Stable Foxp3 Expression Research Articles

Enabling Gene-Edited, Regulatory-like, T Cells (edTreg) for Treatment of IPEX and Other Autoimmune Disorders

IPEX (immunedysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome is a severe congenital autoimmune disorder in males resulting from hemizygous inheritance of a mutant FOXP3 allele. FOXP3 encodes a transcription factor that governs the development, maintenance, and function of regulatory T cells (Treg). We have developed a cell therapy strategy for treatment of IPEX using a gene-editing approach in which CRISPR/Cas9 RNPs are co-delivered with an AAV6 donor template designed to integrate into the FOXP3 locus an expression cassette containing the MND promoter driving expression of a functional FOXP3 cDNA and a surface LNGFR tag linked by a 2A ribosomal skip peptide. This approach enforces heterologous FOXP3 expression in IPEX CD4 effector T cells (Teff), while simultaneously eliminating expression of the endogenous FOXP3 allele. The resultant high level and stable expression of functional FOXP3 converts Teff to Treg-like cells with immunosuppressive activity. Using an optimized protocol, we obtained efficient HDR rates across multiple healthy donors. Edited cells were consistently enriched to >95% purity by a magnetic LNGFR antibody selection and expanded 50-fold in a week. Expression of FOXP3 cDNA in edited cells was sufficient to enforce Treg-like phenotypes including the up-regulation of Treg-associated markers (CD25, CTLA-4, and ICOS), and down-regulation of CD127 and inflammatory cytokines (IL2, IFNgamma, TNFalpha). Importantly, we demonstrate sustained in vivo suppressive activity of these edited Treg-like cells (edTreg) in a xeno-GvHD mouse model. edTreg (as well as expanded natural Treg) limited effector T cell expansion and tissue infiltration and significantly protected mice from xeno-GvHD induced by co-transferred autologous effector T cells. Along with preliminary data showing successful editing in CD4 T cells from IPEX patients, our data provide key pre-clinical proof-of-concept and safety data supporting use of edTreg in a clinical trial for IPEX and, potentially, for use in other autoimmune diseases. Disclosures Torgerson: Shire: Consultancy; CSL Behring: Consultancy; ADMA Biosciences: Consultancy; UCB: Consultancy. Scharenberg:Casebia Therapeutics LLc: Employment, Equity Ownership; Alpine Biosciences: Consultancy, Equity Ownership; Generation Bio: Equity Ownership.

Manufacture and In Vivo Support of Gene-Edited, Regulatory-like, T-Cells (edTreg) Using a Synthetic IL2 Receptor

Regulatory T-cells (Treg) depend on signals from IL2 and their endogenous T-cell receptors to survive, proliferate, and maintain suppressive activity. We have developed a strategy for engineering effector CD4 T-cells into edited, regulatory-like T-cells (edTregs) for treatment of autoimmune disease. edTregs contain a rapamycin-activated synthetic IL2 receptor (designated a chemical-induced signaling complex or CISC) for use in selective cell manufacturing and in vivo trophic support. Our strategy uses gene editing to integrate an MND/ CISC expression cassette into the native FOXP3 locus, resulting in the CISC system cis-linked to expression of the native FOXP3 gene. The resulting high-level and stable expression of FOXP3 converts peripheral T-cells to edTregs with immunosuppressive activity. Cis-linked expression of the CISC system allows for selective cell expansion and in vivo support using rapamycin. Using an optimized protocol, we obtained efficient HDR rates across cells from multiple healthy donors. Edited cells were enriched to >90% purity and expanded 20-50-fold over a 10 day period of culture in rapamycin. Importantly, we demonstrated sustained in vivo suppressive activity of edTregs in a xeno-GvHD mouse model, and successful trophic support of these edTregs by rapamycin (via the CISC) in the absence of effector CD4 T-cells. Along with preliminary data showing successful editing of CD4-positive T-cells from auto-immune disease patients, our data provide pre-clinical proof-of-concept data supporting clinical use of CISC-edTreg in conjunction with rapamycin support for therapy of autoimmune disease. Disclosures Uenishi: Casebia Therapeutics: Employment. Mallari:Casebia Therapeutics: Employment. Gamboa:Casebia Therapeutics: Employment. Boucher:Casebia Therapeutics LLc: Employment. Chin:Casebia Therapeutics LLc: Employment. Aeran:Casebia Therapeutics: Employment. Wodziak:Casebia Therapeutics: Employment. Gebremeskel:Casebia Therapeutics: Employment. Vo:Casebia Therapeutics: Employment. Ito:Casebia Therapeutics: Employment. Patel:Casebia Therapeutics: Employment. Abe:Casebia Therapeutics: Employment. Stankovich:Casebia Therapeutics LLc: Employment. Torgerson:Shire: Consultancy; CSL Behring: Consultancy; ADMA Biosciences: Consultancy; UCB: Consultancy. Scharenberg:Generation Bio: Equity Ownership; Casebia Therapeutics LLc: Employment, Equity Ownership; Alpine Biosciences: Consultancy, Equity Ownership. Cost:Casebia Therapeutics: Employment.

Dynamic Imprinting of the Treg Cell-Specific Epigenetic Signature in Developing Thymic Regulatory T Cells.

Regulatory T (Treg) cells mainly develop within the thymus and arise from CD25+Foxp3− (CD25+ TregP) or CD25−Foxp3+ (Foxp3+ TregP) Treg cell precursors resulting in Treg cells harboring distinct transcriptomic profiles and complementary T cell receptor repertoires. The stable and long-term expression of Foxp3 in Treg cells and their stable suppressive phenotype are controlled by the demethylation of Treg cell-specific epigenetic signature genes including an evolutionarily conserved CpG-rich element within the Foxp3 locus, the Treg-specific demethylated region (TSDR). Here we analyzed the dynamics of the imprinting of the Treg cell-specific epigenetic signature genes in thymic Treg cells. We could demonstrate that CD25+Foxp3+ Treg cells show a progressive demethylation of most signature genes during maturation within the thymus. Interestingly, a partial demethylation of several Treg cell-specific epigenetic signature genes was already observed in Foxp3+ TregP but not in CD25+ TregP. Furthermore, Foxp3+ TregP were very transient in nature and arose at a more mature developmental stage when compared to CD25+ TregP. When the two Treg cell precursors were cultured in presence of IL-2, a factor known to be critical for thymic Treg cell development, we observed a major impact of IL-2 on the demethylation of the TSDR with a more pronounced effect on Foxp3+ TregP. Together, these results suggest that the establishment of the Treg cell-specific hypomethylation pattern is a continuous process throughout thymic Treg cell development and that the two known Treg cell precursors display distinct dynamics for the imprinting of the Treg cell-specific epigenetic signature genes.

Foxp3 Instability Helps tTregs Distinguish Self and Non-self.

Regulatory T cells (Tregs) are small subsets of CD4 T cells that play a central role in the controlling of immune tolerance. Tregs are either generated in the thymus (tTregs) or the periphery (pTregs), and both express the master transcription factor Foxp3. Stable expression of Foxp3 is important for the maintenance of Tregs identity and their suppressive function. Similar to conventional T cells, Tregs can recognize both self- and non-self-antigens, and TCR engagement leads to Treg activation and the generation of effector Tregs. Emerging shreds of evidence suggest Tregs are not always stable, even fully committed mature tTregs, and can lose foxp3 expression and programming to effector-like T cells. In this review, we summarize recent findings in Treg instability and the intrinsic and extrinsic mechanisms in controlling the Foxp3 expression. Finally, we propose a new hypothesis that Foxp3 instability might help tTregs distinguish between self and non-self-antigens.

The role of two common FOXP3 gene promoter polymorphisms in preeclampsia in a Turkish population: a case–control study

Preeclampsia (PE), which occurs in approximately 5% of pregnancies worldwide and constitutes clinically serious complications in 2–3%, is one of the leading causes of maternal and prenatal morbidity and mortality. Recent studies report that regulatory T (Treg) cells, which act as immunosuppressant, are associated with PE. It is clearly defined that FOXP3/Scurfin (Forkhead Box P3) is involved in the development and function of Tregs. However, there are different conclusions regarding the relationship between PE and FOXP3 gene polymorphisms for different populations. For this reason, in this study we investigate the association between FOXP3 gene promoter region polymorphisms and PE in a Turkish population 500 PE patients and 500 healthy pregnant women. Blood samples taken from pregnant women were studied by PCR-RFLP method. As a result, rs2232365 polymorphism was significantly associated with disease (p < .0001) while no significant association was found between rs3761548 polymorphism and the disease (p = .17). Based on these results, it is though that FOXP3 rs2232365 polymorphism may be predisposed to PE development in terms of Turkish population. However, further and functional studies are needed in terms of other polymorphisms and mutations.IMPACT STATEMENTWhat is already known on this subject? A number of recent publications suggest that Tregs may play a role in the pathogenesis of PE. It is known that a stable and high FOXP3 expression is required to maintain the suppressive T cell function of Tregs. Down regulation of FOXP3 in PE has been reported in many previous studies, but the mechanism is still uncertain.What do the results of this study add? Our study has examined two FOXP3 promoter region polymorphisms in terms of Turkish population for the first time. Rs2232365 polymorphism associated with the disease in heterozygous genotype.What are the implications of these findings for clinical practice and/or further research? It has been shown that FOXP3 gene promoter region polymorphisms may be associated with PE for Turkish population. Our results can be a guide for more detailed statistical evaluations and functional studies.

Signaling Through gp130 Compromises Suppressive Function in Human FOXP3+ Regulatory T Cells.

The CD4+FOXP3+ regulatory T cell (Treg) subset is an indispensable mediator of immune tolerance. While high and stable expression of the transcription factor FOXP3 is considered a hallmark feature of Treg cells, our previous studies have demonstrated that the human FOXP3+ subset is functionally heterogeneous, whereby a sizeable proportion of FOXP3+ cells in healthy individuals have a diminished capacity to suppress the proliferation and cytokine production of responder cells. Notably, these non-suppressive cells are indistinguishable from suppressive Treg cells using conventional markers of human Treg. Here we investigate potential factors that underlie loss of suppressive function in human Treg cells. We show that high expression of the IL-6 family cytokine receptor subunit gp130 identifies Treg cells with reduced suppressive capacity ex vivo and in primary FOXP3+ clones. We further show that two gp130-signaling cytokines, IL-6 and IL-27, impair the suppressive capacity of human Treg cells. Finally, we show that gp130 signaling reduces the expression of the transcription factor Helios, whose expression is essential for stable Treg function. These results highlight the role of gp130 in regulating human Treg function, and suggest that modulation of gp130 signaling may serve as a potential avenue for the therapeutic manipulation of human Treg function.

Mapping the generation and fate of Tregs associated with recovery of Autoimmune Disease

Abstract Experimental autoimmune uveitis (EAU) is used to gain a better understanding of human autoimmune uveitis. Resolution of EAU is in part due to emergence of ocular antigen specific regulatory T cells (Post-EAU Tregs) that are found in the spleen of post-EAU mice. However, the stability of these cells and in what tissues these cells migrate to suppress disease is not known. Therefore, we assessed the stability of post-EAU Tregs, asked where these cells migrate to suppress disease, and determined when and where they emerge during EAU. FoxP3GFP-Cre; Rosa26stopfl/fl-Tom mice were used to identify FoxP3 and exFoxP3 T cells. Post-EAU FoxP3 or exFoxP3 cells were adoptively transferred into recipient EAU mice. The post-EAU exFoxP3 cells did not suppress EAU in the recipient mice, but Tregs capable of suppressing uveitis had stable FoxP3 expression and were found in both the eye and lymphoid tissues at the onset of EAU in recipient mice. In donor mice, both FoxP3 and exFoxP3 cells emerged in the eye and were found in the lymphoid tissues throughout EAU. In contrast, neither FoxP3 nor exFoxP3 T cells were found in the eyes of post-EAU mice, rather were found in lymphoid tissues alone. This study elucidates a dynamic relationship of Treg migration from the eye to secondary lymphoid tissues that coincides with the resolution of EAU. Also, this study highlights that FoxP3 is a requirement for the suppressive function of post-EAU Tregs, and these Tregs traffic to the eye to prevent a uveitic relapse but migrate out after resolution. This work suggests that unstable FoxP3 expression or aberrant tissue trafficking may provide an explanation for chronic and relapsing autoimmune uveitis.

The E3 Ligase TRAF6 directs FOXP3 localization and facilitates Treg function through K63‐type ubiquitination

For the immune system to function properly, the activation of leukocytes must be tightly regulated. Failures of immune control mechanisms underlie the aberrant targeting of self‐antigens (i.e. autoimmunity) and the collateral damage of healthy tissues during excessive responses to infectious threats or the unnecessary immune mobilization against innocuous commensal microbes. Foxp3‐expressing CD4+ Regulatory T cells (Tregs) are a major cellular force working to prevent these pathologies of immune dysregulation. These cells characteristically exhibit multiple suppressive function that is absolutely required for the maintenance of immune homeostasis. However, these cells can hamper desirable anti‐tumor immune responses in cancer patients. Accordingly, a relative abundance of Tregs in the tumor microenvironment (TME) is a negative prognostic factor in many cancers. Treg‐depleting or antagonizing strategies show promise as potential interventions capable of unleashing anti‐tumor immunity from suppression. The characteristic gene expression and suppressive function of Tregs depend considerably on the stable expression and activity of the transcription factor FOXP3. While transcriptional regulation of the Foxp3gene has been studied in depth, both the expression and function of this factor are also modulated at the protein level. However, the molecular players involved in posttranslational FOXP3 regulation are just beginning to be elucidated. We have found that in the absence of the E3 ligase TRAF6 Tregs were dysfunctional in vivo. In these studies, mice with Treg‐restricted deletion of TRAF6 were highly resistant to implanted tumors and displayed enhanced anti‐tumor immunity. We further determined that FOXP3 undergoes lysine‐63 linked (K63) ubiquitination at lysine 262, and this posttranslational modification was mediated by TRAF6. When deprived of TRAF6 activity or rendered insensitive to K63 ubiquitination, FOXP3 displayed aberrant, perinuclear accumulation and disrupted regulatory function. Thus, K63 ubiquitination by TRAF6 ensures proper localization of FOXP3 and facilitates the transcription factor's gene‐regulating activity in Tregs. These results implicate TRAF6 as a key, hitherto unappreciated posttranslational facilitator of FOXP3 stabilization and Treg suppressive function that may be targeted by novel tolerance‐breaking immunotherapies to improve anti‐tumor immunity and combat disease progression in cancer patients.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TRAF6 directs FOXP3 localization and facilitates regulatory T‐cell function through K63‐linked ubiquitination

Regulatory T cells (Tregs) are crucial mediators of immune control. The characteristic gene expression and suppressive functions of Tregs depend considerably on the stable expression and activity of the transcription factor FOXP3. Transcriptional regulation of the Foxp3 gene has been studied in depth, but both the expression and function of this factor are also modulated at the protein level. However, the molecular players involved in posttranslational FOXP3 regulation are just beginning to be elucidated. Here, we found that TRAF6‐deficient Tregs were dysfunctional in vivo; mice with Treg‐restricted deletion of TRAF6 were resistant to implanted tumors and displayed enhanced anti‐tumor immunity. We further determined that FOXP3 undergoes K63‐linked ubiquitination at lysine 262 mediated by the E3 ligase TRAF6. In the absence of TRAF6 activity or upon mutation of the ubiquitination site, FOXP3 displayed aberrant, perinuclear accumulation and disrupted regulatory function. Thus, K63‐linked ubiquitination by TRAF6 ensures proper localization of FOXP3 and facilitates the transcription factor's gene‐regulating activity in Tregs. These results implicate TRAF6 as a key posttranslational, Treg‐stabilizing regulator that may be targeted in novel tolerance‐breaking therapies.

Loss of TET proteins in regulatory T cells promotes abnormal proliferation, Foxp3 destabilization and IL-17 expression.

Ten-eleven translocation (TET) proteins regulate DNA methylation and gene expression by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Although Tet2/Tet3 deficiency has been reported to lead to myeloid cell, B-cell and invariant natural killer T (iNKT) cell malignancy, the effect of TET on regulatory T cells (Tregs) has not been elucidated. We found that Tet2/Tet3 deficiency in Tregs led to lethal hyperproliferation of CD4+Foxp3+ T cells in the spleen and mesenteric lymph nodes after 5 months of age. Additionally, in aged Treg-specific Tet2/Tet3-deficient mice, serum IgG1, IgG3, IgM and IgE levels were markedly elevated. High IL-17 expression was observed in both Foxp3+ and Fopx3- CD4+ T cells, and adoptive transfer of Tet2/Tet3-deficient Tregs into lymphopenic mice inhibited Foxp3 expression and caused conversion into IL-17-producing cells. However, the conserved non-coding DNA sequence-2 (CNS2) region of the Foxp3 gene locus, which has been shown to be particularly important for stable Foxp3 expression, was only partly methylated. We identified novel TET-dependent demethylation sites in the Foxp3 upstream enhancer, which may contribute to stable Foxp3 expression. Together, these data indicate that Tet2 and Tet3 are involved in Treg stability and immune homeostasis in mice.

ELEVATED METHYLATION LEVELS OF FOXP3-TSDR IS ASSOCIATED WITH INCREASED RISK FOR ADVERSE OUTCOMES IN THE PATIENTS WITH ACUTE CORONARY SYNDROME

Demethylation of the FOXP3 corresponds with stability of FOXP3 expression and immunosuppressive function of Tregs. We have demonstrated that reduction in Treg cells is associated with acute coronary syndrome (ACS). The aim of this study was to establish the relationship between methylation level of

Mitochondrial complex III is essential for suppressive function of regulatory T cells.

Regulatory T cells (Treg cells), a distinct subset of CD4+ T cells, are necessary for the maintenance of immune self-tolerance and homeostasis1,2. Recent studies have demonstrated that Treg cells exhibit a unique metabolic profile characterized by an increase in mitochondrial metabolism relative to other CD4+ effector subsets3,4. Furthermore, the Treg cell lineage-defining transcription factor, Foxp3, has been shown to promote respiration5,6; however, it remains unknown whether the mitochondrial respiratory chain is required for Treg cell suppressive capacity, stability, and survival. Here we report that Treg cell-specific ablation of mitochondrial respiratory chain complex III results in the development of a fatal inflammatory disease early in life, without impacting Treg cell number. Mice lacking mitochondrial complex III specifically in Treg cells displayed a loss of Treg cell suppressive capacity without altering Treg cell proliferation and survival. Treg cells deficient in complex III had decreased expression of genes associated with Treg function while maintaining stable Foxp3 expression. Loss of complex III in Treg cells increased DNA methylation as well as the metabolites 2-hydroxyglutarate (2-HG) and succinate that inhibit the ten-eleven translocation (TET) family of DNA demethylases7. Thus, Treg cells require mitochondrial complex III to maintain immune regulatory gene expression and suppressive function.

Human Bone Marrow Derived Mesenchymal Stromal Cells Enhance the Number and Function of Umbilical Cord Blood Peripheral Tregs during IL-2 Driven Ex Vivo Expansion

Tumor growth factor β (TGF-β)-induced peripheral regulatory T cells (pTreg) are a promising therapeutic cell source that exhibit Foxp3 expression and suppressive functions similar to natural regulatory T cells. Nonetheless, their clinical potential is limited by the instability of Foxp3 expression and T cell exhaustion that occurs during ex vivo expansion. We postulated that mesenchymal stromal cells (MSCs) could enhance the number, function and Foxp3 expression stability of pTregs during IL-2 driven 21 day expansion due to their diverse immunomodulatory properties. In this study, we observed that use of a human bone marrow mesenchymal stromal cells (hBM-MSC) platform significantly enhanced the number of pTreg during IL-2 driven 21 day ex vivo expansion vs. standard suspension culture condition (MSC platform: 80.2 x 106 vs. IL2/media: 39.3 x 106, n=6; p<0.01). Also the number of pTreg expressing a naive phenotype (CD4+CD45RA+ and CD4+CD62L+ ) were significantly increased (CD45RA+; MSC platform: 74.4 ± 1.6 x 106 vs. IL2/media: 45.9 ± 2.9 x 106, n=6, p<0.001; CD62L+; MSC platform: 79.1 ± 1.3 x 106 vs. IL2/media: 54.5 ± 2.1 x 106, n=6, p<0.001), as well as stability of Foxp3 expression (IL-2/media: 88.2 ± 1.7% vs. MSC platform: 96.2 ± 1.1%, n=7; p<0.05). In addition, pTreg suppressive function was noted to be more potent during 21 day IL-2 driven ex vivo expansion compared to standard IL-2/media culture condition (MSC platform: 79% vs. media: 35% inhibition of T cell proliferation in 10:1 ratio, n=6; p<0.01). pTreg expanded over a hBM-MSC platform exhibited higher surface CD25, CTLA-4, and ICOS MFI expression (CD25; MSC platform: 1410 vs. Media: 774; p<0.001, CTLA-4; MSC platform: 1084 vs. Media: 318; p<0.001, ICOS; MSC platform: 4386 vs. Media: 2641, p<0.01, n=6). Notably, hBM-MSC enhancement of pTreg ex vivo expansion requires direct cell-cell contact, as Foxp3 expression in pTreg was not enhanced by hBM-MSC conditioned media (CM:73.4 ± 6.8% vs. MSC platform: 96.2 ± 1.0%, p<0.001; and IL2/media: 88.8 ± 1.6% vs. MSC platform: 96.2 ± 1.0%, p<0.01) nor in a trans-well culture experiments (Transwell: 83.4 ± 2.5% vs. IL2/media: 88.8 ± 1.6%; and Transwell: 83.4 ± 2.5% vs. MSC platform: 96.2 ± 1.0%, p<0.01). Importantly, optical sectioning microscopy and flow cytometry revealed that hBM-MSC supports Treg number and function via direct contact-dependent mitochondrial transfer (Figure 1A-B). Cytochalasin B treatment blocked mitochondrial transfer, suggesting that tunneling nanotubes (TNT) facilitate mitochondrial transfer from hBM-MSC to pTreg during IL-2 driven ex vivo expansion (Mock: 2208 ± 122.1 vs. Cyto B: 923.8 ± 89 MFI, n=6, p<0.0001). Moreover, the quantity of ATP (n=6; p<0.01) mitochondrial potential of pTreg (MSC platform: 9010 ± 224.5 vs. media: 7316 ± 122.7 MFI, n=6; p<0.01) were significantly enhanced in pTreg during IL-2 driven ex vivo expansion over a hBM-MSC platform. Taken together, hBM-MSC significantly improves the number, maturation, and function of pTreg during 21 day IL-2 driven ex vivo expansion. We have identified one key mechanism of action of hBM-MSC underlying these favorable effects on pTreg during ex vivo expansion to be mitochondrial transfer via TNT. Notably, these studies identify a novel role of hBM-MSC to overcome current limitations in IL-2/media suspension culture conditions including T cell senescence, and loss of Foxp3 expression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Conversion of T-Effector Cells to Immunosuppressive T-Regulatory-like Cells By CRISPR/Cas9-Mediated Integration of a FOXP3 Transgene

Regulatory T cells (Treg) are distinct among T cell subtypes, having the primary role of suppressing adaptive immune responses. The importance of these cells in immune self-tolerance is underscored by the genetically inherited syndrome IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked), which is caused by an inactivating mutation in FOXP3. FOXP3 is a transcription factor that is a determinant of regulatory T cell function. Patients with IPEX syndrome suffer from the rapid and severe onset of multi-organ autoimmunity, including severe enteropathy, Type I diabetes, thyroiditis, skin inflammation and other features. In mouse models of IPEX, neonatal transplantation of wild-type Tregs is sufficient to prevent the development of disease. Less-severe Treg defects have also been implicated in the etiology of a variety of prevalent autoimmune diseases. It is possible that the pivotal role for Tregs in self-tolerance could be exploited clinically to improve therapies for autoimmunity and other diseases of tolerance. However, the use of autologous ex vivo expanded Treg as a clinical cell therapy is problematic: Tregs are present in low numbers in the peripheral blood, they expand slowly in culture ex vivo, and they may lack antigen specificities necessary for efficient suppression in specialized tissues. They may also down-regulate FOXP3 expression and lose functional activity in vivo in the setting of chronic inflammation. Additionally, autologous Tregs from patients with autoimmune disease may exhibit cell intrinsic dysfunction, while IPEX patients do not even have Tregs. To overcome these issues, we developed a gene editing approach to enforce stable expression of FOXP3 in primary human CD4+ peripheral blood T cells. CRISPR/Cas9 ribonucleoprotein and an AAV6-delivered donor template were developed to target a MND promoter-FOXP3 cDNA expression cassette (linked to a cell surface LNGFR tag by a 2A ribosome skip peptide) to the FOXP3 locus by homology directed repair (HDR). Highly efficient HDR rates were achieved across multiple donors (~34%; 5 donors in 9 experiments). For therapy of IPEX caused by FOXP3 missense mutations, integration of the functional coding sequence simultaneously abolishes endogenous FOXP3 expression. Following gene editing, expression of FOXP3 was sufficient to drive Treg-like phenotypic changes, including the up-regulation of CD25 and inhibitory receptors and down-regulation of CD127 and inflammatory cytokines. Further, consistent with the translatability of this approach into clinical manufacturing, FOXP3+ cells could be enriched to >90% purity by a simple LNGFR antibody column and expanded 20-fold within one week. Importantly, transfer of these edited Treg-like cells (edTreg) to NOD-scid-IL2Rγ-/- mice prevented xeno-graft vs. host disease (xeno-GvHD) mediated by co-transferred autologous effector T cells; xeno-GvHD protection correlated with long-term survival of the edTregs, and a marked reduction in effector T cell expansion and tissue infiltration. These data support the development of edited regulatory T cells for the treatment of IPEX and other autoimmune disease. DisclosuresScharenberg:Generation Bio: Equity Ownership; Casebia Therapeutics: Employment; Alpine Immune Sciences: Equity Ownership.

Regulatory T Cell Plasticity and Stability and Autoimmune Diseases.

CD4+CD25+ regulatory T cells (Tregs) are a class of CD4+ T cells with immunosuppressive functions that play a critical role in maintaining immune homeostasis. However, in certain disease settings, Tregs demonstrate plastic differentiation, and the stability of these Tregs, which is characterized by the stable expression or protective epigenetic modifications of the transcription factor Foxp3, becomes abnormal. Plastic Tregs have some features of helper T (Th) cells, such as the secretion of Th-related cytokines and the expression of specific transcription factors in Th cells, but also still retain the expression of Foxp3, a feature of Tregs. Although such Th-like Tregs can secrete pro-inflammatory cytokines, they still possess a strong ability to inhibit specific Th cell responses. Therefore, the plastic differentiation of Tregs not only increases the complexity of the immune circumstances under pathological conditions, especially autoimmune diseases, but also shows an association with changes in the stability of Tregs. The plastic differentiation and stability change of Tregs play vital roles in the progression of diseases. This review focuses on the phenotypic characteristics, functions, and formation conditions of several plastic Tregs and also summarizes the changes of Treg stability and their effects on inhibitory function. Additionally, the effects of Treg plasticity and stability on disease prognosis for several autoimmune diseases were also investigated in order to better understand the relationship between Tregs and autoimmune diseases.

Induction of CD4+CD25+FOXP3+ regulatory T cells by mesenchymal stem cells is associated with modulation of ubiquitination factors and TSDR demethylation

BackgroundMesenchymal stem cells (MSCs) are known for their ability to induce the conversion of conventional T cells (Tconvs) into induced regulatory T cells (iTregs) in specific inflammatory contexts. Stable Foxp3 expression plays a major role in the phenotypic and functional stability of iTregs. However, how MSCs induce stable Foxp3 expression remains unknown.MethodsWe first investigated the role of cell–cell contact and cytokine secretion by bone marrow-derived MSCs (BM-MSCs) on the induction, stability, and suppressive functions of Tregs under various experimental conditions that lead to Foxp3 generation by flow cytometry and ELISA respectively. Second, we studied the effect of MSCs on TRAF6, GRAIL, USP7, STUB1, and UBC13 mRNA expression in CD4+ T cells in correlation with the suppressive function of iTregs by real-time PCR; also, we investigated Foxp3 Treg-specific demethylated region (TSDR) methylation in correlation with Foxp3 stability by the high-resolution melting technique. Third, we studied the effect of ex-vivo-expanded BM-MSCs on the induction of transplant tolerance in a model of fully allogeneic skin transplantation. We further analyzed the cytokine secretion patterns in grafted mice as well as the mRNA expression of ubiquitination genes in CD4+ T cells collected from the spleens of protected mice.ResultsWe found that in-vitro MSC-induced Tregs express high mRNA levels of ubiquitination genes such as TRAF6, GRAIL, and USP7 and low levels of STUB1. Moreover, they have enhanced TSDR demethylation. Infusion of MSCs in a murine model of allogeneic skin transplantation prolonged allograft survival. When CD4+ T cells were harvested from the spleens of grafted mice, we observed that mRNA expression of the Foxp3 gene was elevated. Furthermore, Foxp3 mRNA expression was associated with increased TRAF6, GRAIL, UBC13, and USP7 and decreased STUB1 mRNA levels compared with the levels observed in vitro.ConclusionsOur data suggest a possible ubiquitination mechanism by which MSCs convert Tconvs to suppressive and stable iTregs.

STAT5 and TET2 Cooperate to Regulate FOXP3-TSDR Demethylation in CD4+ T Cells of Patients with Colorectal Cancer.

The tumor-infiltrating Tregs are linked to colorectal cancer progression and outcome. FOXP3 is regarded as a critical developmental and functional factor for Tregs. FOXP3-TSDR demethylation is required for stable expression of FOXP3 and maintenance of Treg function. In our study, we found specific DNA hypomethylation of FOXP3-TSDR in CD4+ T cells from colon tumor tissues as compared with normal colonic tissues. Moreover, we also found that the expression of STAT5 and TET2 was increased in CD4+ T cells from colon tumor tissues, and the superfluous STAT5 and TET2 binding to FOXP3-TSDR resulted in DNA hypomethylation. In conclusion, we have demonstrated that excessive amounts of STAT5 may bind more TET2 to the FOXP3-TSDR and upregulate FOXP3 expression via DNA demethylation. Our study improved the mechanism of FOXP3-TSDR hypomethylation in tumor-infiltrating CD4+ T cells of CRC patients.

A mechanism involving sibling transcription factor assistance preserves the integrity of an active Foxp3 locus in peripherally induced regulatory T cells.

Abstract Regulatory T (Treg) cell mediated dominant negative regulation is critical for suppressing hyper-immune responses. Broadly classified as thymus derived (tTreg) and peripherally induced (iTreg), these cells share a stringent dependence on the transcription factor Foxp3. In spite of significant advances, precise understanding of the molecular events that promote and preserve unceasing expression of Foxp3 in Treg cells is still incompletely understood. Here we show that Foxp1, a forkhead transcription factor and thus a sibling family member of Foxp3, is essential for sustained high level expression of Foxp3 specifically in iTreg cells. By being readily associated with the Foxp3 locus in a TGFβ dependent manner, Foxp1 critically participates during multiple phases of iTreg development and maturity. By employing an inducible model of temporal deletion of Foxp1 in precursor CD4+ T cells, we found that it is required for optimum expression of Foxp3 during the onset of iTreg induction. Even a conditional ablation of Foxp1 in iTreg cells at a later developmental time point, when high level transcription of Foxp3 was already established, resulted in dramatic instability of the differentiated cell state. Accordingly, Treg specific deletion of Foxp1 renders cells gradually lose Foxp3, resulting in dramatically reduced Nrp1−Helios− iTreg compartment in mice, ensuing significantly augmented intestinal inflammation. Interestingly, the stability of Foxp3 expression in tTreg cells remained unaffected in the absence of Foxp1. Thus, our study unravels a mechanistic module of evolutionarily akin members of a transcription factor subfamily in establishing a novel molecular program indispensable to ensure efficient immune homeostasis.

Dissecting the genetic networks that control regulatory T cell stability using pooled CRISPR screens

Abstract Regulatory T cells (Tregs) are a specialized subset of CD4+ T cells that suppress inflammation to maintain homeostasis and prevent autoimmunity. Treg development and function depend on expression of the master transcription factor Foxp3. While Tregs have been thought to be irreversibly committed to suppressive functions, lineage tracing studies have challenged this by revealing that Tregs can exhibit plasticity. Tregs that lose Foxp3 expression, termed ‘exTregs’, have been shown to acquire cytokine production capabilities of pro-inflammatory effector T cells and exacerbate autoimmunity. However, the gene regulatory programs that promote or disrupt Foxp3 stability in Tregs under various physiological conditions are not well understood. Here we have leveraged improved functional genetic tools, including pooled CRISPR screens, to identify nuclear factors that regulate Treg plasticity. A Foxp3 lineage-tracing reporter mouse model was used to confidently distinguish Foxp3− exTregs from contaminating Foxp3−T effectors. Using HiChIP, we identified Treg and exTreg-specific chromatin loops and systematically assessed their contribution to Foxp3 stability. Furthermore, we investigated transcriptional signatures of distinct exTreg populations residing in the peripheral organs using single-cell RNA sequencing. Collectively, this work helps us better understand the genetic networks that control stable expression of Foxp3 in Tregs as they encounter changing inflammatory environments.

Critical Role of TGF-β and IL-2 Receptor Signaling in Foxp3 Induction by an Inhibitor of DNA Methylation.

Under physiological conditions, CD4+ regulatory T (Treg) cells expressing the transcription factor Foxp3 are generated in the thymus [thymus-derived Foxp3+ Treg (tTregs) cells] and extrathymically at peripheral sites [peripherally induced Foxp3+ Treg (pTreg) cell], and both developmental subsets play non-redundant roles in maintaining self-tolerance throughout life. In addition, a variety of experimental in vitro and in vivo modalities can extrathymically elicit a Foxp3+ Treg cell phenotype in peripheral CD4+Foxp3− T cells, which has attracted much interest as an approach toward cell-based therapy in clinical settings of undesired immune responses. A particularly notable example is the in vitro induction of Foxp3 expression and Treg cell activity (iTreg cells) in initially naive CD4+Foxp3− T cells through T cell receptor (TCR) and IL-2R ligation, in the presence of exogenous TGF-β. Clinical application of Foxp3+ iTreg cells has been hampered by the fact that TGF-β-driven Foxp3 induction is not sufficient to fully recapitulate the epigenetic and transcriptional signature of in vivo induced Foxp3+ tTreg and pTreg cells, which includes the failure to imprint iTreg cells with stable Foxp3 expression. This hurdle can be potentially overcome by pharmacological interference with DNA methyltransferase activity and CpG methylation [e.g., by the cytosine nucleoside analog 5-aza-2′-deoxycytidine (5-aza-dC)] to stabilize TGF-β-induced Foxp3 expression and to promote a Foxp3+ iTreg cell phenotype even in the absence of added TGF-β. However, the molecular mechanisms of 5-aza-dC-mediated Foxp3+ iTreg cell generation have remained incompletely understood. Here, we show that in the absence of exogenously added TGF-β and IL-2, efficient 5-aza-dC-mediated Foxp3+ iTreg cell generation from TCR-stimulated CD4+Foxp3− T cells is critically dependent on TGF-βR and IL-2R signaling and that this process is driven by TGF-β and IL-2, which could either be FCS derived or produced by T cells on TCR stimulation. Overall, these findings contribute to our understanding of the molecular mechanisms underlying the process of Foxp3 induction and may provide a rational basis for generating phenotypically and functionally stable iTreg cells.