Abstract Merkel cell carcinoma (MCC) is an aggressive human skin cancer mainly induced by Merkel cell polyomavirus (MCPyV). While the virus is common in humans, the unlikely event of oncogenic transformation occurs only rarely, and development of MCC is furthermore primarily seen in immunosuppressed and elderly. Indicating, that despite oncogenic potential further lack of immunosurveillance is necessary for cancer development. Two mutation events, often inflicted by UV light, allow the clonal integration of the viral genome into the host genome and the translation of the two viral oncogenes large T (LTA) and small T antigen (STA). The importance of immune recognition for clearing disease was segmented by the encouraging results using anti-PD1 for treatment of metastatic MCC. Recently, the first PD-1 checkpoint inhibitor (avelumab) became FDA approved. To further understand the mechanism of action, and to direct T-cell responsiveness specifically to MCC, there is a great interest to identify T-cell epitopes in MCPyV. To date, several T-cell epitopes derived the MCPyV-derived proteins LTA, STA and viral capsid protein1 (VP1) have been described. However, only restricted to a limited number of HLA types (HLA-A01, -A02, -A03, -A11, -A24 and B07). Here we aim to expand the knowledge about T-cell epitopes by including a broader range of HLA restrictions (HLA-B08, -B35 and -B44). Potential T-cell epitopes were selected by in-silico prediction. Given a total of 148 peptides across the nine MHC class I. Peripheral blood mononuclear cells (PBMC) from 49 patients were analyzed using a platform consisting of a magnetic-based enrichment of MHC multimer binding cells, which allowed detection of low frequency T-cell clones after 2-3 weeks of culturing. Followed by detection of T-cell reactivity against the MCPyV-derived epitopes using combinatorial color encoding of MHC multimer. Using this strategy, we identified 60 MCPyV-directed T-cell responses against 34 different peptides in PBMC from 31 of the patients. Furthermore, three patients’ tumor-infiltrating lymphocytes (TIL) were analyzed directly ex vivo for detection of T-cell reactivity against the MCPyV-derived peptides. We identified four MCPyV-directed T-cell responses against four different peptides. Importantly, epitope targets embedded in the LTA and STA proteins were solely observed in the patient samples while T-cell recognition of the capsid protein VP1 was observed in both patients and healthy donors. For selected LTA/STA-derived epitopes processing and presentation were demonstrated in MCC tumor cell lines, as well as the functional capacity of LTA/STA responsive CD8 T-cells. The identification of novel LTA/STA-derived epitopes will facilitate the use of targeted T-cell therapy to enhanced MCC recognition and clearance in combination with checkpoint inhibition. Such strategies should be tailored according to the patient’s HLA type, and hence expanding the epitope repertoire with additional HLA restrictions would benefit a larger cohort of MCC patients. Citation Format: Ulla Kring Hansen, Rikke Lyngaa, Per Thor Straten, Jürgen C. Becker, Candice D Church, Paul Nghiem, Sine Reker Hadrup. T-cell recognition of large T and small T antigen in Merkel cell polyomavirus-associated cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B020.