Analyzing Cytokine- and TCR-Mediated Lymphocyte Responses by RNAi

通过 RNAi 分析细胞因子和 TCR 介导的淋巴细胞反应

• 批准号: 8555902
• 负责人: William Paul
• 金额: $ 29.07万
• 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/8555902
• 关键词: 1-Phosphatidylinositol 3-Kinase; Accounting; Biological Assay; Biological Process; CD4 Positive T Lymphocytes; Candidate Disease Gene; Cell Separation; Cell physiology; Cells; Chemicals; Cyclic AMP; Cytokine Gene; Development; Elements; Extinction (Psychology); Future; Gene Expression; Genes; Genetic; Genome; Genomics; Goals; Immunoglobulins; In Vitro; Interleukin-2; Lentivirus Vector; Libraries; Lymphocyte; Mediating; Methods; Modeling; Mus; Participant; Pathway interactions; Pattern; Peripheral; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphorylation; Play; Population; Preparation; Process; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proto-Oncogene Proteins c-akt; Purinergic P1 Receptors; Puromycin; RNA Interference; Receptor Activation; Regulation; Resistance; Rest; Role; Scientist; Screening procedure; Set protein; Signal Pathway; Signal Transduction; Staging; Structure; Subfamily lentivirinae; System; T cell differentiation; T-Cell Receptor; T-Lymphocyte; Technology; Th1 Cells; Work; base; cytokine; insight; instrument; interest; member; protein expression; response; small hairpin RNA; small molecule; tool

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. MTMR7 normally represses Th2 and Th17 differentiation while MTMR9 represses Th1 differentiation. The differential fnction of these two li9pid 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.

为了更深入地了解淋巴细胞中细胞因子决定的和基于免疫球蛋白/T细胞受体的信号转导的遗传调控，人们努力使用RNA干扰(RNAi)技术作为筛选工具。在带有嘌呤霉素抗性元件的慢病毒载体中，构建了一个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CD4T细胞分化为Th1细胞中的重要性。所使用的模型利用了在单元中准备的指示器小鼠，其中ZS-Green表达标志着主要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，很容易被检测到，并可以通过细胞分选得到纯化。初步筛选的目的是确定在Th分化过程中，哪些磷酸酶对T-bet的表达或消失是重要的。将幼稚的CD4T细胞暴露在与磷酸酶互补的所有shRNA文库(超过1000个成员)中，这些shRNA存在于慢病毒中，因此它们可以被导入到幼稚的CD4T细胞中。然后这些细胞在Th1条件下分化4天，在IL-2中休息，并暴露在嘌呤霉素中，以剔除没有整合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。在额外培养的4天结束时，前Th1细胞中有1/2的T-bet阴性。纯化了T-bet阴性和阳性细胞，并对掺入的shRENAs进行了PCR扩增，避免了发夹结构带来的困难。使用ABI仪器对得到的大量扩增shRNA进行深度测序。有20多个shRNA在T-bet阴性细胞中唯一表达，在T-bet阳性细胞中有类似数量的shRNA表达。用一个外部和一个内部引物进行的聚合酶链式反应扩增表明，技术方面是可重复性的。MTMR7和MTMR9，作用于磷脂酰肌醇的磷酸酯在重复筛选和在限制Th1条件下启动时都显示出可重复的活性。分析表明，这些磷酸酶可以影响PI-3激酶通路，其调节AKT磷酸化的活性表明。MTMR7通常抑制Th2和Th17的分化，而MTMR9抑制Th1的分化。这两种脂肪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，以及基于文库的筛选在鉴定候选细胞以进行进一步分析方面的能力。 与此同时，已经进行了化学基因组筛查，以检查Lopac集合中各种小分子的能力，以改变Th17细胞向Th1细胞转换的速度。这是使用ZS-Green标记完成的，因此抑制或增强CD4T细胞可塑性的小分子有合理的可能性被检测到。已经确定了几个抑制开关的候选基因，目前正在进行二次筛选。其中最有趣的是调节腺苷受体表达的分子，那些导致受体激活和cAMP升高抑制切换到Th1细胞的分子。