Global Gene Regulation in CD4 T Cell Signaling

描述（由申请人提供）：迄今为止，对控制HIV在CD 4淋巴细胞中复制能力的病毒和细胞调节元件之间的复杂相互作用了解甚少。有人提出，HIV复制的调节与T细胞活化事件的控制密切相关，并且HIV有效地利用、转移或抑制T细胞信号传导途径的选择组分来启动和促进其自身的生长周期。在这个研究领域的长期目标是识别，并确定相对重要性，T细胞激活级联控制艾滋病毒复制的主要CD 4淋巴细胞的组件。如果能够鉴定出对病毒复制能力至关重要，但对T细胞活力和功能来说是多余的细胞调节元件，那么这些细胞因子可能为开发创新的治疗干预提供新的靶点。增加对CD 4 T细胞活化调节的基本生物学机制的理解，将大大增加成功鉴定具有这些独特特征的任何细胞调节组分的机会。因此，本文提出的研究的目标是在全球范围内阐明通过特异性T细胞信号转导途径诱导的调控基因级联。为了实现这一目标，将使用分离的幼稚和记忆表型亚群的明确定义的原代CD 4细胞模型来确定T细胞成熟状态和特异性信号参与在产生特征性基因表达谱中的作用。设计实验以实现比较由以下引起的基因表达谱的特定目的：1）在存在或不存在CD 28共刺激的情况下，最大至最小TCR诱导的刺激的条件;和2）与由TCR诱导的刺激产生的刺激相比的“稳态”细胞因子刺激。为了实现所述目的，将使用已知与增强与减少HIV复制相关的选定培养条件来比较通过基因芯片微阵列分析定义的诱导细胞基因表达谱。将使用实时PCR确认和定量观察到的特定细胞活化途径内的基因表达差异。然后可以通过流式细胞术和蛋白质印迹方法在蛋白质产生的翻译水平上进一步研究基因表达中的确认差异。

DESCRIPTION (provided by applicant): To date, little is understood about the complex interactions between viral and cellular regulatory elements that control the replicative capacity of HIV in CD4 lymphocytes. It is proposed that the regulation of HIV replication is linked tightly and specifically to the control of T cell activation events and that HIV efficiently utilizes, diverts, or inhibits select components of T cell signaling pathways to initiate and promote its own growth cycle. The long term objective in this research area is to identify, and determine the relative importance of, components of the T cell activation cascade that control HIV replication in primary CD4 lymphocytes. If cellular regulatory elements can be identified that are critical to viral replicative capacity, but redundant for T cell viability and function, then such cellular factors may provide new targets for the development of innovative treatment interventions. An increased understanding of the basic biologic mechanisms governing the regulation ofCD4 T cell activation would enhance greatly the chances for success in identifying any cellular regulatory components with these unique features. Therefore, the goal of the research proposed herein is to elucidate, on a global scale, the regulatory gene cascade that is induced through specific T cell signal transduction pathways. To address this goal, a well-defined primary CD4 cell model of isolated naive and memory phenotypic subpopulations will be used to determine the roles of T cell maturation state and specific signal engagement in the generation of characteristic gene expression profiles. Experiments are designed to achieve the specific aims of comparing gene expression profiles elicited by: 1) conditions of maximal to minimal TCR-induced stimulation, in the presence or absence of CD28 costimulation; and 2) "homeostatic" cytokine stimulation contrasted to that generated by TCR-induced stimulation. To achieve the stated aims, selected culture conditions that are known to be associated with enhanced vs. reduced HIV replication will be used to compare induced cellular gene expression profiles defined by GeneChip microarray analysis. Observed differences in gene expression within specific cell activation pathways will be confirmed and quantified using real-time PCR. Confirmed differences in gene expression may then be investigated further at the translational level of protein production, by flow cytometric and Western blot methods.