Studies Of Th1/th2 Differentiation

在过去的几年中，控制Th 1分化的分子机制导致了两种主要的Th 1相关信号通路的鉴定，一种涉及IL-12/Stat 4，另一种涉及IFN-γ/Stat 1/T-bet。其中，后者或T-bet途径被认为是最重要的，因为T-bet缺陷小鼠（T-bet-/-小鼠）不能产生Th 1应答，并且在发育和/或发育的Th 2细胞中T-bet的逆转录病毒表达不仅诱导IFN-γ产生，而且抑制IL-4和IL-5产生。此外，T-bet的这种作用已经显示至少部分地不依赖于STAT 4，因为在某些条件下（PMA和离子霉素），T-bet在源自STAT 4缺陷小鼠的发育中的Th 1细胞中的逆转录病毒表达已经显示支持IFN-γ合成。在以前的研究中，我们发现加塔-3通过控制STAT 4水平来调节Th 1分化。因此，我们追求的想法是，T-bet在T细胞分化中的主要作用与其调节加塔-3水平和/或功能的能力有关。 在最初的研究中，我们表明，来自T-bet-/-小鼠的细胞（T-bet-/-细胞）实际上能够产生几乎正常量的IFN-γ，只要满足以下两个条件之一：通过在不存在IL-4的情况下培养细胞来阻断加塔-3积累，或者逆转录病毒STAT 4表达绕过了STAT 4水平的加塔-3负调节。在另外的研究中，我们阐述了T-bet和IL-12 R β 2链表达之间的关系。先前，Afkarian等人报道了在发育中的Th 2细胞中逆转录病毒T-bet表达诱导IL-12 R β 2链表达。使用类似的方法，我们使用完全分化的Th 2克隆证实了这一结果;然而，我们发现逆转录病毒表达和T-bet的内源性表达都不会导致发育中的Th 1细胞中的IL-12 R β 2表达。这与以下事实相关：T-bet-/-细胞在严格的Th 1条件下表达正常水平的IL-12 R β 2链，但在生理Th 1条件下（没有抗IL-4抗体）表达相对低水平的IL-12 R β 2链，在这种情况下，它们产生大量IL-4并优先发育成Th 2细胞。虽然这些数据表明T-bet对于IL-12 R β 2链表达不是必需的，但它们不排除通过其阻断加塔-3信号传导的能力的间接影响。我们目前的结果还表明，STAT 4信号可以诱导IL-12 R β 2链的表达，在T-bet的情况下。这一事实，加上我们先前的观察，即加塔-3通过对STAT 4的作用下调IL-12 R β 2链，强烈表明T-bet对IL-12 R β 2链的作用很可能是通过加塔-3和STAT 4的间接作用。 本研究强调了T-bet在调节加塔-3中的作用，这与Szabo等人先前的报告一致，该报告显示T-bet抑制Th 2细胞中的IL-4和IL-5，并通过显示在这些情况下逆转录病毒T-bet表达抑制加塔-3 mRNA和蛋白质，提供了该发现的机制基础。在我们当前研究的第三部分中，我们表明，虽然表达T-bet的D10细胞产生减少量的IL-4和IL-5，但这种减少显示依赖于TCR信号的强度，表明后者可以通过迄今未知的机制超越T-bet效应。此外，表达T-bet的D10细胞仅产生少量IFN-γ（与正常Th 1细胞相比）。这一结果与早期的研究一致，早期的研究表明，逆转录病毒T-bet表达只能在用非生理刺激物PMA和离子霉素刺激细胞后诱导大量IFN-γ产生。最后，虽然这些数据清楚地表明T-bet调节加塔-3，但它们并没有说明这种调节的机制。在使用环己酰胺处理的细胞的研究中，我们表明，虽然T-bet通过对加塔-3转录产生影响而不是对新合成的加塔-3 mRNA产生影响，至少部分介导了加塔-3抑制，但我们没有排除对加塔-3蛋白的影响。在后者方面，T-bet对加塔-3的影响可能是间接的，因为其活性可能涉及诱导中间体，例如加塔-3的朋友（FOG），其以某种方式影响加塔-3功能。 总之，这些发现提供了T细胞分化的改良模型的基础。在该模型中，假定加塔-3是其水平/活性决定辅助性T细胞分化命运的中心因子，不仅通过其诱导Th 2细胞因子产生的能力，而且通过其通过下调STAT 4和（间接）IL-12 R β 2链阻断Th 1细胞因子产生的能力。因此，在IL-12和IL-4存在下发生的T细胞分化过程中，（初始T细胞的通常环境）而Th 1分化是通过TCR和IL-12信号传导诱导STAT 4而启动的，这种分化不会发生，因为伴随的IL-4对STAT 6和加塔-3的诱导激活了上述加塔-3抑制STAT 4/IL-4的程序。12 R β 2链表达。然而，如果T-bet也以足够的水平被诱导，则这种加塔-3抑制被抵消并且Th 1分化确实发生。应该注意的是，虽然TCR刺激本身可以诱导T-bet，但调节加塔-3所必需的T-bet水平只能通过STAT 4诱导的IFN-γ产生和随后的STAT 1活化来获得。这种对T-bet上调的额外要求确保了在生理条件下，在缺乏强且持续的IL-12信号传导的情况下不会发生潜在有害的Th 1应答。

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.