Discarded Human Thymus Is a Novel Source of Stable and Long-Lived Therapeutic Regulatory T Cells
E. DijkeRomy E. HoeppliT. EllisJ. PearceyQing HuangAlicia N. McMurchyKarin BoerA.M.A. PeetersGeraldine AubertI. LarsenDavid RossIvan M. RebeykaAndrew CampbellCarla C. BaanMegan K. LevingsLori J. West
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Regulatory T cell (Treg)–based therapy is a promising approach to treat many immune-mediated disorders such as autoimmune diseases, organ transplant rejection, and graft-versus-host disease (GVHD). Challenges to successful clinical implementation of adoptive Treg therapy include difficulties isolating homogeneous cell populations and developing expansion protocols that result in adequate numbers of cells that remain stable, even under inflammatory conditions. We investigated the potential of discarded human thymuses, routinely removed during pediatric cardiac surgery, to be used as a novel source of therapeutic Tregs. Here, we show that large numbers of FOXP3+ Tregs can be isolated and expanded from a single thymus. Expanded thymic Tregs had stable FOXP3 expression and long telomeres, and suppressed proliferation and cytokine production of activated allogeneic T cells in vitro. Moreover, expanded thymic Tregs delayed development of xenogeneic GVHD in vivo more effectively than expanded Tregs isolated based on CD25 expression from peripheral blood. Importantly, in contrast to expanded blood Tregs, expanded thymic Tregs remained stable under inflammatory conditions. Our results demonstrate that discarded pediatric thymuses are an excellent source of therapeutic Tregs, having the potential to overcome limitations currently hindering the use of Tregs derived from peripheral or cord blood. Regulatory T cell (Treg)–based therapy is a promising approach to treat many immune-mediated disorders such as autoimmune diseases, organ transplant rejection, and graft-versus-host disease (GVHD). Challenges to successful clinical implementation of adoptive Treg therapy include difficulties isolating homogeneous cell populations and developing expansion protocols that result in adequate numbers of cells that remain stable, even under inflammatory conditions. We investigated the potential of discarded human thymuses, routinely removed during pediatric cardiac surgery, to be used as a novel source of therapeutic Tregs. Here, we show that large numbers of FOXP3+ Tregs can be isolated and expanded from a single thymus. Expanded thymic Tregs had stable FOXP3 expression and long telomeres, and suppressed proliferation and cytokine production of activated allogeneic T cells in vitro. Moreover, expanded thymic Tregs delayed development of xenogeneic GVHD in vivo more effectively than expanded Tregs isolated based on CD25 expression from peripheral blood. Importantly, in contrast to expanded blood Tregs, expanded thymic Tregs remained stable under inflammatory conditions. Our results demonstrate that discarded pediatric thymuses are an excellent source of therapeutic Tregs, having the potential to overcome limitations currently hindering the use of Tregs derived from peripheral or cord blood.Keywords:
Cell therapy
Regulatory T cell
Objective: To observe the expression of CD4~+CD25~+ regulatory T cell and Foxp3 gene from peripheral blood in patients with systemic lupus erythematosus(SLE),and to investigate their roles in the pathogenesis of SLE.Methods: The percentage of CD4~+CD25~+ regulatory T cells was detected by bicolor flow cytometry,and the expression of Foxp3 mRNA on T lymphocytes was determined by RT-PCR in 25 SLE patients and 25 healthy controls.Results: The percentage of CD4~+ cells and CD4~+CD25~+ T cells,and level of Foxp3 mRNA on T lymphocytes were significantly lower in SLE group than those in control group(P0.01).There was a positive correlation between the percentage of CD4~+CD25~+ T cell expression and expression of Foxp3 mRNA(r=0.786,P0.01) in SLE group.Conclusion:There is a disturbance of cellular immune function in SLE.The downexpression of CD4~+CD25~+ regulatory T cell and Foxp3 mRNA on T lymphocytes may be involved in the immunopathogenesis of SLE.
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Abstract Background Appearance of improper immune responses against the fetus and/or inadequate immunoregulatory mechanisms during pregnancy may lead to recurrent spontaneous abortion (RSA). T H 17 cells play a significant role in inducing inflammation, autoimmune disease, and acute transplant rejection, while regulatory T (Treg) cells moderate the function of immune system in order to retain homeostasis. Methods This case-control study was designed to evaluate T H 17 as well as Treg cells in 25 women with RSA and 25 age-matched healthy non-pregnant women. Flow cytometric assay was performed using monoclonal antibodies to detect CD4 + CD25 + Treg cells (CD25 dim and CD25 bright ). FoxP3 and RORγt expressions were compared using real-time PCR, and pro-inflammatory and anti-inflammatory cytokines were measured by ELISA kits. Independent-samples T test was employed for statistical analysis. Results The ratio of CD4 + CD25 bright T cells was remarkably lower in women with RSA ( P <0.05), and CD4 + CD25 dim T cells did not show any significant difference among the groups ( P >0.05). RORγt was up-regulated, and FoxP3 was down-regulated significantly in case group ( P <0.05). The significant increase of IL-6 and IL-17 as well as the decrease of TGF-β was indicated in RSA group ( P <0.05). Also, IL-10 did not vary among the groups ( P >0.05). Conclusion These remarks prove that the decrease in regulatory factors such as CD4 + CD25 bright T-cells, TGF-β and FoxP3 expression may disrupt immune tolerance and homeostasis during pregnancy. Also, the environment rich in RORγt, IL-6, and IL-17 suggests the detrimental role of T H 17 cells, which may lead to fetal rejection.
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Homeostasis
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Objective To study the proportion of CD4+CD25+FoxP3+ regulatory T cell in patients with type 2 diabetes(T2DM) before and after insulin intensive therapy.Methods The propotion of CD4+CD25+FoxP3+ regulatory T cells was measured with flow cytometry in 46 T2DM cases before and after insulin intensive therapy and the relationship between that and other cytokines was analyzed.Results The propotion of CD4+CD25+FoxP3+ regulatory T cell and the amonts of CD3+T and CD4+T cells were significantly higher after treatment than those before(P0.05).The levels of C-reactive protein(C-RP) and tumor necrosis factor-α(TNF-α) were significantly lower after treatment than those before(P0.05).The proportion of CD4+CD25+FoxP3+ regulatory T cells was not significantly correlated with CD3+,CD4+,TNF-α,IL-6,HOMA-IR and HOMA-β.After 2 weeks of insulin treatment,the level of TNF-α was positively correlated with both HOMA-IR and HOMA-β(P0.05),while IL-6 negatively correlated with C-RP(P0.05).Conclusion The proportion of CD4+CD25+FoxP3+ regulatory T cells is increased in T2DM patients after insulin intensive therapy.Reduced level of Treg cells may be the result of hyperglycemia and a factor for the development and progress of T2DM as well.
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Abstract Investigation of the role of regulatory T cells (Treg) in model systems is facilitated by their depletion using anti-CD25 Abs, but there has been considerable debate about the effectiveness of this strategy. In this study, we have compared the depletion and repopulation of CD4+CD25+Foxp3+ Treg in uninfected and malaria-infected mice using 7D4 and/or PC61 anti-CD25 Abs. We find that numbers and percentages of CD25high cells, but not Foxp3+ cells, are transiently reduced after 7D4 treatment, whereas treatment with PC61 alone or in combination with 7D4 (7D4 plus PC61) reduces but does not eliminate Foxp3+ cells for up to 2 wk. Importantly, all protocols fail to eliminate significant populations of CD25−Foxp3+ or CD25lowFoxp3+ cells, which retain potent regulatory capacity. By adoptive transfer we show that repopulation of the spleen by CD25highFoxp3+ cells results from the re-expression of CD25 on peripheral populations of CD25−Foxp3+ but not from the conversion of peripheral Foxp3− cells. CD25highFoxp3+ repopulation occurs more rapidly in 7D4-treated mice than in 7D4 plus PC61-treated mice, reflecting ongoing clearance of emergent CD25+Foxp3+ cells by persistent PC61 Ab. However, in 7D4 plus PC61-treated mice undergoing acute malaria infection, repopulation of the spleen by CD25+Foxp3+ cells occurs extremely rapidly, with malaria infection driving proliferation and CD25 expression in peripheral CD4+CD25−Foxp3+ cells and/or conversion of CD4+CD25−Foxp3− cells. Finally, we reveal an essential role for IL-2 for the re-expression of CD25 by Foxp3+ cells after anti-CD25 treatment and observe that TGF-β is required, in the absence of CD25 and IL-2, to maintain splenic Foxp3+ cell numbers and a normal ratio of Treg:non-Treg cells.
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This study aimed to evaluate the relationship between the amount of CD4+CD25+Foxp3+ regulatory T cells in the circulation of patients with hepatocellular carcinoma(HCC) and the survival rate.We employed flow cytometry to analyze the relative proportions of CD4+CD25+Foxp3+ regulatory T cells in liver tissues and peripheral blood of 20 HCC patients and of healthy volunteers.After 3 years of follow-up,we retrospectively analyzed the patients.We found that the percentage of CD4+CD25+Foxp3+ regulatory T cells around the tumor tissues was 3.24%±1.42%,and the percentage of CD4+CD25+Foxp3+ regulatory T cells in the peripheral blood of HCC patients was 3.19%±1.56%,both of them were higher than those of the healthy volunteers 2.05%±1.20%(P 0.05).Furthermore,the frequency of CD4+CD25+Foxp3+ regulatory T cells is positively correlated the pathological stages of HCC,but negatively correlated with the survival of HCC.We concluded that CD4+CD25+Foxp3+ regulatory T cells may attenuate the anti-tumor immune responses of HCC patients.
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Cell-mediated immunity (CMI) plays a critical role in the control of brucellosis. Regulatory T cells (Tregs) have a functional character in modulating the balance between host immune response and tolerance, which can eventually lead to chronic infection or relapse. The aim of this study was to assess the alteration of Tregs in cases of brucellosis before and after treatment. Thirty cases of acute brucellosis with the mean age of 41.03±15.15 years (case group) and 30 healthy persons with the mean age of 40.63±13.95 years (control group) were selected and assessed. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of all individuals. We analyzed the alteration of Treg cell count using flow cytometry for CD4, CD25, and FoxP3 markers. The level of CD4+ CD25+ FoxP3+ Treg cells was increased in active patients compared with controls (2.5±0.99% vs 1.6±0.84%, p= 0.0004), but it had declined in the treated cases (1.83±0.73%, p=0.02). The level of Tregs was elevated in three relapsed cases. The frequency of Tregs and Treg/Teff (effector T cell) ratio was correlated with inverse serum agglutination test (SAT) and, 2-mercaptoethanol (2-ME) titers as markers of treatment in brucellosis. Based on our findings, we suggest that regulatory cells, such as CD4+ CD25+ FoxP3+ Treg cells, may contribute to the development of infection processes involving immune responses in brucellosis, and evaluation of regulatory T-cell levels may be a potential diagnostic strategy for the treatment outcome in chronic and relapsed cases of brucellosis.
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Objective To investigate the expression of CD4+CD25+FOXP3+regulatory T cells(Tregs), IL-6 and TNF-α in patients with juvenile idiopathic arthritis (JIA) and its clinical significance. Methods The percentages of CD4+CD25+FOXP3+ Tregs in 58 children with JIA and 40 healthy controls were detected by flow cytometry. Serum levels of IL-6 and TNF-α in each patient was detected by chemiluminescence. The correlations between the expression of CD4+CD25+FOXP3+ Tregs and IL-6, TNF-α were analyzed by pearson correlation analysis. We measured the expression level of CD4+CD25+FOXP3+regulatory T cells, IL-6 and TNF-α of 18 cases six weeks after the treatment of tocilizumab (TCZ) in order to figure out the dynamic changes using methods above. Results The percentages of CD4+CD25+FOXP3+Tregs in juvenile idiopathic arthritis were significantly lower than those in 40 healthy volunteers, while levels of IL-6 and TNF-α were significantly higher. However, no obvious difference in the levels of CD4+CD25+FOXP3+Tregs, IL-6 or TNF-α was observed between patients with systemic and poly-articular JIA. Pearson correlation analysis showed that the percentages of CD4+CD25+FOXP3+Tregs negatively correlated with the levels of IL-6 and TNF-α, while levels of IL-6 positively correlated with the levels of TNF-α. Compared with pre-treatment of TCZ, levels of CD4+CD25+FOXP3+Tregs in post-treatment markedly increased, which however were still lower than control group while the levels of IL-6 significantly decreased, yet remained higher than control group. There was no statistical difference between post and pre-treatment in the levels of TNF-α. Conclusion The percentages of CD4+CD25+FOXP3+ Tregs in peripheral blood of JIA children decreases, and it has a negative correlation with IL-6 and TNF-α. Furthermore, the levels of CD4+CD25+FOXP3+ Tregs and IL-6 are partially restored after treatment with TCZ, which may be helpful to assess the activity of systemic JIA and the efficacy of therapy.
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