T-bet down-modulation in tolerized Th1 effector CD4 cells confers a TCR-distal signaling defect that selectively impairs IFN-gamma expression.

When Th1 effector CD4 cells encounter tolerizing Ag in vivo, their capacity to express the effector cytokines IFN-gamma and TNF-alpha is lost more rapidly than noneffector functions such as IL-2 production and proliferation. To localize the relevant intracellular signaling defects, cytokine expression was compared following restimulation with Ag vs agents that bypass TCR-proximal signaling. IFN-gamma and TNF-alpha expression were both partially rescued when TCR-proximal signaling was bypassed, indicating that both TCR-proximal and -distal signaling defects impair the expression of these two effector cytokines. In contrast, bypassing TCR-proximal signaling fully rescued IL-2 expression. T-bet, a transcription and chromatin remodeling factor that is required to direct the differentiation of naive CD4 cells into IFN-gamma-expressing Th1 effectors, was partially down-modulated in tolerized Th1 effectors. Enforcing T-bet expression during tolerization selectively rescued the ability to express IFN-gamma, but not TNF-alpha. Conversely, expression of a dominant-negative T-bet in Th1 effectors selectively impaired the ability to express IFN-gamma, but not TNF-alpha. Analysis of histone acetylation at the IFN-gamma promoter further suggested that down-modulation of T-bet expression during Th1 effector CD4 cell tolerization does not impair IFN-gamma expression potential through alterations in chromatin structure.