T CELL ONTOGENY: MECHANISMS OF IFN GAMMA REGULATION

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DESCRIPTION (Adapted from Applicant's Description): Interferon-gamma (IFN-gamma) is a critical immunomodulatory molecule produced by activated effector T cells, but unlike IL2 it is not efficiently expressed by antigenically naive T cells. This proposal will characterize the molecular mechanisms responsible for this differential expression of IFN-gamma in neonatal T cells compared to adult T cells. The preliminary data demonstrates that transient transfection assays using the IFN-gamma 5' promoter driving luciferase can be successfully accomplished in primary non-transformed human T cells. In addition, the data show that the p38 MAP kinase signal transduction pathway is critical for expression of IFN-gamma and that the IFN-gamma promoter is directly activated by IL12 treatment of the human T cells. Aim 1 will characterize the essential regulatory elements and transcription factors which regulate IFN-gamma gene transcription in primary T cells. This will be accompanied by using transient transfection assays of adult and neonatal T cells with IFN-gamma reporter plasmids. Regulatory elements and associated transcription factors required to direct differences in expression during T cell maturation and differences in expression during T cell maturation and differentiation will be identified using DNASE footprinting and gel-shift assays. Aim 2 will characterize p38 MAP kinase activity in adult and neonatal T cells in response to cellular activation as well as cytokines and co-stimulatory signals. In Aim 3, the molecular mechanisms of IL12 and IL18 induction of IFN-gamma expression in adult and neonatal T cells will be determined. Methods will include ELISA determination of IFN-gamma secretion, Northern blot detection of IFN-gamma mRNA and transient transfection assays with reporter plasmids. Characterization of the transcriptional regulation of IFN-gamma during T cell activation and effector cell differentiation will serve as a model for understanding the ontogeny of T cell maturation in the neonate. This will provide a rationale approach for future therapeutic interventions to reduce the infant s increased susceptibility to disseminated viral and intracellular infections. Furthermore, elucidation of the transcriptional regulation of IFN-gamma during antigen-driven priming will also contribute to the understanding of the pathogenesis of T helper cell type 1 (Th-1) mediated diseases.