NF-kB activation and function in B lymphocytes

National Institute On Aging

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Abstract

During FY12 we accomplished the following: 1. Extended our studies of B cell priming by antigen. Short-term exposure of nave spleen B cells to antigen receptor (BCR) cross-linking makes them susceptible to receive T cell help via. CD40. We have previously shown that the memory of antigen priming lasts 6-9h in vitro and CD40 activation during this time leads to G1 progression as assayed by blast formation. We now extended the times of CD40 stimulation and found that B cell priming resulted in enhanced cell division. We found that the effects of short-term BCR cross-linking were evident 48-60h later in the form of increased cell division of primed cells. In the course of these studies we found that a subset of B cells continued to go through 2-3 cell divisions in the absence of exogenous signaling. We are currently further investigating this novel mode of cell-cycle control. 2. We initiated studies of mRNA half-life of NF-κB regulated genes in B lymphocytes. Additionally, we began to characterize mRNAs that were inducibly degraded as a consequence of BCR cross-linking. 3. We established conditions to carry out chromatin immunoprecipitation studies with anti-p65/RelA antibodies in primary mouse B cells. Using the IKBα promoter as an established NF-κB-dependent target gene we found that p65/RelA ws recruited to the promoter within 45 minutes of BCR cross-linking and rapidly depleted from the promoter thereafter. These kinetics are consistent with IKBα-dependent nuclear export of NF-κB as a primary mode of down-regulating NF-κB activation. We propose to use this technique to study the kinetics of NF-κB binding and removal from genes in response to B cell activating stimuli on a genome-wide scale.