The intent of this proposal is to determine what factors prevent pre-diabetic individuals from developing diabetes. This proposal will investigate the regulation of IL-4 in a subset of T cells, which are implicated in the progression of human type I diabetes. In human disease, incomplete concordance of disease progression between identical twins, both with autoreactive T cells and autoantibodies suggests that other factors contribute to the incidence and progression of IDDM. Investigators have recently described a subset of regulatory V-alpha 24J-alphaQ T cells that are reduced in frequency and are Th1-like (lacking IL-4 secretion) in diabetic twins as compared to non-diabetic twins. In addition, the non- or slow-progressors to disease had high levels of the cytokines IL-4 and gamma-IFN in their circulation; this may reflect a more Th0- or Th2-like phenotype for these individuals. Investigator has set up collaborations with laboratories investigating T cell signaling events to address the question, what signaling events lead to the loss of IL-4 secretion in the invariant V-alpha 24J-alphaQ T cells in individuals with diabetes. Expression of the nuclear factors, GATA-3, c-maf, NIP45, and NF-AT species, that have been identified as IL-4 transcription factors, will be quantitated by Northern blot under conditions of activation and conditions of exogenous factors to bias cells toward a Th1 or Th2 phenotype. This approach will involve transfection of c-maf and GATA-3 into non IL-4 secreting V-alpha 24J-alphaQ T cells to determine if there is a dominant negative signal preventing IL-4 secretion. Further, proximal signaling pathways involving phosphorylation of STAT 6 on tyr-641 in the V-alpha 24J-alphaQ T cells will be examined. In addition, as there are defects in Ca(2+) flux in V-alpha 24J-alphaQ T cells from diabetics that do not secrete IL-4, early TCR associated signaling events will be surveyed. Besides examining targeted pathways, broader approaches, to isolate target genes, which may regulate IL-4 expression in V-alpha 24J-alphaQ T cells from subjects with diabetes, will be used. Subtraction methods coupled with PCR (RDA) and DNA microchip array hybridization will be performed. Lastly, whether the cytokine secretion phenotype of V-alpha 24J-alphaQ T cells from the diabetics can be altered to a Th0/Th2 phenotype using the data generated from the first three aims will be examined. The eventual goal of the project is to develop a therapy that can be administered to pre-diabetics that prevent their progression into diabetics.
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