Over the past several years, the molecular mechanisms governing Th1 differentiation, have led to the identification of two major Th1-related signaling pathways, one involving IL-12/Stat4 and the other IFN-gamma/Stat1/T-bet. Of these, the latter or T-bet pathway has been assumed to be the most important since T-bet deficient mice (T-bet-/- mice) cannot mount Th1 responses and retroviral expression of T-bet in developing and/or developed Th2 cells not only induces IFN-gamma production, but also suppresses IL-4 and IL-5 production. Moreover, such effects of T-bet have been shown to be at least partially independent of STAT4 since under certain conditions (PMA and ionomycin) retroviral expression of T-bet in developing Th1 cells derived from STAT4-deficient mice have been shown to support IFN-gamma synthesis. In previous studies we showed the GATA-3 regulates Th1 differentiation by controlling STAT4 levels. We therefore pursued the idea that the main role of T-bet in T cell differentiation relates to its ability to regulate GATA-3 levels and/or function. In initial studies, we showed that cells from T-bet-/- mice (T-bet-/- cells) are in fact capable of producing virtually normal amounts of IFN-gamma provided one of two conditions are met: GATA-3 accumulation is blocked by culturing cells in the absence of IL-4 or GATA-3 negative regulation of STAT4 levels is by-passed by retroviral STAT4 expression. In additional studies, we addressed the relation between T-bet and IL-12Rbeta2 chain expression. Previously, Afkarian et al. reported that retroviral T-bet expression in developing Th2 cells induces IL-12Rbeta2 chain expression. Using a similar approach, we confirmed this result using a fully differentiated Th2 clone; however, we found that neither retroviral expression nor endogenous expression of T-bet led to IL-12Rbeta2 expression in developing Th1 cells. This correlated with the fact that T-bet-/- cells expressed normal levels of IL-12Rbeta2 chain under strict Th1 conditions, but expressed relatively low levels of IL-12Rbeta2 chain under physiological Th1 conditions (without anti-IL-4 antibody), in which case they produce much IL-4 and preferentially develop into Th2 cells. While these data demonstrate that T-bet is not essential for IL-12Rbeta2 chain expression, they do not rule out an indirect influence through its ability to block GATA-3 signaling. Our current results also show that STAT4 signaling can induce IL-12Rbeta2 chain expression in the absence of T-bet. This fact, plus our previous observation that GATA-3 down-regulation of the IL-12Rbeta2 chain occurs through an effect on STAT4, strongly suggests that the T-bet effect on the IL-12Rbeta2 chain is most likely an indirect effect through GATA-3 and STAT4. The present study emphasizing the role of T-bet in regulating GATA-3 is consonant with previous reports by Szabo et al. showing that T-bet suppresses IL-4 and IL-5 in Th2 cells and provide a mechanistic basis of this finding by showing that GATA-3 mRNA and protein was suppressed by retroviral T-bet expression in these circumstances. In a third component of our current studies, we showed that while T-bet expressing D10 cells produced reduced amounts of IL-4 and IL-5, such reduction was shown to be dependent on the strength of the TCR signal indicating the latter can over-ride T-bet effects by as yet unknown mechanisms. In addition, T-bet-expressing D10 cells produced only a small amount of IFN-gamma (compared to normal Th1 cells). This result is consistent with earlier studies which showed that retroviral T-bet expression can only induce large amounts of IFN-gamma production following stimulation of cells with a non-physiological stimulus, PMA and ionomycin. Finally, while these data clearly establish that T-bet regulates GATA-3, they do not speak to the mechanisms of such regulation. In studies utilizing cyclohexamide-treated cells, we showed that while T-bet at least partially mediates GATA-3 suppression by exerting an effect on GATA-3 transcription rather than an effect on newly synthesized GATA-3 mRNA, we have not ruled out an effect on GATA-3 protein. In the latter regard, the T-bet effect on GATA-3 may be indirect in that its activity can involve the induction of intermediates such as friend of GATA-3 (FOG) that somehow influences GATA-3 function. In summary, these findings provide the basis of a modified model of T cell differentiation. In this model, GATA-3 is assumed to be the central factor whose level/activity decides the fate of helper T cell differentiation, not only through its capacity to induce Th2 cytokine production, but also through its capacity to block Th1 cytokine production via down-regulation of STAT4 and (indirectly) the IL-12Rbeta2 chain. Thus, in the course of T cell differentiation occurring in the presence of IL-12 and IL-4 (the usual milieu of a naive T cell) while Th1 differentiation is initiated by the induction of STAT4 by TCR and IL-12 signaling, such differentiation does not occur because concomitant IL-4 induction of STAT6 and GATA-3 activates the aforementioned program of GATA-3 suppression of STAT4/IL-12Rbeta2 chain expression. If, however, T-bet is also induced at sufficient levels, such GATA-3 suppression is counteracted and Th1 differentiation does occur. It should be noted that while TCR stimulation can itself induce T-bet, the level of T-bet necessary to regulate GATA-3 can only be obtained via STAT4-induced IFN-gamma production and subsequent activation of STAT1. This additional requirement for T-bet up-regulation ensures that under physiologic conditions, potentially harmful Th1 responses do not occur in the absence of strong and persistent IL-12 signaling.
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