scholarly journals Comparing the Effects of the mTOR Inhibitors Azithromycin and Rapamycin on In Vitro Expanded Regulatory T Cells

2019 ◽  
Vol 28 (12) ◽  
pp. 1603-1613 ◽  
Author(s):  
Marcus Bergström ◽  
Malin Müller ◽  
Marie Karlsson ◽  
Hanne Scholz ◽  
Nils Tore Vethe ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Mtor Inhibitors ◽  
Allogeneic Transplantation ◽  
Suppressive Effect ◽  
And Function ◽  
Treg Phenotype ◽  
Suppression Assay

Adoptive transfer of autologous polyclonal regulatory T cells (Tregs) is a promising option for reducing graft rejection in allogeneic transplantation. To gain therapeutic levels of Tregs there is a need to expand obtained cells ex vivo, usually in the presence of the mTOR inhibitor Rapamycin due to its ability to suppress proliferation of non-Treg T cells, thus promoting a purer Treg yield. Azithromycin is a bacteriostatic macrolide with mTOR inhibitory activity that has been shown to exert immunomodulatory effects on several types of immune cells. In this study we investigated the effects of Azithromycin, compared with Rapamycin, on Treg phenotype, growth, and function when expanding bulk, naïve, and memory Tregs. Furthermore, the intracellular concentration of Rapamycin in CD4+ T cells as well as in the culture medium was measured for up to 48 h after supplemented. Treg phenotype was assessed by flow cytometry and Treg function was measured as inhibition of responder T-cell expansion in a suppression assay. The concentration of Rapamycin was quantified with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Azithromycin and Rapamycin both promoted a FoxP3-positive Treg phenotype in bulk Tregs, while Rapamycin also increased FoxP3 and FoxP3+Helios positivity in naïve and memory Tregs. Furthermore, Rapamycin inhibited the expansion of naïve Tregs, but also increased their suppressive effect. Rapamycin was quickly degraded in 37°C medium, yet was retained intracellularly. While both compounds may benefit expansion of FoxP3+ Tregs in vitro, further studies elucidating the effects of Azithromycin treatment on Tregs are needed to determine its potential use.

scholarly journals Trichostatin A Promotes the Generation and Suppressive Functions of Regulatory T Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Cristian Doñas ◽  
Macarena Fritz ◽  
Valeria Manríquez ◽  
Gabriela Tejón ◽  
María Rosa Bono ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Trichostatin A ◽  
Gene Promoter ◽  
Treg Cells ◽  
Specific Subset ◽  
Global Increase ◽  
And Function ◽  
H3 Acetylation

Regulatory T cells are a specific subset of lymphocytes that suppress immune responses and play a crucial role in the maintenance of self-tolerance. They can be generated in the thymus as well as in the periphery through differentiation of naïve CD4+T cells. The forkhead box P3 transcription factor (Foxp3) is a crucial molecule regulating the generation and function of Tregs. Here we show that thefoxp3gene promoter becomes hyperacetylated inin vitrodifferentiated Tregs compared to naïve CD4+T cells. We also show that the histone deacetylase inhibitor TSA stimulated thein vitrodifferentiation of naïve CD4+T cells into Tregs and that this induction was accompanied by a global increase in histone H3 acetylation. Importantly, we also demonstrated that Tregs generated in the presence of TSA have phenotypical and functional differences from the Tregs generated in the absence of TSA. Thus, TSA-generated Tregs showed increased suppressive activities, which could potentially be explained by a mechanism involving the ectonucleotidases CD39 and CD73. Our data show that TSA could potentially be used to enhance the differentiation and suppressive function of CD4+Foxp3+Treg cells.

scholarly journals BHLHE40 Regulates IL-10 and IFN-γ Production in T Cells but Does Not Interfere With Human Type 1 Regulatory T Cell Differentiation

2021 ◽  
Vol 12 ◽  
Author(s):  
Molly Javier Uyeda ◽  
Robert A. Freeborn ◽  
Brandon Cieniewicz ◽  
Rosa Romano ◽  
Ping (Pauline) Chen ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
Ex Vivo ◽  
Surface Expression ◽  
Surface Molecules ◽  
Ifn Γ ◽  
And Function ◽  
Tr1 Cells

Type 1 regulatory T (Tr1) cells are subset of peripherally induced antigen-specific regulatory T cells. IL-10 signaling has been shown to be indispensable for polarization and function of Tr1 cells. However, the transcriptional machinery underlying human Tr1 cell differentiation and function is not yet elucidated. To this end, we performed RNA sequencing on ex vivo human CD49b+LAG3+ Tr1 cells. We identified the transcription factor, BHLHE40, to be highly expressed in Tr1 cells. Even though Tr1 cells characteristically produce high levels of IL-10, we found that BHLHE40 represses IL-10 and increases IFN-γ secretion in naïve CD4+ T cells. Through CRISPR/Cas9-mediated knockout, we determined that IL10 significantly increased in the sgBHLHE40-edited cells and BHLHE40 is dispensable for naïve CD4+ T cells to differentiate into Tr1 cells in vitro. Interestingly, BHLHE40 overexpression induces the surface expression of CD49b and LAG3, co-expressed surface molecules attributed to Tr1 cells, but promotes IFN-γ production. Our findings uncover a novel mechanism whereby BHLHE40 acts as a regulator of IL-10 and IFN-γ in human CD4+ T cells.

scholarly journals Transcriptional heterogeneity in cancer-associated regulatory T cells is predictive of survival

2018 ◽  
Author(s):  
Nicholas Borcherding ◽  
Kawther K. Ahmed ◽  
Andrew P. Voigt ◽  
Ajaykumar Vishwakarma ◽  
Ryan Kolb ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Immune Cells ◽  
Ex Vivo ◽  
Expression Patterns ◽  
Suppressive Effect ◽  
Renal Clear Cell Carcinoma ◽  
Cell Fates ◽  
Treg Subset

Regulatory T cells (Tregs) are a population of T cells that exert a suppressive effect on a variety of immune cells and non-immune cells. The suppressive effects of Tregs are detrimental to anti-tumor immunity. Recent investigations into cancer-associated Tregs have identified common expression patterns for tumor-infiltration, however the functional heterogeneity in tumor-infiltrating (TI) Treg is largely unknown. We performed single-cell sequencing on immune cells derived from renal clear cell carcinoma (ccRCC) patients, isolating 160 peripheral-blood (PB) Tregs and 574 TI Tregs. We identified distinct transcriptional TI Treg cell fates, with a suppressive subset expressing CD177. We demonstrate CD177+ TI-Tregs have preferential suppressive effects in vivo and ex vivo. Gene signatures derived the CD177+ Treg subset had superior ability to predict survival in ccRCC and seven other cancer types. Further investigation into the development and regulation of TI-Treg heterogeneity will be vital to the application of tumor immunotherapies that possess minimal side effects.

scholarly journals IL-2 administration increases CD4+CD25hi Foxp3+ regulatory T cells in cancer patients

Blood ◽  
2006 ◽  
Vol 107 (6) ◽  
pp. 2409-2414 ◽  
Author(s):  
Mojgan Ahmadzadeh ◽  
Steven A. Rosenberg
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Interleukin 2 ◽  
Selective Inhibition ◽  
High Dose ◽  
Protein Levels ◽  
And Function ◽  
The Impact

Abstract Interleukin-2 (IL-2) is historically known as a T-cell growth factor. Accumulating evidence from knockout mice suggests that IL-2 is crucial for the homeostasis and function of CD4+CD25+ regulatory T cells in vivo. However, the impact of administered IL-2 in an immune intact host has not been studied in rodents or humans. Here, we studied the impact of IL-2 administration on the frequency and function of human CD4+CD25hi T cells in immune intact patients with melanoma or renal cancer. We found that the frequency of CD4+CD25hi T cells was significantly increased after IL-2 treatment, and these cells expressed phenotypic markers associated with regulatory T cells. In addition, both transcript and protein levels of Foxp3, a transcription factor exclusively expressed on regulatory T cells, were consistently increased in CD4 T cells following IL-2 treatment. Functional analysis of the increased number of CD4+CD25hi T cells revealed that this population exhibited potent suppressive activity in vitro. Collectively, our results demonstrate that administration of high-dose IL-2 increased the frequency of circulating CD4+CD25hi Foxp3+ regulatory T cells. Our findings suggest that selective inhibition of IL-2-mediated enhancement of regulatory T cells may improve the therapeutic effectiveness of IL-2 administration. (Blood. 2006;107:2409-2414)

The Significance of mTOR Inhibitor, Everolimus in TGF-β-Induced Regulatory T cells from Cord Blood

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2180-2180
Author(s):  
Tokiko Nagamura-Inoue ◽  
Seiichiro Kobayashi ◽  
Kazuo Ogami ◽  
Yuki Yamamoto ◽  
Kiyoko Izawa ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Cord Blood ◽  
Cell Population ◽  
Cd4 T Cells ◽  
Mtor Inhibitor ◽  
Ex Vivo ◽  
Mtor Inhibitors ◽  
Ex Vivo Expansion ◽  
The Impact

Abstract Abstract 2180 Background: Regulatory T cells (Tregs) play an important role in immune-tolerance to allograft. Cord blood (CB) is rich in naïve T cells and is a promising source of inducible Tregs (iTregs), since it was reported that stable iTregs may be derived exclusively from naïve T cells. However, the standard method for iTregs has not yet been established. Here we studied the impact of mTOR inhibitors, rapamycin (Rap) and everolimus (Eve), on ex vivo expansion of iTregs from CB-CD4+ T cells. Methods: CB-CD4+ T cell were isolated using anti-CD4 monoclonal antibody (MAb)-conjugated magnetic beads, and cultured in a flask coated with anti-CD3/CD28 MAbs and supplemented with IL-2 and TGF-β in the presence or absence of Rap or Eve. After two weeks of culture, the total number of CD4+ T cells was calculated, and the incidence of CD25+Foxp3+ cell population among those was estimated by FACS. Results and Discussions: Both Rap and Eve significantly increased the incidence of CD25+Foxp3+ cell population in CD4+ T cells. However, Rap apparently inhibited their growth and did not increase the absolute number of CD25+Foxp3+ cells in comparison to the control. On the other hand, Eve contributed to efficient expansion of iTregs at the concentration between 1 and 50ng/ml without no significant inhibition of their growth. Expansion of CD4+ T cells with TGF-β and Eve yielded 71.5 ±23.5% purity of CD25+Foxp3+ cells which also expressed CTLA-4 as well as the memory phenotype, while the purity obtained with TGF-β only was 47.4±30.0% and that without TGF-β/Eve was 7.3±4.5%. Thus, an average of 2.95±2.8 x107 iTregs were obtained from the initial input of 5×104 CD4+ T cells. The resulting iTregs with TGF-β, TGF-β/Rap and TGF-β/Eve inhibited the proliferation of CFSE-labeled T cells stimulated with allogeneic dendritic cells. The precise mechanism for Foxp3 induction by mTOR inhibitors still remains to be elucidated. Furthermore, we found that expression of CD26 (DPP-IV) was significantly down-regulated in CD4+ T cells expanded with TGF-β and profoundly with TGF-β/Eve, while CD127 was negative after culture in all the conditions. Mean fluorescence intensity of CD26 indicated 67.5 in CD4+ T cells without TGF-β, 1.58 with TGF-β, 0.18 with TGF-β/Rap and 0.12 with TGF-β/Eve, respectively. Accordingly, CD26 negativity may be an indicator of iTregs together with Foxp3. Conclusion: mTOR inhibitor, Eve, is an efficient co-inducer of iTregs and applicable to ex vivo expansion of iTregs in a clinical setting. Disclosures: No relevant conflicts of interest to declare.

Unique Role of mTOR Inhibitor, Everolimus in Inducible Regulatory T Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4349-4349
Author(s):  
Tokiko Nagamura-Inoue ◽  
Yuki Yamamoto ◽  
Seiichiro Kobayashi ◽  
Kazuo Ogami ◽  
Kiyoko Izawa ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Cd4 T Cells ◽  
Mtor Inhibitor ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Foxp3 Expression ◽  
Induction Rate ◽  
The Impact

Abstract Abstract 4349 Background: Regulatory T cells (Tregs) play an important role in immune-tolerance to allograft. Unbalance between Tregs and effector T cells is involved in graft-versus-host disease (GvHD) and other autoimmune disorders. Adoptive use of inducible Tregs (iTregs) is a candidate immunosuppressive therapy, and major concern has been focused on sustained expression of Foxp3 in iTregs. We previously reported that iTregs can be efficiently expanded from cord blood (CB)-derived CD4+ T cells in the presence of IL2, TGFb and a mTOR inhibitor, Everolimus (Eve). However, the effect of Eve on in vitro induction of iTreg remains to be elucidated. Here we studied the impact of Eve on CB-CD4+ T cells. Methods: CD4+ T cells were prepared from CB with a purity of >95% and put into the flask coated with anti-CD3/CD28 MAb. For Treg induction, these cultures were supplemented with IL2, IL-2/TGFb, IL2/TGFb/Eve, or IL2/Eve and kept for two weeks. The resulting CD4+ T cells including variable proportion of iTregs were subjected to mixed lymphocyte reaction (MLR) along with CFSE-labeled autologous responder T cells and allogeneic dendritic cells (DCs) as stimulator. Results: The basal proportion of CD25+Foxp3+ cells in CB-CD4+ T cells was 0.60 ± 0.59%. After two weeks, the induction rate of CD25+Foxp3+CD4+ T cells was higher in the culture with IL2/TGFb/Eve than that with IL2/TGFb, but Eve itself could not significantly induce iTregs in the absence of TGFb (Figure1.). The iTreg ratio (CD25+Foxp3+ cells/total CD4+ T cells) was 79.3 ± 17.4% in the culture with IL2/TGFb/Eve, 53.1 ± 23.8% with IL2/TGFb, 35.5±18.6% with IL2/Eve and 22.7 ± 18.6% with IL2, respectively. There was no significant relationship between the dose of Eve and the iTreg ratio, but the highest ratio and induction rate of iTregs were observed at 10nM Eve. Thus, an average of 2.95 ± 2.8 ×107 iTregs was obtained from 5 ×104 CB-CD4+ T cells after two weeks of culture with IL2/TGFb/Eve. The iTreg-rich population cultured with IL2/TGFb/Eve and IL2/TGFb, but not IL2 alone, efficiently inhibited MLR triggered by allogeneic DCs (Figure 2.). These iTregs were also active in MLR using allogeneic responder T cells. Interestingly, IL2/Eve-treated CB-CD4+ T cells also inhibited MLR, irrespective of the low or moderate iTreg ratio. The inhibitory effect on MLR was much less observed by another mTOR inhibitor, rapamycin, rather than Eve (Figure2). Expression of CD26 on CD4+ T cells was inversely correlated to Foxp3 expression and significantly down-regulated by TGFb with or without Eve. Discussion: Treatment of CB-CD4+ T cells with IL2/TGFb/Eve results in the efficient ex vivo expansion of functional iTregs. Eve enhanced TGFb induction of Foxp3 expression, but did not induce Foxp3 expression by itself. mTOR is a complex of TORC1 and 2. Rapamycin is reported to inhibit TORC1, while Eve inhibits both of them, at general dose. In recent report, mTOR-deficient T cells (TORC1/2, not TORC1 alone) displayed normal activation and IL-2 production upon initial stimulation, but failed to differentiate into effecter T cells, instead, differentiated into Tregs. Although the direct mechanism to inhibit MLR by CB-CD4+ T cells treated with Eve remained to be elucidated, these results suggested the aberrant pathways of immunological inhibition. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Increased Activation and Oligoclonality of Peripheral CD8+ T Cells in the Chronic Human Helminth Infection Alveolar Echinococcosis

2002 ◽  
Vol 70 (3) ◽  
pp. 1168-1174 ◽  
Author(s):  
Burkhard J. Manfras ◽  
Stefan Reuter ◽  
Thomas Wendland ◽  
Peter Kern
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Alveolar Echinococcosis ◽  
Ex Vivo ◽  
Active Role ◽  
Cell Receptor ◽  
T Cell Subsets ◽  
And Function

ABSTRACT Alveolar echinococcosis (AE) in humans is a chronic disease characterized by slowly expanding liver lesions. Cellular immunity restricts the spreading of the extracellular pathogen, but functional contributions of CD4+ and CD8+ T cells are not defined. Here we studied ex vivo the phenotype and function of circulating T-cell subsets in AE patients by means of flow cytometry, T-cell receptor spectratyping, and lymphocyte proliferation. AE patients with parasitic lesions displayed a significant increase of activation of predominantly CD8+ T cells compared to healthy controls and AE patients without lesions. In vitro, proliferative T-cell responses to polyclonal stimulation with recall antigens and Echinococcus multilocularis vesicular fluid antigen were sustained during chronic persisting infection in all AE patients. Only in AE patients with parasitic lesions did T-cell receptor spectratyping reveal increased oligoclonality of CD8+ but not CD4+ T cells, suggesting a persistent antigenic drive for CD8+ T cells with subsequent proliferation of selected clonotypes. Thus, our data provide strong evidence for an active role of CD8+ T cells in AE.

scholarly journals A Comparison of Ex Vivo Expanded Human Regulatory T Cells Using Allogeneic Stimulated B Cells or Monocyte-Derived Dendritic Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Linda M. Lee ◽  
Hong Zhang ◽  
Karim Lee ◽  
Horace Liang ◽  
Alexander Merleev ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
B Cells ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Low Frequencies ◽  
Almost All

Alloreactive regulatory T cells (arTregs) are more potent than polyclonal Tregs at suppressing immune responses to transplant antigens. Human arTregs can be expanded with allogeneic CD40L-stimulated B cells (sBcs) or stimulated-matured monocyte-derived dendritic cells (sDCs). Here, we compared the expansion efficiency and properties of arTregs stimulated ex vivo using these two types of antigen-presenting cells. Compared to sBcs, sDCs stimulated Tregs to expand two times more in number. The superior expansion-inducing capacity of sDCs correlated with their higher expression of CD80, CD86, and T cell-attracting chemokines. sBc- and sDC-arTregs expressed comparable levels of FOXP3, HELIOS, CD25, CD27, and CD62L, demethylated FOXP3 enhancer and in vitro suppressive function. sBc- and sDCs-arTregs had similar gene expression profiles that were distinct from primary Tregs. sBc- and sDC-arTregs exhibited similar low frequencies of IFN-γ, IL-4, and IL-17A-producing cells, and the cytokine-producing arTregs expressed high levels of FOXP3. Almost all sBc- and sDC-arTregs expressed CXCR3, which may enable them traffic to inflammatory sites. Thus, sDCs-arTregs that expand more readily, are phenotypically similar to sBc-arTregs, supporting sDCs as a viable alternative for arTreg production for clinical evaluation.

Sirolimus Inhibits Proliferation but Enhances Suppressive Activity of Rhesus Macaque Natural CD4 Regulatory T Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1008-1008
Author(s):  
Karnail Singh ◽  
Natalia Kozyr ◽  
Linda Stempora ◽  
Allan D Kirk ◽  
Christian P Larsen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Cd8 T Cell ◽  
T Cell Proliferation ◽  
Suppressive Activity ◽  
Suppressive Capacity

Abstract Abstract 1008 Regulatory T cells (Tregs) have been shown to be potent inhibitors of autoimmunity, and to be capable of suppressing alloimmune responses that occur during both allograft rejection and graft-versus host disease. However, they have yet to gain widespread use clinically, due in part to the fact that it remains extremely costly and difficult to produce them in sufficient numbers and with sufficient suppressive capacity to significantly impact the alloimmune response. Here we have used our established non-human primate model to demonstrate that significant Treg expansion (up to 600-fold in 21 days) can be maintained, and suppressive capacity enhanced by exposing Treg cultures to a short burst of sirolimus at the end of the culture period. Using a highly sensitive and specific in vitro CFSE-MLR assay we show that Tregs significantly inhibit allo-proliferation of multiple T cell subpopulations including both CD4+ and CD8+ T cells (3.2 and 2.7-fold inhibition of proliferation, respectively), as well as their CD28+CD95+ and CD28-CD95+ subpopulations (2.2 and 2.1 and 1.9 and 2.7-fold inhibition of CD4+ and CD8+ subpopulation proliferation, respectively). Tregs were able to combine in vitro with the newly FDA-approved CTLA4-Ig analog belatacept to enhance the inhibition of alloproliferation that occurred with either agent alone (4.8-fold inhibition of CD8 T cell proliferation with Tregs + belatacept, compared to 3.0-fold or 1.9-fold inhibition of CD8 T cell proliferation with Tregs or belatacept alone, respectively). Importantly, we have found that the suppressive activity of ex-vivo expanded Tregs could be further enhanced by pulsing with sirolimus. Thus, while long-term culture of Tregs in the presence of sirolimus (1–1000 nM) profoundly inhibited Treg expansion (50–800 fold inhibition of expansion when cultured in the presence of 1–1000 nM sirolimus), a 48 hour pulse of sirolimus (100 nM) on days 20–21 of culture completely preserved Treg yields while doubling their suppressive function against CD8 proliferation when compared to unpulsed Tregs, p<0.01) A mechanistic evaluation of the increase potency observed with sirolimus pulsed Tregs (SPTs) has revealed several key differences that distinguish these cells from the less-potent unpulsed Tregs: SPTs were found to undergo fewer rounds of proliferation in an MLR when compared with unpulsed Tregs (14% proliferation in SPTs versus 37% proliferation in un-pulsed Tregs, p= 0.015), suggesting that the suppressive capability of Tregs may be inversely related to their proliferative capacity. SPTs were also shown to have significantly increased expression of CD25 (p=0.04) and total CTLA4 (p= 0.009) compared to unpulsed Tregs, implicating signaling through both of these molecules in their enhanced function. Our results suggest that the creation of SPTs may provide a novel avenue by which to achieve enhanced Treg-based suppression of alloimmunity, in a manner that is amenable to large-scale ex-vivo expansion and to combinatorial therapy with novel, costimulation-blockade-based immunosuppression strategies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Ets-1 transcription factor controls the development and function of natural regulatory T cells

2010 ◽  
Vol 207 (10) ◽  
pp. 2113-2125 ◽  
Author(s):  
Enguerran Mouly ◽  
Karine Chemin ◽  
Hai Vu Nguyen ◽  
Martine Chopin ◽  
Laurent Mesnard ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
T Cell ◽  
Regulatory T Cells ◽  
Molecular Mechanisms ◽  
Fetal Liver ◽  
Regulatory Sequence ◽  
Activated T Cells ◽  
And Function

Regulatory T cells (T reg cells) constitute a population of CD4+ T cells that limits immune responses. The transcription factor Foxp3 is important for determining the development and function of T reg cells; however, the molecular mechanisms that trigger and maintain its expression remain incompletely understood. In this study, we show that mice deficient for the Ets-1 transcription factor (Ets-1−/−) developed T cell–mediated splenomegaly and systemic autoimmunity that can be blocked by functional wild-type T reg cells. Spleens of Ets-1−/− mice contained mostly activated T cells, including Th2-polarized CD4+ cells and had reduced percentages of T reg cells. Splenic and thymic Ets-1−/− T reg cells expressed low levels of Foxp3 and displayed the CD103 marker that characterizes antigen-experienced T reg cells. Thymic development of Ets-1−/− T reg cells appeared intrinsically altered as Foxp3-expressing cells differentiate poorly in mixed fetal liver reconstituted chimera and fetal thymic organ culture. Ets-1−/− T reg cells showed decreased in vitro suppression activity and did not protect Rag2−/− hosts from naive T cell–induced inflammatory bowel disease. Furthermore, in T reg cells, Ets-1 interacted with the Foxp3 intronic enhancer and was required for demethylation of this regulatory sequence. These data demonstrate that Ets-1 is required for the development of natural T reg cells and suggest a role for this transcription factor in the regulation of Foxp3 expression.

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