The diabetes type 1 locus Idd6 modulates activity of CD4+CD25+ regulatory T-cells

The genetic locus Idd6 confers susceptibility to the spontaneous development of type 1 diabetes in the NOD mouse. Our studies on disease resistance of the congenic mouse strain NOD.C3H 6.VIII showed that Idd6 influences T-cell activities in the peripheral immune system and suggest that a major mechanism by which the Idd6 locus modifies diabetes development is via modulation of regulatory T-cell activities. Our transfer experiments using total splenocytes and purified T-cells demonstrated that the locus specifically controls the efficiency of disease protection mediated by the regulatory CD4(+)CD25(+) T-cell subset. Our data also implicate the Idd6 locus in controlling the balance between infiltrating lymphocytes and antigen-presenting cells within the pancreatic islet.     

Defective suppressor function in CD4(+)CD25(+) T-cells from patients with type 1 diabetes

Type 1 diabetes is a T-cell-mediated disease that is associated with loss of immunological tolerance to self-antigens. The mechanisms involved in maintenance of peripheral tolerance include a specialized subset of regulatory T-cells (Treg) within the CD4(+)CD25(+) T-cell population, but the function and phenotype of these cells in type 1 diabetes have not been investigated. We hypothesized that a deficiency in the CD4(+)CD25(+) Treg population or its function could contribute to the lack of self-tolerance evident in patients with type 1 diabetes. We show that although levels of CD4(+)CD25(+) T-cells are normal in patients with recent-onset adult type 1 diabetes, the ability of the Tregs in this population to suppress T-cell proliferation during in vitro cocultures is markedly reduced compared with control subjects (P = 0.007). Moreover, in patients with type 1 diabetes, these cocultures display a more proinflammatory phenotype, with increased secretion of interferon-gamma (P = 0.005) and decreased interleukin-10 production (P = 0.03). These deficiencies may reflect a disturbance in the balance of the CD4(+)CD25(+) population, because in patients with type 1 diabetes, a higher proportion of these cells coexpress the early activation marker CD69 (P = 0.007) and intracellular CTLA-4 (P = 0.01). These data demonstrate deficiency in function of the CD4(+)CD25(+) Treg population that may influence the pathogenesis of type 1 diabetes.     

In vivo control of diabetogenic T-cells by regulatory CD4+CD25+ T-cells expressing Foxp3

To understand the ability of regulatory T-cells to control diabetes development in clinically relevant situations, we established a new model of accelerated diabetes in young DP-BB rats by transferring purified T-cells from DR-BB rats made acutely diabetic. Transfer of 3, 5, 10, or 23 million pure in vitro-activated T-cells accelerated diabetes onset in >90% of the recipients, with the degree of acceleration being dosage dependent. Cotransfer of unfractionated leukocytes from healthy donors prevented diabetes. Full protection was achieved when protective cells were transferred 3-4 days before diabetogenic cells, whereas transfer 2 days before conferred only partial protection. Protection resided in the CD4(+) fraction, as purified CD4(+) T-cells prevented the accelerated diabetes. When CD25(+) cells were depleted from these cells before they were transferred, their ability to prevent diabetes was impaired. In contrast, two million CD4(+)CD25(+) cells (expressing Foxp3) prevented the accelerated diabetes when transferred both before and simultaneously with the diabetogenic T-cells. In addition, 2 million CD4(+)CD25(+) T-cells prevented spontaneous diabetes, even when given to rats age 42 days, whereas 20 million CD4(+)CD25(-) cells (with low Foxp3 expression) were far less effective. We thus demonstrated that CD4(+)CD25(+) cells exhibit powerful regulatory potential in rat diabetes.     

肝癌病人外周血中CD4+CD25+FOXP3+调节性T淋巴细胞表达水平的临床研究

目的 了解CD4+CD25+FOXP3+T调节性T细胞在肝癌病人外周血中的表达水平并探讨其临床意义.方法 应用流式细胞术测定18例肝癌病人外周血CD4+CD25+FOXP3+调节性T细胞占CD4+T淋巴细胞百分比,并与26例l临床对照者和24例健康对照者进行比较.结果 肝癌病人外周血中CD4+CD25+T细胞占CD4+T细胞百分比(4.25%±3.98%)明显高于临床对照组(1.34%±1.14%)及健康对照组(1.29%±0.95%)(P＜0.01),而两个对照组之间并无显著性差异(P＞0.05).CD4+CD25+FOXP3+T细胞在肝癌病人外周血所占CD4+T细胞比率(2.94%±0.91%)也较临床对照组(0.76%±0.34%)及健康对照组(0.81%±0.29%)显著升高(P＜0.001),且升高幅度强于CD4+CD25+T细胞水平,两个对照组之间并无显著性差异(P＞0.05).结论 CD4+CD25+FOXP3+T细胞是更为准确的调节性T细胞,其在肝癌病人外周血中表达水平明显升高,检测CD4+CD25+FOXP3+T水平对肝癌的预防治疗具有重要临床意义.     

Induction of tolerance in type 1 diabetes via both CD4+CD25+ T regulatory cells and T regulatory type 1 cells

Success in developing novel therapies to recommence self-tolerance in autoimmunity depends on the induction of T regulatory (Tr) cells. Here, we report that rapamycin combined with interleukin (IL)-10 efficiently blocks type 1 diabetes development and induces long-term immunotolerance in the absence of chronic immunosuppression in nonobese diabetic (NOD) mice. Rapamycin mediates accumulation in the pancreas of suppressive CD4(+)CD25(+)FoxP3(+) Tr cells, which prevent diabetes. IL-10 induces Tr type 1 (Tr1) cells, which reside in the spleen and prevent migration of diabetogenic T-cells to the draining lymph nodes. These two Tr cell subsets act in concert to control diabetogenic T-cells that are still present in long-term tolerant mice. Rapamycin plus IL-10 treatment, promoting distinct subsets of Tr cells, may constitute a novel and potent tolerance-inducing protocol for immune-mediated diseases.     

Kinetic analysis reveals potency of CD4+ CD25bright+ regulatory T-cells in kidney transplant patients

Donor-specific hyporesponsiveness as occurs after allogeneic kidney transplantation may be mediated by repression of effector cells by a specific subset of T-cells: the CD4(+) CD25(bright+) FoxP3(+) regulatory T-cells (Tregs). Here, we examined the suppressive capacity of Tregs isolated from the leukafereses product of 6 kidney transplant recipients, by reconstituting Tregs to responder T-cells at several time-points after initiation of proliferation. We show that Tregs derived from kidney transplant patients potently restrain proliferation to donor-antigens and 3rd party-antigens in classic reconstitution assays (i.e. addition of Tregs at the start of the co-incubation). However, when Tregs were added 5 days after initiation of proliferation, they were still capable of suppressing proliferation to donor-antigens (by 38%) but no longer to 3rd party-antigens. Thus, we conclude that the potency of Tregs to suppress reactivity to specific antigens should be determined by reconstitution to ongoing reactions.     

Distribution of CD4+CD25high regulatory T-cells in tumor-draining lymph nodes in patients with gastric cancer

BACKGROUND: Regulatory T-cells (T-regs) can inhibit the immune response mediated by T-cells. There is an increasing evidence that there is an increased proportion of T-regs in PBLs and tumor-infiltrating lymphocytes in several different human malignancies, although the mechanism remains unclear. In the present study, we evaluated the prevalence of CD4+CD25high T-regs in tumor-draining lymph nodes in patients with gastric cancers. MATERIALS AND METHODS: Regional lymph nodes in the stomach of the patients with gastric cancer (n=44) were classified into N1 regional lymph nodes adjacent to the gastric tumor and N2 regional lymph nodes marginally distant from the tumor. The population of CD4+CD25high T-cells as a percentage of total CD4+ cells was evaluated by flow cytometric analysis with triple-color staining. Cytokine production (IL-10 and IFN-gamma) was evaluated by intracellular cytokine staining and the antiproliferative function of CD4+CD25+ cells positively selected by magnetic beads was measured by evaluating the proliferative activity of CD4+CD25- cells in response to anti-CD3 plus anti-CD28 in the presence of autologous CD4+CD25+ cells. RESULTS: The percentage of CD4+CD25high T-cells in N1 regional lymph nodes (3.1 +/- 0.3%) was significantly higher than that of control mesenteric lymph nodes (1.2 +/- 0.3%, P <0.01). Furthermore, a more extended area (N2) of regional lymph nodes, as well as adjacent lymph nodes (N1) to the tumors, was involved in an increased prevalence of CD4+CD25high T-cells according to the disease progression. The functional evaluations confirmed that CD4+CD25high T-cells derived from the lymph nodes have an inhibitory activity corresponding to T-regs. CONCLUSIONS: The populations of CD4+CD25high T-cells in the regional lymph nodes in patients with gastric cancer were significantly higher in comparison to those in control lymph nodes. The increased prevalence of T-regs may be one of the explanations for impaired cell-mediated immunity in cancer-bearing hosts.     

Functional defects and the influence of age on the frequency of CD4+ CD25+ T-cells in type 1 diabetes

CD4+ CD25+ T-cells appear to play a crucial role in regulating the immune response. Therefore, we evaluated the peripheral blood frequency and function of CD4+ CD25+ T-cells in 70 type 1 diabetic patients and 37 healthy individuals. Interestingly, a positive correlation was observed between increasing age and CD4+ CD25+ T-cell frequency in both subject groups. In contrast to previous studies of nonobese diabetic mice and type 1 diabetic patients, similar frequencies of CD4+ CD25+ and CD4+ CD25(+Bright) T-cells were observed in healthy control subjects and type 1 diabetic patients of similar age. There was no difference between type 1 diabetic subjects of recent-onset versus those with established disease in terms of their CD4+ CD25+ or CD4+ CD25(+Bright) T-cell frequency. However, type 1 diabetic patients were markedly defective in their ability to suppress the proliferation of autologous effector T-cells in vitro. This type 1 diabetes-associated defect in suppression was associated with reduced production of interleukin (IL)-2, gamma-interferon, and transforming growth factor-beta, whereas other cytokines including those of adaptive and innate immunity (IL-10, IL-1beta, IL-6, IL-8, IL-12p70, and tumor necrosis factor-alpha) were similar in control subjects and type 1 diabetic patients. These data suggest that age strongly influences the frequency of CD4+ CD25+ T-cells and that function, rather than frequency, may represent the means by which these cells associate with type 1 diabetes in humans.     

Extracorporeal photochemotherapy is accompanied by increasing levels of circulating CD4+CD25+GITR+Foxp3+CD62L+ functional regulatory T-cells in patients with graft-versus-host disease

BACKGROUND: Extracorporeal photochemotherapy (ECP) produces clinical improvements in refractory/resistant graft-versus-host disease (GvHD). Immunological mechanisms of ECP are still under investigation. METHODS: We have evaluated the changes in frequency and immunophenotype of circulating regulatory T cells (T-regs) in 10 patients undergoing allogeneic hematopoietic stem cell transplantation, receiving ECP for acute (n=4) or chronic (n=6) GvHD. T-regs were monitored for expression of surface CD4, CD25, GITR, CD45RO, CD62L and intracytoplasmic Foxp3. T-regs were sorted by fluorescence-activated cell sorting to perform functional assays by interferon (IFN)-gamma enzyme-linked immunospot and real-time quantitative polymerase chain reaction (RQ-PCR) to measure Foxp3, transforming growth factor (TGF)-beta, and interleukin (IL)-10 mRNA. RESULTS: ECP was accompanied by a significant increase of CD4+CD25+ T-regs after six procedures, increasing from 8.9% to 29.1% of total CD4 (P<0.05), with a simultaneous increase of glucocorticoid induced tumor necrosis factor receptor expression on CD4+CD25+ cells (from 15% to 40.8%, P<0.05). This increase was sustained after 12 procedures. T-regs expressed high levels of CD62L, CD45RO, and Foxp3. Sorted CD4+CD25+ T-regs were potently inhibitory toward the CD4+CD25- fraction, when matched with an allogeneic target (IFN-gamma secretion was reduced by 79%). Trans-well experiments showed that cell-to-cell contact was necessary to exert inhibitory activity. RQ-PCR revealed a significant expression of Foxp3 in CD4+CD25+ T-regs, but there was virtually no detection of TGF-beta and IL-10. GvHD improved in all patients, allowing tapering or discontinuation of immunosuppressive drugs. CONCLUSION: Our study shows a time correlation between ECP and increasing percentages of circulating functional T-regs. Albeit suggestive, our results need to be confirmed on larger series to determine the actual role of T-reg in mediating the clinical effect of ECP.     

CD4+CD25+FOXP3+T细胞在肝癌肝移植肿瘤复发中的作用

目的 探讨肝癌肝移植病人移植前后外周血和肿瘤组织中CD4+CD25+FOXP3+T细胞比例变化及其临床意义.方法 用流式细胞仪检测肝癌肝移植病人和其他肝移植病人术后外周血中CD4+CD25+FOXP3+T细胞的比例,并采用正常人作对照.用免疫组化法检测肝癌病人和非肝癌病人肿瘤组织中FoxP3的表达及CD8+T细胞浸润的比例.观察肝癌肝移植病人术后及肿瘤复发后调节性T细胞的变化及其对肿瘤复发的影响.结果 流式细胞检测显示肝癌肝移植、非肝癌肝移植的病人术后外周血中CD4+CD25+FOXP3+T细胞占CD4+T细胞的比例较正常人明显升高,分别为(10.15±1.00)%、(5.30±1.64)%和(3.20±1.18)%,P<0.05.肝癌肝移植肿瘤复发病人较未复发病人外周血CD4+CD25+FOXP3+T比例明显升高,分别为(15.15±1.50)%和(6.80±1.50)%,P<0.01.免疫组化检测显示肿瘤组织中FOXP3+T细胞增多,CD8+T细胞浸润明显减少.结论 肝癌肝移植肿瘤复发的病人外周血中CD4+CD25+FOXP3+T细胞比例升高.调节性T细胞可能通过减少CD8+T细胞浸润,加速肿瘤复发.     

CD4+ CD25+ regulatory T-cells inhibit the islet innate immune response and promote islet engraftment

Early islet cell loss is a significant problem in clinical islet cell transplantation. Diverse stress stimuli induce innate immune responses in islets that contribute to beta-cell dysfunction, inflammation, and loss. Here, we show that cytokine-stimulated murine islets express multiple inflammatory chemokines that recruit T-cells and thereby impair islet function in vitro and in vivo. Both nonislet ductal and exocrine elements and the individual islet cellular components contribute to this innate immune response. CD4+ CD25+ regulatory T-cells inhibit islet chemokine expression through a cell contact-dependent, soluble factor-independent mechanism and inhibit effector T-cell migration to the islet. Regulatory T-cells can also migrate to stimulated islets. Cotransfer of regulatory T-cells with islets in a transplantation model prevents islet innate immune responses and inflammation and preserves normal architecture and engraftment. Regulatory T-cell inhibition of multiple components of innate immune responses may be a fundamental aspect of their function that influences ischemia-reperfusion injury and adaptive immunity.     

GAD65-specific CD4+ T-cells with high antigen avidity are prevalent in peripheral blood of patients with type 1 diabetes

Negative selection of self-reactive T-cells during thymic development, along with activation-induced cell death in peripheral lymphocytes, is designed to limit the expansion and persistence of autoreactive T-cells. Autoreactive T-cells are nevertheless present, both in patients with type 1 diabetes and in at-risk subjects. By using MHC class II tetramers to probe the T-cell receptor (TcR) specificity and avidity of GAD65 reactive T-cell clones isolated from patients with type 1 diabetes, we identified high-avidity CD4+ T-cells in peripheral blood, coexisting with low-avidity cells directed to the same GAD65 epitope specificity. A variety of cytokine patterns was observed, even among T-cells with high MHC-peptide avidity, and the clones utilize a biased set of TcR genes that favor two combinations, Valpha12-beta5.1 and Valpha17-Vbeta4. Presence of these high-avidity TcRs indicates a failure to delete autoreactive T-cells that likely arise from oligoclonal expansion in response to autoantigen exposure during the progression of type 1 diabetes.     

Spontaneous allograft tolerance in B7-deficient mice independent of preexisting endogenous CD4+CD25+ regulatory T-cells

BACKGROUND: Acute cardiac allograft rejection requires host, but not donor, expression of B7-1/B7-2 costimulatory molecules. However, acute cardiac rejection requires direct antigen presentation by donor-derived antigen presenting cells to CD4 T-cells and does not require indirect antigen presentation to CD4 T-cells. Given this discrepancy in the literature and that the consequence of allograft exposure in B7-deficient mice is unknown; the goal of the study was to examine the antidonor status of allografted B7-1/B7-2-deficient hosts. METHODS: C57Bl/6 B7-1/B7-2-/- mice were grafted with heterotopic BALB/c hearts. Recipients bearing long-term surviving allografts were used to examine the status of antidonor reactivity in vitro and in vivo. Tolerance was examined in vivo through adoptive transfer of splenocytes from graft-bearing animals to secondary immune-deficient Rag-1-/- hosts bearing donor-type or third-party cardiac allografts and by regulatory T-cell depletion with anti-CD25 antibody. RESULTS: When transferred to B7-replete Rag-1-/- recipients, cells from na?ve B7-1/B7-2-/- mice readily initiated cardiac allograft rejection. However, splenocytes transferred from long-term allograft acceptor B7-1/B7-2-/- hosts failed to reject donor-type hearts but acutely rejected third-party allografts. In addition, such cells did not reject (donorxthird-party) F1 allografts. Finally, in vivo depletion of regulatory T-cells did not prevent long-term acceptance. CONCLUSIONS: Results demonstrate that B7-deficient T-cells are capable of acute cardiac allograft rejection in a B7-replete environment. Importantly, results also show that B7-deficient hosts do not simply ignore cardiac allografts, but rather spontaneously develop transferable, donor-specific tolerance and linked suppression in vivo. Interestingly, this tolerant state does not require endogenous CD4+CD25+ regulatory T-cells.     

Calcineurin inhibitors, but not rapamycin, reduce percentages of CD4+CD25+FOXP3+ regulatory T cells in renal transplant recipients

BACKGROUND: Immunosuppression in renal transplantation, although manageable in the short-term, is a major hurdle for long-term graft survival. Recently, increased frequencies of CD4CD25 regulatory T cells (Tregs) have been described as an additional mechanism that induces alloimmune tolerance. METHODS: We assessed 64 renal transplant recipients with stable renal function for at least one year. Patients were divided into two groups according to the immunosuppression they were receiving at the moment of the study: one consisted of patients receiving rapamycin (Rapa) but not calcineurin inhibitors (CNI), and the other group received CNI but not Rapa. The Rapa group was further divided into three subgroups according to their previous experience with CNI: CNI-free, CNI withdrawal, and CNI conversion. Frequencies of blood Tregs were studied by flow cytometry after staining with monoclonal antibodies specific for different markers of Tregs. RESULTS: Frequencies of CD4 T cells with regulatory phenotype and function were significantly decreased in peripheral blood of renal transplant patients receiving CNI compared with those receiving Rapa. This effect was independent of an early exposure to CNI because the CNI-free patients in the Rapa group showed similar frequencies of Tregs to the CNI withdrawal and CNI conversion groups. CONCLUSIONS: CNI, but not Rapa, induce a decrease of circulating Tregs in stable renal transplant recipients. Thus, Rapa might be further explored in strategies using preservation of Tregs for transplant tolerance. Furthermore, quantification of blood Tregs may be a suitable tool to identify renal transplant recipients who may be candidates for reduced immunosuppression.     

Increased CD4+ CD25+ T regulatory cell activity in trauma patients depresses protective Th1 immunity

OBJECTIVES: We recently reported increased CD4 CD25 T regulatory (Treg) activity after burn injury in mice. This study sought to determine if Tregs mediate the reduction in TH1-type immunity after serious injury in man and if Treg function is altered by injury. METHODS: Peripheral blood was withdrawn from 19 consenting adult patients (35.1 +/- 16.3 years of age) with Injury Severity Scores (ISS) 36.6 +/- 13.9 on days 1 and 7 after trauma and from 5 healthy individuals. CD4 T cells were purified and sorted into Treg (CD25(high)) and Treg-depleted populations. After activation of cells with anti-CD3/CD28 antibody, production of the TH1-type cytokine IFNgamma, TH2-type cytokines (IL-4 and IL-5), and the inhibitory cytokine IL-10 was measured using cytometric bead arrays. Treg activity was measured by in vitro suppression of autologous CD4 T cell proliferation. RESULTS: All patients survived, 9 (47%) developed infection postinjury. IFNgamma production by patient CD4 T cells was decreased on day 1 and day 7, when compared with healthy controls. However, when Tregs were depleted from the CD4 T cells, the IFNgamma production increased to control levels. Tregs were the chief source of IL-4 and IL-5 as well as IL-10. Treg suppression of T cell proliferation increased significantly from day 1 to day 7 after injury. CONCLUSIONS: We demonstrate for the first time that human Tregs are increased in potency after severe injury. Most significantly, Tregs are important mediators of the suppression of T cell activation and the reduction in TH1 cytokine production found after injury.