GATA3 is a crucial transcription factor in the regulation of T helper cell differentiation. Not only is GATA3 critical for inducing Th2 differentiation, but it is also essential for repressing Th1 differentiation. It has been reported that GATA3 represses the IL-12-STAT4-IFNγ pathway during Th2 differentiation through multiple mechanisms including the suppression of both IL-12Rβ2 and STAT4 (Ouyang et al., 1998
; Usui et al., 2003
). Here, by using a mouse line carrying a Cre transgene that conditionally deletes Gata3
in naïve CD4+
T cells, we demonstrated that GATA3 actively suppresses IFNγ production even in the presence of IL-4, anti-IL-12 and anti-IFNγ. Without GATA3, the expression of STAT4 is increased, but only a modest increase of IL-12Rβ2 mRNA expression was noted. IL-12Rβ2 expression can be regulated by the IL-12-STAT4 and the IFNγ-T-bet pathways (Afkarian et al., 2002
; Lawless et al., 2000
; Ouyang et al., 1998
). Thus, during Th1 differentiation in the presence of IL-12, the direct target suppressed by enforced GATA3 is likely to be STAT4 and through regulating the IL-12-STAT4 pathway, GATA3 indirectly affects IL-12Rβ2, IFNγ, and T-bet expression.
In this report, we describe Runx3-Eomes-mediated IFNγ production in GATA3-deficient “Th2” cells. Such IFNγ production is IL-12-STAT4- and IFNγ-T-bet-independent. The expression of Eomes, but not T-bet, is enhanced in GATA3-deficient CD4+
T cells cultured under Th2 conditions, where both IL-12 and IFNγ are neutralized. Enforced Eomes expression during Th2 differentiation induces the capacity of these cells to produce IFNγ in the absence of T-bet. Moreover, GATA3-single deficient and GATA3-T-bet-double deficient CD4+
T cells cultured under Th2 conditions produce similar amount of IFNγ. Such IFNγ production is inhibited by T-bet DN, which has been reported to suppress the function of both T-bet and Eomes (Pearce et al., 2003
). Although the inhibition of IFNγ production by T-bet DN is only partial, the data clearly imply that Eomes, not T-bet, contributes to IFNγ production in GATA3-deficient “Th2” cells. We have reported earlier that in Gata3
-deleted “Th2” clones, T-bet expression was elevated (Zhu et al., 2006
). Since these clones had been cultured for a long period of time, it is possible that the large amount of IFNγ produced by these clones in the culture, were not completely neutralized and that IFNγ was responsible for T-bet induction.
Expressing Runx3 in activated CD4+
T cells cultured under Th2 conditions strongly suppresses IL-4 production. Although others have argued that the suppression of IL-4 by Runx3 requires T-bet (Djuretic et al., 2007
), our results clearly demonstrate the suppression of IL-4 by Runx3 is as efficient in T-bet-deficient Th2 cells as in WT cells. In addition, the ChIPseq data show that the Runx protein complex but not T-bet binds to the HS IV site of the Il4
locus in Th1 cells. However, T-bet but not Runx3 suppresses GATA3 expression. Thus, Runx3 and T-bet can collaborate in repressing IL-4 production through two different mechanisms--Runx3 directly suppresses IL-4 and T-bet indirectly affects IL-4 by repressing GATA3.
Besides suppressing IL-4 production, Runx3 induces IFNγ production in Th2 cells correlated with strong induction of Eomes, but not T-bet. Although there is only a slight increase of Runx3 expression when GATA3 is absent, deletion of Runx3 from GATA3-T-bet double deficient cells results in dramatic decrease in IFNγ production indicating that Runx3 is needed for IFNγ production in these cells. However, deletion of Runx3 from GATA3-T-bet double deficient cells only causes a modest decrease in Eomes expression suggesting Runx3 is redundant for Eomes induction in GATA3-deficient “Th2” cells. Furthermore, although Runx3 is highly expressed in Th1 cells, only a small percentage of these cells express Eomes even when GATA3 is deleted, suggesting that Runx3 is not sufficient to induce Eomes expression in a Th1 environment. Whether the Eomes-expressing CD4+
T cells represent a unique lineage of Th cells needs to be further studied. Nevertheless, it has been recently reported that CD8+
T cells, in which GATA3 is under-expressed, have adopted the Runx3-Eomes pathway for their optimal IFNγ production (Cruz-Guilloty et al., 2009
Runx3 is highly expressed in Th1 cells and enforced expression of Runx3 DN in Th1 cells results in diminished IFNγ production consistent with an earlier report that Runx3 regulates IFNγ production (Djuretic et al., 2007
). Here we show Runx3-mediated IFNγ production can be blocked by GATA3. Furthermore, by using ChIPseq, we show that, in normal Th1 cells, the Runx protein complex binds to multiple critical regulatory elements of Ifng
gene, some of which are also bound to T-bet. STAT4 has also been reported to directly bind to the Ifng
gene. Therefore, optimal IFNγ production and Th1 differentiation involves three pathways: IL-12-STAT4-, IFNγ-T-bet- and Runx3-mediated pathways. Each pathway makes some contribution to IFNγ production through direct action on the Ifng
gene, and there is also crosstalk among these pathways. For example, STAT4 and T-bet have a synergistic effect on inducing IFNγ production; Runx3 and T-bet can interact with each other and bind to many sites in Ifng
locus; T-bet may be responsible for up-regulating Runx3 expression; IL-12-STAT4 is partially responsible for T-bet up-regulation; T-bet down-regulates GATA3 during Th1 differentiation resulting in the up-regulation of STAT4 as well as the release of the inhibition of Runx3-mediated IFNγ production. Our finding of GATA3 regulating Runx3 function and STAT4, but not T-bet expression, increases our understanding on the cross-regulation of Th1 versus Th2 differentiation.
We also show that GATA3 physically interacts with Runx3 in primary T cells and the level of IFNγ production strongly correlates with the ratio of Runx3 to GATA3. The N-terminus and zinc fingers, but not C-terminus of GATA3, are required for the interaction with Runx3. The GATA3 mutant with the C-terminal deletion substantially loses its ability to induce IL-4, but is fully functional in suppressing Runx3-mediated IFNγ production, suggesting that the positive and negative functions of GATA3 can be separated. This may be critical for guiding effective immune intervention strategies in treating Th1 and Th2-related diseases.
Two reports previously suggest that the association of transcription factors dominant in different Th lineages can mutually regulate their functions (i.e. T-bet and GATA3; Foxp3 and RORγt) (Hwang et al., 2005
; Zhou et al., 2008
). Here, we reported a third example of such mutual regulation, between GATA3 and Runx3 during Th1-Th2 differentiation. Since both GATA3 and Runx3 are also critical during CD4 versus CD8 lineage commitment in thymus (Bosselut, 2004
; Hernandez-Hoyos et al., 2003
; Pai et al., 2003
; Sato et al., 2005
; Setoguchi et al., 2008
; Taniuchi et al., 2002
), the interaction and cross-regulation of these two molecules may also play a role in determining CD4-CD8 fate in the thymus, a subject that needs to be further studied.
In conclusion, Runx3 directly binds to many critical regulatory elements at Ifng locus and induces IFNγ expression even in the absence of T-bet. GATA3 suppresses Runx3-mediated IFNγ production through protein-protein interaction. Therefore, the relative amount of GATA3 and Runx3 expression regulates Th1 versus Th2 responses.