CD79A as a molecular switch regulating B cell activation

B lymphocytes are critical players in the development of protective immunity, as well as in pathological states such as autoimmunity. Careful regulation of the strength and quality of biochemical signals that originate from their antigen receptors (BCR) is essential to prevent the unwanted activation of potentially harmful autoreactive

B cells, and to generate optimally protective antibody responses. Inhibitory signaling mechanisms play a central role in regulating BCR signaling. Best characterized is the role of Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM)-containing inhibitory receptors that become activated upon co-aggregation with actively

signaling BCRs. The BCR-associated Src-family kinase Lyn tyrosine phosphorylates their ITIM motifs, resulting in the recruitment of phosphatases such as the tyrosine phosphatase SHP-1 and the inositol phosphatase SHIP-1 that actively suppress BCR signaling. SHIP-1 activity is increased in autoreactive B cells and plays a

critical role in maintaining B cell tolerance by suppressing PI3K-dependent signaling. How SHIP-1 is activated in autoreactive B cells is still unclear. BCR stimulation by itself, without recruitment of known ITIM-containing inhibitory receptors, activates SHIP-1. In an unbiased screen, detecting tyrosine phosphorylated receptors that

bind SHIP-1, we identified CD79A as containing the SHIP-1 docking site. CD79A and CD79B form a heterodimer that mediates signal transduction following BCR stimulation. Each chain contains a single Immunoreceptor Tyrosine-based Activation Motif (ITAM). Phosphorylation of the two conserved tyrosines in the

ITAMs is the critical first step towards activating signaling. Previously, we showed that in autoreactive B cells CD79A ITAMs are predominantly monophosphorylated. Studies of cell lines expressing chimeric receptors suggest that monophosphorylation of the CD79A Y182 residue biases signaling towards inhibitory signaling.

We hypothesize that CD79A ITAMs can act like a molecular switch wherein ITAM monophosphorylation tips the balance towards inhibitory signaling by preferentially recruiting the regulatory proteins Lyn and SHIP-1. To test this hypothesis, we have generated novel mouse models in which the ability of the CD79A ITAM to

become tyrosine phosphorylated is variably restricted to monophosphorylation of the membrane proximal (Y182) or membrane distal (Y193) residue. In Aim 1 we will determine how individual CD79A ITAM tyrosines impact BCR signalosome composition and downstream signaling, both directly and indirectly via co-

aggregated ITIM-containing receptors. In Aim 2 we will determine the impact of individual CD79A ITAM tyrosines on biological processes such as B cell development, B cell activation, antibody responses, and B cell tolerance. Specifically, we will determine if CD79A ITAM monophosphorylation of CD79A Y182 drives the

anergic phenotype of autoreactive B cells. These studies will provide unique insights into the earliest activating and inhibitory events that shape B cell activation and tolerance. It will also lead to a better understanding of ITAM biology with direct translational implications for chimeric antigen receptor design.