Treg Phenotype Research Articles

Research Highlights

Research Highlights

ВЛИЯНИЕ IL-7 И IL-15 НА T-РЕГУЛЯТОРНЫЕ КЛЕТКИ ЗДОРОВЫХ ДОНОРОВ И ПАЦИЕНТОВ С РЕВМАТОИДНЫМ АРТРИТОМ IN VITRO

The aim of this study was to assess the infl uence of homeostatic factors IL-7 and IL-15, that induced homeostatic proliferation (HP), on the phenotypic characteristics of T-regulatory cells (Treg) from healthy donors and patients with rheumatoid arthritis (RA) in vitro. Analysis of Treg phenotype was performed by fl ow cytometry, using Foxp3, CD127, CD4 and CD25. Isolated from the blood cells were cultured with and without stimulation during 7 days. IL-7, IL-15 and anti-CD3 antibodies supplemented IL-2 were chosen as activators. Purifi ed Treg cells were labeled with CFSE dye to assess proliferation. We estimated that HP factors eff ectively maintain the number and phenotype (CD25 + FoxP3 + ) of Treg in vitro. Also, in patients group we showed an increasing of CD4 + CD25 + FoxP3 + cells under all stimulation condition that may indicate the induction of Treg from conventional T cells. Herewith under IL-7 and IL-15 Treg cells from RA patients had a low proliferative activity than Treg from donors. The reduced proliferative activity of Treg in the group of RA-patients under IL-7 and IL-15 perhaps make a signifi - cant contribution to the development of the disease due to the delay of Treg pool reconstitution in the lymphopenia conditions on the initial stage. Furthermore, increased induction of CD4 + CD25 + FoxP3 + cells in PBMC cultures in patients group associated with pathogenesis of RA, since induced Treg have transient and unstable expression of FoxP3.

The Migratory Properties and Numbers of T Regulatory Cell Subsets in Circulation Are Differentially Influenced by Season and Are Associated With Vitamin D Status.

The control of peripheral immune responses by FOXP3+ T regulatory (Treg) cells is essential for immune tolerance. However, at any given time, Treg frequencies in whole blood can vary more than fivefold between individuals. An understanding of factors that influence Treg numbers and migration within and between individuals would be a powerful tool for cellular therapies that utilize the immunomodulatory properties of Tregs to control pathology associated with inflammation. We sought to understand how season could influence Treg numbers and phenotype by monitoring the proportion of natural thymus-derived Tregs (nTregs) defined as (CD3+CD4+CD25+FOXP3+CD127–/low) cells as a proportion of CD4+ T cells and compared these to all FOXP3+ Tregs (allTregs, CD3+CD25+FOXP3+CD127–/low). We were able to determine changes within individuals during 1 year suggesting an influence of season on nTreg frequencies. We found that, between individuals at any given time, nTreg/CD4+ T cells ranged from 1.8% in February to 8.8% in the summer where median nTreg/CD4 in January and February was 2.4% (range 3.75–1.76) and in July and August was 4.5% (range 8.81–3.17) p = 0.025. Importantly we were able to monitor individual nTreg frequencies throughout the year in donors that started the year with high or low nTregs. Some nTreg variation could be attributed to vitamin D status where normal linear regression estimated that an absolute increase in nTreg/CD4+ by 0.11% could be expected with 10 nmol increase in serum 25 (OH) vitamin D3 (p = 0.005, 95% CI: 0.03–0.19). We assessed migration markers on Tregs for the skin and/or gut. Here cutaneous lymphocyte associated antigen (CLA+) expression on CD25+FOXP3+CD4+/CD4+ was compared with the same population expressing the gut associated integrin, β7. Gut tropic CD25+FOXP3+β7+Tregs/CD4+ had similar dynamics to nTreg/CD4+. Conversely, CD25+FOXP3+CLA+Tregs/CD4+ showed no association with vitamin D status. Important for cellular therapies requiring isolation of Tregs, the absolute number of β7+CD4+CD25+FOXP3+Tregs was positively associated with 25(OH)vitamin D3 (R2 = 0.0208, r = 0.184, p = 0.021) whereas the absolute numbers of CLA+CD4+CD25+FOXP3+Tregs in the periphery were not influenced by vitamin D status. These baseline observations provide new opportunities to utilize seasonal variables that influence Treg numbers and their migratory potential in patients or donors.

Impact of fascioliasis reinfection on Fasciola hepatica egg shedding: relationship with the immune-regulatory response

Fascioliasis is a disease caused by liver flukes. In human fascioliasis hyperendemic areas, reinfection and chronicity are the norm. Control strategies in humans require the use of egg count techniques to calculate the appropriate treatment dose for colic risk prevention. The present study investigates how fascioliasis reinfection affects liver fluke egg shedding and its relationship with the immune-regulatory response. The experimental design reproduced the usual reinfection/chronicity conditions in human fascioliasis endemic areas and included Fasciola hepatica primo-infected Wistar rats (PI) and rats reinfected at 4 weeks (R4), 8 weeks (R8), 12 weeks (R12), and negative control rats. In a longitudinal study (0-20 weeks post-infection, p.i.), serical IgG1 levels and eggs per gram of faeces (epg) were analyzed. In a cross-sectional study, the expression of the genes associated with Th1 (Ifng, Il12a, Il12b, Nos2), Th2 (Il4, Arg1), Treg (Foxp3, Il10, Tgfb, Ebi3), and Th17 (Il17) in the spleen and thymus was analyzed. In R8 and R12, transiently higher averages of epg and epg/worm in reinfected groups vs PI group were detected at least in the weeks following reinfection. The kinetics of IgG1 levels shows that reinfected groups followed a pattern similar to the one in the PI group, but transiently higher averages of IgG1 levels in reinfected groups vs the PI group were detected in the weeks following reinfection. Epg correlated with IgG1 levels and also with systemic Il10 and thymic Ifng, and Il10 expression levels. These results suggest that epg depends on the Th1 and Treg phenotype and that the determination of the fluke burden by epg is likely to be an overestimation in cases of recent reinfection in low burden situations. A strategy to facilitate the implementation of epg count techniques and the subsequent decision on the appropriate treatment dose for each patient to prevent colic risk is required.

In Silico Modeling of Metabolic State in Single Th17 Cells Reveals Novel Regulators of Inflammation and Autoimmunity

Abstract Cellular metabolism is a major regulator of immune response, but it is difficult to study the metabolic status of an individual immune cell using current technologies. Here, we present Compass, an algorithm to characterize the metabolic landscape of single cells in silico based on single-cell RNA-Seq and flux balance analysis. We used Compass to study the landscape of metabolic heterogeneity in Th17 cells and search for novel metabolic regulators of their inflammatory function. In central carbon metabolism, Compass predicted a metabolic switch between glycolysis and fatty acid oxidation that mirrors the Th17 vs. Treg phenotype, which we validated through transcriptomic, metabolic and functional assays. The TCA cycle was predicted to break at two points, both of which have been independently identified by other groups in M1 macrophage polarization. Surprisingly, and contrary to common immunometabolic understanding, Compass predicted that glycolysis too was divided into functional modules, and that one of them supported an anti-inflammatory phenotype. We validate the paradoxical prediction and demonstrate that inhibition of this module promotes a pro-inflammatory transcriptional program in Th17 cells, resulting in neuroinflammation in an adoptive transfer model of autoimmune disease. In conclusion, Compass is a widely applicable algorithm to characterize metabolic states at single cell resolution. It allows associating cellular metabolic states with effector functions and detection of metabolic targets that regulate effector phenotypes. We expect it to become a widely used tool with the increasing availability of single-cell RNA-Seq data, spurred by efforts such as the human cell atlas.

Anergy-derived Foxp3+ T regulatory cell differentiation in the neonatal mouse

Abstract The neonatal period is a natural scenario to study the effects of peripheral self-antigen (Ag) recognition by autoreactive conventional CD4+ T cells under conditions of low Foxp3+ T regulatory cell (Treg) numbers and function. Moreover, the neonatal environment is an interesting scenario to study the relationship between the induction of CD4+ T cell anergy and the peripheral generation of anergy-derived Tregs, given that anergy induction relies on normal Treg function whereas anergy-derived Treg generation is optimal in the setting of Treg cell deficiency. We have hypothesized that the neonatal period is both necessary and sufficient to allow for the induction of anergy as well as the peripheral differentiation of anergy-derived Foxp3+ Tregs from naive conventional CD4+ T cells during self-Ag recognition. To test our hypotheses we developed a system of neonatal peripheral tolerance induction. Neonatal pups engineered to express the experimental “self” peptide Ag chicken ovalbumin (OVAp) were given OVAp/I-Ab–specific OT-II CD4+ T cells, and the pups were later examined for anergy induction, Treg differentiation, and development of autoimmunity. Our data demonstrate that in the neonatal period: 1) previously naive CD4+ T cells express markers of anergy following exposure to self-Ag, and a small fraction of these cells can be seen to differentiate to a Foxp3+ Treg phenotype, and 2) previously anergic CD4+ T cells typically retain their anergic phenotype but also give rise to a substantial fraction of Foxp3+ Tregs, and 3) animals remain free of autoimmune disease. These findings suggest that during the neonatal period peripheral Foxp3+ Treg cell differentiation may proceed through a conventional anergic CD4+ T cell intermediate.

Alpha-synuclein specific regulatory T cells expanded with bee venom phospholipase A2 improves the neuroprotective effects in MPTP-induced mouse model of Parkinson’s disease

Abstract Parkinson’s disease (PD) is a long-term degenerative autoimmune disease that mainly affects the motor system. Cell therapy using regulatory T cells (Tregs) has been shown to be effective on PD progression. To improve the efficiency of Treg therapy on PD, alpha-synuclein (a-syn) was used as a self-antigen for enrichment of a-syn specific Tregs during ex vivo expansion. In addition, to solve the problem of changing the phenotype of Tregs during expansion, bee venom phospholipase A2 (bvPLA2) was treated. The results showed that treatment of bvPLA2 remains Tregs to naïve phenotype that possessed optimal suppressive activities. And a-syn presenting changed the TCR repertoire in Tregs. As a result, these changes in Tregs by bvPLA2 and a-syn improve the neuroprotective effects on MPTP-induced mouse model of PD. Motor function deficits induced MPTP intoxication was palliated by adoptive transfer of a-syn specific and bvPLA2 treated Treg. Adoptive transfer of a-syn specific and bvPLA2 treated Treg also reduced activated microglia, especially pro-inflammatory microglia in MPTP-induced PD model. Dopaminergic neurons in substantia nigra (SN) was dramatically protected from MPTP intoxication by the Tregs that has treated with a-syn and bvPLA2. Thus, adoptive transfer of a-syn specific and bvPLA2 treated Tregs shows substantial neuroprotective effects on MPTP-induced PD mice.

Obesity contributes to a dysfunctional regulatory T Cell phenotype within adipose tissue

Abstract Adipose tissue (AT) regulatory T Cells (Tregs) play a key role in regulating inflammatory and metabolic responses. Obesity is associated with a decrease in AT Tregs as well as an increase in tissue inflammation and systemic insulin resistance in both humans and mice. We hypothesize that, in addition to the decrease in AT Treg number, there is a dysfunctional Treg phenotype caused by the increased inflammatory state within obese AT. We isolated Tregs from AT and peripheral blood(PB) from lean and obese individuals. Real-time PCR analysis was used to measure the expression of several known markers for T cell exhaustion. Protein expression of each marker was validated using FACS analysis. When compared to PB, there was a significant increase in PD-1 expression on AT Tregs. PD-1 and TIGIT were increased in Tregs isolated from obese vs. lean individuals, while CD127 expression was reduced. To determine if PD-1 expression correlates with Treg dysfunction, isolated AT and PB Tregs were added to CFSE-labeled PBMC. After 4 days culture, PB Tregs significantly inhibited proliferation but there was less suppression by AT Tregs. RNAseq analyses of PD-1 high vs. PD-1 negative AT and PB Tregs revealed a unique expression phenotype within AT Tregs. AT PD-1 high Tregs express altered metabolic and inflammatory pathways promoting loss of Tregs and increased inflammation. These data suggest that Treg exhaustion may explain the decreased Treg abundance in AT of obese individuals, which has important metabolic implications. (Partially supported by Foods for Health, a focus area of the Discovery Themes Initiative at The Ohio State University)

New Insights about Regulatory T Cells Distribution and Function with Exercise: The Role of Immunometabolism.

A lack of physical activity is linked to the development of many chronic diseases through a chronic low-grade inflammation state. It is now well accepted that the immune system plays a central role in the development of several chronic diseases, including insulin resistance, type 2 diabetes, atherosclerosis, heart failure and certain types of cancer. Exercise elicits a strong anti-inflammatory response independently of weight loss and can be a useful non-pharmacologic strategy to counteract the low-grade inflammation. The CD4+CD25+CD127- FoxP3+ Regulatory T (Treg) cells are a unique subset of helper T-cells, which regulate immune response and establish self-tolerance through the secretion of immunoregulatory cytokines, such as IL-10 and TGF-β, and the suppression of the function and activity of many immune effector cells (including monocytes/macrophages, dendritic cells, CD4+ and CD8+ T cells, and Natural Killers). The metabolic phenotype of Tregs are regulated by the transcription factor Foxp3, providing flexibility in fuel choice, but a preference for higher fatty acid oxidation. In this review, we focus on the mechanisms by which exercise - both acute and chronic - exerts its antiinflammatory effects through Treg cells mobilization. Furthermore, we discuss the implications of immunometabolic changes during exercise for the modulation of Treg phenotype and its immunosuppressive function. This narrative review focuses on the current knowledge regarding the role of Treg cells in the context of acute and chronic exercise using data from observational and experimental studies. Emerging evidence suggests that the immunomodulatory effects of exercise are mediated by the ability of exercise to adjust and improve Tregs number and function.

Ionizing radiation modulates the phenotype and function of human CD4+ induced regulatory T cells

BackgroundThe use of immunotherapy strategies for the treatment of advanced cancer is rapidly increasing. Most immunotherapies rely on induction of CD8+ tumor-specific cytotoxic T cells that are capable of directly killing cancer cells. Tumors, however, utilize a variety of mechanisms that can suppress anti-tumor immunity. CD4+ regulatory T cells can directly inhibit cytotoxic T cell activity and these cells can be recruited, or induced, by cancer cells allowing escape from immune attack. The use of ionizing radiation as a treatment for cancer has been shown to enhance anti-tumor immunity by several mechanisms including immunogenic tumor cell death and phenotypic modulation of tumor cells. Less is known about the impact of radiation directly on suppressive regulatory T cells. In this study we investigate the direct effect of radiation on human TREG viability, phenotype, and suppressive activity.ResultsBoth natural and TGF-β1-induced CD4+ TREG cells exhibited increased resistance to radiation (10 Gy) as compared to CD4+ conventional T cells. Treatment, however, decreased Foxp3 expression in natural and induced TREG cells and the reduction was more robust in induced TREGS. Radiation also modulated the expression of signature iTREG molecules, inducing increased expression of LAG-3 and decreased expression of CD25 and CTLA-4. Despite the disconcordant modulation of suppressive molecules, irradiated iTREGS exhibited a reduced capacity to suppress the proliferation of CD8+ T cells.ConclusionsOur findings demonstrate that while human TREG cells are more resistant to radiation-induced death, treatment causes downregulation of Foxp3 expression, as well as modulation in the expression of TREG signature molecules associated with suppressive activity. Functionally, irradiated TGF-β1-induced TREGS were less effective at inhibiting CD8+ T cell proliferation. These data suggest that doses of radiotherapy in the hypofractionated range could be utilized to effectively target and reduce TREG activity, particularly when used in combination with cancer immunotherapies.

Foxp3+ Tregs from Langerhans cell histiocytosis lesions co-express CD56 and have a definitively regulatory capacity

Langerhans cell histiocytosis (LCH) lesions contain myeloid lineage ‘LCH’ cells. Regulatory T cells (Tregs) are also enriched within lesions, although their role in LCH pathogenesis is unknown. LCH cells are thought to produce the transforming growth factor beta (TGF-β) within lesions, however whether Tregs contribute is unestablished. Using flow cytometry, we analyzed relative frequencies of live Tregs from LCH patients and identified CD56 expression and TGF-β production by lesion Tregs. While CD56+ Tregs were enriched in lesions, overall CD56+ T cells were reduced in the blood from active LCH patients compared to non-active disease patients, and there was a negative correlation between CD8+CD56+ T cells and Tregs. We propose that inducing a Treg phenotype in T cells such as CD56+ T cells may be a mechanism by which LCH cells divert inflammatory T cell responses. Thus, Tregs within LCH lesions are likely an important component in LCH pathogenesis.

PD47-05 REGULATORY T CELLS PLAY A KEY ROLE IN BLADDER CANCER DEVELOPMENT

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology IV (PD47)1 Apr 2020PD47-05 REGULATORY T CELLS PLAY A KEY ROLE IN BLADDER CANCER DEVELOPMENT Karen Wheeler*, Niannan Ji, Neelam Mukherjee, and Robert Svatek Karen Wheeler*Karen Wheeler* More articles by this author , Niannan JiNiannan Ji More articles by this author , Neelam MukherjeeNeelam Mukherjee More articles by this author , and Robert SvatekRobert Svatek More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000934.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Regulatory T cells (Treg) are vital in the development and maintenance of immune tolerance. They function to dampen immune responses and their dysfunction leads to autoimmune disease. Treg are known to play key roles in the development of cancers including lung, breast, head and neck, and others. They are believed to aid the tumors in immune evasion. In bladder cancer (BCa), clinical studies have shown an increase in Treg in patients’ tumors or peripheral blood correlates with a worse prognosis. Herein we determine whether Treg play a key role in the development of BCa. METHODS: Female wild type C57BL/6 mice or transgenic DEREG mice (express a diptheria toxin [DT] receptor whenever Foxp3, the driver of a Treg phenotype, is expressed) were used. Metastatic cancer was induced by injecting 0.5 x 106 MB49 bladder tumor cells via tail vein into mice on the day of surgery. Orthotopic (bladder) cancer was induced by catheterizing the bladders of female mice and injecting poly-L-lysine followed by 0.08x106 MB49 cells at day 0. Treg were depleted in DEREG mice with administration of 50g/kg DT given intraperitoneally every 4 days. Mice were sacrificed at days 21-26 and bladders, lungs, and bladder draining lymph nodes (BLN) were harvested for weight and flow cytometric analysis. RESULTS: In mice that received both orthotopic and intravenous bladder tumor challenge, Treg were increased in the bladder tumors but not the lymph nodes or lung (Fig 1). In DEREG mice challenged with orthotopic BCa, depletion of Treg with DT administration abrogated the development of BCa (Fig 2A). Additionally, Treg depleted mice had an increase in the number and percentage of antigen specific CD8+ T cells in the bladder draining lymph nodes as well as an increase in activated CD4+ T cells (Fig 2B). CONCLUSIONS: Regulatory T cells are necessary for BCa development in the orthotopic model of BCa. Treg appear to function in an antigen-specific manner, but pan-T cell activation cannot be excluded. Development of immunotherapy targeting Treg in the bladder may represent a novel cancer therapeutic. Source of Funding: AUA Care Foundation Award, CPRIT Training Grant © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e927-e927 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Karen Wheeler* More articles by this author Niannan Ji More articles by this author Neelam Mukherjee More articles by this author Robert Svatek More articles by this author Expand All Advertisement PDF downloadLoading ...

Human Leukocyte Antigen (HLA) Class II Expression on Regulatory T Cells (Tregs) Isolated from Discarded Human Thymus is Induced by In Vitro Expansion Conditions

Tregs modulate immune responses and are a promising therapeutic tool to limit graft rejection in organ transplantation. We showed that potent Tregs can be isolated and expanded from human thymuses, routinely removed during pediatric cardiac surgery, indicating potential for an 'off-the-shelf' tolerogenic therapy. Upregulation of HLA on expanded Tregs may increase their immunogenicity, which is a possible obstacle for clinical purpose. Here, we studied HLA class II expression on thymic Tregs under various expansion conditions. FOXP3+CD25+ Tregs were isolated from infant thymuses (n=5) by mechanical dissociation and magnetic-bead-based cell separation and cultured under various conditions for 14 days (TABLE 1). Anti-IFN-γ antibodies were added to condition B to study the role of IFN-γ. Treg phenotype and HLA class II expression were assessed by flow cytometry. Cytokine production was analyzed by multiplex assay. All culture conditions expanded the Treg population, except condition H. Cultures with artificial antigen-presenting cells (conditions A, B, C, D) contained fewer HLA class II-expressing Tregs than T Cell Activator cultures (conditions E, F, G) [range: 2-45% vs. 18-84%, respectively; p<0.001]. Additionally, CST OpTmizer medium (conditions C, D) induced less class II-expressing Tregs than ImmunoCult-XF medium (conditions A, B) [range: 2-29% vs. 17-45%, respectively; p=0.004]. Addition of rapamycin had no effect. The % class II+ Tregs was positively correlated to IFN-γ production (p=0.02) and decreased in the presence of anti-IFN-γ antibodies in a dose-dependent manner. The conditions used for Treg expansion affected the percentage of thymic Tregs that upregulate HLA class II, which at least in part was due to IFN-γ production by the cells. Expansion protocols that limit IFN-γ production may therefore generate less immunogenic Tregs for tolerogenic therapy in organ transplantation.

Use of Tregs as a cell-based therapy via CD39 for benign prostate hyperplasia with inflammation.

Benign prostatic hyperplasia (BPH) occurs most commonly among older men, often accompanied by chronic tissue inflammation. Although its aetiology remains unclear, autoimmune dysregulation may contribute to BPH. Regulatory T cells (Tregs) prevent autoimmune responses and maintain immune homeostasis. In this study, we aimed to investigate Tregs frequency, phenotype, and function in BPH patients and to evaluate adoptive transfer Tregs for immunotherapy in mice with BPH via CD39. Prostate specimens and peripheral blood from BPH patients were used to investigate Treg subsets, phenotype and Treg‐associated cytokine production. Sorted CD39+/− Tregs from healthy mice were adoptively transferred into mice before or after testosterone propionate administration. The Tregs percentage in peripheral blood from BPH patients was attenuated, exhibiting low Foxp3 and CD39 expression with low levels of serum IL‐10, IL‐35 and TGF‐β. Immunohistochemistry revealed Foxp3+ cells were significantly diminished in BPH prostate with severe inflammatory. Although the Tregs subset was comprised of more effector/memory Tregs, CD39 was still down‐regulated on effector/memory Tregs in BPH patients. Before or after testosterone propionate administration, no alterations of BPH symptoms were observed due to CD39‐ Tregs in mice, however, CD39+Tregs existed more potency than Tregs to regulate prostatic hyperplasia and inhibit inflammation by decreasing IL‐1β and PSA secretion, and increasing IL‐10 and TGF‐β secretion. Furthermore, adoptive transfer with functional Tregs not only improved prostate hyperplasia but also regulated muscle cell proliferation in bladder. Adoptive transfer with Tregs may provide a novel method for the prevention and treatment of BPH clinically.

Visceral adipose tissue Tregs and the cells that nurture them

Visceral adipose tissue (VAT) is a primary site for storage of excess energy, but it also serves as an important endocrine organ that impacts organismal metabolism. Chronic, low-grade inflammation of VAT, and eventually systemically, is one of the major drivers of obesity-associated insulin resistance and metabolic abnormalities. A unique population of regulatory T cells (Tregs), with a distinct transcriptional profile and antigen receptor repertoire resides in VAT, keeps inflammation in check and regulates organismal metabolism. Accumulation of these cells depends on interactions with other local immunocytes and, importantly, subtypes of VAT mesenchymal stromal cells (VmSCs) that are either immunomodulators or adipogenic. We summarize our current understanding of the phenotype, function, dependencies, derivation, and modulations of VAT Tregs, and review the heterogeneity and regulation of VmSCs as well as their cross talk with VAT Tregs. Lastly, we discuss imperative questions remaining to be answered.

Inhibiting the coregulator CoREST impairs Foxp3+ Treg function and promotes antitumor immunity.

Foxp3+ Tregs are key to immune homeostasis, but the contributions of various large, multiprotein complexes that regulate gene expression remain unexplored. We analyzed the role in Tregs of the evolutionarily conserved CoREST complex, consisting of a scaffolding protein, Rcor1 or Rcor2, plus Hdac1 or Hdac2 and Lsd1 enzymes. Rcor1, Rcor2, and Lsd1 were physically associated with Foxp3, and mice with conditional deletion of Rcor1 in Foxp3+ Tregs had decreased proportions of Tregs in peripheral lymphoid tissues and increased Treg expression of IL-2 and IFN-γ compared with what was found in WT cells. Mice with conditional deletion of the gene encoding Rcor1 in their Tregs had reduced suppression of homeostatic proliferation, inability to maintain long-term allograft survival despite costimulation blockade, and enhanced antitumor immunity in syngeneic models. Comparable findings were seen in WT mice treated with CoREST complex bivalent inhibitors, which also altered the phenotype of human Tregs and impaired their suppressive function. Our data point to the potential for therapeutic modulation of Treg functions by pharmacologic targeting of enzymatic components of the CoREST complex and contribute to an understanding of the biochemical and molecular mechanisms by which Foxp3 represses large gene sets and maintains the unique properties of this key immune cell.

Mesenchymal stem cells regulate the Th17/Treg cell balance partly through hepatocyte growth factor in vitro

IntroductionMesenchymal stem cells (MSCs) exert immunomodulatory functions by inducing the development and differentiation of naive T cells into T cells with an anti-inflammatory regulatory T cell (Treg) phenotype. Our previous study showed that hepatocyte growth factor (HGF) secreted by MSCs had immunomodulatory effects in the context of lipopolysaccharide (LPS) stimulation. We hypothesized that HGF is a key factor in the MSC-mediated regulation of the T helper 17 (Th17) cell/regulatory T (Treg) cell balance.MethodsWe investigated the effects of MSCs on the differentiation of CD4+ T cells and the functions of Th17/Treg cells in response to LPS stimulation by performing in vitro coculture experiments. MSCs were added to the upper chambers of cell culture inserts, and CD4+ T cells were plated in the lower chambers, followed by treatment with LPS or an anti-HGF antibody. Th17 (CD4+CD3+RORrt+) and Treg (CD4+CD25+Foxp3+) cell frequencies were analysed by flow cytometry, and the expression of Th17 cell- and Treg cell-related cytokines in the CD4+ T cells or culture medium was measured by quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Neutrophil functions were determined by flow cytometry after a coculture with Th17/Treg cells.ResultsThe percentage of CD4+CD25+Foxp3+ cells was significantly increased in the CD4+ T cell population, while the percentage of CD4+CD3+RORrt+ cells was significantly decreased after MSC coculture. However, the MSC-induced effect was significantly inhibited by the anti-HGF antibody (p < 0.05). Furthermore, MSCs significantly inhibited the CD4+ T cell expression of IL-17 and IL-6 but increased the expression of IL-10 (p < 0.05 or p < 0.01); these effects were inhibited by the anti-HGF antibody (p < 0.05). In addition, CD4+ T cells cocultured with MSCs significantly inhibited neutrophil phagocytic and oxidative burst activities (p < 0.05 or p < 0.01); however, these MSC-induced effects were inhibited by the anti-HGF antibody (p < 0.05).ConclusionThese data suggested that MSCs induced the conversion of fully differentiated Th17 cells into functional Treg cells and thereby modulated the Th17/Treg cell balance in the CD4+ T cell population, which was partly attributed to HGF secreted by the MSCs.

CD4+FOXP3+ T Cells in Rheumatoid Arthritis Bone Marrow Are Partially Impaired.

There is evolving evidence that dysregulation of immune homeostasis in the bone marrow (BM) adjacent to the inflamed joints is involved in the pathogenesis of. In this study, we are addressing the phenotype and function of regulatory T cells (Tregs) residing in the BM of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). BM and peripheral blood samples were obtained from RA and OA patients undergoing hip replacement surgery. The number and phenotype of Tregs were analyzed by flow cytometry and immunohistochemistry. The function of Tregs was investigated ex vivo, addressing their suppressive activity on effector T cells. [3H]-Thymidine incorporation assay and specific enzyme-linked immunosorbent assay were used for quantification of cell proliferation and pro-inflammatory (TNF, IFN-γ) cytokine release, respectively. Significantly lower numbers of CD4+FOXP3+ T cells were found in the BM of patients with RA compared to control patients with OA. High expression of CD127 (IL-7α receptor) and relatively low expression of CXCR4 (receptor for stromal cell-derived factor CXCL12) are characteristics of the CD4+FOXP3+ cells residing in the BM of RA patients. The BM-resident Tregs of RA patients demonstrated a limited suppressive activity on the investigated immune response. Our results indicate that the reduced number and impaired functional properties of CD4+FOXP3+ T cells present in the BM of RA patients may favor the inflammatory process, which is observed in RA BM.

Early reduction of regulatory T cells is associated with acute rejection in liver transplantation under tacrolimus-based immunosuppression with basiliximab induction.

Regulatory T (Treg) cells are important in preventing acute rejection (AR) in solid organ transplantation, but the clinical relevance of the different kinetics early after liver transplantation (LT) in acute rejectors and non-rejectors is unclear. We analyzed peripheral blood samples of 128 LT recipients receiving basiliximab induction plus tacrolimus immunosuppression. Samples were obtained at pretransplant, D7, and D30 after LT. Frequency and phenotype of Tregs were analyzed by flow cytometry. The predictive value of Treg frequency at D7 was assessed for suspected acute rejection (SAR) and was validated for biopsy-proven AR (BPAR). We found that the frequencies of total and activated Tregs at D7 were significantly lower in recipients with SAR and BPAR. Treg was more reduced in BPARs by in vitro tacrolimus treatment in the presence of basiliximab. Moreover, an early reduction of Treg frequency in rejectors was associated with a greater increase in Treg apoptosis and further attenuated IL-2 signaling. D7 Treg frequency was an independent risk factor for SAR, which was also validated for BPAR. In conclusion, first-week peripheral blood Treg frequency correlates with AR after LT under tacrolimus-based immunosuppression, which needs to be proven in larger, geographically and clinically diverse populations.

MicroRNA miR-155 is required for expansion of regulatory T cells to mediate robust pregnancy tolerance in mice.

The immune-regulatory microRNA miR-155 is reduced in recurrent miscarriage, suggesting that miR-155 contributes to immune tolerance in pregnancy. Here we show miR-155 is induced in the uterine mucosa and draining lymph nodes (dLN) during the female immune response to male seminal fluid alloantigens. Mice with null mutation in miR-155 (miR-155-/-) exhibited a reduced CD4+ T cell response after mating, with a disproportionate loss of CD25+FOXP3+ Treg cells. miR-155 deficiency impaired expansion of both peripheral and thymic Treg cells, distinguished by neuropilin-1 (NRP1), and fewer Treg cells expressed Ki67 proliferation marker and suppressive function marker CTLA4. Altered Treg phenotype distribution in miR-155-/- mice was confirmed by t-distributed neighbor embedding (tSNE) analysis. Fewer dendritic cells (DCs) and macrophages trafficked to the dLN of miR-155-/- mice, associated with lower CCR7 on DCs, and reduced uterine Ccl19 expression, implicating compromised antigen presentation in the stunted Treg cell response. miR-155-/- mice exhibited elevated susceptibility to inflammation-induced fetal loss and fetal growth restriction compared with miR-155+/+ controls, but outcomes were restored by transfer of wild-type Tregs. Thus miR-155 is a key regulator of immune adaptation to pregnancy and is necessary for sufficient Tregs to achieve robust pregnancy tolerance and protect against fetal loss.