B-LCL-HROC69 Cells

B-LCL-HROC69 is an Epstein-Barr virus (EBV)-immortalized B lymphoblastoid cell line established from tumor-infiltrating B cells (TiBc) isolated from a primary colorectal carcinoma specimen designated HROC69. The parental tumor originated from an adult male patient with right-sided colorectal carcinoma of conventional sporadic type and advanced stage disease. B cells were isolated from freshly resected tumor tissue and immortalized ex vivo using supernatant from the EBV-producing B95/8 marmoset cell line in the presence of cyclosporin A to suppress T- and NK-cell outgrowth. Outgrowth of EBV-transformed B-cell clones typically occurred within several weeks, and clonality was confirmed by immunoglobulin heavy- and light-chain gene rearrangement analysis using BIOMED-2 multiplex PCR protocols.

B-LCL-HROC69 secretes immunoglobulin A (IgA), as determined by isotype-specific ELISA of long-term culture supernatants. In contrast to several IgG-producing TiBc lines established in parallel, IgA derived from HROC69 was not further characterized for tumor cell binding in the initial functional screening assays. Importantly, no spontaneous outgrowth of B-cell cultures occurred in the absence of exogenous EBV, indicating that the immortalization is an in vitro event rather than the consequence of latent EBV infection in vivo. B-LCL-HROC69 therefore represents a monoclonal, antigen-experienced tumor-infiltrating B-cell model suitable for investigating humoral immune responses within the colorectal carcinoma microenvironment and for the potential identification of tumor-associated antigens recognized by locally expanded B-cell clones.