the peptide-MHC-I complex. Which peptides are presented by MHC-I is therefore highly important. Peptide selection is usually assisted by the tapasin protein, although some MHC-I molecules can select peptides independently of tapasin, but it is not known how this occurs. Here, we provide an atomistic description of the tapasin-independent peptide selection mechanism. Our mechanism applies to multiple MHC-I allotypes and illustrates how an immunogenic peptide is presented by one MHC-I molecule, but not by another closely related molecule. This new insight provides a rational basis for therapeutic treatments. ### Competing Interest Statement The authors have declared no competing interest. The datasets presented in this study are available upon request and will be made available in online repositories following peer review. Cancer Research UK, https://ror.org/054225q67, A28279

Pancreatic ductal adenocarcinoma has a dismal prognosis. A comprehensive analysis of single-cell multi-omic data from matched tumour-infiltrated CD45+ cells and peripheral blood in 12 patients, and two published datasets, reveals a complex immune infiltrate. Patients have either a myeloid-enriched or adaptive-enriched tumour microenvironment. Adaptive immune cell-enriched is intrinsically linked with highly distinct B and T cell clonal selection, diversification, and differentiation. Using TCR data, we see the largest clonal expansions in CD8 effector memory, senescent cells, and highly activated regulatory T cells which are induced within the tumour from naïve cells. We identify pathways that potentially lead to a suppressive microenvironment, including investigational targets TIGIT/PVR and SIRPA/CD47. Analysis of patients from the APACT clinical trial shows that myeloid enrichment had a shorter overall survival compared to those with adaptive cell enrichment. Strategies for rationale therapeutic development in this disease include boosting of B cell responses, targeting immunosuppressive macrophages, and specific Treg cell depletion approaches. Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis involving evasion of immune control. Here, the authors perform a comprehensive analysis of single-cell multi-omic data revealing either a myeloid-enriched or adaptive-enriched tumour microenvironment, linked to distinct B and T cell clonal selection and differentiation, distinct overall survival, and potential therapeutic approaches.

Introduction:Antigen processing and presentation are vital processes of the adaptive immunity. These processes involve a series of intracellular and extracellular events, including the enzymology within cells during antigen processing, the loading and presentation of antigenic peptides on major histocompatibility complexes, the recruitment of T cells, their interaction with antigen-presenting cells, and the expression of adhesion, co-stimulatory and co-inhibitory molecules at the T cell immunological synapse. These events collectively fine-tune and sustain antigen recognition and T cell function. Dysregulation of this machinery can profoundly impact the efficacy of cancer immunotherapy. Imaging technologies have emerged as powerful tools for elucidating the mechanisms underlying antigen processing and presentation. By providing complementary perspectives into the cellular and molecular interactions at play, imaging has significantly enhanced our understanding of these complex immunological events in cancer. Such insights can improve the monitoring of immunotherapy responses, facilitate the identification of effective treatments, and aid in predicting patient outcomes. Methods:This review explores the role of imaging in studying antigen processing and presentation in the context of cancer. Conclusion:It highlights key considerations for developing imaging tools and biomarkers to detect components of these pathways. Additionally, it examines the strengths and limitations of various imaging approaches and discusses their potential for clinical translation.

Major Histocompatibility Complex class I (MHC-I) molecules bind and present peptides to cytotoxic T cells, protecting against pathogens and cancer. MHC-I is highly polymorphic and each allotype is promiscuous, and capable of binding a unique and diverse repertoire of peptide ligands. The peptide editing chaperone tapasin optimizes this allotype specific repertoire of peptides, resulting in the selection of high affinity peptides. MHC-I allotypes differ in the extent they engage tapasin. This suggests that tapasin-dependent MHC-I allotypes should present a less diverse repertoire that is enriched in higher-affinity peptides, and which are present in higher abundance, than tapasin independent MHC-I allotypes, which should present a broader repertoire containing peptides with a lower average affinity. Experimental verification of this hypothesis has been confounded by the different peptide binding specificities of MHC-I allotypes. Here, we independently investigated the peptide focusing function of tapasin by introducing a point mutation into a tapasin independent MHC-I allotype that dramatically increased its tapasin dependence without substantially altering its peptide binding specificity. This allowed us to demonstrate ligand focusing by tapasin at both the repertoire level in cellulo, and by using an in vitro system in which tapasin was artificially tethered to MHC-I, at the individual peptide level. We found that tapasin had a greater influence on tapasin dependent MHC-I molecules, and that tapasin modulated peptide selection according to peptide-MHC-I complex stability, disfavoring short-lived peptide-MHC-I complexes. Thus, tapasin dependent MHC-I molecules experience greater tapasin filtering, resulting in less diverse MHC-I immunopeptidomes that are enriched in high affinity peptide-MHC-I complexes.

Oesophageal adenocarcinoma (OAC) is the 7th most common cancer in the United Kingdom (UK) and remains a significant health challenge. This study presents a proteomic analysis of seven OAC donors complementing our previous neoantigen identification study of their human leukocyte antigen (HLA) immunopeptidomes. Our small UK cohort were selected from donors undergoing treatment for OAC. We used label-free mass spectrometry proteomics to compare OAC tumour tissue to matched normal adjacent tissue (NAT) to quantify expression of 3552 proteins. We identified differential expression of a number of proteins previously linked to OAC and other cancers including common markers of tumourigenesis and immunohistological markers, as well as enrichment of processes and pathways relating to RNA processing and the immune system. Our findings also offer insight into the role of the protein stability in the generation of an OAC neoantigen we previously identified. These results provide independent corroboration of existing oesophageal adenocarcinoma biomarker studies that may inform future diagnostic and therapeutic research.

ObjectiveTo understand the CD8+ tumour infiltrating lymphocyte (TIL) compartment of oesophageal adenocarcinoma (OAC) with regards to markers of lymphocyte exhaustion, tissue residency and to identify possible reasons behind differential responses to therapy.DesignTumour samples from 44 patients undergoing curative resection for OAC were assessed by flow cytometry for presence of antigen-experienced TILs and markers of activation and exhaustion. Populations of PD-1 and CD39 positive OAC TILs were sorted, and bulk RNA sequencing undertaken using a modified SmartSeq2 protocol. Flow cytometric assessment of functionality was completed.ResultsA higher proportion of antigen experienced CD8+ OAC TILs was associated with improved survival following surgery; while, high double positivity (DP) for PD-1 and CD39 among these TILs also correlated significantly with outcome. These DP TILs possess a minority population which is positive for the markers of exhaustion TIM3 and LAG3. Transcriptomic assessment of the PD-1 and CD39 DP TILs demonstrated enrichment for a tissue resident memory T lymphocyte (TRM) phenotype associated with improved survival in other cancers, reinforced by positivity for the canonical TRM marker CD103 by flow cytometry. This population demonstrated maintained functional capacity both in their transcriptomic profile, and on flow cytometric assessment, as well as preserved proliferative capacity.ConclusionResected OAC are variably infiltrated by PD-1 and CD39 DP TILs, an abundance of which among lymphocytes is associated with improved survival. This DP population has an increased, but still modest, frequency of TIM3 and LAG3 positivity compared to DN, and is in keeping with a functionally competent TRM phenotype.

Abstract Background The development of novel treatments for oesophageal adenocarcinoma (OAC) represents an area of significant unmet need. Despite the application of immune checkpoint blockade alongside chemotherapy as standard of care for some patients with metastatic OAC, responses to immunotherapies are modest and 5-year survival is 17%. The reasons for this remains unclear and while tumour infiltration with CD8+ T lymphocytes (TILs) at the time of resection is associated with improved outcomes, this heterogeneous population remains incompletely characterised in OAC. Tissue resident memory cells (TRM) are increasingly understood as key mediators of immunotherapy response but their role in OAC is poorly understood. Methods Tumour samples were accessed by punch biopsy from the central portion of cancers resected from 44 patients undergoing curative surgery for OAC. Samples were assessed by multi-parametric flow cytometry for the presence of antigen-experienced TILs and markers of activation and exhaustion. Populations of antigen-experienced PD-1 and CD39 positive CD8+ OAC TILs were sorted, and bulk RNA sequencing (RNAseq) undertaken using a modified SmartSeq2 protocol allowing differential gene expression analysis and gene set enrichment analysis. Flow cytometric assessment of functionality was completed. Results Resected OAC are often highly infiltrated with antigen experienced CD8+ TILs expressing high levels of PD-1 and CD39 and an abundance of this population correlates with improved survival (%PD-1/CD39+ >Median, OS HR 0.12). RNAseq of sorted TILs identified this PD-1+ and CD39+ lymphocyte population as enriched for precursor exhausted-like CD8+ TRM (NES 2.01 p=0.00). This is correlated by their CD103+ and TIM3- predominance on flow cytometry in keeping with TRMsassociated with immunotherapy response in other cancers. This population demonstrated a maintained proliferative potential, an ability to degranulate and produce the key effector molecules IFN-γ, TNF-α and Granzyme B. Conclusions An abundance of a PD-1 and CD39 positive CD8+ TIL population, consistent with a precursor exhausted-like TRM population with maintained proliferative and functional capacity, is observed to correlate with improved outcomes following curative surgery for OAC. This TRM population is consistent with analogous populations observed to be responsible for immunotherapy response in other cancers and their identification may predict benefit from such approaches in OAC and allow better patient selection for treatment with immune checkpoint inhibitors.

Non-small cell lung cancer (NSCLC) has poor survival even for those receiving modern checkpoint inhibitor therapies. Personalised vaccines based on short peptide neoantigens containing tumour mutations, presented to cytotoxic T-cells by human leukocyte antigen (HLA) molecules, are an attractive strategy. However, identifying therapeutically relevant neoantigens is challenging, with existing methods yielding positive responses in only 6% of candidates tested, and neoantigen-based vaccines in melanoma, glioblastoma and pancreatic cancer producing an immune responses in about 50% of patients. Here we report a proteogenomics approach to identify neoantigens in tumours from a cohort of 24 NSCLC patients: 15 adenocarcinoma, 9 squamous cell carcinoma. We characterised the mutational and HLA immunopeptide landscapes of NSCLC using whole exome sequencing, transcriptomics and mass spectrometry immunopeptidomics. We directly identified one neoantigen, and additional predicted neoantigens were generated using an existing in silico neoantigen prediction workflow. Using the immunopeptidomes to filter for candidate predicted neoantigens we identified positive functional assay responses for 5 out of the 6 patients we tested, with an overall success rate of 13%, inclusive of the directly observed neoantigen. Finally, for one patient using scRNAseq we identified a CD8+ effector T-cell clonotype expanded only in response to the putative class I HLA neoantigen. These results represent an improvement in both the quantity of neoantigens identified and the specificity of immune responses to neoantigens, utilising knowledge of the HLA peptides presented on a tumour. Thus immunopeptidomics has the potential to improve the efficacy of neoantigen based personalised cancer vaccine workflows.

The most effective responses to intracellular pathogens have a breadth of T-cell clones with different affinities for their cognate peptide, and a diversity of functional phenotypes, from effector to long-lived memory cells. While high- and low-affinity T-cells are inherently skewed towards becoming effector and memory, respectively, overall, both functional subsets exploit a wide range of affinities. How the breadth of affinities and functionalities are coordinated is therefore unclear. In this study, we provide evidence that direct sensing of the cytokine IFN-γ by CD8 + T-cells is a factor controlling the integration of T-cell affinity and differentiation during infection. IFN-γ increases the expansion of low-affinity T-cells, allowing them to overcome the selective advantage of high-affinity T-cells. Concomitantly, IFN-γ reinforces high-affinity T-cell entry into the memory pool. As a result, direct IFN-γ sensing by CD8 + T-cells increases the avidity of the memory response. This comes at the expense of the primary T-cell response, for which IFN-γ decreases the avidity, leading to sub-optimum immunity to infection. IFN-γ sensing by CD8 + T-cells is paracrine, provided by a distinct subset of CD8 + T-cells called Virtual Memory T-cells, an antigen inexperienced subset that harbors memory features. Overall, we propose that IFN-γ and Virtual Memory T-cells fulfil a critical immunoregulatory role by enabling the coordination of T-cell avidity and fate.