In vitro immunization of naive mouse B cells: establishment of IgM secreting hybridomas specific for souble protein or hapten from B cells cultured on CD40 ligand transfected mouse fibroblasts.

CD40 has been shown to play an important role in the regulation of B cell survival, proliferation and Ig class switching. The natural partner for CD40 is CD40 ligand, gp 39, which is transiently expressed on activated T cells. In vitro, CD40 ligation leads to polyclonal B cell proliferation and, in the presence of appropriate cytokines, to the secretion of Ig of various isotypes. In the present study we show that naive B cells cultured in vitro on CD40L-transfected mouse fibroblasts in the presence of two different soluble antigens (beta-galactosidase and phenyloxazolone coupled to ovalbumin) can be specifically immunized as shown by direct single cell Elispot assays or after establishment of B cell hybridomas. However, under the conditions of in vitro immunization used, all hybridomas analysed produced specific IgM antibodies only and we failed to detect cells that had switched to other isotypes. The data suggests that CD40 ligation can be used for efficient in vitro immunization against soluble antigens for IgM production but that CD40 signals even in conjunction with cytokines are insufficient to induce high rate switching.