Analyzing Cytokine- and TCR-Mediated Lymphocyte Responses by RNAi

为了更深入地了解淋巴细胞中由细胞因子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，已经开始努力使用RNA干扰（RNAi）技术作为筛选工具。在带嘌呤霉素耐药元件的慢病毒载体上制备了包含所有小鼠磷酸酶的shRNA文库。单位科学家选择分析磷酸酶在外周血CD4 T细胞向Th1细胞分化中的重要性。所使用的模型利用了在Unit中制备的指示小鼠，其中ZS-Green的表达标志着主Th1转录调控因子T-bet的存在。当T细胞在体外分化为Th1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。最初筛选的目的是确定在Th分化过程中哪些磷酸酶对T-bet的表达或消失很重要。将未成熟的CD4 T细胞暴露于一个包含所有与磷酸酶互补的shrna（超过1000个成员）的文库中，这些shrna位于慢病毒中，因此它们可以被引入未成熟的CD4 T细胞中。然后细胞在Th1条件下分化4天，在IL-2中休息，并暴露于嘌呤霉素中，以消除未合并和表达文库成员的细胞。在这个阶段，绝大多数细胞已经是ZS-Green+。然后将细胞转移到Th2培养条件下。在另外4天的培养结束时，1/2的前Th1细胞为T-bet阴性。纯化T-bet阴性和阳性细胞，利用避免发夹结构带来的困难的方法对合并的shRENAs进行PCR扩增。由此产生的大量扩增shrna使用ABI仪器进行深度测序。超过20个shrna在T-bet阴性细胞中唯一表达，在T-bet阳性细胞中也有相似的数量。技术方面被证明是可重复使用PCR扩增与一个外部和一个内部引物。MTMR7和MTMR9，作用于磷脂酰肌醇的磷酸酯在筛选和限制性Th1条件下的引物中都显示出可重复的活性。实验表明，这些磷酸酶可以影响PI-3激酶通路，如它们调节AKT磷酸化的活性。MTMR7通常抑制Th2和Th17的分化，而MTMR9则抑制Th1的分化。这两种脂质磷酸酶的不同功能可以通过它们的表达模式来解释。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，以及基于文库筛选确定候选细胞以供进一步分析的能力。

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 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. MTMR7 normally represses Th2 and Th17 differentiation while MTMR9 represses Th1 differentiation. The differential fnction of these two lipid phosphatases can be accounted for by their pattern of expression. 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. Among the most interesting are molecules that regulate expression of adenosine receptors, with those that cause activation of receptors and elevation of cAMP repressing switching to Th1 cells.