IN-VITRO IMMUNIZATION OF NAIVE MOUSE B-CELLS - ESTABLISHMENT OF IGM SECRETING HYBRIDOMAS SPECIFIC FOR SOLUBLE-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 c ell survival, proliferation and Ig class switching, The natural partne r for CD40 is CD40 ligand, gp 39, which is transiently expressed on ac tivated T cells, In vitro, CD40 ligation leads to polyclonal B cell pr oliferation and, in the presence of appropriate cytokines, to the secr etion of Ig of various isotypes. In the present study we show that nai ve B cells cultured in vitro on CD40L-transfected mouse fibroblasts in the presence of two different soluble antigens (beta-galactosidase an d phenyloxazolone coupled to ovalbumin) can be specifically immunized as shown by direct single cell Elispot assays or after establishment o f B cell hybridomas. However, under the conditions of in vitro immuniz ation used, all hybridomas analysed produced specific IgM antibodies o nly and we failed to detect cells that had switched to other isotypes. The data suggest that CD40 ligation can be used for efficient in vitr o immunization against soluble antigens for IgM production but that CD 40 signals even in conjunction with cytokines are insufficient to indu ce high rate switching.