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Abstract
Regulating B cell activation and apoptosis is critical so that effective antimicrobial immunity occurs without autoimmunity. The death receptor CD95/Fas is critical for removing self-reactive and low-affinity B cells. B cells that encounter cognate antigen can be costimulated by innate and adaptive signals to protect them from CD95 apoptosis. CpG DNA signaling through TLR9 induces polyclonal activation of B cells and can protect from apoptosis. CD40 engagement on B cells by CD 154 on T cells causes proliferation and differentiation, including upregulation of CD95. However, sustained CD40 signals rescue B cells from CD95 killing. This dissertation describes the mechanisms of rapid TLR9 and CD40 rescue from CD95 apoptosis. We found that CD40 signals inhibited CD95-induced caspase 8 and 3 activation but did not block association of CD95 with FADD, the step proximal to caspase activation. CD40 rescue was independent of activation of NF-κB or new protein synthesis, and efficacy was lost if CD40 signaling was delayed. Intracellular TRAF6 was required for CD40 rescue, but normal association of TRAF6 with the CD40 cytoplasmic tail was not. Inhibitors of the P13K-Akt pathway were able to block CD40-mediated rescue from CD95 apoptosis. As expected, TLR9 rescue was dependent on endosome acidification and could be blocked with an oligonucleotide inhibitor of TLR9 signaling, confirming receptor specificity. Similar to CD40 rescue, TLR9 rescue was independent of new protein synthesis and inhibited caspase activation in CD95-treated B cells. TLR9 rescue also required TRAF6 and the PI3K-Akt pathway. Because both rescue signals required TRAF6 and P13K, we tested TRAF6-/- B cells and found that they had reduced phospho-Akt levels after CD40 or CpG stimulation compared to wild type cells, suggesting that TRAF6 is critical for linking CD40 and TLR9 to P13K activation.
To mimic B cell encounter with antigen, we prestimulated through the BCR before adding other activation signals. BCR pretreatment lowered levels of CD95 apoptosis, but CD40 or TLR9 signaling provided an additional protective effect, indicating that costimulation can cooperate with the BCR to rescue B cells from apoptosis.
