Interleukin-7 is a survival factor for CD4+ CD25+ T-cells and is expressed by diabetes-suppressive dendritic cells

Dendritic cells can facilitate allograft survival and prevent autoimmunity via direct and indirect cell-mediated mechanisms. Recent studies demonstrate that immunoregulatory dendritic cells (iDCs) confer immune hyporesponsiveness in part through CD4(+) CD25(+) T regulatory cells (Tregs). Herein, we provide evidence to support the hypothesis that dendritic cells derived from NOD mice and engineered ex vivo to exhibit suppressed expression of the CD40, CD80, and CD86 costimulatory molecules motivate an increase in the prevalence of regulatory CD4(+) CD25(+) T-cells via interleukin (IL)-7. Unlike control dendritic cells, these dendritic cells expressed significant levels of IL-7. Exogenous addition of IL-7 to NOD T-cells did not promote expansion or proliferation, but instead selectively maintained the number of CD4(+) CD25(+) T-cells by inhibiting activation of apoptosis in these cells. In vitro, IL-7 receptor alpha-chain (IL-7Ralpha) was expressed at significantly higher levels on CD4(+) CD25(+) T-cells compared with CD4(+) CD25(-) T-cells irrespective of resting or stimulated state. In vivo, CD4(+) CD25(+) T-cells obtained from NOD-scid mice reconstituted with ex vivo engineered iDCs and NOD splenocytes expressed significantly higher levels of IL-7Ralpha compared with levels in the CD4(+) CD25(-) subset, especially in diabetes-suppressive dendritic cell-administered NOD-scid recipients. Taken together, our data suggest a novel mechanism by which iDCs delay autoimmunity through the CD4(+) CD25(+) Treg pathway and suggest IL-7 as a survival factor for these putative Tregs, which express the alpha-chain of its receptor at considerably higher levels than CD4(+) CD25(-) T-cells.     

Triazolopyrimidine derivative NK026680 and donor-specific transfusion induces CD4+CD25+Foxp3+ T cells and ameliorates allograft rejection in an antigen-specific manner

We have previously demonstrated the unique properties of a new triazolopyrimidine derivative, NK026680, which exerts immunosuppressive effects in rat heart transplant model and confers tolerogeneic properties on ex vivo-conditioned dendritic cells in mice. We herein demonstrate that NK026680 promotes the expansion of regulatory T cells (Tregs) with potent immunoregulatory effects when used in combination with donor-specific transfusion (DST).BALB/c (H-2^d) heart graft were transplanted into C57BL/6 (H-2^b) mice following intravenous injection of donor splenocytes (DST) and oral administration of NK026680. The NK026680 plus DST treatment markedly prolonged the survival time of the donor-graft, but not that of the 3rd party-graft (C3H; H-2^k). Treg cells in the recipient spleen on day 0 expanded when stimulated with donor-antigens in vivo and in vitro. After heart transplantation, Treg cells accumulated into the graft and increased in the spleen. NK026680 plus DST also decreased activated CD8⁺ T cells in the spleen and inhibited infiltration of CD8⁺ T cells into the graft. Depletion of CD25⁺ cells inhibited the graft prolonging effect of the NK026680 plus DST treatment.NK026680 administration together with DST induces potent immunoregulatory effects in an antigen-specific manner, likely due to the in vivo generation of donor-specific Tregs.     

GITR Ligation Blocks Allograft Protection by Induced CD25+CD4+ Regulatory T Cells without Enhancing Effector T-Cell Function

The induction of operational tolerance prior to transplant could provide a solution to the complications of current immunosuppression in transplantation. In rodents, operational tolerance frequently correlates with the presence of CD25(+)CD4(+) regulatory T cells (Tregs) but their function is usually demonstrated by adoptive transfer into lymphopenic hosts leading some to question their relevance to normal immunocompetent recipients. The role of these cells in primary transplant recipients has been explored using anti-CD25 antibody but specific targeting of Treg is not possible since CD25 is also up-regulated on activated effector T cells. To overcome this limitation we targeted the Treg associated molecule GITR in tolerized primary transplant recipients. This reverses regulation resulting in acute allograft rejection. This is not due to co-stimulation of effector cells since rejection mediated by isolated populations of CD4(+)CD25(-) or CD8(+)CD25(-) T cells transferred into Rag(-/-) mice was not enhanced by anti-GITR antibody. Furthermore, GITR cross-linking does not provide co-stimulation for in vitro proliferation of the same CD4(+)CD25(-) or CD8(+)CD25(-) T-cell populations in response donor-strain APC. Thus, CD4(+)CD25(+)GITR(+) Treg play an essential role in early graft protection in primary transplant recipients following tolerance induction providing further support for protocols that might generate similar populations in clinical transplantation.     

Characterization of CD3+CD4-CD8- (double negative) T cells reconstitution in patients following hematopoietic stem-cell transplantation

Background

CD3+CD4−CD8−double negative (DN) T cells, as a distinct subset of regulatory T cells (Tregs), played a pivotal role in patients following hematopoietic stem-cell transplantation.

Methods

This study examines the behavior of CD3+CD4−CD8− double negative (DN) T cells in 73 patients at days 30, 60, 90 and 180 after allo-HSCT.

Results

There was no significant difference in neutrophil and platelet engraftment between the higher and lower absolute counts of 30 days DN Tregs (p = 0.674, 0.863, respectively). The reconstitution of DN Tregs was significantly slower than that of CD8+, CD4+, and CD3+CD8+CD28− T cells (p < 0.001), but significantly faster than that of CD19+ and CD4+CD25+ T cells (p < 0.001, p = 0.032, respectively). Importantly, in the HLA mismatched group, DN Tregs reconstitution had significant effect on aGVHD (p = 0.027) and there was significant correlation between aGVHD and DN Tregs reconstitution (p = 0.035). DN Tregs reconstitution was significantly faster in the patients who were devoid of aGVHD than that of patients who developed aGVHD. Furthermore, we compared the absolute value of DN Tregs at 30 days, 60 days, 90 days and 180 days after allo-HSCT with grade aGVHD and found an inverse linear relationship in the HLA mismatched group (n = 37, P < 0.001, r = − 0.573).

Conclusions

The successful expansion of DN Tregs at 60 days after allo-HCST may help avoid severe manifestations of aGVHD in the HLA mismatched group, suggesting that DN Tregs have potential protection effect against aGVHD.     

Monotherapy rapamycin allows an increase of CD4+ CD25bright+ FoxP3+ T cells in renal recipients

CD4⁺ CD25^bright+ FoxP3⁺ regulatory T cells (Tregs) may control donor-specific allogeneic responses in kidney transplant recipients. Recent evidence demonstrated that three phenotypical Treg-subsets, naive (CCR7⁺CD45RO⁻), central-memory (CCR7⁺CD45RO⁺) and effector-memory (CCR7⁻CD45RO⁺), are essential for the development and function of antigen-specific suppression in the lymphoid and peripheral tissues. Also, it has been appreciated that Tregs are affected by immunosuppressive agents. In clinical practice, however, the effect of a single drug remains to be determined. Therefore, we analyzed the effect of several immunosuppressive agents on the number, phenotype and function of peripheral Tregs from 46 stable kidney transplant recipients. These patients were converted to monotherapy with tacrolimus ( n  = 15), rapamycin ( n  = 17) or mycophenolate mofetil ( n  = 14). Blood was obtained at inclusion and 6 months thereafter. The number of Tregs increased significantly in patients on monotherapy with rapamycin ( P  < 0.001), which was caused by increased numbers of Tregs with a central-memory and an effector-memory phenotype (both P  < 0.05). At 6 months after conversion, however, the suppressive function of Tregs did not significantly change in co-cultures stimulated with donor-Ag. Therefore, monotherapy with rapamycin allows the signals that are needed to increase the number of functional Tregs with a memory phenotype, thereby enhancing the potential capacity to regulate donor-specific responses in the lymphoid and the peripheral tissues.     

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.     

High frequency of CD4+ CD25- CD69+ T cells is correlated with a low risk of acute graft-versus-host disease in allotransplants

Regulatory T cells (Tregs) maintain transplantation tolerance and suppress graft-versus-host disease (GvHD) in humans. We monitored 17 subjects with acute GvHD to determine whether Treg frequency correlates with acute GvHD. We found the percent of CD4(+) CD25(-) CD69(+) Tregs decreases when acute GvHD develops and increases after acute GvHD is controlled. We next sequentially studied 50 subjects receiving conventional allotransplants. We show a high frequency and increased numbers of CD4(+) CD25(-) CD69(+) Tregs are associated with a reduced risk of acute GvHD. We also show that CD4(+) CD25(-) CD69(+) Treg numbers increase substantially early after allografts and that a low percent of CD4(+) CD25(-) CD69(+) Tregs is associated with an increased risk of acute GvHD. Reconstitution of Tregs early post-transplant is associated with less acute GvHD. These data imply that CD4(+) CD25(-) CD69(+) Tregs are a novel subset of regulatory T cells that may protect against acute GvHD after allotransplants.     

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.     

肝移植急性排斥反应者外周血CD4+CD25+Foxp3+调节性T淋巴细胞的变化

目的 探讨良性终末期肝病患者肝移植术后外周血CD4+CD25+叉状头螺旋转录因子(Foxp3)+调节性T淋巴细胞在急性排斥反应期的变化及意义.方法 2004年12月至2008年1月间,符合入选条件的良性终末期肝病患者共55例,按照术后是否发生急性排斥反应分为排斥组(14例)和无排斥组(41例).肝移植术前用流式细胞仪检测患者外周血CD4+CD25+Foxp3+T淋巴细胞占CD4+T淋巴细胞的百分率(简称CD4+CD25+Foxp3+T细胞百分率),出院后1年内每隔3～6个月复查;发生急性排斥反应时,于治疗前和治疗缓解后(3～6个月)复查.比较两组患者外周血CD4+CD25+Foxp3+T细胞百分率的变化,对排斥组发生急性排斥反应时外周血CD4+CD25+Foxr3+T细胞百分率与排斥反应活动指数(RAI的相关性进行统计学分析.结果 肝移植术前,排斥组与无排斥组外周血CD4+CD25+Foxp3+T细胞百分率的差异无统计学意义(P＞0.05).排斥组患者发生急性排斥反应时外周血CD4+CD25+Foxp3+T细胞百分率为(2.23±0.54)%,低于无排斥组的(2.99±0.86)%,差异有统计学意义(P＜0.01).排斥组中,患者发生急性排斥反应时外周血CD4+CD25+Foxp3+T细胞百分率低于未发生急性排斥反应时的(3.67±0.70)%,差异有统计学意义(P＜0.01).排斥组患者发生急性排斥反应时外周血CD4+CD25+Foxp3+T细胞百分率与RAI呈负相关(r=-0.80,P＜0.01).结论 监测肝移植受者外周血CD4+CD25+Foxp3+调节性T淋巴细胞的变化,可辅助诊断急性排斥反应及判断其严重程度.

Abstract：

Objective To investigate the expression of peripheral blood (PB) CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) in patients with benign end-stage liver disease after liver transplantation and the relationship between levels of PB Tregs and acute rejection. Methods A prospective analysis was performed on 55 consecutive patients who underwent liver transplantation.Fourteen out of 55 cases suffered from acute rejection after liver transplantation were defined as rejection group,while the rest patients were classified into no acute rejection group. PB was obtained from liver transplant patients at different time points longitudinally: pre-transplant, post-transplant within one year and acute rejection. The circulating CD4+ CD25+ Foxp3+ Tregs in PB were measured by flow cytometry. Blood samples were drawn during acute rejection, at the same time, liver biopsies were performed. The circulating CD4+ CD25+ Foxp3+ Tregs were compared between two groups.Results There was no difference between two groups in levels of circulating CD4+ CD25+ Foxp3 + Tregs cells pre-transplant. However, the levels of circulating CD4+ CD25+ Foxp3+ Tregs in rejection group were decreased significantly as compared with no-rejection group (2. 23 % ± 0. 54 % vs. 2. 99 % ±0. 86 %,P＜0.01). The frequency of CD4+ CD25+ Foxp3+ T cells was negatively correlated with rejection activity index (RAI) (r = - 0. 80, P＜0. 01 ). Conclusion Monitoring PB CD4+ CD25+ Foxp3+ Tregs levels may be helpful in evaluating the immune state and act as a more sensitive marker for acute rejection diagnosis in the patients following liver transplantation.     

Quantitative in situ analysis of FoxP3+ T regulatory cells on transplant tissue using laser scanning cytometry

There is abundant evidence that immune cells infiltrating into a transplanted organ play a critical role for destructive inflammatory or regulatory immune reactions. Quantitative in situ analysis (i.e., in tissue sections) of immune cells remains challenging due to a lack of objective methodology. Laser scanning cytometry (LSC) is an imaging-based methodology that performs quantitative measurements on fluorescently and/ or chromatically stained tissue or cellular specimens at a single-cell level. In this study, we have developed a novel objective method for analysis of immune cells, including Foxp3(+) T regulatory cells (Tregs), on formalin-fixed /paraffin-embedded (FFPE) transplant biopsy sections using iCys? Research Imaging Cytometer. The development of multiple immunofluorescent staining was established using FFPE human tonsil sample. The CD4/CD8 ratio and the population of Tregs among CD4(+) cells were analyzed using iCys and compared with the results from conventional flow cytometry analysis (FCM). Our multiple immunofluorescent staining techniques allow obtaining clear staining on FFPE sections. The CD4/CD8 ratio analyzed by iCys was concordant with those obtained by FCM. This method was also applicable for liver, small intestine, kidney, pancreas, and heart transplant biopsy sections and provide an objective quantification of Tregs within the grafts.     

TGF-β Induces Foxp3 + T-Regulatory Cells from CD4 + CD25 − Precursors

CD4 + CD25 + regulatory T cells (Tregs) are potent suppressors, playing important roles in autoimmunity and transplantation tolerance. Understanding the signals necessary for the generation and expansion of Tregs is important for clinical cellular therapy, but only limited progress has been made. Recent reports suggest a role for TGF-beta in the generation of Tregs from CD4 + CD25 - precursors, but the mechanism remains unknown. Here, we demonstrate that TGF-beta2 triggers Foxp3 expression in CD4 + CD25 - precursors, and these Foxp3 + cells act like conventional Tregs. The generation of Foxp3 + Tregs requires stimulation of the T-cell receptor, the IL-2R and the TGF-beta receptor. More importantly, strong costimulation through CD28 prevents Foxp3 expression and suppressive function in an IL-4-dependent manner. Furthermore, TGF-beta-driven Tregs inhibit innate inflammatory responses to syngeneic transplanted pancreatic islets and enhance islet transplant survival. Thus, TGF-beta is a key regulator of the signaling pathways that initiate and maintain Foxp3 expression and suppressive function in CD4 + CD25 - precursors. TGF-beta and signaling through TGF-beta receptor, CD28 costimulation and IL-4 may be key components for the manipulation of Treg. The de novo generation of Foxp3 + cells from CD4 + cells has the potential to be used for treatment of autoimmune diseases and induction of transplant tolerance.     

Reduced CD4+ CD25++ CD45RA− Foxp3hi activated regulatory T cells and its association with acute rejection in patients with kidney transplantation

BackgroundIt was found that regulatory T cells (Tregs) importantly affect the maintenance of the kidney graft. However, Tregs are a heterogeneous population with less to more suppressive activity. The aim of this study was to determine the effects of different subsets of Tregs, as well as their ratio to effector T cells (Teff), on kidney transplantation outcomes.MethodsA total of 58 participants were enrolled in this study and divided into four groups: (i) first kidney transplant recipients (stable 1); (ii) second kidney transplant recipients (stable 2); (iii) transplant recipients with acute rejection (AR); and (iv) healthy control subjects. By using flow cytometer, the frequencies of CD4⁺ CD25⁺⁺ CD45RA⁻ Foxp3^hi activated Tregs (aTregs), CD4⁺ CD25⁺ CD45RA⁺ Foxp3^lo resting Tregs (rTregs), CD4⁺ CD25⁺ CD45RA⁻ Foxp3^lo non-suppressive T cells, CD4⁺ CD25⁺ Foxp3⁻ cells Teff, and total Tregs were analyzed in all subjects.ResultsThe frequency of aTregs (as well as the ratio of aTregs/Tregs) was significantly lower in the AR patients than the other three groups. In contrast to AR patients, stables 1 and 2 had a higher aTreg/Treg ratio than those in the control group. Although patients with AR had a significantly lower total Tregs than the other three groups, the balance of total Tregs and Teff was similar between patients with and without AR.ConclusionPatients with AR had poorer immunoregulatory properties than those with normal graft functioning, as well as those in the control group. These reduced immunoregulatory properties in patients with AR could lead to graft rejection.     

Short-term anti-CD25 monoclonal antibody treatment and neogenetic CD4(+)CD25(high) regulatory T cells in kidney transplantation

CD4(+)CD25(high) T cells named regulatory T (Treg) cells are generated and play a key role in the induction and maintenance of transplant tolerance in organ recipients. Interleukin-2 (IL-2) enhance the development of effector cells and is essential for generation of Treg cells. The effect of the anti-CD25 monoclonal antibody (anti-CD25mAb) induction therapy on the neogenetic CD4(+)CD25(high)Treg cells is important for therapeutic strategies in kidney transplant. To clarify the question, a prospective study was conducted in 21 living donor kidney transplant recipients who randomly divided into the anti-CD25mAb group (Daclizumab) with 11 patients and the control group with 10 patients. The frequency of CD4(+)CD25(high)Treg cells in total CD4(+) T cells was analyzed by flow cytometry and FoxP3 expression by RT-PCR in peripheral blood, and results were compared at day 0, 3, 13, 17, 27 posttransplantation. There was no significant difference in patient characteristics and allograft survival. The present study showed that in vivo antigen-specific Treg cells population were generated and expanded after transplant. Both groups showed a significant increase in the frequency of CD4(+)CD25(high)Treg cells and higher level of FoxP3 mRNA after transplantation while the serum creatinine declined. Compared with the control group, recipients with anti-CD25mAb injection had significantly lower percentage of CD4(+)CD25(high) in total CD4(+) cells (1.13%+/-0.13% vs 1.94%+/-0.22%, P=0.00; 3.75%+/-0.28% vs 7.11%+/-0.51%, P=0.00) on day 3, 17 after transplantation. While, the percentage was not significantly different on day 10, 27 (3.72%+/-0.19% vs 4.36%+/-0.28%, P=0.08; 7.84%+/-0.35% vs 8.56%+/-0.36%, P=0.16). However, there was not obvious difference in Foxp3 expression level associated with the source of the CD4(+)CD25(high)Treg cells at the different time point after transplant. Our data indicated that CD4(+)CD25(high)Treg cells were transiently affected by anti-CD25mAb, without depletion. In conclusion, the short-term treatment with anti-CD25mAb might not prevent the production, proliferation of neogenetic Treg cells in organ transplant.     

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.     

Modified CD4(+) T-cell response in recipients of old cardiac allografts

With an increasing demand, organs from elderly donors are more frequently utilized for transplantation. Herein, we analyzed the impact of donor age on CD4(+) T-cell responses with regard to regulatory and effector mechanisms. Young (3months) BM12 recipients were engrafted with young or old (18months) B6 cardiac allografts. Systemic CD4(+) T-cell responses and intragraft changes were monitored and compared to age-matched syngenic transplant controls. While elderly, nonmanipulated hearts contained significantly elevated frequencies of donor-derived leukocytes prior to transplantation, allograft survival was age-independent. T-cell activation, however, was delayed and associated with a compromised immune response in mixed lymphocyte cultures (MLR; P=0.0002) early after transplantation (day 14). During the time course after transplantation, recipients of old grafts demonstrated an augmented immune response as shown by significantly higher frequencies of activated CD4(+) T-cells and a stronger in vitro alloreactivity (MLR; ELISPOT; P<0.01). In parallel, frequencies of regulatory T-cells had increased systemically and overall fewer CD4(+) T-cells were detected intragraft. Interestingly, changes in the CD4(+) T-cell response were not reflected by graft morphology. Of note, transplantation of young and old syngenic hearts did not show age-related differences of the CD4(+) T-cells response suggesting that old grafts can recover from a period of short cold ischemia time. Our data suggest that donor age is associated with an augmented CD4(+) T-cells response which did not affect graft survival in our model. These findings contribute to a better understanding of the immune response following the engraftment of older donor organs.     

Long time follow up of CD28- CD4+ T cells in living kidney transplant patients

We previously reported that the CD28(-) CD4(+) T cell subpopulation was expanded in the kidney allograft patients with long graft survival, although these T cells were rarely found in patients with graft survival <5 yr. To understand the CD28(-) CD4(+) T cells in the long-term acceptance of kidney allografts, we examined functions of this population and performed a 4 yr follow up study. Peripheral blood mononuclear cells (PBMC) were obtained from 47 long-term living related kidney allograft recipients. CD28(+) CD4(+) and CD28(-) CD4(+) T cells purified by cell sorting were analyzed for expression of V(beta) repertoire. Donor-specific response was examined in mixed lymphocyte reaction (MLR). A follow up study with long-term kidney allograft patients was performed for 4 yr about the rate of CD28(-) CD4(+) T cells. Eleven patients were examined by MLRs against donors and third party. Four patients with a marked increase of CD28(-) CD4(+) T cells showed the donor-specific responses appeared to be lower when compared with third party-specific responses. Freshly sorted CD28(-) CD4(+) T cells showed a restricted V(beta) repertoire, whereas the V(beta) usage of CD28(+) CD4(+) T cells from the same patients was much diversified. Such difference in V(beta) repertoire was not evident between the two populations from healthy control. A follow up study showed the ratio of CD28(-) CD4(+) T cells appeared to be lower in patients who were suspected of chronic rejection. These unusual CD4(+) T cells might be related to the long-term acceptance of human transplant allografts.     

The roles of CD25+CD4+ regulatory T cells in operational tolerance after living donor liver transplantation

Recent evidence suggests that CD4+CD25+ regulatory T cells (Tregs) affect immune responses, including those to alloantigens in organ transplants. We have followed a group of liver allograft recipients with good liver graft function who have been weaned off immunosuppression (IS). The purpose of this study was to determine whether Tregs contributed functionally to the mechanisms of graft acceptance. MATERIAL AND METHODS: The functional assay used peripheral blood obtained from LTx recipients free of immunosuppression. The Whole population of CD4+ T cells and the CD4+ T cells depleted of CD4+CD25 high cells were tested for proliferation against donor versus third party stimulators. Moreover to determine the antigen-specificity of the Tregs, serially diluted numbering of CD4+CD25+ T cells were co-cultured with CD4+CD25- T cells. The proliferation responses were examined toward donor versus third party stimulators. RESULT: CD4+ T cells from all LTx recipients off immunosuppression showed hyporesponsiveness to the donor but not to third party stimulators. However, even after depletion of the CD4+CD25 high population, the cells continued to be hyporesponsive toward the donor. In four out of five cases, the suppression exhibited by CD4+CD25+ T cells was more specific for the donor. DISCUSSION: These findings suggest that donor alloantigen specific regulation by Tregs is one of multiple mechanisms that may contribute to the maintenance of liver graft survival in the absence of immunosuppression.     

Requirement of CD28 signaling in homeostasis/survival of TGF-beta converted CD4+CD25+ Tregs from thymic CD4+CD25- single positive T cells

BACKGROUND: The thymus is a major organ that generates "natural" CD4+CD25+ T regulatory cells (Tregs). However, the detailed pathway(s) by which Tregs are developed remain a mystery. CD28-/- mice have profound decrease in Tregs, but the underlying molecular events remain largely undefined. METHODS: CD4+CD25+ thymocytes from wildtype and CD28-/- mice were cultured with T-cell receptor (TCR) and transforming growth factor (TGF)-beta stimulation to generate CD25+ Tregs and their phenotype and function were studied in vitro and in vivo. RESULTS: TGF-beta induced Foxp3 expression in thymic CD4+CD25+ cells and converted them to CD25+ Tregs. The converted Tregs expressed high levels of CD25, whereas the non-suppressive CD4+ T cells from the control cultures expressed CD25(low). CD28-/- thymic CD4+CD25+ cells showed transit lower levels of Foxp3 upon TCR and TGF-beta stimulation early in culture, but the defect in Foxp3 expression was restored to normal levels after 60-72 hr. Consequently, TGF-beta converted CD28-/- CD25+ cells to CD25+ Tregs that were indistinguishable from those of the wildtype mice. However, the total number of TGF-beta converted CD28-/- Tregs was significantly lower than that of wildtype mice. In vivo, TGF-beta converted CD28-/- CD25+ Tregs were less viable than those from the wildtype mice. Importantly, TGF-beta induced alloantigen specific CD4+CD25+ Tregs from thymic CD25-SP cells which also required CD28 to maintain their survival. CONCLUSIONS: TGF-beta and TCR co-stimulation converts thymic CD4+CD25+ T cells into CD4+CD25+ Tregs by inducing Foxp3, and the contribution of CD28 stimulation to this process is mainly through maintaining survival of the induced Tregs.