CD40 intracellular TRAF binding domains modulate B cell activation and experimental autoimmune encephalomyelitis development

Abstract CD40 is a costimulatory surface protein expressed on multiple immune cells, including B cells, where it is required for their full activation. Upon interacting with CD40 ligand, CD40 initiates downstream signaling that culminates in activation of naïve B cells. Previous work from our lab revealed that cell autonomous expression of CD40 on B cells regulates their activity, including germinal center development and antibody class switching. Intracellular (IC) domains of the CD40 protein are crucial for signaling. Several of these domains are known to be associated with TNF receptor associated factor proteins (TRAFs) which facilitate further downstream signaling. These include a TRAF6 binding domain and a TRAF2/3 binding domain. We analyzed the CD40 IC domain requirements for CD40 signaling in B cells and for induction of experimental autoimmune encephalomyelitis (EAE). To do so, multiple mouse strains were generated with elimination of the putative TRAF6 or TRAF2/3 domains by CRISPR technology. Whereas eliminating the putative TRAF6 binding site (Domain 1) had little to no effect on B cell viability and in vitro activation by anti-CD40 antibody, eliminating the putative TRAF2/3 binding site (Domain 2) impaired both these functions. Similarly, generation of high affinity IgG in vivo was also dependent on Domain 2 but not Domain 1. In contrast, both Domain 1 and Domain 2 were required for full development of EAE. Interestingly, mixed BM chimeras revealed that the ameliorating effect of CD40 mutations on EAE was mediated by hematopoietic derived non-B cells. Our results indicate that the CD40 TRAF2/3 binding Domain 2 is critical for CD40 dependent B cell function, while both Domain 1 and Domain 2 expression by other APCs function in EAE induction.