Analyzing Cytokine- and TCR-Mediated Lymphocyte Response

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。已获得选定的shRNAs和siRNAs文库。利用Amaxa转基因技术将siRNAs导入静止的CD4T细胞中，在测试系统中，JAK3等激酶的表达和功能得到了显著的抑制。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，并将很快开始测试在没有主要的IL-4诱导剂的情况下，从幼稚细胞中诱导产生IL-4的CD4T细胞。 用小鼠GeneNet？进行了全基因组siRNA筛选的初步努力。慢病毒siRNA文库？包括从System Bioscience Inc.购买的39,000个基因。[SBI]，靶向IL-4诱导M12.4.1 B淋巴瘤细胞中的CD23。通过感染成功地将文库导入M12.4.1细胞，并根据感染细胞对嘌呤霉素的抗性消除了非感染细胞。然后用IL-4刺激感染的M12细胞，通过基于荧光的细胞分选将表达CD23的细胞从CD23阴性的细胞中分离出来。对阴性细胞进行再刺激，将表达细胞和非表达细胞再次分离。第三轮后，收集CD23+和CD23-细胞，从载体侧翼序列扩增慢病毒siRNA插入片段，并用Affymetrix基因芯片芯片进行芯片分析。一系列候选序列在CD23阴性细胞中显著富含，推测反映了那些更有可能在非CD23表达者中发现的siRNA，由此推断，它们在阻止细胞表达CD23方面发挥了一定的作用。这些基因包括：JAK1、Map4k2(一种MAP、KNASE、KEK)、SYK和一种非受体类型的蛋白酪氨酸磷酸酶。我们现在正在验证单独使用这些基因的siRNAs是否会削弱IL-4诱导CD23的能力，并排除这种抑制是否反映了siRNA的非靶点效应。JAK1的鉴定是令人放心的，因为IL-4信号依赖于IL-4ra；IL-4ra的胞浆结构域与h Jak-1相关，JAK1对IL-4介导的信号转导是必不可少的。任何似乎具有特异性的基因都将被更详细地研究，以深入了解它们可能如何阻止CD23的诱导。

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.