Global Gene Regulation in CD4 T Cell Signaling

描述(由申请人提供)：到目前为止，对控制HIV在CD4淋巴细胞中复制能力的病毒和细胞调控元件之间的复杂相互作用知之甚少。有人认为，HIV复制的调控与T细胞激活事件的控制密切相关，并且HIV有效地利用、转移或抑制T细胞信号通路的特定成分来启动和促进其自身的生长周期。这一研究领域的长期目标是确定并确定控制HIV在初级CD4淋巴细胞中复制的T细胞激活级联成分的相对重要性。如果能够确定对病毒复制能力至关重要，但对T细胞活性和功能是多余的细胞调控元件，那么这种细胞因子可能为创新治疗干预的发展提供新的靶点。对调节CD4T细胞活化的基本生物学机制的深入了解将极大地提高成功识别具有这些独特特征的细胞调节成分的机会。因此，本文提出的研究目标是在全球范围内阐明通过特定的T细胞信号转导途径诱导的调控基因级联。为了达到这一目标，我们将使用一个定义明确的分离的幼稚和记忆表型亚群的原代CD4细胞模型来确定T细胞成熟状态和特定信号参与在特征基因表达谱生成中的作用。实验旨在实现以下特定目的：1)在存在或不存在CD28共刺激的情况下，最大或最小TCR诱导刺激的条件；以及2)与TCR诱导刺激所产生的“内稳态”细胞因子刺激形成对比。为了实现上述目标，选定的已知与艾滋病毒复制增强和减少相关的培养条件将用于比较基因芯片微阵列分析定义的诱导细胞基因表达谱。观察到的特定细胞激活途径中基因表达的差异将通过实时荧光聚合酶链式反应(Real-time PCR)得到确认和量化。然后，可以通过流式细胞仪和Western印迹方法，在蛋白质生产的翻译水平上进一步研究基因表达的差异。

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.