Perspectives in Melanoma: meeting report from the Melanoma Bridge (December 1st-3rd, 2022-Naples, Italy).

Outcomes for patients with melanoma have improved over the past decade with the clinical development and approval of immunotherapies targeting immune checkpoint receptors such as programmed death-1 (PD-1), programmed death ligand 1 (PD-L1) or cytotoxic T lymphocyte antigen-4 (CTLA-4). Combinations of these checkpoint therapies with other agents are now being explored to improve outcomes and enhance benefit-risk profiles of treatment. Alternative inhibitory receptors have been identified that may be targeted for anti-tumor immune therapy, such as lymphocyte-activation gene-3 (LAG-3), as have several potential target oncogenes for molecularly targeted therapy, such as tyrosine kinase inhibitors. Unfortunately, many patients still progress and acquire resistance to immunotherapy and molecularly targeted therapies. To bypass resistance, combination treatment with immunotherapies and single or multiple TKIs have been shown to improve prognosis compared to monotherapy. The number of new combinations treatment under development for melanoma provides options for the number of patients to achieve a therapeutic benefit. Many diagnostic and prognostic assays have begun to show clinical applicability providing additional tools to optimize and individualize treatments. However, the question on the optimal algorithm of first- and later-line therapies and the search for biomarkers to guide these decisions are still under investigation. This year, the Melanoma Bridge Congress (Dec 1st-3rd, 2022, Naples, Italy) addressed the latest advances in melanoma research, focusing on themes of paramount importance for melanoma prevention, diagnosis and treatment. This included sessions dedicated to systems biology on immunotherapy, immunogenicity and gene expression profiling, biomarkers, and combination treatment strategies.

Cellular and Humoral Immunity to SARS-CoV-2 Infection in Multiple Sclerosis Patients on Ocrelizumab and Other Disease-Modifying Therapies: A Multi-Ethnic Observational Study.

OBJECTIVE: The objective of this study was to determine the impact of multiple sclerosis (MS) disease-modifying therapies (DMTs) on the development of cellular and humoral immunity to severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection. METHODS: Patients with MS aged 18 to 60 years were evaluated for anti-nucleocapsid and anti-Spike receptor-binding domain (RBD) antibody with electro-chemiluminescence immunoassay; antibody responses to Spike protein, RBD, N-terminal domain with multiepitope bead-based immunoassays (MBI); live virus immunofluorescence-based microneutralization assay; T-cell responses to SARS-CoV-2 Spike using TruCulture enzyme-linked immunosorbent assay (ELISA); and IL-2 and IFNγ ELISpot assays. Assay results were compared by DMT class. Spearman correlation and multivariate analyses were performed to examine associations between immunologic responses and infection severity. RESULTS: Between January 6, 2021, and July 21, 2021, 389 patients with MS were recruited (mean age 40.3 years; 74% women; 62% non-White). Most common DMTs were ocrelizumab (OCR)-40%; natalizumab -17%, Sphingosine 1-phosphate receptor (S1P) modulators -12%; and 15% untreated. One hundred seventy-seven patients (46%) had laboratory evidence of SARS-CoV-2 infection; 130 had symptomatic infection, and 47 were asymptomatic. Antibody responses were markedly attenuated in OCR compared with other groups (p ≤0.0001). T-cell responses (IFNγ) were decreased in S1P (p = 0.03), increased in natalizumab (p <0.001), and similar in other DMTs, including OCR. Cellular and humoral responses were moderately correlated in both OCR (r = 0.45, p = 0.0002) and non-OCR (r = 0.64, p <0.0001). Immune responses did not differ by race/ethnicity. Coronavirus disease 2019 (COVID-19) clinical course was mostly non-severe and similar across DMTs; 7% (9/130) were hospitalized. INTERPRETATION: DMTs had differential effects on humoral and cellular immune responses to SARS-CoV-2 infection. Immune responses did not correlate with COVID-19 clinical severity in this relatively young and nondisabled group of patients with MS. ANN NEUROL 2022;91:782-795.

Perspectives in Melanoma: meeting report from the Melanoma Bridge (December 2nd - 4th, 2021, Italy).

Advances in immune checkpoint and combination therapy have led to improvement in overall survival for patients with advanced melanoma. Improved understanding of the tumor, tumor microenvironment and tumor immune-evasion mechanisms has resulted in new approaches to targeting and harnessing the host immune response. Combination modalities with other immunotherapy agents, chemotherapy, radiotherapy, electrochemotherapy are also being explored to overcome resistance and to potentiate the immune response. In addition, novel approaches such as adoptive cell therapy, oncogenic viruses, vaccines and different strategies of drug administration including sequential, or combination treatment are being tested. Despite the progress in diagnosis of melanocytic lesions, correct classification of patients, selection of appropriate adjuvant and systemic theràapies, and prediction of response to therapy remain real challenges in melanoma. Improved understanding of the tumor microenvironment, tumor immunity and response to therapy has prompted extensive translational and clinical research in melanoma. There is a growing evidence that genomic and immune features of pre-treatment tumor biopsies may correlate with response in patients with melanoma and other cancers, but they have yet to be fully characterized and implemented clinically. Development of novel biomarker platforms may help to improve diagnostics and predictive accuracy for selection of patients for specific treatment. Overall, the future research efforts in melanoma therapeutics and translational research should focus on several aspects including: (a) developing robust biomarkers to predict efficacy of therapeutic modalities to guide clinical decision-making and optimize treatment regimens, (b) identifying mechanisms of therapeutic resistance to immune checkpoint inhibitors that are potentially actionable, (c) identifying biomarkers to predict therapy-induced adverse events, and (d) studying mechanism of actions of therapeutic agents and developing algorithms to optimize combination treatments. During the Melanoma Bridge meeting (December 2nd-4th, 2021, Naples, Italy) discussions focused on the currently approved systemic and local therapies for advanced melanoma and discussed novel biomarker strategies and advances in precision medicine as well as the impact of COVID-19 pandemic on management of melanoma patients.

Serial single-cell profiling analysis of metastatic TNBC during Nab-paclitaxel and pembrolizumab treatment.

PURPOSE: Immunotherapy has recently been shown to improve outcomes for advanced PD-L1-positive triple-negative breast cancer (TNBC) in the Impassion130 trial, leading to FDA approval of the first immune checkpoint inhibitor in combination with taxane chemotherapy. To further develop predictive biomarkers and improve therapeutic efficacy of the combination, interrogation of the tumor immune microenvironment before therapy as well as during each component of treatment is crucial. Here we use single-cell RNA sequencing (scRNA-seq) on tumor biopsies to assess immune cell changes from two patients with advanced TNBC treated in a prospective trial at predefined serial time points, before treatment, on taxane chemotherapy and on chemo-immunotherapy. METHODS: Both patients (one responder and one progressor) received the trial therapy, in cycle 1 nab-paclitaxel given as single agent, in cycle 2 nab-paclitaxel in combination with pembrolizumab. Tumor core biopsies were obtained at baseline, 3 weeks (after cycle 1, chemotherapy alone) and 6 weeks (after cycle 2, chemo-immunotherapy). Single-cell RNA sequencing (scRNA-seq) of both cancer cells and infiltrating immune cells isolated were performed from fresh tumor core biopsy specimens by 10 × chromium sequencing. RESULTS: ScRNA-seq analysis showed significant baseline heterogeneity of tumor-infiltrating immune cell populations between the two patients as well as modulation of the tumor microenvironment by chemotherapy and immunotherapy. In the responding patient there was a population of PD-1high-expressing T cells which significantly decreased after nab-paclitaxel plus pembrolizumab treatment as well as a presence of tissue-resident memory T cells (TRM). In contrast, tumors from the patient with rapid disease progression showed a prevalent and persistent myeloid compartment. CONCLUSIONS: Our study provides a deep cellular analysis of on-treatment changes during chemo-immunotherapy for advanced TNBC, demonstrating not only feasibility of single-cell analyses on serial tumor biopsies but also the heterogeneity of TNBC and differences in on-treatment changes in responder versus progressor.

Perspectives in Melanoma: meeting report from the Melanoma Bridge (December 3rd-5th, 2020, Italy).

Advances in immune checkpoint therapy and targeted therapy have led to improvement in overall survival for patients with advanced melanoma. Single agent checkpoint PD-1 blockade and combination with BRAF/MEK targeted therapy demonstrated benefit in overall survival (OS). Superior response rates have been demonstrated with combined PD-1/CTLA-4 blockade, with a significant OS benefit compared with single-agent PD-1 blockade. Despite the progress in diagnosis of melanocytic lesions, correct classification of patients, selection of appropriate adjuvant and systemic therapies, and prediction of response to therapy remain real challenges in melanoma. Improved understanding of the tumor microenvironment, tumor immunity and response to therapy has prompted extensive translational and clinical research in melanoma. Development of novel biomarker platforms may help to improve diagnostics and predictive accuracy for selection of patients for specific treatment. There is a growing evidence that genomic and immune features of pre-treatment tumor biopsies may correlate with response in patients with melanoma and other cancers but they have yet to be fully characterized and implemented clinically. Overall, the progress in melanoma therapeutics and translational research will help to optimize treatment regimens to overcome resistance and develop robust biomarkers to guide clinical decision-making. During the Melanoma Bridge meeting (December 3rd-5th, 2020, Italy) we reviewed the currently approved systemic and local therapies for advanced melanoma and discussed novel biomarker strategies and advances in precision medicine.

Baseline antibody profiles predict toxicity in melanoma patients treated with immune checkpoint inhibitors.

BACKGROUND: Immune checkpoint inhibitors (anti-CTLA-4, anti-PD-1, or the combination) enhance anti-tumor immune responses, yielding durable clinical benefit in several cancer types, including melanoma. However, a subset of patients experience immune-related adverse events (irAEs), which can be severe and result in treatment termination. To date, no biomarker exists that can predict development of irAEs. METHODS: We hypothesized that pre-treatment antibody profiles identify a subset of patients who possess a sub-clinical autoimmune phenotype that predisposes them to develop severe irAEs following immune system disinhibition. Using a HuProt human proteome array, we profiled baseline antibody levels in sera from melanoma patients treated with anti-CTLA-4, anti-PD-1, or the combination, and used support vector machine models to identify pre-treatment antibody signatures that predict irAE development. RESULTS: We identified distinct pre-treatment serum antibody profiles associated with severe irAEs for each therapy group. Support vector machine classifier models identified antibody signatures that could effectively discriminate between toxicity groups with > 90% accuracy, sensitivity, and specificity. Pathway analyses revealed significant enrichment of antibody targets associated with immunity/autoimmunity, including TNFα signaling, toll-like receptor signaling and microRNA biogenesis. CONCLUSIONS: Our results provide the first evidence supporting a predisposition to develop severe irAEs upon immune system disinhibition, which requires further independent validation in a clinical trial setting.

Constitutive Lck Activity Drives Sensitivity Differences between CD8+ Memory T Cell Subsets.

CD8(+) T cells develop increased sensitivity following Ag experience, and differences in sensitivity exist between T cell memory subsets. How differential TCR signaling between memory subsets contributes to sensitivity differences is unclear. We show in mouse effector memory T cells (TEM) that >50% of lymphocyte-specific protein tyrosine kinase (Lck) exists in a constitutively active conformation, compared with <20% in central memory T cells (TCM). Immediately proximal to Lck signaling, we observed enhanced Zap-70 phosphorylation in TEM following TCR ligation compared with TCM Furthermore, we observed superior cytotoxic effector function in TEM compared with TCM, and we provide evidence that this results from a lower probability of TCM reaching threshold signaling owing to the decreased magnitude of TCR-proximal signaling. We provide evidence that the differences in Lck constitutive activity between CD8(+) TCM and TEM are due to differential regulation by SH2 domain-containing phosphatase-1 (Shp-1) and C-terminal Src kinase, and we use modeling of early TCR signaling to reveal the significance of these differences. We show that inhibition of Shp-1 results in increased constitutive Lck activity in TCM to levels similar to TEM, as well as increased cytotoxic effector function in TCM Collectively, this work demonstrates a role for constitutive Lck activity in controlling Ag sensitivity, and it suggests that differential activities of TCR-proximal signaling components may contribute to establishing the divergent effector properties of TCM and TEM. This work also identifies Shp-1 as a potential target to improve the cytotoxic effector functions of TCM for adoptive cell therapy applications.

Imprinting of the mature T cell response by self.

T cell development requires recognition of self-peptides in the thymus. Two recent studies by Allen and colleagues shed new light into the connection between self-recognition during positive selection and recognition of foreign antigen in the periphery.

Impact of aging on host immune response and survival in melanoma: an analysis of 3 patient cohorts.

BACKGROUND: Age has been reported as an independent prognostic factor for melanoma-specific survival (MSS). We tested the hypothesis that age impacts the host anti-tumor immune response, accounting for age-specific survival outcomes in three unique melanoma patient cohorts. METHODS: We queried the U.S. population-based Surveillance, Epidemiology, and End Results Program (SEER), the prospective tertiary care hospital-based Interdisciplinary Melanoma Cooperative Group (IMCG) biorepository, and the Cancer Genome Atlas (TCGA) biospecimen database to test the association of patient age at time of melanoma diagnosis with clinicopathologic features and survival outcomes. Age groups were defined as ≤45 (young), 46-65 (intermediate), and >65 (older). Each age group in the IMCG and TCGA cohorts was stratified by tumor infiltrating lymphocyte (TIL) measurements and tested for association with MSS. Differential expression of 594 immunoregulatory genes was assessed in a subset of primary melanomas in the IMCG and TCGA cohorts using an integrative pathway analysis. RESULTS: We analyzed 304, 476 (SEER), 1241 (IMCG), and 292 (TCGA) patients. Increasing age at melanoma diagnosis in both the SEER and IMCG cohorts demonstrated a positive correlation with tumor thickness, ulceration, stage, and mortality, however age in the TCGA cohort did not correlate with mortality. Older age was associated with shorter MSS in all three cohorts. When the young age group in both the IMCG and TCGA cohorts was stratified by TIL status, there were no differences in MSS. However, older IMCG patients with brisk TILs and intermediate aged TCGA patients with high lymphocyte scores (3-6) had improved MSS. Gene expression analysis revealed top pathways (T cell trafficking, communication, and differentiation) and top upstream regulators (CD3, CD28, IFNG, and STAT3) that significantly changed with age in 84 IMCG and 43 TCGA primary melanomas. CONCLUSIONS: Older age at time of melanoma diagnosis is associated with shorter MSS, however age's association with clinicopathologic features is dependent upon specific characteristics of the study population. TIL as a read-out of the host immune response may have greater prognostic impact in patients older than age 45. Recognition of age-related factors negatively impacting host immune responses may provide new insights into therapeutic strategies for the elderly.

TCR Microclusters pre-exist and contain molecules necessary for TCR signal transduction.

TCR-dependent signaling events have been observed to occur in TCR microclusters. We found that some TCR microclusters are present in unstimulated murine T cells, indicating that the mechanisms leading to microcluster formation do not require ligand binding. These pre-existing microclusters increase in absolute number following engagement by low-potency ligands. This increase is accompanied by an increase in cell spreading, with the result that the density of TCR microclusters on the surface of the T cell is not a strong function of ligand potency. In characterizing their composition, we observed a constant number of TCRs in a microcluster, constitutive exclusion of the phosphatase CD45, and preassociation with the signaling adapters linker for activation of T cells and growth factor receptor-bound protein 2. The existence of TCR microclusters prior to ligand binding in a state that is conducive for the initiation of downstream signaling could explain, in part, the rapid kinetics with which TCR signal transduction occurs.

Specific increase in potency via structure-based design of a TCR.

Adoptive immunotherapy with Ag-specific T lymphocytes is a powerful strategy for cancer treatment. However, most tumor Ags are nonreactive "self" proteins, which presents an immunotherapy design challenge. Recent studies have shown that tumor-specific TCRs can be transduced into normal PBLs, which persist after transfer in ∼30% of patients and effectively destroy tumor cells in vivo. Although encouraging, the limited clinical responses underscore the need for enrichment of T cells with desirable antitumor capabilities prior to patient transfer. In this study, we used structure-based design to predict point mutations of a TCR (DMF5) that enhance its binding affinity for an agonist tumor Ag-MHC (peptide-MHC [pMHC]), Mart-1 (27L)-HLA-A2, which elicits full T cell activation to trigger immune responses. We analyzed the effects of selected TCR point mutations on T cell activation potency and analyzed cross-reactivity with related Ags. Our results showed that the mutated TCRs had improved T cell activation potency while retaining a high degree of specificity. Such affinity-optimized TCRs have demonstrated to be very specific for Mart-1 (27L), the epitope for which they were structurally designed. Although of somewhat limited clinical relevance, these studies open the possibility for future structural-based studies that could potentially be used in adoptive immunotherapy to treat melanoma while avoiding adverse autoimmunity-derived effects.

Polyreactivity increases the apparent affinity of anti-HIV antibodies by heteroligation.

During immune responses, antibodies are selected for their ability to bind to foreign antigens with high affinity, in part by their ability to undergo homotypic bivalent binding. However, this type of binding is not always possible. For example, the small number of gp140 glycoprotein spikes displayed on the surface of the human immunodeficiency virus (HIV) disfavours homotypic bivalent antibody binding. Here we show that during the human antibody response to HIV, somatic mutations that increase antibody affinity also increase breadth and neutralizing potency. Surprisingly, the responding naive and memory B cells produce polyreactive antibodies, which are capable of bivalent heteroligation between one high-affinity anti-HIV-gp140 combining site and a second low-affinity site on another molecular structure on HIV. Although cross-reactivity to self-antigens or polyreactivity is strongly selected against during B-cell development, it is a common serologic feature of certain infections in humans, including HIV, Epstein-Barr virus and hepatitis C virus. Seventy-five per cent of the 134 monoclonal anti-HIV-gp140 antibodies cloned from six patients with high titres of neutralizing antibodies are polyreactive. Despite the low affinity of the polyreactive combining site, heteroligation demonstrably increases the apparent affinity of polyreactive antibodies to HIV.

Genetic associations of the interleukin locus at 1q32.1 with clinical outcomes of cutaneous melanoma.

BACKGROUND: Due to high melanoma immunogenicity, germline genetic variants in immune pathways have been studied for association with melanoma prognosis. However, limited candidate selection, inadequate power, or lack of independent validation have hampered the reproducibility of these prior findings, preventing personalised clinical applicability in melanoma prognostication. Our objective was to assess the prognostic utility of genetic variants in immunomodulatory pathways for prediction of melanoma clinical outcomes. METHODS: We genotyped 72 tag single nucleotide polymorphisms (SNPs) in 44 immunomodulatory genes in a population sample of 1022 melanoma patients and performed Cox regression analysis to test the association between SNPs and melanoma recurrence-free (RFS) and overall survival (OS). We have further investigated the most significant associations using a fine mapping strategy and followed with functional analyses in CD4+ T cells in a subset of 75 melanoma patients. RESULTS: The most significant associations were found with melanoma OS for rs3024493 in IL10 at chromosome 1q32.1 (heterozygous HR 0.58, 95% CI 0.39 to 0.86; p=0.0006), a variant previously shown to be linked with autoimmune conditions. Multiple additional SNPs at 1q32.1 were also nominally associated with OS confirming at least two independent association signals in this locus. In addition, we found rs3024493 associated with the downregulation of interleukin 10 (IL10) secretion in CD4+ T cells. CONCLUSIONS: We discovered novel associations of IL10 with melanoma survival at 1q32.1, suggesting this locus should be considered as a novel melanoma prognostic biomarker with potential for aiding melanoma patient management. Our findings also provide further support for an alternative role of IL10 in stimulation of anti-tumour immune response.

Strength of PD-1 signaling differentially affects T-cell effector functions.

High surface expression of programmed death 1 (PD-1) is associated with T-cell exhaustion; however, the relationship between PD-1 expression and T-cell dysfunction has not been delineated. We developed a model to study PD-1 signaling in primary human T cells to study how PD-1 expression affected T-cell function. By determining the number of T-cell receptor/peptide-MHC complexes needed to initiate a Ca(2+) flux, we found that PD-1 ligation dramatically shifts the dose-response curve, making T cells much less sensitive to T-cell receptor-generated signals. Importantly, other T-cell functions were differentially sensitive to PD-1 expression. We observed that high levels of PD-1 expression were required to inhibit macrophage inflammatory protein 1 beta production, lower levels were required to block cytotoxicity and IFN-γ production, and very low levels of PD-1 expression could inhibit TNF-α and IL-2 production as well as T-cell expansion. These findings provide insight into the role of PD-1 expression in enforcing T-cell exhaustion and the therapeutic potential of PD-1 blockade.

T-cell receptor affinity and avidity defines antitumor response and autoimmunity in T-cell immunotherapy.

T cells expressing antigen-specific T-cell receptors (TCRs) can mediate effective tumor regression, but they often also are accompanied by autoimmune responses. To determine the TCR affinity threshold defining the optimal balance between effective antitumor activity and autoimmunity in vivo, we used a unique self-antigen system comprising seven human melanoma gp100(209-217)-specific TCRs spanning physiological affinities (1-100 µM). We found that in vitro and in vivo T-cell responses are determined by TCR affinity, except in one case that was compensated by substantial CD8 involvement. Strikingly, we found that T-cell antitumor activity and autoimmunity are closely coupled but plateau at a defined TCR affinity of 10 µM, likely due to diminished contribution of TCR affinity to avidity above the threshold. Together, these results suggest that a relatively low-affinity threshold is necessary for the immune system to avoid self-damage, given the close relationship between antitumor activity and autoimmunity. The low threshold, in turn, indicates that adoptive T-cell therapy treatment strategies using in vitro-generated high-affinity TCRs do not necessarily improve efficacy.

2D TCR-pMHC-CD8 kinetics determines T-cell responses in a self-antigen-specific TCR system.

Two-dimensional (2D) kinetic analysis directly measures molecular interactions at cell-cell junctions, thereby incorporating inherent cellular effects. By comparison, three-dimensional (3D) analysis probes the intrinsic physical chemistry of interacting molecules isolated from the cell. To understand how T-cell tumor reactivity relates to 2D and 3D binding parameters and to directly compare them, we performed kinetic analyses of a panel of human T-cell receptors (TCRs) interacting with a melanoma self-antigen peptide (gp100209 -217 ) bound to peptide-major histocompatibility complex in the absence and presence of co-receptor CD8. We found that while 3D parameters are inadequate to predict T-cell function, 2D parameters (that do not correlate with their 3D counterparts) show a far broader dynamic range and significantly improved correlation with T-cell function. Thus, our data support the general notion that 2D parameters of TCR-peptide-major histocompatibility complex-CD8 interactions determine T-cell responsiveness and suggest a potential 2D-based strategy to screen TCRs for tumor immunotherapy.

Vitellogenin recognizes cell damage through membrane binding and shields living cells from reactive oxygen species.

Large lipid transfer proteins are involved in lipid transportation and diverse other molecular processes. These serum proteins include vitellogenins, which are egg yolk precursors and pathogen pattern recognition receptors, and apolipoprotein B, which is an anti-inflammatory cholesterol carrier. In the honey bee, vitellogenin acts as an antioxidant, and elevated vitellogenin titer is linked to prolonged life span in this animal. Here, we show that vitellogenin has cell and membrane binding activity and that it binds preferentially to dead and damaged cells. Vitellogenin binds directly to phosphatidylcholine liposomes and with higher affinity to liposomes containing phosphatidylserine, a lipid of the inner leaflet of cell membranes that is exposed in damaged cells. Vitellogenin binding to live cells, furthermore, improves cell oxidative stress tolerance. This study can shed more light on why large lipid transfer proteins have a well conserved α-helical domain, because we locate the lipid bilayer-binding ability of vitellogenin largely to this region. We suggest that recognition of cell damage and oxidation shield properties are two mechanisms that allow vitellogenin to extend honey bee life span.

Melanoma expression of matrix metalloproteinase-23 is associated with blunted tumor immunity and poor responses to immunotherapy.

BACKGROUND: Matrix metalloproteinase-23 (MMP-23) can block the voltage-gated potassium channel Kv1.3, whose function is important for sustained Ca(2+) signaling during T cell activation. MMP-23 may also alter T cell activity and phenotype through cleavage of proteins affecting cytokine and chemokine signaling. We therefore tested the hypothesis that MMP-23 can negatively regulate the anti-tumor T cell response in human melanoma. METHODS: We characterized MMP-23 expression in primary melanoma patients who received adjuvant immunotherapy. We examined the association of MMP-23 with the anti-tumor immune response - as assessed by the prevalence of tumor-infiltrating lymphocytes and Foxp3(+) regulatory T cells. Further, we examined the association between MMP-23 expression and response to immunotherapy. Considering also an in trans mechanism, we examined the association of melanoma MMP-23 and melanoma Kv1.3 expression. RESULTS: Our data revealed an inverse association between primary melanoma MMP-23 expression and the anti-tumor T cell response, as demonstrated by decreased tumor-infiltrating lymphocytes (TIL) (P = 0.05), in particular brisk TILs (P = 0.04), and a trend towards an increased proportion of immunosuppressive Foxp3(+) regulatory T cells (P = 0.07). High melanoma MMP-23 expression is also associated with recurrence in patients treated with immune biologics (P = 0.037) but not in those treated with vaccines (P = 0.64). Further, high melanoma MMP-23 expression is associated with shorter periods of progression-free survival for patients receiving immune biologics (P = 0.025). On the other hand, there is no relationship between melanoma MMP-23 and melanoma Kv1.3 expression (P = 0.27). CONCLUSIONS: Our data support a role for MMP-23 as a potential immunosuppressive target in melanoma, as well as a possible biomarker for informing melanoma immunotherapies.

Structural basis for self-discrimination by neoantigen-specific TCRs.

T cell receptors (TCR) are pivotal in mediating tumour cell cytolysis via recognition of mutation-derived tumour neoantigens (neoAgs) presented by major histocompatibility class-I (MHC-I). Understanding the factors governing the emergence of neoAg from somatic mutations is a major focus of current research. However, the structural and cellular determinants controlling TCR recognition of neoAgs remain poorly understood. This study describes the multi-level analysis of a model neoAg from the B16F10 murine melanoma, H2-Db/Hsf2 p.K72N68-76, as well as its cognate TCR 47BE7. Through cellular, molecular and structural studies we demonstrate that the p.K72N mutation enhances H2-Db binding, thereby improving cell surface presentation and stabilizing the TCR 47BE7 epitope. Furthermore, TCR 47BE7 exhibited high functional avidity and selectivity, attributable to a broad, stringent, binding interface enabling recognition of native B16F10 despite low antigen density. Our findings provide insight into the generation of anchor-residue modified neoAg, and emphasize the value of molecular and structural investigations of neoAg in diverse MHC-I contexts for advancing the understanding of neoAg immunogenicity.

Longitudinal study of immunity to SARS-CoV2 in ocrelizumab-treated MS patients up to 2 years after COVID-19 vaccination.

OBJECTIVES: (1) To plot the trajectory of humoral and cellular immune responses to the primary (two-dose) COVID-19 mRNA series and the third/booster dose in B-cell-depleted multiple sclerosis (MS) patients up to 2 years post-vaccination; (2) to identify predictors of immune responses to vaccination; and (3) to assess the impact of intercurrent COVID-19 infections on SARS CoV-2-specific immunity. METHODS: Sixty ocrelizumab-treated MS patients were enrolled from NYU (New York) and University of Colorado (Anschutz) MS Centers. Samples were collected pre-vaccination, and then 4, 12, 24, and 48 weeks post-primary series, and 4, 12, 24, and 48 weeks post-booster. Binding anti-Spike antibody responses were assessed with multiplex bead-based immunoassay (MBI) and electrochemiluminescence (Elecsys®, Roche Diagnostics), and neutralizing antibody responses with live-virus immunofluorescence-based microneutralization assay. Spike-specific cellular responses were assessed with IFNγ/IL-2 ELISpot (Invitrogen) and, in a subset, by sequencing complementarity determining regions (CDR)-3 within T-cell receptors (Adaptive Biotechnologies). A linear mixed-effect model was used to compare antibody and cytokine levels across time points. Multivariate analyses identified predictors of immune responses. RESULTS: The primary vaccination induced an 11- to 208-fold increase in binding and neutralizing antibody levels and a 3- to 4-fold increase in IFNγ/IL-2 responses, followed by a modest decline in antibody but not cytokine responses. Booster dose induced a further 3- to 5-fold increase in binding antibodies and 4- to 5-fold increase in IFNγ/IL-2, which were maintained for up to 1 year. Infections had a variable impact on immunity. INTERPRETATION: Humoral and cellular benefits of COVID-19 vaccination in B-cell-depleted MS patients were sustained for up to 2 years when booster doses were administered.