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Adoptive immunotherapy with Tregs is a promising approach for preventing or treating type 1 diabetes. Islet Antigen-specific Tregs have more potent therapeutic effects than polyclonal cells, but their low frequency is a barrier for clinical application. To generate Tregs that recognize islet antigens, we engineered a chimeric antigen receptor (CAR) derived from a monoclonal antibody with specificity for the insulin B chain 10-23 peptide presented in the context of the I[A.sup.g7] MHC class II allele present in NOD mice. Peptide specificity of the resulting InsB-g7 CAR was confirmed by tetramer staining and T cell proliferation in response to recombinant or islet-derived peptide. The InsB-g7 CAR redirected NOD Treg specificity such that insulin B 10-23-peptide stimulation enhanced suppressive function, measured via reduction of proliferation and IL-2 production by BDC2.5 T cells and CD80 and CD86 expression on dendritic cells. Cotransfer of InsB-g7 CAR Tregs prevented adoptive transfer diabetes by BDC2.5 T cells in immunodeficient NOD mice. In WT NOD mice, InsB-g7 CAR Tregs prevented spontaneous diabetes. These results show that engineering Treg specificity for islet antigens using a T cell receptor-like CAR is a promising therapeutic approach for the prevention of autoimmune diabetes., Introduction Autoimmune type 1 diabetes (T1D) is caused by T cell-mediated destruction of the insulin-producing [beta] cells of the islets of Langerhans in the pancreas. Tregs, defined by expression of [...]

Suppression of pathogenic immune responses is a major goal in the prevention and treatment of type 1 diabetes. Adoptive cell therapy using regulatory T cells (Tregs), a naturally suppressive immune subset that is often dysfunctional in type 1 diabetes, is a promising approach to achieving localised and specific immune suppression in the pancreas or site of islet transplant. However, clinical trials testing administration of polyclonal Tregs in recent-onset type 1 diabetes have observed limited efficacy despite an excellent safety profile. Several barriers to efficacy have been identified, including lack of antigen specificity, low cell persistence post-administration and difficulty in generating sufficient cell numbers. Fortunately, the emergence of advanced gene editing techniques has opened the door to new strategies to engineer Tregs with improved specificity and function. These strategies include the engineering of FOXP3 expression to produce a larger source of suppressive cells for infusion, expressing T cell receptors or chimeric antigen receptors to generate antigen-specific Tregs and improving Treg survival by targeting cytokine pathways. Although these approaches are being applied in a variety of autoimmune and transplant contexts, type 1 diabetes presents unique opportunities and challenges for the genetic engineering of Tregs for adoptive cell therapy. Here we discuss the role of Tregs in type 1 diabetes pathogenesis and the application of Treg engineering in the context of type 1 diabetes. [ABSTRACT FROM AUTHOR]

Summary: Since their discovery, CD4+CD25hiFOXP3hi regulatory T cells (Tregs) have been firmly established as a critical cell type for regulating immune homeostasis through a plethora of mechanisms. Due to their immunoregulatory power, delivery of polyclonal Tregs has been explored as a therapy to dampen inflammation in the settings of transplantation and autoimmunity. Evidence shows that Treg therapy is safe and well‐tolerated, but efficacy remains undefined and could be limited by poor persistence in vivo and lack of antigen specificity. With the advent of new genetic engineering tools, it is now possible to create bespoke "designer" Tregs that not only overcome possible limitations of polyclonal Tregs but also introduce new features. Here, we review the development of designer Tregs through the perspective of three 'eras': (1) the era of FOXP3 engineering, in which breakthroughs in the biological understanding of this transcription factor enabled the conversion of conventional T cells to Tregs; (2) the antigen‐specificity era, in which transgenic T‐cell receptors and chimeric antigen receptors were introduced to create more potent and directed Treg therapies; and (3) the current era, which is harnessing advanced genome‐editing techniques to introduce and refine existing and new engineering approaches. The year 2022 marked the entry of "designer" Tregs into the clinic, with exciting potential for application and efficacy in a wide variety of immune‐mediated diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Activation‐induced marker (AIM) assays have proven to be an accessible and rapid means of antigen‐specific T‐cell detection. The method typically involves short‐term incubation of whole blood or peripheral blood mononuclear cells with antigens of interest, where autologous antigen‐presenting cells process and present peptides in complex with major histocompatibility complex (MHC) molecules. Recognition of peptide–MHC complexes by T‐cell receptors then induces upregulation of activation markers on the T cells that can be detected by flow cytometry. In this review, we highlight the most widely used activation markers for assays in the literature while identifying nuances and potential downfalls associated with the technique. We provide a summary of how AIM assays have been used in both discovery science and clinical studies, including studies of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) immunity. This review primarily focuses on AIM assays using human blood or peripheral blood mononuclear cell samples, with some considerations noted for tissue‐derived T cells and nonhuman samples. AIM assays are a powerful tool that enables detailed analysis of antigen‐specific T‐cell frequency, phenotype and function without needing to know the precise antigenic peptides and their MHC restriction elements, enabling a wider analysis of immunity generated following infection and/or vaccination. [ABSTRACT FROM AUTHOR]

For the past century, insulin injections have saved millions of lives, but glycemic instability is still a persistent challenge for people with diabetes, leading to tremendous morbidity and premature mortality. Research in the field of islet transplantation has demonstrated that replacing insulin-producing β cells can restore euglycemia comparable to individuals without diabetes. However, a short supply of cadaveric islet donors, the technically challenging process of isolating islets, and the requirement for chronic immune suppression have impeded widespread clinical adoption. Rather than relying on cadaveric cells, pluripotent stem cells could serve as a virtually unlimited supply of insulin-producing β cells. Protocols have been developed that mimic the normal in vivo development of the human pancreas to generate pancreatic progenitor cells in vitro. Ongoing investigations have yielded progressively more mature β-like cells in vitro that produce insulin but do not yet fully mimic healthy mature β cells. Alongside development of differentiation protocols, other work has provided insight into potential implantation sites for stem cell–derived islet cells including the subcutaneous space, portal vein, and omentum. To optimize implanted cell survival and function, development of immune modulation therapies is ongoing, including selection of immunomodulatory medications and genetic modification of implanted cells to evade immune responses. Further, macroencapsulation or microencapsulation devices could be used to contain and/or immunoprotect implanted cells from the immune response including by using 3-dimensional bioprinting to facilitate the process. Remarkably, ongoing clinical trials have now yielded the first patient relying on differentiated stem cells rather than syringes as their insulin replacement therapy. [ABSTRACT FROM AUTHOR]

Adoptive immunotherapy with regulatory T cells (Tregs) is a promising treatment for allograft rejection and graft- versushost 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.sup.-]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.sup.+] 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., Introduction The essential role of regulatory T cells (Tregs) in preventing autoimmunity and controlling responses to alloantigens is well established. Multiple Treg-based cell therapy approaches are now being tested in [...]

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.sup.+] and [CD8.sup.+] 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.sup.-/-] 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., Introduction T regulatory cells (Tregs) are essential for preventing immunity to self antigens (Ags) and nonharmful foreign Ags; they also play an active role in suppressing immunity to tumors and [...]

Defining the immunological landscape of human tissue is an important area of research, but challenges include the impact of tissue disaggregation on cell phenotypes and the low abundance of immune cells in many tissues. Here, we describe methods to troubleshoot and standardize Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) for studies involving enzymatic digestion of human tissue. We tested epitope susceptibility of 92 antibodies commonly used to differentiate immune lineages and cell states on human peripheral blood mononuclear cells following treatment with an enzymatic digestion cocktail used to isolate islets. We observed CD4, CD8a, CD25, CD27, CD120b, CCR4, CCR6, and PD1 display significant sensitivity to enzymatic treatment, effects that often could not be overcome with alternate antibodies. Comparison of flow cytometry-based CITE-seq antibody titrations and sequencing data supports that for the majority of antibodies, flow cytometry accurately predicts optimal antibody concentrations for CITE-seq. Comparison by CITE-seq of immune cells in enzymatically digested islet tissue and donor-matched spleen not treated with enzymes revealed little digestion-induced epitope cleavage, suggesting increased sensitivity of CITE-seq and/or that the islet structure may protect resident immune cells from enzymes. Within islets, CITE-seq identified immune cells difficult to identify by transcriptional signatures alone, such as distinct tissue-resident T cell subsets, mast cells, and innate lymphoid cells (ILCs). Collectively this study identifies strategies for the rational design and testing of CITE-seq antibodies for single-cell studies of immune cells within islets and other tissues. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease (IBD) pathogenesis is associated with dysregulated [CD4.sup.+] Th cell responses, with intestinal homeostasis depending on the balance between IL-17-producing Th17 and [Foxp3.sup.+] 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-P1. Pharmacological inhibition ofG9A 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., Introduction The inflammatory bowel diseases (IBDs) are a group of chronic intestinal inflammatory diseases that include ulcerative colitis (UC) and Crohn disease (CD). IBD is thought to occur as a [...]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Type 1 diabetes is characterized by destruction of insulin-producing β cells in the pancreatic islets by effector T cells. Tregs, defined by the markers CD4 and FoxP3, regulate immune responses by suppressing effector T cells and are recruited to sites of action by the chemokine CCL22. Here, we demonstrate that production of CCL22 in islets after intrapancreatic duct injection of double-stranded adeno-associated virus encoding CCL22 recruits endogenous Tregs to the islets and confers long-term protection from autoimmune diabetes in NOD mice. In addition, adenoviral expression of CCL22 in syngeneic islet transplants in diabetic NOD recipients prevented β cell destruction by autoreactive T cells and thereby delayed recurrence of diabetes. CCL22 expression increased the frequency of Tregs, produced higher levels of TGF-β in the [CD4.sup.+] T cell population near islets, and decreased the frequency of circulating autoreactive [CD8.sup.+] T cells and [CD8.sup.+] IFN-γ-producing T cells. The protective effect of CCL22 was abrogated by depletion of Tregs with a CD25-specific antibody. Our results indicate that islet expression of CCL22 recruits Tregs and attenuates autoimmune destruction of β cells. CCL22-mediated recruitment of Tregs to islets may be a novel therapeutic strategy for type 1 diabetes., Introduction The complex pathogenesis of autoimmune diabetes in NOD mice and humans involves multiple immune components. The autoimmune response recurs when βcells are transplanted into type 1 diabetic patients or [...]

The authors of this article, all women who have been deeply committed to the Federation of Clinical Immunology Societies (FOCIS), performed a retrospective analysis of gender equality practices of FOCIS to identify areas for improvement and make recommendations accordingly. Gender data were obtained and analyzed for the period from January 2010 to July 2021. Outcome measures included numbers of men and women across the following categories: membership enrollment, meeting and course faculty and attendees, committee and leadership composition. FOCIS' past and present leaders, steering committee members, FCE directors, individual members, as well as education, annual meeting scientific program and FCE committee members and management staff of FOCIS were surveyed by email questionnaire for feedback on FOCIS policies and practice with respect to gender equality and inclusion. Although women represent 50% of the membership, they have been underrepresented in all leadership, educational, and committee roles within the FOCIS organization. Surveying FOCIS leadership and membership revealed a growing recognition of disparities in female leadership across all FOCIS missions, leading to significant improvement in multiple areas since 2016. We highlight these changes and propose a number of recommendations that can be used by FOCIS to improve gender equality. [ABSTRACT FROM AUTHOR]

The autoimmune disease immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) is caused by mutations in the forkhead box protein P3 (FOXP3) gene. In the mouse model of FOXP3 deficiency, the lack [...]

Cytometric immunophenotyping is a powerful tool to discover and implement T‐cell biomarkers of type 1 diabetes (T1D) progression and response to clinical therapy. Although many discovery‐based T‐cell biomarkers have been described, to date, no such markers have been widely adopted in standard practice. The heterogeneous nature of T1D and lack of standardized assays and experimental design across studies is a major barrier to the broader adoption of T‐cell immunophenotyping assays. There is an unmet need to harmonize the design of immunophenotyping assays, including those that measure antigen‐agnostic cell populations, such that data collected from different clinical trial sites and T1D cohorts are comparable, yet account for cohort‐specific features and different drug mechanisms of action. In these Guidelines, we aim to provide expert advice on how to unify aspects of study design and practice. We provide recommendations for defining cohorts, method implementation, as well as tools for data analysis and reporting by highlighting and building on selected successes. Harmonization of cytometry‐based T‐cell assays will allow researchers to better integrate findings across trials, ultimately enabling the identification and validation of biomarkers of disease progression and treatment response in T1D. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (Heliosmid)‐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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Transforming growth factor‐beta (TGF‐β) family proteins mediate many vital biological functions in growth, development and regulation of the immune system. TGF‐β itself controls immune homeostasis and inflammation, including conversion of naïve CD4+ T cells into Foxp3+ regulatory T cells (Tregs) in the presence of interleukin‐2 and T‐cell receptor ligands. The helminth parasite Heligmosomoides polygyrus exploits this pathway through a structurally novel TGF‐β mimic (Hp‐TGM), which binds to mammalian TGF‐β receptors and induces Tregs. Here, we performed detailed comparisons of Hp‐TGM with mammalian TGF‐β. Compared with TGF‐β, Hp‐TGM induced greater numbers of Foxp3+ Tregs (iTregs), with more intense Foxp3 expression. Both ligands upregulated Treg functional markers CD73, CD103 and programmed death‐ligand 1, but Hp‐TGM induced significantly higher CD39 expression than did TGF‐β. Interestingly, in contrast to canonical TGF‐β signaling through Smad2/3, Hp‐TGM stimulation was slower and more sustained. Gene expression profiles induced by TGF‐β and Hp‐TGM were remarkably similar, and both types of iTregs suppressed T‐cell responses in vitro and experimental autoimmune encephalomyelitis‐driven inflammation in vivo. In vitro, both types of iTregs were equally stable under inflammatory conditions, but Hp‐TGM‐induced iTregs were more stable in vivo during dextran sodium sulfate‐induced colitis, with greater retention of Foxp3 expression and lower conversion to a ROR‐γt+ phenotype. Altogether, results from this study suggest that the parasite cytokine mimic, Hp‐TGM, may deliver a qualitatively different signal to CD4+ T cells with downstream consequences for the long‐term stability of iTregs. These data highlight the potential of Hp‐TGM as a new modulator of T‐cell responses in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Immune homeostasis in the intestine is tightly controlled by FOXP3+ regulatory T cells (Tregs), defects of which are linked to the development of chronic conditions, such as inflammatory bowel disease (IBD). As a mechanism of immune evasion, several species of intestinal parasites boost Treg activity. The parasite Heligmosomoides polygyrus is known to secrete a molecule (Hp‐TGM) that mimics the ability of TGF‐β to induce FOXP3 expression in CD4+ T cells. The study aimed to investigate whether Hp‐TGM could induce human FOXP3+ Tregs as a potential therapeutic approach for inflammatory diseases. CD4+ T cells from healthy volunteers were expanded in the presence of Hp‐TGM or TGF‐β. Treg induction was measured by flow cytometric detection of FOXP3 and other Treg markers, such as CD25 and CTLA‐4. Epigenetic changes were detected using ChIP‐Seq and pyrosequencing of FOXP3. Treg phenotype stability was assessed following inflammatory cytokine challenge and Treg function was evaluated by cellular co‐culture suppression assays and cytometric bead arrays for secreted cytokines. Hp‐TGM efficiently induced FOXP3 expression (> 60%), in addition to CD25 and CTLA‐4, and caused epigenetic modification of the FOXP3 locus to a greater extent than TGF‐β. Hp‐TGM‐induced Tregs had superior suppressive function compared with TGF‐β‐induced Tregs, and retained their phenotype following exposure to inflammatory cytokines. Furthermore, Hp‐TGM induced a Treg‐like phenotype in in vivo differentiated Th1 and Th17 cells, indicating its potential to re‐program memory cells to enhance immune tolerance. These data indicate Hp‐TGM has potential to be used to generate stable human FOXP3+ Tregs to treat IBD and other inflammatory diseases. [ABSTRACT FROM AUTHOR]