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Due to their suppressive capacity, regulatory T cells (Tregs) have attracted growing interest as an adoptive cellular therapy for the prevention of allograft rejection, but limited Treg recovery and lower quality of adult-derived Tregs could represent an obstacle to success. To address this challenge, we developed a new approach that provides large quantities of Tregs with high purity and excellent features, sourced from thymic tissue routinely removed during pediatric cardiac surgeries (thyTregs). We report on a 2-year follow-up of the first patient treated worldwide with thyTregs, included in a phase I/II clinical trial evaluating the administration of autologous thyTreg in infants undergoing heart transplantation. In addition to observing no adverse effects that could be attributed to thyTreg administration, we report that the Treg frequency in the periphery was preserved during the 2-year follow-up period. These initial results are consistent with the trial objective, which is to confirm safety of the autologous thyTreg administration and its capacity to restore the Treg pool., (© 2023 Bernaldo-de-Quirós et al.)

Regulatory T cells (Tregs) are essential for immune homeostasis and suppression of pathological autoimmunity but can also play a detrimental role in cancer progression via inhibition of anti-tumor immunity. Thus, there is broad applicability for therapeutic Treg targeting, either to enhance function, for example, through adoptive cell therapy (ACT), or to inhibit function with small molecules or antibody-mediated blockade. For both of these strategies, the metabolic state of Tregs is an important consideration since cellular metabolism is intricately linked to function. Mounting evidence has shown that targeting metabolic pathways can selectively promote or inhibit Treg function. This review aims to synthesize the current understanding of Treg metabolism and discuss emerging metabolic targeting strategies in the contexts of transplantation, autoimmunity, and cancer. We discuss approaches to gene editing and cell culture to manipulate Treg metabolism during ex vivo expansion for ACT, as well as in vivo nutritional and pharmacological interventions to modulate Treg metabolism in disease states. Overall, the intricate connection between metabolism and phenotype presents a powerful opportunity to therapeutically tune Treg function., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Background: Acute cellular rejection (ACR), an alloimmune response involving CD4+ and CD8+ T cells, occurs in up to 20% of patients within the first year following heart transplantation. The balance between a conventional versus regulatory CD4+ T cell alloimmune response is believed to contribute to developing ACR. Therefore, tracking these cells may elucidate whether changes in these cell populations could signal ACR risk., Methods: We used a CD4+ T cell gene signature (TGS) panel that tracks CD4+ conventional T cells (Tconv) and regulatory T cells (Treg) on longitudinal samples from 94 adult heart transplant recipients. We evaluated combined diagnostic performance of the TGS panel with a previously developed biomarker panel for ACR diagnosis, HEARTBiT, while also investigating TGS' prognostic utility., Results: Compared with nonrejection samples, rejection samples showed decreased Treg- and increased Tconv-gene expression. The TGS panel was able to discriminate between ACR and nonrejection samples and, when combined with HEARTBiT, showed improved specificity compared with either model alone. Furthermore, the increased risk of ACR in the TGS model was associated with lower expression of Treg genes in patients who later developed ACR. Reduced Treg gene expression was positively associated with younger recipient age and higher intrapatient tacrolimus variability., Conclusions: We demonstrated that expression of genes associated with CD4+ Tconv and Treg could identify patients at risk of ACR. In our post hoc analysis, complementing HEARTBiT with TGS resulted in an improved classification of ACR. Our study suggests that HEARTBiT and TGS may serve as useful tools for further research and test development., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

The metabolic milieu is emerging as a major contributing factor in the maintenance of the immunosuppressive microenvironment within tumors. In particular, the presence of lactic acid produced by highly glycolytic cancer cells is known to suppress antitumor immune cell subsets while promoting immunosuppressive cell populations, such as regulatory T cells (Tregs). Unlike conventional T cells, Tregs have a unique, potent ability to take up lactic acid to fuel both mitochondrial metabolism and gluconeogenesis, thus supporting suppressive function and proliferation. In this issue of the European Journal of Immunology [Eur. J. Immunol. 2023.53:2250258], Rao et al. uncover a novel mechanism by which lactic acid can support Treg accumulation within tumors in mice. This study shows that lactic acid, through a pH-dependent mechanism rather than lactate itself, promotes TGFβ-induced differentiation of Tregs from conventional CD4 + T cells. These findings build on the already multifaceted role of lactic acid in maintaining an immunosuppressive tumor microenvironment., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Regulatory T-cell (Treg) therapy has shown promise in treating autoimmune diseases, transplant rejection, or graft-versus-host disease in early clinical trials. These trials have demonstrated that cell therapy using polyclonal Tregs is feasible and safe, however, the field has been limited by the lack of polyclonal cell specificity and consequent large cell numbers required, and the difficulty in generating autologous products for some patients. Thus, the field is moving toward 'next generation' Treg cell therapies that include genetic modification strategies to engineer specificity and/or modify function, as well as methods to generate Tregs in vitro. In this review, we describe how genetic modification of Tregs using viral transduction or gene editing may be incorporated into Treg manufacturing protocols. We also describe how Tregs may be generated via FOXP3 gene editing or overexpression, or by differentiation from pluripotent stem cells. The application of these various types of engineered Tregs is discussed., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Given the promising results from phase 1/2 clinical trials of therapy involving regulatory T cells (Tregs), it is critical to develop Treg manufacturing methods that use well-defined reagents., Methods: Seeking to maximize expansion of human thymic Tregs activated with anti-CD3/CD28 antibody-coated beads and cultured in serum-free medium, the authors investigated the effect of adjusting process parameters including cell density and cell concentration, and feeding strategy on Treg yield and quality., Results: The authors found that levels of expansion and viability varied with cell density on the day of restimulation. Tregs restimulated at low cell densities (1 × 10 5 cells/cm 2 ) initially had high growth rates, viability and FOXP3 expression, but these parameters decreased with time and were less stable than those observed in cultures of Tregs restimulated at high cell densities (5 × 10 5 cells/cm 2 ), which had slower growth rates. High-density expansion was associated with expression of inhibitory molecules and lower intracellular oxygen and extracellular nutrient concentrations as well as extracellular lactate accumulation. Experiments to test the effect of low oxygen revealed that transient exposure to low oxygen levels had little impact on expansion, viability or phenotype. Similarly, blockade of inhibitory molecules had little effect. By contrast, replenishing nutrients by increasing the feeding frequency between 2 days and 4 days after restimulation increased FOXP3, viability and expansion in high-density cultures., Conclusion: These data show the previously undescribed consequences of adjusting cell density on Treg expansion and establish a Good Manufacturing Practice-relevant protocol using non-cell-based activation reagents and serum-free media that supports sustained expansion without loss of viability or phenotype., (Copyright © 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Regulatory T-cell (Treg) therapy is under clinical investigation for the treatment of transplant rejection, autoimmune disease, and graft-versus-host disease. With the advent of genome editing, attention has turned to reinforcing Treg function for therapeutic benefit. A hallmark of Tregs is dampened activation of PI3K-AKT signaling, of which PTEN is a major negative regulator. Loss-of-function studies of PTEN, however, have not conclusively shown a requirement for PTEN in upholding Treg function and stability. Using CRISPR-based genome editing in human Tregs, we show that PTEN ablation does not cause a global defect in Treg function and stability; rather, it selectively blocks their ability to suppress antigen-presenting cells. PTEN-KO Tregs exhibit elevated glycolytic activity, upregulate FOXP3, maintain a Treg phenotype, and have no discernible defects in lineage stability. Functionally, PTEN is dispensable for human Treg-mediated inhibition of T-cell activity in vitro and in vivo but is required for suppression of costimulatory molecule expression by antigen-presenting cells. These data are the first to define a role for a signaling pathway in controlling a subset of human Treg activity. Moreover, they point to the functional necessity of PTEN-regulated PI3K-AKT activity for optimal human Treg function., (© 2022 Wiley-VCH GmbH.)

Treg therapy holds promise as a potentially curative approach to establish immune tolerance in transplantation and autoimmune disease. An outstanding question is whether therapeutic Tregs have the potential to transdifferentiate into effector T-cells and, thus, exacerbate rather than suppress immune responses. In mice, the transcription factor Helios is thought to promote Treg lineage stability in a range of inflammatory contexts. In humans, the role of Helios in Tregs is less clear, in part, due to the inability to enrich and study subsets of Helios-positive versus Helios-negative Tregs. Using an in vitro expansion system, we found that loss of high Helios expression and emergence of an intermediate Helios (Helios mid )-expressing population correlated with Treg destabilization. We used CRISPR/Cas9 to genetically ablate Helios expression in human naive or memory Tregs and found that Helios-KO and unedited Tregs were equivalent in their suppressive function and stability in inflammation. Thus, high Helios expression is a marker, but not a driver, of human Treg stability in vitro. These data highlight the importance of monitoring Helios expression in therapeutic Treg manufacturing and provide new insight into the biological function of this transcription factor in human T-cells., (© 2021 Wiley-VCH GmbH.)

Gene editing of living cells is a cornerstone of present-day medical research that has enabled scientists to address fundamental biologic questions and identify novel strategies to treat diseases. The ability to manipulate adoptive cell therapy products has revolutionized cancer immunotherapy and promises similar results for the treatment of autoimmune diseases, inflammatory disorders, and transplant rejection. Clinical trials have recently deemed polyclonal regulatory T (Treg) cell therapy to be a safe therapeutic option, but questions remain regarding the efficacy of this approach. In this review, we discuss how gene editing technologies are being applied to transform the future of Treg cell therapy, focusing on the preclinical strategies that are currently being investigated to enhance the efficacy, function, and survival of human Treg cells. We explore approaches that may be used to generate immunoregulatory cells ex vivo, detail emerging strategies that are being used to modify these cells (such as using chimeric antigen receptors to confer antigen specificity), and outline concepts that have been explored to repurpose conventional T cells to target and destroy autoreactive and alloreactive lymphocytes. We also describe the key hurdles that currently hinder the clinical adoption of Treg cell therapy and propose potential future avenues of research for this field., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Type 1 diabetes results from defects in immune self-tolerance that lead to inflammatory infiltrate in pancreatic islets, beta cell dysfunction and T cell-mediated killing of beta cells. Although therapies that broadly inhibit immunity show promise to mitigate autoinflammatory damage caused by effector T cells, these are unlikely to permanently reset tolerance or promote regeneration of the already diminished pool of beta cells. An emerging concept is that certain populations of immune cells may have the capacity to both promote tolerance and support the restoration of beta cells by supporting proliferation, differentiation and/or regeneration. Here we will highlight three immune cell types-macrophages, regulatory T cells and innate lymphoid cells-for which there is evidence of dual roles of immune regulation and tissue regeneration. We explore how findings in this area from other fields might be extrapolated to type 1 diabetes and highlight recent discoveries in the context of type 1 diabetes. We also discuss technological advances that are supporting this area of research and contextualise new therapeutic avenues to consider for type 1 diabetes., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Regulatory T cell (Treg) therapy is a promising curative approach for a variety of immune-mediated conditions. CRISPR-based genome editing allows precise insertion of transgenes through homology-directed repair, but its use in human Tregs has been limited. We report an optimized protocol for CRISPR-mediated gene knockin in human Tregs with high-yield expansion. To establish a benchmark of human Treg dysfunction, we target the master transcription factor FOXP3 in naive and memory Tregs. Although FOXP3-ablated Tregs upregulate cytokine expression, effects on suppressive capacity in vitro manifest slowly and primarily in memory Tregs. Moreover, FOXP3-ablated Tregs retain their characteristic protein, transcriptional, and DNA methylation profile. Instead, FOXP3 maintains DNA methylation at regions enriched for AP-1 binding sites. Thus, although FOXP3 is important for human Treg development, it has a limited role in maintaining mature Treg identity. Optimized gene knockin with human Tregs will enable mechanistic studies and the development of tailored, next-generation Treg cell therapies., Competing Interests: Declaration of interests M.K.L. received research funding from Sangamo Therapeutics, Bristol-Myers Squibb, Pfizer, Takeda, and CRISPR Therapeutics for work unrelated to this study. All other authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Adoptive cell therapy with genetically modified regulatory T cells (Tregs) is under clinical investigation for the treatment of transplant rejection and various autoimmune conditions. A limitation of modelling this approach in mice is the lack of optimized protocols for expanding and transducing mouse Tregs. Here we describe a protocol for purifying, expanding and retrovirally transducing mouse Tregs with a vector encoding a chimeric antigen receptor as a model transgene. We found that isolation of Tregs from C57Bl/6J Foxp3 E GFP mice solely based on eGFP expression resulted in sufficiently pure cells; co-sorting of CD25 hi cells was not essential. Although expansion with rapamycin reduced Treg expansion, it promoted maximal in vitro suppressive activity. Retroviral transduction of Tregs following 2 days of stimulation with anti-CD3/CD28 beads achieved a transduction efficiency of ~40% and did not impair their suppressive capacity. When injected into a conventional T cell (Tconv)-transfer-induced colitis model, transduced Tregs inhibited colitis progression at ratios as low as 1 Treg to 100 Tconvs, and maintained Foxp3 and transgene expression throughout an 8-week period. This method facilitates the study of transduced Tregs in animal models and will enable the study of genetically engineered Treg therapy for a variety of inflammatory diseases., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Regulatory T cells (Tregs) have a critical role in maintaining self-tolerance and immune homeostasis. There is much interest in using Tregs as a cell therapy to re-establish tolerance in conditions such as inflammatory bowel disease and type 1 diabetes, with many ongoing clinical studies testing the safety and efficacy of this approach. Manufacturing of Tregs for therapy typically involves ex vivo expansion to obtain sufficient cell numbers for infusion and comes with the risk of altering the activity of key biological processes. However, this process also offers an opportunity to tailor Treg function to maximize in vivo activity. In this review, we focus on the roles of antigen-presenting cells (APCs) in the generation and function of Tregs in humans. In addition to stimulating the development of Tregs, APCs activate Tregs and provide signals that induce specialized functional and homing marker expression. Cross talk between Tregs and APCs is a critical, often under-appreciated, aspect of Treg biology, with APCs mediating the key properties of infectious tolerance and bystander suppression. Understanding the biology of human Treg-APC interactions will reveal new ways to optimize Treg-based therapeutic approaches., (© 2020 Wiley-VCH GmbH.)

Regulatory T cells (Tregs) control immune homeostasis and prevent exacerbated immune responses, and can be used as cell therapy to dampen a variety of autoimmune or autoinflammatory responses. Treg therapy is significantly more effective if the cells are antigen-specific. One way to re-direct the specificity of Tregs is to engineer them to express a Chimeric Antigen Receptor (CAR). Proof-of-concept studies have shown the potential for "basic" models of CAR-Tregs to be used as cellular therapy in autoimmunity, organ transplantation and hematopoietic stem cell transplantation. In parallel, work in the context of cancer has significantly advanced knowledge of how to optimise CAR-T cell structure and function for more precise and potent function. In this review, we summarize the current state of knowledge about important considerations when generating CAR-Tregs. We also extrapolate from emerging findings with CAR-T cells about strategies to further improve CAR-Treg function, creating "luxury" models with refined activity., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Inflammatory bowel diseases (IBD) are chronic conditions that result from uncontrolled intestinal inflammation. Pathogenic Th17 cells, characterized by production of IL-17A in the absence of IL-10, are thought to contribute to this inflammation, but in humans, antibody-mediated blockade of IL-17A is an ineffective IBD therapy whereas IL-23 blockade is effective. Here, we investigated the effects of pharmacological inhibition of RORC2, the Th17 cell lineage-defining transcription factor, on in vivo-differentiated human Th17 cells and Th17-like Tregs (Th17-Tregs). BMS-336, a small molecule RORC2 inverse agonist, inhibited expression of RORC2-regulated genes in peripheral Th17 cells (CD4 + CD25 - CD127 + CXCR3 - CCR4 + CCR6 + ) in a dose-dependent manner, with similar inhibitory effects on laminar propria mononuclear cells from IBD and non-IBD subjects. Exposure of peripheral Th17-Tregs (CD4 + CD25 hi CD127 lo CXCR3 - CCR4 + CCR6 + ) to BMS-336 also inhibited IL-17A production and prevented inflammatory cytokine-induced destabilization, as evidenced by preserved FOXP3 expression and epigenetic status of the Treg-specific demethylation region. In parallel, RORC2 inhibition increased the production of IL-10 in Th17-Tregs, resulting in enhanced suppression of inflammatory cytokines from myeloid cells. Thus, via its ability to simultaneously inhibit Th17 cells and enhance the stability and function of Th17-Tregs, pharmacological inhibition of RORC2 is a promising approach to suppress inflammation and promote immune regulation in IBD., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Adipose tissue (AT) regulatory T cells (T regs) control inflammation and metabolism. Diet-induced obesity causes hyperinsulinemia and diminishes visceral AT (VAT) T reg number and function, but whether these two phenomena were mechanistically linked was unknown. Using a T reg-specific insulin receptor (Insr) deletion model, we found that diet-induced T reg dysfunction is driven by T reg-intrinsic insulin signaling. Compared with Foxp3cre mice, after 13 wk of high-fat diet, Foxp3creInsrfl/fl mice exhibited improved glucose tolerance and insulin sensitivity, effects associated with lower AT inflammation and increased numbers of ST2+ T regs in brown AT, but not VAT. Similarly, Foxp3creInsrfl/fl mice were protected from the metabolic effects of aging, but surprisingly had reduced VAT T regs and increased VAT inflammation compared with Foxp3cre mice. Thus, in both diet- and aging-associated hyperinsulinemia, excessive Insr signaling in T regs leads to undesirable metabolic outcomes. Ablation of Insr signaling in T regs represents a novel approach to mitigate the detrimental effects of hyperinsulinemia on immunoregulation of metabolic syndrome., Competing Interests: Disclosures: M. Levings reported grants from TxCell SpA, grants from Pfizer, grants from Takeda, grants from CRISPR Therapeutics, and grants from Bristol-Myers Squibb outside the submitted work. No other disclosures were reported., (© 2020 Wu et al.)

Regulatory T (Treg) cell-specific deletion of a gene of interest is a procedure widely used to study mechanisms controlling Treg development, homeostasis and function. Accordingly, several transgenic mouse lines have been generated that bear the Cre recombinase under control of the Foxp3 promoter either as a random transgene insertion or knocked into the endogenous Foxp3 locus, with the Foxp3 YFP-Cre strain of mice being one of the most widely used. In an attempt to generate Treg cells that lacked expression of the insulin receptor (Insr), we crossed Foxp3 YFP-Cre mice with Insr fl/fl mice. Using a conventional two-band PCR genotyping method we found that offspring genotypes did not correspond to the expected Mendelian ratios. We therefore developed a quantitative PCR-based genotyping method to investigate possible ectopic recombination outside the Treg lineage. With this method we found that ~50% of the F 1 -generation mice showed evidence of ectopic recombination and that ~10% of the F 2 -generation mice had germline Cre recombination activity leading to a high frequency of offspring with global Insr deletion. Use of the quantitative PCR genotyping method enabled accurate selection of mice without ectopic recombination and only the desired Treg cell-specific Insr deletion. Our data highlight the need to use genotyping methods that allow for assessment of possible ectopic recombination driven by the Foxp3 YFP-Cre allele, particularly when studying genes that are systemically expressed., (© 2019 John Wiley & Sons Ltd.)

Background & Aims: T-regulatory (Treg) cells suppress the immune response to maintain homeostasis. There are 2 main subsets of Treg cells: FOXP3 (forkhead box protein 3)-positive Treg cells, which do not produce high levels of effector cytokines, and type 1 Treg (Tr1) cells, which are FOXP3-negative and secrete interleukin (IL) 10. IL10 is an anti-inflammatory cytokine, so Tr1 cells might be used in the treatment of inflammatory bowel diseases. We aimed to develop methods to isolate and expand human Tr1 cells and define their functions., Methods: We obtained blood and colon biopsy samples from patients with Crohn's disease or ulcerative colitis or healthy individuals (controls). CD4 + T cells were isolated from blood samples and stimulated with anti-CD3 and anti-CD28 beads, and Tr1 cells were purified by using an IL10 cytokine-capture assay and cell sorting. FOXP3-positive Treg cells were sorted as CD4 + CD25 high CD127 low cells from unstimulated cells. Tr1 and FOXP3-positive Treg cells were expanded, and phenotypes and gene expression profiles were compared. T cells in peripheral blood mononuclear cells from healthy donors were stimulated with anti-CD3 and anti-CD28 beads, and the suppressive abilities of Tr1 and FOXP3-positive Treg cells were measured. Human colon organoid cultures were established, cultured with supernatants from Tr1 or FOXP3-positive cells, and analyzed by immunofluorescence and flow cytometry. T84 cells (human colon adenocarcinoma epithelial cells) were incubated with supernatants from Tr1 or FOXP3-positive cells, and transepithelial electrical resistance was measured to determine epithelial cell barrier function., Results: Phenotypes of Tr1 cells isolated from control individuals vs patients with Crohn's disease or ulcerative colitis did not differ significantly after expansion. Tr1 cells and FOXP3-positive Treg cells suppressed proliferation of effector T cells, but only Tr1 cells suppressed secretion of IL1B and tumor necrosis factor from myeloid cells. Tr1 cells, but not FOXP3-positive Treg cells, isolated from healthy individuals and patients with Crohn's disease or ulcerative colitis secreted IL22, which promoted barrier function of human intestinal epithelial cells. Tr1 cell culture supernatants promoted differentiation of mucin-producing goblet cells in intestinal organoid cultures., Conclusions: Human Tr1 cells suppress proliferation of effector T cells (adaptive immune response) and production of IL1B and TNF by myeloid cells (inmate immune response). They also secrete IL22 to promote barrier function. They might be developed as a cell-based therapy for intestinal inflammatory disorders., (Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Regulatory T cells (Tregs) are a promising therapy for several immune-mediated conditions but manufacturing a homogeneous and consistent product, especially one that includes cryopreservation, has been challenging. Discarded pediatric thymuses are an excellent source of therapeutic Tregs with advantages including cell quantity, homogeneity and stability. Here we report systematic testing of activation reagents, cell culture media, restimulation timing and cryopreservation to develop a Good Manufacturing Practice (GMP)-compatible method to expand and cryopreserve Tregs. By comparing activation reagents, including soluble antibody tetramers, antibody-conjugated beads and artificial antigen-presenting cells (aAPCs) and different media, we found that the combination of Dynabeads Treg Xpander and ImmunoCult-XF medium preserved FOXP3 expression and suppressive function and resulted in expansion that was comparable with a single stimulation with aAPCs. Cryopreservation tests revealed a critical timing effect: only cells cryopreserved 1-3 days, but not >3 days, after restimulation maintained high viability and FOXP3 expression upon thawing. Restimulation timing was a less critical process parameter than the time between restimulation and cryopreservation. This systematic testing of key variables provides increased certainty regarding methods for in vitro expansion and cryopreservation of Tregs. The ability to cryopreserve expanded Tregs will have broad-ranging applications including enabling centralized manufacturing and long-term storage of cell products., (Copyright © 2019 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Little is known about how Treg cells mediate suppression, especially in tissues. In this issue of Immunity, Schmidleithner et al. report that Treg cells catabolize PGE 2 into 15-keto-PGE 2 , a T cell-inhibitory metabolite, and that this mechanism controls inflammation in adipose tissue., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Regulatory T cell (Treg) therapy is a potential curative approach for a variety of immune-mediated conditions, including autoimmunity and transplantation, in which there is pathological tissue damage. In mice, IL-33R (ST2)-expressing Tregs mediate tissue repair by producing the growth factor amphiregulin, but whether similar tissue-reparative Tregs exist in humans remains unclear. We show that human Tregs in blood and multiple tissue types produced amphiregulin, but this was neither a unique feature of Tregs nor selectively upregulated in tissues. Human Tregs in blood, tonsil, synovial fluid, colon, and lung tissues did not express ST2, so ST2 + Tregs were engineered via lentiviral-mediated overexpression, and their therapeutic potential for cell therapy was examined. Engineered ST2 + Tregs exhibited TCR-independent, IL-33-stimulated amphiregulin expression and a heightened ability to induce M2-like macrophages. The finding that amphiregulin-producing Tregs have a noneffector phenotype and are progressively lost upon TCR-induced proliferation and differentiation suggests that the tissue repair capacity of human Tregs may be an innate function that operates independently from their classical suppressive function., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Background: Multiple therapeutic strategies to restore immune regulation and slow type 1 diabetes (T1D) progression are in development and testing. A major challenge has been defining biomarkers to prospectively identify subjects likely to benefit from immunotherapy and/or measure intervention effects. We previously found that, compared with healthy controls, Tregs from children with new-onset T1D have an altered Treg gene signature (TGS), suggesting that this could be an immunoregulatory biomarker., Methods: nanoString was used to assess the TGS in sorted Tregs (CD4+CD25hiCD127lo) or peripheral blood mononuclear cells (PBMCs) from individuals with T1D or type 2 diabetes, healthy controls, or T1D recipients of immunotherapy. Biomarker discovery pipelines were developed and applied to various sample group comparisons., Results: Compared with controls, the TGS in isolated Tregs or PBMCs was altered in adult new-onset and cross-sectional T1D cohorts, with sensitivity or specificity of biomarkers increased by including T1D-associated SNPs in algorithms. The TGS was distinct in T1D versus type 2 diabetes, indicating disease-specific alterations. TGS measurement at the time of T1D onset revealed an algorithm that accurately predicted future rapid versus slow C-peptide decline, as determined by longitudinal analysis of placebo arms of START and T1DAL trials. The same algorithm stratified participants in a phase I/II clinical trial of ustekinumab (αIL-12/23p40) for future rapid versus slow C-peptide decline., Conclusion: These data suggest that biomarkers based on measuring TGSs could be a new approach to stratify patients and monitor autoimmune activity in T1D., Funding: JDRF (1-PNF-2015-113-Q-R, 2-PAR-2015-123-Q-R, 3-SRA-2016-209-Q-R, 3-PDF-2014-217-A-N), the JDRF Canadian Clinical Trials Network, the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (UM1AI109565 and FY15ITN168), and BCCHRI.

Chimeric antigen receptor (CAR) technology can be used to engineer the antigen specificity of regulatory T cells (Tregs) and improve their potency as an adoptive cell therapy in multiple disease models. As synthetic receptors, CARs carry the risk of immunogenicity, particularly when derived from nonhuman antibodies. Using an HLA-A*02:01-specific CAR (A2-CAR) encoding a single-chain variable fragment (Fv) derived from a mouse antibody, we developed a panel of 20 humanized A2-CARs (hA2-CARs). Systematic testing demonstrated variations in expression, and ability to bind HLA-A*02:01 and stimulate human Treg suppression in vitro. In addition, we developed a new method to comprehensively map the alloantigen specificity of CARs, revealing that humanization reduced HLA-A cross-reactivity. In vivo bioluminescence imaging showed rapid trafficking and persistence of hA2-CAR Tregs in A2-expressing allografts, with eventual migration to draining lymph nodes. Adoptive transfer of hA2-CAR Tregs suppressed HLA-A2+ cell-mediated xenogeneic graft-versus-host disease and diminished rejection of human HLA-A2+ skin allografts. These data provide a platform for systematic development and specificity testing of humanized alloantigen-specific CARs that can be used to engineer specificity and homing of therapeutic Tregs.

Obesity-associated visceral adipose tissue (AT) inflammation promotes insulin resistance and type 2 diabetes (T2D). In mice, lean visceral AT is populated with anti-inflammatory cells, notably regulatory T cells (Tregs) expressing the IL-33 receptor ST2. Conversely, obese AT contains fewer Tregs and more proinflammatory cells. In humans, however, there is limited evidence for a similar pattern of obesity-associated immunomodulation. We used flow cytometry and mRNA quantification to characterize human omental AT in 29 obese subjects, 18 of whom had T2D. Patients with T2D had increased proportions of inflammatory cells, including M1 macrophages, with positive correlations to body mass index. In contrast, Treg frequencies negatively correlated to body mass index but were comparable between T2D and non-T2D individuals. Compared to human thymic Tregs, omental AT Tregs expressed similar levels of FOXP3, CD25, IKZF2, and CTLA4, but higher levels of PPARG, CCR4, PRDM1, and CXCL2. ST2, however, was not detectable on omental AT Tregs from lean or obese subjects. This is the first comprehensive investigation into how omental AT immunity changes with obesity and T2D in humans, revealing important similarities and differences to paradigms in mice. These data increase our understanding of how pathways of immune regulation could be targeted to ameliorate AT inflammation in humans., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose of Review: Adoptive cell therapy using CD4FOXP3 regulatory T cells (Treg) has emerged as a promising therapeutic strategy to treat autoimmunity and alloimmunity. Preclinical studies suggest that the efficacy of Treg therapy can be improved by modifying the antigen specificity, stability and function of therapeutic Tregs. We review recent innovations that considerably enhance the possibilities of controlling these parameters., Recent Findings: Antigen-specific Tregs can be generated by genetically modifying polyclonal Tregs to express designated T-cell receptors or single-chain chimeric antigen receptors. The benefits of this approach can be further extended by using novel strategies to fine-tune the antigen-specificity and affinity of Treg in vivo. CRISPR/Cas 9 technology now enables the modification of therapeutic Tregs so they are safer, more stable and long lived. The differentiation and homing properties of Tregs can also be modulated by gene editing or modifying ex-vivo stimulation conditions., Summary: A new wave of innovation has considerably increased the number of strategies that could be used to increase the therapeutic potential of Treg therapy. However, the increased complexity of these approaches may limit their wide accessibility. Third-party therapy with off-the-shelf Treg products could be a solution.

We optimized a method to detect FOXP3 by mass cytometry and compared the resulting data to conventional flow cytometry. We also demonstrated the utility of the protocol to profile antigen-specific Tregs from whole blood, or Tregs from tissues such as cord blood, thymus and synovial fluid., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose of Review: The application of regulatory T cell (Treg) therapy in organ transplantation is actively being pursued using unmodified, typically polyclonal cells. As the results of these ongoing clinical trials emerge, it is time to plan the next wave of clinical trials of Tregs. Here we will review a key strategy to improve Treg effectiveness and reduce side effects, namely increasing Treg specificity - both in terms of antigen recognition and localization to the allograft., Recent Findings: Study of chemokine signatures accompanying acute rejection has revealed several chemokines that could be targeted to increase Treg homing. For example, Tregs possessing a Th1-like phenotype and expressing CXCR3 are better able to migrate towards local inflammation. Allografts themselves can be modified to increase Treg-attracting chemokines and Tregs themselves can produce chemokines, facilitating local proximity to their targets of suppression. Finally, tailoring Treg antigen specificity by T-cell or chimeric antigen receptor engineering is another approach to increase the specificity of suppression and optimize localization., Summary: Treg localization to the graft is important, but the important role of lymph node and germinal center homing cannot be overlooked. There is an opportunity to learn from advances made in cancer immunotherapy to optimize Treg therapy for transplantation.

Background: Celiac disease is a chronic immune-mediated inflammatory disorder of the gut triggered by dietary gluten. Although the effector T-cell response in patients with celiac disease has been well characterized, the role of regulatory T (Treg) cells in the loss of tolerance to gluten remains poorly understood., Objective: We sought to define whether patients with celiac disease have a dysfunction or lack of gluten-specific forkhead box protein 3 (FOXP3) + Treg cells., Methods: Treated patients with celiac disease underwent oral wheat challenge to stimulate recirculation of gluten-specific T cells. Peripheral blood was collected before and after challenge. To comprehensively measure the gluten-specific CD4 + T-cell response, we paired traditional IFN-γ ELISpot with an assay to detect antigen-specific CD4 + T cells that does not rely on tetramers, antigen-stimulated cytokine production, or proliferation but rather on antigen-induced coexpression of CD25 and OX40 (CD134)., Results: Numbers of circulating gluten-specific Treg cells and effector T cells both increased significantly after oral wheat challenge, peaking at day 6. Surprisingly, we found that approximately 80% of the ex vivo circulating gluten-specific CD4 + T cells were FOXP3 + CD39 + Treg cells, which reside within the pool of memory CD4 + CD25 + CD127 low CD45RO + Treg cells. Although we observed normal suppressive function in peripheral polyclonal Treg cells from patients with celiac disease, after a short in vitro expansion, the gluten-specific FOXP3 + CD39 + Treg cells exhibited significantly reduced suppressive function compared with polyclonal Treg cells., Conclusion: This study provides the first estimation of FOXP3 + CD39 + Treg cell frequency within circulating gluten-specific CD4 + T cells after oral gluten challenge of patients with celiac disease. FOXP3 + CD39 + Treg cells comprised a major proportion of all circulating gluten-specific CD4 + T cells but had impaired suppressive function, indicating that Treg cell dysfunction might be a key contributor to disease pathogenesis., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Obesity and type-2 diabetes (T2D) are associated with metabolic defects and inflammatory processes in fat depots. FoxP3 + regulatory T cells (Tregs) control immune tolerance, and have an important role in controlling tissue-specific inflammation. In this mini-review we will discuss current insights into how cross-talk between T cells and adipose tissue shapes the inflammatory environment in obesity-associated metabolic diseases, focusing on the role of CD4 + T cells and Tregs. We will also highlight potential opportunities for how the immunoregulatory properties of Tregs could be harnessed to control inflammation in obesity and T2D and emphasize the critical need for more research on humans to establish mechanisms that are conserved in both mice and humans., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cellular therapy with CD4FOXP3 T regulatory (Treg) cells is a promising strategy to induce tolerance after solid-organ transplantation or prevent graft-versus-host disease after transfer of hematopoietic stem cells. Treg cells currently used in clinical trials are either polyclonal, donor- or antigen-specific. Aside from variations in isolation and expansion protocols, however, most therapeutic Treg cell-based products are much alike. Ongoing basic science work has provided considerable new insight into multiple facets of Treg cell biology, including their stability, homing, and functional specialization; integrating these basic science discoveries with clinical efforts will support the development of next-generation therapeutic Treg cells with enhanced efficacy. In this review, we summarize recent advances in knowledge of how Treg cells home to lymphoid and peripheral tissues, and control antibody production and tissue repair. We also discuss newly appreciated pathways that modulate context-specific Treg cell function and stability. Strategies to improve and tailor Treg cells for cell therapy to induce transplantation tolerance are highlighted.

Cellular therapy with T-regulatory cells (Tregs) is a promising strategy to control immune responses and restore immune tolerance in a variety of immune-mediated diseases, such as transplant rejection and autoimmunity. Multiple clinical trials are currently testing this approach, typically by infusing a single dose of polyclonal Tregs that have been expanded in vitro. However, evidence from animal models of Treg therapy has clearly shown that antigen-specific Tregs are vastly superior to polyclonal cells, meaning that fewer cells are needed for the desired therapeutic effect. Traditional methods to obtain antigen-specific Tregs include antigen-stimulated expansion or T-cell receptor (TCR) overexpression. However, these methods are limited by low cell numbers, complex manufacturing procedures, and knowledge of patient-specific TCRs which recognize disease-relevant MHC-peptide complexes. Recently, several groups have explored the potential to use chimeric antigen receptors (CARs) to generate antigen-specific Tregs. Here, we discuss the progress in this field and highlight the major outstanding questions that remain to be addressed as this approach moves toward clinical applications., (Copyright © 2017. Published by Elsevier Inc.)

FOXP3 controls the development and function of T regulatory cells (Tregs). Autoimmunity is linked to changes in FOXP3 activity that can occur at multiple levels and lead to Treg dysfunction. For example, changes in IL-2 signaling, FOXP3 transcription and/or post-translational modifications can all contribute to loss of self-tolerance. As additional pathways of FOXP3 regulation are elucidated, new therapeutic approaches to increase Treg activity either by cell therapy or pharmacological intervention are being tested. Early success from pioneering studies of Treg-based therapy in transplantation has promoted the undertaking of similar studies in autoimmunity, with emerging evidence for the effectiveness of these approaches, particularly in the context of type 1 diabetes., (Copyright © 2016. Published by Elsevier Ltd.)

Type 1 diabetes (T1D) is caused by immune-mediated destruction of insulin-producing β-cells. Insufficient control of autoreactive T cells by regulatory T cells (Tregs) is believed to contribute to disease pathogenesis, but changes in Treg function are difficult to quantify because of the lack of Treg-exclusive markers in humans and the complexity of functional experiments. We established a new way to track Tregs by using a gene signature that discriminates between Tregs and conventional T cells regardless of their activation states. The resulting 31-gene panel was validated with the NanoString nCounter platform and then measured in sorted CD4(+)CD25(hi)CD127(lo) Tregs from children with T1D and age-matched control subjects. By using biomarker discovery analysis, we found that expression of a combination of six genes, including TNFRSF1B (CD120b) and FOXP3, was significantly different between Tregs from subjects with new-onset T1D and control subjects, resulting in a sensitive (mean ± SD 0.86 ± 0.14) and specific (0.78 ± 0.18) biomarker algorithm. Thus, although the proportion of Tregs in peripheral blood is similar between children with T1D and control subjects, significant changes in gene expression can be detected early in disease process. These findings provide new insight into the mechanisms underlying the failure to control autoimmunity in T1D and might lead to a biomarker test to monitor Tregs throughout disease progression., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Adoptive immunotherapy with regulatory T cells (Tregs) is a promising treatment for allograft rejection and graft-versus-host disease (GVHD). Emerging data indicate that, compared with polyclonal Tregs, disease-relevant antigen-specific Tregs may have numerous advantages, such as a need for fewer cells and reduced risk of nonspecific immune suppression. Current methods to generate alloantigen-specific Tregs rely on expansion with allogeneic antigen-presenting cells, which requires access to donor and recipient cells and multiple MHC mismatches. The successful use of chimeric antigen receptors (CARs) for the generation of antigen-specific effector T cells suggests that a similar approach could be used to generate alloantigen-specific Tregs. Here, we have described the creation of an HLA-A2-specific CAR (A2-CAR) and its application in the generation of alloantigen-specific human Tregs. In vitro, A2-CAR-expressing Tregs maintained their expected phenotype and suppressive function before, during, and after A2-CAR-mediated stimulation. In mouse models, human A2-CAR-expressing Tregs were superior to Tregs expressing an irrelevant CAR at preventing xenogeneic GVHD caused by HLA-A2+ T cells. Together, our results demonstrate that use of CAR technology to generate potent, functional, and stable alloantigen-specific human Tregs markedly enhances their therapeutic potential in transplantation and sets the stage for using this approach for making antigen-specific Tregs for therapy of multiple diseases.

T regulatory cells (Tregs) control immune homeostasis by preventing inappropriate responses to self and nonharmful foreign antigens. Tregs use multiple mechanisms to control immune responses, all of which require these cells to be near their targets of suppression; however, it is not known how Treg-to-target proximity is controlled. Here, we found that Tregs attract CD4+ and CD8+ T cells by producing chemokines. Specifically, Tregs produced both CCL3 and CCL4 in response to stimulation, and production of these chemokines was critical for migration of target T cells, as Tregs from Ccl3-/- mice, which are also deficient for CCL4 production, did not promote migration. Moreover, CCR5 expression by target T cells was required for migration of these cells to supernatants conditioned by Tregs. Tregs deficient for expression of CCL3 and CCL4 were impaired in their ability to suppress experimental autoimmune encephalomyelitis or islet allograft rejection in murine models. Moreover, Tregs from subjects with established type 1 diabetes were impaired in their ability to produce CCL3 and CCL4. Together, these results demonstrate a previously unappreciated facet of Treg function and suggest that chemokine secretion by Tregs is a fundamental aspect of their therapeutic effect in autoimmunity and transplantation.

The two best-characterized types of CD4(+) regulatory T cells (Tregs) are Foxp3(+) Tregs and Foxp3(-) type 1 regulatory (Tr1) cells. The ability of Foxp3(+) Tregs and Tr1 cells to suppress adaptive immune responses is well known, but how these cells regulate innate immunity is less defined. We discovered that CD44(hi)Foxp3(-) T cells from unmanipulated mice are enriched in Tr1 cell precursors, enabling differentiation of cells that express IL-10, as well as Tr1-associated cell surface markers, CD49b and LAG-3, and transcription factors, cMaf, Blimp-1, and AhR. We compared the ability of Tr1 cells versus Foxp3(+) Tregs to suppress IL-1β production from macrophages following LPS and ATP stimulation. Surprisingly, Tr1 cells, but not Foxp3(+) Tregs, inhibited the transcription of pro-IL-1β mRNA, inflammasome-mediated activation of caspase-1, and secretion of mature IL-1β. Consistent with the role for IL-10 in Tr1 cell-mediated suppression, inhibition of inflammasome activation and IL-1β secretion was abrogated in IL-10R-deficient macrophages. Moreover, IL-1β production from macrophages derived from Nlrp3(A350V) knockin mice, which carry a mutation found in cryopyrin-associated periodic syndrome patients, was suppressed by Tr1 cells but not Foxp3(+) Tregs. Using an adoptive transfer model, we found a direct correlation between Tr1 cell engraftment and protection from weight loss in mice expressing a gain-of-function NLRP3. Collectively, these data provide the first evidence for a differential role of Tr1 cells and Foxp3(+) Tregs in regulating innate immune responses. Through their capacity to produce high amounts of IL-10, Tr1 cells may have unique therapeutic effects in disease-associated inflammasome activation., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Obesity is associated with insulin resistance and inflammation thought to be caused by a visceral adipose tissue (VAT)-localized reduction in immunoregulatory cells and increase in proinflammatory immune cells. We previously found that VAT regulatory T cells (Tregs) normally express high levels of IL-10 and that expression of this cytokine in VAT Tregs is specifically reduced in mice fed a high-fat diet. In this study, we further investigated the phenotype of VAT Tregs and found that the majority of IL-10-expressing Tregs in the VAT of lean mice also expressed the ST2 chain of the IL-33R. In addition to high expression of IL-10, ST2(+) Tregs in lean VAT expressed higher proportions of Th2-associated proteins, including GATA3 and CCR4, and Neuropillin-1 compared with ST2(-) Tregs. The proportion of ST2(+) Tregs in VAT was severely diminished in obese mice that had been fed a high-fat/sucrose diet, and this effect could be completely reversed by treatment with IL-33. IL-33 treatment also reversed VAT inflammation in obese mice and resulted in a reduction of hyperinsulinemia and insulin resistance. These data suggest that IL-33 contributes to the maintenance of the normal pool of ST2(+) Tregs in the VAT, and that therapeutic administration of IL-33 results in multiple anti-obesity effects, including the reversal of VAT inflammation and alleviation of insulin resistance., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Background: Systemic sclerosis (SSc) is an autoimmune disorder characterized by fibrosis of the skin and internal organs. Pathologic conversion of regulatory T (Treg) cells into inflammatory cytokine-producing cells is thought to be an important step in the progression of autoimmunity, but whether loss of normal Treg cell function contributes to SSc is unknown., Objective: We sought to determine whether Treg cells in the blood and skin of patients with SSc acquired abnormal production of effector cytokines., Methods: Peripheral blood and skin biopsy specimens were collected from control subjects and patients with limited or diffuse SSc. Flow cytometry was used to evaluate expression of cell-surface proteins and the cytokine production profile of forkhead box protein 3-positive Treg cells compared with forkhead box protein 3-negative conventional T cells., Results: Treg cells in the blood of patients with SSc had a normal phenotype and did not produce T-effector cytokines. In contrast, Treg cells from skin affected by SSc produced significant amounts of IL-4 and IL-13. Although Treg cells in the blood of patients with SSc did not make TH2 cytokines, they contained a significantly higher proportion of skin-homing cells expressing TH2 cell-associated chemokine receptors. Evidence that IL-33 caused the differentiation of skin Treg cells into TH2-like cells, combined with high tissue-localized expression of this cytokine in patients with SSc and expression of the ST2 chain of the IL-33 receptor on skin-localized Treg cells, suggests that IL-33 might be an important stimulator of tissue-localized loss of normal Treg cell function., Conclusion: These data are the first evidence for the presence of TH2-like Treg cells in human autoimmunity and show that Treg cell plasticity can be tissue specific. Localized dysfunction of Treg cells is a previously unknown factor that might contribute to fibrosis in patients with SSc., (Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Treg cells control immune responses to self and nonharmful foreign antigens. Emerging data from animal models indicate that Treg cells function in both secondary lymphoid organs and tissues, and that these different microenvironments may contain specialized subsets of Treg cells with distinct mechanisms of action. The design of therapies for the restoration of tissue-localized immune homeostasis is dependent upon understanding how local immune responses are influenced by Treg cells in health versus disease. Here we review the current state of knowledge about human Treg cells in four locations: the skin, lung, intestine, and joint. Despite the distinct biology of these tissues, there are commonalities in the biology of their resident Treg cells, including phenotypic and functional differences from circulating Treg cells, and the presence of cytokine-producing (e.g. IL-17(+)) FOXP3(+) cells. We also highlight the challenges to studying tissue Treg cells in humans, and opportunities to use new technologies for the detailed analysis of Treg cells at the single-cell level. As emerging biological therapies are increasingly targeted toward tissue-specific effects, it is critical to understand their potential impact on local immune regulation., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Manipulation of regulatory T cell (Treg) migration by islet expression of the chemokine CCL22 prevents diabetes in NOD mice and delays recurrent autoimmunity in syngeneic islet transplants. We sought to determine whether attracting Tregs with CCL22 also prevents islet allograft rejection. Isolated Bl/6 mouse islets were transduced overnight with adenovirus expressing CCL22 (Ad-CCL22) downstream of the CMV promoter. Islets were transplanted under the renal capsule of Balb/c recipients made diabetic by streptozotocin. To assess immunologic tolerance, graft-bearing kidneys from recipients of CCL22-expressing islet grafts were removed, and mice received a second transplant of naive islets from the same donor strain or third-party islets into the contralateral kidney. Adenoviral expression of CCL22 conferred prolonged protection of islet allografts in MHC-mismatched, diabetic recipients, maintaining normoglycemia in 75% of recipients for at least 80 days. Increased frequency of Treg cells was observed in islet grafts transduced with Ad-CCL22 compared with untreated grafts. Normoglycemic recipients of CCL22-expressing islet grafts showed complete absence of antidonor antibodies and no lymphocyte proliferation after exposure to donor splenocytes. After removal of the primary graft at day 80, mice that received a second transplant with untreated islets from the same donor strain did not reject the grafts, suggesting the development of tolerance. Expression of CCL22 recruits Treg cells to transplanted islets, prevents activation of alloreactive T-cells and islet allograft failure and induces alloantigen-specific tolerance. Manipulation of Treg cells by CCL22 in transplanted islets may be a novel therapeutic strategy for diabetes.

Inflammatory bowel disease (IBD) pathogenesis is associated with dysregulated CD4⁺ Th cell responses, with intestinal homeostasis depending on the balance between IL-17-producing Th17 and Foxp3⁺ Tregs. Differentiation of naive T cells into Th17 and Treg subsets is associated with specific gene expression profiles; however, the contribution of epigenetic mechanisms to controlling Th17 and Treg differentiation remains unclear. Using a murine T cell transfer model of colitis, we found that T cell-intrinsic expression of the histone lysine methyltransferase G9A was required for development of pathogenic T cells and intestinal inflammation. G9A-mediated dimethylation of histone H3 lysine 9 (H3K9me2) restricted Th17 and Treg differentiation in vitro and in vivo. H3K9me2 was found at high levels in naive Th cells and was lost following Th cell activation. Loss of G9A in naive T cells was associated with increased chromatin accessibility and heightened sensitivity to TGF-β1. Pharmacological inhibition of G9A methyltransferase activity in WT T cells promoted Th17 and Treg differentiation. Our data indicate that G9A-dependent H3K9me2 is a homeostatic epigenetic checkpoint that regulates Th17 and Treg responses by limiting chromatin accessibility and TGF-β1 responsiveness, suggesting G9A as a therapeutic target for treating intestinal inflammation.

Regulatory T cells (Tregs) act in trans to control immune responses. The suppressive function of Tregs relies heavily on high and stable expression of the transcription factor FOXP3, which, together with other transcription factors, activates anti-inflammatory genes and represses proinflammatory genes. FOXP3 is required to shape the unique signaling mechanisms in Tregs, creating a positive-feedback pathway to further enhance its own expression. In addition, FOXP3 is thought to switch on a complex transcriptional network that leads to the stabilization of the Treg phenotype. Emerging data reveal that FOXP3 achieves this function in concert with several other transcription factors, many of which are associated with lineages of conventional T cells. In this review, we will discuss the structural features of FOXP3 and how it functions by interacting with other transcription factors. We will also summarize the role of FOXP3 in establishing the unique signaling cascades in Tregs. Finally, we will dissect the cooperative roles of FOXP3 and other T-cell lineage-defining transcription factors and discuss how these networks not only control the ability to Tregs to suppress different types of immune responses, but also enable Treg plasticity.

Chronic inflammation is known to promote metabolic dysregulation in obesity and type 2 diabetes. Although the precise origin of the unchecked inflammatory response in obesity is unclear, it is known that overproduction of proinflammatory cytokines by innate immune cells affects metabolism. For example, TNF-α contributes to the inability of cells to respond to insulin and to the increase in levels of insulin. Whether this hyperinsulinemia itself is part of a feedback loop that affects the progression of chronic adipose inflammation is unknown. In this article, we show that regulatory T cells (Tregs) express the insulin receptor, and that high levels of insulin impair the ability of Tregs to suppress inflammatory responses via effects on the AKT/mTOR signaling pathway. Insulin activated AKT signaling in Tregs, leading to inhibition of both IL-10 production and the ability of Tregs to suppress the production of TNF-α by macrophages in a contact-independent manner. The effect of insulin on Treg suppression was limited to IL-10 production and it did not alter the expression of other proteins associated with Treg function, including CTLA-4, CD39, and TGF-β. In a model of diet-induced obesity, Tregs from the visceral adipose tissue of hyperinsulinemic, obese mice showed a similar specific decrease in IL-10 production, as well as a parallel increase in production of IFN-γ. These data suggest that hyperinsulinemia may contribute to the development of obesity-associated inflammation via a previously unknown effect of insulin on the IL-10-mediated function of Tregs.

FOXP3-expressing T regulatory cells (Tregs) can be divided into two distinct subsets: naturally occurring Tregs (nTregs) that develop in the thymus, and induced Tregs (iTregs) that differentiate in peripheral tissues upon exposure to Ag in a tolerogenic environment. Recently it has been proposed that expression of Helios, an Ikaros family transcription factor, may specifically identify nTregs, allowing specific tracking of Tregs from different origins in health and disease. Surprisingly, we found that Helios(-) cells can be readily identified within naive (CD45RA(+)CD31(+)CCR7(+)CD62L(+)) FOXP3(+) Tregs, a finding inconsistent with the notion that lack of Helios expression identifies Ag-experienced iTregs that should express memory markers. To investigate the phenotype and function of naive Helios(+) and Helios(-) Tregs within the nTreg population, we isolated single-cell clones from each subset. We found that both Helios(+) and Helios(-) nTreg clones have a similar suppressive capacity, as well as expression of FOXP3 and cell surface proteins, including CD39 and CTLA-4. Helios(-) nTregs, however, produced significantly more CCL3 and IFN-γ compared with Helios(+) nTregs. Despite increased cytokine/chemokine production, Helios(-) FOXP3(+) nTreg clones were demethylated at the FOXP3 Treg-specific demethylated region, indicative of Treg lineage stability. When cultured under Th1-polarizing conditions, Helios(+) and Helios(-) nTreg clones had an equal ability to produce IFN-γ. Collectively, these data show that a lack of Helios expression does not exclusively identify human iTregs, and, to our knowledge, the data provide the first evidence for the coexistence of Helios(+) and Helios(-) nTregs in human peripheral blood.

T regulatory cells (Tregs) suppress immune responses and therefore have potential to be used in the clinic as a cellular therapy for autoimmune disease and to prevent rejection of transplanted organs. Obtaining sufficient numbers of these cells for therapeutic use is a challenge, however, since there are currently no Treg cell-specific markers, and they have a poor in vitro expansion potential. Tregs express high levels of FOXP3, a transcription factor that is critical for their function. We have shown that lentivirus-based overexpression of FOXP3 can reprogram naïve or memory human CD4(+) T cells into cells which possess a phenotype and function similar to ex vivo Tregs. Here we will review the methodology involved in generating, expanding, and testing FOXP3-transduced cells and their ex vivo Treg counterparts.

Purpose of Review: There is great hope that cellular therapy with regulatory T cells (Tregs) will be an effective way to induce alloantigen specific tolerance, ultimately allowing for reduction or elimination of nonspecific immunosuppression. In the past, considerable effort was focused on defining the optimal ways to isolate and expand Tregs from peripheral or cord blood. Now that expansion of therapeutically relevant numbers of Tregs is feasible, we need to consider what is going to happen to the cells when they are transferred in vivo., Recent Findings: For optimal function, Tregs must be able to traffic to the correct location(s) and, despite the presence of immunosuppressive therapy, live long enough to transfer their regulatory function to recipient T cells. Within the Treg pool, there are also functionally specialized subsets, identified by chemokine receptor expression and/or cytokine production, which control their trafficking and relative ability to suppress different types of T helper cells, respectively. Recent findings imply that the plasticity of appropriately obtained populations of Tregs may not be of as great concern as previously suggested. Experimental data have also provided evidence as to how one might design adjunctive treatment that best supports the viability and function of Tregs after transfer., Summary: Knowledge of how Tregs work in transplantation comes from studies that do not recapitulate how these cells will be used in humans. There is a need to develop better preclinical models to study how the in-vivo function of human Tregs can be optimized to ensure they can meet the challenge of inducing transplantation tolerance.

T regulatory (Treg) cells are critical for maintaining immune homeostasis and establishing tolerance to foreign, non-pathogenic antigens including those found in commensal bacteria and food. Because of their multiple suppressive mechanisms, Tregs represent a promising strategy for engineering tolerance to self and non-self antigens in chronic inflammatory diseases. Already in clinical trials in the transplantation setting, the question remains whether this therapy would be effective for the treatment of mucosal inflammatory diseases that do not pose an immediate threat to life. In this review we will discuss evidence from both animal models and patients suggesting that Treg therapy would be beneficial in the context of inflammatory bowel disease (IBD). We will examine the role of T-cell versus Treg dysfunction in IBD and discuss the putative antigens that could be potential targets of antigen-directed Treg therapy. Finally, the challenges of using Treg therapy in IBD will be discussed, with a specific emphasis on the role that the microbiota may play in the outcome of this treatment. As Treg therapy becomes a bedside reality in the field of transplantation, there is great hope that it will soon also be deployed in the setting of IBD and ultimately prove more effective than the current non-specific immunosuppressive therapies., (© 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.)

While natural CD4(+)Foxp3(+) regulatory T (nT(REG)) cells have long been viewed as a stable and distinct lineage that is committed to suppressive functions in vivo, recent evidence supporting this notion remains highly controversial. We sought to determine whether Foxp3 expression and the nT(REG) cell phenotype are stable in vivo and modulated by the inflammatory microenvironment. Here, we show that Foxp3(+) nT(REG) cells from thymic or peripheral lymphoid organs reveal extensive functional plasticity in vivo. We show that nT(REG) cells readily lose Foxp3 expression, destabilizing their phenotype, in turn, enabling them to reprogram into Th1 and Th17 effector cells. nT(REG) cell reprogramming is a characteristic of the entire Foxp3(+) nT(REG) population and the stable Foxp3(NEG) T(REG) cell phenotype is associated with a methylated foxp3 promoter. The extent of nT(REG) cell reprogramming is modulated by the presence of effector T cell-mediated signals, and occurs independently of variation in IL-2 production in vivo. Moreover, the gut microenvironment or parasitic infection favours the reprogramming of Foxp3(+) T(REG) cells into effector T cells and promotes host immunity. IL-17 is predominantly produced by reprogrammed Foxp3(+) nT(REG) cells, and precedes Foxp3 down-regulation, a process accentuated in mesenteric sites. Lastly, mTOR inhibition with the immunosuppressive drug, rapamycin, stabilizes Foxp3 expression in T(REG) cells and strongly inhibits IL-17 but not RORγt expression in reprogrammed Foxp3(-) T(REG) cells. Overall, inflammatory signals modulate mTOR signalling and influence the stability of the Foxp3(+) nT(REG) cell phenotype.