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Background Merkel cell carcinoma (MCC) is an aggressive skin cancer with roughly 80% of cases driven by the Merkel cell polyomavirus. Despite frequent CD8+ T cell infiltration, MCC tumors often evade immune-mediated killing. While anti-PD-1/PD-L1 therapies are efficacious, MCC patients resistant or relapsing after immunotherapy have a poor prognosis. While many immune regulatory mechanisms are known, and there is evidence of T cell dysfunction in MCC, few studies have performed in-depth analyses of the tumor microenvironment. Here, we use 15-color multiplex immunofluorescence (mIF) and spatial transcriptomics to interrogate immune regulatory pathways in MCC tumors.Methods We analyzed seven resected MCC tumors from treatment-naïve patients using spatial biology approaches. We performed mIF using the Rarecyte Orion™ system (CD3e, CD4, CD8a, CD11b, CD20, CD56, CD163, FoxP3, GzmB, Ki-67, LAG3, Pan-CK, PD-1, PD-L1, and SOX10). Data was analyzed using QuPath. We also performed spatial transcriptomics using the 10X Genomics Visium platform. The data were analyzed using the Seurat package in R.Results The mIF analyses of MCC revealed prominent infiltration of FoxP3+ regulatory T cells (Tregs). We observed varying degrees of tertiary lymphoid structure (TLS) formation, comprising CD20+ B cells, Tregs, non-Treg CD4+ T cells, CD8+ T cells, CD11b+ myeloid cells, and CD163+ macrophages. In the TLS, CD11b+ cells expressed PD-L1, and non-Treg CD4 T cells expressed PD-1. Outside the TLS, PD-L1 expression was found in stromal cells but not on the tumor cells. We observed frequent examples of close proximity between PD1+CD8+ T cells and PD-L1+CD163 macrophages. LAG3 expression was mainly restricted to CD8 T cells outside TLS and peritumoral and intra-tumoral regions. We also performed spatial transcriptomics and observed a similar pattern of expression consistent with the immunofluorescence staining. We observed upregulation of multiple inhibitory pathways, including TIGIT, CTLA-4, and LAG3. Our data also revealed that some T cells express cutaneous lymphocyte-associated antigen (CLA) and CD39. Elevated frequencies of CD39+CLA+ CD8+ T cells in the periphery of MCC patients has been previously reported to be associated with improved response to PD-1 therapy, and notably this population was enriched for tumor-reactive T cells. Our analyses are ongoing to identify immune regulatory pathways and their association with different lymphocyte populations.Conclusions Our studies reveal a tumor microenvironment in MCC characterized by heavy immune infiltration and associated with expression of multiple immune inhibitory molecules. Our analyses are ongoing and may suggest opportunities for augmenting immune-based approaches and achieving improved patient outcomes.Acknowledgements Research reported in this Abstarct was supported by The Ohio State University Comprehensive Cancer Center (OSUCCC) and the National Institutes of Health (NIH) under grant P30CA016058. This research was made possible through resources, expertise, and support provided by the Pelotonia Institute for Immuno-Oncology (PIIO), which is funded by the Pelotonia community and the OSUCCC. We thank the PIIO and the Immune Monitoring and Discovery Platform for 10X Genomics- Visium experiment.