B-CELL ACTIVATION VIA CD40 IS REQUIRED FOR SPECIFIC ANTIBODY-PRODUCTION BY ANTIGEN-STIMULATED HUMAN B-CELLS

Costimulatory signals provided by T cells are required for B cells to produce specific antibody (Ab) to T-dependent antigen (Ag) bacteriophage phix 174. In this study, we demonstrate that if cultured in the presence of anti-CD40, interleukin 10 (IL-10), and Ag, purified B cells can produce antiphage Ab in quantities comparable to those synthesized by B cells cocultured with Ag and T cells. Isotypes produced by B cells in this culture system correspond to those observed in sera of B cell donors. Culture of immunoglobulin (Ig)D- and IgD+ B cells reveals that Ag-induced production of antiphage Ab is restricted to IgD- subset of B cells. In the absence of Ag, anti-CD40/IL-10-stimulated B cells produce only minute amounts of antiphage Ab, indicating that Ag stimulation is indispensable and provides a signal that is synergistic with anti-CD40 and IL-10. Addition of a soluble form of the CD40 ligand (sgp39) to the culture system has a similar effect on specific Ab synthesis as anti-CD40; addition of the soluble construct, CD40 Ig, known to inhibit gp39/CD40 interaction, suppresses in vitro antiphage Ab production by Ag exposed peripheral blood mononuclear cells. Finally, in vivo requirement of gp39/CD40 interaction for specific Ab production was demonstrated by the finding that activated T cells from patients with x-linked hyper IgM syndrome express functionally defective gp39 and respond with depressed Ab titers and fail to switch from IgM to IgG after multiple phage immunizations. These observations illustrate that in vitro and possibly in vivo Ag-specific Ab synthesis requires the presence of Ag and IL-10, and activation signals via CD40.