Background Merkel cell carcinoma (MCC) is a highly immunogenic, rare but aggressive skin cancer. Approximately 80% of MCCs are driven by the Merkel cell polyomavirus (MCPyV), with ~50% objective response rate to PD–(L)1 blockade. It is hypothesized that this exceptional response rate is likely due to CD8+ T cell responses against MCPyV T antigens. We and others have shown that mutant neoantigen–specific tumor–infiltrating lymphocytes (TIL) express a tissue resident memory (TRM) transcriptional signature characterized by high CXCL13 and ENTPD1 (CD39) levels and low IL–7 receptor (IL7R) levels. However, it is unknown if MCPyV–reactive TIL, which are specific for a foreign viral antigen, share transcriptomic features with mutant neoantigen–specific TIL found in other solid tumors, which typically recognize peptides generated from missense point mutations.

Methods We performed scTCRseq/RNAseq coupled with barcoded multimers against HLA–restricted MCPyV epitopes on CD8+ TIL from treatment–naïve metastatic lymph node lesions in 3 MCC patients. MCPyV–specific TIL were identified by barcoded multimer reads and distribution patterns. MCC data were integrated with scTCRseq/RNAseq data from 3 anti–PD–1–treated primary lung cancers in which neoantigen–specific TIL were detected previously (2,057 neoantigen–specific TIL from 51,799 CD8+ TIL).

Results From 3 MCC specimens, we generated single–cell transcriptomic data for 39,794 CD8+ TIL, among which 4,473 MCPyV–specific TIL were identified. Integrated UMAP showed both lung neoantigen– and MCPyV–specific TIL localized to TRM regions, with 82% of MCPyV–specific TIL found in clusters having high CD103 and ZNF683 (HOBIT) expression. Consistent with published reports on neoantigen–specific TIL, MCPyV–specific TIL also exhibited high CXCL13 and ENTPD1 and low IL7R expression. Despite sharing certain transcriptional features with neoantigen–specific TIL, MCPyV–specific TIL exhibited differential expression of key genes implicating their functional state. Relative to neoantigen–specific TIL, MCPyV–specific TIL expressed higher levels of CCR7, TCF7, GZMK, and, surprisingly, LAG3 (p<2.22e–16 for all) but not PDCD1 (PD–1). Importantly, the majority (53%) of all MCPyV–specific TIL co–expressed GZMK and LAG3. This transcriptional program has been associated with a highly proliferative stem–like pool capable of rapid antigen targeting upon immune checkpoint blockade (ICB).

Conclusions While TIL recognizing viral oncogenes vs mutant neoantigens displayed common features consistent with tissue residence, MCPyV–specific MCC TIL exhibited distinct transcriptional patterns compared to neoantigen–specific TIL from lung cancers. These differences, which may be influenced by treatment status and tumor site, have potential therapeutic implications. Further investigation of combined PD–(L)1 and LAG–3 inhibition in MCC may be warranted, considering the shared and unique TIL characteristics observed.