DGK in T Cell Regulation and Tolerance

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Program Director/Principal Investigator (Last, First, Middle): Gajewski, Thomas F. 1R01 AI080745-01A1 Revised Abstract Section Evidence suggests that one mechanism of peripheral immunologic tolerance occurs through the process of T cell anergy. Anergic I cells have been characterized to have a defect in TCR/CD28mediated Ras activation. Recent data have indicated that upregulated expression of lipid kinases of the diacylglycerol kinase (DGK) family, in particular DGK-u, participate in the suppression of (Ti j-<-0 L]' ADZ .Ti U,0 0(Q 0-0 COD c-0 'D- 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-farnily binding sites in the putative DGK-ci promoter. The major goal of this proposal is to gain a detailed understanding of DGKs and EGR2 in controlling T cell activation and peripheral ._0 5a' coo u�r tow tolerance. In the first specific aim, the transcriptional regulation of the DGK-u 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, ... OUP adenoviruses for EGR2 expression, and a Cre adenovirus for conditional deletion of EGR2 in peripheral I cells. Supportive data will come from ChIP assays and gene expression profiling. In O-0 the second specific aim, the functional role of DGK-u tyrosine phosphorylabon will :E, �c' N-0 0-0 aux. be o03�-' targeted genes in the post-thymic I 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 (gyp m='(Dm 3m03 a�fl` .., -'1 ascertained. Mutants of DGK-z will be analyzed functionally using adenoviral vectors and CAR Tg T cells in vitro, and transgenic mice expressing key DGK-u 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 directly in peripheral T cells on peripheral tolerance will be investigated. These experiments will take advantage of the Cre-adenovirus approach to delete conditionally _=c� l17 immunologic tolerance in disease situations. 111 CDG'O V,12 oar coo COB