Biochemistry Of Lymphocyte Activation

Arthritis, Musculoskeletal, Skin Dis

Investigators

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Abstract

Cytokines are secreted proteins that regulate cell growth and differentiation. These factors are especially important in regulating immune and inflammatory responses. as well as lymphoid development, differentiation, homeostasis, tolerance, and memory. Understanding the molecular basis of cytokine action provides important insights into the pathogenesis of immune-mediated disease and offers new therapeutic targets. We discovered Jak3, a kinase essential for signaling by cytokines 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 Jak3 results in severe combined immunodeficiency (SCID). We received a patent for targeting Jak3 as a new type of immunosuppressant drug and established a CRADA with Pfizer to generate a selective Jak3 antagonist. The compound, CP 690,550, was produced by Pfizer and found to be effective in preclinical models. The drug is being tested presently in Phase I/II studies in rheumatoid arthritis, psoriasis and transplant rejection, where it appears to be efficacious. [unreadable] [unreadable] 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 showed that mice completely lacking in Stat5A and Stat5B have major disruptions of T and B cell development. Stat5A/B is also critical for lymphoid stem cells and for CD8+ T cell survival. Thus, loss of Stat5A/B results in a SCID, similar to Jak3 . [unreadable] [unreadable] To more carefully dissect the function of Stat5A/B in the immune system, we selectively deleted in Stat5A/B in CD4+ T cells to understand its role in T cell differentiation. In addition to the well-known helper T cell fates, T helper 1 and T helper 2 cells, additional fates are now recognized. Regulatory T cells (Treg cells) are an essential subset that maintains peripheral tolerance. Tregs are generated in the thymus from CD4+ T cells (natural Treg cells or nTregs) and can be induced in the periphery (iTreg cells). Both nTreg and iTreg cells express a transcription factor, forkhead box protein 3 (Foxp3). Deficiency of Foxp3 results in lethal autoimmunity in mouse models and in humans; the disease in humans is termed IPEX. We found that deficiency of Stat5A/B in CD4+ T cells abrogated Treg development in vivo and blocked conversion of peripheral cells to become iTregs. We further found that Stat5A/B directly bind the Foxp3 gene, particularly in conserved sites in the first intron. CD4-cre, Stat5fl/fl mice also develop widespread autoimmune disease including inflammatory bowel disease. In related studies, we found that Stat3 is a critical negative regulator of Foxp3 expression. [unreadable] [unreadable] An even more recently recognized subset of CD4+ T cells is cells that preferentially produce the cytokine IL-17 (Th17 cells). IL-17 is a major inflammatory cytokine, which appears to contribute to the pathogenesis of many autoimmune and autoinflammatory disorders including rheumatoid arthritis, spondyloarthropathy, multiple sclerosis and inflammatory bowel disease. Of interest is that Th17 cells are thought to be developmentally related to regulatory T cells (Tregs) as both subsets can be induced from naive CD4+ T cells in the presence of transforming growth factor-beta (TGFb-1) in the context of different cytokines. Using CD4-Cre Stat3fl/fl mice, we showed that Stat3 is essential for Th17 differentiation. We further showed using chromatin immunoprecipitation that Stat3 binds to the Il17 gene. Conversely, we found that tissue-specific deletion of a negative regulator of Stat3 termed SOCS3 resulted in increased generation of Th17 cells and widespread autoimmune disease characterized by increased expression of IL-17 and IL-17-target genes. By performing microarrays, we found that Th17 cells express the cytokine IL-21. We found that IL-21 production was also dependent upon Stat3 and also found that IL-21 promotes IL-17 production; in this respect, the autocrine production of IL-17 by Th17 is analogous to other Th subset (e.g. Th2 cells and IL-4). We also found that IL-2, produced by activated T cells inhibits Th17 differentiation and promotes Treg differentiation. Conversely, blockade of IL-2 promoted Th17 differentiation and attenuated Treg differentiation. We found that in IL-2-deficient mice, which have systemic autoimmune disease, also have expansion of Th17 cells. IL-2 activates Stat5 and we found that Stat5-deficient T cells had increased tendency to become Th17 cells. In related studies, we found that IL-2 inhibited its own production in a Stat5-dependent manner. We also showed that another cytokine, IL-27, is a potent inhibitor of Th17 cells. IL-27 signals through Stat1 to inhibit IL-17 production. We extended these studies to examine the factors that govern Th17 in human CD4+ T cells. We provided evidence for a key role of IL-23 but also showed that human CD4+ T cells have much greater plasticity with respect to cytokine production. [unreadable] [unreadable] Th17 cells express the orphan retinoid receptor Rorgt, expression of which is dependent upon Stat3. We found that agonists of another class of retinoid receptor, retinoic acid, inhibited Th1, Th2 and Th17 differentiation. Retinoic acid downregulated expression of Rorgt but importantly, retinoic acid also upregulated Foxp3 and induced functional Tregs. Conversely, antagonist of retinoic acid receptor alpha (Rar alpha) blocked Foxp3 expression. Interestingly, immunosuppressive mucosal dendritic cells produce retinoic acid and this is now thought to be a mechanism by which they promote immune tolerance in the gut. [unreadable] [unreadable] 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 are selectively induced by IL-12. One gene we identified a small, hematopoietic-specific regulator of G-proteins, we termed Cybr as an IL-12-regulated gene. We generated Cybr knockout mice, but found that Cybr is dispensable for IL-12 signaling and IFN-gamma regulation. Another gene identified in our microarray studies was 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 IFN-gamma gene regulation. [unreadable] [unreadable] We have also identified the proprotein converstase, furin, as an IL-12 regulated gene. We generated mice that lack furin in T cells using Cre-lox strategy and found that these mice develop autoimmune disease characterized by gastritis, ileitis and colitis. The mice also produce anti-nuclear antibodies, a characteristic feature of systemic lupus erythematosus. The T cells in these mice overproduce cytokines, especially Th2 cytokines. TGF-beta is a known target of furin and the mice have impaired production of TGF-beta. Thus these mice represent a new model of autoimmune disease and illustrate the essential function of furin in T cells.