Studies Of Th1/Th2 Differentiation

在此期间，我们进一步利用IL-12beta2链转基因小鼠和用来制造这种小鼠的转基因构建物，广泛地研究了STAT4在Th1分化中的作用。在这些研究中，我们最初注意到STAT4缺陷细胞表现出IL-12Rbeta2链的低表达，从而提出了STAT4的主要功能是维持IL-12Rbeta2链的表达，而不是对干扰素-γ转录的下游影响。为了检验这种可能性，我们首先证明了表达转基因IL-12Rbeta2链的STAT4-/-CD4+T细胞不能经历IL-12诱导的干扰素-γ的产生或增殖。这一结果表明，在没有STAT4的情况下，即使存在合格的IL-12R，Th1分化也是不可能的。在进一步的实验中，我们发现，内源性IL-12Rbeta2链缺陷的CD4+T细胞感染了表达β2链胞浆酪氨酸突变位点的逆转录病毒，导致细胞表达无法支持STAT4酪氨酸磷酸化的Beta2链，也不能经历IL-12诱导的干扰素-γ的产生或增殖。IL-12Rbeta2不能激活STAT4(但能够为其他Th1功能提供信号)的存在再次表明，激活的STAT4对于Th1分化是必要的，因为STAT4可能作用于干扰素-伽马转录。在相关研究中，我们还利用这个系统确定了Beta2链胞质内片段上的酪氨酸位点，它介导了STAT4和STAT3的激活，因此是Th1T细胞分化和增殖所必需的。我们发现了STAT4和STAT3必需的三个酪氨酸位点，其中两个是重叠的。这一结果与在人类细胞中发现的不同，在人类细胞中，只有一种酪氨酸能够介导STAT4的磷酸化。最后，我们发现在没有激活的STAT4的情况下，IL-12诱导的Th1T细胞的增殖可以发生，尽管水平降低。这可能是由于未激活的STAT4的作用，因为在STAT4缺陷的细胞中没有看到IL-12的增殖。

In the current period we made further use of IL-12beta2 chain transgenic mice and the transgenic construct used to create this mouse in an extensive examination of the role of STAT4 in Th1 differentiation. In these studies we noted initially that STAT4 deficient cells exhibit poor expression of the IL-12Rbeta2 chain and thus raised the possibility that the major function of STAT4 is the maintenance of IL-12Rbeta2 chain expression rather than down-stream effects on IFN-gamma transcription. To examine this possibility, we first showed that STAT4-/- CD4+ T cells expressing transgenic IL-12Rbeta2 chains are unable to undergo IL-12-induced IFN-gamma production or proliferation. This result showed that in the absence of STAT4, Th1 differentiation is not possible in spite of the presence of a competent IL-12R. In further experiments, we showed that CD4+ T cells deficient in endogenous IL-12Rbeta2 chain and infected with retroviruses expressing mutated beta2 chain cytoplasmic tyrosine sites that resulted in cells expressing a beta2 chain that was unable to support STAT4 tyrosine phosphorylation are also unable to undergo IL-12 induced IFN-gamma production or proliferation. The presence of an IL-12Rbeta2 unable to activate STAT4 (but able to provide signals for other Th1 functions) again showed that activated STAT4 is necessary for Th1 differentiation in that STAT4 acts "downstream," presumably on IFN-gamma transcription. In related studies we also used this system to define the tyrosine sites on the intracytoplasmic segment of the beta2 chain that mediate both STAT4 and STAT3 activation and thus are necessary for Th1 T cell differentiation and proliferation. We found three tyrosine sites necessary for STAT4 and STAT3, two which are overlapping. This result differs from those found in human cells where only one tyrosine is able to mediate STAT4 phosphorylation. Finally, we showed that Th1 T cell proliferation induced by IL-12 could occur in the absence of activated STAT4 albeit at a reduced level. This may be due to effects of unactivated STAT4 as no IL-12 proliferation was seen in STAT4 deficient cells.