Requirements of class II-mediated B cell differentiation for class II cross-linking and cyclic AMP

Cells of the mouse B cell clone, CH12.LX, receive Ag-dependent differentiative signals through their surface membrane class II molecules. The present study was performed to determine the role of class II cross-linking and cAMP in the successful delivery of these signals. Delivery of differentiative signals by anti-Ek mAb was increased by further cross-linking with a secondary anti-isotype antibody. Intact or (Fab')2, but not Fab forms of anti-Ek successfully delivered the Ag-dependent differentiative signal. Inability of monovalent Fab fragments to deliver the signal could not be attributed to an inability to adequately bind Ek molecules. The requirement for cAMP for class II-mediated signaling was also examined, because previous studies have implicated elevated cAMP levels as necessary for class II signaling. Both Ag-dependent, Ek-mediated differentiation and the Ek-mediated inhibition of Ag-independent LPS-induced differentiation were inhibited by the adenyl cyclase inhibitor 2'5'ddA, although elevation of cAMP was not in itself sufficient to deliver the differentiative signal. Inhibition of LPS-induced differentiation could be mediated by mAb binding to either Ek, Abk, or Abb on CH12.LX or an Ab-bearing transfectant, CH12.ABB1. This inhibition was abrogated by 2'5'ddA in the case of Ek or Abb, both of which deliver Ag-dependent differentiative signals to CH12.LX cells. In the case of Abk, which does not deliver such signals to CH12.LX, 2'5'ddA did not abrogate anti-Abk-mediated inhibition of the LPS response. The effects of 2'5'ddA were reversed by the cAMP analog, dibutyryl cAMP, and Ag-dependent-induced differentiation of CH12.LX or CH12.ABB1 was accompanied by an increase in cAMP levels.