NF-kB activation and function in B lymphocytes

National Institute On Aging

Investigators

Linked publications & trials

Abstract

The NF-kB family of transcription factors are stress sensors in a wide variety of cell types. Their nuclear expression can be induced in response to diverse stimuli including reactive oxygen species (ROS), DNA damage, endoplasmic reticulum (ER) stress, and inflammatory cytokines such as TNF and IL-1. NF-kB target genes include inflammatory cytokines such as IL-6, TNF and IL-1, cell adhesion molecules, and molecules involved in maintenance of cell viability. The fundamental goal of our studies is to identify molecular mechanisms that underlie cell-specificity and stimulus-specificity of NF-kB-dependent gene expression. In some cell systems NF-kB has been shown to intersect with functions of the general transcription factor GTFIIi. Thus, in parallel, we explore the role of GTFIIi-driven transcription in B and T lymphocytes. During FY25 we accomplished the following: 1)We analyzed NF-kB-dependent gene expression in activated B cells by evaluating the consequences of 2 initiating signals on purified populations of follicular (Fo) and marginal zone (MZ) B cells from mouse spleen. We found that nuclear translocation of RelA was induced only in Fo but not MZ B cells in response to B cell receptor activation. LPS induced NF-kB translocation in both subsets. This difference was reflected in inducible gene expression in Fo versus MZ B cells. We extended these studies using B cells with conditional deletion of RelA and germline deletion of Rel. To delete both Rel and RelA we used pharmacologic inhibition if IkB kinase that we previously showed would reduce short-term and long-term Rel/RelA induction in BCR-activated B cells. Time-dependent RNasAse-seq was carried out; data anlysis is ongoing. 2)We initiated studies to interrogate NF-kB genomic binding using Cut and Run procedure. This will enable us to work with small cell populations obtained from aging mice. Optimal conditions have not yet been identified and we continue to strive to accomplish this important experimental goal. 3)We initiated studies to identify Rel and RelA targets in T helper cell subsets. TH skewing has proved to be problematic and we are still working out the best conditions for each of TH1, TH2 and TH17 subsets. 4)We examined the effects of hypoxia on NF-kB responses. We have preliminary evidence that GC-derived B cell lymphomas behave differently from ABC-type lymphomas in their responsiveness to hypoxia and NF-B. We are extending these observations to screen additional cell lines of each type. Once generality of the phenomemenon has ben established our goal is to uncover the underlying mechanism. 5)We optimized conditions for chromatin immunoprecipitation with anti-TFII-I antibodies as a means to study crosstalk between NF-kB and TFII-I. 6)We bred TFII-I floxed mice to CD4-cre to obtain conditional deletion of GTFII-I in the T lineage. Immunophenotyping by flow cytometry has been completed, activation studies with CD4+ and CD8+ cells are underway.