Defective Treg Research Articles

TIGIT signaling restores suppressor function of Th1 Tregs.

Th1 Tregs are characterized by the acquisition of proinflammatory cytokine secretion and reduced suppressor activity. Th1 Tregs are found at increased frequency in autoimmune diseases, including type 1 diabetes and multiple sclerosis (MS). We have previously reported that in vitro stimulation with IL-12 recapitulates the functional and molecular features of MS-associated Th1 Tregs, revealing a central role for hyperactivation of the Akt pathway in their induction. TIGIT is a newly identified coinhibitory receptor that marks Tregs that specifically control Th1 and Th17 responses. Here, we report that signaling through TIGIT counteracts the action of IL-12 in inducing the Th1 program. Specifically, TIGIT signaling represses production of IFN-γ and T-bet expression and restores suppressor function in Tregs treated with IL-12. FoxO1 functional inhibition abolishes the protective effect of TIGIT, indicating that TIGIT signaling promotes FoxO1 nuclear localization. Consistent with this observation, signaling through TIGIT leads to a rapid suppression of Akt function and FoxO1 phosphorylation. Finally, TIGIT stimulation reduces the production of IFN-γ and corrects the suppressor defect of Tregs from patients with MS. Our results indicate an important role for TIGIT in controlling the functional stability of Tregs through repression of Akt, suggesting that the TIGIT pathway could be targeted for immunomodulatory therapies in human autoimmune disorders.

Past, Present, and Future of Regulatory T Cell Therapy in Transplantation and Autoimmunity.

Regulatory T cells (Tregs) are important for the induction and maintenance of peripheral tolerance therefore, they are key in preventing excessive immune responses and autoimmunity. In the last decades, several reports have been focussed on understanding the biology of Tregs and their mechanisms of action. Preclinical studies have demonstrated the ability of Tregs to delay/prevent graft rejection and to control autoimmune responses following adoptive transfer in vivo. Due to these promising results, Tregs have been extensively studied as a potential new tool for the prevention of graft rejection and/or the treatment of autoimmune diseases. Currently, solid organ transplantation remains the treatment of choice for end-stage organ failure. However, chronic rejection and the ensuing side effects of immunosuppressants represent the main limiting factors for organ acceptance and patient survival. Autoimmune disorders are chronic diseases caused by the breakdown of tolerance against self-antigens. This is triggered either by a numerical or functional Treg defect, or by the resistance of effector T cells to suppression. In this scenario, patients receiving high doses of immunosuppressant are left susceptible to life-threatening opportunistic infections and have increased risk of malignancies. In the last 10 years, a few phase I clinical trials aiming to investigate safety and feasibility of Treg-based therapy have been completed and published, whilst an increasing numbers of trials are still ongoing. The first results showed safety and feasibility of Treg therapy and phase II clinical trials are already enrolling. In this review, we describe our understanding of Tregs focussing primarily on their ontogenesis, mechanisms of action and methods used in the clinic for isolation and expansion. Furthermore, we will describe the ongoing studies and the results from the first clinical trials with Tregs in the setting of solid organ transplantation and autoimmune disorders. Finally, we will discuss strategies to further improve the success of Treg therapy.

Absence of MHC-II expression by lymph node stromal cells results in autoimmunity

How lymph node stromal cells (LNSCs) shape peripheral T-cell responses remains unclear. We have previously demonstrated that murine LNSCs, lymphatic endothelial cells (LECs), blood endothelial cells (BECs), and fibroblastic reticular cells (FRCs) use the IFN-γ-inducible promoter IV (pIV) of the MHC class II (MHCII) transactivator CIITA to express MHCII. Here, we show that aging mice (>1 yr old) in which MHCII is abrogated in LNSCs by the selective deletion of pIV exhibit a significant T-cell dysregulation in LNs, including defective Treg and increased effector CD4+ and CD8+ T-cell frequencies, resulting in enhanced peripheral organ T-cell infiltration and autoantibody production. The proliferation of LN-Tregs interacting with LECs increases following MHCII up-regulation by LECs upon aging or after exposure to IFN-γ, this effect being abolished in mice in which LECs lack MHCII. Overall, our work underpins the importance of LNSCs, particularly LECs, in supporting Tregs and T-cell tolerance.

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.

Decreased regulatory function of CD4+CD25+CD45RA+ T cells and impaired IL-2 signalling pathway in patients with type 2 diabetes mellitus.

In this study, the frequency and function of CD4+CD25+CD45RA+ regulatory T cells (Treg) and intracellular IL-2 signalling molecules in patients with type 2 diabetes mellitus (T2DM) were investigated. Tregs and responder T cells (Tresp, CD4+CD25- T cells) were sorted and suppression assays were performed using flow cytometry. Phosphorylation of signal transducer and activator of transcription-5 (pSTAT5) were assessed using flow cytometry. Gene expression of FOXP3 was performed with the SYBR green Real Time PCR method. Production of IL-2 from cultured cells was assessed using ELISA. We observed a functional defect of CD4+CD25+CD45RA+ Tregs in T2DM patients with higher proliferation of Tresp cells, in response to anti-CD3 and anti CD28 stimulation in the presence of Tregs invitro. The results showed that the proliferation of Tresps in the absence of Treg cells was higher in T2DM patients than in healthy controls. Decreased FOXP3 mRNA expression and pSTAT5 were observed within the Tregs of the patients, whereas the level of secreted IL-2 from PBMCs culture was not statically different between T2DM patients and healthy individuals. Changes in intracellular IL-2 pathways and FOXP3 gene expression may contribute to the defect of Tregs in T2DM patients. These findings indicating that the purified CD4+CD25+CD45RA+ Treg cells have reduced functional capacity together with impaired IL-2 pathway in T2DM, and the Tregs could be used for a potential novel therapeutic target.

Diagnosis of DOCK8 deficiency using Flow cytometry Biomarkers: an Egyptian Center experience

In the past few years, several genes were shown to be implicated in various forms of the Hyper Immunoglobulin E syndrome. The present study is the first to describe a cohort of DOCK8 deficiency patients from Egypt. The study included 15 patients with features of combined immunodeficiency (CID) suggestive of DOCK8 deficiency. Flow cytometry was used for evaluation of DOCK8 expression and studying different immunological characteristics of those patients including evaluation of Th17, Tregs, T and B lymphocytes differentiation and the effect of the DOCK8 deficiency on the activation of the STAT3. Diagnosis was confirmed by mutational analysis.Profound defects in Th17 cells and Tregs were observed in all patients with impaired STAT3 phosphorylation, indicating that DOCK8 plays a pivotal role in the STAT3 signaling pathway. These findings together with decrease in memory B cells and defective DOCK8 expression by flow cytometry can confirm the diagnosis.

Defective Treg response in acute kidney injury was caused by a reduction in TIM-3+ Treg cells

ABSTRACTBackground: Despite years of research, the treatment of acute kidney injury (AKI) remains a significant challenge. Animal studies presented causal links between elevated regulatory T cell (Treg) response and better prognosis in AKI. Previous studies in mice and humans showed that TIM-3+ Treg cells were more potent than TIM-3- Treg cells. In this study, we investigated the role of TIM-3 in Treg in AKI patients.Methods: Peripheral blood from AKI patients and healthy controls were gathered, and TIM-3+ Treg subset was examined.Results: Compared to healthy controls, the AKI patients presented a significant upregulation in the frequency of circulating CD4+CD25+ T cells; however, the majority of this increase was from the CD4+CD25+TIM-3- subset, and the frequency of CD4+CD25+TIM-3+ T cells was downregulated in AKI patients. In both healthy controls and AKI patients, the CD4+CD25+TIM-3+ T cells expressed higher levels of Foxp3, and were more potent at expressing LFA-1, LAG-3, CTLA-4, IL-10 and TGF-β. In addition, the CD4+CD25+TIM-3+ T cells from both healthy controls and AKI patients presented higher capacity to suppress CD4+CD25- T cell proliferation than the CD4+CD25+TIM-3- T cells. Interestingly, the total CD4+CD25+ T cells from AKI patients presented significantly lower inhibitory capacity than those from healthy controls, indicating that the low frequency of CD4+CD25+TIM-3+ T cells was restricting the efficacy of the Treg responses in AKI patients.Conclusions: We demonstrated that TIM-3 downregulation impaired the function of Treg cells in AKI. The therapeutic potential of CD4+CD25+TIM-3+ T cells in AKI should be investigated in future studies.

“Alarming" T-Regulatory Cells (Tregs) to Protect from Type 1 Diabetes (T1D)

Defects in IL-2 signaling and Tregs correlate with susceptibility to T1D. Helminth burden also negatively correlates with the incidence of T1D. The alarmin IL-33 and its receptor ST2 are elicited in anti-helminth immunity, which also increase Tregs, M2 macrophages and Th2 response. We hypothesized that IL-33 may cooperate with IL-2 in promoting Tregs to protect from T1D. Indeed, a major subset of Tregs express ST2, thus enabling a role for IL-33 in Treg biology. Non-obese diabetic (NOD) mice, which have less Tregs than the nondiabetic NOD.Idd3 congenic mice, also exhibit less ST2+ Tregs (13±3%) than NOD.Idd3 (38±11%). For better cooperativity and preferential targeting of Tregs, we generated a hybrid cytokine (IL233) bearing IL-2 and IL-33 activities in a single molecule. Recent-hyperglycemia onset NOD mice (n≥6/group) were treated with daily injections (3.3pmol/g) of IL-2 and/or IL-33 or IL233 for 5 days. IL-33 alone offered a similar, if not better reversal of T1D with a 70% cumulative incidence (CI) compared to IL-2 alone (80% CI), whereas IL233 offered the best protection with 50% of mice remaining in remission 10-weeks post-treatment. There was a robust increase in the proportion of Tregs in pancreatic lymph node in IL233 group (25±3.7%) compared to 10±3.9%, 16±4.1%, 14±4.3% and 19±4.3% in Saline, IL-2 only, IL-33 only and IL-2 and IL-33 groups respectively. IL233 treatment also increased the levels of Tregs and group-2 innate lymphoid cells in an alternate species (rats) and blocked high-dose streptozotocin (STZ)-induced beta-cell death. Interestingly, the average islet size was bigger in the rats treated with IL233 in the STZ model, indicating a role of IL233 in islet growth and repair. Thus, IL233 utilizes the synergy of IL-2 and IL-33 to promote Tregs and bears strong therapeutic potential for T1D. Disclosure R. Sharma: None. V. Sabapathy: None.

2028 Discovery and evaluation of FOXP3 dimerization inhibitors

OBJECTIVES/SPECIFIC AIMS: Immuno-oncology (IO) strategies are promising new approaches for the treatment of a variety of malignancies, including multiple myeloma (MM). Regulatory T cells (Tregs), which suppress effector T cell function, are a limitation to durable IO responses. The transcription factor FOXP3 is critical for the mature Treg phenotype. FOXP3 homodimerization is required for DNA binding and transcriptional activity, and mutations mapping to the dimerization region are associated with IPEX syndrome, resulting in dysfunctional Tregs in humans. We therefore hypothesize that inhibitors of FOXP3 dimerization will repress Treg suppression and enhance the anti-MM activity of IO. METHODS/STUDY POPULATION: To discover FOXP3 dimerization inhibitors, we are modeling FOXP3 homodimerization in vitro. Currently, we are optimizing an ALPHA screen and an ELISA-based dimerization assay using recombinant full length and truncated versions of FOXP3 to discover peptidomimetics that inhibit homodimerization. Induced Tregs expanded from human PBMCs will be treated with lead biologics and functional assays will be performed. RESULTS/ANTICIPATED RESULTS: Here we demonstrate Treg suppression of T cell proliferation and IFN-γ secretion after 5 days of co-culture under basal conditions. Additionally, we developed a MM/T cell co-culture system to measure anti-MM T cell responses and show decreased anti-MM T cell activity in the presence of Tregs. We expect to exploit the assays outlined here to demonstrate defective Treg suppression when FOXP3 dimerization is inhibited. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies support drug discovery efforts that will ultimately improve IO therapies for patients with MM.

Expression of Eos is critical for T regulatory cell (Treg) survival and function

Abstract Eos is a transcription factor belonging to the Ikaros transcription factor family and is preferentially expressed in Treg and activated T-conventional (T conv) cells. Our previous studies had shown that young mice with a global deletion of Eos did not appear to have defective Treg. To fully characterize the role of Eos in Treg function, we generated Eosfl/flx Foxp3Cre (Eos cKO) mice. Beginning at 3–4 months of age, both CD4+ and CD8+ T cells, as well as Treg in the Eos cKO mice expressed an activated/memory phenotype. The percentage of Tregs in the Eos cKO mice was higher than controls, suggesting that the Eos deficient Tregs were attempting to inhibit the activated immune response. H&E staining revealed severe lymphoid infiltration of lungs, kidneys, liver, small intestine, salivary glands of Eos cKO mice. The autoimmune state in the Eos cKO mice was characterized by enhanced levels of IFN-g production by CD4+ Foxp3− T cells, elevated percentages of T-follicular helper cells, and elevated levels of serum anti-nuclear antibodies. Tregs from Eos cKO mice failed to protect in the IBD transfer model and cKO mice also demonstrated enhanced susceptibility to EAE. Nevertheless, the in vitro suppressive function of Treg from cKO was normal. We determined the phenotype of Eos deficient Tregs under non-inflammatory conditions in heterozygous Eosfl/fl Foxp3cre/+ female mice. Although the thymic development of Eos deficient Tregs was not impaired, a marked reduction in Eos deficient Tregs was present in secondary lymphoid tissues indicating a defect in Treg survival in a competitive environment. We conclude that Eos is not required for the development of Tregs, but plays a critical role in controlling many, but not all, of the suppressive functions of Treg.

Lupus susceptibility gene Esrrg regulates Treg development and suppressive function through mitochondrial metabolism

Abstract Estrogen-Related Receptor Gamma (Esrrg) is the candidate gene for the Sle1c2 susceptibility locus, which encodes the orphan nuclear receptor ERR-g, in the NZM2410 mouse model of Systemic Lupus Erythematosus (SLE). Congenic C57BL/6 (B6) mice expressing this locus have an increased IFN-g production and dysregulated metabolism in their CD4 T cells, but the role of ERR-g in CD4 T cells is still unclear. In muscle, heart, iPSCs, pancreatic β cells and neurons, Esrrg has been identified as a major regulator of mitochondrial metabolism. We generated Treg conditional Esrrg knockout mice Foxp3cre Esrrgf/f (Esrrg-KO) and compared CD4+ T cells between Esrrg-KO and B6 mice. Tregs from Esrrg-KO mice showed a reduced expression of Foxp3 and CD25, and other markers associated with Treg suppressive activity. Esrrg-KO mice demonstrated non-Treg global CD4 T cell activation, including the increased frequency and number of CD69 and T effector memory cells, as well as increased spontaneous T follicular helper (Tfh) cells and decreased T follicular regulatory (Tfr) cell frequencies. In the T-dependent NP-KLH immunization, Esrrg-KO mice produced increased Tfh and germinal center B cells and less Tfr, which led to high affinity anti-NP IgG. Esrrg-deficiency in Tregs significantly altered the expression of genes and metabolites, ATP, NAD+ and ROS, involved in mitochondria function. Esrrg-KO mice also exhibited a high expression of mTORC1 and Hif1-a, a pathway that is known to destabilize Foxp3 expression. Finally, Esrrg-deficiency decreased both respiration and glycolysis in Tregs. These results demonstrate that Esrrg-deficiency results in defective mitochondrial function in Tregs, which leads to defective Tregs and global CD4 T cell hyperactivation.

C-Maf and STAT3 are essential but distinct regulators of RORγt+ and follicular regulatory T cells

Abstract Foxp3+ regulatory T cells (Tregs) must adapt to function within a wide range of tissue environments and types of immune response. This flexibility is achieved through specialization, whereby context-dependent transcriptional changes integrate unique functionality into general Treg programming. In the most well-studied examples, this involves the co-expression of Foxp3 and another “lineage-defining” transcription factor (TF) which confers expression of specific functional genes. Two such specialized populations are RORγt+ Tregs and Bcl-6+ T follicular regulatory (Tfr) cells, which have important functions in regulating colitis and humoral immune responses, respectively. Although the importance of Treg specialization is now clear, the underlying network of signals and TFs driving this process is largely undefined. Here, we identify the TF c-Maf as an essential regulator of Treg specialization for both RORγt+ Tregs and Tfr cells. Importantly, the role of c-Maf in these subsets is highly specific, because deletion of Maf does not affect general Treg development, generation of other specialized Tregs, or expression of suppressive molecules. In vitro, we show that addition of IL-6 to Treg-promoting conditions is sufficient to drive both c-Maf expression and c-Maf-dependent conversion of naïve T cells to RORγt+ Tregs, suggesting that IL-6 might drive c-Maf in vivo. Surprisingly, although STAT3 – the primary downstream effector of IL-6 – is required for development of both RORγt+ Tregs and Tfr cells, we find that Stat3 deletion in vivo does not ablate c-Maf expression and results in Treg defects distinct from Maf deletion. Therefore, c-Maf and STAT3 are cooperative, but distinct regulators of RORγt+ and follicular Treg development.

Low-dose IL-2 expands CD4+ regulatory T cells with a suppressive function in vitro via the STAT5-dependent pathway in patients with chronic kidney diseases

Background: Patients with chronic kidney disease (CKD) often have CD4+ regulatory T cells (Tregs) dysfunction and chronic inflammation. We aim to investigate the effect, function, and related mechanism of low-dose IL-2 on CD4+ regulatory T cells expansion in vitro from patients with CKD. Methods: A total of 148 newly diagnosed patients with CKD at Stage III and 35 healthy volunteer subjects were recruited into our studies. The number of peripheral Tregs in peripheral blood mononuclear cells isolated from CKD patients, which were characterized by FACS as CD4+CD25hi and CD4+CD25+FoxP3+. The effect of low-dose IL-2 on expansion of Tregs, and the suppressive function of expanded Tregs were also analyzed by FACS. The levels of FoxP3 mRNA were detected by qRT-PCR. The activation of IL-2 induced Stat5 and blocking experiments were assessed by Western Blotting and FACS. Results: We found that the frequency of peripheral Tregs from CKD patients was significantly lower than that in healthy volunteer subjects. We also showed that IL-2 selectively expanded CD4+CD25hi and CD4+CD25+FoxP3+ regulatory T cells, and also upregulated the expression of FoxP3 mRNA. Our in vitro studies demonstrated that expanded CD4+ regulatory T cells from CKD patients suppressed proinflammatory Th1 and Th17 cell response. Furthermore, STAT5 activation is required for IL-2-induced expansion of regulatory T cells and expression of FoxP3 mRNA from CKD patients. Conclusions: Our findings support the clinical Treg defects in CKD patients with glomerular diseases, and the rationale of evaluating low-dose IL-2 treatment for selectively modulating CD4+ Tregs.

Regulatory T Cells As Supporters of Psychoimmune Resilience: Toward Immunotherapy of Major Depressive Disorder.

There is growing evidence that inflammation plays a role in major depressive disorder (MDD). As the main role of regulatory T cells (Tregs) is to control inflammation, this might denote a Treg insufficiency in MDD. However, neither a qualitative nor a quantitative defect of Tregs has been ascertained and no causality direction between inflammation and depression has been established. Here, after reviewing the evidence supporting a relation between Treg insufficiency and MDD, we conclude that a novel therapeutic approach based on Treg stimulation could be valuable in at least the subset of patients with inflammatory MDD. Low-dose interleukin-2 appears to be a good candidate as it is not only a safe stimulator of Tregs in humans but also an inhibitor of pro-inflammatory Th17 lymphocytes. Here, we discuss that a thorough immune investigation as well as immunotherapy will be heuristic for deciphering the pathophysiology of MDD.

Functional Defects of Regulatory T Cell Through Interleukin 10 Mediated Mechanism in the Induction of Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM) is a metabolic and low-grade inflammatory disease most commonly found in pregnant women with high body mass index and non-Caucasian ethnicities; however, not all women of high-risk groups develop GDM. We hypothesized that regulatory T cells (Tregs) might present a role in suppressing GDM development. To this end, 55 high-risk women at early pregnancy (first trimester) were recruited, and 21 of them developed GDM while the other 34 did not. Compared to those subjects who did not develop GDM (non-GDM), the patients who developed GDM presented reduced levels of Tregs and elevated levels of serum interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha). The Tregs in the GDM group also presented reduced levels of transforming growth factor beta and IL-10, compared with the non-GDM group. The frequency of circulating Tregs and serum TNF-alpha level were inversely correlated. In addition, addition of Tregs from non-GDM patients, but not those from GDM patients, significantly suppressed the interferon gamma and TNF-alpha production by effector T cells through IL-10-mediated mechanisms, suggesting a functional defect in Tregs from GDM subjects. Together, these data indicated that the presence of functional Tregs could protect the pregnant women from GDM development by suppressing pro-inflammatory responses and that the dysregulation of Tregs early in pregnancy elevated the risk of GDM.

All-Trans Retinoic Acid Induces CD4+CD25+FOXP3+ Regulatory T Cells by Increasing FOXP3 Demethylation in Systemic Sclerosis CD4+ T Cells.

Background Retinoic acid (RA) is an active metabolite of vitamin A and has been reported to improve the clinical symptoms of patients with systemic sclerosis (SSc). However, the mechanism of RA in the prevention of SSc remains unclear. Regulatory T cells (Tregs) are a subpopulation of T cells with immunosuppressive activity. The quantitative and functional defects of Tregs may mediate the immune dysfunction in SSc. The addition of all-trans retinoic acid (ATRA) to human naïve CD4+ cells could promote the maturation of Tregs and increase the stable expression of Foxp3. In this study, we explored the role of RA on Tregs in SSc CD4+ T cells and its possible epigenetic mechanisms, so as to further understand the mechanisms of RA on SSc. Methods CD4+ T cells were isolated from peripheral blood of SSc and treated with or without ATRA and/or transforming growth factor-β (TGF-β). The percentage of CD4+CD25+FOXP3+ Tregs was counted by flow cytometry. FOXP3 mRNA and protein levels were measured by quantitative real-time reverse transcriptase polymerase chain reaction and Western blotting, respectively. Bisulfite sequencing was performed to determine the methylation status of the FOXP3 proximal promoter sequences. Results The expression of Tregs and FOXP3 in CD4+ T cells from patients with SSc increased in response to ATRA. Moreover, combined stimulation with ATRA and TGF-β resulted in the enhancement of these effects. Further studies revealed that stimulation with ATRA increased the expression of FOXP3 in SSc CD4+ T cells by downregulating FOXP3 promoter methylation levels. Conclusions ATRA acts as an inducer of Treg response in SSc CD4+ T cells via demethylation of the FOXP3 promoter and activation of FOXP3 expression. This may be one of the molecular mechanisms for ATRA, and therefore, RA can be used for the treatment of SSc.

Autoimmunity and its association with regulatory T cells and B cell subsets in patients with common variable immunodeficiency

BackgroundCommon variable immunodeficiency (CVID) is one of the most prevalent symptomatic primary immunodeficiencies (PIDs), which manifests a wide clinical variability such as autoimmunity, as well as T cell and B cell abnormalities. MethodsA total of 72 patients with CVID were enrolled in this study. Patients were evaluated for clinical manifestations and classified according to the presence or absence of autoimmune disease. We measured regulatory T cells (Tregs) and B-cell subsets using flow cytometry, as well as specific antibody response (SAR) to pneumococcal vaccine, autoantibodies and anti-IgA in patients. ResultsTwenty-nine patients (40.3%) have shown at least one autoimmune manifestation. Autoimmune cytopenias and autoimmune gastrointestinal diseases were the most common. A significant association was detected between autoimmunity and presence of hepatomegaly and splenomegaly. Among CVID patients, 38.5% and 79.3% presented a defect in Tregs and switched memory B-cells, respectively, whereas 69.0% presented CD21low B cell expansion. Among patients with a defect in Treg, switched memory and CD21low B cell, the frequency of autoimmunity was 80.0%, 52.2% and 55.0%, respectively. A negative correlation was observed between the frequency of Tregs and CD21low B cell population. 82.2% of patients had a defective SAR which was associated with the lack of autoantibodies. ConclusionsAutoimmunity may be the first clinical manifestation of CVID, thus routine screening of immunoglobulins is suggested for patients with autoimmunity. Lack of SAR in CVID is associated with the lack of specific autoantibodies in patients with autoimmunity. It is suggested that physicians use alternative diagnostic procedures.

Pediatric autoimmune hepatitis shows a disproportionate decline of regulatory T cells in the liver and of IL-2 in the blood of patients undergoing therapy.

Background & AimsThe autoimmune hepatitis (AIH) is a chronic hepatitis driven by the adaptive immunity that affects all age groups. A functional and numerical regulatory T cell (Treg) defect has been reported in pediatric AIH (pAIH), while an intrahepatic increase in adult AIH (aAIH) patients has been detected in current research findings.MethodsTherefore, we quantified the intrahepatic numbers of Treg, T and B cells, as well as serum cytokine levels before and during therapy in pAIH.ResultsWe found a disproportional intrahepatic enrichment of Tregs in untreated pAIH compared to pediatric non-alcoholic fatty liver disease. The increase of Treg/total T cells was even more pronounced than in aAIH due to fewer infiltrating T and B cells. Portal densities of Treg, as well as total T and B cells, declined significantly during therapy. However, portal Treg densities decreased disproportionately, leading to even decreasing ratios of Treg to T and B cells during therapy. Out of 28 serum cytokines IL-2 showed the strongest (10fold) decrease under therapy. This decline of IL-2 was associated with decreasing intrahepatic Treg numbers under therapy. None of the baseline T and B cell infiltration parameters were associated with the subsequent treatment response in pAIH.ConclusionsIntrahepatic Tregs are rather enriched in untreated pAIH. The disproportional decrease of Tregs during therapy may be caused by a decrease of IL-2 levels. New therapies should, therefore, aim in strengthening intrahepatic immune regulation.

Elevated levels of Bcl-3 inhibits Treg development and function resulting in spontaneous colitis

Bcl-3 is an atypical NF-κB family member that regulates NF-κB-dependent gene expression in effector T cells, but a cell-intrinsic function in regulatory T (Treg) cells and colitis is not clear. Here we show that Bcl-3 expression levels in colonic T cells correlate with disease manifestation in patients with inflammatory bowel disease. Mice with T-cell-specific overexpression of Bcl-3 develop severe colitis that can be attributed to defective Treg cell development and function, leading to the infiltration of immune cells such as pro-inflammatory γδT cells, but not αβ T cells. In Treg cells, Bcl-3 associates directly with NF-κB p50 to inhibit DNA binding of p50/p50 and p50/p65 NF-κB dimers, thereby regulating NF-κB-mediated gene expression. This study thus reveals intrinsic functions of Bcl-3 in Treg cells, identifies Bcl-3 as a potential prognostic marker for colitis and illustrates the mechanism by which Bcl-3 regulates NF-κB activity in Tregs to prevent colitis.

CTLA-4 haploinsufficiency in a patient with an autoimmune lymphoproliferative disorder

CTLA-4 haploinsufficiency in a patient with an autoimmune lymphoproliferative disorder