Regulation of T cell Differentiation

在将要描述的一系列研究中，我们定义了一种新的il -10产生调节性T细胞的诱导机制和功能，我们称之为Tr2 T细胞。

In the series of studies to be described we define the mechanism of induction and the function of a new type of IL-10-producing regulatory T cell that we have termed Tr2 T cells. In initial studies we showed that Tr2 cells are induced by DCs stimulated 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. The T cells so stimulated undergo two interlocking molecular processes that together result in Tr2 cells. The first involves activation of GATA3, a factor that binds to the 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 involves activation of the TORC1 arm of the mTOR signaling pathway and the expression of a particular C/EBP isoform. These conclusions were initiated by micro-array analyses of Tr2 cells in which we showed that Tr2 gene expression was distinct from that in Tr1 and Th2 expression and that C/EBP signaling was among the several signaling pathways that could underlie this distinct expression pattern. In follow-up studies we showed 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 retroviral expression of isoforms of C/EBP led to recovery of IL-10 production, but only if the repleting retrovirus expressed the LIP isoform of C/EBP but not the LAP isoform of C/EBP. Finally, we showed C/EBP processing into LIP or LAP isoforms was dependent on mTOR signaling in that phosphorylation of eukaryote initiation factor ((elf)-4E) resulting from TORC1 activity regulated C/EBP translation into LIP and LAP and is necessary for 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 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. 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. To explore the regulatory function of Tr2 cells we conducted studies of Tr2 cell regulation of experimental asthma induced by house-dust mite antigen (HDM. We found that ZD administration (IP) during initial asthma induction by HDM gives rise to a dramatic reduction in total BAL cells, BAL eosinophils and CD4-positive cells; in addition, total IgE and HDM-specific IgE in the circulation are dramatically reduced. These studies thus showed that Tr2 can be induced by ZD during a Th2-driven inflammation such as asthma and may therefore have efficacy in treating asthma. In our initial studies we showed that DCs stimulated by ZD via Dectin-1 release a soluble factor to induce Tr2 cells. To determine the identity of this factor we conducted numerous studies in which T cells were cultured under conditions that would allow identification of a specific cytokine as a Tr2-inducing factor but no such factor could be so identified. However, in recent studies we showed that Dectin-1 stimulation causes up-regulation of glutamine transporter (CD98 and LAT1) expression in stimulated DCs and that glutamine induces T cells to produce IL-10 via TCA cycle conversion to alpha-ketoglutamate, an mTOR stimulant. Thus, the picture that emerges is that Tr2 induction is largely due to glutaminolysis and resultant TORC1 generation of the LIP isoform of C/EBPbeta.