Generation of heterohybridomas secreting human immunoglobulins; pokeweed mitogen prestimulation is highly effective but phytohemagglutinin drives most B cells into apoptosis

Abstract

Human monoclonal antibodies have commonly been generated by forming hybridomas of stable lymphoblastoid cell lines and Epstein-Barr virus (EBV)-transformed human B cells that have been exposed to phytohaemagglutin (PHA)-stimulated T cells. However, this technique has predominantly given rise to IgM- but very rarely IgG- or IgA-producing clones. We now report that, regardless of prior EBV infection, pokeweed mitogen (PWM) stimulation of human peripheral blood mononuclear cells (PBMCs) generated much higher numbers of IgM-, IgA- and IgG-producing B cells than did stimulation with PHA. Fusion of PWM-stimulated PBMCs with a mouse myeloma cell line also gave rise to 7- to 12-fold higher numbers of IgG- and IgA-producing heterohybridomas than PBMCs that were prestimulated with PHA. Judged by Annexin V staining, stimulation with PHA induced a very high rate of B cell apoptosis within 24 h, whereas, even after 7 days, PWM stimulation only induced marginal B cell apoptosis. This should explain why PHA is much inferior to PWM in stimulating immunoglobulin (Ig) production in vitro and in generating immunoglobulin-producing human B cell hybridomas. It is concluded that PWM stimulation may greatly facilitate the generation of human monoclonal antibodies of all isotypes.