The data presented in this study show that Ets-1 is required for normal development and function of T reg cells and that defects in this cell subset were responsible for some of the immunological disorders in Ets-1−/−
mice. Viable young mutant animals had reduced numbers of T reg cells in the spleen, but the frequency in the thymus appeared normal. In both sites, Ets-1−/−
T reg cells had an unusual phenotype in that they expressed CD103 (), a marker typical of cells that experienced antigen under certain inflammatory conditions (Huehn et al., 2004
; Suffia et al., 2005
). This raised the possibility that the majority of thymic Ets-1−/−
T reg cells were antigen-experienced recirculating cells, thereby masking an important quantitative deficit in thymic development of these cells. This was indeed the case, as supported by the very low frequency of thymic T reg cells in FTOCs (), in 5-d-old newborns (not depicted), and in mixed WT/KO chimeras analyzed at relative early time points ( and ).
Ets-1−/− mice have a severe phenotype that affects other lymphocyte lineages. In such context, the T reg cell deficit could be secondary to T reg cell–independent abnormalities, such as the T cell lymphopenia, the hyperactivation status of conventional T cells, and dysfunctions resulting thereof. As demonstrated in this study using mixed WT/KO fetal liver chimeras, normal T reg cell development was achieved in a thymic environment even with an excess of Ets-1−/− cells. In contrast, the same environment did not rescue the deficit of T reg cell development of Ets-1−/− precursors, ruling out a potential role of extrinsic factors, e.g., IL-2 or TGF-β, in this deficit. These results strongly supported a T reg cell–intrinsic role of Ets-1.
Normal T reg cells were shown to prevent Ets-1−/− T cell–mediated splenomegaly and B cell dysfunctions observed in viable mutants. This block correlated with decreased accumulation of Ets-1−/− T cells, drastic reduction in the capacity of mutant CD4+ T cells to produce IL-4, and significant decrease of the proportion of CD44highCD62-L− cells to the benefit of activated/memory cells coexpressing high levels of these two molecules (Fig. S7). Moreover, cells with a naive phenotype (CD44lowCD62-Lhigh) were now clearly detected, but their frequency was still significantly lower than in controls. Thus, Ets-1−/− conventional T cells were highly susceptible to key T reg cell–mediated negative signals, but the failure to accumulate high numbers of naive cells could indicate that Ets-1−/− conventional CD4 T cells did not respond to T reg cell–mediated signals inhibiting activation or that they were in an intrinsic state of activation caused by the lack of Ets-1.
Although this issue is difficult to assess given the lymphopenic context of Ets-1−/−
and mixed WT/KO chimeras, our experiments also provided evidence suggestive of cell intrinsic defects in conventional Ets-1−/−
CD4 T cells, which provide possible explanations for the distinct spectrum of immune disorders in these mice as compared with other T reg cell–defective models with partial or complete impaired Foxp3 function. First, these cells had reduced growth potential and/or survival in vitro (Bories et al., 1995
; Muthusamy et al., 1995
). This could explain the T cell lymphopenia, despite the severe T reg cell deficit, instead of uncontrolled T cell expansion. Second, they showed enhanced Th2 polarization but poor capacity to produce IFN-γ ( and Fig. S2) or to induce IBD in Rag2-deficient hosts (not depicted). Together with the absence of IBD in viable Ets-1−/−
mice, these results suggest that inactivation of Ets-1 might negatively impact Th1 polarization, which is in line with the finding that T-bet function was markedly impaired in Ets-1 mutated T cells, stimulated under Th1-skewing conditions (Grenningloh et al., 2005
). Collectively, these results suggest that Ets-1 inactivation also affects conventional T cells in a cell-intrinsic manner and provide an explanation for the absence of massive T cell expansions and enhanced Th2 polarization in mutant animals.
Ets-1 may regulate Foxp3
expression trough several nonmutually exclusive ways. It could have an indirect effect by regulating the transcription of genes encoding direct regulators of Foxp3
or by controlling expression of signaling molecules acting upstream of Foxp3
(i.e., IL-2R or TCR). However, our finding that Ets-1 binds to the CNS2 region and that the enhancer activity of this element relies on ETS-binding sites rather supports a more direct effect. Histone modification and DNA demethylation have been associated with stable expression of Foxp3 in both thymic and peripherally generated T reg cells (Floess et al., 2007
; Polansky et al., 2008
). In this context, inactivation of nuclear factors such as Runx–CBF-β that are involved in chromatin remodeling processes markedly impaired the T reg cell compartment (Kitoh et al., 2009
; Klunker et al., 2009
; Rudra et al., 2009
). Interestingly, Runx–CBF-β−/−
mice both displayed T reg cell deficit associated with hyper IgE secretion and enhanced Th2 polarization (Kitoh et al., 2009
). Thus, as the Runx–CBF-β complex, Ets-1 could contribute to trigger the epigenetic changes required for stable Foxp3 expression. This hypothesis is strengthened by the highly methylated status of the CNS2 region in splenic Ets-1−/−
T reg cells that correlated with the reduced level of Foxp3 transcripts and proteins ( and ). However, although CBF-β was required to maintain high levels of Foxp3 in the periphery, unlike Ets-1−/−
mice, CBF-β–deficient mice had normal thymic T reg cell development (Kitoh et al., 2009
; Rudra et al., 2009
). This suggests that Ets-1 function may not be restricted to the context of such epigenetic modifier nuclear complexes.
We report in this study that functional Ets-binding sites within the CNS2 region are located near sequences occupied by STAT5 and CREB nuclear factors in T reg cells (; Kim and Leonard, 2007
). Interestingly, Ets-1 has been shown to interact with STAT5 in T cells that were activated in vitro in the presence of IL-2 and, upon those conditions, to bind DNA target sites (Rameil et al., 2000
). Thus, during thymic T reg cell development, Ets-1–STAT5 complexes could interact with Foxp3
regulatory sequences, thereby ensuring optimal expression of the gene. Furthermore, upon cellular activation, phosphorylated Ets-1 and CREB are both able to associate with CBP/p300, two proteins know to carry histone acetyl transferase activities (Chrivia et al., 1993
; Yang et al., 1998
; Foulds et al., 2004
). Thus, for thymocytes that experience strong TCR/CD28 and IL-2 signals, Ets-1 activity could be required to efficiently target a large complex of nuclear factors to the Foxp3
intronic enhancer. However, gene-targeted deletion of the CNS2 region was shown to impair heritable Foxp3 expression in dividing cells but, unlike Ets-1 inactivation, not to affect the development of thymic T reg cells (Zheng et al., 2010
). These data suggest that Ets-1 could interact with additional regulatory sequences within the Foxp3 locus.
The CNS1 sequence appears to be essential for TGF-β–mediated Foxp3 induction in conventional T cells but dispensable for T reg cell thymic development. We show in this study that TGF-β could induce Foxp3 expression in Ets-1−/−
T cells in vitro (), and because we failed to detect Ets-1 binding to the Foxp3
promoter (), it seems unlikely that neither the CNS1 nor the promoter would be required for Ets-1–mediated regulation of the Foxp3
gene. An additional region, CNS3, was shown to increase the frequency of T reg cells generated in the thymus and in the spleen, but its deletion left the levels of Foxp3 unaffected (Zheng et al., 2010
). Thus, Ets-1 may not act through a single regulatory region but rather interact at several sites, and only from these combined activities would proper expression and epigenetic modification at the Foxp3 locus be achieved.
Altogether, our data support a model in which Ets-1 participates in a nucleoprotein complex whose function would be regulated by TCR and/or γ-chain signaling pathways to induce and lock up Foxp3 expression during the development of thymic T reg cells. These results identify Ets-1 as a key regulator of the immune system and of the biology of T reg cells.