Spontaneous EBV-positive B cell line from rectal tumor: characterization, interaction with autologous tumor-infiltrating T lymphocytes, comparison with B lymphoma cell lines

Recent studies underscore herpesvirus-associated risks, especially HHV6 and Epstein–Barr virus (EBV), in CAR-T and tumor-infiltrating lymphocyte (TIL) therapies, partly due to ex vivo cell manipulation. EBV-driven B cell outgrowth can occur during TIL expansion, and EBV-transformed B cells may exert immunomodulatory functions, potentially impacting TIL therapy efficacy — areas that remain underexplored. Here, we characterized a spontaneously arising wild-type EBV-transformed B cell line, lcl_burn0214, derived from rectal cancer tumor tissue during ex vivo culture. The lcl_burn0214 cell line has undergone 70 passages and was confirmed as a monoclonal B cell line of patient origin via HLA genotyping, immunophenotyping (CD19, CD20), B cell receptor clonotype analysis. RNA-seq analysis comparing EBV-positive and -negative B lymphoma and lymphoblastoid cell lines revealed that lcl_burn0214’s gene expression closely resembles that of lymphoblastoid cells rather than malignant B lymphomas. In immunocompromised mice, lcl_burn0214 exhibited limited tumorigenicity. The cell line expressed immune checkpoint genes (CD274, CSF1, TNFSF4). Nevertheless, autologous TILs were negative for B cells and exhibited specific anti-EBV activity, as demonstrated by the interferon-gamma response in the ELISPOT assay during coculture with lcl_burn0214. Our findings underscore the importance of monitoring TIL products for EBV-positive B cell contamination during manufacturing to ensure safety and therapeutic efficacy.