Human CD19(+)CD25(high) B regulatory cells suppress proliferation of CD4(+) T cells and enhance Foxp3 and CTLA-4 expression in T-regulatory cells

Studies in both animal models and humans have shown a subset of B cells behaving as immuno-regulatory cells, being a source of inhibitory cytokines such as IL-10 and TGF-β. Our aims were to establish the presence of human B regulatory (Breg) cells and to assess their ability to suppress proliferation of CD4(+) T cells and to mediate T regulatory (Treg) cells' properties. For this purpose, human Breg, CD4(+) T and Treg cells were purified using magnetic microbeads. CFSE-labeled CD4(+) T cells were stimulated and cultured alone or with Breg cells. Their proliferative response was determined 72 hours later based on the CFSE staining. In parallel, Treg cells were cultured alone or with Breg cells in different conditions for 24 hours, and then stained and analyzed for Foxp3 and CTLA-4 expression. We found that, the co-culture of Breg cells (defined as CD25(high) CD27(high) CD86(high) CD1d(high) IL-10(high) TGF-β(high)) with autologous stimulated CD4(+) T cells decreased significantly (in a dose-dependent way) the proliferative capacity of CD4(+) T cells. Furthermore, Foxp3 and CTLA-4 expression in Treg cells were enhanced by non-stimulated and further by ODN-CD40L stimulated Breg cells. The regulatory function of Breg cells on Treg cells was mainly dependent on a direct contact between Breg and Treg cells, but was also TGF-β but not IL-10 dependent. In conclusion, human Breg cells decrease the proliferation of CD4(+) T cells and also enhance the expression of Foxp3 and CTLA-4 in Treg cells by cell-to-cell contact.     

IL-2 induces in vivo suppression by CD4(+)CD25(+)Foxp3(+) regulatory T cells

Interleukin-2 (IL-2) treatment is currently used to enhance T cell-mediated immune responses against tumors or in viral infections. At the same time, IL-2 is essential for the peripheral homeostasis of CD4(+)CD25(+)Foxp3(+ )regulatory T cells (Treg). In our study, we show that IL-2 is also an important activator of Treg suppressive activity in vivo. IL-2 treatment induces Treg expansion as well as IL-10 production and increases their suppressive potential in vitro. Importantly, in vivo application of IL-2 via gene-gun vaccination using IL-2 encoding DNA plasmids (pIL-2) inhibited naive antigen-specific T cell proliferation as well as a Th1-induced delayed type hypersensitivity response. The suppressive effect can be transferred onto naive animals by Treg from IL-2-treated mice and the suppression depends on the synergistic action of IL-10 and TGF-beta. These data highlight that during therapeutic treatment with IL-2 the concomitant activation of Treg may indeed counteract the intended activation of cellular immunity.     

Total glucosides of paeony induces regulatory CD4(+)CD25(+) T cells by increasing Foxp3 demethylation in lupus CD4(+) T cells

Total glucosides of paeony (TGP), an active compound extracted from Paeony root, has been used in therapy for autoimmune diseases. However the molecular mechanism of TGP in the prevention of autoimmune response remains unclear. In this study, we found that TGP treatment significantly increased the percentage and number of Treg cells in lupus CD4(+) T cells. Further investigation revealed that treatment with TGP increased the expression of Foxp3 in lupus CD4(+) T cells by down-regulating Foxp3 promoter methylation levels. However, we couldn't observe similar results in healthy control CD4(+) T cells treated by TGP. Moreover, our results also showed that IFN-γ and IL-2 expression was enhanced in TGP-treated lupus CD4(+) T cells. These findings indicate that TGP inhibits autoimmunity in SLE patients possibly by inducing Treg cell differentiation, which may in turn be due to its ability to regulate the methylation status of the Foxp3 promoter and activate IFN-γ and IL-2 signaling.     

Natural and TGF-beta-induced Foxp3(+)CD4(+) CD25(+) regulatory T cells are not mirror images of each other

Foxp3(+) CD4(+) CD25(+) regulatory cell (Treg) subsets that maintain immunologic homeostasis have been considered to be a homogeneous population of naturally occurring, thymus-derived CD4(+)CD25(+) cells (nTregs). However, similar Foxp3+ Tregs can be induced from CD25(-) precursors in vivo, and ex vivo with interleukin 2 (IL-2) and transforming growth factor beta (TGF-beta) (iTregs). These two subsets differ in their principal antigen specificities and in the T-cell receptor signal strength and co-stimulatory requirements needed for their generation. However, whether iTregs have any unique functions in vivo has been unclear. Although IL-6 can convert nTregs to Th17 cells, iTregs induced by IL-2 and TGF-beta are resistant to this cytokine and thereby might retain suppressive function at inflammatory sites. Thus, nTregs and iTregs may have different roles in the adaptive immune response.     

Immunomodulation of human CD19+CD25high regulatory B cells via Th17/Foxp3 regulatory T cells and Th1/Th2 cytokines

Regulatory B (Breg) cells are a special subset of immunoregulatory cells with unique phenotypes and functions. In this study, human CD19⁺CD25^high Breg cells were purified from human peripheral blood. Based on the coculture system of Breg cells and CD4⁺ T cells in vitro, Breg cells were found to promote the increase in regulatory T (Treg) cells while decreasing the number of Th17 cells. Breg cells regulate Treg cells through two processes: cell-cell contact and cytokines. TGF-βsRII, a blocker of transforming growth factor-β (TGF-β), can attenuate the effects of Treg elevation, suggesting that TGF-β is the main cytokine, while Breg cells rather than interleukin-10 (IL-10) regulate the differentiation of Treg cells. However, Th17 cells were mainly regulated by cytokines, without an obvious regulatory effect on cell-cell contacts. Breg cells may regulate Th17 cells by a pathway independent of TGF-β and IL-6. The coculture of Breg cells and CD4⁺ T cells led to changes in the cytokine spectrum, which included significant increases in IL-4, IL-6 and IL-10 but not obvious changes in IL-2, IFN-γ and TNF. The inhibitory effect of Breg cells was weakened by blocking cell-cell contacts in cultures separated with the Transwell chamber because IL-10 decreased while IL-6 increased when compared with cocultured Breg and CD4⁺ T cells. When the IL-10 inhibitor IL-10sRα was added, IL-6 and TNF levels significantly increased, while treatment with the TGF-β inhibitor TGF-βsRII did not result in similar changes, suggesting that IL-10 is an important molecule to inhibit the proinflammatory factors IL-6 and TNF in this culture system.     

Inhibition of viral replication downregulates CD4(+)CD25(high) regulatory T cells and programmed death-ligand 1 in chronic hepatitis B

Chronic hepatitis B is characterized by an impaired immune response to hepatitis B virus (HBV). Telbivudine treatment has significantly improved the clinical outcome of chronic HBV infection. However, the underlying mechanism behind the antiviral response of patients treated with nucleoside analogs remains unclear. To gather more evidence about the mechanism responsible for the weak immune response, in this study we analyzed the effects on HBV viral load of treatment with the nucleoside analogue telbivudine and the percentage of Tregs, programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) expression, and related cytokine production. Peripheral blood mononuclear cells (PBMCs) and serum of 28 patients with chronic hepatitis B were collected at baseline, and 3?mo and 6?mo after therapy was begun. In parallel with the decline in viral load and serum ALT normalization, we found a decline in circulating CD4(+)CD25(high) Tregs, PD-L1 on CD4(+) T cells, and IL-9 production. The expression of PD-1 on CD4(+) T cells and the production of IFN-γ did not increase during therapy. Our findings suggest that the antiviral effect of the nucleoside analogs may be attributable not only to their direct effect on virus suppression, but also to their immunoregulatory capabilities.     

Circulating and thymic CD4 CD25 T regulatory cells in myasthenia gravis: effect of immunosuppressive treatment

Accumulating evidence indicates an immunosuppressive role of the thymus-derived CD4+ T-cell population constitutively expressing high level of CD25, T regulatory (Treg) cells, in autoimmune diseases. Here we show that the number of Treg cells in the blood is significantly lower in untreated myasthenia gravis patients than in age-matched healthy subjects, whereas it is normal or elevated in patients on immunosuppressive therapy (prednisone frequently associated with azathioprine). Therapeutic thymectomy (Tx) for either the thymoma or non-neoplastic thymic alterations that are often associated with myasthenia gravis provided unique material for studying intrathymic Treg cells and correlating them with their peripheral counterparts. We observed that Tx prevents the increase of Treg cells in the circulation that follows immunosuppressive therapy (particularly evident if the thymus is not neoplastic), indicating that the thymus contributes to Treg-cell normalization. However, thymic Treg cells are not modulated by immunosuppressive therapy and even in thymectomized patients Treg-cell numbers in the blood eventually recover. The present findings suggest that a deficiency in Treg cells favours the development of myasthenia gravis and that their normalization is an important clinical benefit of immunosuppressive therapy. Treg normalization appears to be largely thymus independent and possibly reflects the reported capacity of corticosteroids to promote Treg-cell development.     

Estrogen enhances the functions of CD4(+)CD25(+)Foxp3(+) regulatory T cells that suppress osteoclast differentiation and bone resorption in vitro

Cross-talk has been shown to occur between the immune system and bone metabolism pathways. In the present study, we investigated the impact of CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells on osteoclastogenesis and bone resorption. Treg cells that were isolated and purified from peripheral blood mononuclear cells (PBMCs) of healthy adults inhibited both the differentiation of osteoclasts (OCs) from human embryo bone marrow cells (BMCs) and the pit formation in a dose-dependent manner. In cell cocultures, the production levels of both interleukin-10 (IL-10) and transforming growth factor-beta 1 (TGF-β1) were proportionally upregulated as the ratio of Treg cells to BMCs was increased, and the inhibition of OC differentiation and bone resorption by Treg cells was completely reversed by anti-IL-10 and anti-TGF-β1 antibodies. Treatment of BMC and Treg cell cocultures with 17β-estradiol (E2) at concentrations between 10⁻⁷ and 10⁻⁹ mol/l suppressed OC differentiation and bone resorption more efficiently than it did in cultures of BMCs alone; this enhanced suppression occurred via the stimulation of Treg cell IL-10 and TGF-β1 expression. These data suggest that Treg cells suppress OC differentiation and bone resorption by secreting IL-10 and TGF-β1. E2 enhances the suppressive effects of Treg cells on OC differentiation and bone resorption by stimulating IL-10 and TGF-β1 secretion from these cells. Therefore, Treg cell-derived IL-10 and TGF-β1 are likely involved in the regulation of E2 on bone metabolism and represent potential therapeutic targets for the treatment of postmenopausal osteoporosis (PMO).     

In vitro-expanded CD4CD25Foxp3 regulatory T cells controls corneal allograft rejection

Aims

Natural CD4⁺CD25⁺ regulatory cells (nTregs) have been implicated in maintaining peripheral immune tolerance. This study aims to test whether immunotherapy using in vitro-expanded Treg (iTregs) could suppress allograft rejection in corneal transplantation model.

Methods

Natural CD4⁺CD25⁺ T cells were freshly purified from naïve mice and expanded in vitro by culturing with anti-CD3/CD28-coated Dynabeads, interleukin (IL)-2 and transforming growth factor (TGF-β1). Suppression ability of iTregs was assayed by co-culturing with CD4⁺CD25⁻ T cells (Teff) in vitro and by targeting corneal allograft rejection in vivo. Tracking of iTreg after adoptive transfer in vivo were examined by CFSE labeling.

Results

Natural Treg cells were expanded by culturing with anti-CD3/CD28-coated Dynabeads in the presence of IL-2 and TGF-β1. Compared with nTregs, iTregs had similar expression of CD62L, and PD- L1, lower expression of CD69, higher levels of PD-1, CD25, and Foxp3. iTreg cells exerted stronger suppression function than natural Treg cells when cocultured with CD4⁺CD25⁻ T cells in vitro and prevented fully MHC-mismatched corneal allograft rejection. Survival of iTreg cells could suppress alloimmune reaction and most prone to migrate to graft draining LNs and spleens. Moreover, maintaining CD25 expression on iTregs was indicative for preservation of allosuppression.

Conclusion

Therapeutic use of in vitro-expanded CD4⁺CD25⁺ T cells may be a effective and safe tool for controlling allograft rejection and may help induce allograft tolerance.     

Association between CD4(+)CD25(high) T cells and atopy in children

BACKGROUND: There is evidence that CD4(+)CD25(high) T-regulatory cells are important for establishing tolerance to allergens, but information in children is limited. OBJECTIVE: To test the hypothesis that greater numbers and function of CD4(+)CD25(high) T cells are associated with a reduced risk of childhood allergies and wheezing. METHODS: A cohort of 151 six-year-old children from atopic families was analyzed for peripheral blood CD4(+)CD25(high) and CD4(+)CD25(int) T cells by flow cytometry and for clinical and immunologic correlates of atopy. The associations between these variables were assessed by regression analysis. RESULTS: Factors positively associated with % CD4(+)CD25(high)/CD4 T cells were male sex, number of positive allergen-specific IgE tests, total IgE, season, and 1-month average total pollen count preceding blood draw. The percentage of CD4(+)CD25(high)/total CD4 T cells did not correlate with induced cytokine production, and correlated negatively with suppressive capacity of CD4(+)CD25(+) T cells (r = -0.45; P = .034). The percentage of CD4(+)CD25(int)/CD4 T cells was 54% higher in pollen-sensitized children compared with nonsensitized children in spring (P = .023 for interaction), and correlated positively with IL-5, IL-10, and IL-13 (P < or = .001 for all). CONCLUSION: Our findings suggest that blood CD4(+)CD25(high) cells are a mixture of activated and regulatory T cells, and that these cells could be seasonally regulated by environmental factors such as pollen exposure. CLINICAL IMPLICATIONS: Seasonal increases in CD4CD25(high) expression in children with allergy may represent systemic immune activation caused by pollen exposures.     

Interleukin-7 matures suppressive CD127(+) forkhead box P3 (FoxP3)(+) T cells into CD127(-) CD25(high) FoxP3(+) regulatory T cells

We have identified a novel interleukin (IL)-7-responsive T cell population [forkhead box P3 (FoxP3(+) ) CD4(+) CD25(+) CD127(+) ] that is comparably functionally suppressive to conventional FoxP3(+) CD4(+) CD25(+) regulatory T cells (T(regs) ). Although IL-2 is the most critical cytokine for thymic development of FoxP3(+) T(regs) , in the periphery other cytokines can be compensatory. CD25(+) CD127(+) T cells treated with IL-7 phenotypically 'matured' into the known 'classical' FoxP3(+) CD4(+) CD25(high) CD127(-) FoxP3(+) T(regs) . In freshly isolated splenocytes, the highest level of FoxP3 expression was found in CD127(+) CD25(+) T cells when compared with CD127(-) CD25(+) or CD127(+) CD25(-) cells. IL-7 treatment of CD4(+) CD25(+) T cells induced an increase in the accumulation of FoxP3 in the nucleus in vitro. IL-7-mediated CD25 cell surface up-regulation was accompanied by a concurrent down-regulation of CD127 in vitro. IL-7 treatment of the CD127(+) CD25(+) FoxP3(+) cells also resulted in up-regulation of cytotoxic T lymphocyte antigen 4 without any changes in CD45RA at the cell surface. Collectively, these data support emerging evidence that FoxP3(+) T cells expressing CD127 are comparably functionally suppressive to CD25(+) CD127(-) FoxP3(+) T cells. This IL-7-sensitive regulation of FoxP3(+) T(reg) phenotype could underlie one peripheral non-IL-2-dependent compensatory mechanism of T(reg) survival and functional activity, particularly for adaptive T(regs) in the control of autoimmunity or suppression of activated effector T cells.     

CD4+CD25+Foxp3+ regulatory T cells induce cytokine deprivation-mediated apoptosis of effector CD4+ T cells

A key issue in mammalian immunology is how CD4+CD25+Foxp3+ regulatory T cells (T(reg) cells) suppress immune responses. Here we show that T(reg) cells induced apoptosis of effector CD4+ T cells in vitro and in vivo in a mouse model of inflammatory bowel disease. T(reg) cells did not affect the early activation or proliferation of effector CD4+ T cells. Cytokines that signal through the common gamma-chain suppressed T(reg) cell-induced apoptosis. T(reg) cell-induced effector CD4+ T cell death required the proapoptotic protein Bim, and effector CD4+ T cells incubated with T(reg) cells showed less activation of the prosurvival kinase Akt and less phosphorylation of the proapoptotic protein Bad. Thus, cytokine deprivation-induced apoptosis is a prominent mechanism by which T(reg) cells inhibit effector T cell responses.     

Expression of CD25(high) regulatory T cells and PD-1 in gastric infiltrating CD4(+) T lymphocytes in patients with Helicobacter pylori infection

We observed by flow cytometry that the frequency of both gastric infiltrating Tregs and PD-1-positive CD4 T cells is correlated with the density of Helicobacter pylori, suggesting that cellular immunity against this pathogen is inhibited.     

Equine CD4(+) CD25(high) T cells exhibit regulatory activity by close contact and cytokine-dependent mechanisms in vitro

Horses are particularly prone to allergic and autoimmune diseases, but little information about equine regulatory T cells (Treg) is currently available. The aim of this study therefore was to investigate the existence of CD4(+) Treg cells in horses, determine their suppressive function as well as their mechanism of action. Freshly isolated peripheral blood mononuclear cells (PBMC) from healthy horses were examined for CD4, CD25 and forkhead box P3 (FoxP3) expression. We show that equine FoxP3 is expressed constitutively by a population of CD4(+) CD25(+) T cells, mainly in the CD4(+) CD25(high) subpopulation. Proliferation of CD4(+) CD25(-) sorted cells stimulated with irradiated allogenic PBMC was significantly suppressed in co-culture with CD4(+) CD25(high) sorted cells in a dose-dependent manner. The mechanism of suppression by the CD4(+) CD25(high) cell population is mediated by close contact as well as interleukin (IL)-10 and transforming growth factor-β1 (TGF-β1) and probably other factors. In addition, we studied the in vitro induction of CD4(+) Treg and their characteristics compared to those of freshly isolated CD4(+) Treg cells. Upon stimulation with a combination of concanavalin A, TGF-β1 and IL-2, CD4(+) CD25(+) T cells which express FoxP3 and have suppressive capability were induced from CD4(+) CD25(-) cells. The induced CD4(+) CD25(high) express higher levels of IL-10 and TGF-β1 mRNA compared to the freshly isolated ones. Thus, in horses as in man, the circulating CD4(+) CD25(high) subpopulation contains natural Treg cells and functional Treg can be induced in vitro upon appropriate stimulation. Our study provides the first evidence of the regulatory function of CD4(+) CD25(+) cells in horses and offers insights into ex vivo manipulation of Treg cells.     

CD4(+)CD25(+)Foxp3(+) regulatory T cells promote Th17 cells in vitro and enhance host resistance in mouse Candida albicans Th17 cell infection model

Th17 cells and CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells are thought to promote and suppress inflammatory responses, respectively. Here we explore why under Th17 cell polarizing conditions, Treg cells did not suppress, but rather upregulated, the expression of interleukin-17A (IL-17A), IL-17F, and IL-22 from responding CD4(+) T?cells (Tresp cells). Upregulation of IL-17 cytokines in Tresp cells was dependent on?consumption of IL-2 by Treg cells, especially at early time points both in?vitro and in?vivo. During an oral Candida albicans infection in mice, Treg cells induced IL-17 cytokines in Tresp cells, which markedly enhanced fungal clearance and recovery from infection. These findings show how Treg cells can promote acute Th17 cell responses to suppress mucosal fungus infections and reveal that Treg cells?have a powerful capability to fight infections besides their role in maintaining tolerance or immune homeostasis.     

Expansion and activation of CD4(+)CD25(+) regulatory T cells in Heligmosomoides polygyrus infection

Regulatory T cell responses to infectious organisms influence not only immunity and immunopathology, but also responses to bystander antigens. Mice infected with the gastrointestinal nematode parasite Heligmosomoides polygyrus show an early Th2-dominated immune response (days 7-14), but by day 28 a strongly regulatory profile is evident with antigen-specific IL-10 release and elevated frequency of CD4(+) T cells bearing surface TGF-beta. CD4(+)CD25(+) T cells from infected mice show enhanced capacity to block in vitro effector T cell proliferation. CD4(+)CD25(+) cell numbers expand dramatically during infection, with parallel growth of both CD25(+)Foxp3(+) and CD25(+)Foxp3(-) subsets. CTLA-4 and glucocorticoid-induced tolerance-associated receptor, also associated with regulatory T cell function, become more prominent, due to both expanded CD25(+) cell numbers and increased expression among the CD25(-) population. Both intensity and frequency of CD103 expression by CD4(+) T cells rise significantly, with greatest expansion among CD25(+)Foxp3(+) cells. While TGF-beta expression is observed among both CD25(+)Foxp3(+) and CD25(+)Foxp3(-) subsets, it is the latter population which shows higher TGF-beta staining following infection. These data demonstrate in a chronic helminth infection that Foxp3(+) regulatory T cells are stimulated, increasing CD103 expression in particular, but that significant changes occur to other populations including expansion of CD25(+)TGF-beta(+)Foxp3(-) cells, and induction of CTLA-4 on CD25(-) non-regulatory lymphocytes.     

CD25(+)CD4(+) regulatory T cells exert in vitro suppressive activity independent of CTLA-4

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is constitutively expressed on CD25(+)CD4(+) regulatory T cells (Treg) and is suggested to play a role in Treg-mediated suppression. However, the results of analysis with anti-CTLA-4 have been controversial. We addressed this issue by analyzing mice over-expressing or deficient in CTLA-4. For over-expression, CTLA-4 transgenic mice expressing a full-length (FL) or a truncated (TL) mutant of CTLA-4 were analyzed. FL T cells expressed similar levels of CTLA-4 to Treg, whereas TL T cells expressed much higher levels on the cell surface. The number of Treg in both mice was decreased, although Foxp3 expression was not altered. Treg from both mice exerted suppressive activity, whereas CD25(-) T cells from FL mice showed no suppression. Furthermore, CD25(+)CD4 thymocytes from young CTLA-4-deficient mice were analyzed and found to exhibit suppressive activity. These results indicate that Treg exert in vitro suppressive activity independent of CTLA-4 expression.     

CD4~+CD25~+调节性T细胞/Th17细胞失衡与婴幼儿脓毒症

目的 观察不同免疫状态下婴幼儿脓毒症CD4~+CD25~+Foxp3~(high)岫调节性T细胞(Tr)/Th17细胞的变化,探讨婴幼儿脓毒症适应性免疫紊乱可能的机制.方法 婴幼儿脓毒症48例,健康同龄儿童对照组26例.用流式细胞术榆测CD14~+单核细胞HLA-DR表达率,CD4~+CD25~+Foxp3~(high)Tr 比例及Th17细胞比例;用ELISA法检测细胞因子IL-1β、IL-6、IL-10、TNF-α、TGF-β及IL-17A等浓度,计算IL-10/TNF-α比值;实时荧光定量PCR(real-time PCR)检测CD4~+T细胞Foxp3、ROR-γt mRNA表达及IL-17A mRNA表达.以CD14~+单核细胞HLA-DR表达＞或＜30%为阈值,将患儿分为免疫激活组(DR-H组)和免疫抑制组(DR-L组).结果 DR-L组IL-10/TNF-α比值明显高于DR-H组(P＜0.05)及对照组(P＜0.05).CD4~+CD25~+Foxp3~(high) Tr细胞比例及转录因子Foxp3基因表达DR-L组明显高于对照组及DR-H组(P＜O.05).Th17细胞比例、IL-17A血浓度、Th17细胞IL-17A基因表达及转录因子ROR-γt基因表达DR-H组及DR-L组均明显高于对照组(P＜0.05),两组之间差异无统计学意义(P＞0.05).DR-H组和DR-L组Th17细胞主要分化调控因子IL-6、IL-1β血清浓度明显高于正常对照组(P＜0.05),两组问IL-6、IL-1β浓度差异无统计学意义(P＞0.05),DB-L组CD4~+CD25~+Foxp3~(high) Tr细胞主要调节因子TGF-β血浓度明显高于DR-H组及对照组(P＜0.05).结论 Th17持续过度活化可能是导致脓毒症前炎症细胞因子/趋化因子持续增高的原因之一;CD4~+CD25~+Foxp3~(high) Tr细胞/Th17细胞失衡可能参与脓毒症混合性拮抗反应综合征(MARS)的发生发展,婴幼儿脓毒症细胞因子微环境变化可能是导致CD4~+CD25~+Foxp3~(high) Tr细胞/Thl7细胞失衡的原因之一.