In these studies we define a new type of IL-10-producing regulatory T cell that is induced by Dectin receptor-stimulated dendritic cells. These cells differ from previously described IL-10-producing regulatory cells both with respect to their mode of induction and the molecular events that underlie their activation of IL-10 gene transcription. On this basis we have termed them Tr2 regulatory T cells. During the current periodfunction of this With respect to their mode of induction we showed that Tr2 cells are induced by zymogen-depleted yeast extracts (ZD)and by the hyphal form of C. albicans, both of which express 1,3-beta glucan, the ligand of Dectin-1. With respect to IL-10 gene transcription, Tr2 cells undergo two interlocking molecular processes that together result in high level IL-10 production. The first involves T cell production of IL-4 and activation of Th2 genes including STAT6 and GATA3. On this basis, T cells that cannot produce IL-4, or express STAT6 and GATA3 cannot be induced to become high level IL-10 producers following Dectin-1 stimulation of inducing DCs. The key element in this signaling pathway is most likely GATA3 since we show that GATA3 binds to the Tr2 cell IL-10 promoter at two sites, i.e., at a distal site where it acts as a direct transcription factor and at the proximal site where it acts indirectly on transcription as a epigenetic factor that augments histone acetylation. The second and more unique process guiding IL-10 transcription in Tr2 cells was revealed in studies showing that the TORC1 arm of the mTOR signaling pathway is a critical component of IL-10 production in such cells. This was shown quite definitively by the fact that IL-10 production in T cells stimulated by Dectin-1-activated DCs is subject to dose-dependent inhibition by the presence of rapamycin. The mechanism of this inhibitory effect was found to involve another quite distinctive aspect of IL-10 transcription in Tr2 cells, namely that such transcription depends on expression of a particular C/EBP isoform whose induction depends on TORC1 signaling. The series of studies that led to these conclusions began with micro-array analyses in which we examined gene expression in Tr2 cells as well as in Tr1 and Th2 cells. These studies showed that gene expression in Tr2 cells was distinct from that in Tr1 and Th2 cells and that IPA analysis of such expression showed that C/EBP signaling was among the several signaling pathways that could underlie this distinct expression pattern. In subsequent studies to examine this possibility we showed first that stimulation of T cells from mice with targeted deletion of C/EBP stimulated under Tr2 conditions led to greatly decreased IL-10 production as compared to similarly stimulated WT cells. In addition, we showed that T cells from C/EBP-deficient mice stimulated under Tr2 conditions in which C/EBP levels were partially repleted by culture with a retrovirus expressing isoforms of C/EBP led to recovery of IL-10 production if the repleting retrovirus expressed the LIP isoform of C/EBP but not if the virus expressed the LAP isoform of C/EBP. These studies thus provided strong evidence that the LIP, but not the LAP isoform of C/EBP is the isoform of C/EBP that is a necessary component of IL-10 transcription in Tr2 cells. Finally, we could relate the relation of TORC1 signaling to IL-10 production in Tr2 cells with studies that showed that TORC1 signaling led to the phosphorylation of eukaryote initiation factor ((elf)-4E), a factor that has been shown to regulate C/EBP translation into LAP and LIP and is necessary for the LIP expression. Thus, in the absence of TORC-1 signaling due to the presence of rapamycin, LIP translation from C/EBP is virtually absent and, as a result, IL-10 production in Tr2 cells is greatly inhibited. In parallel studies, we investigated the mechanism of how LIP regulates IL-10 production in Tr2 cells. These initially centered around studies with an IL-10 promoter-luciferase construct already alluded to above and showed that promoter activity was maximally stimulated by the presence of plasmids expressing CREB1 and LIP and in fact deletion of binding sites for these factors led to greatly reduced promoter activity. Since the CREB1 and LIP binding sites in the promoter are adjacent to one another and CREB1 had been shown previously to bind to C/EBP we reasoned that the LIP1/CREB1 cooperativity was due to facilitated binding of one or both factors to the IL-10 promoter. This hypothesis was subsequently supported by EMSA studies that showed that CREB1-LIP protein complexes extracted from the nucleus of HEK293 cells (pre-transfected with CREB1 and LIP expressing plasmids) bound to the DNA sequence found in the IL-10 promoter binding these transcription factors under physiologic conditions; in contrast, a similarly obtained CREB1-LAP complex had a poor capacity to bind to this sequence. These findings were accompanied by studies showing that C/EBP and CREB1 binding to the IL-10 promoter in Tr2 cells as determined by CHiP studies was enhanced in cells expressing LIP and LAP as compared to cells expressing only LAP, indicating the CREB1 binding is enhanced by complex formation with LIP. These studies support the conclusion that TORC1 signaling in nascent Tr2 cells leads to high IL-10 production because such signaling generates LIP-CREB1 complexes and augmented binding of these transcription factors to the IL-10 promoter. In summary, these studies establish that Dectin-1 stimulation of DCs elicits an new type of regulatory T cell that this is exquisitively dependent on mTor (TORC1) signaling. During a previous study period we showed that C. albicans renal infection is associated with IL-10 producing CD4 T cells in the renal tissue and mice that have CD4 T cells that cannot produce IL-10 (CD4Cre/ IL:-10 flox mice)i.e., mice that cannot produce Tr2 cells, exhibit better survival of infection than wild type mice. Conversely, treatment of mice with Celecoxib, an agent that augments Tr2 cell generation, have reduced survival. Thus, generation of Tr2 cells limits the pro-inflammatory (protective) effect of C. albicans infection. During the current we evaluated the function of Tr2 cells in the regulation of experimental allergic encephalitis (EAE) a model of multiple sclerosis. We found that administration of ZD during the course of EAE development had no effect on the CNS inflammation whereas administration of relatively small numbers of Tr2 cells at the time of symptom initiation brought about a very significant decrease in CNS inflammation which was partially reversible by co-administration of anti-IL-10. We concluded from these studies that Tr2 cells cannot be generated during a Th1/Th17-mediated inflammation such as EAE, but that once generated independently have potent regulatory capacity. We then turned our attention to Tr2 regulation of experimental asthma induced by house-dust mite antigen (HDM), In this Th2-mediated inflammation we found that ZD administration (IP) at the initial of asthma induction by HDM gave rise to a dramatic reduction in total BAL cells, BAL eosinophils and CD4-pos cells; in addition, total IgE and HDM-specific IgE in the circulation were dramatically reduced. These studies showed that Tr2 can be induced by ZD during a Th2-driven inflammation such as asthma and may therefore have efficacy in treating asthma.
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