Analyzing Cytokine- and TCR-Mediated Lymphocyte Response

分析细胞因子和 TCR 介导的淋巴细胞反应

• 批准号: 7313476
• 负责人: wiliam e paul
• 金额: --
• 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/7313476
• 关键词: Analyzing; Cytokine; TCR; Mediated; Lymphocyte

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. Selected libraries of shRNAs and siRNAs have been obtained. Introduction of siRNAs into resting CD4 T cells using Amaxa transfection technology has been achieved and, in test systems, impressive inhibition of expression and function of kinases such as Jak3 has been obtained. Currently, we have assembled a protein tyrosine kinase library and a protein tyrosine phosphatase library and will shortly begin testing this on the induction of IL-4-producing CD4 T cells from naive cells cultured in the absence of the principle IL-4 inducer, which is IL-4 itself. An initial effort at genome-wide siRNA screening has been performed with a mouse ?GeneNet? lentiviral siRNA Library? comprising 39,000 genes purchased from System Biosciences Inc. [SBI], targeting the induction of CD23 in M12.4.1 B lymphoma cells by IL-4. The library was successfully introduced in M12.4.1 cells by infection and non-infected cells eliminated based on the resistance of the infected cells to puromycin. The infected M12 cells were then stimulated with IL-4 and the cells that expressed CD23 were separated from the CD23-negative cells by fluorescence-based cell sorting. The negative cells were restimulated; expressing and non-expressing cells were separated again. After a third round, the CD23+ and CD23- cells were harvested, the lentiviral siRNA inserts amplified by PCR using primers from the flanking sequences in the vector and the amplified sequences evaluated by microarray analysis using Affymetrix gene chips. A series of candidate sequences were found that were strikingly enriched in the CD23-negative cells, presumably reflecting those siRNAs more likely to be found in non-CD23-expressers and thus, by inference, that have played some role in preventing cells from expressing CD23. Among these are the genes for: Jak1, Map4k2(a Map kinase, knase, kinase kinase), syk and a non-receptor-type protein tyrosine phosphatase. We are now in the process of validating whether siRNAs for these genes used independently will diminish the ability of IL-4 to induce CD23 and to also exclude whether the inhbition reflects an off-target effect of the siRNA. The identification of Jak1 is reassuring in that IL-4 signaling depends upon IL-4Ra; the cytosolic domain of IL-4Ra is associated withh Jak-1 and Jak1 is essential for IL-4-mediated signaling. Any genes that appear to be specific will be studied in greater detail to get insight into how they may block CD23 induction.

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。已获取选定的shrna和sirna文库。使用Amaxa转染技术将sirna引入静息CD4 T细胞已经实现，并且在测试系统中，已经获得了令人印象深刻的Jak3等激酶的表达和功能抑制。目前，我们已经组装了一个蛋白酪氨酸激酶文库和一个蛋白酪氨酸磷酸酶文库，并将很快开始在缺乏IL-4诱导剂（即IL-4本身）的幼稚细胞中诱导产生IL-4的CD4 T细胞。