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Regulation of T cell Differentiation

T 细胞分化的调节

基本信息

批准号：
7732554
负责人：
WARREN STROBER
金额：
$ 91.29万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/7732554
关键词：
Area Binding Binding Sites Biological Assay CD4 Positive T Lymphocytes Cell Differentiation process Cells Chronic Chronic Phase of Disease Clinical Research Co-Immunoprecipitations Colitis Complex Condition Coupled Crohn&apos s disease Cultured Cells Data Distal Dominant-Negative Mutation Down-Regulation Enhancers Event Functional RNA Genetic Transcription Goals IL17 gene Inflammation Inflammatory Bowel Diseases Interferon Type II Interleukin-17 Ionomycin Jurkat Cells Laboratories Lead Modeling Molecular Mucosal Immune Responses Mucositis Pathway interactions Patients Play Production Regulation Reporter Retroviridae Role Series Site Small Interfering RNA Stream T-Lymphocyte Tetradecanoylphorbol Acetate Time Transcription Initiation Site Transfection Ulcerative Colitis cellular transduction cytokine interleukin-23 mutant promoter transcription factor

项目摘要

In an initial series of studies we generated a series of reporter constructs containing regions of the IL-17 gene upstream of this transcription start site and then assessed the activity of these constructs following their transfection into Jurkat cells and stimulation with PMA and ionomycin. We found that whereas the lineage-specific IL-17 transcription factor, RORgamma-t. is present in a 1.1kb promoter fragment and this factor is necessary for IL-17 transcription, optimal IL-17 transcription requires the presence of a 2kb promoter fragment containing up-stream sequence that does not bind RORgamma-t. In further studies we identified Runx (particularly Runx1) as another IL-17 transcription factor that binds to the IL-17 promoter at an up-stream site. However, whereas this transcription factor was critically necessary for optimal IL-17 transcription it is totally dependent on RORgamma-t binding and activity. In yet other studies we identified at least one distal (up-stream) conserved non-coding sequence (CNS sequence) that contains binding sites for both RORgamma-t and Runx and which is necessary for IL-17 transcription. To substantiate the above findings in CD4+ T cells undergoing Th17 differentiation we conducted extensive studies in which we evaluated the effect of Runx1 (or Runx2) down-regulation of IL-17 expression. These studies involved the transfection of cells with Runx-specific siRNA or the transduction of cells with a retrovirus expressing either a short-hairpin siRNA or a dominant-negative Runx construct. The results of these studies were congruent in that they showed that down-regulation of Runx led to greatly decrease IL-17 production. However, as presaged by the reporter studies discussed above, down-regulation of Runx1 in cells cultured under conditions leading to the absence of RORgammat or only small amounts of RORgammat, had no or little effect on IL-17 expression. In related studies we showed using ChIP assays that both RORgamma-t and Runx1 bind to the IL-17 promoter and the CNS enhancer of T cells undergoing Th17 differentiation. Recently it has been shown that Foxp3, the lineage-specific factor of regulatory T cells interacts with RORgamma-t and thereby down-regulates the latters capacity to induce IL-17 expression. To explore the regulatory activity of Foxp3 in relation to the role of Runx in IL-17 transcription we performed co-transfection studies of CD4+ T cells of Runx and Foxp3 mutants. We found that whereas wild type Foxp3 suppressed RORgamma-t-induced IL-17 expression, a Foxp3 mutant lacking the ability to bind to Runx1 had no effect. These data revealed the critical fact that Foxp3 inhibition of Th17 depends both on its ability to bind to Runx and to RORgamma-t. In a final series of studies related to such binding we performed co-immunoprecipitation studies in which we showed first that Runx1 physically interacts with RORgammat and second that Foxp3 intereacts with RORgt. These data, coupled with previous data that Foxp3 physically interacts with Runx1 lead to the conclusion that a complex, three-way interaction between Runx1, Foxp3 and RORgamma-t determines the differentiation of CD4 cells into Th17 cells, or alternatively into regulatory T cells. In addition, they strongly imply that regulation of Runx1 is another way that IL-17 transcription is regulated.

在最初的一系列研究中，我们产生了一系列的报告构建体，其含有该转录起始位点上游的IL-17基因的区域，然后在将这些构建体转染到Jurkat细胞中并用PMA和离子霉素刺激后评估这些构建体的活性。我们发现，尽管谱系特异性IL-17转录因子ROR γ-t.存在于1.1kb启动子片段中，并且该因子是IL-17转录所必需的，最佳IL-17转录需要存在含有不结合ROR γ-t的上游序列的2kb启动子片段。在进一步的研究中，我们确定Runx（特别是Runx 1）作为另一种IL-17转录因子，在上游位点与IL-17启动子结合。然而，尽管该转录因子对于最佳IL-17转录是至关重要的，但它完全依赖于ROR γ-t结合和活性。在其他研究中，我们鉴定了至少一个远端（上游）保守的非编码序列（CNS序列），其含有ROR γ-t和Runx的结合位点，并且其是IL-17转录所必需的。为了证实在经历Th 17分化的CD 4 + T细胞中的上述发现，我们进行了广泛的研究，其中我们评估了Runx 1（或Runx 2）下调IL-17表达的作用。这些研究涉及用Runx特异性siRNA转染细胞或用表达短发夹siRNA或显性阴性Runx构建体的逆转录病毒转导细胞。这些研究的结果是一致的，因为它们表明Runx的下调导致IL-17的产生大大减少。然而，如上述报告基因研究所预示的，在导致ROR γ mat不存在或仅少量ROR γ mat的条件下培养的细胞中Runx 1的下调对IL-17表达没有影响或影响很小。在相关的研究中，我们使用ChIP检测表明RORgamma-t和Runx 1都与IL-17启动子和经历Th 17分化的T细胞的CNS增强子结合。最近已经显示，Foxp 3（调节性T细胞的谱系特异性因子）与ROR γ-t相互作用，从而下调ROR γ-t诱导IL-17表达的能力。为了探索Foxp 3的调节活性与Runx在IL-17转录中的作用的关系，我们进行了Runx和Foxp 3突变体的CD 4 + T细胞的共转染研究。我们发现野生型Foxp 3抑制ROR γ-t诱导的IL-17表达，而缺乏与Runx 1结合能力的Foxp 3突变体则没有影响。这些数据揭示了Foxp 3对Th 17的抑制依赖于其结合Runx和ROR γ-t的能力这一关键事实。在与这种结合相关的最后一系列研究中，我们进行了免疫共沉淀研究，其中我们首先表明Runx 1与RORgammat物理相互作用，其次是Foxp 3与RORgt相互作用。这些数据，加上以前的数据，Foxp 3与Runx 1的物理相互作用，导致的结论是，一个复杂的，三路之间的相互作用Runx 1，Foxp 3和RORgamma-t决定的CD 4细胞分化为Th 17细胞，或可替代地为调节性T细胞。此外，他们强烈暗示Runx 1的调节是IL-17转录调节的另一种方式。