Latent EBV reactivation drives aberrant B-cell proliferation during ex vivo tumor-infiltrating lymphocyte expansion from EBV-negative rectal cancer tumor tissue.

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 and safety — areas that remain underexplored. Here, we characterized a spontaneously arising wild-type EBV-transformed B cell line, lcl_burn0214, established during ex vivo culture of a tumor specimen from a rectal cancer patient who was EBV-negative. 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 revealed that lcl_burn0214’s gene expression closely resembles that of lymphoblastoid cells (r = 0.88) rather than malignant B lymphomas. In immunocompromised mice, lcl_burn0214 exhibited limited tumorigenicity. Autologous TILs were negative for B cells and exhibited a strong interferon-gamma response in the ELISPOT assay during coculture with lcl_burn0214 or with the EBV peptide pool (1000–1800 spots per million of TILs). Our findings underscore the importance of monitoring TIL products for EBV-positive B cell contamination during manufacturing to ensure safety and therapeutic efficacy even for EBV-negative patients.