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 ly mphocytes with a human/mouse heteromyeloma line. In addition to a fusion pr otocol optimised for the fusion partners, the activation of B lymphocytes i s crucial for fusion and hybrid efficiency. In this study, we initially tre ated peripheral blood mononuclear cells (PBMC) from normal blood donors wit h a large panel of known stimulants and determined the yield of human antib ody-secreting hybridomas after electrofusion with the heteromyeloma cell li ne H73C11; 3- to 5-day incubation with phytohaemagglutinin L (PHA-L) result ed in the highest number of secreting hybrids. In a second set of experimen ts, PBMC were depleted from various cell populations, including CD14(+) mon ocytes, 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 experime nts, CD19(+) B lymphocytes were identified as the major source of secreting hybridomas. For optimal fusion efficiency, CD19(+) B cells were shown to r equire direct physical contact with other cell populations, most probably T lymphocytes, during the stimulation process. Our data highlight the import ance of an adequate stimulation prior to electrofusion and may be helpful t o further facilitate the development of human monoclonal antibodies. (C) 20 01 Elsevier Science B.V. All rights reserved.