NF-kB activation and function in B lymphocytes

National Institute On Aging

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Abstract

During FY16 we accomplished the following: 1. We completed the first phase of studies on signal-independent B cell proliferation. Of note, during this year we carried out CyTOF analysis of flow cytometrically sorted B cells that are undergoing signal-independent proliferation or not. SPADE analysis of the data revealed a clear distinction of the G1 stage between these cells, which is consistent with the hypothesis that the decision to divide without further stimulation may be made at the M/G1 transition. Our working model is that persistent survivin expression in G1 cells may underlie this phenotype. Ongoing experiments will test whether survivin interacts with G1 cell division kinases (Cdk4 and 6) to promote G1 progression. 2. We examined transcriptional responses of B cells that carry conditional deletion of the GtfIIi gene (that encodes the signal responsive transcription factor TFII-I. B cells were purified from spleens of GtfIIifl/fl x CD19-cre mice and stimulated with anti-Ig or LPS. RNA analysis by qRT-PCR was used to examine the effects on the very few predicted targets of TFII-I. A broader RNA-Seq analysis is underway. We also examined the response of TFII-I-null B cells to the survival cytokine BAFF, and found that TFII-I was not required for survival signaling. 3. We developed cell lines that inducibly express a dominant negative form of IB (dnIB) to rigorously identify NF-B target genes. We created these cell lines with PMA and ionomycin to activate NF-B in the presence or absence of dnIB. Test qRT-PCR studies showed varying degrees of suppression of putative NF-B target genes. RNA-Seq analyses are underway. 4. A significant amount of time was devoted to correlating the results from kinetic studies of inducible gene expression, RNA stability and NF-B recruitment to the genome of spleen B cells activated via the BCR to obtain a comprehensive view of NF-B-dependent transcriptional response of mouse B cells. 5. We generated mice that contain a constantly active IB kinase beta (caIKK) gene flanked by a floxed terminator. The gene can be induced by breeding to an appropriate Cre and, when expressed, should stimulate NF-B activation in the absence of external stimuli. We propose to use this mouse strain to activate NF-B signaling in different tissues to evaluate the effects of inflammatory signaling on aging and age-associated conditions.