Regulation of T cell Differentiation

T 细胞分化的调节

• 批准号: 9354759
• 负责人: Warren Strober
• 金额: $ 53.79万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9354759
• 关键词: Antigen Presentation; Binding; Binding Sites; CCAAT-Enhancer-Binding Proteins; CD3 Antigens; CREB1 gene; Candida albicans; Carbohydrates; Cell Nucleus; Cell physiology; Cell surface; Cells; Coculture Techniques; Complex; Crohn' s disease; Crude Extracts; DNA Sequence; Dendritic Cells; Distal; Dose; EMSA; Effector Cell; Enzyme Precursors; Epigenetic Process; Eukaryota; Event; Exhibits; GATA3 gene; Gene Expression; Generations; Genes; Genetic Transcription; Glucans; Histone Acetylation; Host Defense; Indium; Individual; Infection; Inflammatory; Inflammatory Bowel Diseases; Interleukin-10; Interleukin-4; Laboratories; Ligands; Luciferases; Mediating; Molecular; Mus; Nature; Organism; Pathogenesis; Patients; Pattern; Peptide Initiation Factors; Phosphorylation; Physiological; Plants; Plasmids; Process; Production; Protein Isoforms; Recovery; Regulation; Regulatory T-Lymphocyte; Reporting; Research; Retroviridae; STAT6 gene; Series; Signal Pathway; Signal Transduction; Sirolimus; Site; T cell differentiation; T cell response; T-Lymphocyte; Th2 Cells; Time; Tissues; Translations; Virus; Yeasts; Zymosan; arm; base; beta-Glucans; cytokine; dectin 1; defense response; gut microbiome; mTOR Signaling Pathway; promoter; protein complex; receptor; response; transcription factor

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. With respect to their mode of induction we showed that Tr2 cells are induced by zymogen-depleted yeast extracts and by the hyphal form of C. albicans, both of which express 1,3-glucan, the ligand of Dectin-1; in contrast, it is not induced by dendritic cells stimulated by the various TLR ligands or, indeed, the yeast form of C. albicans that also expresses TLR ligands. In fact, TLR ligand stimulation of DCs profoundly inhibits the induction of Tr2 cells. As discussed below, this mode of induction greatly influences host defense responses to C. albicans infection since it leads to an effector cell response to the infection that is regulated by a regulatory T cell response induced by the hyphal form despite the fact that this is the form that mediates tissue invasion. 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 and that at the distal site GATA3 acts as a direct transcription factor whereas at the proximal site it acts indirectly on transcription as a epigenetic factor that augments histone acetylation. Such GATA3 activity appears to differ from GATA3 activity in Th2 cells where it has been shown to lack the ability to act as a direct transcription factor. 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 and that of TORC1 signaling regulates the expression of this isoform. 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 th LAP isoform of C/EBP; in fact, repletion with the LAP isoform actually reduced baseline IL-10 production. 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 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 because of the presence of rapamycin, LIP translation from C/EBP is virtually asent 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 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. A second and perhaps equally important way in which the LIP and LAP isoforms of C/EBP regulates IL-10 transcrlption in Tr2 cells relates to the previously discussed positive effect of GATA3 on such transcription. Both isoforms bind to GATA3 but the consequences of such binding are different. LAP binding results in enhanced proteosomal degradation of GATA3 whereas LIP binding results in inhibition of proteosomal degradation. Thus, the increased LIP/LAP ratio jn Tr2 cells favor increased GATA3 levels and its attendant effects of IL-10 gene transcription whereas a decreased LIP/LAP ration has the opposite effect. The studies described above establish that Dectin-1 stimulation of DCs elicits an new type of regulatory T cell that this eexquisitively dependent on mTor (TORC1) signaling. The question that remains is what is being produced by Dectin-1 stimulated cells that results in such signaling. Currently, we are subjecting supernatents derived from Dectin-1 stimulated DCs that induce Tr2 cells to mass specrometric analysis to determine the molecular nature of the stimulatory material.

在这些研究中，我们定义了一种新型的IL-10产生调节T细胞，该细胞是由dectin受体刺激的树突状细胞诱导的。 这些细胞不同于先前描述的IL-10产生调节细胞的诱导方式和其激活IL-10基因转录的分子事件。 在此基础上，我们将它们称为TR2调节T细胞。 关于它们的诱导方式，我们表明TR2细胞是由扎伊斯原耗血的酵母提取物和白色念珠菌的菌丝形式诱导的，均表达1,3-葡聚糖，Dectin-1的配体。相比之下，它不是由各种TLR配体刺激的树突状细胞或也表达TLR配体的酵母菌形式所诱导的。 实际上，DC的TLR配体刺激深刻抑制了TR2细胞的诱导。 如下所述，这种诱导方式极大地影响了对白色念珠菌感染的宿主防御反应，因为它导致了对感染的效应细胞反应，该反应受到菌丝形式引起的调节性T细胞反应调节，尽管这是介导组织入侵的形式。 关于IL-10基因转录，TR2细胞经历了两个互锁的分子过程，共同导致高水平的IL-10产生。 首先涉及IL-4的T细胞产生以及包括STAT6和GATA3在内的Th2基因的激活。 在此基础上，无法产生IL-4的T细胞，也不能诱导表达STAT6和GATA3成为诱导DC的dectin-1刺激后成为高级IL-10生产者。 该信号通路中的关键要素很可能是GATA3，因为我们表明GATA3在两个位点与TR2细胞IL-10启动子结合，并且在远端位点GATA3在直接转录因子上起着直接转录因子的作用，而在接近位点，它在转录中是间接起作用的，它作为一种表皮因子而起作用，该因子是一种表观型因子，该因子促进了Histone actone actone actone actone actone actone actyletation。 这种GATA3活性似乎与Th2细胞中的GATA3活性有所不同，在Th2细胞中缺乏充当直接转录因子的能力。 在研究中揭示了TR2细胞中的第二个也是更独特的过程指导IL-10转录，表明MTOR信号传导途径的Torc1臂是此类细胞中IL-10产生的关键组成部分。 这一事实非常明确地表明，由Dectin-1激活的DC刺激的T细胞中的IL-10产生受到雷帕霉素的抑制作用依赖性抑制作用。发现这种抑制作用的机制涉及TR2细胞中IL-10转录的另一个非常独特的方面，即这种转录取决于特定的C/EBP同工型的表达，而TORC1信号传导调节该同工型的表达。 导致这些结论的一系列研究始于微阵列分析，其中我们检查了TR2细胞以及TR1和TH2细胞中的基因表达。 这些研究表明，TR2细胞中的基因表达与TR1和TH2细胞中的基因表达不同，并且对这种表达的IPA分析表明，C/EBP信号传导是几种可能是这种不同表达模式的信号传导途径之一。 在随后的研究中，为了检查这种可能性，我们首先表明，与类似刺激的WT细胞相比，在TR2条件下，在TR2条件下刺激C/EBP的T细胞的刺激导致IL-10产生大大降低。 此外，我们表明，在TR2条件下刺激C/EBP水平的T细胞用培养物培养用逆转录病毒表达C/EBP的逆转录病毒会导致IL-10产生的恢复，如果逆转导致逆转录病毒表达C/EBP的唇部相结合，则会导致IL-10产生的恢复，但不会表达Cirus Iseform iSForem iSform iSoform iSoforgorm iSoforgormeform。实际上，用圈型同工型的充气实际上降低了基线IL-10产生。因此，这些研究提供了有力的证据，表明C/EBP的LIP（而不是C/EBP的同工型）是C/EBP的同工型，这是TR2细胞中IL-10转录的必要组成部分。最后，我们可以将TORC1信号传导与TR2细胞中IL-10产生的关系与研究相关联，这表明TORC1信号信号是真核生物启动因子（（ELF）-4E）的磷酸化，该因子已显示出来调节C/EBP转化为LAP和LIP的C/EBP转换，并且是LIP表达的所必需的。 因此，在没有TORC-1信号传导的情况下，由于存在雷帕霉素，C/EBP的唇翻译实际上是非常出色的，因此，TR2细胞中的IL-10产生极大地抑制了。 在平行研究中，我们研究了LIP如何调节TR2细胞中IL-10产生的机制。 这些最初以IL-10启动子 - 卢西酶构建体的研究为中心，并表明启动子活性受到表达CREB1和唇质的质粒的存在最大程度地刺激了这些因素的结合位点，从而大大降低了启动子活性。由于启动子中的CREB1和唇部结合位点彼此相邻，并且先前已证明CREB1与C/EBP结合，因此我们认为LIP1/CREB1协作性是由于一个或两种因素与IL-10启动子的促进结合所致。 EMSA研究随后证明了这一假设，该研究表明，从HEK293细胞核中提取的CREB1-LIP蛋白复合物（用CREB1和唇部表达质粒预先感染与IL-10启动子在物理学条件下这些转录因子结合的IL-10启动子中的DNA序列结合的质量）；相比之下，类似获得的CREB1-LAP复合物的结合能力较差。这些发现伴随着研究表明，通过芯片研究确定的C/EBP和CREB1与TR2细胞中IL-10启动子的结合增强了与仅表达膝盖的细胞相比，表达唇部和lap的细胞中增强了CREB1的结合，表明CREB1结合通过与LIP的复合物形成增强。这些研究支持以下结论：新生TR2细胞中的TORC1信号传导导致高IL-10产生，因为这种信号传导会产生唇-CREB1复合物以及这些转录因子与IL-10启动子的增强结合。 C/EBP的LIP和LAP同工型调节TR2细胞中IL-10转移的第二种也许同样重要的方式与先前讨论的GATA3对这种转录的阳性作用有关。 两种同工型都与GATA3结合，但这种结合的后果是不同的。 LAP结合导致GATA3的蛋白质体降解增强，而唇部结合导致抑制蛋白质体降解。 因此，增加的唇/lap比率JN TR2细胞有利于GATA3水平升高及其对IL-10基因转录的伴随效应，而唇部/lap降低的降低具有相反的作用。 上面描述的研究表明，DCS的Dectin-1刺激引起了一种新型的调节T细胞，该细胞依赖于MTOR（TORC1）信号传导。 剩下的问题是导致这种信号传导的Dectin-1刺激细胞产生的问题。 目前，我们正在对源自dectin-1刺激的DC衍生的上清液进行诱导TR2细胞进行质量规格分析，以确定刺激材料的分子性质。