γδ T cells in Merkel cell carcinomas have a proinflammatory profile prognostic of patient survival

Abstract

Merkel cell carcinomas (MCC) are immunogenic skin cancers associated with viral infection or UV-mutagenesis. To study T-cell infiltrates in MCC, we analyzed 58 MCC lesions from 39 patients using multiplex-immunohistochemistry/immunofluorescence (m-IHC/IF). CD4+ or CD8+ T cells comprised the majority of infiltrating T lymphocytes in most tumors. However, almost half of the tumors harbored prominent CD4/CD8 double negative (DN) T-cell infiltrates (>20% DN T cells) and in 12% of cases, DN T cells represented the majority of T cells. Flow cytometric analysis of single-cell suspensions from fresh tumors identified DN T cells as predominantly Vδ2- γδ T cells. In the context of γδ T-cell inflammation, these cells expressed PD1 and LAG3, which is consistent with a suppressed or exhausted phenotype, and CD103, which indicates tissue-residency. Furthermore, single-cell RNA sequencing (scRNA-seq) identified a transcriptional profile of γδ T cells suggestive of proinflammatory potential. T-cell receptor (TCR) analysis confirmed clonal expansion of Vδ1 and Vδ3 clonotypes and functional studies using cloned γδ TCRs demonstrated restriction of these for CD1c and MR1 antigen-presenting molecules. Based on a 13-gene γδ T cell-signature derived from scRNA-seq analysis, gene-set enrichment on bulk RNA-seq data showed a positive correlation between enrichment scores and DN T-cell infiltrates. An improved disease-specific survival was evident for patients with high enrichment scores and complete responses to anti-PD1/PD-L1 treatment were observed in three of four cases with high enrichment scores. Thus, γδ T-cell infiltration may serve as a prognostic biomarker and should be explored for therapeutic interventions.