: The regulation of helper T-cell clonal expansion, and differentiation influences the outcome of infections and the susceptibility to autoimmune diseases. Signal transduction pathways activated by cell surface receptors converge on transcriptional factors which determine effector responses by helper T-cells. A long-term goal of this work is to characterize transcriptional mechanisms that regulate the potency of effector T-cell responses in vivo as well as in vitro. Effector T-cells express restricted profiles of cytokines which in the extremes mark them as being T helper 1 (ThI) or T helper 2 (Th2) cells. When naive, uncommitted T helper precursors become Th1 effectors that produce TNFs and IFN-gamma, they provide help essential to inflammation and the host defense against pathogens in phagocytic cells. Because of this key role in host defense, the mechanisms by which signaling and transcriptional regulatory pathways control Th 1 development in vivo are of critical importance. The strength of an inflammatory effector response must be determined not only by the activation of specific cytokine genes (e.g., IFN-gamma) but also by efficiency of clonal expansion of antigen-specific T-cells and rates of gene transcription after differentiation. Using a T-cell-specific transgenic model to investigate the role of the NF-k/Rel pathway in vivo, we discovered a preferential requirement for NF-kB in the type 1 (inflammatory) T-cell-dependent response as compared to a type 2 (allergic) response. Preliminary studies provide evidence that clonal expansion in vivo is impaired, but also support the existence of a specific requirement for NF-kB in IFN-g gene activation. Using transgenic T-cells and retrovector-mediated transduction, in Aim 1 we will distinguish the relative contributions of clonal expansion vs. effector cytokine gene activation, and identify mechanism(s) of NF-kB in interferon gamma gene activation. In this Aim, one hypothesis is that NF-kB preferentially promotes Thi clonal expansion by mediating protection against restimulation-induced death. A second hypothesis is that the NF-kB/Rel pathway regulates induction of the Th1-specific transcription factor T-bet, and collaborates with T-bet in regulating IFN-gamma transcription rates. To complement these studies, we will use knockout mice to test the hypothesis that one specific NF-kB subunit, Rem, plays a T-cell-intrinsic role in Th1 development (Aim 2). Finally, in Aim 3 we will investigate the interplay between the T-bet and NF-kB/Rel pathways in effecting epigenetic changes associated with the initial activation of IFN-y gene expression (chromatin restructuring and CpG methylation). Together, the proposed experiments will provide important insights into the transcriptional regulation of T-cell-dependent inflammatory responses.
