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: 8336199
• 负责人: William Paul
• 金额: $ 57.24万
• 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/8336199
• 关键词: 1-Phosphatidylinositol 3-Kinase; Biological Assay; Biological Process; CD4 Positive T Lymphocytes; Candidate Disease Gene; Cell Separation; Cell physiology; Cells; Chemicals; 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; Peripheral; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphorylation; Play; Population; Preparation; Process; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proto-Oncogene Proteins c-akt; Puromycin; RNA Interference; 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. 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.

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。已在带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。 该单位科学家选择分析磷酸酶在外周 CD4 T 细胞分化为 Th1 细胞中的重要性。 所使用的模型利用了在该单元中制备的指示小鼠，其中 ZS-Green 表达标记了主 Th1 转录调节因子 T-bet 的存在。 当 T 细胞在体外分化为 Th1 细胞时，它们表达大量 GFP，很容易检测到，并且可以通过细胞分选进行纯化。 进行的初始筛选旨在确定哪些磷酸酶对于 Th 分化过程中 T-bet 的表达或消亡至关重要。 将幼稚 CD4 T 细胞暴露于与磷酸酶互补的所有 shRNA 文库（超过 1000 个成员）。这些 shRNA 位于慢病毒中，以便可以将它们引入幼稚 CD4 T 细胞中。 然后将细胞在 Th1 条件下分化 4 天，在 IL-2 中静置并暴露于嘌呤霉素，以消除尚未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+。 然后将细胞转移至Th2培养条件。 在另外四天的培养结束时，1/2 的前 Th1 细胞呈 T-bet 阴性。 纯化 T-bet 阴性和阳性细胞，并利用避免发夹结构带来的困难的方法对掺入的 shRENA 进行 PCR 扩增。 使用 ABI 仪器对所得的大量扩增 shRNA 进行深度测序。 超过 20 个 shRNA 被发现在 T-bet 阴性细胞中独特表达，而在 T-bet 阳性细胞中也有类似数量的表达。 使用一种外部引物和一种内部引物的 PCR 扩增表明技术方面是可重复的。 作用于磷脂酰肌醇的磷酸酶 MTMR7 和 MTMR9 在重复筛选和在限制 Th1 条件下进行的引发试验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发挥的关键作用，以及基于文库的筛选在识别候选物以供进一步分析方面的作用。 与此同时，还进行了化学基因组筛选，以检查 LOPAC 组中各种小分子改变 Th17 细胞转变为 Th1 细胞的速率的能力。 这是使用 ZS-Green 标记完成的，因此抑制或增强 CD4 T 细胞可塑性的小分子有合理的可能性被检测到。 已经确定了几种抑制转换的候选药物，目前正在进行二次筛选。