Presentation Of Peptides Research Articles

MHC-II dynamics are maintained in HLA-DR allotypes to ensure catalyzed peptide exchange

Presentation of antigenic peptides by major histocompatibility complex class II (MHC-II) proteins determines T helper cell reactivity. The MHC-II genetic locus displays a large degree of allelic polymorphism influencing the peptide repertoire presented by the resulting MHC-II protein allotypes. During antigen processing, the human leukocyte antigen (HLA) molecule HLA-DM (DM) encounters these distinct allotypes and catalyzes exchange of the placeholder peptide CLIP by exploiting dynamic features of MHC-II. Here, we investigate 12 highly abundant CLIP-bound HLA-DRB1 allotypes and correlate dynamics to catalysis by DM. Despite large differences in thermodynamic stability, peptide exchange rates fall into a target range that maintains DM responsiveness. A DM-susceptible conformation is conserved in MHC-II molecules, and allosteric coupling between polymorphic sites affects dynamic states that influence DM catalysis. As exemplified for rheumatoid arthritis, we postulate that intrinsic dynamic features of peptide–MHC-II complexes contribute to the association of individual MHC-II allotypes with autoimmune disease.

Effects of HLA single chain trimer design on peptide presentation and stability.

MHC class I "single-chain trimer" molecules, coupling MHC heavy chain, β2-microglobulin, and a specific peptide into a single polypeptide chain, are widely used in research. To more fully understand caveats associated with this design that may affect its use for basic and translational studies, we evaluated a set of engineered single-chain trimers with combinations of stabilizing mutations across eight different classical and non-classical human class I alleles with 44 different peptides, including a novel human/murine chimeric design. While, overall, single-chain trimers accurately recapitulate native molecules, care was needed in selecting designs for studying peptides longer or shorter than 9-mers, as single-chain trimer design could affect peptide conformation. In the process, we observed that predictions of peptide binding were often discordant with experiment and that yields and stabilities varied widely with construct design. We also developed novel reagents to improve the crystallizability of these proteins and confirmed novel modes of peptide presentation.

Inhibited MHC Class I and MHC Class II antigen processing and presentation upon Sars-CoV-2 infection

Abstract Major histocompatibility (MHC) bound viral peptides are the immune signatures to initiate the selective and cross-reactive T cell responses. Identifying the antigenic targets of adaptive immunity to SARS-CoV-2 is a key towards understanding the pathogenesis of Sars-CoV-2 and the rapidly evolving mutants. We isolated the immunopeptidomes to identify naturally processed and presented MHC-II (HLA-DR, HLA-DP) and MHC-I (HLA-ABC) bound canonical and out-of-frame viral peptides. We identified 5 HLA-DR canonical epitopes (13 peptides) from spike protein, 2 HLA-DP canonical epitopes (4 peptides) from spike and membrane proteins, 1 canonical MHC-Class I peptides and 5 out-of-frame MHC-I peptides from different ORF proteins. The viral peptides were presented in lower abundances as compared to host peptides obscuring the identification of the MHC bound viral peptides. Some of these peptides were shown to recall T cell responses in COVID-19 donors in other reported studies. We observed the downregulation of surface and total HLA-ABC, HLA-DR and HLA-DP expression upon Sars-CoV-2 infection possibly restricting the presentation of viral peptides and therefore delaying the immune response. The mechanisms by which SARS-CoV-2 evades adaptive immune responses mediated by MHC-I are studied in detail by different groups. Hence, we further investigating the modulators of the MHC-II pathway using proteomics-based approach. We observed significant downregulation of CD74 protein, CIITA protein and cathepsins in infected cells affecting the processing and presentation of viral peptides. These results may help in understanding significantly altered antigen presentation in Sars-CoV-2 infected cells.

Endo-lysosomal assembly variations among Human Leukocyte Antigen class I (HLA-I) allotypes

Abstract The extreme polymorphisms of HLA-I proteins enable the presentation of diverse peptides to cytotoxic T lymphocytes (CTL). The canonical endoplasmic reticulum (ER) HLA-I assembly pathway enables presentation of cytosolic peptides, but effective intracellular surveillance requires multi-compartmental antigen sampling. Endo-lysosomes are generally sites of HLA class II assembly, but human monocytes and monocyte-derived dendritic cells (moDCs) also contain significant reserves of endo-lysosomal HLA-I molecules. We hypothesized variable influences of HLA-I polymorphisms upon outcomes of endo-lysosomal trafficking, as the stabilities and peptide occupancies of cell surface HLA-I are variable. Consistent with this model, when the endo-lysosomal pH of moDCs is disrupted, HLA-B allotypes display varying propensities for reductions in surface expression, with HLA-B* 08:01 or HLA-B*35:01 being among the most resistant or sensitive respectively, among eight tested HLA-B allotypes. Perturbations of moDC endo-lysosomal pH result in redistribution of HLA-B*35:01, but not HLA-B* 08:01, to LAMP1 +compartments and increase HLA-B*35:01 peptide receptivity. These findings reveal the intersection of the vacuolar cross-presentation pathway with a constitutive assembly pathway for some HLA-B allotypes. Notably, cross-presentation of epitopes derived from two soluble antigens was also more efficient for B*35:01 compared to B*08:01, even when matched for T cell response sensitivity, and more affected by cathepsin inhibition. Thus, HLA-I polymorphisms dictate the degree of endo-lysosomal assembly, which can supplement ER assembly for constitutive HLA-I expression and increase the efficiency of cross-presentation. Supported by grants NIH R01 AI044115 T32GM008353 and T32AI007413

Expanded presentation of Lyme autoantigens and identification of a novel immunogenic CD4+ T cell epitope from Borrelia burgdorferiMCP4 in murine Lyme arthritis

Abstract Lyme arthritis (LA), caused by Borrelia burgdorferi (Bb), is often accompanied by autoimmune T and B cell responses, but the mechanisms of infection-induced autoimmunity are unclear. We used an immunopeptidomics approach to identify potential Lyme autoantigens and immunogenic Bb antigens, which were validated by histology and testing of T cell reactivity. C57BL/6 (B6) mice, which develop mild inflammatory LA, and B6 Il10−/− (IL10 KO) mice, which develop severe, persistent LA, were infected with Bb for 4 or 16 weeks, and MHCII-bound peptides from inguinal and popliteal lymph nodes were identified by LC-MS/MS. Joint inflammation, fibrosis, and vascular remodeling were analyzed by H&E, Masson’s trichrome, and anti-CD31 immunohistochemistry, respectively. Six Bb peptides were identified by LC-MS/MS, of which one epitope from methyl-accepting chemotaxis protein 4 (MCP4) was an immunogenic CD4+ T cell antigen. Over 10,000 self peptides, particularly from proteins involved in cholesterol metabolism, tissue damage, and vascular inflammation were identified in infected mice. Presentation of peptides from previously identified human Lyme autoantigens apolipoprotein B-100, fibronectin, and type V collagen were expanded in infected mice, suggestive of epitope spreading. Consistent with immunopeptidomics data, joints showed increased inflammatory infiltrate, fibrosis and neovascularization in infected mice, compared with joints from uninfected mice. In conclusion, this immunopeptidomics approach revealed key insights into potential mechanisms of infection-induced autoimmunity in LA and indentified a novel immunogenic CD4+ T cell antigen from Bb MCP4. Supported by grants from NIAID (R21AI148982)

Deletion of Vβ3 +CD4 +T-cells by endogenous mouse mammary tumor virus 3 prevents type 1 diabetes induction by autoreactive CD8 +T-cells

Abstract In both humans and NOD mice, type 1 diabetes (T1D) develops from the autoimmune destruction of pancreatic beta cells by T-cells. Interactions between both helper CD4 +and cytotoxic CD8 +T-cells are essential for T1D development in NOD mice. Previous work has indicated that pathogenic T-cells arise from deleterious interactions between relatively common genes which regulate aspects of T-cell activation/effector function (Ctla-4, Tnfrsf9, Il2/Il21), peptide presentation (H2-A g7, B2m), and T-cell receptor signaling (Ptpn22). In this study, we used a combination of subcongenic mapping and a CRISPR/Cas9 screen to identify the NOD-encoded Mtv3 provirus as a genetic element affecting CD4 +/CD8 +T-cell interactions through an additional mechanism, altering the T-cell receptor (TCR) repertoire. Mtv3 encodes a superantigen that deletes the majority of Vβ3 +thymocytes in NOD mice. Ablating Mtv3 and restoring Vβ3 +T-cells has no effect on spontaneous T1D development in NOD mice. However, transferring Mtv3 to C57BL/6 (B6) mice congenic for the NOD H2 g7MHC haplotype (B6.H2 g7) completely blocks their normal susceptibility to T1D mediated by transferred CD8 +T-cells transgenically expressing AI4 or NY8.3 TCRs. The entire genetic effect is manifest by Vβ3 +CD4 +T-cells, which unless deleted by Mtv3, accumulate in insulitic lesions and trigger in B6 background mice the pathogenic activation of diabetogenic CD8 +T-cells. Our findings provide evidence that endogenous Mtv superantigens can influence autoimmune responses. Furthermore, since most common mouse strains have gaps in their TCR Vβ repertoire due to Mtvs, it raises questions about the role of Mtvs in other mouse models designed to reflect human disorders. Supported by grants from the American Diabetes Association (1-14-BS-051) and NIH (AI130656)

Autophagy-dependent regulation of MHC-I molecule presentation.

The major histocompatibility complex (MHC) class I molecules present peptide antigens to MHC class I-restricted CD8+ T lymphocytes to elicit an effective immune response. The conventional antigen-processing pathway for MHC-I presentation depends on proteasome-mediated peptide generation and peptide loading in the endoplasmic reticulum by members of the peptide loading complex. Recent discoveries in this field highlight the role of alternative MHC-I peptide loading and presentation pathways, one of them being autophagy. Autophagy is a cell-intrinsic degradative pathway that ensures cellular homoeostasis and plays critical roles in cellular immunity. In this review article, we discuss the role of autophagy in MHC class I-restricted antigen presentation, elucidating new findings on the crosstalk of autophagy and ER-mediated MHC-I peptide presentation, dendritic cell-mediated cross-presentation and also mechanisms governing immune evasion. A detailed molecular understanding of the key drivers of autophagy-mediated MHC-I modulation holds promising targets to devise effective measures to improve T cell immunotherapies.

Elucidating the role of heat shock protein receptor CD91 on antigen presenting cells during cancer immunosurveillance

Abstract The premise of immunosurveillance is that the immune system, particularly by T cells, allows detection and elimination of emerging tumor cells. However, the mechanisms that allow efficient T cell priming against nascent tumors despite limited antigen availability remain unclear. We have shown that immunogenic heat shock proteins (HSPs) such as gp96 and their receptor CD91 are one such mechanism. HSPs efficiently chaperone minute levels of peptides from tumors to elicit antigen-specific immunity. Cross presentation of HSP-chaperoned peptides by CD91 +dendritic cells is a key mechanism for priming T cell responses. Diverse DC subsets vary in their capacity to modulate such anti-tumor immune responses. In this project, we aim to understand which CD91 +DC subset(s) is/are important for HSP-mediated cancer immunosurveillance. We generated mouse strains with deletion of CD91 on specific DC subsets. cDC1s are thought to be superior cross-presenters that excel in activation of cytotoxic lymphocytes. CD91 deletion in cDC1 render mice highly susceptible to chemically induced tumors. To better understand the CD91-HSP pathway, we tracked the dispersal of tumor-derived gp96 among myeloid cells using tumor cells that express EGFP-tagged gp96. We show that migratory DC subpopulations, such as CD103 +(cDC1) dendritic cells acquire gp96 from the tumor microenvironment, while a different DC subset (CD11b +CD11c +) transfers protective antitumor immunity. The hand-off of tumor-derived gp96 between migratory and lymph node resident DCs is currently under investigation. These results advance our understanding of the HSP-CD91 pathway and its role in DC function which can have significant implications for cancer prognosis and therapy. 5R01CA233803-04

Calibrating T cell help by antigen cross-linking as a universal vaccine platform against rapidly evolving pathogenic viruses

Abstract The effectiveness of influenza vaccine is limited by the heterogeneous immune response it elicits. Factors such as age, sex, and pre-existing antibody titers only partially explain this heterogeneity. Using a novel deconvolution analysis we find subtype-specific bias as a major source of this inter-individual variation. Since the 1940’s it has been proposed that this phenomenon is due to prior strain exposure, referred to as “Original Antigenic Sin”. But our analysis of flu vaccine responses in a monozygotic twin cohort showed that antigenic imprinting from past exposures can only partially explain strain preference (r 2=0.25, p=4.7×10 −5). Thus, we hypothesized that host genetics may play a major role in subtype-bias. Consistent with this is our finding that strain-preferences are evident after vaccination in an influenza naïve infant cohort indicating that prior exposure is not essential. We determined the source of this genetic component to the wide variation in influenza hemagglutinin peptide presentation by different HLA class-II alleles resulting in an imbalanced, subtype-specific CD4 +T follicular help after vaccination. To address this, we developed a tunable, plug-and-play vaccine platform to better calibrate T cell help. In a human tonsil organoid system, we demonstrated that engineered, cross-linked HA induces a significantly higher response against multiple influenza subtypes in comparison to the inactivated influenza vaccine. In summary, we have developed an effective strategy to overcome response bias in a population with diverse genetic backgrounds and pre-existing immunity. Furthermore, we successfully adapted this platform to generate a pan-coronavirus vaccine. Supported by grants from NIH to MMD (5U19AI090019, 5U19AI057229). Additional support was provided by ITI-YIA to VM.

Quantifying how TCR sequence variation affects T cell fate at single-cell resolution

Abstract Mucosal-Associated Invariant T (MAIT) and Natural Killer T (NKT) cells demonstrate that some T cell transcriptional fates are driven by the T cell antigen receptor (TCR). Though activation through the TCR is integral to T cell differentiation, the contribution of TCR sequence features to other T cell transcriptional fates remains yet to be defined. In this study, we identify how αβ V(D)J recombination affects T cell differentiation at single-cell resolution. By applying Canonical Correlation Analysis (CCA) to 340,557 TCR clones collected from 256 individuals, we define a set of TCR scoring functions that quantify transcriptional fate predispositions conferred by the TCR. Unsurprisingly, the strongest fate predispositions correspond to cognate peptide presentation molecules: MR1- or CD1d-restricted PLZFhigh innate-like (MAIT/NKT) transcriptional fate (72.6-fold increase in fate likelihood for top percentile compared to bottom percentile TCRs, AUC = 0.85) and MHC class I-restricted CD8 fate versus MHC class II-restricted CD4 fate (18.7-fold increase, AUC = 0.75). However, our results also uncover that hydrophobic CDR3 residues promote regulatory T cell transcriptional states in both the CD8 and CD4 lineages, and that TCR sequence features preferred by thymic positive selection continue to promote memory formation in the periphery. Applying our TCR scoring functions to 10× dextramer-labeled T cells reveals that not all antigen-specific TCRs are equally capable of mounting an effector response. Even among T cells that recognize the same antigen, biophysical variation in the TCR sequence directs which T cells undergo transcriptional reprogramming to form immunological memory. Supported by grant from NIH (T32GM007753)

Precise activation of antiviral memory T cells in solid tumors triggers a multi-cytokine response that ultimately drives tumor clearance.

Abstract The reactivation of CD8 +tissue-resident memory T cells (T RM) triggers a broad, yet local response that mobilizes adaptive and innate immune cells. This immunostimulatory process can be initiated in solid tumors when infiltrating antiviral memory T cells are reactivated. The intratumoral delivery of viral peptides is sufficient to clear poorly immunogenic tumors in mice and synergizes with immune checkpoint therapy. This study investigated the mechanisms required for this anti-tumor response and novel “Peptide Alarm Therapy” that is currently being tested in a Phase I clinical trial. In contrast to expectations, viral peptide presentation by cancer cells was not required for treatment efficacy, and while NK and CD4 T cells are activated and/or recruited to the tumor microenvironment (TME) following antiviral memory T cell activation, depletion of either population did not limit tumor clearance. Interestingly, inflammatory monocytes were greatly increased in both the TME and tumor-draining lymph node (tdLN) following therapy. Moreover, antigen presenting cells (APCs) containing tumor-derived antigen accumulated within the tdLN. TNFα, IFNγ, and IL-2 are cytokines that can signal to APC populations and are produced by activated T RM. When these cytokines were blocked collectively, but not independently, the treatment efficacy of Peptide Alarm Therapy was greatly reduced. Further, when these cytokines were intratumorally injected, they recapitulated some phenomena achieved by antiviral T cell activation. Thus, local reactivation of antiviral memory T cells, which infiltrate tumors and possess known specificities, drives a multi-cytokine response that promotes the immune-stimulation that effectuates tumor clearance. 1R01CA238439-01A1, 1F30CA253992-01

Immunopeptidomics reveals determinants of Mycobacterium tuberculosis antigen presentation on MHC class I.

CD8+ T cell recognition of Mycobacterium tuberculosis (Mtb)-specific peptides presented on major histocompatibility complex class I (MHC-I) contributes to immunity to tuberculosis (TB), but the principles that govern presentation of Mtb antigens on MHC-I are incompletely understood. In this study, mass spectrometry (MS) analysis of the MHC-I repertoire of Mtb-infected primary human macrophages reveals that substrates of Mtb's type VII secretion systems (T7SS) are overrepresented among Mtb-derived peptides presented on MHC-I. Quantitative, targeted MS shows that ESX-1 activity is required for presentation of Mtb peptides derived from both ESX-1 substrates and ESX-5 substrates on MHC-I, consistent with a model in which proteins secreted by multiple T7SSs access a cytosolic antigen processing pathway via ESX-1-mediated phagosome permeabilization. Chemical inhibition of proteasome activity, lysosomal acidification, or cysteine cathepsin activity did not block presentation of Mtb antigens on MHC-I, suggesting involvement of other proteolytic pathways or redundancy among multiple pathways. Our study identifies Mtb antigens presented on MHC-I that could serve as targets for TB vaccines, and reveals how the activity of multiple T7SSs interacts to contribute to presentation of Mtb antigens on MHC-I.

Peptide Hamming Graphs: A network representation of peptides presented through specific HLAs to identify potential epitope clusters

BackgroundClass I Major Histocompatibility Complex plays a critical role in the adaptive immune response by binding to peptides processed by Proteasome and Transporter associated with antigen processing complex and presenting them on the cell surface to cytotoxic T-cells. Understanding the process of peptide presentation and studying how presented peptides are distributed in the huge space of all potential epitopes could have a dramatic impact in the context of vaccine design, transplantation, autoimmunity, and cancer development. MethodsIn the present work we propose a graph-driven approach to investigate the landscape of both self (human) and viral (254 organisms) peptides presented on cell surface through class I Major Histocompatibility Complex considering specific HLAs. For each considered HLA (N = 89) we designed a network, namely Peptide Hamming Graph, where nodes are peptides predicted to be presented by a given HLA and an edge is set when the Hamming distance between two peptides is equal or smaller than 2 (i.e. the same amino acid occurs in at least 7 positions of the two sequences). ResultsThrough the analysis of Peptide Hamming Graphs we studied how predicted presented peptides are distributed in the whole configurational space for different HLAs, identifying sets of viral peptides that can constitute a potential target for the immune system. In particular we selected connected components of the graph made exclusively of viral peptides and sets of viral peptides with high node degree interacting exclusively with viral neighbours. ConclusionsThis work constitutes an innovative approach to study potential cytotoxic T-cell epitopes relying on a network approach, overcoming the classical paradigm based on the identification of potential epitopes only considering their features as single peptides. T-cell cross-reactivity plays a focal role for the efficacy of this strategy increasing the probability of recognition, and consequently a stronger immune response, of presented peptides far from self, sharing a common pattern in terms of sequence similarity.

Abstract 6437: Development of therapeutic vaccines against ovarian cancer

Abstract Epithelial ovarian cancer (EOC) has a devastating impact on the health of women and has not significantly benefited from advances in immunotherapy mainly because of the lack of well-defined actionable antigen targets. It is therefore essential to discover tumor-specific antigens (TSAs) that are specific to ovarian cancer, shared by a significant proportion of patients and capable of being targeted by the immune system. Using a groundbreaking method, we have previously identified 91 aberrantly expressed TSAs (aeTSAs), 18% of which were shared by at least 80% of the TCGA cohort (The Cancer Genome Atlas). These TSAs originate from unmutated non-exonic genomic sequences and their expression results from cancer-specific epigenetic changes. In the present study, our goal was to evaluate the immunogenicity of these aeTSAs. To do so, 49/91 antigenswere selected based on their presentation by high-frequency HLA allotypes (9 alleles included) and their expression in a large proportion of EOCs patients. Using functional in vitro expansion of naive CD8 T cells by co-culture with TSA-pulsed dendritic cells (DC) followed by CDR3 TCR sequencing, we first assessed the ability of aeTSAs in stimulating the immune system. Notably, 98% of our antigens were able to significantly expand CD8 T cell clonotypes, indicating that their repertoires are available for vaccination. In addition, tetramer staining of CD8 T cell populations after culture with TSA-pulsed DCs revealed that 32% of our aeTSAs tested (13/40) could expand specific CD8 T cells at levels that are detectable by flow cytometry. When selecting an optimal vaccination strategy, DC vaccines are particularly attractive. Using mass spectrometry to measure the abundance of TSAs presented at the cell surface, we next compared two modalities for engineering TSA-based DC vaccines: synthetic peptide pulsing vs TSA-encoding RNA minigenes transfection. Our preliminary results show that synthetic peptide pulsing leads to higher amounts of peptides presented at the surface of the dendritic cells compared to RNA minigenes electroporation. Moreover, we show a direct correlation between the abundance of peptides detected by MS immediately after pulsing on DCs and their predicted binding affinity. This correlation is not maintained with time (24h after pulsing), suggesting that the detection of peptides 24h post-pulsing is linked to the stability of peptide-MHC complexes rather than peptide binding affinity. In conclusion, we show that aeTSAs are attractive targets for EOC immunotherapy, as most of them can expand sizeable populations of CD8 T cells. We also reveal that direct pulsing of aeTSAs on DCs leads to better peptide presentation than RNA minigene transfection. These results are of capital importance, as optimal TSA presentation by DCs leads to stronger anti-tumor responses. We believe that our approach could have a significant impact on immunotherapy of EOC, and eventually of other cancer types. Citation Format: Leslie Hesnard, Catherine Thériault, Maxime Cahuzac, Chantal Durette, Krystel Vincent, Marie-Pierre Hardy, Gabriel Ouellet Lavallée, Joël Lanoix, Pierre Thibault, Claude Perreault. Development of therapeutic vaccines against ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6437.

Abstract 3467: GRWD5769: A first-in-class inhibitor of ERAP1, generating novel cancer antigens to drive de novo anti-tumor T cell responses

Abstract Immune checkpoint inhibitors (ICI) have changed the cancer treatment paradigm, yet significant unmet need remains. Clinical data shows that increasing antigen presentation diversity, genomic instability, tumor mutational burden and HLA diversity, as well as preventing permanent exhaustion of pre-existing cytotoxic T cells, caused by chronic cancer antigen stimulation, are all factors that improve clinical response to ICI. Additionally, increasing presentation of cancer-specific target peptides on cancer cells will enhance T cell receptor (TCR)-mediated therapeutics such as ImmTACs or TCR-Engineered T cells. Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims peptides loaded into classical and non-classical MHC Class I. In cancer, knockout or inhibition of ERAP1 changes a proportion of the antigen repertoire, generating and upregulating cancer antigens. This leads to the activation of de novo anti-tumor T cell responses, causing tumor growth inhibition and bypassing T cell exhaustion. We report the preclinical development, characterization and mechanistic analysis of the first-in-class ERAP1 inhibitor, GRWD5769. Extensive analysis of the immunopeptidomes of diverse cancer cell lines robustly showed that GRWD5769 modulates the cancer-related antigen repertoire across genotypes and cancer-type backgrounds. Novel or upregulated neoantigens generated by ERAP1 inhibition are conserved across cancer cell types and genetic backgrounds. ERAP1 inhibition in combination with an ICI (anti-PD1 mAb) diversified the TCR repertoire, upregulated prognostic immune gene markers in the tumor, including markers for recently activated (thus non-exhausted) T cells, and drove infiltration of T cells into syngeneic mouse model tumors. This combination is efficacious across syngeneic models, including different mouse strains and the effects of ERAP1 inhibition on the T cell response correlate with efficacy. Further, ex vivo human T cell co-cultures with cancer cell lines demonstrate that novel and upregulated neoantigens generated by ERAP1 inhibition drive tumor cell killing. In addition, these studies showed enhanced tumor cell killing when an ImmTAC was used in combination with ERAP1 inhibition to enhance presentation of the cancer-specific target peptide. In conclusion, the first-in-class, ERAP1 inhibitor clinical candidate, GRWD5769, drives novel anti-tumor T cell responses through neoantigen creation and circumventing T cell exhaustion. GRWD5769 has demonstrated a good safety profile in GLP toxicology studies and robust proof of mechanism and proof of principle biomarkers have been developed to provide a clear path to establish the activity and efficacy of GRWD5769 in patients in 2023. Citation Format: Andrew Leishman, Wayne Paes, Emma Sparrow, Ana Ribeiro, Michael Cundell, Milos Aleksic, Michael P. Pinggera, Jessica Sette, Kate Anderton, Nicola Ternette, Juliet Morgan, Jason J. Shiers, Martin Quibell, Peter I. Joyce. GRWD5769: A first-in-class inhibitor of ERAP1, generating novel cancer antigens to drive de novo anti-tumor T cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3467.

Abstract 5364: Language modeling of peptide-HLA interactions achieves state-of-the-art performance on prediction of peptide presentation by HLA Class II

Abstract Precise and sensitive prediction of neoantigen presentation to the immune system via human leukocyte antigen (HLA) class II molecules remains a challenge despite the early success of neural networks applied to HLA class I. However, it is necessary to address this modeling challenge because presentation of a neoantigen epitope by both classes of HLA molecules may be valuable to induce a sustained immune response with therapeutic cancer vaccines. Previously we have developed a machine-learning based platform, EDGETM, that provides a state-of-the-art model to predict presentation of peptides by HLA Class I. Here we propose a new addition to our EDGETM platform: a model that leverages structural information of putative epitopes and HLA class II alleles from their in-situ context to predict presentation of peptides by HLA class II. Our model achieves this by leveraging the Evolutionary Scale Model pre-trained protein language model (LM), which has been demonstrated to embed protein sequences with rich structural information. The input to the model is a linear peptide consisting of an epitope and its flanking amino acids, concatenated with structurally relevant amino acids from each HLA allele. This allows our model to treat the modeling problem entirely as a natural language processing task, which minimizes imputation of covariates found in prior approaches when performing inference in the context of vaccine design, while maximizing the richness of the LM embeddings on longer linear peptides. Crucially, this also allows our model to generalize to any allele that has a known sequence. Additionally, DR-, DP-, and DQ-specific immunoaffinity purified mass spectrometry multi-allelic (MA) presentation data were generated per tumor or cell line sample, spanning 89 alleles in aggregate. We demonstrate that incrementally decreasing HLA class II allele MA resolution during training results in substantially improved predictions for situations where MA presentation data has completely ambiguous epitope presentation across DR/DP/DQ alleles. Our model achieves an Average Precision (AP) of 0.92 and ROC-AUC of 0.98 on the same benchmark validation data as the current state-of-the-art model BERTMHC, which achieved an AP of 0.81 and ROC-AUC of 0.95. These are the best AP and ROC-AUC for an HLA Class II presentation model on this benchmark dataset to the best of our knowledge. Our model is a significant advancement in HLA class II epitope prediction that allows our EDGETM platform to bring neoantigen vaccine design optimized for both class I and class II presentation within reach. Citation Format: Daniel Sprague, Joshua Klein, Italo Faria do Valle, Olivia Petrillo, Melissa Rotunno, Matthew Davis, Monica Lane, Karin Jooss, Ankur Dhanik. Language modeling of peptide-HLA interactions achieves state-of-the-art performance on prediction of peptide presentation by HLA Class II. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5364.

Abstract 6635: Lipid and glycolipid antigen presentation faults as a mechanism of resistance to immune checkpoint therapy in poorly immunogenic melanomas

Abstract Introduction: Cold tumors are characterized by lacking infiltration of antitumor T cells. These tumors are universally refractory to immune checkpoint therapies (ICT). While poor T cell infiltration has been partly ascribed to low tumor mutational burden (TMB), TMB doesn’t necessarily exclude immunogenic responses nor ensure a response to ICT. Mechanisms of T cell exclusion in tumors with sufficient TMB may be caused by molecular failure of the antigen processing and presentation (APP), which impacts T cell generation. It was previously described that loss of heterozygosity (LOH) of an essential component of the MHC class I molecule, the beta-2-microglobulin (B2M), is associated with low antigen presentation, tumor T cell infiltration, and ICT resistance in melanoma. Here we provide further insight into APP and ICT response in melanoma. Methods: Transcriptomic analysis and visualization of genomic data from the GDC-TCGA skin cutaneous melanoma (SKCM) study were performed using the Xena platform. Four anti-PD1 metastatic melanoma cohorts were normalized with transcripts per million units. STRING protein-protein interaction analysis was performed in the human database with 11 nodes and 39 edges. Nanostring transcriptomic data from metastatic uveal melanoma (mUM) was revisited from our previous publications for Spearman’s correlational and group analysis of APP signatures amongst ICT responders and non-responders. Results: In an expanded analysis of 4 normalized anti-PD1 treated metastatic melanoma cohorts (n=272), we confirmed that transcriptomic levels of B2M are significantly reduced in non-responders. Moreover, STRING protein-protein interaction analysis of B2M revealed CD1D as a potential molecular partner of B2M. We, therefore, observed that CD1D transcriptomic expression is not only reduced in anti-PD1 poor responders but is a survival prognosticator in primary tumors from the GDC-TCGA study. Interestingly, CD1D expression is also significantly reduced in poorly immunogenic mUM tumors, a cancer type regarded as universally refractory to ICT. Conclusions: These findings suggest that APP faults in ICT-resistant tumors are not restricted to faults in peptide presentation to T lymphocytes (via B2M) but may also impact the presentation of tumor-associated lipid and glycolipid antigens via CD1d, which can impact antitumor responses of natural killer T cells (NKT). Adjuvant therapies that restore MHC-I and CD1d-mediated antigen presentation may unleash the response to ICT via NKT. Understanding CD1d expression faults locally and systemically may provide valuable prognostication information likely to impact how cancer is treated by optimally selecting patients for ICT and validating novel therapeutic targets for immunotherapy development. Citation Format: Carlos Rogerio Figueiredo. Lipid and glycolipid antigen presentation faults as a mechanism of resistance to immune checkpoint therapy in poorly immunogenic melanomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6635.

Cover Story: Eur. J. Immunol. 4'23

European Journal of ImmunologyVolume 53, Issue 4 2370041 Cover PictureFree Access Cover Story: Eur. J. Immunol. 4'23 First published: 10 April 2023 https://doi.org/10.1002/eji.202370041AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract Our cover image features immunostaining of classical and non-classical MHC class I molecules on HPV6/11 induced respiratory papilloma epithelia. HLA-A, B, and C are absent on these epithelial cells but are expressed in the papilloma stroma. However, non-classical MHC class I molecules, HLA-E and -G, are strongly expressed on papilloma epithelial cells and stroma. The image are from Israr et al. This is the strategy used by HPV6/11 infection to block NK activation and reduce HPV6/11 peptide presentation to T-cells. The authors also show that papilloma cells express increased amounts of PGE2 that directly inhibits NK cell function by signaling through the NK cell PGE2 receptors EP2 and EP4. The original image was digitally modified for the cover. Volume53, Issue4April 20232370041 RelatedInformation

HLA-DMB alleles and haplotypes in Ecuador (Cuenca) Amerindians: Importance for HLA and disease studies.

HLA-DMB allele frequencies and HLA-DBM-DRB1-DQB1 extended haplotypes were studied for the first time in Amerindians (Cuenca city area, Ecuador). It was found that most common extended haplotypes gathered the most frequent HLA-DRB1 Amerindian alleles. HLA-DMB polymorphism studies may be important to uncover HLA and diseases pathogenesis and also in an extended HLA haplotype frameshift. HLA-DM molecule has a crucial role together with CLIP protein in HLA class II peptide presentation. HLA extended haplotypes including complement and non classical genes alleles are proposed to HLA and disease studies.

Arg-tRNA synthetase links inflammatory metabolism to RNA splicing and nuclear trafficking via SRRM2.

Cells respond to perturbations such as inflammation by sensing changes in metabolite levels. Especially prominent is arginine, which has known connections to the inflammatory response. Aminoacyl-tRNA synthetases, enzymes that catalyse the first step of protein synthesis, can also mediate cell signalling. Here we show that depletion of arginine during inflammation decreased levels of nuclear-localized arginyl-tRNA synthetase (ArgRS). Surprisingly, we found that nuclear ArgRS interacts and co-localizes with serine/arginine repetitive matrix protein 2 (SRRM2), a spliceosomal and nuclear speckle protein, and that decreased levels of nuclear ArgRS correlated with changes in condensate-like nuclear trafficking of SRRM2 and splice-site usage in certain genes. These splice-site usage changes cumulated in the synthesis of different protein isoforms that altered cellular metabolism and peptide presentation to immune cells. Our findings uncover a mechanism whereby an aminoacyl-tRNA synthetase cognate to a key amino acid that is metabolically controlled during inflammation modulates the splicing machinery.