Immunoglobulin isotype switching by B cells requires two signals. The first signal is delivered by cytokines which target specific CH genes for transcription and subsequent recombination. The second signal is delivered by interaction of the B cell surface antigen CD40 with its ligand (CD40L). CD40 is the only physiologic B cell receptor known to activate class switching. The intracellular (IC) domain of CD40 has a binding site for Jak3, one for TRAF6 and one for the TRAF proteins TRAF2 and TRAF3 (TRAF2/3 site). We have shown that CD40 isotype switching proceeds normally in B cells from Jak3 deficient patients, ruling out a role for Jak3 in isotype switching. To assess the role of TRAF proteins in CD40 mediated isotype switching, we have examined the ability of mutant CD40 transgenes that lack one or both TRAF binding sites (deltaTRAF6, deltaTRAF2/3 and deltaTRAFs respectively) to reconstitute isotype switching in CD40-/- B cells. Isotype switching was normal in the deltaTRAF6 mutant, but was markedly diminished or absent in the two mutants that fail to bind TRAF2 and TRAF3 (deltaTRAF2/3 and deltaTRAFs). CD40 activation of NFkappaB and of the MAP kinases p38 and Jnk was intact in deltaTRAF6 B cells, but was virtually absent in deltaTRAF2/3 B cells. Furthermore, our data in B cells from boys with IKKgamma mutations and in B cells treated with MAP kinase inhibitors suggest that NFkappaB, p38 and Jnk, are important for CD40 isotype switching. Our first hypothesis is that CD40 mediated isotype switching requires both TRAF2 and TRAF3 and that CD40 mutant transgenes that selectively lack the ability to bind TRAF2 or TRAF3 will be unable to drive isotype switching. Secondly, we propose that oligomerization of TRAF2 and TRAF3 may be sufficient to induce isotype switching following CD40 ligation, in the absence of additional signals from the intracellular domain of CD40. Thirdly, we propose that the TRAF2 associated protein RIP, which binds to IKK, and the MAP kinases p38 and Jnk are downstream of CD40 in the signaling pathway that activates isotype switching. To test this mechanistic model of CD40 mediated isotype switching, we propose to use transgenic mice and B cells from patients with mutations in IKKgamma to: 1. Perform a functional analysis of CD40 signaling by mutant CD40 proteins that includes a) molecular analysis of IgE and IgG1 isotype switching, b) analysis of B cell activation, proliferation and survival, and c) a biochemical analysis of signal transduction. 2. Examine the structural requirements for CD40 isotype switching by examining the capacity of novel CD40 mutant transgenes to reconstitute the isotype switch defect in CD40-/- B cells. We will a) examine the role of TRAF2 versus TRAF3, b) assess whether binding to TRAF2 and TRAF3 sufficient for CD40 isotype switching and c) determine the role of the transmembrane and extracellular domains of CD40 in isotype switching. 3. Define the role of molecules downstream of CD40 in isotype switching. We will a) examine the role of a) the TRAF interacting protein RIP, b) of the scaffold subunit of the Ikappakinase IKKgamma, and c) the MAP kinases p38 and JNK. The results of the proposed studies should have an important impact on the understanding and treatment of allergic and immunologic diseases.
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