Regulation of T cell Differentiation

在一系列的研究中，我们描述了一种新型的IL-10产生调节性T细胞，我们称之为Tr 2 T细胞的诱导机制和功能。 在最初的研究中，我们表明Tr 2细胞是由酶原耗尽的酵母提取物（ZD）和菌丝形式的C.白色念珠菌，两者都表达Dectin-1的配体1，3-β葡聚糖。 如此刺激的T细胞经历两个连锁的分子过程，共同导致Tr 2细胞。 第一个涉及GATA 3的激活，GATA 3是一种在两个位点结合IL-10启动子的因子，即，在远端位点，它作为直接转录因子发挥作用，在近端位点，它作为增强组蛋白乙酰化的表观遗传因子间接作用于转录。 第二个涉及mTOR信号通路的TORC 1臂的激活和特定C/EBP同种型的表达。 这些结论是由Tr 2细胞的微阵列分析开始的，我们发现Tr 2基因表达与Tr 1和Th 2表达不同，C/EBP信号传导是可能构成这种不同表达模式的几种信号传导途径之一。 在后续研究中，我们显示，与类似刺激的WT细胞相比，在Tr 2条件下刺激的来自具有靶向缺失C/EBP的小鼠的T细胞的刺激导致IL-10产生大大降低。 此外，我们发现，在Tr 2条件下，C/EBP水平被C/EBP亚型的逆转录病毒表达部分补充的C/EBP缺陷小鼠的T细胞刺激导致IL-10产生的恢复，但只有当补充的逆转录病毒表达C/EBP的LIP亚型而不是C/EBP的LIP亚型时。最后，我们表明C/EBP加工成LIP或LIPs亚型依赖于mTOR信号传导，因为TORC 1活性导致的真核生物起始因子（（elf）-4E）的磷酸化调节C/EBP翻译成LIP和LIPs，并且是LIP表达所必需的。 因此，在由于雷帕霉素的存在而不存在TORC-1信号传导的情况下，实际上不存在来自C/EBP的LIP翻译，因此，IL-10的产生被极大地抑制。 在平行研究中，我们研究了LIP如何调节Tr 2细胞中IL-10产生的机制。这些研究最初集中在上面已经提到的IL-10启动子-荧光素酶构建体的研究上，并显示启动子活性被表达CREB 1和LIP的质粒的存在最大程度地刺激，事实上，这些因子的结合位点的缺失导致启动子活性大大降低。由于CREB 1和LIP在启动子中的结合位点彼此相邻，并且CREB 1先前已显示与C/EBP结合，我们推断LIP 1/CREB 1协同性是由于一种或两种因子与IL-10启动子的促进结合。 这一假设随后得到EMSA研究的支持，该研究表明，从HEK 293细胞（用CREB 1和LIP表达质粒预转染）的细胞核中提取的CREB 1-LIP蛋白复合物与在IL-10启动子中发现的DNA序列结合，该启动子在生理条件下结合这些转录因子;相反，类似获得的CREB 1-LIP复合物与该序列结合的能力较差。这些发现伴随着研究显示，如通过CHiP研究确定的，在Tr 2细胞中C/EBP和CREB 1与IL-10启动子的结合在表达LIP和CREB的细胞中与仅表达LIP的细胞相比增强，表明CREB 1结合通过与LIP形成复合物而增强。这些研究支持以下结论：新生Tr 2细胞中的TORC 1信号传导导致高IL-10产生，因为这种信号传导产生LIP-CREB 1复合物并增强这些转录因子与IL-10启动子的结合。 在以前的研究中，我们发现C。白色念珠菌肾感染与肾组织中产生IL-10的CD 4 T细胞和具有不能产生IL-10的CD 4 T细胞的小鼠（CD 4Cre/ IL：-10flox小鼠）有关，即，不能产生Tr 2细胞的小鼠表现出比野生型小鼠更好的感染存活率。 相反，用塞来昔布（一种增加Tr 2细胞生成的药物）治疗小鼠，存活率降低。 因此，Tr 2细胞的产生限制了C.白色念珠菌感染 为探讨Tr 2细胞的调节功能，本实验研究了Tr 2细胞对屋尘螨抗原（HDM）诱导的实验性哮喘的调节作用。 我们发现，ZD管理（IP）在最初的哮喘诱导HDM引起显着减少总BAL细胞，BAL嗜酸性粒细胞和CD 4阳性细胞;此外，总IgE和HDM特异性IgE在循环中显着减少。 因此，这些研究表明，Tr 2可以在Th 2驱动的炎症如哮喘期间由ZD诱导，因此可能具有治疗哮喘的功效。 在我们的初步研究中，我们表明，DC刺激ZD通过Dectin-1释放可溶性因子诱导Tr 2细胞。为了确定该因子的身份，我们进行了许多研究，其中T细胞在允许将特定细胞因子鉴定为Tr 2诱导因子的条件下培养，但没有这样的因子可以被如此鉴定。然而，在最近的研究中，我们表明，Dectin-1刺激导致在刺激的DC中谷氨酰胺转运蛋白（CD 98和LAT 1）表达上调，谷氨酰胺诱导T细胞通过TCA循环转化为α-酮谷氨酸（一种mTOR刺激剂）产生IL-10。 因此，出现的图片是Tr 2诱导主要是由于β-内酰胺酶解和由此产生的C/EBP β的LIP同种型的TORC 1产生。

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.