Merkel Cell Polyomavirus-Specific CD8+ and CD4+ T-cell Responses Identified in Merkel Cell Carcinomas and Blood

Abstract

PURPOSE Merkel cell polyomavirus (MCPyV) is prevalent in the general population, integrates into most Merkel cell carcinomas (MCCs) and encodes oncoproteins required for MCC tumor growth. We sought to characterize T-cell responses directed against viral proteins that drive this cancer as a step toward immunotherapy. METHODS Intracellular cytokine cytometry, IFN-γ-ELISPOT, and a novel HLA-A*2402-restricted MCPyV tetramer were used to identify and characterize T-cell responses against MCPyV oncoproteins in tumors and blood of MCC patients and control subjects. RESULTS We isolated virus-reactive CD8 or CD4 T cells from MCPyV-positive MCC tumors (2 of 6), but not from virus-negative tumors (0 of 4). MCPyV-specific T-cell responses were also detected in the blood of MCC patients (14 of 27) and control subjects (5 of 13). These T cells recognized a broad range of peptides derived from capsid proteins (2 epitopes) and oncoproteins (24 epitopes). HLA-A*2402-restricted MCPyV oncoprotein processing and presentation by mammalian cells led to CD8-mediated cytotoxicity. Virus-specific CD8 T cells were markedly enriched among tumor-infiltrating lymphocytes as compared to blood, implying intact T-cell trafficking into the tumor. While tetramer-positive CD8 T cells were detected in the blood of 2 of 5 HLA-matched MCC patients, these cells failed to produce IFN-γ when challenged ex vivo with peptide. CONCLUSIONS Our findings suggest that MCC tumors often develop despite the presence of T cells specific for MCPyV T-Ag oncoproteins. The identified epitopes may be candidates for peptide-specific vaccines and tumor- or virus-specific adoptive immunotherapies to overcome immune evasion mechanisms in MCC patients.

Rationale For Immune-Based Therapies in Merkel Polyomavirus-Positive and -Negative Merkel Cell Carcinomas

“…MCPyV-specific T cells isolated from MCC tumors and peripheral blood have been shown to express elevated levels of the inhibitory markers PD-1 and TIM-3 relative to control CMV-or EBV-specific cells (Figure 2: process 'E') [49]. Additionally, MCPyV-specific CD8 T cells often have a limited ability to secrete the effector cytokine IFN-γ following antigenic stimulation and TILs within MCC tumors have markedly lower expression of the early activation marker CD69 relative to T cells isolated from normal skin, supporting the notion that these cells are dysfunctional and exhausted [43,69,70].…”

“…In an effort to identify MCPyV-specific T cells, Iyer et al described an initial set of 24 epitopes within the persistently expressed region of the T-antigen oncoproteins [43]. Five of the 24 were recognized specifically by CD4 T cells and subsequently two additional CD4 epitopes have been reported [43][44][45].…”

“…Furthermore, expression of genes encoding granzyme A, B, H and K, CCL19, lymphocyte activation genes (SLAMF1 and NKG2D) and CD8α are associated with favorable prognoses, independent of stage [46]. To date, 17 MCPyV-specific CD8 epitopes have been identified, for which 14 HLA-I tetramers have been generated, enabling functional and phenotypic analysis [43,45,49,50]. Importantly, while robust CD8 responses have been associated with improved outcome in MCC, only 4-18% of MCC patients present with significant CD8 infiltration, suggesting that most MCCs block intratumoral CD8 infiltration as a means of evading immune detection [46,51].…”

“…Th1 CD4s also serve to recruit NK and type-I macrophages (proinflammatory) to the tumor site, thereby orchestrating robust antitumor immunity [72]. In the setting of MCC, secretion of Th1 and Th2 type cytokines by bulk intratumoral CD4 T cells was observed from one MCC patient [43]. Whether a significant bias toward a particular subtype occurs in MCPyV-specific CD4s or in additional MCC tumors has not been investigated.…”

Analysis of Spontaneous Tumor-Specific CD4 T-cell Immunity in Lung Cancer Using Promiscuous HLA-DR Telomerase-Derived Epitopes: Potential Synergistic Effect with Chemotherapy Response

“…For the recall response against viruses, cells were similarly cultured with the mix of 32 peptides from cytomegalovirus, influenza virus, and Epstein-Barr virus (CTL). After 1 week of cell culture, the presence of UCP-specific T cells was measured by IFN-g ELISPOT as detailed above (30)(31)(32).…”