TRAF dependence of CD40 signaling in B cells

The activation of B lymphocytes in response to protein antigens may require a signal from T cells. That signal is provided by the cell surface molecule CD154, and is mediated by the receptor CD40. The interaction of this ligand-receptor pair is critical for developing thymus-dependent humoral immune responses. The cellular events that occur after CD40 ligation are mediated by cytoplasmic signaling functions that are largely unknown. Studies have focused on a family of intracellular proteins that bind to CD40 called TNF Receptor Associating Factors (TRAFs).; The functions of TRAF family proteins have been defined by overexpression of the proteins in cell lines, and deletion of the genes in mice. In those studies, potential roles for TRAF proteins in B cell activation have been established. Prior to the work shown here, no data have shown the specific roles of different TRAF binding domains on CD40, with respect to B cell activation and humoral immunity in vivo.; The experiments shown here culminate in the development of a novel system for studying TRAF dependence of CD40 in vivo. Preliminary characterization of TRAF function in CD40 signaling is undertaken by overexpression, and by mutating TRAF binding sites in the CD40 cytoplasmic domain. The mutations in CD40 that disrupt TRAF recruitment were finally engineered into a human-murine chimeric CD40 molecule (X-CD40) that could be expressed as a transgene in mice.; Studies of the biochemical signaling pathways in naïve murine B cells expressing mutant forms of X-CD40 showed that much of the signaling capacity of CD40 resides in the binding site for TRAF2 and TRAF3, while no signaling function is mediated through TRAF6. These experiments also revealed a TRAF-independent signaling activity. Studies of humoral immune responses in mice expressing mutant X-CD40 showed that many CD40 dependent immune functions rely on recruitment of TRAF6. The reliance of CD40 on TRAF6 in vivo was not predicted by previously existing data. The data demonstrate the utility of studying the immune system in a healthy animal, and also grant a novel understanding of the role of TRAF proteins in CD40 function.