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Cell lineage specification depends on both gene activation and gene silencing, and in the differentiation of T helper progenitors to Th1 or Th2 effector cells, this requires the action of two opposing transcription factors, T-bet and GATA-3. T-bet is essential for the development of Th1 cells, and GATA-3 performs an equivalent role in Th2 development. We report that T-bet represses Th2 lineage commitment through tyrosine kinase-mediated interaction between the two transcription factors that interferes with the binding of GATA-3 to its target DNA. These results provide a novel function for tyrosine phosphorylation of a transcription factor in specifying alternate fates of a common progenitor cell.

The transcription factors signal transducer and activator of transcription (STAT)1 and T-bet control the differentiation of interferon (IFN)-gamma-producing T helper type (Th)1 cells. Here we compare the role of T-bet and STAT1 in the initiation and regulation of experimental autoimmune encephalomyelitis (EAE), a disease initiated by Th1 cells. T-bet-deficient mice immunized with myelin oligodendrocyte glycoprotein (MOG) were resistant to the development of EAE. This protection was also observed when T-bet(-/-) mice were crossed to the MOG-specific 2D2 T cell receptor transgenic strain. In contrast, although T-bet is downstream of STAT1, STAT1(-/-) mice were highly susceptible to EAE and developed more severe and accelerated disease with atypical neuropathologic features. The function of T-bet was dominant as mice deficient in both T-bet and STAT1 were also protected from EAE. CD4(+) CD25(+) regulatory T cells from these two mice strains were fully competent and do not explain the difference in disease susceptibility. However, enhanced EAE in STAT1(-/-) mice was associated with continued generation of IFN-gamma-producing Th1 cells and up-regulation of selective chemokines responsible for the increased recruitment of macrophages and neutrophils in the central nervous system. Although the two transcription factors, STAT1 and T-bet, both induce IFN-gamma gene transcription, our results demonstrate marked differences in their function in regulating pathogenic Th1 cell responses.

gammadelta T cells predominantly produce IFN-gamma upon activation. To determine the basis for default production of IFN-gamma by gammadelta T cells, we analyzed the transcription factors T-box expressed in T cells (T-bet) and GATA-3. T-bet, absent in naive cells, was induced upon TCR signaling, with IFN-gamma production. T-bet also regulated IL-4 synthesis, as gammadelta cells isolated from T-bet-deficient mice displayed enhanced IL-4 levels with reduced IFN-gamma production. Notably, T-bet expression after TCR signaling in gammadelta cells was not down-regulated by IL-4, in conjunction with a higher ratio of T-bet:GATA-3 expression than that found in CD4(+) T cells. Indeed, overexpression of GATA-3 failed to inhibit IFN-gamma secretion in gammadelta cells to the degree seen in CD4(+) T cells. These results indicate that T-bet enhances IFN-gamma secretion and suppresses IL-4 secretion in gammadelta cells, and that GATA-3 fails to counterbalance T-bet-mediated IFN-gamma production, accounting for the default synthesis of IFN-gamma by these T lymphocytes.

Much of our focus in understanding Th1/Th2 development has been on the signals delivered by IL-12 and IL-4 as final determinants of terminal T cell differentiation. Because extinction of IL-12 signaling in early Th2 development could potentially be important in imprinting a more permanent Th2 phenotype on a population of T cells, we have also examined various parameters regulating the IL-12 signaling pathway. Whereas IL-4 appears to repress functional IL-12 signaling through inhibition of IL-12R beta 2 expression, IFN-gamma in the mouse, and IFN-alpha in the human appear to induce IL-12R beta 2 expression and promote IL-12 responsiveness. We propose that Th1 development can be considered in two stages, capacitance and development. Capacitance would simply involve expression of IL-12R beta 1 and beta 2 subunits, regulated by TCR, IL-4 and IFNs. The second stage, development, we propose is the true IL-12 induced developmental stage, involving expression of Stat4 inducible proteins. In the human, this may also occur via IFN-alpha, which is able to activate Stat4. It is perhaps possible that all of Stat4 actions on Th1 development may be exert directly by Stat4 at the IFN-gamma gene, however we suggest that, more likely, Stat4 may act to induce Th1 development through the induction of other non-cytokine genes, whose stable expression maintains the transcriptional state of a Th1 cell.

Interleukin 12 (IL-12) initiates the differentiation of naive CD4+ T cells to T helper type 1 (Th1) cells critical for resistance to intracellular pathogens such as Leishmania major. To explore the basis of IL-12 action, we analyzed induction of nuclear factors in Th1 cells. IL-12 selectively induced nuclear DNA-binding complexes that contained Stat3 and Stat4, recently cloned members of the family of signal transducers and activators of transcription (STATs). While Stat3 participates in signaling for several other cytokines, Stat4 was not previously known to participate in the signaling pathway for any natural ligand. The selective activation of Stat4 provides a basis for unique actions of IL-12 on Th1 development. Thus, this study presents the first identification of the early events in IL-12 signaling in T cells and of ligand activation of Stat4.