Cytokines are secreted proteins that regulate cell growth and differentiation. These factors are especially important in regulating immune and inflammatory responses. Specific cytokines are critical for lymphoid development, differentiation, homeostasis, tolerance, and memory. Thus understanding the molecular basis of cytokine action can provide important insights on the pathogenesis of immune-mediated disease as well as offer new therapeutic targets. We discovered the human tyrosine kinase, Jak3, which is essential for signaling by a major class of immunoregulatory cytokines, those that bind the common gamma chain, gc (IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21). We found that mutation of gc or Jak3 results in severe combined immunodeficiency (SCID) and in this fiscal year, we reported 10 new individuals from 7 unrelated families who exhibited new Jak3 mutations. We described the clinical presentation of these patients and assessed the structure/function consequences of the specific mutations. Nine of the 10 patients are currently well following stem cell transplantation, with all exhibiting normal T cell function. However, restoration of antibody function was only noted in a minority of patients and natural killer function was severely depressed at presentation and remained impaired in most patients. A major objective of the laboratory is the understanding the structure and function of Jak3, in vitro and in vivo. Using patient derived mutations as a tool, we have studied the trafficking of Jak3 and gc in living cell using fluorescent fusion proteins. We found that Jak3-GFP is cytosolic and cannot traffic to the plasma membrane without its cognate receptor, gc. The structural requirements for the proper localization of Jak3 are quite stringent. We demonstrated that the amino-terminal band four point one, ezrin, radixin, moiesin (FERM) domain is necessary for receptor association, it is not sufficient; indeed the entire Jak3 molecule is required. Kinase activity is not required, but a number of mutants that disrupt kinase activity do affect trafficking. In addition, several additional patient-derived and artificial mutants were found to disrupt trafficking, including mutations in the pseudokinase domain and the putative SH2 domain. In related studies, we have also defined the major sites of autophosphorylation within Jak3. We have recently mapped one site as being an important means by which the adapter molecule SH2Bb is bound. After the discovery of Jak3, our group established a CRADA with Pfizer, the goal being the generation of a selective, clinically usefully Jak3 antagonist as a new class of immunosuppressant. Such a compound has now been generated and was tested in two transplant models, mouse and primate; in both settings the drug was effective in blocking transplant rejection. Importantly, the drug has an approximately 20-100 fold selectivity for Jak3 compared to other Jaks. This is important because inhibition of Jak1 and especially Jak2 would be expected to be associated with significant clinical toxicities including anemia, thrombocytopenia and leucopenia. Both in vitro and in cell-based assays, Jak2 signaling was relatively spared. In animals, the drug was associated with only mild anemia. Following the activation of Jaks by cytokines, the next step in signaling is the activation of a family of transcription factors called Stats (signal transducers and activators of transcription). We have recently found that gene targeted mice that completely lack Stat5A and Stat5B, have major disruptions of T and B cell development. Thus, we conclude, Stat5, like Jak3 and gc, is essential for proper lymphoid development in large measure as a major mediator of IL-7?s actions. More detailed analysis of these mice and the role of Stat5 in immunity is underway. The second major area of investigation is the control of helper T (Th) cell differentiation and the regulation of cell-mediated immunity. Several factors regulate this process including the cytokine IL-12, which we discovered to activate the Janus kinases, Tyk2 and Jak2 and the transcription factor Stat4. The activation of this pathway leads to production of another key cytokine, IFN-gamma, which is critical for host defense against intracellular pathogens. To delineate ithe transcriptional control mechanism(s) that regulate IFN-gamma mRNA expression, we mapped a DNase hypersensitivity site approximately 3.5 - 4.0 kb upstream of the transcriptional start site. Using chromatin immunoprecipitation assays we identified cytokine-inducible histone H3 acetylation in this region. Within this distal region we found a Stat5 binding site, which we demonstrated using chromatin immunoprecipitation. These data lead us to conclude that this distal region serves as both a target of chromatin remodeling in the IFNG locus as well as a cytokine-induced transcriptional enhancer that binds Stat5 proteins. We next compared and contrasted histone acetylation and mRNA expression for three key Th1-expressed genes, IFNG, TBET, and IL18RAP and found them to be distinctly regulated. The TBET and the IFNG genes, but not the IL18RAP gene showed preferential acetylation of histones H3 and H4 during TH1 differentiation. Analysis of acetylation of specific histone residues revealed that H3(K9), H4(K8), and H4(K12) were preferentially modified in TH1 cells, suggesting a possible contribution of acetylation of these residues for induction of these genes. On the other hand, the acetylation of IL18RAP gene occurred both in TH1 and TH2 cells with the similar kinetics and on the same residues. In addition, histone H3 acetylation of IFNG and TBET genes occurred with different kinetics however, and was distinctively regulated by cytokines indicating that histone acetylation during TH1 differentiation is a process that is regulated by various factors at multiple levels. We also found that histone acetylation of the IFNG could occur independently of TBET through the use of Tbet knockout mice. Finally, we found that treating Th2 cells with a histone deacetylation inhibitor,restored histone acetylation of the IFNG and TBET genes, but not fully restore their expression in TH2 cells, indicating that histone acetylation explains one but not all the aspects of TH1 specific gene expression. In efforts to better understand target genes activated by cytokines, we utilized microarray technology to identify genes selectively induced by IL-12. We demonstrated that a relatively small number of genes is selectively induced by IL-12, compared to other cytokines like IL-2 and Type I interferon, a finding that argues for an instructive model of cytokine action as opposed to the stochastic models proposed by some workers. One gene that we identified in these microarray studies that was of particular interest is the serine/threonine kinase Cot/Tpl2. This kinase is directly inducible by IL-12 and inhibited by IL-4; accordingly, it is preferentially expressed in Th1 cells. Using Cot/Tp12 knock out mice and siRNA to knock down Cot/Tpl2 levels, we showed that Cot/Tpl2 is important in interferon gamma gene regulation. We identified a number of other genes that we hope will improve our understanding of the cell biology of helper T cell differentiation.
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| O'Shea, John J; Murray, Peter J (2008) Cytokine signaling modules in inflammatory responses. Immunity 28:477-87 |
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| Ramos, Haydee L; O'Shea, John J; Watford, Wendy T (2007) STAT5 isoforms: controversies and clarifications. Biochem J 404:e1-2 |
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