T cell activation as well as IL-4 stimulation transiently induces Gfi-1 expression, resulting in optimal Th2 cell expansion (37
). In the absence of Gfi-1, Th2 expansion is affected both in vitro and in vivo (41
). Th1 cells from Gfi1
cKO mice expand normally but make more IFN-γ than WT Th1 cells. In addition, Gfi1
cKO Th2 cells display a different pattern of chromatin histone modification than wild-type Th2 cells. These observations led us to reassess the function of Gfi-1 during T cell differentiation, particularly because two new Th lineages, Th17 and iTreg cells, have been recently defined. In this paper, we showed that Gfi-1 is down-regulated by TGF-β stimulation during Th17 and iTreg cell–inducing conditions, and enforced expression of Gfi-1 inhibits both Th17 and iTreg cell differentiation. Furthermore, in the absence of Gfi-1, the percentage of Foxp3+
cells that express CD103 was substantially higher than in wild-type mice, and this subset expanded in vivo upon immunization.
Gfi-1 is a transcriptional repressor (51
). Gfi-1 and its congener Gfi-1b have been recently reported to associate with the histone demethylase LSD1 and CoREST to form a repressive complex (46
). Gfi-1 directs the complex to the promoter of target genes where LSD1 can demethylate H3K4 residues and thus repress transcription. Knockdown of LSD1 in erythroid cells leads to increased H3K4 dimethylation and trimethylation of Gfi-1b targets. In this paper, we showed that Il17a/Il17f
are the targets for Gfi-1 in T cells. Furthermore, LSD1 is recruited to those sites in a Gfi-1–dependent manner.
promoter regions are heavily methylated on H3K4 residues in Gfi1
cKO Th2 cells but not in WT Th2 cells; methylation levels are comparable to those found in WT Th17 cells. However, we were not able to locate Gfi-1 binding sites within the Rorc
loci, suggesting that they may not be the direct targets of Gfi-1. A future goal will be to understand the mechanism through which Gfi-1 regulates the chromatin modification at the Rorc
loci. RORγt is also expressed in the cells cultured under iTreg cell–inducing conditions, correlating with the H3K4 methylation in such cells, although iTreg cells fail to express IL-17 (unpublished data). These results indicate that RORγt can be up-regulated by TGF-β alone (28
) and that down-regulation of Gfi-1 by TGF-β is critical for RORγt expression during Th17 cell differentiation.
In a model of EAE involving immunization with MOG, the percentage of IL-17–producing cells found in Gfi1 cKO mice was modestly higher than in wild-type controls. In addition, Toxoplasma gondii infection or Schistosoma mansoni egg injection induced higher levels of IL-17 production in Gfi1 cKO than in wild-type mice (unpublished data). Thus, similar to our in vitro results, the optimal appearance of IL-17–producing cells induced by TGF-β in vivo requires down-regulation of Gfi-1 expression, which is transiently induced by T cell activation and/or IL-4 stimulation.
Gfi-1 expression is also down-regulated by TGF-β during iTreg cell differentiation, and enforced expression of Gfi-1 partially suppresses the induction of Foxp3+
Treg cells. By down-regulating Gfi-1, Treg cells become more active and also display a tissue-seeking phenotype. In Gfi1
cKO mice, the proportion of Foxp3+
effector/memory Treg cells is already high, but this population is further increased after MOG immunization. In addition, Foxp3+
are also expanded during T. gondii
infection or in response to S. mansoni
egg injection (Fig. S4, available at http://www.jem.org/cgi/content/full/jem.20081666/DC1
). We have not yet determined whether the increased percentage of Foxp3+
cells in the Gfi1
cKO mice and the expansion of this subset after immunization reflects a unique role for Gfi-1 in the development of Foxp3+
T cells, or whether the higher percentage of Foxp3+
T cells is secondary to an increased contribution of peripherally generated Foxp3+
in the cKO mice that is further potentiated after immunization. In normal mice, the Foxp3+
cell population is actively cycling and contains a high proportion of apoptotic cells (7
), so it remains possible that Gfi-1 may play a role in the homeostasis of this subset.
A selective effect of Gfi-1 on Treg cells with a modest effect on Th17 cell differentiation in vivo could be explained by the differential requirement of TGF-β in the induction of these two lineages. A low concentration of TGF-β is sufficient for Th17 cell differentiation, whereas iTreg cell differentiation requires a higher concentration of TGF-β (54
). In the absence of Gfi-1, Treg cell numbers may increase in response to levels of TGF-β that normally only induce Th17 cell differentiation in wild-type mice after MOG immunization.
The selective expansion of the Foxp3+
cells correlates with delayed EAE onset. Treg cells have been suggested to suppress autoimmune diseases by several mechanisms: by suppressing the activation and expansion of the effector cells (6
); by suppressing functions of effector cells, such as cytokine production (55
); and by limiting recruitment of effector cells to sites of inflammation (56
). In our model, the disease onset was delayed in the Gfi1
cKO mice, yet the number of effector cells that are capable of making IL-17 was not only not reduced but was modestly increased. Thus, it is most likely that the Foxp3+
cells suppress disease by affecting recruitment and/or activation of the effector cells at inflammation sites consistent with the tissue-seeking phenotype of the Foxp3+
T cells that are increased in frequency in Gfi1
cKO mice. The majority of Treg cells found in the CNS are Foxp3+
, even in wild-type mice. In addition, Treg cells are a much larger proportion of CNS CD4 T cells in Gfi1
cKO than in wild-type mice.
Given the dual effects of Gfi-1 on effector and Treg cells, modulation of its expression and function could have opposite effects depending on the timing, location, and cell type in which it is expressed, just as is the case with the administration of TGF-β or anti–TGF-β (57
). However, blockade of Gfi-1 function during early stages of autoimmune diseases may be beneficial through the resultant expansion of Treg cells. Likewise, the specific targeting of Gfi-1 in Treg cells may enhance their migration to sites of inflammation as well as increase their suppressive activity. Thus, the predominant effect of Gfi-1 on Treg cells in vivo suggests Gfi-1 and its pathway of action could be considered as a target for modulating Treg cells.