γδ T Cells in Merkel Cell Carcinomas Have a Proinflammatory Profile Prognostic of Patient Survival

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-IHC/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 V delta 2(-) gamma delta T cells. In the context of gamma delta T-cell inflammation, these cells expressed PD-1 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 gamma delta T cells suggestive of proinflammatory potential. T-cell receptor (TCR) analysis confirmed clonal expansion of V delta 1 and V delta 3 clonotypes, and functional studies using cloned gamma delta TCRs demonstrated restriction of these for CD1c and MR1 antigen-presenting molecules. On the basis of a 13-gene gamma delta 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-PD-1/PD-L1 treatment were observed in three of four cases with high enrichment scores. Thus, gamma delta T-cell infiltration may serve as a prognostic biomarker and should be explored for therapeutic interventions.