Characterization of the roles of TNF receptor-associated factor 6 in CD40-mediated B lymphocyte effector functions

Signaling through CD40 in B cells leads to B cell proliferation, Ig and IL- 6 secretion, isotype switching, and up-regulation of surface molecules, TNF receptor-associated factor (TRAF) proteins associate with the cytoplasmic tail of CD40 and act as adapter molecules. Of the six TRAFs identified to d ate, TRAFs 2, 3, 5, and 6 are reported to associate directly with the cytop lasmic tail of CD40, but previous studies have principally examined transie nt overexpression of TRAF6 in cells that do not normally express CD40. Thus , we examined the role of TRAF6 in CD40-mediated B lymphocyte effector func tions using two approaches. We produced and stably expressed in mouse B cel l lines a human CD40 molecule with two cytoplasmic domain point mutations ( hCD40EEAA); this mutant fails to bind TRAF6, while showing normal associati on,vith TRAFs 2 and 3, We also inducibly expressed in B cells a transfected "dominant-negative'' TRAF6 molecule which contains only the C-terminal TRA F-binding domain of TRAF6. Using both molecules, we found that TRAF6 associ ation with CD40 is important for CD40-induced IL-6 and Ig secretion, and th at TRAF6 mediates its effects on CD40-stimulated Ig secretion principally t hrough its effects on IL-6 production by the B cell. TRAF6 association with CD40 was also found to be important for B7-1 up-regulation, but not for up -regulation of other surface molecules, Interestingly, however, although we could show TRAF6 dependent CD40-mediated activation of NF-kappa B in 293 k idney epithelial cells, no such effect was seen in B cells, suggesting that TRAF6 has cell-type-specific functions.