Humoral immune and autoimmune responses are governed by the interactions of CD154 expressed on helper T cells and CD40 on B cells. Extensive efforts have been made to unravel how CD40 engagement transduces the diversity of signals to trigger B cell expansion and differentiation. Like many of the other TNF Receptor family members, it is believed that the recruitment of TNF Receptor Associate Factors (TRAFs) to the cytoplasmic domain of CD40 plays a critical role in regulating CD40 biology. This proposal focuses on the role that TRAFs, and other signaling elements play in regulating B cell function in vivo. We present a unique cohort of Tg/knock-out mice that express mutations in the cytoplasmic tail of CD40 which disrupt the interaction of the cytoplasmic domain with specific TRAFs. B cells from these mice will be studied in vitro (Specific Aim #1) and in vivo (Specific Aim #2) to address the causal relationships between signaling domains of the cytoplasmic tail of CD40 and the induction of in vitro and in vivo B cell biochemistry and function. Preliminary data shows that the loss in TRAF recruitment in B cells exerts minimal or no impact on early IgM and lgG responses in vivo, however, a major effect of TRAF recruitment is observed on the durability of humoral immune responses in vivo. While loss in TRAF recruitment results in defined lesions in humoral immunity, many aspects of the humoral immune response are intact in the absence of TRAF recruitment. We propose to identify (Specific Aim #3) non-TRAF binding domains in CD40 that are critical for early B cell activation. Genetic and proteonomic approaches are presented that will identify novel, functionally significant sites in the CD40 tail and the factors they bind. Enhanced TRAF recruitment and chronic CD40 signaling in the B cell compartment is believed to contribute to the development of lupus. We propose that the expression of a constitutively active form of CD40 (CD40zip; a leucine-zippered, myristoylated CD40 tail), within the B cell compartment will drive B cell expansion and differentiation in vivo and result in the development of a lupus-like syndrome (Specific Aim#4). Assuming the development of autoimmunity in Tg CD40zip mice, mutations in the TRAF binding sites of CD40zip, will be engineered to evaluate the contribution of specific TRAFs to the development of autoimmunity.