B-CELL DIFFERENTIATION-INDUCED BY HELPER T-CELL MEMBRANES - EVIDENCE FOR SEQUENTIAL ISOTYPE SWITCHING AND A REQUIREMENT FOR LYMPHOKINES DURING PROLIFERATION

Small dense B cells are stimulated to proliferate by membranes prepare d from activated helper T (Th) cell clones. In combination with Th2 ly mphokines, Th membranes stimulate B cells to differentiate to secrete predominantly immunoglobulin (Ig)M, IgG1 and IgE. The activity in Th m embrane requires the expression of CD40 ligand by the T cells, and ini tiation of the B cell response occurs through the ligation of CD40 on the B cell surface. We have further characterized the properties of th e B cell response and found that Th membranes stimulated B cell prolif eration and Ig secretion in a cell density independent manner and the majority of the stimulated B cells underwent a limited number of divis ion rounds between day 2 and 5 of culture. IgM-secreting cells appeare d in culture by day 3 and increased to reach a maximum, comprised of 3 0 % of the viable cells, on day 5. IgG1-secreting cells appeared 12-24 h after IgM-secreting cells, and IgE-secreting cells did not appear u ntil day 5. These data are consistent with a sequential model of isoty pe switching related to cell division. As lymphokines were absolutely required for antibody production we were able to determine when during culture they were essential. Lymphokines needed to be present prior t o and during B cell proliferation for differentiation to Ig-secreting cells to proceed. This period corresponded closely to the time of maxi mum DNA synthesis. Consistent with sequential switching of Ig isotypes , differentiation to IgM secretion required the shortest exposure to l ymphokines and IgE the longest. These experiments strongly suggest tha t the lymphokine-induced commitment to differentiate is made before DN A synthesis begins, although the lymphokine-delivered signals are nece ssary during DNA synthesis to support Ig class switching.