Analyzing Cytokine- and TCR-Mediated Lymphocyte Response

为了更深入地了解淋巴细胞中由精氨酸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，已经进行了使用RNA干扰（RNAi）技术作为筛选工具的努力。已经获得了选择的shRNA和siRNA文库。已经实现了使用Amaxa转染技术将siRNA引入静息CD 4 T细胞，并且在测试系统中，已经获得了对激酶如Jak 3的表达和功能的令人印象深刻的抑制。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，并将很快开始测试其对在不存在主要IL-4诱导剂（即IL-4本身）的情况下培养的幼稚细胞诱导产生IL-4的CD 4 T细胞的作用。 在全基因组siRNA筛选的初步努力已经进行了小鼠？基因网？慢病毒siRNA文库？包含39，000个购自System Biosciences Inc.的基因。[SBI]靶向IL-4对M12.4.1 B淋巴瘤细胞中CD 23的诱导。通过感染将该文库成功地引入M12.4.1细胞中，并基于感染细胞对嘌呤霉素的抗性消除未感染的细胞。然后用IL-4刺激感染的M12细胞，并通过基于荧光的细胞分选将表达CD 23的细胞与CD 23阴性细胞分离。再次刺激阴性细胞;再次分离表达和非表达细胞。第三轮后，收获CD 23+和CD 23-细胞，使用来自载体中侧翼序列的引物通过PCR扩增慢病毒siRNA插入物，并使用Affyphase基因芯片通过微阵列分析评估扩增的序列。发现一系列候选序列在CD 23阴性细胞中显著富集，推测反映了那些siRNA更可能在非CD 23表达者中发现，因此，通过推断，它们在阻止细胞表达CD 23中发挥了一些作用。其中包括Jak 1、Map 4k 2（一种Map激酶、knase、激酶激酶）、syk和一种非受体型蛋白酪氨酸磷酸酶的基因。我们现在正在验证独立使用的这些基因的siRNA是否会降低IL-4诱导CD 23的能力，并排除这种抑制是否反映了siRNA的脱靶效应。Jak 1的鉴定令人放心，因为IL-4信号传导依赖于IL-4 Ra; IL-4 Ra的胞质结构域与Jak-1相关，并且Jak 1对于IL-4介导的信号传导是必需的。将更详细地研究任何似乎是特异性的基因，以了解它们如何阻断CD 23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.