Gewurz, BenjaminSchnabel, Julia2026-10-1920262026-10-192026Schnabel, Julia. 2026. Tumor-infiltrating B cells target intracellular and tumor-associated autoantigens in Merkel cell carcinoma. Doctoral Dissertation, Harvard University Graduate School of Arts and Sciences.32862771https://dash.harvard.edu/handle/1/42760629Tumor-infiltrating B cells (TIL-Bs) are emerging as important immune cells in immunotherapy response and clearance of tumors. However, the mechanisms and antigen specificities of TIL-Bs in the tumor microenvironment (TME) are not well understood. In this dissertation, I characterize TIL-Bs and identify their cognate antigens in Merkel cell carcinoma (MCC) to understand the question: what is an antigen to the adaptive immune system in the TME? MCC is a rare and aggressive neuroendocrine carcinoma of the skin caused by either Merkel cell polyomavirus (MCPyV) in virus-positive MCC (MCCP) or by high UV damage in virus-negative MCC (MCCN). Approximately half of patients with advanced or metastatic disease respond to immunotherapy (anti-PD1/PD-L1) as a first-line treatment regardless of viral status, suggesting strong immune activation within the TME. Recent studies in MCC identified that TIL-Bs correlate with immunotherapy response and are associated with tumor control. To investigate TIL-Bs in MCC, I combined publicly available MCC single-cell RNA sequencing (scRNAseq) datasets with my own dataset to create an MCC TIL-B dataset with 44 samples and 12,402 cells. Memory B cells make up the largest proportion of TIL-Bs in MCCP and MCCN and show strong evidence of a germinal center-derived response. I also identified that the proportion of plasma cells is significantly higher in responders to immunotherapy, suggesting antibodies could be assisting in the immune response to MCC. To determine the antigen specificity of TIL-Bs, I recombinantly expressed and characterized 36 antibodies from B cell receptors of TIL-Bs from an MCCP patient biopsy. Flow cytometry and immunoprecipitation mass spectrometry revealed most antibodies bound intracellular proteins that consisted of known autoantigens and one tumor-associated antigen. Known autoantigens consisted of tRNA synthetases, p97/VCP and RNA-binding proteins. These antibodies were derived from plasma cells and had evidence of moderate to high levels of somatic hypermutation (SHM), suggesting they were derived from a germinal center response. I further validated that several RNA-binding proteins, SND1 and DNA-PKcs, targeted by antibodies with high SHM were localized to the cell surface in MCC. This finding supports a potential mechanism for how autoantigens that are mostly characterized as intracellular could be exposed to TIL-Bs. Lysine-specific demethylase 1 (LSD1)-CoREST is an essential epigenetic regulator complex in MCCP that is transactivated by the viral oncoprotein small T antigen. This complex restricts neuroendocrine differentiation to maintain tumor identity and is a therapeutic vulnerability. I identified 2 distinct clonally expanded antibodies from plasma cells with low SHM that recognized LSD1-CoREST in complex with myelin transcription factor 1 (MYT1). These antibodies also shared the same V gene on the heavy chain (\textit{IGHV6-1}, V\textsubscript{H}6-1) and light chain \textit{(IGKV1-33}, V\textsubscript{K}1-33), which in combination with low SHM suggests a convergent, germline-encoded response to endogenous antigen. To further validate this observation, I used TRUST4 on my TIL-B dataset to computationally derive antibodies and performed BCR sequencing on additional patient tumor biopsies, selecting for antibodies with the V\textsubscript{H}6-1/V\textsubscript{K}1-33 pairing that ranged from low to high SHM. This resulted in the discovery of 5 additional antibodies, 1 computationally derived and 4 from BCR-seq, that recognized MYT1-LSD1-CoREST from 3 distinct MCCP tumor samples. This demonstrates that MYT1-LSD1-CoREST is a tumor-associated autoantigen that elicits a convergent B cell response in MCCP. In summary, this work challenges what constitutes an antigen in the TME and how TIL-Bs respond to these antigens. I show that germinal center-derived TIL-Bs target classical intracellular autoantigens and a novel tumor-associated autoantigen important for maintaining MCC identity, suggesting positive selection for autoantibodies in the TME that may contribute to anti-tumor immunity.application/pdfenAutoantibodiesAutoantigensCancer ImmunologyTumor-infiltrating B cellsV gene biasVirologyImmunologyBiologyThesis or Dissertation2026-10-190000-0001-6755-8672