The goal of this dissertation was to determine the proximal molecular mechanism by which CD86 engagement signals directly to the B cell to increase the level of IgG1 antibody produced. Previous reports showed that CD86 engagement signals directly to a CD40L/IL-4 primed B cell to increase the level of IgG1 produced in vitro and in vivo, without affecting the level of class switch recombination (CSR). The CD86-induced increase in IgG1 production occurs due to the activation of a unique intracellular signaling network that increases the rate of IgG1 transcription via an Oct-2- and NF-κB-dependent increase in 3’-IgH enhancer activity. The CD86-dependent NF-κB activation is regulated via two proximal signaling networks that induce IκBα phosphorylation, subsequent degradation, and release of NF-κB (p50/p65), and subsequent NF-κB (p65) phosphorylation. Although it was understood how CD86 engagement increases the level of IgG1 produced, the proximal molecular mechanism to one of the most proximal CD86-induced signaling intermediates, PLCγ2, remained unknown. Prior reports showed that PLCγ2 was recruited to phosphorylated tyrosine residues, which are absent within the cytoplasmic domain of CD86. Therefore, the hypothesis tested in this dissertation was that the CD86 cytoplasmic domain associated directly with a protein/protein complex capable of undergoing tyrosine phosphorylation to activate PLCγ2 and was required for the CD86-dependent increase in IgG1 production. To test our hypothesis we designed the following specific aims: 1) To determine if a functional signaling protein(s) associated directly with CD86 to mediate the CD86-induced increase in PLCγ2 activation and IgG1 and 2) To evaluate if the CD86 cytoplasmic domain was required for the CD86-induced increase in IgG1.
Using a proteomics-based identification approach, we show for the very first time that the tyrosine-containing transmembrane adaptor proteins, prohibitin-1 (Phb1) and prohibitin-2 (Phb2), bind to CD86. The expression of Phb1/2 and association with CD86 increased primarily after priming with CD40. The CD86-induced increase in Oct-2 and IgG1 was less when either Phb1/2 expression was reduced by shRNA or the cytoplasmic domain of CD86 was truncated or mutated at serine/threonine PKC-phosphorylation sites, which did not affect Phb1/2 binding to CD86. In addition, we show that an intact CD86 cytoplasmic domain is necessary for an optimal IgG1 response against a T-dependent antigen in vivo. Furthermore, we show that Phb1/2 and the CD86 cytoplasmic domain are required for the CD86-induced phosphorylation and subsequent degradation of IκBα, which we reported leads to NF-κB p50/p65 activation; whereas, only Phb1/2 was required for the CD86-induced phosphorylation of PLCγ2 and PKCα/βII, which we reported leads to NF-κB (p65) phosphorylation and subsequent nuclear translocation. Thus, our findings suggest that Phb1/2 and the CD86 cytoplasmic domain cooperate to mediate CD86 signaling in a B cell through differential phosphorylation of distal signaling intermediates required to increase IgG1. The significance of this dissertation is that it is the first to identify the proximal CD86-induced intracellular signaling mechanism in a B cell that regulates the level of IgG1 produced. Knowledge gained from this work will provide novel therapeutic targets either to elevate or suppress the level of IgG1 produced.