Improvement of EBV transformation and cloning efficiency of human B cells using culture supernatants from lymphoblastoid cell lines

Exposure of human B cells to Epstein-Barr virus (EBV) usually results in low frequencies of transformed cells. The transformed cells can be cloned poorly by limiting dilution, even when feeder cells are used. In recent years it has become clear that growth and antibody production of EBV-transformed cells are influenced by auto- and paracrine growth factors. Therefore, supernatants from the lymphoblastoid B cell lines JY and Raji were used as a source of growth factors to investigate their effect during EBV transformation of human B cells and consequently on cloning by limiting dilution of these transformed cells. Initial experiments to clone three established EBV-transformed B cell lines showed a strong increase in outgrowth of the number of cells in the presence of the supernatant (range: 1 per 2–8 of the originally plated cells) as compared to cells cultured without the supernatant (range: 1 per 17–100 of the originally plated cells). Transformation efficiencies of freshly isolated tonsil B cells were not influenced by the supernatant and were generally less than 1%. In contrast, transformation efficiency was increased up to 9.4% if B cells were both transformed and cultured in the presence of the supernatant. Cloning efficiencies increased if the cells used were transformed in the presence of the supernatant. Best results were seen when the supernatant was present during transformation and cloning of the cells. The presence of the supernatant during transformation and/or cloning of the B cells dramatically enhanced the number of B cells secreting IgG. Cloning of two established tonsil B cell lines resulted in a large number of B cell clones. Nineteen randomly chosen B cell clones were tested for their immunoglobulin and cytokine production patterns. It was found that neither one of these tested clones showed the same production pattern. IL-4 and IL-5 producing B cell clones were found. The secretion of IgG was correlated with the capacity to produce IL-1 (8 out of 10 clones), while IgM secretion was correlated with a lack of IL-1 production (4 out of 4 clones). These results indicate that supernatants derived from continuously growing B cell lines can be used to obtain large numbers of IgG-secreting transformed B cells. The use of these supernatants probably does not selectively favor the growth of a specific subpopulation of B cells.