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 and affect natural killer cell biology. We are investigating the role of STAT, NFkappaB and T-bet proteins in regulating interferon-gamma expression and how a highly conserved element in the 3' untranslated region may affect interferon-gamma mRNA stability. We are also 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, IL-2 + IL-12, IL-2 + IL-18 and PMA/bryostatin + interferon-alpha. 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. 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 preliminary data indicates that this mouse produces significantly more interferon-gamma upon treatment with IL-12 or IL-18. In addition, low levels of interferon-gamma are detected in the serum of knockout mice but not wild type control mice. Furthermore, 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. Furthermore, the architecture of lymph nodes, spleen and thymus is disrupted and the liver exhibits signs of chronic inflammation. Ongoing studies are currently being conducted to investigate if the host B and T cell response to antigen challenge is altered. We have also identified a putative RNA binding protein that may interact with this region and are beginning experiments to determine the specificity of this interaction. As indicated above, we are also utilizing bioinformatics/evolutionary conservation analysis to determine if microRNAs (miRNAs) may target the interferon-gamma mRNA. To address this possibility, we have generated a stable small interfering RNA (siRNA) transfectant of the dicer gene in the human natural killer cell line NK92. Dicer is an enzyme critical for overall miRNA processing and thus decreased dicer levels may alter interferon-gamma gene expression if miRNAs are involved in gene expression. Preliminary results indicate that interferon-gamma expression may be increased in response to specific stimuli in the dicer knockdown NK92 transfectant, thus implicating miRNAs in the regulation of interferon-gamma expression. Furthermore, we have identified an evolutionarily conserved miRNA binding site in the interferon-gamma 3' untranslated region and preliminary data indicates that this site contributes to regulating interferon-gamma expression. 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.
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