CD19⁺ B lymphocytes are the major source of human antibody-secreting hybridomas generated by electrofusion

Human monoclonal antibodies may be generated by electrofusion of human B lymphocytes with a human/mouse heteromyeloma line. In addition to a fusion protocol optimised for the fusion partners, the activation of B lymphocytes is crucial for fusion and hybrid efficiency. In this study, we initially treated peripheral blood mononuclear cells (PBMC) from normal blood donors with a large panel of known stimulants and determined the yield of human antibody-secreting hybridomas after electrofusion with the heteromyeloma cell line H73C11; 3- to 5-day incubation with phytohaemagglutinin L (PHA-L) resulted in the highest number of secreting hybrids. In a second set of experiments, PBMC were depleted from various cell populations, including CD14⁺ monocytes, CD8⁺ T lymphocytes, and CD2⁺ T cells, respectively. Undepleted PBMC stimulated with PHA-L were shown to give rise to the highest number of secreting hybridomas when subjected to electrofusion, whereas depletion of CD2⁺ T lymphocytes greatly reduced the yield. In a final set of experiments, CD19⁺ B lymphocytes were identified as the major source of secreting hybridomas. For optimal fusion efficiency, CD19⁺ B cells were shown to require direct physical contact with other cell populations, most probably T lymphocytes, during the stimulation process. Our data highlight the importance of an adequate stimulation prior to electrofusion and may be helpful to further facilitate the development of human monoclonal antibodies.