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1.  Blockade of LFA-1 augments in vitro differentiation of antigen-induced Foxp3+ Treg cells 
Adoptive transfer of antigen-specific, in vitro-induced Foxp3+ Treg (iTreg) cells protects against autoimmune disease. To generate antigen-specific iTreg cells at high purity, however, remains a challenge. Whereas polyclonal T cell stimulation with anti-CD3 and anti-CD28 antibody yields Foxp3+ iTreg cells at a purity of 90–95%, antigen-induced iTreg cells typically do not exceed a purity of 65–75%, even in a TCR-transgenic model. In a similar vein to thymic Treg cell selection, iTreg cell differentiation is influenced not only by antigen recognition and the availability of TGF-β but also by co-factors including costimulation and adhesion molecules. In this study, we demonstrate that blockade of the T cell integrin Leukocyte Function-associated Antigen-1 (LFA-1) during antigen-mediated iTreg cell differentiation augments Foxp3 induction, leading to approximately 90% purity of Foxp3+ iTreg cells. This increased efficacy not only boosts the yield of Foxp3+ iTreg cells, it also reduces contamination with activated effector T cells, thus improving the safety of adoptive transfer immunotherapy.
Highlights
•iTreg cells can be generated in an antigen-specific manner, even if specific Tconv cells are present at low frequency.•Blockade of anti-LFA-1 during iTreg cell differentiation augments Foxp3 induction.•The blockade of LFA-1 alters the iTreg cell phenotype but does not impair stability or function.
doi:10.1016/j.jim.2014.07.012
PMCID: PMC4265731  PMID: 25108241
Foxp3; LFA-1; Treg cell; Immunotherapy; Autoimmunity
2.  Systems Modeling of Molecular Mechanisms Controlling Cytokine-driven CD4+ T Cell Differentiation and Phenotype Plasticity 
PLoS Computational Biology  2013;9(4):e1003027.
Differentiation of CD4+ T cells into effector or regulatory phenotypes is tightly controlled by the cytokine milieu, complex intracellular signaling networks and numerous transcriptional regulators. We combined experimental approaches and computational modeling to investigate the mechanisms controlling differentiation and plasticity of CD4+ T cells in the gut of mice. Our computational model encompasses the major intracellular pathways involved in CD4+ T cell differentiation into T helper 1 (Th1), Th2, Th17 and induced regulatory T cells (iTreg). Our modeling efforts predicted a critical role for peroxisome proliferator-activated receptor gamma (PPARγ) in modulating plasticity between Th17 and iTreg cells. PPARγ regulates differentiation, activation and cytokine production, thereby controlling the induction of effector and regulatory responses, and is a promising therapeutic target for dysregulated immune responses and inflammation. Our modeling efforts predict that following PPARγ activation, Th17 cells undergo phenotype switch and become iTreg cells. This prediction was validated by results of adoptive transfer studies showing an increase of colonic iTreg and a decrease of Th17 cells in the gut mucosa of mice with colitis following pharmacological activation of PPARγ. Deletion of PPARγ in CD4+ T cells impaired mucosal iTreg and enhanced colitogenic Th17 responses in mice with CD4+ T cell-induced colitis. Thus, for the first time we provide novel molecular evidence in vivo demonstrating that PPARγ in addition to regulating CD4+ T cell differentiation also plays a major role controlling Th17 and iTreg plasticity in the gut mucosa.
Author Summary
CD4+ T cells can differentiate into different phenotypes depending on the cytokine milieu. Due to the complexity of this process, we have constructed a computational and mathematical model with sixty ordinary differential equations representing a CD4+ T cell differentiating into either Th1, Th2, Th17 or iTreg cells. The model includes cytokines, nuclear receptors and transcription factors that define fate and function of CD4+ T cells. Computational simulations illustrate how a proinflammatory Th17 cell can undergo reprogramming into an anti-inflammatory iTreg phenotype following PPARγ activation. This modeling-derived hypothesis has been validated with in vitro and in vivo experiments. Experimental data support the modeling-derived prediction and demonstrate that the loss of PPARγ enhances a proinflammatory response characterized by Th17 in colitis-induced mice. Moreover, pharmacological activation of PPARγ in vivo can affect the Th17/iTreg balance by upregulating FOXP3 and downregulating IL-17A and RORγt. In summary, we demonstrate that computational simulations using our CD4+ T cell model provide novel unforeseen hypotheses related to the molecular mechanisms controlling differentiation and function of CD4+ T cells. In vivo findings validated the modeling prediction that PPARγ modulates differentiation and plasticity of CD4+ T cells in mice.
doi:10.1371/journal.pcbi.1003027
PMCID: PMC3617204  PMID: 23592971
3.  Optimal Isolation of Functional Foxp3+ Induced Regulatory T Cells Using DEREG Mice 
PLoS ONE  2012;7(9):e44760.
Foxp3 reporter mice including DEREG (DEpletion of REGulatory T cells) mice have greatly helped in exploring the biology of Foxp3+ Tregs. DEREG mice express a DTR-eGFP fusion protein under the control of a bacterial artificial chromosome (BAC)-encoded Foxp3 promoter, allowing the viable isolation and inducible depletion of Foxp3+ Tregs. Adaptive Tregs differentiated in vitro to express Foxp3 (iTregs) are gaining high interest as potential therapeutics for inflammatory conditions such as autoimmunity, allergy and transplant rejection. However, selective isolation of Foxp3+ iTregs with a stable phenotype still remains to be a problem, especially in the human setting. While screening for culture conditions to generate stable CD4+Foxp3+ iTregs from DEREG mice, with maximum suppressive activity, we observed an unexpected dichotomy of eGFP and Foxp3 expression which is not seen in ex vivo isolated cells from DEREG mice. Further characterization of eGFP+Foxp3− cells revealed relatively lower CD25 expression and a lack of suppressive activity in vitro. Similarly, eGFP− cells isolated from the same cultures were not suppressive despite of a broad CD25 expression reflecting mere T cell activation. In contrast, eGFP+Foxp3+ iTregs exhibited potent suppressive activity comparable to that of natural eGFP+Foxp3+ Tregs, emphasizing the importance of isolating Foxp3 expressing iTregs. Interestingly, the use of plate-bound anti-CD3 and anti-CD28 or Flt3L-driven BMDC resulted in considerable resolution of the observed dichotomy. In summary, we defined culture conditions for efficient generation of eGFP+Foxp3+ iTregs by use of DEREG mice. Isolation of functional Foxp3+ iTregs using DEREG mice can also be achieved under sub-optimal conditions based on the magnitude of surface CD25 expression, in synergy with transgene encoded eGFP. Besides, the reported phenomenon may be of general interest for exploring Foxp3 gene regulation, given that Foxp3 and eGFP expression are driven from distinct Foxp3 loci and because this dichotomy preferentially occurs only under defined in vitro conditions.
doi:10.1371/journal.pone.0044760
PMCID: PMC3434173  PMID: 22957107
4.  Interaction with activated monocytes enhances cytokine expression and suppressive activity of human CD4+CD45RO+CD25+CD127low regulatory T cells 
Arthritis and rheumatism  2013;65(3):627-638.
Objective
Despite the high frequency of CD4+ T cells with a regulatory phenotype (CD25+CD127lowFoxP3+) in the joints of patients with rheumatoid arthritis (RA), inflammation persists. One possible explanation is that human Tregs are converted into pro-inflammatory IL-17-producing cells by inflammatory mediators and thereby lose their suppressive function. We investigated whether activated monocytes, which are potent producers of inflammatory cytokines and abundantly present in the rheumatic joint, induce pro-inflammatory cytokine expression in human Tregs and impair their regulatory function.
Methods
The presence and phenotype of CD4+CD45RO+CD25+CD127low T cells (memory Tregs) and CD14+ monocytes in the peripheral blood (PB) and synovial fluid (SF) from patients with RA was investigated by flow cytometry. FACS-sorted memory Tregs from healthy controls were co-cultured with autologous activated monocytes and stimulated with anti-CD3 monoclonal antibody. Intracellular cytokine expression, phenotype and function of cells were determined by flow cytometry, ELISA and proliferation assays.
Results
Patients with RA showed higher frequencies of CD4+CD45RO+CD25+CD127low Tregs and activated CD14+ monocytes in SF relative to PB. In vitro-activated monocytes induced an increase in the percentage of IL-17+, IFNγ+ and TNF-α+, but also IL-10+ Tregs. The observed increase in IL-17+ and IFNγ+ Tregs was driven by monocyte-derived IL-1β, IL-6 and TNF-α and was mediated by both CD14+CD16− and CD14+CD16+ monocyte subsets. Despite enhanced cytokine expression, cells maintained their CD25+FoxP3+CD39+ Treg phenotype and showed enhanced capacity to suppress proliferation and IL-17 production by effector T cells.
Conclusion
Tregs exposed to a pro-inflammatory environment show increased cytokine expression as well as enhanced suppressive activity.
doi:10.1002/art.37832
PMCID: PMC3947722  PMID: 23280063
5.  Generation and large-scale expansion of human inducible regulatory T cells that suppress graft-versus-host disease 
Adoptive transfer of thymus-derived natural regulatory T-cells (nTregs) effectively suppresses disease in murine models of autoimmunity and graft-versus-host disease (GVHD). TGFβ induces Foxp3 expression and suppressive function in stimulated murine CD4+25- T cells, and these induced Treg (iTregs), like nTreg, suppress auto- and allo-reactivity in vivo. However, while TGFβ induces Foxp3 expression in stimulated human T-cells, the expanded cells lack suppressor cell function. Here we show that Rapamycin (Rapa) enhances TGFβ-dependent Foxp3 expression and induces a potent suppressor function in naïve (CD4+25-45RA+) T cells. Rapa/TGFβ iTregs are anergic, express CD25 at levels higher than expanded nTregs, and few cells secrete IL-2, IFNγ or IL-17 even after PMA and Ionomycin stimulation in vitro. Unlike other published methods of inducing Treg function, Rapa/TGFβ induces suppressive function even in the presence of memory CD4+ T-cells. A single apheresis unit of blood yields an average ~240×109 (range ~70–560×109) iTregs from CD4+25- T-cells in ≤ 2 weeks of culture. Most importantly, Rapa/TGFβ iTregs suppress disease in a xenogeneic model of GVHD. This study opens the door for iTreg cellular therapy for human diseases.
doi:10.1111/j.1600-6143.2011.03558.x
PMCID: PMC3552455  PMID: 21564534
GVHD; Treg; Foxp3; Rapamycin; TGFβ
6.  Antigen-specific iTreg impair DC function via an IL-10/MARCH 1–Dependent Mechanism1 
Journal of immunology (Baltimore, Md. : 1950)  2013;191(12):10.4049/jimmunol.1301693.
Foxp3+ T regulatory cells (Treg) are critically important for the maintenance of immunological tolerance, immune homeostasis and prevention of autoimmunity. Dendritic cells (DCs) are one of the major targets of Treg-mediated suppression. Some studies have suggested that Treg-mediated suppression of DC function is mediated by the interaction of CTLA-4 on Treg with CD80/CD86 on the DC resulting in down- regulation of CD80/CD86 expression and a decrease in co-stimulation. We have re-examined the effects of Treg on mouse DC function in a model in which antigen-specific, induced Treg (iTreg) are co-cultured with DC in the absence of Teff cells. iTreg-treated DC are markedly defective in their capacity to activate naïve T cells. iTreg from CTLA-4 deficient mice failed to induce downregulation of CD80/CD86, but DCs treated with CTLA-4 deficient iTreg still exhibited impaired capacity to activate naïve T cells. The iTreg-induced defect in DC function could be completely reversed by anti-IL-10 and IL-10 deficient iTregs failed to down-regulate DC function. iTreg-treated DCs expressed high levels of MARCH1, an E3 ubiquitin ligase, recently found to degrade CD86 and MHC-II on the DC and expressed lower levels of CD83, a molecule involved in neutralizing the function of MARCH1. Both the enhanced expression of MARCH1 and the decreased expression of CD83 were mediated by IL-10 produced by the iTreg. Taken together, these studies demonstrate that a major suppressive mechanism of DC function by iTreg is secondary to the effects of IL-10 on MARCH1 and CD83 expression.
doi:10.4049/jimmunol.1301693
PMCID: PMC3858537  PMID: 24218453
7.  TGF-β-induced Myelin Peptide-Specific Regulatory T Cells Mediate Antigen-Specific Suppression of Induction of Experimental Autoimmune Encephalomyelitis1 
The low number of natural regulatory T cells (nTreg cells) in the circulation specific for a particular antigen and concerns about the bystander suppressive capacity of expanded nTregs presents a major clinical challenge for nTreg-based therapeutic treatment of autoimmune diseases. In the present study, we demonstrate that naïve CD4+CD25-Foxp3- T cells specific for the myelin proteolipid protein (PLP)139-151 peptide, can be converted into CD25+Foxp3+ induced Treg cells (iTreg cells) when stimulated in the presence of transforming growth factor-β (TGF-β), retinoic acid and interleukin-2. These PLP139-151-specific iTreg cells (139-iTreg cells) have a phenotype similar to natural Treg cells, but additionally express an intermediate level of CD62L and a high level of CD103. Upon transfer into SJL/J mice, 139-iTreg cells undergo antigen-driven proliferation and are effective at suppressing induction of experimental autoimmune encephalomyelitis induced by the cognate PLP139-151 peptide, but not PLP178-191 or a mixture of the two peptides. Furthermore, 139-iTregs inhibit delayed-type-hypersensitivity (DTH) responses to PLP139-151, but not PLP178-191, MOG35-55 or OVA323-339 in mice primed with a mixture of PLP139-151 and the other respective peptides. Additionally, 139-iTreg cells suppress the proliferation and activation of PLP139-151-, but not MOG35-55-specific CD4+ T cells in SJL/B6 F1 mice primed with a combination of PLP139-151 and MOG35-55. These findings suggest that antigen-specific-iTreg cells are amplified in vivo when exposed to cognate antigen under inflammatory conditions, and these activated iTreg cells suppress CD4+ responder T cells in an antigen-specific manner.
doi:10.4049/jimmunol.0904044
PMCID: PMC2882517  PMID: 20483764
8.  Tumor antigen specific iTreg accumulate in the tumor microenvironment and suppress therapeutic vaccination 
Oncoimmunology  2012;1(5):642-648.
Tumor specific antigens (TSA) provide an opportunity to mobilize therapeutic immune responses against cancer. To evade such responses, tumor development in immunocompetent hosts is accompanied by acquisition of both active and passive mechanisms of immune suppression, including recruitment of CD4+FoxP3+ regulatory T cells (Treg). Thymic derived Treg (nTreg) may recognize self-antigens in the tumor microenvironment, while peripherally induced Treg (iTreg) may preferentially recognize the same TSA which provide an opportunity for therapeutic immunity from peripheral T cells. In this study we provide a systematic analysis of nTreg and iTreg accumulation in the tumor microenvironment (TME) at the cellular level. iTreg accumulation to the TME was influenced by the abundance of a known TSA, and in the absence of a known TSA intratumoral Treg displayed a unique TCR repertoire from peripheral Treg. In vivo suppression assays demonstrate that cognate-antigen matched iTreg are more potent suppressors of CD4+ than are polyclonal iTreg or nTreg, but were unable to suppress CD8+ T cell proliferation. Suppression occurred only locally at the site of immunization, and correlated with decreased expression of CD80 and CD86 on CD11c positive cells. Although established tumors facilitated the induction of TSA-specific iTreg, these iTreg suppressed CD4+ T cell accumulation only locally to the TME. Tumor mediated suppression of CD8+ T cell immunity appeared independent of TSA-specific iTreg.
doi:10.4161/onci.20298
PMCID: PMC3429568  PMID: 22934256
FoxP3; iTreg; nTreg; regulatory T cell; tumor microenvironment
9.  Efficient and Selective Prevention of GVHD by Antigen-Specific Induced Tregs via Linked-Suppression in Mice 
Naturally occurring regulatory T cells (nTregs) suppress the development of GVHD and may spare graft-versus-leukemia (GVL) effect. Because nTreg is a rare population in a healthy individual, the limited source and the non-selective suppression are major hurdles towards the application of nTregs in the control of clinical GVHD after allogeneic HCT. An alternative approach is to generate induced Tregs (iTregs) from naïve CD4 precursors, but the effectiveness of iTregs in the control of GVHD is highly controversial and requires further investigation. The other critical but unsolved issue on Treg therapy is how to achieve antigen (Ag)-specific tolerance that distinguishes GVHD and GVL effect. To address the important issues on the effectiveness of iTregs and Ag-specificity of Tregs, we generated Ag-specific iTregs and tested their potential in the prevention of GVHD in pre-clinical BMT model. CD4+CD25+Foxp3+ iTregs generated from OT-II TCR transgenic T cells specific for OVA target Ag efficiently prevented GVHD induced by polyclonal T effector cells (Teffs) only in the allogeneic recipients that express OVA protein but not in OVA− recipients. The efficacy of these Ag-specific iTregs was significantly higher than polyclonal iTregs. As controls, OT-II CD4+Foxp3− cells had no effect on GVHD development in OVA− recipients and exacerbated GVHD in OVA+ recipients when transplanted together with polyclonal Teffs. Because the iTregs recognize OVA whereas Teffs recognize alloAg bm12, our data reveal for the first time that Tregs prevent GVHD through a linked suppression. Mechanistically, OT-II iTregs expanded extensively, and significantly suppressed expansion and infiltration of Teffs in OVA+ but not in OVA− recipients. These results demonstrate that Ag-specific iTregs can prevent GVHD efficiently and selectively, providing a proof of principle that Ag-specific iTregs may represent a promising cell therapy for their specificity and higher efficacy in allogeneic HCT.
doi:10.1016/j.bbmt.2010.12.710
PMCID: PMC3039088  PMID: 21224010
10.  Alterations of peripheral CD4+CD25+Foxp3+ T regulatory cells in mice with STZ-induced diabetes 
Complications arising from abnormal immune responses are the major causes of mortality and morbidity in diabetic patients. CD4+CD25+ T regulatory cells (Tregs) play pivotal roles in controlling immune homeostasis, immunity and tolerance. The effect of hyperglycemia on CD4+CD25+ Tregs has not yet been addressed. Here we used streptozotocin (STZ)-induced diabetic mice to study the effects of long-term hyperglycemia on CD4+CD25+ Tregs in vivo. Four months after the onset of diabetes, the frequency of CD4+CD25+Foxp3+ T regulatory cells was significantly elevated in the spleen, peripheral blood lymphocytes (PBLs), peripheral lymph nodes (pLNs) and mesenteric LNs (mLNs). CD4+CD25+ Tregs obtained from mice with diabetes displayed defective immunosuppressive functions and an activated/memory phenotype. Insulin administration rescued these changes in the CD4+CD25+ Tregs of diabetic mice. The percentage of thymic CD4+CD25+ naturally occurring Tregs (nTregs) and peripheral CD4+Helios+Foxp3+ nTregs were markedly enhanced in diabetic mice, indicating that thymic output contributed to the increased frequency of peripheral CD4+CD25+ Tregs in diabetic mice. In an in vitro assay in which Tregs were induced from CD4+CD25− T cells by transforming growth factor (TGF)-β, high glucose enhanced the efficiency of CD4+CD25+Foxp3+ inducible Tregs (iTregs) induction. In addition, CD4+CD25− T cells from diabetic mice were more susceptible to CD4+CD25+Foxp3+ iTreg differentiation than those cells from control mice. These data, together with the enhanced frequency of CD4+Helios−Foxp3+ iTregs in the periphery of mice with diabetes, indicate that enhanced CD4+CD25+Foxp3+ iTreg induction also contributes to a peripheral increase in CD4+CD25+ Tregs in diabetic mice. Our data show that hyperglycemia may alter the frequency of CD4+CD25+Foxp3+ Tregs in mice, which may result in late-state immune dysfunction in patients with diabetes.
doi:10.1038/cmi.2011.37
PMCID: PMC4002930  PMID: 21983870
CD4+CD25+Foxp3+ regulatory T cells;  diabetes; hyperglycemia; immune disorder; mice
11.  Rapamycin-Conditioned Donor Dendritic Cells Differentiate CD4+CD25+Foxp3+ T Cells In Vitro with TGF-β 1 for Islet Transplantation 
Dendritic cells (DCs) conditioned with the mammalian target of rapamycin (mTOR) inhibitor rapamycin have been previously shown to expand naturally existing regulatory T cells (nTregs). This work addresses whether rapamycin-conditioned donor DCs could effectively induce CD4+CD25+Foxp3+ Tregs (iTregs) in cell cultures with alloantigen specificities, and whether such in vitro-differentiated CD4+CD25+Foxp3+ iTregs could effectively control acute rejection in allogeneic islet transplantation. We found that donor BALB/c bone marrow-derived DCs (BMDCs) pharmacologically modified by the mTOR inhibitor rapamycin had significantly enhanced ability to induce CD4+CD25+Foxp3+ iTregs of recipient origin (C57BL/6 (B6)) in vitro under Treg driving conditions compared to unmodified BMDCs. These in vitro-induced CD4+CD25+Foxp3+ iTregs exerted donor-specific suppression in vitro, and prolonged allogeneic islet graft survival in vivo in RAG−/− hosts upon coadoptive transfer with T-effector cells. The CD4+CD25+Foxp3+ iTregs expanded and preferentially maintained Foxp3 expression in the graft draining lymph nodes. Finally, the CD4+CD25+Foxp3+ iTregs were further able to induce endogenous naïve T cells to convert to CD4+CD25+Foxp3+ T cells. We conclude that rapamycin-conditioned donor BMDCs can be exploited for efficient in vitro differentiation of donor antigen-specific CD4+CD25+Foxp3+ iTregs. Such in vitro-generated donor-specific CD4+CD25+Foxp3+ iTregs are able to effectively control allogeneic islet graft rejection.
doi:10.1111/j.1600-6143.2010.03199.x
PMCID: PMC3995630  PMID: 20626386
Dendritic cells; graft rejection; induced regulatory T cells; islet transplantation; rapamycin; transforming growth factor-β 1
12.  IL-2 Controls the Stability of Foxp3 expression in TGF-β-induced Foxp3+ T cells in vivo 
Stimulation of naïve mouse CD4+Foxp3− T cells in the presence of TGF-β results in the induction of Foxp3 expression and T suppressor function. However, Foxp3 expression in these induced T regulatory cells (iTreg) is unstable raising the possibility that iTreg would not be useful for treatment of autoimmune diseases. To analyze the factors that control the stability of Foxp3 expression in iTreg, we generated OVA-specific iTreg from OT-II Foxp3-GFP knock in mice. Following transfer to normal C57BL/6 mice, OT-II GFP+ cells maintained high levels of Foxp3 expression for 8 days. However, they rapidly lost Foxp3 expression upon stimulation with OVA in IFA in vivo. This unstable phenotype was associated with a strong methylation of the Treg-specific demethylated region (TSDR) within the Foxp3 locus. Administration of IL-2/anti-IL-2 complexes expanded the numbers of transferred Foxp3+ iTreg in the absence of antigen challenge. Notably, when the iTreg were stimulated with antigen, treatment with IL-2/anti-IL-2 complexes stabilized Foxp3 expression and resulted in enhanced demethylation of the TSDR. Conversely, neutralization of IL-2 or disruption of its signaling by deletion of Stat5 diminished the level of Foxp3 expression resulting in decreased suppressor function of the iTreg in vivo. Our data suggest that stimulation with TGF-β in vitro is not sufficient for imprinting T cells with stable expression of Foxp3. Administration of IL-2 in vivo results in stabilization of Foxp3 expression and may prove to be a valuable adjunct for the use of iTreg for the treatment of autoimmune diseases.
doi:10.4049/jimmunol.1100061
PMCID: PMC3098943  PMID: 21525380
13.  Generation and Function of Induced Regulatory T Cells 
CD4+ CD25+ Foxp3+ regulatory T (Treg) cells are essential to the balance between pro- and anti-inflammatory responses. There are two major subsets of Treg cells, “natural” Treg (nTreg) cells that develop in the thymus, and “induced” Treg (iTreg) cells that arise in the periphery from CD4+ Foxp3− conventional T cells and can be generated in vitro. Previous work has established that both subsets are required for immunological tolerance. Additionally, in vitro-derived iTreg cells can reestablish tolerance in situations where Treg cells are decreased or defective. This review will focus on iTreg cells, drawing comparisons to nTreg cells when possible. We discuss the molecular mechanisms of iTreg cell induction, both in vivo and in vitro, review the Foxp3-dependent and -independent transcriptional landscape of iTreg cells, and examine the proposed suppressive mechanisms utilized by each Treg cell subset. We also compare the T cell receptor repertoire of the Treg cell subsets, discuss inflammatory conditions where iTreg cells are generated or have been used for treatment, and address the issue of iTreg cell stability.
doi:10.3389/fimmu.2013.00152
PMCID: PMC3685796  PMID: 23801990
Treg cells; Treg stability; immunotherapy; Treg function; gene expression profiling; TCR repertoire
14.  Induced T Regulatory Cells Suppress Osteoclastogenesis and Bone Erosion in Collagen-induced Arthritis Better than Natural T Tegulatory Cells 
Annals of the rheumatic diseases  2012;71(9):1567-1572.
Objective
Osteoclasts are responsible for bone destruction in rheumatoid arthritis (RA) and natural CD4+Foxp3+regulatory T cells (nTregs) can inhibit osteoclastogenesis. This study aims to determine whether TGF-β-induced CD4+Foxp3+regulatory T cells (iTregs) also suppress osteolastogenesis and bone erosion in collagen induced arthritis (CIA).
Methods
Osteoclasts were induced from bone-marrow CD11b+ cells with RANKL and macrophage colony-stimulating factor (M-CSF), and assessed with tartrate-resistant acid phosphatase (TRAP) staining. CD4+ iTregs were generated with TGF-β and added to cultures with different ratios with CD11b+ cells. Transwell and antibody blockade experiments were performed to define the mechanism of action. NF-kB activation was determined by western blot. 3×106 CD4+ iTregs, nTregs or control cells were adoptively transferred to DBA1/J mice on day 14 after immunization with CII/CFA. CIA onset and severity were monitored and bone erosion was examined by CT scan.
Results
Both CD4+ Tregs almost completely suppressed osteoclastogenesis but only iTregs sustained the effect in the presence of IL-6 in vitro. CD4+ iTregs but not nTregs and control cells injected after immunization and before of onset of CIA significantly suppressed disease development. Of note, CT scan showed that the joints in CD4+ iTregs but not nTregs or control cells infused CIA had less bone erosion. Treatment with CD4+ iTregs but not other cells dramatically decreased the levels of NF-kB p65/p50 in osteoclasts in vitro and P65/50 and RANKL expression by synovial tissues in vivo.
Conclusion
Manipulation of CD4+ iTregs may have therapeutic effects on rheumatoid arthritis and other bone erosion related diseases.
doi:10.1136/annrheumdis-2011-201052
PMCID: PMC4038329  PMID: 22764040
15.  CD8+ FOXP3+ REGULATORY T CELLS ARE INDUCED DURING GRAFT VERSUS HOST DISEASE AND MITIGATE DISEASE SEVERITY 
Regulatory T cells (Tregs), in particular CD4+ Foxp3+ T cells, have been shown to play an important role in the maintenance of tolerance after allogeneic stem cell transplantation. In the current study, we have identified a population of CD8+ Foxp3+ T cells that are induced early during GVHD, constitute a significant percentage of the entire Treg population, and are present in all major GVHD target organs. These cells expressed many of the same cell surface molecules as found on CD4+ Tregs and potently suppressed in vitro alloreactive T cell responses. Induction of these cells correlated positively with the degree of MHC disparity between donor and recipient and was significantly greater than that observed for CD4+ induced Tregs (iTregs) in nearly all tissue sites. Mice that lacked the ability to make both CD8+ and CD4+ iTregs had accelerated GVHD mortality compared to animals that were competent to make both iTreg populations. The absence of both iTreg populations was associated with significantly greater expansion of activated donor T cells and increased numbers of CD4+ and CD8+ T cells that secreted IFN-γ and IL-17. The presence of CD8+ iTregs, however, was sufficient to prevent increased GVHD mortality in the complete absence of CD4+ Tregs, indicating at least one functional iTreg population was sufficient to prevent an exacerbation in GVHD severity, and that CD8+ iTregs could compensate for CD4+ iTregs. These studies define a novel population of CD8+ Tregs that play a role in mitigating the severity of GVHD after allogeneic stem cell transplantation.
doi:10.4049/jimmunol.1200886
PMCID: PMC3381996  PMID: 22649199
16.  Progesterone suppresses the mTOR pathway and promotes generation of induced regulatory T cells with increased stability 
European journal of immunology  2012;42(10):2683-2696.
Summary
While induced FoxP3+ T cells (iTregs) are promising cellular therapeutics to treat inflammatory diseases, a limitation in utilizing iTregs prepared in vitro is their low stability in inflammatory conditions. Progesterone (P4) is an immune regulatory nuclear hormone with a potent Treg induction activity. We reasoned that this function of progesterone would be utilized to generate iTregs with highly suppressive activity and improved stability in vivo. We generated iTregs with progesterone in vitro and found that progesterone generates iTregs that are highly stable in inflammatory conditions. Moreover, P4-induced iTregs highly express latency-associated peptide TGFβ1 and are efficient in regulating inflammation in multiple tissues, whereas control iTregs induced with TGFβ1 alone are less stable and ineffective in suppressing inflammation. The function of progesterone in inducing iTregs with improved regulatory activity is associated with the function of P4 in suppressing the mTOR pathway. Moreover, the function of progesterone in inducing FoxP3+ T cells is decreased but not completely abolished on nuclear progesterone receptor-deficient T cells, suggesting that both nuclear and non-nuclear progesterone receptors are involved in mediating the function. We conclude that P4 can be utilized to generate iTregs with a high therapeutic potential in treatment of tissue inflammation.
doi:10.1002/eji.201142317
PMCID: PMC3668644  PMID: 22740122
FoxP3; progesterone; inflammation
17.  In Vitro Induced Regulatory T Cells Are Unique from Endogenous Regulatory T Cells and Effective at Suppressing Late Stages of Ongoing Autoimmunity 
PLoS ONE  2014;9(8):e104698.
Strategies to boost the numbers and functions of regulatory T cells (Tregs) are currently being tested as means to treat autoimmunity. While Tregs have been shown to be effective in this role, strategies to manipulate Tregs to effectively suppress later stages of ongoing diseases need to be established. In this study, we evaluated the ability of TGF-β-induced Tregs (iTregs) specific for the major self-antigen in autoimmune gastritis to suppress established autoimmune gastritis in mice. When transferred into mice during later stages of disease, iTregs demethylated the Foxp3 promoter, maintained Foxp3 expression, and suppressed effector T cell proliferation. More importantly, these iTregs were effective at stopping disease progression. Untreated mice had high numbers of endogenous Tregs (enTregs) but these were unable to stop disease progression. In contrast, iTregs, were found in relatively low numbers in treated mice, yet were effective at stopping disease progression, suggesting qualitative differences in suppressor functions. We identified several inhibitory receptors (LAG-3, PD-1, GARP, and TNFR2), cytokines (TGF-β1 and IL12p35), and transcription factors (IRF4 and Tbet) expressed at higher levels by iTregs compared to enTregs isolated form mice with ongoing disease, which likely accounts for superior suppressor ability in this disease model. These data support efforts to use iTregs in therapies to treat establish autoimmunity, and show that iTregs are more effective than enTregs at suppressing inflammation in this disease model.
doi:10.1371/journal.pone.0104698
PMCID: PMC4131893  PMID: 25119105
18.  Regulation of Foxp3+ Inducible Regulatory T Cell Stability by SOCS2 
Suppressor of cytokine signaling (SOCS) proteins are key regulators of CD4+ T cell differentiation, and in particular, we have recently shown that SOCS2 inhibits the development of Th2 cells and allergic immune responses. Interestingly, transcriptome analyses have identified SOCS2 as being preferentially expressed in both natural regulatory T cells (Tregs) and inducible Tregs (iTregs); however, the role of SOCS2 in Foxp3+ Treg function or development has not been fully elucidated. In this study, we show that despite having no effect on natural Treg development or function, SOCS2 is highly expressed in iTregs and required for the stable expression of Foxp3 in iTregs in vitro and in vivo. Indeed, SOCS2-deficient CD4+ T cells upregulated Foxp3 following in vitro TGF-β stimulation, but failed to maintain stable expression of Foxp3. Moreover, in vivo generation of iTregs following OVA feeding was impaired in the absence of SOCS2 and could be rescued in the presence of IL-4 neutralizing Ab. Following IL-4 stimulation, SOCS2-deficient Foxp3+ iTregs secreted elevated IFN-γ and IL-13 levels and displayed enhanced STAT6 phosphorylation. Therefore, we propose that SOCS2 regulates iTreg stability by downregulating IL-4 signaling. Moreover, SOCS2 is essential to maintain the anti-inflammatory phenotype of iTregs by preventing the secretion of proinflammatory cytokines. Collectively, these results suggest that SOCS2 may prevent IL-4–induced Foxp3+ iTreg instability. Foxp3+ iTregs are key regulators of immune responses at mucosal surfaces; therefore, this dual role of SOCS2 in both Th2 and Foxp3+ iTregs reinforces SOCS2 as a potential therapeutic target for Th2-biased diseases.
doi:10.4049/jimmunol.1201396
PMCID: PMC3607399  PMID: 23455506
19.  CD4+CD62L+ Central Memory T Cells Can Be Converted to Foxp3+ T Cells 
PLoS ONE  2013;8(10):e77322.
The peripheral Foxp3+ Treg pool consists of naturally arising Treg (nTreg) and adaptive Treg cells (iTreg). It is well known that naive CD4+ T cells can be readily converted to Foxp3+ iTreg in vitro, and memory CD4+ T cells are resistant to conversion. In this study, we investigated the induction of Foxp3+ T cells from various CD4+ T-cell subsets in human peripheral blood. Though naive CD4+ T cells were readily converted to Foxp3+ T cells with TGF-β and IL-2 treatment in vitro, such Foxp3+ T cells did not express the memory marker CD45RO as do Foxp3+ T cells induced in the peripheral blood of Hepatitis B Virus (HBV) patients. Interestingly, a subset of human memory CD4+ T cells, defined as CD62L+ central memory T cells, could be induced by TGF-β to differentiate into Foxp3+ T cells. It is well known that Foxp3+ T cells derived from human CD4+CD25- T cells in vitro are lack suppressive functions. Our data about the suppressive functions of CD4+CD62L+ central memory T cell-derived Foxp3+ T cells support this conception, and an epigenetic analysis of these cells showed a similar methylation pattern in the FOXP3 Treg-specific demethylated region as the naive CD4+ T cell-derived Foxp3+ T cells. But further research showed that mouse CD4+ central memory T cells also could be induced to differentiate into Foxp3+ T cells, such Foxp3+ T cells could suppress the proliferation of effector T cells. Thus, our study identified CD4+CD62L+ central memory T cells as a novel potential source of iTreg.
doi:10.1371/journal.pone.0077322
PMCID: PMC3796486  PMID: 24155942
20.  Murine CD4+CD25- cells activated in vitro with PMA/ionomycin and anti-CD3 acquire regulatory function and ameliorate experimental colitis in vivo 
BMC Gastroenterology  2012;12:172.
Background
Induced regulatory T (iTreg) lymphocytes show promise for application in the treatment of allergic, autoimmune and inflammatory disorders. iTreg cells demonstrate advantages over natural Treg (nTreg) cells in terms of increased number of starting population and greater potential to proliferate. Different activation methods to generate iTreg cells result in iTreg cells that are heterogeneous in phenotype and mechanisms of suppression. Therefore it is of interest to explore new techniques to generate iTreg cells and to determine their physiological relevance.
Methods
Using phorbol myristate acetate (PMA)/ionomycin and anti-CD3 activation of CD4+CD25- cells we generated in vitro functional CD4+CD25+ iTreg (TregPMA) cells. Functionality of the generated TregPMA cells was tested in vivo in a mouse model of inflammatory bowel disease (IBD) - CD45RB transfer colitis model.
Results
TregPMA cells expressed regulatory markers and proved to ameliorate the disease phenotype in murine CD45RB transfer colitis model. The body weight loss and disease activity scores for TregPMA treated mice were reduced when compared to diseased control group. Histological assessment of colon sections confirmed amelioration of the disease phenotype. Additionally, cytokine analysis showed decreased levels of proinflammatory colonic and plasma IL-6, colonic IL-1 β and higher levels of colonic IL-17 when compared to diseased control group.
Conclusions
This study identifies a new method to generate in vitro iTreg cells (TregPMA cells) which physiological efficacy has been demonstrated in vivo.
doi:10.1186/1471-230X-12-172
PMCID: PMC3536706  PMID: 23198878
IBD; CD45RB transfer; Treg; PMA/ionomycin
21.  Foxp3+ Inducible Regulatory T cells Suppress Endothelial Activation and Leukocyte Recruitment1 
The ability of regulatory T cells (Treg) to traffic to sites of inflammation supports their role in controlling immune responses. This feature supports the idea that adoptive transfer of in vitro expanded human Treg could be used for treatment of immune/inflammatory diseases. However, the migratory behavior of Treg as well as their direct influence at the site of inflammation remains poorly understood. To explore the possibility that Treg may have direct anti-inflammatory influences on tissues, independent of their well-established suppressive effects on lymphocytes, we studied the adhesive interactions between mouse Treg and endothelial cells, as well as their influence on endothelial function during acute inflammation. We show that Foxp3+ adaptive/inducible Treg (iTreg) but not naturally occurring Treg (nTreg) efficiently interact with endothelial selectins and transmigrate through endothelial monolayers in vitro. In response to activation by endothelial antigen presentation or immobilized anti-CD3ε, Foxp3+ iTreg suppressed TNFα and IL-1β mediated endothelial selectin expression and adhesiveness to effector T cells. This suppression was contact independent, rapid acting, and mediated by TGFβ-induced activin receptor like kinase [ALK]5 signaling in endothelial cells. In addition, Foxp3+ iTreg adhered to inflamed endothelium in vivo and their secretion products blocked acute inflammation in a model of peritonitis. These data support the concept that Foxp3+ iTreg help to regulate inflammation independently of their influence on effector T cells by direct suppression of endothelial activation and leukocyte recruitment.
doi:10.4049/jimmunol.1003947
PMCID: PMC3217244  PMID: 21873519
T cells; adhesion molecules; endothelial cells; inflammation; suppression
22.  Efficient induction of CD25- iTreg by co-immunization requires strongly antigenic epitopes for T cells 
BMC Immunology  2011;12:27.
Background
We previously showed that co-immunization with a protein antigen and a DNA vaccine coding for the same antigen induces CD40low IL-10high tolerogenic DCs, which in turn stimulates the expansion of antigen-specific CD4+CD25-Foxp3+ regulatory T cells (CD25- iTreg). However, it was unclear how to choose the antigen sequence to maximize tolerogenic antigen presentation and, consequently, CD25- iTreg induction.
Results
In the present study, we demonstrated the requirement of highly antigenic epitopes for CD25- iTreg induction. Firstly, we showed that the induction of CD25- iTreg by tolerogenic DC can be blocked by anti-MHC-II antibody. Next, both the number and the suppressive activity of CD25- iTreg correlated positively with the overt antigenicity of an epitope to activate T cells. Finally, in a mouse model of dermatitis, highly antigenic epitopes derived from a flea allergen not only induced more CD25- iTreg, but also more effectively prevented allergenic reaction to the allergen than did weakly antigenic epitopes.
Conclusions
Our data thus indicate that efficient induction of CD25- iTreg requires highly antigenic peptide epitopes. This finding suggests that highly antigenic epitopes should be used for efficient induction of CD25- iTreg for clinical applications such as flea allergic dermatitis.
doi:10.1186/1471-2172-12-27
PMCID: PMC3107816  PMID: 21542943
23.  Tolerogenic Splenic IDO+ Dendritic Cells from the Mice Treated with Induced-Treg Cells Suppress Collagen-Induced Arthritis 
Journal of Immunology Research  2014;2014:831054.
TGF-β-induced regulatory T cells (iTregs) retain Foxp3 expression and immune-suppressive activity in collagen-induced arthritis (CIA). However, the mechanisms whereby transferred iTregs suppress immune responses, particularly the interplay between iTregs and dendritic cells (DCs) in vivo, remain incompletely understood. In this study, we found that after treatment with iTregs, splenic CD11c+DCs, termed “DCiTreg,” expressed tolerogenic phenotypes, secreted high levels of IL-10, TGF-β, and IDO, and showed potent immunosuppressive activity in vitro. After reinfusion with DCiTreg, marked antiarthritic activity improved clinical scores and histological end-points were observed. The serological levels of inflammatory cytokines and anti-CII antibodies were low and TGF-β production was high in the DCiTreg-treated group. DCiTreg also induced new iTregs in vivo. Moreover, the inhibitory activity of DCiTreg on CIA was lost following pretreatment with the inhibitor of indoleamine 2,3-dioxygenase (IDO). Collectively, these findings suggest that transferred iTregs could induce tolerogenic characteristics in splenic DCs and these cells could effectively dampen CIA in an IDO-dependent manner. Thus, the potential therapeutic effects of iTregs in CIA are likely maintained through the generation of tolerogenic DCs in vivo.
doi:10.1155/2014/831054
PMCID: PMC4227353  PMID: 25405209
24.  A Central Role for Induced Regulatory T Cells in Tolerance Induction in Experimental Colitis1 
In addition to thymus-derived or natural T regulatory (nTreg) cells, a second subset of induced T regulatory (iTreg) cells arises de novo from conventional CD4+ T cells in the periphery. The function of iTreg cells in tolerance was examined in a CD45RBhighCD4+ T cell transfer model of colitis. In situ-generated iTreg cells were similar to nTreg cells in their capacity to suppress T cell proliferation in vitro and their absence in vivo accelerated bowel disease. Treatment with nTreg cells resolved the colitis, but only when iTreg cells were also present. Although iTreg cells required Foxp3 for suppressive activity and phenotypic stability, their gene expression profile was distinct from the established nTreg “genetic signature,” indicative of developmental and possibly mechanistic differences. These results identified a functional role for iTreg cells in vivo and demonstrated that both iTreg and nTreg cells can act in concert to maintain tolerance.
doi:10.4049/jimmunol.0802535
PMCID: PMC2763205  PMID: 19265124
25.  Progranulin Facilitates Conversion and Function of Regulatory T Cells under Inflammatory Conditions 
PLoS ONE  2014;9(11):e112110.
The progranulin (PGRN) is known to protect regulatory T cells (Tregs) from a negative regulation by TNF-α, and its levels are elevated in various kinds of autoimmune diseases. Whether PGRN directly regulates the conversion of CD4+CD25-T cells into Foxp3-expressing regulatory T cells (iTreg), and whether PGRN affects the immunosuppressive function of Tregs, however, remain unknown. In this study we provide evidences demonstrating that PGRN is able to stimulate the conversion of CD4+CD25-T cells into iTreg in a dose-dependent manner in vitro. In addition, PGRN showed synergistic effects with TGF-β1 on the induction of iTreg. PGRN was required for the immunosuppressive function of Tregs, since PGRN-deficient Tregs have a significant decreased ability to suppress the proliferation of effector T cells (Teff). In addition, PGRN deficiency caused a marked reduction in Tregs number in the course of inflammatory arthritis, although no significant difference was observed in the numbers of Tregs between wild type and PGRN deficient mice during development. Furthermore, PGRN deficiency led to significant upregulation of the Wnt receptor gene Fzd2. Collectively, this study reveals that PGRN directly regulates the numbers and function of Tregs under inflammatory conditions, and provides new insight into the immune regulatory mechanism of PGRN in the pathogenesis of inflammatory and immune-related diseases.
doi:10.1371/journal.pone.0112110
PMCID: PMC4230946  PMID: 25393765

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