Proliferation Of Treg Cells Research Articles

Promising effect of rapamycin on multiple sclerosis

The routine therapies for relapsing-remitting multiple sclerosis (RRMS) are common disease-modifying medications, yet are not effective in all patients. The aim of the present clinical trial was to evaluate the therapeutic effects of rapamycin on the clinical and radiological aspects, regulatory T cells proliferation and FOXP3 and GARP gene expression in the patients with RRMS. In this study, eight patients with RRMS were chosen and included in the trial. Patients received rapamycin (Rapacan, Biocon, India) for six months. Magnetic resonance imaging (MRI) of the patients’ brain was taken before and after the therapy. Patients’ expanded disability status scale (EDSS), and FoxP3 and GARP gene expression, and Treg cell proliferation were also been evaluated. All the patients had some degrees of significant reduction in mean plaque area size (P = 0.012, Z = −2.520), and minimum and maximum size of the plaques (P = 0.012, Z = −2.521). EDSS of 50% of patients was decreased after the treatment, yet it was not significant (P = 0.059, Z = −1.89). The expression rate of FOXP3 (P = 0.003) and GARP genes in Tregs increased after the therapy. We found a promising response to rapamycin among our cases with minor side effects and it may be considered as a therapeutic option of this disease.

Abstract 3754: The novel hexavalent human CD137/4-1BB agonist HERA-CD137L promotes anti-cancer immunity by activating CD8 T cells while regulatory T cells are not affected

Abstract CD137/4-1BB is an inducible costimulatory receptor mainly expressed on immune cells following activation. Binding of the cognate ligand CD137L/4-1BBL leads to receptor trimerization and activation of signaling cascades involved in expansion and survival of T cells and myeloid cells as well as memory formation and protection against autoimmunity. Apogenix´ HERA technology is based on trivalent single-chain molecular mimics of the TNF-SF receptor binding domains fused to a silenced human IgG1-Fc-domain which serves exclusively as a dimerization scaffold. This results in hexavalent agonists replicating the natural receptor binding mode. Due to their inherent high receptor clustering capacity, HERA compounds are true agonists and their biological activity is, in contrast to bivalent anti-TNFR-SF antibodies, independent of FcR-mediated crosslinking events. HERA-CD137L was produced in CHO-S cells and purified resulting in highly pure non-aggregating protein lots. PK studies in mice demonstrated a terminal half-life of 75 hours indicating excellent in vivo stability. To study the effects on immune cells in vitro, T cells were isolated from healthy-donor buffy coats and stimulated with anti-CD3 antibody alone or in combination with HERA-CD137L. Using multicolor flow cytometry, we confirmed that expression of CD137 increased on CD8+ T cells following stimulation with anti-CD3 antibody. In accord with upregulation of the activation markers CD25 and CD69 and the memory marker CD45RO, treatment with HERA-CD137L enhanced proliferation of both CD4+ and CD8+ T cells, as determined by CFSE analysis. Intracellular accumulation of IFN-γ, TNF-α, Granzyme B and Perforin upon CD137 ligation was observed in CD8+ but not CD4+ T cells. HERA-CD137L treatment of THP-1 monocytes co-cultured with primary T cells also increased their cytotoxic activity against multiple tumor cell lines, including colorectal and mammary, as shown in a real-time live cell analysis (RTCA) assay. Primary human monocytes express low levels of CD137 and differentiation to macrophages in vitro did not increase expression. However, pro-inflammatory cytokines such as TNF-α and the chemokine CCL4 were secreted after stimulation with HERA-CD137L. RTCA assays further demonstrated enhanced antigen-specific killing of MDA-MB231 tumor cells by HERA-CD137L treated T cells. HERA-CD137L conveys its activity through effector T cell proliferation while regulatory T (Treg) cell proliferation or production of anti-inflammatory cytokines are not altered in Treg cell cultures. In contrast, HERA-CD137L prevented Treg-mediated suppression of effector T cells. Based on the in vitro data presented, HERA-CD137L is a promising candidate to promote anti-tumor immune responses either as single agent or in combination with other IO-compounds. Citation Format: Meinolf Thiemann, Jaromir Sykora, David M. Richards, Christian Merz, Viola Marschall, Mauricio Redondo Mueller, Julian P. Sefrin, Karl Heinonen, Harald Fricke, Christian Gieffers, Oliver Hill. The novel hexavalent human CD137/4-1BB agonist HERA-CD137L promotes anti-cancer immunity by activating CD8 T cells while regulatory T cells are not affected [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3754.

Effect of TGF-β1 on blood CD4+CD25high regulatory T cell proliferation and Foxp3 expression during non-small cell lung cancer blood metastasis.

Metastatic circulating tumor cells in non-small cell lung cancer (NSCLC) metastasis have been reported to be associated with an immune response. The present study aimed to provide a theoretical basis for the immunomodulatory processes during NSCLC blood metastasis. NSCLC blood and normal peripheral blood mononuclear cells (PBMCs) were collected. The quantity of cluster of differentiation (CD)4+CD25high regulatory T (Treg) cells and the intracellular forkhead box protein 3 (Foxp3) expression in CD4+CD25high Treg cells were determined by flow cytometry. Furthermore, the effect of transforming growth factor β1 (TGF-β1) on NSCLC blood CD4+CD25+ Treg cell proliferation was explored by activating blood mononuclear cells with an anti-CD3 monoclonal antibody, interleukin-2 and different doses of TGF-β1. Reverse transcription-quantitative polymerase chain reaction assays were used to detect the mRNA expression of Foxp3. Carboxyfluorescein succinimidyl ester staining was used to analyze the proliferation dynamics of lymphocyte subsets. Results indicate that the proportion of CD4+ T cells in the blood of patients with NSCLC was significantly higher compared with normal peripheral blood (P<0.01). Foxp3 expression in NSCLC blood Treg cells was significantly decreased compared with normal peripheral blood (P<0.01). NSCLC blood mononuclear cells treated with TGF-β1 at 1, 5 and 25 ng/ml significantly induced Foxp3 expression in CD4+CD25+ Treg cells compared with the control group (P<0.05). The proportion of CD4+CD25+ Treg and CD8+ T cells were elevated in generation 6, 7, 8 after 6 days of TGF-β1 treatment compared with untreated cells. The proportion of CD4+CD25+ Treg and CD8+ T cells were elevated in generation 8, 9 and with TGF-β1 treatment after 8 days compared with untreated cells. These results indicate that CD4+CD25+ Treg cells proliferate at a greater rate compared with CD8+ T cells after 4, 6 or 8 days of treatment. The proportion of CD4+CD25high Treg cells in NSCLC blood was significantly higher (P<0.05) compared with normal peripheral blood. The number of Foxp3+ T cells was significantly lower (P<0.05) compared with normal peripheral blood. The data presented in this study suggest that NSCLC blood CD4+CD25high Treg cells are functionally immature and that TGF-β1 may promote maturation.

Oral immunotherapy with omalizumab reverses the Th2 cell-like programme of regulatory T cells and restores their function.

Oral immunotherapy (OIT) successfully desensitizes patients with food allergies, but the immune mechanisms mediating its efficacy remain obscure. We tested the hypothesis that allergen-specific regulatory T (Treg) cell function is impaired in food allergy and is restored by anti-IgE antibody (omalizumab)-supplemented OIT. Peanut-specific T effector (Teff) and Treg cell proliferative responses, activation markers and cytokine expression were analysed by flow cytometry in 13 peanut-allergic subjects before the start of omalizumab-supplemented OIT and periodically in some subjects thereafter for up to 2years. Peripheral blood regulatory T cells (Treg cells) were analysed for their peanut-specific suppressor function before and at 1 year following OIT. This study was registered on ClinicalTrials.gov (NCT01290913). Proliferation of allergen-specific Teff and Treg cells precipitously declined following the initiation of omalizumab therapy prior to OIT, followed by partial recovery after the initiation of OIT. At baseline, peanut-specific Treg cells exhibited a Th2 cell-like phenotype, characterized by increased IL-4 expression, which progressively reversed upon OIT. Peanut-specific Treg cell suppressor activity was absent at the start of omalizumab/OIT therapy but became robust following OIT. Absent peanut-specific Treg cell function could also be recovered by the acute blockade of IL-4/IL-4R receptor signalling in Treg cells, which inhibited their IL-4 production. OIT supplemented by omalizumab promotes allergen desensitization through an initial omalizumab-dependent step that acutely depletes allergen-reactive T cells, followed by an increase in allergen-specific Treg cell activity due to the reversal of their Th2 cell-like programme. Improved Treg cell function may be a key mechanism by which OIT ameliorates food allergy.

The IL‐33‐ST2‐MyD88 axis promotes regulatory T cell proliferation in the murine liver

Hepatic Foxp3+ Treg cells are crucial for maintaining local immune homeostasis in the liver. However, the environmental cues required for hepatic Treg cell homeostasis are unclear. In this study, we showed that the IL-33 receptor ST2 was preferentially expressed on Treg cells in the mouse liver, but it was more lowly expressed in the spleen, mesenteric lymph nodes, and blood. More importantly, we found that IL-33 promoted the proliferation of hepatic Treg cells through myeloid differentiation factor MyD88 signaling concomitant with increased cyclin-dependent kinase 4 and cyclin D1 expression. These results suggest that IL-33 is a potential tissue-specific factor controlling Treg cell homeostasis via increased Treg proliferation in the liver.

Janus Kinase 3 modulates the tolerogenic immunology of liver through Kupffer cells.

BackgroundJanus kinase 3 (Jak3) is a non‐receptor protein kinase involved in a diverse array of processes like differentiation, development, cell‐growth and hematopoietic signaling cascades. We have previously shown that loss of Jak3 exacerbates symptoms of inflammatory bowel disease (IBD) in a murine model of ulcerative colitis. The compromised intestinal barrier functions in ulcerative colitis have been shown to lead to systemic chronic low grade inflammation and subsequent hepatic disease. In this report, we investigated the tissue‐specific role of Jak3 in maintaining hepatic homeostasis during ulcerative colitis.MethodsIn order to test our hypothesis, we used a global Jak3 KO (Jak3−/−) mice model and compared its effect with either the intestinal epithelial specific Jak3 KO (Int Jak3 KO) mice or immune cell specific Jak3 KO (Imm Jak3 KO) mice. These mice were treated with 2.5% 40 kda DSS in our study. The organs were collected at the end of the study period under aseptic conditions and analyzed using a combination of flowcytometric, confocal microscopy, and western analysis techniques.ResultsOur results show increased susceptibility to colitis in DSS‐treated Jak3−/− mice. They also show a high‐degree of association between the disease scores and hepatic inflammation in Imm Jak3 KO mice, indicating a prominent role of hematopoietic Jak3 in maintaining hepatic immunological homeostasis. A differential flowcytometric assay showed an increased influx of monocytes, but reduced differentiation of residential kupffer cells in imm Jak3 KO mice compared to its floxed control during ulcerative colitis. Further, the same group of mice also showed an increased TLR‐4 expression and higher IL‐6, IL‐17 and Tnf‐α as compared to the control mice. Together, these results confirm the lack of Jak3 in immune cells leads to a compromise in the activation of kupffer cells coupled with increased extravasation of the circulating monocytes into the liver. This could lead to liver disease and inflammation in ulcerative colitis. In addition, we also found an increased Th17 effector function and simultaneous suppression of Treg cell proliferation in the absence of immune cell Jak3, when compared to its control mice, on treatment with DSS.ConclusionCollectively, these results indicate that Jak3 plays a major role in regulating the macrophage based tolerogenic immunology of the hepatic system.Support or Funding InformationThis study in part was supported by NIH grant R43GM109528This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulatory T Cell Metabolism in the Hepatic Microenvironment.

Thymic-derived naturally occurring regulatory T cells (tTreg) are crucial for maintaining peripheral immune homeostasis. They play a crucial role in preventing autoimmunity and maintaining organ transplant without requiring immunosuppression. Cellular metabolism has recently emerged as an important regulator of adaptive immune cell balance between Treg and effector T cells. While the metabolic requirements of conventional T cells are increasingly understood, the role of Treg cellular metabolism is less clear. The continuous exposure of metabolites and nutrients to the human liver via the portal blood flow influences the lineage fitness, function, proliferation, migration, and survival of Treg cells. As cellular metabolism has an impact on its function, it is crucial to understand the metabolic pathways wiring in regulatory T cells. Currently, there are ongoing early phase clinical trials with polyclonal and antigen-specific good manufacturing practice (GMP) Treg therapy to treat autoimmune diseases and organ transplantation. Thus, enhancing immunometabolic pathways of Treg by translational approach with existing or new drugs would utilize Treg cells to their full potential for effective cellular therapy.

Taking aim at regulatory T cells

Cancer Immunotherapy Cancer immunotherapy attempts to stimulate the patient's immune system against a tumor, but it can be limited by the suppressive effects of the patient's own regulatory T (Treg) cells. Nie et al. showed that coinhibiting a TNF (tumor necrosis factor) receptor reduced Treg cell activity and proliferation, stimulated antitumor immune memory, and even shrank colon and breast tumors in mice that were unresponsive to common single-agent immunotherapies. Thus, adding anti-TNF therapeutics may help to increase and broaden the efficacy of immunotherapy for cancer patients. Sci. Signal. 10 , eaan0790 (2018).

The Kynurenine Pathway As a Novel Link between Allergy and the Gut Microbiome.

In the past few decades, the indoleamine 2,3 dioxygenase (IDO) subset of the kynurenine (KYN) pathway of tryptophan (TRP) metabolism has been the subject of much research in the area of immune tolerance. In this review, we aim to incorporate new findings on this pathway in relation to allergy and the gut microbiome, while providing a comprehensive overview of the pathway itself. Stimulated by interferon gamma, IDO acts as a tolerogenic, immunosuppressive enzyme to attenuate allergic responses by the induction of the KYN-IDO pathway, resultant depletion of TRP, and elevation in KYN metabolites. Acting through the aryl hydrocarbon receptor, KYN metabolites cause T-cell anergy and apoptosis, proliferation of Treg and Th17 cells, and deviation of the Th1/Th2 response, although the outcome is highly dependent on the microenvironment. Moreover, new evidence from germ-free mice and human infants shows that gut microbiota and breast milk are key in determining the functioning of the KYN-IDO pathway. As such, we recommend further research on how this pathway may be a critical link between the microbiome and development of allergy.

Immunometabolism and regulatory T cells

Recent studies have shown that immune cells play an important role in the development of non-immune diseases. Regulatory T cells (Treg) are a group of T cell subsets which have immunoregulatory and immunosuppressive functions. Abnormalities in the number and function of Treg cells may cause immune imbalance, which leads to the development of diseases. Immunometabolic disorders may in turn affect the generation, proliferation and function of Treg cells. Clarification of the relationship and interaction between immunometabolism and Treg cells will make it possible to treat metabolic diseases with cellular immunotherapy. Key words: Immunometabolism; Regulatory T cell; Metabolic disease

The Effect of TGF-β on Treg Cells in Adverse Pregnancy Outcome upon Toxoplasma gondii Infection.

Toxoplasma gondii (T. gondii) is a ubiquitous intracellular protozoan parasite that causes adverse pregnancy outcomes. Its infection downregulates the Treg cell population and TGF-β level, which may contribute to adverse pregnancy outcomes. TGF-β plays a critical role in Treg cell development, but whether TGF-β treatment can affect the number and function of Treg cells and hence alleviate adverse pregnancy outcomes caused by T. gondii infection remains elusive. In this study, T. gondii-infected pregnant mice were treated with TGF-β or TGF-β-neutralizing antibody. The pregnancy outcomes were observed on gestational day 14. The numbers of Treg cells and pSmad3, programmed death 1 (PD-1), and Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) expression of Treg cells were analyzed by flow cytometry. Histological changes were assessed using HE staining, while IL-10 and TNF-α levels were measured using ELISA. The results indicated that TGF-β treatment improved the T. gondii-induced adverse pregnancy outcomes, with alleviation of hemorrhage, restoration of uterine spiral arteries of the placenta, and increased Treg cell numbers; meanwhile, TGF-β neutralization resulted in more serious adverse pregnancy outcomes, with serious hemorrhage, more dilated uterine spiral arteries, and decreased Treg cell numbers. pSmad3 expression in CD4+ cells and CTLA-4 and PD-1 levels on Treg cells were upregulated by TGF-β treatment, but downregulated by TGF-β neutralization. The ratio of IL-10/TNF-α also increased after TGF-β treatment, but decreased after TGF-β neutralization. Our data indicate that TGF-β treatment could improve adverse pregnancy outcomes caused by T. gondii infection by upregulating Treg cell differentiation and function via the TGF-β/Smad3 signaling pathway, but not the proliferation of Treg cells.

Forkhead-box transcription factor 1 affects the apoptosis of natural regulatory T cells by controlling Aven expression

BackgroundRegulatory T (Treg) cells play important roles in autoimmune diseases, cancer, and organ transplantation. Forkhead box protein o1 (Foxo1) and IL-7Rα(CD127) are closely related to the homeostasis of Treg cells. However, the mechanism underlying Treg proliferation and activation remains unclear. Here, we evaluated how the over-expression of Foxo1 affects Treg cell proliferation via intracellular signaling. nTreg cells were transfected separately with Foxo1 and Aven small-interfering RNA (siRNA) or over-expression plasmid. The expression of signaling pathway genes and CD127 was confirmed using RT-qPCR and western blot analysis. The expression of cell surface molecules and apoptosis was confirmed by Flow Cytometry 3-(4, 5-Dimethylthiazol-2-yl) 2,5- diphenyltetrazolium bromide for cell proliferation assays.ResultsFoxo1 strengthened the proliferative ability of Treg cells by activating IL-7/CD127 signaling. In addition, Foxo1 suppressed Treg cell apoptosis by regulating Aven expression.ConclusionsThe results in this study indicated that Foxo1 is a positive regulatory factor for the proliferation and activity of Treg cells. Foxo1 might be a potential target for the activation of nTreg cells in vivo and in vitro.

Effect of EBI3 on radiation-induced immunosuppression of cervical cancer HeLa cells by regulating Treg cells through PD-1/PD-L1 pathway.

This study aimed to investigate the effect of EBI3 on radiation-induced immunosuppression of cervical cancer HeLa cells by regulating Treg cells through PD-1/PD-L1 signaling pathway. A total of 43 adult female Wistar rats were selected and injected with HeLa cells in the caudal vein to construct a rat model of cervical cancer. All model rats were randomly divided into the radiotherapy group ( n = 31) and the control group ( n = 12). The immunophenotype of Treg cells was detected by the flow cytometry. The protein expressions of EBI3, PD-1, and PD-L1 in cervical cancer tissues were tested by the streptavidin-peroxidase method. HeLa cells in the logarithmic growth phase were divided into four groups: the blank, the negative control group, the EBI3 mimics group, and the EBI3 inhibitors group. Western blotting was used to detect PD-1 and PD-L1 protein expressions. MTT assay was performed to measure the proliferation of Treg cells. Flow cytometry was used to detect cell cycle and apoptosis, and CD4+/CD8+ T cell ratio in each group. Compared with before and 1 week after radiotherapy, the percentages of CD4+T cells and CD8+T cells were significantly decreased in the radiotherapy group at 1 month after radiotherapy. Furthermore, down-regulation of EBI3 and up-regulation of PD-1 and PD-L1 were observed in cervical cancer tissues at 1 month after radiotherapy. In comparison to the blank and negative control groups, increased expression of EBI3 and decreased expressions of PD-1 and PD-L1 were found in the EBI3 mimics group. However, the EBI3 inhibitors group had a lower expression of EBI3 and higher expressions of PD-1 and PD-L1 than those in the blank and negative control groups. The EBI3 mimics group showed an increase in the optical density value (0.43 ± 0.05), while a decrease in the optical density value (0.31 ± 0.02) was found in the EBI3 inhibitors group. Moreover, compared with the blank and negative control groups, the apoptosis rates of Treg/CD4+T/CD8+T cells were decreased in the EBI3 mimics group, but the EBI3 inhibitors group exhibited an increase in apoptosis rate. In conclusion, over-expression of EBI3 could reduce the apoptosis of Treg/CD4+T/CD8+T cells and prevent radiation-induced immunosuppression of cervical cancer HeLa cells by inhibiting the activation of PD-1/PD-L1 signaling pathway.

Toll-Like Receptor 4 Signaling in High Mobility Group Box-1 Protein 1 Mediated the Suppression of Regulatory T-Cells.

BackgroundTreg cells play a central role in the suppression of immune response, and their suppressive capacity can be modulated by toll-like receptor (TLR) ligands. However, the detailed pathway of TLR ligand modulation is still unknown. The present study aimed to evaluate the effect of the high mobility group box-1 protein 1 (HMGB1) and lipopolysaccharide (LPS) on Treg cells through TLR4 signaling.Material/MethodsTreg cells were purified from healthy human peripheral blood mononuclear cells (PBMCs) by magnetic-bead activity cell sorting (MACS), blocked by anti-TLR4 monoclonal antibody, and then incubated with different concentration of LPS or HMGB1. The level of gene expression of IL-1β, IL-10, IFN-γ, and TGF-β were detected using quantitative real-time polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA), and the proliferation of Treg cells after treating by LPS and HMGB1 was analyzed by flow cytometry. The NF-κB expression in Treg cells was examined by Western blotting.ResultsLPS treated CD4 CD25 Treg cells directly increased the expression of IL-1β and IL-10 and decreased the expression of IFN-γ and TGF-β. However, HMGB1 treatment resulted in a marked decreased expression of IL-1β, IL-10, IFN-γ, and TGF-β. The proliferation of CD4+ T cells was significantly inhibited by Treg cells in the LPS treatment group, but weaken in the HMGB1 treatment group. These data suggest that HMGB1 and LPS stimulation could downregulate the expression NF-κB p65 in cytoplasmic proteins and increase the expression in nuclear proteins, thus leading to modulation of IL-1β, IL-10, IFN-γ, and TGF-β expression; moreover, the suppressive function of Treg cells could be regulated by TLR4.ConclusionsTLR4 signaling in HMGB1 mediated the suppressive function of Treg cells through the activation of the NF-κB pathway.

Construction and Functional Analysis of Luciferase Reporter Plasmid Containing CD25 Gene Promoter

CD25 is the alpha chain of IL-2R and IL-2R is distributed on the surface of activated T cells, B cells, and NK cell. The transcriptional factor CD25 plays an important role in the proliferation and differentiation of Treg cells. MKL-1 is also called MAL (megakaryocytic acute leukemia) and belongs to the Myocardin family which also includes MKL-2 (megakaryoblastic leukemia 2) and Myocardin. Studies have shown that in the process of mouse megakaryocyte differentiation, the expression of MKL-1 is up-regulated, and MKL-1 promotes the physiological maturation of human and mouse megakaryocytes. However, the exact mechanism of the CD25 on megakaryocytes is unclear. In this study, human CD25 promoter luciferase reporter construct was generated by PCR amplification. Then the PCR fragment was digested and cloned into pGL3 vector, and this promoter sequence was verified by sequencing. The result showed that luciferase reporter with CD25 promoter was successfully constructed. The expression vectors of human MKL-1 were bought from addgene. Then the activation of the CD25 promoter was detected in 293T cells by luciferase reporter assay after transfected expression vectors of human MKL-1. The result showed that transfected the expression vectors can enhance the transcriptional activity of CD25. Our research will reveal the effect of CD25 on megakaryocytes and may provide a theoretical basis and therapeutic method for diseases caused by megakaryocytes.

TGF-β1 along with other platelet contents augments Treg cells to suppress anti-FVIII immune responses in hemophilia A mice

AbstractPlatelets are a rich source of many cytokines and chemokines including transforming growth factor β 1 (TGF-β1). TGF-β1 is required to convert conventional CD4+ T (Tconv) cells into induced regulatory T (iTreg) cells that express the transcription factor Foxp3. Whether platelet contents will affect Treg cell properties has not been explored. In this study, we show that unfractionated platelet lysates (pltLys) containing TGF-β1 efficiently induced Foxp3 expression in Tconv cells. The common Treg cell surface phenotype and in vitro suppressive activity of unfractionated pltLys-iTreg cells were similar to those of iTreg cells generated using purified TGF-β1 (purTGFβ-iTreg) cells. However, there were substantial differences in gene expression between pltLys-iTreg and purTGFβ-iTreg cells, especially in granzyme B, interferon γ, and interleukin-2 (a 30.99-, 29.18-, and 17.94-fold difference, respectively) as determined by gene microarray analysis. In line with these gene signatures, we found that pltLys-iTreg cells improved cell recovery after transfer and immune suppressive function compared with purTGFβ-iTreg cells in factor VIII (FVIII)–deficient (F8null, hemophilia A model) mice after recombinant human FVIII (rhF8) infusion. Acute antibody-mediated platelet destruction in F8null mice followed by rhF8 infusion increased the number of Treg cells and suppressed the antibody response to rhF8. Consistent with these data, ex vivo proliferation of F8-specific Treg cells from platelet-depleted animals increased when restimulated with rhF8. Together, our data suggest that pltLys-iTreg cells may have advantages in emerging clinical applications and that platelet contents impact the properties of iTreg cells induced by TGF-β1.

Foxp3 and Toll-like receptor signaling balance Treg cell anabolic metabolism for suppression.

CD4+ effector T cells (Teff cells) and regulatory T cells (Treg cells) undergo metabolic reprogramming to support proliferation and immunological function. Although signaling via the lipid kinase PI(3)K (phosphatidylinositol-3-OH kinase), the serine-threonine kinase Akt and the metabolic checkpoint kinase complex mTORC1 induces both expression of the glucose transporter Glut1 and aerobic glycolysis for Teff cell proliferation and inflammatory function, the mechanisms that regulate Treg cell metabolism and function remain unclear. We found that Toll-like receptor (TLR) signals that promote Treg cell proliferation increased PI(3)K-Akt-mTORC1 signaling, glycolysis and expression of Glut1. However, TLR-induced mTORC1 signaling also impaired Treg cell suppressive capacity. Conversely, the transcription factor Foxp3 opposed PI(3)K-Akt-mTORC1 signaling to diminish glycolysis and anabolic metabolism while increasing oxidative and catabolic metabolism. Notably, Glut1 expression was sufficient to increase the number of Treg cells, but it reduced their suppressive capacity and Foxp3 expression. Thus, inflammatory signals and Foxp3 balance mTORC1 signaling and glucose metabolism to control the proliferation and suppressive function of Treg cells.

Development of Regulatory T Cells Mediated by NF-κB and Its Role in GVHD -Review

Graft versus host disease (GVHD) is the major complication of allogenic hematopoietic stem cell transplantation (allo-HSCT), and is a mainly responsible result for the non-recurrence-related mortality of long-term survived patient after transplantation. Studies indicated that regulatory T (Treg) cells can inhibit early-GVHD. Since the transcriptional factor NF-κB has been implicated in the regulation of Treg cell proliferation and activation, it becomes a potential target of GVHD therapy. Thus, in this review the function and signaling pathway of NF-κB, as well as the relationship between GVHD and NF-κB, are discussed and summarized.

IL-7 Mediated Homeostatic Expansion of Human CD4+CD25+FOXP3+ Regulatory T Cells After Depletion With Anti-CD25 Monoclonal Antibody.

The maintenance or expansion of regulatory T (Treg) cells has a fundamental role in the achievement of immunological tolerance after transplantation. Here we aimed to determine mechanisms of human Treg cell depletion and reconstitution after anti-CD25 monoclonal antibody (mAb) treatment. Seventeen patients with type 1 diabetes who received pancreatic islet transplantation and anti-CD25 mAb as induction therapy were studied. We observed an almost complete depletion of Treg cells after injection of anti-CD25 mAb. The kinetic of Treg cell depletion did not parallel the disappearance of CD25+ T cells as CD25 is also rapidly downregulated and internalized. Regulatory T cell reconstitution is completed within 6 months posttransplantation and appeared to be driven by IL-7-mediated homeostatic T cell proliferation. Anti-CD25 mAb treatment sensitizes Treg cell to the biological effect of IL-7, possibly rendering more common γc-chain available to interact with CD127. Homeostatic Treg cell proliferation is resistant to the inhibitory effect of rapamycin and FK506 but can be blocked by the presence of mycophenolate mofetil. Our data suggest that a compensatory mechanism of IL-7-mediated homeostatic proliferation can restore the inhibitory network of Treg cell after anti-CD25 induction therapy in islet allotransplantation.

Role of Treg Cells in Pathogensis of Mouse ITP

To explore the role of Treg cells in the pathogenesis of idiopathic thrombocytopenic purpura (ITP). The ITP mouse model was established, the Treg cell ratio in peripheral blood and spleen was detected by flow cytometry, the CD4+ CD25+ T cells were sorted by immunomagnetic beads, the Treg cell associated transcription factors (Foxp3, Smad7, STAT5 and Akt-1) and cytokines (IL-10, TGF-β) in CD4+ CD25+ T cells were enriched from spleen mononuclear cells, and the mRNA expression of Treg cell was measured by real-time PCR. The ratio of Tregs in peripheral blood and spleen decreased significantly in ITP mouse, as compared with the controls (P<0.01). In addition, the mRNA expression of IL-10, TGF-β and Foxp3 decreased significantly in spleen CD4+ CD25+ T cells (P<0.05). Expression of Smad 7 mRNA was higher than that of controls. The alteration in Treg frequency and function may be responsible for the immune dysfunction in ITP disease. It is also speculated that the lower mRNA expression of Foxp3 and higher mRNA expression of Smad 7 may inhibit the proliferation and differentiation of Treg cells.