UNIVERSITY OF CHICAGO, THE
Evidence suggests that one mechanism of peripheral immunologic tolerance occurs through the process of T cell anergy. Anergic T cells have been characterized to have a defect in TCR/CD28-mediated Ras activation. Recent data have indicated that upregulated expression of lipid kinases of the diacylglycerol kinase (DGK) family, in particular DGK-(, participate in the suppression of RasGRP-mediated Ras activation in the anergic state. Correlating with these changes is increased expression of the transcriptional regulator EGR2, and we have identified EGR-family binding sites in the putative DGK-( promoter. The major goal of this proposal is to gain a detailed understanding of DGKs and EGR2 in controlling T cell activation and peripheral tolerance. In the first specific aim, the transcriptional regulation of the DGK-( will be elucidated, with a focus centered on EGR2. Novel tools for the genetic manipulation of primary T cells will be employed using the CAR Tg system. This includes transcriptional reporter adenoviral vectors, adenoviruses for EGR2 expression, and a Cre adenovirus for conditional deletion of EGR2 in peripheral T cells. Supportive data will come from ChIP assays and gene expression profiling. In the second specific aim, the functional role of DGK-( tyrosine phosphorylation will be ascertained. Mutants of DGK-( will be analyzed functionally using adenoviral vectors and CAR Tg T cells in vitro, and transgenic mice expressing key DGK-( mutants in peripheral T cells will be studied for altered immune function in vivo. In the third specific aim, the consequence of elimination of EGR2 or DGK-( directly in peripheral T cells on peripheral tolerance will be investigated. These experiments will take advantage of the Cre-adenovirus approach to delete conditionally targeted genes in the post-thymic T cell compartment. T cell activation properties, in vivo autoimmunity and anergy susceptibility, and possible improved tumor rejection in vivo will be addressed. In total, this work will characterize in detail a critical pathway in the control of peripheral tolerance, paving the way for development of pharmacologic agents to manipulate immunologic tolerance in disease situations. PUBLIC HEALTH RELEVANCE: T cell anergy is a mechanism by which the immune system regulates tolerance to tissues, a process relevant for cancer as well as for autoimmunity. The overall goal of this proposal is to understand in detail the critical molecular changes that we believe control whether T cells become anergic or not. The results of these experiments should pave the way for the development of pharmacologic approaches for the manipulation of immunologic tolerance in relevant disease situations in vivo.