Global Gene Regulation in CD4 T Cell Signaling

点击翻译按钮获取中文摘要

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.