We are analyzing the regulation of cytokine and chemokine gene expression in lymphoid cells. We have chosen interferon-gamma (IFN-gamma) gene expression as a model system for analysis of the control of gene expression in natural killer cells (NK cells) and T cells. We are continuing to dissect the regions of the human interferon-gamma gene to determine which regions enhance/repress gene transcription in response to extracellular signals. In particular, we are utilizing natural killer cell lines to elucidate the mechanisms, both transcriptional and post-transcriptional, by which interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18) or interferon-alpha (IFN-alpha) induce or inhibit interferon-gamma gene expression. We are characterizing the biochemical pathways involved in the synergistic induction of interferon-gamma gene expression in response to phorbol 12-myristate 13-acetate (PMA) or bryostatin + IL-12. Our initial results indicate that combining bryostatin and IL-12, two drugs currently being tested in clinical trials, results in synergistic induction of interferon-gamma both in vitro and in in vivo mouse model systems, thus suggesting that the combination of these drugs could represent a powerful new approach towards cancer immunotherapy. Current studies have revealed that the signaling through Protein Kinase C may be extended when IL-12 is combined with bryostatin. We have used a bioinformatics approach to identify conserved regions of the 3'untranslated portion of the interferon-gamma mRNA. It is believed that these conserved regions represent important regulatory elements in the gene structure as there would be no inherent region for conservation through evolution unless the non-coding regions of the mRNA provided some evolutionary advantage. Based on this analysis, we have targeted a 160-bp region of the murine interferon-gamma 3'untranslated region for deletion, as this region is rich in AUUA sequences and such regions have been previously shown to be important in the regulation of cytokine gene expression. The knockout (KO) mouse has been successfully created and our data indicates that this mouse produces significantly more interferon-gamma upon treatment with IL-12. In addition, low levels of interferon-gamma are detected in the serum of knockout mice but not wild type control mice. Furthermore, the architecture of lymph nodes, spleen and thymus is disrupted and the liver exhibits signs of chronic inflammation. T cell homeostasis has been disrupted as increased CD4+ and CD8+ T cells are present and the T reg cells in the mouse have more potent suppressor activity. There is also an increased TH1 response and a decreased TH2 response to antigenic stimulation. The B cell population is also altered with the apparently loss of marginal zone B cells. In addition to the phenotypic consequences, the B cell response to antigen is also disrupted as increased IgM and Ig2a ab responses are seen with a decrease in the IgG1 response. Strong anti-DNA and anti-nuclear antigen antibody responses are also observed, suggesting that chronic IFN-gamma expression may play a role in the development of lupus. We are also attempting to identify a putative RNA binding protein that may interact with this region. In summary, our approaches towards elucidating the multiple mechanisms involved in the regulation of interferon-gamma demonstrates the complexity by which interferon-gamma gene expression is regulated in immune effector cells. Furthermore we now have developed a mouse model for understanding and elucidating the systems biology effects of long term, chronic IFN-gamma gene expression.
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