We are analyzing the regulation of cytokine and chemokine gene expression in lymphoid cells. We have chosen IFN-gamma gene expression as a model system for analysis of the control of gene expression in T cells and NK cells. We are continuing to dissect the regions of the human IFN-g genomic DNA to determine which regions enhance/repress gene transcription in response to extracellular signals. In particular, we are utilizing NK cell lines to elucidate the mechanisms, both transcriptional and post-transcriptional, by which interleukins 2,4,12,13,15,18 or activation of LY49 activating receptors (murine models) induce or inhibit IFN-g gene expression. Overall, our data indicate that multiple DNA binding protein family members interact with the human IFN-g genomic DNA and that control of IFN-g gene expression involves 5' and intronic transcriptional control regions as well as DNA methylation and mRNA stability/nuclear localization. We are now investigating the role of STAT proteins in regulating IFN-g expression. We are also characterizing the biochemical pathways involved in the synergistic induction of IFN-g gene expression in response to IL-2 + IL-12, IL-2 + IL-18 and antibodies to the LY49 activating receptors + cytokines. In an extension of these studies, the NCI Laboratory of Genomic Diversity has identified polymorphisms in the IFN-g genomic DNA and other cytokine/chemokine genes that might result in altered gene expression. We are currently evaluating the effect of these single nucleotide changes on the transcriptional regulation of these genes. In another aspect of CMIS research, we have utilized IFN-g KO mice to identify a novel NK cell population that produces IL-13 and IL-5 in response to IL-2 and IL-18. This cytokine expression profile suggests that this NK population may play a role in influencing humoral immunity. Ongoing studies are focused on characterizing the biochemical/molecular signaling pathways utilized by these cells, determining their function in vivo and defining the molecular pathways involved in the IL-2 and IL-18 induction of the IL-13 gene. In a new series of studies begun this year, we have been analyzing the biochemical and molecular consequences of introducing the chemokine receptor, CXCR2, into human and mouse T cells and NK cells. Our initial results suggest that when this receptor is introduced into human peripheral blood T cells and these cells are then treated with the receptor ligand gro alpha, the T cells demonstrate significant changes in their gene expression profile. In contrast, introduction of this receptor into a human NK cell line does not seem to result in changes in the transcription of cytokine/chemokine genes upon stimulation with either gro alpha or IL-8.
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