Summary Forkhead Box P3 (Foxp3)-expressing regulatory T (Treg) cells are central to maintaining self-tolerance and immune homeostasis. How Treg cell function and Foxp3 expression are regulated is an important question under intensive investigation. Here, we have demonstrated an essential role for the transcription factor GATA-3, a previously recognized Th2 cell master regulator, in controlling Treg cell function. Treg cell-specific GATA-3 deletion led to a spontaneous inflammatory disorder in mice. GATA-3-null Treg cells were defective in peripheral homeostasis and suppressive function, gained Th17 cell phenotypes and expressed reduced amounts of Foxp3. In addition, GATA-3 controlled Foxp3 expression by binding to and promoting the activity of cis-acting elements of Foxp3. Furthermore, the combined function of GATA-3 and Foxp3 was essential for Foxp3 expression. These findings provide insights into immune regulatory mechanisms and uncover a critical function of GATA-3 in Treg cells and immune tolerance.

GATA-3 is a master regulator for TH2 differentiation. It is however poorly understood if GATA-3 controls mature T cell function beyond TH2 determination. We show that TCR signals and cytokine stimulation promoted GATA-3 expression in CD8 T cells, which controlled cell proliferation. While GATA-3-deficient CD8 T cells were generated, their peripheral maintenance was impaired with reduced IL-7R expression. GATA-3-deficient T cells had defective responses to viral infection and alloantigen. c-Myc was a critical target of GATA-3 in promoting the proliferation of activated T cells. This study thus demonstrates an essential role of GATA-3 in controlling T cell maintenance and expansion beyond TH2 differentiation, providing insights into immune regulation.

IntroductionHuman ␥␦ T cells appear to belong to nonclassical T cells that contribute to both innate and adaptive immune responses. Circulating V␥2V␦2 (also termed V␥9V␦2) T cells exist only in primates and, in humans, constitute 60% to 95% of total blood ␥␦ T cells. V␥2V␦2 T cells in primates can be activated by nonpeptidic phosphorylated metabolites of isoprenoid biosynthesis (eg, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate [HMBPP], isopentenyl pyrophosphate [IPP], and its isomer dimethylallyl pyrophosphate [DMAPP]). [1][2][3] We have recently shown that HMBPP is associated with antigen-presenting cell (APC) membrane and specifically recognized by V␥2V␦2 T-cell receptor (TCR) expressed on V␥2V␦2 T cells. 4 Although HMBPP produced by mycobacteria/other microbes is a potent activator for V␥2V␦2 T cells, these ␥␦ T cells possess both innate and adaptive immune features. [5][6][7][8] The finding that "unprimed" V␥2V␦2 T cells can recognize and react to wide ranges of nonpeptide phospholigands with the capability of "naive" production of cytokines has been interpreted as a pattern recognition-like feature of innate immune cells. On the other hand, the capacity of V␥2V␦2 T cells to undergo major clonal expansion in primary infection and to mount rapid recall-like expansion upon reinfection has been proposed as adaptive immune response of these ␥␦ T cells. 8 Consistent with these memory-type responses is the demonstration of memory phenotypes of V␥2V␦2 T cells in the blood of humans. 9 Accumulating evidence suggests that V␥2V␦2 T cells play a role in mediating immunity against microbial pathogens 8 and tumors. 10 Foxp3-expressiong CD4 ϩ CD25 ϩ regulatory T cells (Tregs) control immune responses to self-antigens and foreign antigens and play a major role in maintaining the balance between immunity and tolerance. 11-14 Murine CD4 ϩ CD25 ϩ regulatory T cells are induced by transforming growth factor ␤ (TGF-␤), although TGF-␤ plus IL-6 favors the development of Th17 cells. 15 [23][24][25][26] We and others have also shown that IL-2 plus phospholigand treatment can induce remarkable expansion of V␥2V␦2 T cells in nonhuman primates. 1,27,28 We therefore took advantage of the IL-2-based in vivo model systems to assess potential interplay or mutual regulation between V␥2V␦2 T cells and Tregs during early mycobacterial infection in nonhuman primates. We found that phosphoantigenactivated V␥2V␦2 T cells were able to down-regulate IL-2-induced expansion of Tregs, and antagonize Treg-driven suppression of in vivo immune responses. Methods AnimalsFour-to 8-year-old, 3-to 4-kg cynomolgus macaques (Macaca fascicularis) were used in this study. A total of 18 monkeys were divided into 3 groups, 6 for each. All animals were maintained and used in accordance with the guidelines of the institutional animal care and use committee of all participating institutions. Animals were anesthetized with 10 mg/kg ketamine HCl (Fort Dodge Animal Health, Fort Dodge, IA) intramuscularly for all blood sampling and treatments. EDTA-anticoagulated...

Dihydroartemisinin (DHA) is an important derivative of an herb medicine Artemisia annua L., used in ancient China. DHA is currently used world-wide to treat malaria by killing malaria-causing parasites. In addition to this prominent effect, DHA is suggested to regulate cellular functions, such as angiogenesis, tumor cell growth and immunity. Nonetheless, how DHA affects T cell function remains poorly understood. We found that DHA potently suppressed Th cell differentiation in vitro. Unexpectedly however, DHA greatly promoted Treg cell generation, in a manner dependent on TGF-βR:Smad signal. In addition, DHA treatment effectively reduced EAE onset and ameliorated ongoing EAE in mice. Administration of DHA significantly decreased Th but increased Treg cells in EAE-inflicted mice without apparent global immune suppression. Moreover, DHA modulated mTOR pathway, because mTOR signal was attenuated in T cells upon DHA treatment. Importantly, enhanced Akt activity neutralized DHA-mediated effects on T cells in an mTOR dependent fashion. This study therefore reveals a novel immune regulatory function of DHA to reciprocally regulate Th and Treg cell generation through modulating mTOR pathway. It addresses how DHA regulates immune function and suggests a new type of drug for treating diseases where mTOR activity to be tempered.

Naturally occurring regulatory T (nTreg) cells express Foxp3 and were originally discovered as immune suppressors critical for self-tolerance and immune homeostasis. Through yet-to-be-defined mechanisms, nTreg cells were recently shown to convert into proinflammatory cells. Particularly, attenuation of Foxp3 expression led to Th2 conversion of nTreg cells in vivo. In this paper, we demonstrated an nTreg-specific mechanism controlling their Th2 conversion. We found that wild-type nTreg cells expressing reduced levels of Foxp3 but not those expressing no Foxp3 produced the Th2 cytokine IL-4. Intriguingly, IL-4 production by converted nTreg cells is required for Th2 differentiation of coexisting naive CD4 T cells in vivo, suggesting that Th2 conversion of nTreg cells might be critical for directing Th2 immune responses. Th2 conversion of nTreg cells was not due to their inability to become Th1 cells, because IFN-γ was produced by Foxp3-low–expressing cells when IL-4/STAT-6 signaling was abrogated. Surprisingly, however, unlike naive CD4 T cells whose IL-4 production is dependent on STAT-6, Foxp3-low–expressing cells generated IL-4 independent of STAT-6, indicating an intrinsic mechanism that favors nTreg-to-Th2 differentiation. Indeed, compared with naive CD4 T cells, nTreg expressed elevated levels of GATA-3 independent of STAT-6. And GATA-3 was required for nTreg-to-Th2 conversion. Foxp3 may account for this GATA-3 upregulation in nTreg cells, because ectopic expression of Foxp3 preferentially promoted GATA-3 but not T-bet expression. Thus, we have identified an intrinsic mechanism that imposes a Th2/Th1 imbalance and predisposes Foxp3-expressing cells to IL-4 production independent of STAT-6 signaling.