Analyzing Cytokine- and TCR-Mediated Lymphocyte Responses by RNAi

为了更深入地了解淋巴细胞中由精氨酸决定的和基于免疫球蛋白/ T细胞受体的信号传导的遗传调控，已经进行了使用RNA干扰（RNAi）技术作为筛选工具的努力。已经在具有嘌呤霉素抗性元件的慢病毒载体中制备了由所有小鼠磷酸酶组成的shRNA文库。 单位科学家选择分析磷酸酶在外周CD 4 T细胞分化为Th 1细胞中的重要性。 利用的模型利用了在该单位中制备的指示小鼠，在该单位中，Th 1-Green表达标志着主Th 1转录调节因子T-bet的存在。 当T细胞在体外分化为Th 1细胞时，它们表达非常大量的GFP，很容易检测到，并且可以通过细胞分选纯化。 进行的初始筛选旨在确定在Th分化过程中哪些磷酸酶对于T-bet的表达或消退是重要的。 将幼稚CD 4 T细胞暴露于与磷酸酶互补的所有shRNA（超过1000个成员）的文库。shRNA在慢病毒中，使得它们可以被引入幼稚CD 4 T细胞中。 然后将细胞在Th 1条件下分化4天，置于IL-2中并暴露于嘌呤霉素，以消除未掺入和表达文库成员的细胞。 在这个阶段，绝大多数细胞已经是绿色+。 然后将细胞转移至Th 2培养条件。 在另外4天的培养结束时，1/2的前Th 1细胞为T-bet阴性。 纯化T-bet阴性和阳性细胞，并利用避免发夹结构导致的困难的方法对掺入的shRENAs进行PCR扩增。 使用ABI仪器对所得的大量扩增的shRNA进行深度测序。 在T-bet阴性细胞中发现了超过20个shRNAs的独特表达，在T-bet阳性细胞中发现了类似数量的shRNAs。 使用一种外部引物和一种内部引物进行PCR扩增，技术方面显示出可重现性。 MTMR 7和MTMR 9，作用于磷脂酰肌醇的磷酸盐，在筛选和在限制性Th 1条件下进行的引发中使用的测定的重复中均显示出可重复的活性。 分析表明，这些磷酸酶可以影响PI-3激酶途径，如其调节AKT磷酸化的活性所示。 这项工作强调了磷酸酶在调节淋巴细胞分化中所起的关键作用，以及基于文库的筛选在识别候选人以供进一步分析中的作用。 与此同时，已经进行了化学基因组筛选，以检查来自LOPAC组的各种小分子改变Th 17细胞转换为Th 1细胞的速率的能力。 这已经使用了绿色标记物，使得抑制或增强CD 4 T细胞可塑性的小分子具有被检测到的合理可能性。 已经确定了几种抑制转换的候选药物，目前正在进行二次筛选。

In order to gain a deeper insight into the genetic regulation of cytokine-determined and immunoglobulin/ T cell receptor based signaling in lymphocytes, efforts to use RNA interference (RNAi) technology as a screening tool have been undertaken. An shRNA library consisting of all the mouse phosphatases has been prepared in lentivirus vectors with a puromycin resistance element. Unit scientists have chosen to analyze the importance of phosphatases in peripheral CD4 T cell differentiation to Th1 cells. The model utilized takes advantage of an indicator mouse prepared in the Unit in which ZS-Green expression marks the presence of the master Th1 transcriptional regulator T-bet. When T cells differentiate in vitro into Th1 cells, they express very large amounts of GFP, are easily detectable and can be purified by cell sorting. The initial screen carried out was aimed at determining what phosphatases were important for the expression or the extinction of T-bet during the Th differentiation process. Nave CD4 T cells were exposed to a library of all shRNAs complementary to phosphatases (over 1000 members), The shRNAs were in lentiviruses so that they could be introduced into naive CD4 T cells. The cells were then differentiated under Th1 conditions for 4 days, rested in IL-2 and exposed to puromycin, to eliminate cells that had not incorporated and expressed a member of the library. At this stage the great majority of the cells were already ZS-Green+. The cells were then shifted to Th2 culture conditions. At the end of four additional days of culture , 1/2 of the former Th1 cells were T-bet negative. The T-bet negative and positive cells were purified and the incorporated shRENAs were PCR amplified, utilizing a method to avoid the difficulties resulting from the hairpin structure. The resulting large set of amplified shRNAs were subjected to deep sequencing using an ABI instrument. More than 20 shRNAs were found to be uniquely expressed in the T-bet negative cells and a similar number in the T-bet positives. The technical aspects were were shown to be reproducible using PCR amplification with one external and one internal primer. MTMR7 and MTMR9, phosphateses that act on phosphatidyl inositols showed reproducible activity both in repetition of the assay used in screening and in priming done under limiting Th1 conditions. Assays indicate that these phosphatases can effect the PI-3 kinase pathway, as shown by their activity to regulate AKT phosphorylation. This work emphasis the key role played by phosphatases in regulating lymphocyte differentiation and the power of library based screening in identifying candiates for further analysis. In parallel, a chemical genomic screen has been undertaken to examine the cpacity of various small molecules from the LOPAC set to alter the rate at which Th17 cells switch to Th1 cells. This has been done using the ZS-Green marker so that small molecules that inhibit or enhance CD4 T cell plasticity have a reasonable likelihood of being detected. Several candidates that inhibit switching have been identified and are now in the course of secondary screening.