Androgen receptor modulates Foxp3 expression in CD4+CD25+Foxp3+ regulatory T-cells

CD4+CD25+Foxp3+ regulatory T (Treg) cells are able to inhibit proliferation and cytokine production in effector T-cells and play a major role in immune responses and prevention of autoimmune disease. A master regulator of Treg cell development and function is the transcription factor Foxp3. Several cytokines, such as TGF-β and IL-2, are known to regulate Foxp3 expression as well as methylation of the Foxp3 locus. We demonstrated previously that testosterone treatment induces a strong increase in the Treg cell population both in vivo and in vitro. Therefore we sought to investigate the direct effect of androgens on expression and regulation of Foxp3. We show a significant androgen-dependent increase of Foxp3 expression in human T-cells from women in the ovulatory phase of the menstrual cycle but not from men and identify a functional androgen response element within the Foxp3 locus. Binding of androgen receptor leads to changes in the acetylation status of histone H4, whereas methylation of defined CpG regions in the Foxp3 gene is unaffected. Our results provide novel evidence for a modulatory role of androgens in the differentiation of Treg cells.

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Prostaglandin E2 directly inhibits the conversion of inducible regulatory T cells through EP2 and EP4 receptors via antagonizing TGF-β signalling

10.1101/2021.04.19.440391 ◽

2021 ◽

Author(s):

Marie Goepp ◽

Siobhan Crittenden ◽

You Zhou ◽

Adriano G Rossi ◽

Shuh Narumiya ◽

...

Keyword(s):

T Cells ◽

Cell Differentiation ◽

T Cell ◽

Prostaglandin E2 ◽

Th17 Cells ◽

Foxp3 Expression ◽

Treg Cells ◽

Itreg Cell

Background and Purpose: Regulatory T (Treg) cells are essential for control of inflammatory processes by suppressing Th1 and Th17 cells. The bioactive lipid mediator prostaglandin E2 (PGE2) promotes inflammatory Th1 and Th17 cells and exacerbates T cell-mediated autoimmune diseases. However, the actions of PGE2 on the development and function of Treg cells, particularly under inflammatory conditions, are debated. In this study, we examined whether PGE2 had a direct action on T cells to modulate de novo differentiation of Treg cells. Experimental Approach: We employed an in vitro T cell culture system of TGF-β-dependent Treg induction from naive T cells. PGE2 and selective agonists for its receptors, and other small molecular inhibitors were used. Mice with specific lack of EP4 receptors in T cells were used to assess Treg cell differentiation in vivo. Human peripheral blood T cells from healthy individuals were used to induce differentiation of inducible Treg cells. Key Results: TGF-β-induced Foxp3 expression and Treg cell differentiation in vitro was markedly inhibited by PGE2, which was due to interrupting TGF-β signalling. EP2 or EP4 agonism mimicked suppression of Foxp3 expression in WT T cells, but not in T cells deficient in EP2 or EP4, respectively. Moreover, deficiency of EP4 in T cells impaired iTreg cell differentiation in vivo. PGE2 also appeared to inhibit the conversion of human iTreg cells. Conclusion and Implications: Our results show a direct, negative regulation of iTreg cell differentiation by PGE2, highlighting the potential for selectively targeting the PGE2-EP2/EP4 pathway to control T cell-mediated inflammation.

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Vitamin A Impairs the Reprogramming of Tregs into IL-17-Producing Cells during Intestinal Inflammation

BioMed Research International ◽

10.1155/2015/137893 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 21

Author(s):

Gabriela Tejón ◽

Valeria Manríquez ◽

Jaime De Calisto ◽

Felipe Flores-Santibáñez ◽

Yessia Hidalgo ◽

...

Keyword(s):

T Cells ◽

Vitamin A ◽

Intestinal Inflammation ◽

Foxp3 Expression ◽

Treg Cells ◽

Transfer Model ◽

Effector Cells ◽

T Effector Cells

Maintaining the identity of Foxp3+regulatory T cells (Tregs) is critical for controlling immune responses in the gut, where an imbalance between Tregs and T effector cells has been linked to inflammatory bowel disease. Accumulating evidence suggests that Tregs can convert into Th17 cells and acquire an inflammatory phenotype. In this study, we used an adoptive transfer model of Ag-specific T cells to study the contribution of different factors to the reprogramming ofin vitro-generated Treg cells (iTreg) into IL-17-producing cells in a mouse model of gut inflammationin vivo. Our results show that intestinal inflammation induces the reprogramming of iTreg cells into IL-17-producing cells and that vitamin A restrains reprogramming in the gut. We also demonstrate that the presence of IL-2 during thein vitrogeneration of iTreg cells confers resistance to Th17 conversion but that IL-2 and retinoic acid (RA) cooperate to maintain Foxp3 expression following stimulation under Th17-polarizing conditions. Additionally, although IL-2 and RA differentially regulate the expression of different Treg cell suppressive markers, Treg cells generated under different polarizing conditions present similar suppressive capacity.

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Basiliximab impairs regulatory T cell (TREG) function and could affect the short-term graft acceptance in children with heart transplantation

Scientific Reports ◽

10.1038/s41598-020-80567-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jacobo López-Abente ◽

Marta Martínez-Bonet ◽

Esther Bernaldo-de-Quirós ◽

Manuela Camino ◽

Nuria Gil ◽

...

Keyword(s):

T Cells ◽

Heart Transplant ◽

Solid Organ Transplantation ◽

Foxp3 Expression ◽

Treg Cells ◽

Protective Role ◽

Regulatory Function ◽

Solid Organ

AbstractCD25, the alpha chain of the IL-2 receptor, is expressed on activated effector T cells that mediate immune graft damage. Induction immunosuppression is commonly used in solid organ transplantation and can include antibodies blocking CD25. However, regulatory T cells (Tregs) also rely on CD25 for their proliferation, survival, and regulatory function. Therefore, CD25-blockade may compromise Treg protective role against rejection. We analysed in vitro the effect of basiliximab (BXM) on the viability, phenotype, proliferation and cytokine production of Treg cells. We also evaluated in vivo the effect of BXM on Treg in thymectomized heart transplant children receiving BXM in comparison to patients not receiving induction therapy. Our results show that BXM reduces Treg counts and function in vitro by affecting their proliferation, Foxp3 expression, and IL-10 secretion capacity. In pediatric heart-transplant patients, we observed decreased Treg counts and a diminished Treg/Teff ratio in BXM-treated patients up to 6-month after treatment, recovering baseline values at the end of the 12-month follow up period. These results reveal that the use of BXM could produce detrimental effects on Tregs, and support the evidence suggesting that BXM induction could impair the protective role of Tregs in the period of highest incidence of acute graft rejection.

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The relationship between human thymus-derived regulatory T cells, TGF-β-induced regulatory T cells and conventional CD4+ T cells as revealed by proteomics

10.1101/2021.10.30.466569 ◽

2021 ◽

Author(s):

Mark Mensink ◽

Ellen Schrama ◽

Maartje van den Biggelaar ◽

Derk Amsen ◽

Jannie Borst ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Protein Expression ◽

Treg Cell ◽

Ex Vivo ◽

Foxp3 Expression ◽

Treg Cells ◽

Cell Populations ◽

The Relationship

The CD4+ regulatory T (Treg) cell lineage, as defined by FOXP3 expression, comprises thymus-derived (t)Treg cells and peripherally induced (p)Treg cells. In human, naive tTreg cells can be purified from blood, but occur in low abundance, while effector pTreg and tTreg cell populations cannot be purified for lack of discriminating cell surface markers. Therefore, studies often employ TGF-β-induced (i)Treg cells that are generated from CD4+ conventional T (Tconv) cells in vitro. Here, we describe the relationship of iTreg cells to tTreg and Tconv cells, as optimally purified from human blood. Global proteomic analysis revealed that iTreg, tTreg and Tconv cell populations each have a unique protein expression pattern. We next used as a benchmark a previously defined proteomic signature that discerns ex vivo naive and effector phenotype Treg cells from Tconv cells and reflects unique Treg cell properties. This Treg cell core signature was largely absent from iTreg cells, while clearly present in simultaneously analyzed tTreg cells. In addition, we used a proteomic signature that distinguishes ex vivo effector Treg cells from Tconv cells and naive Treg cells. This effector Treg cell signature was partially present in iTreg cells. Thus, iTreg cells are distinct from tTreg cells and largely lack the common Treg cell proteomic signature. However, they do have certain protein expression features in common with ex vivo effector Treg cells. These data demonstrate the utility of the core and effector Treg cell signatures as tools to define Treg cell populations and encourage the use of ex vivo Treg cells for functional analyses.

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PD-L1 regulates the development, maintenance, and function of induced regulatory T cells

Journal of Experimental Medicine ◽

10.1084/jem.20090847 ◽

2009 ◽

Vol 206 (13) ◽

pp. 3015-3029 ◽

Cited By ~ 1075

Author(s):

Loise M. Francisco ◽

Victor H. Salinas ◽

Keturah E. Brown ◽

Vijay K. Vanguri ◽

Gordon J. Freeman ◽

...

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Cell Function ◽

Foxp3 Expression ◽

Cell Development ◽

Peripheral Tolerance ◽

Cell Conversion ◽

And Function

Both the programmed death (PD) 1–PD-ligand (PD-L) pathway and regulatory T (T reg) cells are instrumental to the maintenance of peripheral tolerance. We demonstrate that PD-L1 has a pivotal role in regulating induced T reg (iT reg) cell development and sustaining iT reg cell function. PD-L1−/− antigen-presenting cells minimally convert naive CD4 T cells to iT reg cells, showing the essential role of PD-L1 for iT reg cell induction. PD-L1–coated beads induce iT reg cells in vitro, indicating that PD-L1 itself regulates iT reg cell development. Furthermore, PD-L1 enhances and sustains Foxp3 expression and the suppressive function of iT reg cells. The obligatory role for PD-L1 in controlling iT reg cell development and function in vivo is illustrated by a marked reduction in iT reg cell conversion and rapid onset of a fatal inflammatory phenotype in PD-L1−/−PD-L2−/− Rag−/− recipients of naive CD4 T cells. PD-L1 iT reg cell development is mediated through the down-regulation of phospho-Akt, mTOR, S6, and ERK2 and concomitant with the up-regulation of PTEN, all key signaling molecules which are critical for iT reg cell development. Thus, PD-L1 can inhibit T cell responses by promoting both the induction and maintenance of iT reg cells. These studies define a novel mechanism for iT reg cell development and function, as well as a new strategy for controlling T reg cell plasticity.

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UCP3 reciprocally controls CD4+ Th17 and Treg cell differentiation

PLoS ONE ◽

10.1371/journal.pone.0239713 ◽

2020 ◽

Vol 15 (11) ◽

pp. e0239713

Author(s):

Emma B. O’Connor ◽

Natalia Muñoz-Wolf ◽

Gemma Leon ◽

Ed C. Lavelle ◽

Kingston H. G. Mills ◽

...

Keyword(s):

T Cells ◽

Treg Cell ◽

Th17 Cell ◽

Mrna Level ◽

Foxp3 Expression ◽

Intermembrane Space ◽

Activated T Cells ◽

Cell Balance

Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier superfamily that can mediate the transfer of protons into the mitochondrial matrix from the intermembrane space. We have previously reported UCP3 expression in thymocytes, mitochondria of total splenocytes and splenic lymphocytes. Here, we demonstrate that Ucp3 is expressed in peripheral naive CD4+ T cells at the mRNA level before being markedly downregulated following activation. Non-polarized, activated T cells (Th0 cells) from Ucp3-/- mice produced significantly more IL-2, had increased expression of CD25 and CD69 and were more proliferative than Ucp3+/+ Th0 cells. The altered IL-2 expression observed between T cells from Ucp3+/+ and Ucp3-/- mice may be a factor in determining differentiation into Th17 or induced regulatory (iTreg) cells. When compared to Ucp3+/+, CD4+ T cells from Ucp3-/- mice had increased FoxP3 expression under iTreg conditions. Conversely, Ucp3-/- CD4+ T cells produced a significantly lower concentration of IL-17A under Th17 cell-inducing conditions in vitro. These effects were mirrored in antigen-specific T cells from mice immunized with KLH and CT. Interestingly, the altered responses of Ucp3-/- T cells were partially reversed upon neutralisation of IL-2. Together, these data indicate that UCP3 acts to restrict the activation of naive T cells, acting as a rheostat to dampen signals following TCR and CD28 co-receptor ligation, thereby limiting early activation responses. The observation that Ucp3 ablation alters the Th17:Treg cell balance in vivo as well as in vitro suggests that UCP3 is a potential target for the treatment of Th17 cell-mediated autoimmune diseases.

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Decreased Generation and Function of CD4+CD25hi T Regulatory Cells in Human STAT5b Deficiency.

Blood ◽

10.1182/blood.v106.11.768.768 ◽

2005 ◽

Vol 106 (11) ◽

pp. 768-768

Author(s):

Aileen Cleary ◽

Kari Nadeau ◽

Wenwei Tu ◽

Vivian Hwa ◽

Kira Y. Dionis ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Gene Dosage ◽

Foxp3 Expression ◽

Intermediate Phenotype ◽

Gamma Chain ◽

Gene Dosage Effect ◽

And Function

Abstract CD4+ CD25+ regulatory T cells (Tregs) are a well characterized population of cells that play an important role in limiting inflammation and in the maintenance of tolerance to self. Here we describe a patient with a homozygous missense mutation (A630P) in the STAT5b gene who clinically displays immune dysregulation in association with decreased numbers and function of Tregs. Freshly isolated or in vitro-derived CD4+CD25high Treg cells from this patient had low Foxp3 expression, did not suppress non Treg T-cell proliferation, and were unable to kill autologous CD4+CD25neg T cells compared to controls. CD25 expression in response to IL-2 did not increase on freshly isolated CD4 T cells and was decreased on T-cell blasts derived from the patient. The patients mother who was heterozygous for this mutation had an intermediate phenotype for all of these immune abnormalities, indicating a gene dosage effect. In contrast, IL-2 upregulated expression of the common gamma chain (γc) cytokine receptor and perforin by T cells normally. Activation-induced T-cell expression of CD40-ligand (CD154) and interferon-gamma (IFN-γ) were also normal in the patient. These results suggest that the STAT5 pathway propagates an important IL-2 mediated signal that is necessary for Treg generation and function in humans in vivo.

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G Protein-Coupled Receptor 83 Is Dispensable for the Development and Function of Regulatory T Cells

Molecular and Cellular Biology ◽

10.1128/mcb.01075-07 ◽

2007 ◽

Vol 27 (23) ◽

pp. 8065-8072 ◽

Cited By ~ 16

Author(s):

Li-Fan Lu ◽

Marc A. Gavin ◽

Jeffrey P. Rasmussen ◽

Alexander Y. Rudensky

Keyword(s):

T Cells ◽

G Protein ◽

Treg Cell ◽

Cell Biology ◽

Molecular Mechanisms ◽

Foxp3 Expression ◽

Treg Cells ◽

G Protein Coupled Receptor ◽

And Function ◽

G Protein Coupled

ABSTRACT Global analyses of gene expression in regulatory T (Treg) cells, whose development is critically dependent upon the transcription factor Foxp3, have provided many clues as to the molecular mechanisms these cells employ to control immune responses and establish immune tolerance. Through these studies, G protein-coupled receptor 83 (GPR83) was found to be expressed at high levels in Treg-cell populations. However, its function remained unclear. Recently, it has been suggested that GPR83 is involved in the induction of Foxp3 expression in the peripheral nonregulatory Foxp3− CD4 T cells. To examine a role for GPR83 in Treg-cell biology, we generated and characterized GPR83-deficient mice. We have shown that GPR83 abolition does not result in measurable pathology or changes in the numbers or function of Foxp3+ Treg cells. Furthermore, while in vitro analysis suggested a potential involvement of GPR83 in transforming growth factor β-dependent Foxp3 induction, there was no difference in the ability of nonregulatory GPR83-deficient and nondeficient Foxp3− T cells to acquire Foxp3 expression in vivo. Collectively, our results demonstrate that GPR83 is dispensable for Treg-cell development and function.

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Gene editing to induce FOXP3 expression in human CD4+ T cells leads to a stable regulatory phenotype and function

Science Translational Medicine ◽

10.1126/scitranslmed.aay6422 ◽

2020 ◽

Vol 12 (546) ◽

pp. eaay6422 ◽

Cited By ~ 6

Author(s):

Yuchi Honaker ◽

Nicholas Hubbard ◽

Yufei Xiang ◽

Logan Fisher ◽

David Hagin ◽

...

Keyword(s):

T Cells ◽

Gene Editing ◽

Foxp3 Expression ◽

Cell Therapies ◽

Homology Directed Repair ◽

Engineering Strategy ◽

And Function ◽

Regulatory Phenotype

Thymic regulatory T cells (tTregs) are potent inhibitors of autoreactive immune responses, and loss of tTreg function results in fatal autoimmune disease. Defects in tTreg number or function are also implicated in multiple autoimmune diseases, leading to growing interest in use of Treg as cell therapies to establish immune tolerance. Because tTregs are present at low numbers in circulating blood and may be challenging to purify and expand and also inherently defective in some subjects, we designed an alternative strategy to create autologous Treg-like cells from bulk CD4+ T cells. We used homology-directed repair (HDR)–based gene editing to enforce expression of FOXP3, the master transcription factor for tTreg. Targeted insertion of a robust enhancer/promoter proximal to the first coding exon bypassed epigenetic silencing, permitting stable and robust expression of endogenous FOXP3. HDR-edited T cells, edTregs, manifested a transcriptional program leading to sustained expression of canonical markers and suppressive activity of tTreg. Both human and murine edTregs mediated immunosuppression in vivo in models of inflammatory disease. Further, this engineering strategy permitted generation of antigen-specific edTreg with robust in vitro and in vivo functional activity. Last, edTreg could be enriched and expanded at scale using clinically relevant methods. Together, these findings suggest that edTreg production may permit broad future clinical application.

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Loss of FOXP3 Expression and Emergence of Cytokine-Producing Cells after In Vitro Expansion of Human CD4+CD25+CD127 - Regulatory T Cells.

Blood ◽

10.1182/blood.v110.11.63.63 ◽

2007 ◽

Vol 110 (11) ◽

pp. 63-63 ◽

Cited By ~ 4

Author(s):

Petra Hoffmann ◽

Ruediger Eder ◽

Tina J. Boeld ◽

Jochen Huehn ◽

Stefan Floess ◽

...

Keyword(s):

T Cells ◽

Treg Cell ◽

Cell Cultures ◽

Methylation Status ◽

Foxp3 Expression ◽

Treg Cells ◽

Culture Conditions ◽

Cell Therapies ◽

Over Time

Abstract We and others previously showed that the adoptive transfer of donor-type CD4+CD25+ regulatory T (Treg) cells protects from graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT) in animal models. Exploring this strategy in human SCT, we currently perform a first Phase I clinical trial using freshly isolated Treg cells. For future trials requiring large Treg cell numbers for repetitive treatments, we described in vitro culture conditions that permit a more than 3-log expansion of polyclonal Treg cells (Blood 104:895; 2004). A highly enriched starting population proved to be crucial for the generation of pure Treg cell products and we could show that the naive, CD45RA+ subpopulation of CD4+CD25high T cells fulfills this criterion (Blood108:4260; 2006). A recently proposed alternative approach for the isolation of pure Treg cells relies on the exclusion of CD127+ cells, as activated (i.e. CD25+) conventional T cells express high levels of CD127 while CD4+CD25+ Treg cells show no or only weak expression levels (Seddiki et al. and Liu et al., JEM203:1693; 2006). To directly compare these two approaches, we isolated CD4+CD25+CD127low/neg T cells (CD127-Treg) and CD45RA+CD4+CD25high T cells (RA+ Treg) from the same leukapheresis products and analyzed the cells after 2 and 3 weeks of expansion. Whereas both populations were > 94% FOXP3+ upon isolation, only RA+ Treg maintained this high level of FOXP3+ cells throughout the expansion period (93% (range: 78 to 97%; n=11) FOXP3+ after 2 and 87% (range: 71 to 97%; n=9) FOXP3+ after 3 weeks). In contrast, the proportion of FOXP3+ cells in CD127-Treg cultures was already reduced after 2 weeks (82% (range: 56 to 96%; n=11)) and highly variable and significantly lower than that of RA+ Treg cultures after 3 weeks (57% (range: 18 to 93%; n=9; p=0.006)). When further subdivided into CD45RA+ and CD45RA- subpopulations before expansion, cultures of CD45RA-CD127-Treg cells lost FOXP3 expression and comprised substantial numbers of FOXP3- cytokine producing cells (on average 54% and 38% IL-2 and IFN-γ producers, respectively) after 3 weeks, whereas CD45RA+ CD127- Treg behaved similar to RA+ Treg cells, as they maintained FOXP3 expression over time and contained only low numbers of cytokine producers. Furthermore, when we analyzed the DNA methylation status of Treg cells, we found the Treg-specific CpG demethylation pattern within the FOXP3 gene (EJI 37, 2007) in RA+ Treg cell lines, while CD127- Treg cell cultures showed increased methylation over time and even more so RA- Treg cell cultures. Based on these findings, we suggest that isolation and expansion of CD45RA+CD4+CD25high T cells at present represents the best strategy for adoptive cell therapies requiring in vitro expanded Treg cells.

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