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Melanoma, a skin cancer that develops from pigment cells, has been studied intensively, particularly in terms of the immune response to tumours, and has been used as a model for the development of immunotherapy. This is due, in part, to the high mutational burden observed in melanomas, which increases both their immunogenicity and the infiltration of immune cells into the tumours, compared with other types of cancers. The immune response to melanomas involves a complex set of components and interactions. As the tumour evolves, it accumulates an increasing number of genetic and epigenetic alterations, some of which contribute to the immunogenicity of the tumour cells and the infiltration of immune cells. However, tumour evolution also enables the development of resistance mechanisms, which, in turn, lead to tumour immune escape. Understanding the interactions between melanoma tumour cells and the immune system, and the evolving changes within the melanoma tumour cells, the immune system and the microenvironment, is essential for the development of new cancer therapies. However, current research suggests that other extrinsic factors, such as the microbiome, may play a role in the immune response to melanomas. Here, we review the mechanisms underlying the immune response in the tumour and discuss recent advances as well as strategies for treatment development.This Review discusses the mechanisms underlying the immune response to melanomas, as well as the mechanisms of response and resistance of these tumours to immunotherapies. The lessons learned in melanoma may apply to other tumour types.

Predicting the outcome of immunotherapy treatment in melanoma patients is challenging. Alterations in genes involved in antigen presentation and the interferon gamma (IFNγ) pathway play an important role in the immune response to tumors. We describe here that the overexpression of PSMB8 and PSMB9 , two major components of the immunoproteasome, is predictive of better survival and improved response to immune-checkpoint inhibitors of melanoma patients. We study the mechanism underlying this connection by analyzing the antigenic peptide repertoire of cells that overexpress these subunits using HLA peptidomics. We find a higher response of patient-matched tumor infiltrating lymphocytes against antigens diferentially presented after immunoproteasome overexpression. Importantly, we find that PSMB8 and PSMB9 expression levels are much stronger predictors of melanoma patientsʼ immune response to checkpoint inhibitors than the tumors’ mutational burden. These results suggest that PSMB8 and PSMB9 expression levels can serve as important biomarkers for stratifying melanoma patients for immune-checkpoint treatment. The response to immunotherapy of melanoma patients is heterogeneous. Here, the authors demonstrate that a high expression of the two major components of the immunoproteasome, PSMB8 and PSMB9, modulates the production of HLA peptides and it is predictive of better survival and improved response to immune-checkpoint inhibitors of melanoma patients.

BackgroundThe query for tumor shared and neo-antigens as a therapeutic approach has been the focus of cancer immunology for the past two decades. Notably, these peptide sequences can bind to HLA molecules and present on the cell surface, subsequently to be recognized by T-cell receptors (TCRs), activating the immune system and so facilitating in tumor rejection.1–3 The search for new origins of targetable types of HLA peptides is consistently growing, and new studies show peptides that are derived from non-canonical open reading frames (ORFs), altered translation, proteasome splicing, viral proteins and more.4–6 In light of the new findings, showing the important role of intra-tumor and gut bacteria in tumor-genesis and their effect on the immune response,7–10 we went on a quest for discovering whether intracellular bacteria antigens can be presented by tumor cells, and whether these antigens may elicit an immune response.MethodsCombination of HLA peptidomics with 16S rDNA sequencing.ResultsCombination of HLA peptidomics with 16S rDNA sequencing of 17 melanoma metastasis derived from 9 different patients, lead us to the unbiased identification of an intracellular bacterial peptide repertoire presented on HLA-I and HLA-II molecules. We were able to validate these results by co-culturing the bacterial species identified by 16S sequencing with the patient derived melanoma cells, further validating the peptide’s presentation by preforming HLA peptidomics on the infected cells. Importantly, we were able to identify common bacterial peptides from different metastases of the same patient as well as from different patients. Some of the common bacterial peptides, as well as others, were able to elicit an immune response by the autologous tumor infiltrating lymphocytes (TILs), suggesting potential therapeutic implications of these peptides.ConclusionsThe insights gathered through this study elucidate the effect of intra-tumor bacteria on the immune response and so, may lead to the development of novel clinical applications.ReferencesNeefjes J, Jongsma ML, Paul P, Bakke O. Towards a systems understanding of MHC class I and MHC class II antigen presentation. Nat Rev Immunol 2011;11: 823–836.Stronen E, Toebes M, Kelderman S, et al. Targeting of cancer neoantigens with donor-derived T cell receptor repertoires. Science 2016; 352: 1337–1341.Rosenberg SA, Restifo NP. Adoptive cell transfer as personalized immunotherapy for human cancer. Science 2015; 348: 62–68.Chen J, Brunner AD, Cogan JZ, et al. Pervasive functional translation of noncanonical human open reading frames. Science 2020; 367: 1140–1146.Starck SR, Shastri N. Nowhere to hide: unconventional translation yields cryptic peptides for immune surveillance. Immunol Rev 2016;272:8–16.Croft NP, Smith SA, Pickering J, et al. Most viral peptides displayed by class I MHC on infected cells are immunogenic. Proc Natl Acad Sci U S A 2019; 116: 3112–3117.Gopalakrishnan V, Spencer CN, Nezi L, et al. Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients. Science 2018;359:97–103.Routy B, Le Chatelier E, Derosa L, et al. Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors. Science 2018;359:91–97.Matson V, Fessler J, Bao R, et al. The commensal microbiome is associated with anti-PD-1 efficacy in metastatic melanoma patients. Science 2018;359:104–108.10. Nejman D, Livyatan I, Fuks G et al. The human tumor microbiome is composed of tumor type-specific intracellular bacteria. Science 2020;368:973–980.

Neoantigens are now recognized drivers of the antitumor immune response. Recurrent neoantigens, shared among groups of patients, have thus become increasingly coveted therapeutic targets. Here, we report on the data-driven identification of a robustly presented, immunogenic neoantigen that is derived from the combination of HLA-A*01:01 and RAS.Q61K. Analysis of large patient cohorts indicated that this combination applies to 3% of patients with melanoma. Using HLA peptidomics, we were able to demonstrate robust endogenous presentation of the neoantigen in 10 tumor samples. We detected specific reactivity to the mutated peptide within tumor-infiltrating lymphocytes (TILs) from 2 unrelated patients, thus confirming its natural immunogenicity. We further investigated the neoantigen-specific clones and their T cell receptors (TCRs) via a combination of TCR sequencing, TCR overexpression, functional assays, and single-cell transcriptomics. Our analysis revealed a diverse repertoire of neoantigen-specific clones with both intra- and interpatient TCR similarities. Moreover, 1 dominant clone proved to cross-react with the highly prevalent RAS.Q61R variant. Transcriptome analysis revealed a high association of TCR clones with specific T cell phenotypes in response to cognate melanoma, with neoantigen-specific cells showing an activated and dysfunctional phenotype. Identification of recurrent neoantigens and their reactive TCRs can promote \"off-the-shelf\" precision immunotherapies, alleviating limitations of personalized treatments.

The immune response against tumor cells is affected by many different factors. For it to occur, the patient’s T-cells need to recognize the antigens presented by the tumor cells as well as get activated. Knowing the identity (sequence) of those antigens is crucial in order to target them. In tumors, such as melanoma, the high mutational load enables the tumor to grow, spread and resist treatment. But it is also the tumors’ achilles heel when these mutations are presented by the cells’ HLA molecules and are recognized by the immune system as being non-self-antigens. In this study, we developed a pipeline that combine HLA peptidomics and whole exome sequencing in order to identify the neoantigens presented by melanoma cells. We were able to characterize not only the antigenic repertoire, but also to identify their T-cell counterparts and sequence their T-cell receptor (TCR). We show that neo-antigens are more reactive than other tumor-associated antigens (TAAs), and how combination of HLA peptidomics and prediction algorithms, can give a complementary and more comprehensive view of the tumor antigens. In addition to TAAs and neo-antigens we were able to show for the first time the antigens presented by melanoma cells and derived from intra-tumor bacteria, and their ability to elicit immune response by the tumor infiltrating lymphocytes (TILs). In order to evade the immune system the tumor cells exploit different mechanisms such as reducing the presentation of cancer antigens or inhibiting the activation of the effector T-cells. Recently, many studies showed how alterations of the antigen presentation and Interferon gamma (IFNγ) pathways could allow the tumor cells to be resistant to immune checkpoint inhibitor (CPI) therapy. We showed how overexpression of immunoproteasome subunits, PSMB8 and PSMB9, affect the reactivity of the TILs, and response to CPIs. This suggest we can use immunoproteasome expression levels in order to predict the treatment outcome and select the patient that will benefit from this treatment.

Neoantigens are now recognized drivers of the antitumor immune response. Recurrent neoantigens, shared among groups of patients, have thus become increasingly coveted therapeutic targets. Here, we report on the data-driven identification of a robustly presented, immunogenic neoantigen that is derived from the combination of HLA-A·01:01 and RAS.Q61K. Analysis of large patient cohorts indicated that this combination applies to 3% of patients with melanoma. Using HLA peptidomics, we were able to demonstrate robust endogenous presentation of the neoantigen in 10 tumor samples. We detected specific reactivity to the mutated peptide within tumor-infiltrating lymphocytes (TILs) from 2 unrelated patients, thus confirming its natural immunogenicity. We further investigated the neoantigen-specific clones and their T cell receptors (TCRs) via a combination of TCR sequencing, TCR overexpression, functional assays, and single-cell transcriptomics. Our analysis revealed a diverse repertoire of neoantigen-specific clones with both intra- and interpatient TCR similarities. Moreover, 1 dominant clone proved to cross-react with the highly prevalent RAS.Q61R variant. Transcriptome analysis revealed a high association of TCR clones with specific T cell phenotypes in response to cognate melanoma, with neoantigen-specific cells showing an activated and dysfunctional phenotype. Identification of recurrent neoantigens and their reactive TCRs can promote \"off-the-shelf\" precision immunotherapies, alleviating limitations of personalized treatments.

Neuronal activity shapes brain development and refines synaptic connectivity in part through dynamic changes in gene expression. While activity-regulated transcriptional programs have been extensively characterized, the holistic effects of neuronal activity on the full RNA life cycle remain relatively unexplored. Here, we show that neuronal activity influences multiple stages of RNA metabolism and . Among these, RNA stability emerges as a previously underappreciated regulator of gene expression, exerting a stronger influence than transcription on total RNA levels for ∼15% of activity-dependent genes. We go on to profile 3'UTR mRNA motifs that are sufficient to modulate activity-dependent mRNA stability and employ machine learning to identify the neuronal-specific RNA-binding protein HuD as a key regulator of activity-dependent mRNA stabilization. We demonstrate that HuD shapes activity-dependent mRNA abundance of hundreds of transcripts in both soma and distal neuronal processes and that neuronal activity drives the reorganization of HuD-interacting proteins, thereby stabilizing HuD-bound mRNAs and directing them into translationally active granules. Finally, we find that many variants associated with autism spectrum disorder (ASD) and other neurodevelopmental disorders disrupt or promote aberrant activity-dependent changes in mRNA stability. These findings reveal mRNA stability as a widespread mechanism of stimulus-responsive gene regulation in neurons with direct implications for the understanding of neurodevelopmental disorders.

Neuronal activity shapes brain development and refines synaptic connectivity in part through dynamic changes in gene expression. While activity-regulated transcriptional programs have been extensively characterized, the holistic effects of neuronal activity on the full RNA life cycle remain relatively unexplored. Here, we show that neuronal activity influences multiple stages of RNA metabolism in vitro and in vivo . Among these, RNA stability emerges as a previously underappreciated regulator of gene expression, exerting a stronger influence than transcription on total RNA levels for ∼10% of activity-dependent genes. We go on to profile 3'UTR mRNA motifs that are sufficient to modulate activity-dependent mRNA stability and employ machine learning to identify the neuronal-specific RNA-binding protein HuD as a key regulator of activity-dependent mRNA stabilization. We demonstrate that HuD shapes activity-dependent mRNA abundance of hundreds of transcripts in both soma and distal neuronal processes and that neuronal activity drives the reorganization of HuD-interacting proteins, thereby stabilizing HuD-bound mRNAs and directing them into translationally active granules. Finally, we find that many variants associated with autism spectrum disorder (ASD) and other neurodevelopmental disorders disrupt or promote aberrant activity-dependent changes in mRNA stability. These findings reveal mRNA stability as a widespread mechanism of stimulus-responsive gene regulation in neurons with direct implications for the understanding of neurodevelopmental disorders.Neuronal activity shapes brain development and refines synaptic connectivity in part through dynamic changes in gene expression. While activity-regulated transcriptional programs have been extensively characterized, the holistic effects of neuronal activity on the full RNA life cycle remain relatively unexplored. Here, we show that neuronal activity influences multiple stages of RNA metabolism in vitro and in vivo . Among these, RNA stability emerges as a previously underappreciated regulator of gene expression, exerting a stronger influence than transcription on total RNA levels for ∼10% of activity-dependent genes. We go on to profile 3'UTR mRNA motifs that are sufficient to modulate activity-dependent mRNA stability and employ machine learning to identify the neuronal-specific RNA-binding protein HuD as a key regulator of activity-dependent mRNA stabilization. We demonstrate that HuD shapes activity-dependent mRNA abundance of hundreds of transcripts in both soma and distal neuronal processes and that neuronal activity drives the reorganization of HuD-interacting proteins, thereby stabilizing HuD-bound mRNAs and directing them into translationally active granules. Finally, we find that many variants associated with autism spectrum disorder (ASD) and other neurodevelopmental disorders disrupt or promote aberrant activity-dependent changes in mRNA stability. These findings reveal mRNA stability as a widespread mechanism of stimulus-responsive gene regulation in neurons with direct implications for the understanding of neurodevelopmental disorders.