Atypical acute myeloid leukemia-specific transcripts generate shared and immunogenic MHC class-I-associated epitopes.

Acute myeloid leukemia (AML) has not benefited from innovative immunotherapies, mainly because of the lack of actionable immune targets. Using an original proteogenomic approach, we analyzed the major histocompatibility complex class I (MHC class I)-associated immunopeptidome of 19 primary AML samples and identified 58 tumor-specific antigens (TSAs). These TSAs bore no mutations and derived mainly (86%) from supposedly non-coding genomic regions. Two AML-specific aberrations were instrumental in the biogenesis of TSAs, intron retention, and epigenetic changes. Indeed, 48% of TSAs resulted from intron retention and translation, and their RNA expression correlated with mutations of epigenetic modifiers (e.g., DNMT3A). AML TSA-coding transcripts were highly shared among patients and were expressed in both blasts and leukemic stem cells. In AML patients, the predicted number of TSAs correlated with spontaneous expansion of cognate T cell receptor clonotypes, accumulation of activated cytotoxic T cells, immunoediting, and improved survival. These TSAs represent attractive targets for AML immunotherapy.

BioCanRx Summit for Cancer Immunotherapy 2022 Proceedings.

From 19 to 21 November 2022, BioCanRx held its first post-pandemic in-person Summit for Cancer Immunotherapy in Montreal, Canada. The meeting was well attended by patients, trainees, researchers, clinicians, and industry professionals, who came together to discuss the current state and future of biotherapeutics for cancer in Canada and beyond. Three plenaries, three keynote speakers, a lively debate, and panel discussions, together with poster sessions and a social event, made the event memorable and productive. The current state of cellular therapies, cellular engineering, clinical trials, and the role of the cancer microbiome were discussed in plenary session, and the patient voice was welcomed and present throughout the meeting, in large part due to the Learning Institute, a BioCanRx initiative to include patient partners in research. In this meeting review, we highlight the platform presentations, keynote speakers, debate combatants, panellists, and the patient perspective on the annual meeting.

MAIT cells accumulate in ovarian cancer-elicited ascites where they retain their capacity to respond to MR1 ligands and cytokine cues.

The low mutational burden of epithelial ovarian cancer (EOC) is an impediment to immunotherapies that rely on conventional MHC-restricted, neoantigen-reactive T lymphocytes. Mucosa-associated invariant T (MAIT) cells are MR1-restricted T cells with remarkable immunomodulatory properties. We sought to characterize intratumoral and ascitic MAIT cells in EOC. Single-cell RNA sequencing of six primary human tumor specimens demonstrated that MAIT cells were present at low frequencies within several tumors. When detectable, these cells highly expressed CD69 and VSIR, but otherwise exhibited a transcriptomic signature inconsistent with overt cellular activation and/or exhaustion. Unlike mainstream CD8+ T cells, CD8+ MAIT cells harbored high transcript levels of TNF, PRF1, GZMM and GNLY, suggesting their arming and cytotoxic potentials. In a congenic, MAIT cell-sufficient mouse model of EOC, MAIT and invariant natural killer T cells amassed in the peritoneal cavity where they showed robust IL-17A and IFN-γ production capacities, respectively. However, they gradually lost these functions with tumor progression. In a cohort of 23 EOC patients, MAIT cells were readily detectable in all ascitic fluids examined. In a sub-cohort in which we interrogated ascitic MAIT cells for functional impairments, several exhaustion markers, most notably VISTA, were present on the surface. However, ascitic MAIT cells were capable of producing IFN-γ, TNF-α and granzyme B, but neither IL-17A nor IL-10, in response to an MR1 ligand, bacterial lysates containing MR1 ligands, or a combination of IL-12 and IL-18. In conclusion, ascitic MAIT cells in EOC possess inducible effector functions that may be modified in future immunotherapeutic strategies.

Immunogenic stress and death of cancer cells: Contribution of antigenicity vs adjuvanticity to immunosurveillance.

Cancer cells are subjected to constant selection by the immune system, meaning that tumors that become clinically manifest have managed to subvert or hide from immunosurveillance. Immune control can be facilitated by induction of autophagy, as well as by polyploidization of cancer cells. While autophagy causes the release of ATP, a chemotactic signal for myeloid cells, polyploidization can trigger endoplasmic reticulum stress with consequent exposure of the "eat-me" signal calreticulin on the cell surface, thereby facilitating the transfer of tumor antigens into dendritic cells. Hence, both autophagy and polyploidization cause the emission of adjuvant signals that ultimately elicit immune control by CD8+ T lymphocytes. We investigated the possibility that autophagy and polyploidization might also affect the antigenicity of cancer cells by altering the immunopeptidome. Mass spectrometry led to the identification of peptides that were presented on major histocompatibility complex (MHC) class I molecules in an autophagy-dependent fashion or that were specifically exposed on the surface of polyploid cells, yet lost upon passage of such cells through immunocompetent (but not immunodeficient) mice. However, the preferential recognition of autophagy-competent and polyploid cells by the innate and cellular immune systems did not correlate with the preferential recognition of such peptides in vivo. Moreover, vaccination with such peptides was unable to elicit tumor growth-inhibitory responses in vivo. We conclude that autophagy and polyploidy increase the immunogenicity of cancer cells mostly by affecting their adjuvanticity rather than their antigenicity.

Exploiting non-canonical translation to identify new targets for T cell-based cancer immunotherapy.

Cryptic MHC I-associated peptides (MAPs) are produced via two mechanisms: translation of protein-coding genes in non-canonical reading frames and translation of allegedly non-coding sequences. In general, cryptic MAPs are coded by relatively short open reading frames whose translation can be regulated at the level of initiation, elongation or termination. In contrast to conventional MAPs, the processing of cryptic MAPs is frequently proteasome independent. The existence of cryptic MAPs derived from allegedly non-coding regions enlarges the scope of CD8 T cell immunosurveillance from a mere ~2% to as much as ~75% of the human genome. Considering that 99% of cancer-specific mutations are located in those allegedly non-coding regions, cryptic MAPs could furthermore represent a particularly rich source of tumor-specific antigens. However, extensive proteogenomic analyses will be required to determine the breath as well as the temporal and spatial plasticity of the cryptic MAP repertoire in normal and neoplastic cells.

Meeting report--9th IRIC International Symposium on Molecular Targets in Cancer Genomics.

Graduate students and postdoctoral fellows at the Institute for Research in Immunology and Cancer (IRIC) organized the 9th IRIC International Symposium on 14-15 May, 2015. The symposium was held at the IRIC, an ultra-modern research hub and training center located on the hilltop of the Université de Montréal campus in Montreal, Canada. This year's title was 'Molecular Targets in Cancer Genomics', reflecting the common interest of the IRIC student community. Through four broadly themed sessions, organizers sought to highlight the new generation of anti-cancer strategies including targeted therapies directed against actionable cancer-specific mutations, and immunotherapies, which enhance immune responses against cancer. Both targeted and immunotherapies are tailored to cancer-specific features, and require precise knowledge of cancer cells, from their genome to their proteome. The focus of this symposium was on translating the molecular basis of cancer into a functional understanding of aberrant pathways, and to uncover novel targets to be exploited for cancer therapeutic strategies.

Rejection of leukemic cells requires antigen-specific T cells with high functional avidity.

In a context where injection of antigen (Ag)-specific T cells probably represents the future of leukemia immunotherapy, identification of optimal target Ags is crucial. We therefore sought to discover a reliable marker for selection of the most potent Ags. To this end, (1) we immunized mice against 8 individual Ags: 4 minor histocompatibility Ags (miHAs) and 4 leukemia-associated Ags (LAAs) that were overexpressed on leukemic relative to normal thymocytes; (2) we assessed their ability to reject EL4 leukemic cells; and (3) we correlated the properties of our Ags (and their cognate T cells) with their ability to induce protective antileukemic responses. Overall, individual miHAs instigated more potent antileukemic responses than LAAs. Three features had no influence on the ability of primed T cells to reject leukemic cells: (1) MHC-peptide affinity; (2) the stability of MHC-peptide complexes; and (3) epitope density at the surface of leukemic cells, as assessed using mass spectrometry. The cardinal feature of successful Ags is that they were recognized by high-avidity CD8 T cells that proliferated extensively in vivo. Our work suggests that in vitro evaluation of functional avidity represents the best criterion for selection of Ags, which should be prioritized in clinical trials of leukemia immunotherapy.

MHC I-associated peptides preferentially derive from transcripts bearing miRNA response elements.

MHC I-associated peptides (MIPs) play an essential role in normal homeostasis and diverse pathologic conditions. MIPs derive mainly from defective ribosomal products (DRiPs), a subset of nascent proteins that fail to achieve a proper conformation and the physical nature of which remains elusive. In the present study, we used high-throughput proteomic and transcriptomic methods to unravel the structure and biogenesis of MIPs presented by HLA-A and HLA-B molecules on human EBV-infected B lymphocytes from 4 patients. We found that although HLA-different subjects present distinctive MIPs derived from different proteins, these MIPs originate from proteins that are functionally interconnected and implicated in similar biologic pathways. Secondly, the MIP repertoire of human B cells showed no bias toward conserved versus polymorphic genomic sequences, were derived preferentially from abundant transcripts, and conveyed to the cell surface a cell-type-specific signature. Finally, we discovered that MIPs derive preferentially from transcripts bearing miRNA response elements. Furthermore, whereas MIPs of HLA-disparate subjects are coded by different sets of transcripts, these transcripts are regulated by mostly similar miRNAs. Our data support an emerging model in which the generation of MIPs by a transcript depends on its abundance and DRiP rate, which is regulated to a large extent by miRNAs.

Transposable elements regulate thymus development and function.

Transposable elements (TEs) are repetitive sequences representing ~45% of the human and mouse genomes and are highly expressed by medullary thymic epithelial cells (mTECs). In this study, we investigated the role of TEs on T-cell development in the thymus. We performed multiomic analyses of TEs in human and mouse thymic cells to elucidate their role in T-cell development. We report that TE expression in the human thymus is high and shows extensive age- and cell lineage-related variations. TE expression correlates with multiple transcription factors in all cell types of the human thymus. Two cell types express particularly broad TE repertoires: mTECs and plasmacytoid dendritic cells (pDCs). In mTECs, transcriptomic data suggest that TEs interact with transcription factors essential for mTEC development and function (e.g., PAX1 and REL), and immunopeptidomic data showed that TEs generate MHC-I-associated peptides implicated in thymocyte education. Notably, AIRE, FEZF2, and CHD4 regulate small yet non-redundant sets of TEs in murine mTECs. Human thymic pDCs homogenously express large numbers of TEs that likely form dsRNA, which can activate innate immune receptors, potentially explaining why thymic pDCs constitutively secrete IFN ɑ/ß. This study highlights the diversity of interactions between TEs and the adaptive immune system. TEs are genetic parasites, and the two thymic cell types most affected by TEs (mTEcs and pDCs) are essential to establishing central T-cell tolerance. Therefore, we propose that orchestrating TE expression in thymic cells is critical to prevent autoimmunity in vertebrates.

N-MYC impairs innate immune signaling in high-grade serous ovarian carcinoma.

High-grade serous ovarian cancer (HGSC) is a challenging disease, especially for patients with immunologically "cold" tumors devoid of tumor-infiltrating lymphocytes (TILs). We found that HGSC exhibits among the highest levels of MYCN expression and transcriptional signature across human cancers, which is strongly linked to diminished features of antitumor immunity. N-MYC repressed basal and induced IFN type I signaling in HGSC cell lines, leading to decreased chemokine expression and T cell chemoattraction. N-MYC inhibited the induction of IFN type I by suppressing tumor cell-intrinsic STING signaling via reduced STING oligomerization, and by blunting RIG-I-like receptor signaling through inhibition of MAVS aggregation and localization in the mitochondria. Single-cell RNA sequencing of human clinical HGSC samples revealed a strong negative association between cancer cell-intrinsic MYCN transcriptional program and type I IFN signaling. Thus, N-MYC inhibits tumor cell-intrinsic innate immune signaling in HGSC, making it a compelling target for immunotherapy of cold tumors.

Immunological and molecular features of the tumor microenvironment of long-term survivors of ovarian cancer.

BACKGROUND: Despite an overall poor prognosis, about 15% of patients with advanced-stage tubo-ovarian high-grade serous carcinoma (HGSC) survive ten or more years after standard treatment. METHODS: We evaluated the tumor microenvironment of this exceptional, understudied group using a large international cohort enriched for long-term survivors (LTS; 10+ years; n = 374) compared to medium-term (MTS; 5-7.99 years; n = 433) and short-term survivors (STS; 2-4.99 years; n = 416). Primary tumor samples were immunostained and scored for intra-epithelial and intra-stromal densities of 10 immune-cell subsets (including T cells, B cells, plasma cells, myeloid cells, PD-1+ cells, and PD-L1+ cells) and epithelial content. RESULTS: Positive associations with LTS compared to STS were seen for 9/10 immune-cell subsets. In particular, the combination of intra-epithelial CD8+ T cells and intra-stromal B cells showed near five-fold increased odds of LTS compared to STS. All of these associations were stronger in tumors with high epithelial content and/or the C4/Differentiated molecular subtype, despite immune-cell densities generally being higher in tumors with low epithelial content and/or the C2/Immunoreactive molecular subtype. CONCLUSIONS: The tumor microenvironment of HGSC long-term survivors is distinguished by the intersection of T and B cell co-infiltration, high epithelial content and C4/Differentiated molecular subtype, features which may inspire new approaches to immunotherapy. FUNDING: Ovarian Cancer Research Program (OCRP) of the Congressionally Directed Medical Research Program (CDMRP), U.S. Department of Defense (DOD); American Cancer Society; BC Cancer Foundation; Canada's Networks of Centres of Excellence; Canadian Cancer Society; Canadian Institutes of Health Research; Cancer Councils of New South Wales, Victoria, Queensland, South Australia and Tasmania, Cancer Foundation of Western Australia; Cancer Institute NSW; Cancer Research UK; Deutsche Forschungsgesellschaft; ELAN Funds of the University of Erlangen-Nuremberg; Fred C. and Katherine B. Andersen Foundation; Genome BC; German Cancer Research Center; German Federal Ministry of Education and Research, Programme of Clinical Biomedical Research; Instituto de Salud Carlos III; Mayo Foundation; Minnesota Ovarian Cancer Alliance; Ministerio de Economía y Competitividad; MRC; National Center for Advancing Translational Sciences; National Health and Medical Research Council of Australia (NHMRC); Ovarian Cancer Australia; Peter MacCallum Foundation; Sydney West Translational Cancer Research Centre; Terry Fox Research Institute; The Eve Appeal (The Oak Foundation); UK National Institute for Health Research Biomedical Research Centres at the University of Cambridge; University of Pittsburgh School of Medicine; U.S. National Cancer Institute of the National Institutes of Health; VGH & UBC Hospital Foundation; Victorian Cancer Agency.

B cells in the tumor microenvironment: Multi-faceted organizers, regulators, and effectors of anti-tumor immunity.

Our understanding of tumor-infiltrating lymphocytes (TILs) is rapidly expanding beyond T cell-centric perspectives to include B cells and plasma cells, collectively referred to as TIL-Bs. In many cancers, TIL-Bs carry strong prognostic significance and are emerging as key predictors of response to immune checkpoint inhibitors. TIL-Bs can perform multiple functions, including antigen presentation and antibody production, which allow them to focus immune responses on cognate antigen to support both T cell responses and innate mechanisms involving complement, macrophages, and natural killer cells. In the stroma of the most immunologically "hot" tumors, TIL-Bs are prominent components of tertiary lymphoid structures, which resemble lymph nodes structurally and functionally. Additionally, TIL-Bs participate in a variety of other lympho-myeloid aggregates and engage in dynamic interactions with the tumor stroma. Here, we summarize our current understanding of TIL-Bs in human cancer, highlighting the compelling therapeutic opportunities offered by their unique tumor recognition and effector mechanisms.

Tumour-infiltrating B cells: immunological mechanisms, clinical impact and therapeutic opportunities.

Although immunotherapy research to date has focused largely on T cells, there is mounting evidence that tumour-infiltrating B cells and plasma cells (collectively referred to as tumour-infiltrating B lymphocytes (TIL-Bs)) have a crucial, synergistic role in tumour control. In many cancers, TIL-Bs have demonstrated strong predictive and prognostic significance in the context of both standard treatments and immune checkpoint blockade, offering the prospect of new therapeutic opportunities that leverage their unique immunological properties. Drawing insights from autoimmunity, we review the molecular phenotypes, architectural contexts, antigen specificities, effector mechanisms and regulatory pathways relevant to TIL-Bs in human cancer. Although the field is young, the emerging picture is that TIL-Bs promote antitumour immunity through their unique mode of antigen presentation to T cells; their role in assembling and perpetuating immunologically 'hot' tumour microenvironments involving T cells, myeloid cells and natural killer cells; and their potential to combat immune editing and tumour heterogeneity through the easing of self-tolerance mechanisms. We end by discussing the most promising approaches to enhance TIL-B responses in concert with other immune cell subsets to extend the reach, potency and durability of cancer immunotherapy.

IgA transcytosis: A new weapon in the immune response to cancer?

Tumor-infiltrating B cells and plasma cells have emerged as critical players in anti-tumor immunity. A recent report in Nature shows that IgA antibodies produced by these cells can enter tumor cells by transcytosis, impede oncogenic signals, and facilitate T cell-mediated cytotoxicity. These findings reveal a promising new mechanism to exploit for immunotherapy.

Single-cell Profiles and Prognostic Impact of Tumor-Infiltrating Lymphocytes Coexpressing CD39, CD103, and PD-1 in Ovarian Cancer.

PURPOSE: Tumor-infiltrating lymphocytes (TIL) are strongly associated with survival in most cancers; however, the tumor-reactive subset that drives this prognostic effect remains poorly defined. CD39, CD103, and PD-1 have been independently proposed as markers of tumor-reactive CD8+ TIL in various cancers. We evaluated the phenotype, clonality, and prognostic significance of TIL expressing various combinations of these markers in high-grade serous ovarian cancer (HGSC), a malignancy in need of more effective immunotherapeutic approaches. EXPERIMENTAL DESIGN: Expression of CD39, CD103, PD-1, and other immune markers was assessed by high-dimensional flow cytometry, single-cell sequencing, and multiplex immunofluorescence of primary and matched pre/post-chemotherapy HGSC specimens. RESULTS: Coexpression of CD39, CD103, and PD-1 ("triple-positive" phenotype) demarcated subsets of CD8+ TIL and CD4+ regulatory T cells (Treg) with a highly activated/exhausted phenotype. Triple-positive CD8+ TIL exhibited reduced T-cell receptor (TCR) diversity and expressed genes involved in both cytolytic and humoral immunity. Triple-positive Tregs exhibited higher TCR diversity and a tumor-resident phenotype. Triple-positive TIL showed superior prognostic impact relative to TIL expressing other combinations of these markers. TIGIT was uniquely upregulated on triple-positive CD8+ effector cells relative to their CD4+ Treg counterparts. CONCLUSIONS: Coexpression of CD39, CD103, and PD-1 demarcates highly activated CD8+ and CD4+ TIL with inferred roles in cytolytic, humoral, and regulatory immune functions. Triple-positive TIL demonstrate exceptional prognostic significance and express compelling targets for combination immunotherapy, including PD-1, CD39, and TIGIT.

Proteogenomics Uncovers a Vast Repertoire of Shared Tumor-Specific Antigens in Ovarian Cancer.

High-grade serous ovarian cancer (HGSC), the principal cause of death from gynecologic malignancies in the world, has not significantly benefited from advances in cancer immunotherapy. Although HGSC infiltration by lymphocytes correlates with superior survival, the nature of antigens that can elicit anti-HGSC immune responses is unknown. The goal of this study was to establish the global landscape of HGSC tumor-specific antigens (TSA) using a mass spectrometry pipeline that interrogated all reading frames of all genomic regions. In 23 HGSC tumors, we identified 103 TSAs. Classic TSA discovery approaches focusing only on mutated exonic sequences would have uncovered only three of these TSAs. Other mutated TSAs resulted from out-of-frame exonic translation (n = 2) or from noncoding sequences (n = 7). One group of TSAs (n = 91) derived from aberrantly expressed unmutated genomic sequences, which were not expressed in normal tissues. These aberrantly expressed TSAs (aeTSA) originated primarily from nonexonic sequences, in particular intronic (29%) and intergenic (22%) sequences. Their expression was regulated at the transcriptional level by variations in gene copy number and DNA methylation. Although mutated TSAs were unique to individual tumors, aeTSAs were shared by a large proportion of HGSCs. Taking into account the frequency of aeTSA expression and HLA allele frequencies, we calculated that, in Caucasians, the median number of aeTSAs per tumor would be five. We conclude that, in view of their number and the fact that they are shared by many tumors, aeTSAs may be the most attractive targets for HGSC immunotherapy.

Noncoding regions are the main source of targetable tumor-specific antigens.

Tumor-specific antigens (TSAs) represent ideal targets for cancer immunotherapy, but few have been identified thus far. We therefore developed a proteogenomic approach to enable the high-throughput discovery of TSAs coded by potentially all genomic regions. In two murine cancer cell lines and seven human primary tumors, we identified a total of 40 TSAs, about 90% of which derived from allegedly noncoding regions and would have been missed by standard exome-based approaches. Moreover, most of these TSAs derived from nonmutated yet aberrantly expressed transcripts (such as endogenous retroelements) that could be shared by multiple tumor types. Last, we demonstrated that, in mice, the strength of antitumor responses after TSA vaccination was influenced by two parameters that can be estimated in humans and could serve for TSA prioritization in clinical studies: TSA expression and the frequency of TSA-responsive T cells in the preimmune repertoire. In conclusion, the strategy reported herein could considerably facilitate the identification and prioritization of actionable human TSAs.

Global proteogenomic analysis of human MHC class I-associated peptides derived from non-canonical reading frames.

In view of recent reports documenting pervasive translation outside of canonical protein-coding sequences, we wished to determine the proportion of major histocompatibility complex (MHC) class I-associated peptides (MAPs) derived from non-canonical reading frames. Here we perform proteogenomic analyses of MAPs eluted from human B cells using high-throughput mass spectrometry to probe the six-frame translation of the B-cell transcriptome. We report that ∼ 10% of MAPs originate from allegedly noncoding genomic sequences or exonic out-of-frame translation. The biogenesis and properties of these 'cryptic MAPs' differ from those of conventional MAPs. Cryptic MAPs come from very short proteins with atypical C termini, and are coded by transcripts bearing long 3'UTRs enriched in destabilizing elements. Relative to conventional MAPs, cryptic MAPs display different MHC class I-binding preferences and harbour more genomic polymorphisms, some of which are immunogenic. Cryptic MAPs increase the complexity of the MAP repertoire and enhance the scope of CD8 T-cell immunosurveillance.

The nature of self for T cells-a systems-level perspective.

T-cell development and function are regulated by MHC-associated self peptides, collectively referred to as the immunopeptidome. Large-scale mass spectrometry studies have highlighted three key features of the immunopeptidome. First, it is not a mirror of the proteome or the transcriptome, and its content cannot be predicted with currently available bioinformatic tools. Second, the immunopeptidome is more plastic than previously anticipated, and is molded by several cell-intrinsic and cell-extrinsic factors. Finally, the complexity of the immunopeptidome goes beyond the 20-amino acids alphabet encoded in the germline, and is not restricted to canonical reading frames. The large amounts of 'dark matter' in the immunopeptidome, such as polymorphic, cryptic and mutant peptides, can now be explored using novel proteogenomic approaches that combine mass spectrometry and next-generation sequencing.