Regulation of T cell Differentiation

在最初的一系列研究中，我们生成了一系列包含IL-17基因转录起始点上游区域的报告构建体，然后评估了这些构建体在转染Jurkat细胞以及用PMA和离子霉素刺激后的活性。我们发现，而谱系特异性的IL-17转录因子RORGamma-t。存在于1.1kb的启动子片段中，该因子是IL-17转录所必需的，因此，最佳的IL-17转录需要2kb的启动子片段，该片段包含不结合RORGamma-t的上游序列。在进一步的研究中，我们发现RUNX(特别是RUNX1)是另一种IL-17转录因子，它在上游位置与IL-17启动子结合。然而，尽管这种转录因子对于IL-17的最佳转录是至关重要的，但它完全依赖于RORGamma-t的结合和活性。在其他研究中，我们发现了至少一个末端(上游)保守的非编码序列(CNS序列)，它包含RORGamma-t和RUNX的结合位点，是IL-17转录所必需的。 为了在经历Th17分化的CD4+T细胞中证实上述发现，我们进行了广泛的研究，评估了RUNX1(或Runx2)下调IL-17表达的效果。这些研究包括用RUNX特异性siRNA转染细胞或用逆转录病毒转导表达短发夹状siRNA或显性阴性RUNX结构的细胞。这些研究的结果是一致的，因为他们表明RUNX的下调导致IL-17的产生大大减少。然而，如上所述，在缺乏RORGammat或仅有少量RORGammat的条件下培养的细胞中RUNX1的下调对IL-17的表达没有或几乎没有影响。在相关研究中，我们用芯片分析表明，RORGamma-t和RUNX1都与经历Th17分化的T细胞的IL-17启动子和CNS增强子结合。 最近有研究表明，调节性T细胞的谱系特异性因子Foxp3与RORGamma-t相互作用，从而下调后者诱导IL-17表达的能力。为了探讨Foxp3的调节活性与RUNX在IL-17转录中的作用，我们对RUNX和Foxp3突变体的CD4+T细胞进行了共转染研究。我们发现，野生型Foxp3抑制了RORGamma-t诱导的IL-17的表达，而缺乏与RUNX1结合能力的Foxp3突变体则没有影响。这些数据揭示了一个关键事实，即Foxp3对Th17的抑制取决于它与RUNX和RORGamma-t的结合能力。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RUNX1与RORGammat物理上相互作用，其次Foxp3与RORgt相互作用。这些数据，再加上之前Foxp3与RUNX1物理上相互作用的数据，得出的结论是，RUNX1、Foxp3和RORGamma-t之间复杂的三向相互作用决定了CD4细胞分化为Th17细胞，或者替代地分化为调节性T细胞。此外，他们强烈暗示，RUNX1的调节是IL-17转录调节的另一种方式。

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.