Antigen presentation in cancer: insights into tumour immunogenicity and immune evasion.

BackgroundLimited data exist on the role of alterations in HLA Class I antigen processing and presentation machinery in mediating response to immune checkpoint blockade (ICB).MethodsThis retrospective cohort study analyzed transcriptional...

3121 Background: Treatment of non-small cell lung cancer (NSCLC) with immune checkpoint blockade (ICB) has resulted in striking clinical responses, but only in a subset of patients (pts), underscoring the need to identify genomic and molecular determinants of immune evasion. Limited data exist on the potential role of alterations in HLA Class I antigen processing and presentation machinery (APM) in mediating response to ICB. Methods: We conducted a retrospective cohort study analyzing transcriptional profiles from pre-treatment tumor samples of chemotherapy-refractory advanced NSCLC pts treated with ICB. RNA was analyzed using the AmpliSeq transcriptomic platform. An APM signature was generated utilizing 8 genes associated with antigen processing ( B2M, CALR, NLRC5, PSMB9, PSME1, PSME3, RFX5, HSP90AB1) and was examined for its association with response to therapy and progression-free and overall survival (PFS, OS). The APM signature was then evaluated in two independent melanoma cohorts treated with ICB. Results: We analyzed pre-treatment tumor samples from 51 advanced NSCLC pts treated with ICB, median age 64 (range 31-92), smokers (n = 43), adenocarcinoma (n = 31). There were 23 responders and 28 non-responders. The APM signature was significantly higher in responders compared to non-responders (average z-score 2.69 vs. -2.49, p = 0.0001). An APM score above the median value for the entire cohort was significantly associated with improved PFS (HR 0.24, 95% CI, 0.12-0.47, log-rank = 0.001) and OS (HR 0.34, 95% CI, 0.18-0.67, log-rank = 0.005). The APM score was significantly correlated with the well-validated T-cell-inflamed resistance gene expression profile (GEP) score (R2 = 0.32, p < 0.0001). However, the APM score demonstrated improved ability to predict response to ICB relative to the GEP score with AUCs of 0.83 and 0.69, respectively. In an independent cohort of 14 high-risk resectable stage III/IV melanoma pts treated with neoadjuvant anti-PD1 therapy, upregulation of genes involved in antigen processing was associated with improved disease free survival (HR: 0.08, 95% CI, 0.01-0.50, p = 0.0065). In an additional independent melanoma cohort of 28 metastatic pts treated with ICB, a higher APM score was associated with improved overall survival (HR 0.31, 95% CI, 0.09-0.89, log-rank = 0.044). Conclusions: Our data demonstrate that defects in antigen presentation may be an important feature in predicting outcomes to ICB in both lung cancer and melanoma.

The human leukocyte antigen (HLA) processing and presentation machinery (APPM) is altered in various diseases and in response to drug treatments. Defects in the machinery may change presentation levels or alter the repertoire of presented peptides, globally or in an HLA allele-restricted manner, with direct implications for adaptive immunity. In this study, we investigated the immunopeptidome landscape across a panel of isogenic HAP1 cell line clones, each with a KO of a single gene encoding a key protein in the APPM, including B2M, TAP1, TAP2, TAPBP, IRF2, PDIA3, ERAP1, GANAB, SPPL3, CANX, and CALR. We applied immunopeptidomics and proteomics to assess the successful gene KOs on the protein level, understand how these proteins participate in antigen presentation, and contextualize protein expression and antigen presentation. We validated the absence of the KO proteins in the respective samples and found that knocking-out an APPM component leads to the loss of peptide subsets that are normally presented on the control wildtype cells. We assessed the immunopeptidomes qualitatively and quantitatively, considering factors like peptide diversity, peptide length distribution, and binding affinity to the endogenously expressed HLA alleles in HAP1 cells. We demonstrated prominent HLA allele-restricted alterations in several KO conditions. The absence of CALR, CANX, and TAP1 led to significant changes in HLA allele-specific presentation levels. Overall, this work represents the first systematic analysis of how the absence of individual APPM components, knocked out in a single cell line under controlled conditions, affects the immunopeptidome. This approach could facilitate the creation of predictive tools capable of prioritizing HLA-bound peptides likely to be presented when presentation defects occur, such as in cancer and viral infections.

Background: We evaluated the activity of neoadjuvant ICB in localized resectable DDLPS (n=17) and undifferentiated pleomorphic sarcomas (UPS; n=10). DDLPS and UPS patients were randomized to neoadjuvant nivolumab or ipilimumab+nivolumab, with UPS patients receiving concurrent radiotherapy. We assessed genomic markers of early relapse (progression before surgery or relapse within 52 weeks following surgery) using longitudinally acquired tumor samples. Methods: RNA sequencing (RNAseq) and whole genome sequencing (WGS) were performed on longitudinally acquired samples (baseline biopsies and surgical specimens). Differential gene expression between any two groups of patients (i.e., non-early relapse [non-relapsers] vs early relapse [relapsers]) were selected (fold change>1.5 and p value<0.05). Gene set enrichment analyses (GSEA) of KEGG pathways were performed and a network-based approach used to identify genes/pathways associated with MHC-I. Results: At a median follow-up of 23 months, 12 patients (9 DDLPS, 3 UPS) relapsed, including 7 early relapses (relapsers: 5 DDLPS, 2 UPS). The median relapse-free survival was 22 months in DDLPS patients (6 months in relapsers; not reached [NR] in non-relapsers) and NR in UPS patients. At baseline, the most differentially upregulated pathways in non-relapsers compared to relapsers were “graft versus host disease” (GSEA Normalized Enrichment Score[NES]=2.25; False Discovery Rate[FDR] q= 0.009), “natural killer cell mediated cytotoxicity” (NES=2.17; FDR q=0.009), “antigen processing and presentation” (NES=2.16; FDR q=0.009), “allograft rejection” (NES=1.99; FDR q=0.019) and “B-cell receptor signaling pathway” (NES=1.87; FDR q=0.018). In DDLPS patients, the antigen presentation and processing pathway was the most upregulated pathway in non-relapsers compared to relapsers (NES=2.01; FDR q=0.025) while it was not significantly upregulated in UPS (NES=1.15; FDR q=0.62). When looking at pathways longitudinally, the antigen presentation and processing pathway was significantly upregulated at surgery compared to baseline in DDLPS. As antigen presentation and processing was significantly upregulated in DDLPS patients and associated with relapse, we looked for expressed neoantigens that may be processed and presented. Using WGS, we detected 5712 rearrangements at baseline in DDLPS, of which 230 were found in more than one tumor specimen. We also sought to identify genes associated with MHC-I. We selected genes upregulated during ICB comparing baseline to surgical specimens in DDLPS relapsers and looked at the top 10% of genes associated with MHC-I in order to identify potential therapeutic targets for combination. We identified 41 genes upregulated during ICB and associated with MHC-I in relapsers, for which up to 275 inhibitory compounds were found in drug databases. Conclusion: Antigen presentation and processing is a major driver of response to immunotherapy. Future efforts should focus on identifying which antigens are presented to find synergizing compounds in order to increase the clinical benefit of ICB. Citation Format: Elise F. Nassif, Chia-Chin Wu, Kadir Akdemir, Russell G. Witt, Raymond Traweek, Brandon Cope, Prapassorn Thirasastr, Taylor Tate, Grace Mathew, Shadarra Crosby, Randy Chu, Mohammad Mohammad, Kenna Shaw, Ingram Davis, Khalida Wani, Alexander J. Lazar, Wei-Lien Wang, Sheila Duncan, Ashleigh B. Guadagnolo, Andrew J. Bishop, Valerae Lewis, Justin E. Bird, Keila E. Torres, Kelly K. Hunt, Barry W. Feig, Christopher P. Scally, Ravin Ratan, Shreyaskumar Patel, Robert S. Benjamin, Robert Satcher, Kevin McBride, Wolf H. Fridman, Ignacio Wistuba, Andrew Futreal, Jennifer A. Wargo, Neeta Somaiah, Christina L. Roland, Emily Z. Keung. Antigen presentation and processing pathway is associated with early relapse after neoadjuvant immune checkpoint blockade (ICB) in dedifferentiated liposarcomas (DDLPS) [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr PR002.

Simple SummaryA new class of drugs, termed Immune Checkpoint Inhibitors, has revolutionized cancer therapy during the last few years. Unfortunately, these drugs are only effective for a subset of patients and cancer types. Recent work has suggested that how well cancer cells present some of their molecules to the immune system is critical for patient responses to immunotherapy with immune checkpoint inhibitors. Here, we review the role of the biochemical pathway of antigen presentation in cancer and discuss how it can be modulated to enhance the efficacy of cancer immunotherapy.Recent clinical successes of cancer immunotherapy using immune checkpoint inhibitors (ICIs) are rapidly changing the landscape of cancer treatment. Regardless of initial impressive clinical results though, the therapeutic benefit of ICIs appears to be limited to a subset of patients and tumor types. Recent analyses have revealed that the potency of ICI therapies depends on the efficient presentation of tumor-specific antigens by cancer cells and professional antigen presenting cells. Here, we review current knowledge on the role of antigen presentation in cancer. We focus on intracellular antigen processing and presentation by Major Histocompatibility class I (MHCI) molecules and how it can affect cancer immune evasion. Finally, we discuss the pharmacological tractability of manipulating intracellular antigen processing as a complementary approach to enhance tumor immunogenicity and the effectiveness of ICI immunotherapy.

Until a few years ago, lymphatic vessels and lymphatic endothelial cells (LEC) were viewed as part of a passive conduit for lymph and immune cells to reach lymph nodes (LN). However, recent work has shown that LEC are active immunological players whose interaction with dendritic cells and T cells is of important immunomodulatory relevance. While the immunological interaction between LEC and other immune cells has taken a center stage, molecular analysis of LEC antigen processing and presentation machinery is still lagging. Herein we review the current knowledge of LEC MHC I and MHC II antigen processing and presentation pathways, Including the role of LEC in antigen phagocytosis, classical, and non-classical MHC II presentation, proteasome processing and MHC I presentation, and cross-presentation. The ultimate goal is to provide an overview of the LEC antigen processing and presentation machinery that constitutes the molecular basis for their role in MHC I and MHC II-restricted immune responses.

In recent years, therapy with immune modulating antibodies, termed immune checkpoint blockade (ICB), has revolutionized the treatment of advanced metastatic melanoma, yielding long-lasting clinical responses in a subgroup of patients. But despite this remarkable progress, resistance to therapy represents a major clinical challenge. ICB efficacy is critically dependent on cytotoxic CD8+ T cells targeting tumor cells in an HLA class I (HLA-I) antigen-dependent manner. Transcriptional suppression of the HLA-I antigen processing and presentation machinery (HLA-I APM) in melanoma cells leads to HLA-I-low/-negative tumor cell phenotypes escaping CD8+ T cell recognition and contributing to ICB resistance. In general, HLA-I-low/-negative tumor cells can be re-sensitized to T cells by interferons (IFN), augmenting HLA-I APM expression. However, this mechanism fails when melanoma cells acquire resistance to IFN, which recently turned out as a key resistance mechanism in ICB, besides HLA-I APM suppression. Seeking for a strategy to overcome these barriers, we identified a novel mechanism that restores HLA-I antigen presentation in tumor cells independent of IFN (Such et al. (2020) J Clin Invest, doi: 10.1172/JCI131572). We demonstrated that tumor cell-intrinsic activation of the cytosolic innate immunoreceptor RIG-I by its synthetic ligand 3pRNA overcomes transcriptional HLA-I APM suppression in patient-derived IFN-resistant melanoma cells. De novo HLA-I APM expression is IRF1/IRF3-dependent and re-sensitizes melanoma cells to autologous cytotoxic CD8+ T cells. Notably, synthetic RIG-I ligands and ICB synergize in T cell activation, suggesting combinational therapy could be an efficient strategy to improve patient outcomes in melanoma.

Innovative therapies for solid tumors are urgently needed. Recently, therapies that harness the host immune system to fight cancer cells have successfully treated a subset of patients with solid tumors. These responses have been strong and durable but observed in subsets of patients. Work from our group and others has shown that epigenetic therapy, specifically inhibiting the silencing DNA methylation mark, activates immune signaling in tumor cells and can sensitize to immune therapy in murine models. Here we show that colon and ovarian cancer cell lines exhibit lower expression of transcripts involved in antigen processing and presentation to immune cells compared to normal tissues. In addition, treatment with clinically relevant low doses of DNMT inhibitors (that remove DNA methylation) increases expression of both antigen processing and presentation and Cancer Testis Antigens in these cell lines. We confirm that treatment with DNMT inhibitors upregulates expression of the antigen processing and presentation molecules B2M, CALR, CD58, PSMB8, PSMB9 at the RNA and protein level in a wider range of colon and ovarian cancer cell lines and treatment time points than had been described previously. In addition, we show that DNMTi treatment upregulates many Cancer Testis Antigens common to both colon and ovarian cancer. This increase of both antigens and antigen presentation by epigenetic therapy may be one mechanism to sensitize patients to immune therapies.

Understanding tumor resistance to T cell immunotherapies is critical to improve patient outcomes. Our study revealed a role for transcriptional suppression of the tumor-intrinsic HLA class I (HLA-I) antigen processing and presentation machinery (APM) in therapy resistance. Low HLA-I APM mRNA levels in melanoma metastases before immune checkpoint blockade (ICB) correlated with nonresponsiveness to therapy and poor clinical outcome. Patient-derived melanoma cells with silenced HLA-I APM escaped recognition by autologous CD8+ T cells. However, targeted activation of the innate immunoreceptor RIG-I initiated de novo HLA-I APM transcription, thereby overcoming T cell resistance. Antigen presentation was restored in interferon-sensitive (IFN-sensitive) but also immunoedited IFN-resistant melanoma models through RIG-I-dependent stimulation of an IFN-independent salvage pathway involving IRF1 and IRF3. Likewise, enhanced HLA-I APM expression was detected in RIG-Ihi (DDX58hi) melanoma biopsies, correlating with improved patient survival. Induction of HLA-I APM by RIG-I synergized with antibodies blocking PD-1 and TIGIT inhibitory checkpoints in boosting the antitumor T cell activity of ICB nonresponders. Overall, the herein-identified IFN-independent effect of RIG-I on tumor antigen presentation and T cell recognition proposes innate immunoreceptor targeting as a strategy to overcome intrinsic T cell resistance of IFN-sensitive and IFN-resistant melanomas and improve clinical outcomes in immunotherapy.

Processing of antigens within antigen presenting cells (APCs) is necessary for an immune response. Two pathways exist to present antigens to T cells: the major histocompatibility complex class I (MHC I) pathway to activate cytotoxic T cells (CTLs) and the MHC II route to stimulate T helper cells (Ths). Prior to efficient antigen presentation to MHC II, antigens have to be proteolytically degraded by proteases, the cathepsins, inside the endocytic compartment of APCs. Cathepsins process both antigens and self-antigens to antigenic peptides; the latter are critical for autoimmunity. Remarkably, distribution, substrate specificity, and function of cathepsins located in the antigen processing machinery depend on the cell type, primary or cultured cells, or species analyzed. However, a precise understanding of the antigen processing and presentation machinery is needed to generate specific immune modulators since the MHC antigen- processing pathway is subsequently regulated during tumorigenesis, infection, or autoimmunity. In this review, the latest finding regarding function and regulation of the MHC II proteolytic machinery and its possible target for immunomodulation will be discussed. Keywords: Antigen processing and presentation, major histocompatibility complex, cathepsin, cathepsin-specific inhibitors, dendritic cells, B cells, immunomodulation.

Human tumour cells express mutated and non-mutated proteins that can be processed and presented by these cells as peptides bound to human leukocyte antigen (HLA). Some of these peptides are recognized by cognate T cell receptors as 'non-self', leading to specific killing of tumour cells by T cells. This process is fundamental to the success of cancer immunotherapy, which exploits the ability of the immune system to eliminate transformed cells. Mutated antigens (neoantigens) have been implicated in the remarkable therapeutic efficacy of immune checkpoint inhibitors (ICIs), which boost endogenous antitumour immune responses. In recent years, the combination of ICIs with personalized cancer vaccines that target neoantigens and other tumour-specific antigens has emerged as a new therapeutic strategy. However, the robust immune pressure that ICIs exert on cancer cells inevitably amplifies the phenomenon of immune editing, which can allow cancer cells to develop resistance mechanisms that subvert surveillance by the immune system. Diminished antigenicity can be due to defects in the antigen processing and presentation machinery, such as HLA-I/II loss of heterozygosity and loss of functional β2-microglobulin. This poses a considerable challenge for combination therapies that include ICIs and for the design of cancer-specific vaccines.

Universally acceptable donor cells have been developed to address the unmet need for immunotypically matched materials for regenerative medicine. Since forced expression of hypoimmunogenic genes represses the immune response, we established universal pluripotent stem cells (PSCs) by replacing endogenous β2-microglobulin (β2m) with β2m directly conjugated to human leukocyte antigen (HLA)-G, thereby simultaneously suppressing HLA-I expression and the natural killer (NK) cell-mediated immune response. These modified human PSCs retained their pluripotency and differentiation capacity; however, surface presentation of HLA-G was absent from subsequently differentiated cells, particularly cells of neural lineages, due to the downregulation of antigen processing and presentation machinery (APM) genes. Induction of APM genes by overexpression of NLR-family CARD domain-containing 5 (NLRC5) or activator subunit of nuclear factor kappa B (NF-κB) heterodimer (RelA) recovered the surface expression of HLA-G and the hypoimmunogenicity of neural cells. Our findings enhance the utility of hypoimmunogenic cells as universal donors and will contribute to the development of off-the-shelf stem-cell therapeutics.

Little is known about the pathways regulating MHC antigen presentation and the identity of treatment-specific T cell antigens induced by ionizing radiation. For this reason, we investigated the radiation-specific changes in the colorectal tumor cell proteome. We found an increase in DDX58 and ZBP1 protein expression, two nucleic acid sensing molecules likely involved in induction of the dominant interferon response signature observed after genotoxic insult. We further observed treatment-induced changes in key regulators and effector proteins of the antigen processing and presentation machinery. Differential regulation of MHC allele expression was further driving the presentation of a significantly broader MHC-associated peptidome postirradiation, defining a radiation-specific peptide repertoire. Interestingly, treatment-induced peptides originated predominantly from proteins involved in catecholamine synthesis and metabolic pathways. A nuanced relationship between protein expression and antigen presentation was observed where radiation-induced changes in proteins do not correlate with increased presentation of associated peptides. Finally, we detected an increase in the presentation of a tumor-specific neoantigen derived from Mtch1. This study provides new insights into how radiation enhances antigen processing and presentation that could be suitable for the development of combinatorial therapies. Data are available via ProteomeXchange with identifier PXD032003.

The majority of T-cell responses involve proteasome-dependent protein degradation and the downstream presentation of oligopeptide products complexed with major histocompatibility complex (MHC) class I (MHC-I) molecules to peptide-restricted CD8+ T-cells. However, evasion of host immunity is a cancer hallmark that is achieved by disruption of host antigen processing and presentation machinery (APM). Consequently, mechanisms of immune evasion promote cancer growth and survival as well as de novo and acquired resistance to immunotherapy. A multitude of cell signaling pathways modulate the APM and MHC-I-dependent antigen presentation. Pharmacologics that specifically target and modulate proteasome structure and activity represent a novel emerging strategy to improve the treatment of cancers and other diseases characterized by aberrant protein accumulation. FDA-approved pharmacologics that selectively activate proteasomes and/or immunoproteasomes can be repositioned to overcome the current bottlenecks that hinder drug development to enhance antigen presentation, modulate the immunopeptidome, and enhance the cytotoxic activity of endogenous or engineered T-cells. Strategies to enhance antigen presentation may also improve the antitumor activity of T-cell immunotherapies, checkpoint inhibitors, and cancer vaccines. Proteasomes represent actionable therapeutic targets to treat difficult-to-treat infectious processes and neurodegenerative diseases that are characterized by the unwanted accrual of insoluble, deleterious, and potentially toxic proteins. Taken together, we highlight the breadth and magnitude of the proteasome and the immense potential to amplify and unmask the immunopeptidomic landscape to improve the treatment of a spectrum of human diseases.

Response to many immunotherapies depends on effective recognition and eradication of tumor cells by the immune system. This is mediated by the successful presentation of tumor-specific antigens on major histocompatibility complex class I (MHC I) molecules on the surface of tumor cells. Studies have focused on alterations in MHC I complex proteins (e.g. HLA, B2M) and gene regulatory elements (e.g. NLRC5). However, the impact on tumor response of individual defects in components regulating and composing the MHC I antigen processing and presentation machinery (APM) have not been extensively studied. Using a harmonized dataset of six clinical trials of patients with melanoma treated with ICB, whole-exome sequencing (WES, n= 244) and RNA sequencing (RNAseq, n= 426) data from untreated tumor samples were assessed to identify mutations in genes associated with MHC I APM and their regulation. Somatic alterations were correlated with clinical response to ICB and with gene expression profiles in tumor-matched RNAseq data. Mutations in MHC I and MHC I-related genes were found in 63% of patients (n= 154/244). The most recurrently mutated genes were NCAM1 (11%, n=28), TRIM48 (7%, n=17), and CAMK2B (6.6%, n=16), and the transcriptional regulators NLRC5 (9.4%, n=23) and CIITA (9.4%, n=23). Mutations in NLRC5 and CIITA trended toward being mutually exclusive from one another (Fisher’s exact test, p= 0.157) and mutations were overrepresented in tumors with stable or progressive disease following ICB therapy. In addition, lower expression of NLRC5 and CIITA were associated with stable or progressive disease status. NLRC5-mutant tumors that progressed following ICB therapy trended towards lower expression of transcriptional target HLA-A (Kruskal-Wallis, p=0.27); however, there was no difference in HLA-A expression across NLRC5-wildtype tumors. For class II transactivating factor, CIITA, which can induce MHC I expression, mutational status was not associated with HLA-A expression across all clinical response groups. There are genetic alterations spanning MHC I and MHCI-associated genes that may be responsible for inactivating class I antigen presentation by melanoma cells. The impact of these alterations may result in varied clinical responses to ICB therapies. The landscape of these alterations needs to be further delineated to understand how tumors are directly evading class I-mediated recognition and elimination. Citation Format: Mildred Galvez, Katie M. Campbell, Egmidio Medina, Antoni Ribas. Mutations in transcriptional activators of MHC I antigen presentation, NLRC5 and CIITA, are mutually exclusive and have different effects on melanoma gene transcripts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1388.