Faculty Bibliography

Ultraviolet (UV)-induced DNA mutations generate genetic drivers of cutaneous melanoma and numerous neoantigens that can trigger anti-tumor immunity. Melanoma cells must therefore rapidly evade immune detection by modulating cell-autonomous epigenetic mechanisms and tumor-microenvironment interactions. Although angiogenesis typically facilitates immune infiltration, solid tumors increase vascularization while limiting immune cell entry. By comparing transcription factor (TF) expression across early-stage melanoma, naevi, and other cancers, we found that the homeodomain TF HOXD13 drives a melanoblast-like program upregulated in melanoma and strongly correlated with angiogenesis and immune cell exclusion. Using transcriptomics, 3D chromatin profiling, and in vivo models, we show that HOXD13 promotes tumor growth by enhancing angiogenesis and suppressing T-cell infiltration. HOXD13 orchestrates 3D enhancer-promoter contacts activating VEGFA, SEMA3A, and CD73, which remodel vasculature and elevate immunosuppressive adenosine. Consistently, HOXD13-induced tumor growth is reversed by combined VEGFR and adenosine receptor (AdR) inhibition, revealing a dual pro-angiogenic and immunosuppressive HOXD13 axis with therapeutic relevance.

BACKGROUND/UNASSIGNED:There is a greater risk of complications from severe COVID-19 in immunocompromised patients with multiple sclerosis (pwMS) treated with certain disease-modifying therapies (DMTs), as well as a diminished vaccine response. METHODS/UNASSIGNED:In this exploratory, observational study, we recruited 28 patients with Relapsing Remitting MS (RRMS, n=24) or Secondary Progressive MS (SPMS, n=4), that were receiving treatment with either natalizumab or fumarates (diroximel or dimethyl) prior to baseline sample collection. Blood samples were collected before vaccination (baseline), between 4 weeks and 6 months post vaccination, and post booster administration. A multiplex bead immunoassay (MBI) was used to measure anti-Spike IgG, while IFNγ and IL-2 ELISpot assays were used to determine T cell activation. A 35-color spectral flow cytometry panel was used to phenotype bulk B and T cells and SARS-CoV-2-specific T cells, while dimensionality reduction was performed for further phenotypic analysis. RESULTS/UNASSIGNED:We observed a significantly increased absolute lymphocyte count (ALC) (p=0.0003) in natalizumab-treated pwMS when compared to fumarate-treated pwMS primarily due to increased circulating CD19+ B cells. Fumarate-treated pwMS exhibited a diminished Th1/Th2 ratio when compared to natalizumab-treated pwMS (p=0.0004) or healthy controls (p=0.0745), while natalizumab treatment marginally increased the Th1/Th2 ratio compared to healthy controls (p=0.1311). The observed increase in B cells in natalizumab-treated pwMS were predominantly memory B cells, and double negative (DN) B cells. However, no significant differences between the treatment groups were seen in terms of Spike IgG titers following the initial vaccination course or booster dose, nor in SARS-CoV-2-specific CD4+ responses, all of which remained robust for at least 6 months post-vaccination. The magnitude of humoral and cellular immune responses in both treatment groups were comparable to vaccinated healthy controls. Additionally, SARS-CoV-2 spike-specific CD4+ T cell phenotyping revealed a Th2 dominant response to booster dose in natalizumab-treated pwMS (p=0.0485) but not fumarate-treated pwMS. CONCLUSION/UNASSIGNED:pwMS treated with natalizumab or fumarates exhibit similarly robust and durable SARS-CoV-2 specific T cell and humoral responses following vaccination and booster dose. DMT-treated pwMS showing comparable responses to healthy individuals following initial vaccination supports the notion that treatment with these specific DMTs does not diminish strong, long-lasting immunity conferred by COVID-19 vaccination, despite the phenotypic differences modulated by each therapy.

The yellow fever (YF) vaccine is known to elicit strong CD8+ T cell immune responses, predominantly targeting an HLA-A2-restricted immunodominant epitope within the NS4B protein. We aimed to characterize these cells and explore their functional utility when redirected to a target unrelated to YF. We performed single-cell TCR and mRNA sequencing on YF-specific CD8+ T cells from five vaccinated donors, 21 days post-vaccination to characterize their clonal diversity and transcriptional profiles. An HLA-restricted bispecific T cell engager, Redirector of Vaccine-induced Effector Responses (RoVER), was used to redirect YF-specific CD8+ T cells toward target cells including HIV-1-infected CD4+ T cells and CD19+ B cells. The vaccine elicited a robust CD8+ T cell response characterized by a diverse set of differentiated YF-specific cells spanning activated naïve-like, memory and effector phenotypes. Despite biases in the TCR repertoires, antigen-specificity did not promote the development of unique phenotypes following vaccination. Our findings suggest phenotypic overlap causing redundancy in CD8+ T cell immunity across YF epitopes. Using the recombinant RoVER, YF-specific CD8+ T cells could be redirected toward other target cells, leading to efficient target cell elimination. Together, this study enhances our understanding of the cytotoxic T cell response to viral infections and its implications for vaccine development, while also supporting the development of personalised immunotherapies tailored to individual HLA alleles.

Response to immune checkpoint inhibitors (ICIs) in metastatic melanoma (MM) varies among patients, and current baseline biomarkers predicting treatment outcomes are limited. As mitochondrial (MT) metabolism has emerged as an important regulator of host immune function, we explored the association of host MT genetics (MT haplogroups) with ICI efficacy in 1,225 ICI-treated patients with MM from the clinical trial CheckMate-067 and the International Germline Immuno-Oncology Melanoma Consortium. We discovered and validated significant associations of MT haplogroup T (HG-T) with resistance to anti-programmed cell death protein-1-based ICI (both single-agent and combination) and have shown that HG-T is independent from established tumor predictors. We also found that patients belonging to HG-T exhibit a unique nivolumab-resistant baseline peripheral CD8⁺ T cell repertoire compared to other MT haplogroups, providing, to our knowledge, the first link between MT inheritance, host immunity and ICI resistance. The study proposes a host blood-based biomarker with stand-alone clinical value predicting ICI efficacy and points to an ICI-resistance mechanism associated with MT metabolism, with clinical relevance in immuno-oncology.

The pancreas tumor microbiota may influence tumor microenvironment and influence survival in early-stage pancreatic ductal adenocarcinoma (PDAC); however, current studies are limited and small. We investigated the relationship of tumor microbiota to survival in 201 surgically resected patients with localized PDAC (Stages I-II), from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) cohorts. We characterized the tumor microbiome using RNA-sequencing data. We examined the association of the tumor microbiome with overall survival (OS), via meta-analysis with the Cox PH model. A microbial risk score (MRS) was calculated from the OS-associated microbiota. We further explored whether the OS-associated microbiota is related to host tumor immune infiltration. PDAC tumor microbiome α- and β-diversities were not associated with OS; however, 11 bacterial species, including species of Gammaproteobacteria, confirmed by extensive resampling, were significantly associated with OS (all Q < 0.05). The MRS summarizing these bacteria was related to a threefold change in OS (hazard ratio = 2.96 per standard deviation change in the MRS, 95% confidence interval = 2.26-3.86). This result was consistent across the two cohorts and in stratified analyses by adjuvant therapy (chemotherapy/radiation). Identified microbiota and the MRS also exhibited association with memory B cells and naïve CD4⁺ T cells, which may be related to the immune landscape through BCR and TCR signaling pathways. Our study shows that a unique tumor microbiome structure, potentially affecting the tumor immune microenvironment, is associated with poorer survival in resected early-stage PDAC. These findings suggest that microbial mechanisms may be involved in PDAC survival, potentially informing PDAC prognosis and guiding personalized treatment strategies.IMPORTANCEMuch of the available data on the PDAC tumor microbiome and survival are derived from relatively small and heterogeneous studies, including those involving patients with advanced stages of pancreatic cancer. There is a critical knowledge gap in terms of the tumor microbiome and survival in early-stage patients treated by surgical resection; we expect that advancements in survival may initially be best achieved in these patients who are treated with curative intent.

The extracellular molecular organization of the individual CD3 subunits around the αβ T cell receptor (TCR) is critical for initiating T cell signaling. In this study, we incorporate photo-crosslinkers at specific sites within the TCRα, TCRβ, CD3δ, and CD3γ subunits. Through crosslinking and docking, we identify a CD3ε'-CD3γ-CD3ε-CD3δ arrangement situated around the αβTCR in situ within the cell surface environment. We demonstrate the importance of cholesterol in maintaining the stability of the complex and that the 'in situ' complex structure mirrors the structure from 'detergent-purified' complexes. In addition, mutations aimed at stabilizing extracellular TCR-CD3 interfaces lead to poor signaling, suggesting that subunit fluidity is indispensable for signaling. Finally, employing photo-crosslinking and CD3 tetramer assays, we show that the TCR-CD3 complex undergoes minimal subunit movements or reorientations upon interaction with activating antibodies and pMHC tetramers. This suggests an absence of 'inactive-active' conformational states in the TCR constant regions and the extracellular CD3 subunits, unlike the transmembrane regions of the complex. This study contributes a nuanced understanding of TCR signaling, which may inform the development of therapeutics for immune-related disorders.

OBJECTIVES/OBJECTIVE:(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/CONCLUSIONS: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.

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-D^b/Hsf2 p.K72N_68-76, as well as its cognate TCR 47BE7. Through cellular, molecular and structural studies we demonstrate that the p.K72N mutation enhances H2-D^b 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.

BACKGROUND:We previously reported a higher incidence of a pathogenic germline variant in the kinase insert domain receptor (KDR) in melanoma patients compared to the general population. Here, we dissect the impact of this genotype on melanoma tumor growth kinetics, tumor phenotype, and response to treatment with immune checkpoint inhibitors (ICIs) or targeted therapy. METHODS:The KDR genotype was determined and the associations between the KDR Q472H variant (KDR-Var), angiogenesis, tumor immunophenotype, and response to MAPK inhibition or ICI treatment were examined. Melanoma B16 cell lines were transfected with KDR-Var or KDR wild type (KDR-WT), and the differences in tumor kinetics were evaluated. We also examined the impact of KDR-Var on the response of melanoma cells to a combination of VEGFR inhibition with MAPKi. RESULTS:= 0.0159), and KDR-Var cells showed synergistic cytotoxicity to the combination of dabrafenib and lenvatinib. CONCLUSIONS:Our data demonstrate a role of germline KDR-Var in modulating melanoma behavior, including response to treatment. Our data also suggest that anti-angiogenic therapy might be beneficial in patients harboring this genotype, which needs to be tested in clinical trials.

Patients with preexisting autoimmune disease (pAID) are generally excluded from clinical trials for immune checkpoint inhibitors (ICIs) for cancer due to concern of flaring pAID. In this multi-center, retrospective observational study, we compared safety of ICI combination (two ICI agents) versus monotherapy in cancer patients with pAIDs. The primary outcome was time to AEs (immune-related adverse events (irAEs) and/or pAID flares), with progression-free survival (PFS) and overall survival as secondary outcomes. Sixty-four of 133 patients (48%) received ICI combination and 69 (52%) monotherapy. Most had melanoma (32%) and lung cancer (31%). Most common pAIDs were rheumatic (28%) and dermatologic (23%). Over a median follow-up of 15 months (95%CI, 11-18 mo), the cumulative incidence of any-grade irAEs was higher for combination compared to monotherapy (subdistribution hazard ratio (sHR) 2.27, 95%CI 1.35-3.82). No statistically significant difference was observed in high-grade irAEs (sHR 2.31 (0.95-5.66), P = .054) or the cumulative incidence of pAID flares. There was no statistically significant difference for melanoma PFS between combination versus monotherapy (23.2 vs. 17.1mo, P = .53). The combination group was more likely to discontinue or hold ICI, but > 50% of the combination group was still able to continue ICI therapy. No treatment-related deaths occurred. In our cohort with pAIDs, patients had a tolerable toxicity profile with ICI combination therapy. Our results support the use of ICI combination if deemed necessary for cancer therapy in patients with pAIDs, since the ICI toxicities were comparable to monotherapy, able to be effectively managed and mostly did not require ICI interruption.