Immunosuppressive drugs on inducing Ag-specific CD4(+)CD25(+)Foxp3(+) Treg cells during immune response in vivo

A variety of immunosuppressive drugs are currently used in patients with allo-grafts or autoimmune diseases. Though the effects of rapamycin (RPM) and other immunosuppressant on the CD4(+)CD25(+)Foxp3(+) T regulatory cells (Tregs) were studied, their impact on Ag-specific Tregs during immune response was not well defined. In our studies, we adoptively transferred TCR-transgenic CD4(+)KJ1-26(+) T cells, CD4(+)KJ1-26(+)CD25(-) na?ve T cells or CD4(+)KJ1-26(+)CD25(+) Tregs into syngeneic BALB/c mice. 24h later, we treated the recipients with OVA immunization and immunosuppressant including rapamycin (RPM), fingolimod (FTY720), cyclosporin A (CsA), mycophenolate mofetil (MMF), leflunomide (LEF), cyclophosphamide (Cy) or none, respectively. The levels and function of CD4(+)KJ1-26(+)CD25(+)Foxp3(+) Tregs in draining lymph nodes (dLNs) and spleens were determined at different time points. Significantly higher percentage and cell number of Ag-specific CD4(+)KJ1-26(+)CD25(+)Foxp3(+) Tregs were observed in OVA immunized mice treated with RPM or FTY720 compared with mice that received OVA immunization alone. Furthermore, RPM augmented the population of functional iTregs in dLNs and spleens whereas inhibited nTregs during immune response. In contrast to RPM and FTY720, MMF, LEF, CsA, and Cy markedly decreased the levels of Ag-specific CD4(+)KJ1-26(+)CD25(+)Foxp3(+) Tregs during immune response. Thus, different immunosuppressive drugs have distinct effects on the Ag-specific CD4(+)CD25(+)Foxp3(+) Tregs during immune response. The stronger inhibiting effects of MMF, LEF, CsA and Cy on CD4(+)CD25(+)Foxp3(+) Tregs than on T effectors may block the host immune tolerance potentiality.     

Regulatory T cells and T cell depletion: role of immunosuppressive drugs

Allogeneic immune responses are modulated by a subset of host T cells with regulatory function (Treg) contained within the CD4(+)CD25(high) subset. Evidence exists that Treg expand after peritransplantation lymphopenia, inhibit graft rejection, and induce and maintain tolerance. Little, however, is known about the role of Treg in the clinical setting. IL-2 and activation by T cell receptor engagement are instrumental to generate and maintain Treg, but the influence of immunosuppressants on Treg homeostasis in humans in vivo has not been investigated. This study monitored Treg phenotype and function during immune reconstitution in renal transplant recipients who underwent profound T cell depletion with Campath-1H and received sirolimus or cyclosporine (CsA) as part of their maintenance immunosuppressive therapy. CD4(+)CD25(high) cells that expressed FOXP3 underwent homeostatic peripheral expansion during immune reconstitution, more intense in patients who received sirolimus than in those who were given CsA. T cells that were isolated from peripheral blood long term after transplantation were hyporesponsive to alloantigens in both groups. In sirolimus- but not CsA-treated patients, hyporesponsiveness was reversed by Treg depletion. T cells from CsA-treated patients were anergic. Thus, lymphopenia and calcineurin-dependent signaling seem to be primary mediators of CD4(+)CD25(high) Treg expansion in renal transplant patients. These findings will be instrumental in developing "tolerance permissive" immunosuppressive regimens in the clinical setting.     

不同免疫抑制剂对肝移植受者外周血中调节性T淋巴细胞比例的影响

目的 探讨西罗莫司(SRL)和钙调磷酸酶抑制剂(CNI)对肝移植受者外周血中CD4+CD25high T淋巴细胞水平的影响.方法 排除肝移植远期移植肝功能异常的受者,将移植肝功能长期(超过2年)稳定的受者47例纳入研究,其中免疫抑制方案使用SRL者15例(SRL组),使用CNI(均为他克莫司)者32例(CNI组).以同期38名健康成人志愿者作为正常对照.使用流式细胞仪检测各组受试者外周血中单个核细胞CD4、CD25及Foxp3的表达水平,比较各组间外周血中CD4+CD25high调节性T淋巴细胞(Treg细胞)的差异.结果 与正常对照组相比,CNI组外周血淋巴细胞中CD4+ CD25high T淋巴细胞的比例显著减少(P<0.05),SRL组CD4+ CD25high T淋巴细胞的比例显著升高(P<0.05).SRL组、正常对照组和CNI组受试者外周血中CD4+ CD25high Foxp3+ Treg 细胞占CD4+ T淋巴细胞的比例依次降低,分别为1.88%(1.56%～2.60%)、1.15%(0.57%～1.48%)和0.84%(0.46%～1.45%),3组间两两比较,差异均有统计学意义(P<0.01或P<0.05).CD4+ CD25 high T淋巴细胞表达Foxp3的阳性率超过95%,CD4+ CD25 low T淋巴细胞表达Foxp3的阳性率低于20%,CD4+ CD25-T淋巴细胞不表达Foxp3.结论 SRL可促进肝移植受者外周血中Treg细胞水平的升高,而CNI可降低Treg细胞的水平.     

Rapamycin enhances the number of alloantigen-induced human CD103+CD8+ regulatory T cells in vitro

BACKGROUND: Regulatory T cells (T(reg) cells) may be operational in both the induction and maintenance of transplantation tolerance. We recently showed that alloantigen-induced CD103+ CD8+ T cells strongly suppressed T-cell proliferation in mixed lymphocyte culture (MLC) via a contact-dependent mechanism. CD103 directs T lymphocytes to their ligand E-cadherin, which is expressed on renal tubular epithelial cells, and CD103+ CD8+ T cells have been described to be present in late renal allograft rejection. METHODS: We studied the influence of prednisolone, cyclosporin, tacrolimus, CD25 monoclonal antibodies, rapamycin, and mycophenolate mofetil (MMF) on the development and functional activity of alloantigen-activated CD103+ CD8+ T cells in MLC. RESULTS: Calcineurin inhibitors, MMF, and CD25mAb did not influence the number of CD103 expressing CD8+ T cells. In contrast, corticosteroids diminished CD103 expression on alloactivated CD8+ T cells, which appeared to be caused by their inhibitory action on myeloid dendritic cells. Addition of rapamycin to allocultures led to an increased percentage of CD103+ CD8+ alloreactive T cells. Moreover, in the presence of rapamycin, these cells tended to show higher suppressive capacity. CONCLUSIONS: Alloreactive CD103+ CD8+ T(reg) cells may expand and exert their suppressive function during immunosuppressive treatment with rapamycin. These data are relevant in the design of immunosuppressive drug regimens intended to induce and/or maintain transplantation tolerance.     

The effect of immunosuppressive drug rapamycin on regulatory CD4+CD25+Foxp3+T cells in mice

CD4(+)CD25(+)Regulatory T (Treg) cells are crucial for negatively regulating immune responses. Rapamycin (rapa) is an immunosuppressive agent which is widely used for preventing acute graft rejection in patients and has been used to induce operational tolerance in mouse models. The aim of the present study was to determine the effect of rapa on CD4(+)CD25(+)Foxp3(+)Treg cells in a mouse model. After C57BL/6 mice were intraperitoneally given 1.5 mg/kg/day of rapa for 14 days, the percentages, cell numbers, phenotype and function of CD4(+)CD25(+)Treg cells were determined by flow cytometry as well as the in vitro and in vivo functional assays. The cell numbers of CD4(+) and CD4(+)CD25(+)Treg cell subsets were markedly decreased in rapa-treated mice as reported. However, rapa significantly enhanced the ratios of CD4(+)CD25(+)Treg cells or CD4(+)CD25(+)Foxp3(+)Treg cells to CD4(+)T cells in spleens and thymi of mice (P<0.01) respectively. Furthermore, splenic CD4(+)CD25(+)Treg cells in rapa-treated mice showed immunosuppressive ability on the immune response of T effector cells to alloantigens or mitogen as efficiently as the control CD4(+)CD25(+)Treg cells in vitro and in vivo. Thus, rapa could significantly enhance the percentages of CD4(+)CD25(+)Foxp3(+)Treg cells in the thymus and the periphery while keeping these cells functional, indicating that CD4(+)CD25(+)Treg cells are more resistant to rapa than other CD4(+)T cells. The different effects of rapa on CD4(+)CD25(+)Treg and T effector cells make rapa to be a favorable choice for inducing immune tolerance to self-, allo-, or xeno-antigens.     

Development of mouse CD4(+)CD25(+)Foxp3(+) regulatory T cells in xenogeneic pig thymic grafts

Xenogeneic thymus transplantation is an effective strategy to induce tolerance to donors mainly by clonal depletion of reactive T cells. Recent studies have shown that functional mouse CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) could efficiently populate in the periphery of athymic mice after grafting with neonatal pig thymus. However, it is still unknown whether xenogeneic thymus grafts could directly support the development of mouse CD4(+)CD25(+)Foxp3(+) Treg cells as an autogeneic counterpart. Our results show that the percentages of mouse CD4(+)CD8(-)CD25(+) thymocytes are similar among auto- and xenogeneic thymic grafts in thymic mouse recipients. Mouse CD4(+)CD8(-)CD25(+) thymocytes maturing in xenogeneic thymic grafts exhibit similar expressions of Foxp3, TCR, CTLA-4 and GITR as those in autogeneic thymic grafts. Moreover, mouse CD4(+)CD8(-)CD25(+) thymocytes maturing in xenogeneic thymic grafts showed a significant immunosuppressive function on the proliferation of CD4(+)CD25(-) T cells stimulated with Con A or allogeneic antigens, although they showed weaker effects than those maturing in autogeneic thymic grafts. Therefore, the present data provides direct evidence for the ability of xenogeneic porcine thymus grafts to support the development of mouse naturally occurring CD4(+)CD25(+)Foxp3(+) Treg cells.     

Depleting anti-CD4 monoclonal antibody (GK1.5) treatment: influence on regulatory CD4+CD25+Foxp3+ T cells in mice

BACKGROUND: CD4(+)CD25(+) regulatory T (Treg) cells are often essential for the maintenance of immunologic self-tolerance and transplant tolerance in some cases. The effects of depleting anti-CD4 monoclonal antibody (GK1.5), which was used in transplant tolerance induction, on CD4(+)CD25(+) Treg cells have not been investigated. METHODS: Three weeks after BALB/c mice were injected with GK1.5 or phosphate-buffered saline, the levels, phenotype and immunosuppressive function of CD4(+)CD25(+) Treg cells in these mice were detected. RESULTS: The numbers of CD4 and CD4(+)CD25(+) Treg cells in the periphery were markedly decreased in GK1.5-treated mice. However, GK1.5 treatment significantly enhanced the ratios of CD4(+)CD25(+) T cells or CD4(+)CD25(+)Foxp3 T cells to CD4(+) T cells in the periphery (P<0.01). Compared with the control mice, more CD4(+)CD25(+) T cells in GK1.5-treated mice showed CD45RB and CD62L phenotype. Furthermore, enriched CD4(+)CD25(+) Treg cells in GK1.5-treated mice show immunosuppressive ability on the immune response of T effector cells to alloantigens or mitogen as efficiently as those from the control mice in vitro. CONCLUSIONS: GK1.5 could significantly enhance the percentage of CD4(+)CD25(+)Foxp3(+) Treg cells in the periphery while keeping these cells functional, indicating that GK1.5 might affect the potential induction of immune tolerance by different influences on CD4(+)CD25(+)Treg cells and CD4(+)CD25(-) T cells in periphery.     

CD4+ CD25bright+ Regulatory T Cells Can Mediate Donor Nonreactivity in Long-Term Immunosuppressed Kidney Allograft Patients

CD4+ CD25bright+ FoxP3+ T cells are potent regulators of T-cell reactivity, but their possible involvement in donor-specific nonresponsiveness after clinical kidney transplantation remains to be elucidated. We assessed the proliferative donor-reactivity in 33 kidney allograft recipients who were maintained on a combination of proliferation inhibitors (mycophenolate mofetil (MMF) or Azathioprine (Aza)) and prednisone, long (> 5 years) after transplantation. Of the 33 patients, 8 still exhibited donor-reactivity, whereas 25 were classified as donor nonreactive patients. Within these 25 donor nonreactive patients, we assessed the involvement of CD4+ CD25bright+ regulatory T cells both by depleting them from the responder population as well as by reconstituting them to the CD25(-/dim) effector population. The absence of proliferation in these 25 patients, was abolished in 7 (28%) recipients upon depletion of the CD4+ CD25bright+ T cells. Reconstitution of these cells suppressed the donor-reactivity in a dose-dependent manner. Adding-back CD4+ CD25bright+ T cells inhibited the anti-third party response in all recipients, indicating that functional CD4+ CD25bright+ T cells circulate despite more then 5 years of immunosuppressive treatment. Altogether, we conclude that in long-term immunosuppressed kidney allograft patients functional regulatory CD4+ CD25bright+ T cells circulate but that these cells mediate donor non reactivity only in a subset of patients.     

Expression of CD39 by Human Peripheral Blood CD4+CD25+ T Cells Denotes a Regulatory Memory Phenotype

We have shown that CD39 and CD73 are coexpressed on the surface of murine CD4⁺Foxp3⁺ regulatory T cells (Treg) and generate extracellular adenosine, contributing to Treg immunosuppressive activity. We now describe that CD39, independently of CD73, is expressed by a subset of blood‐derived human CD4⁺CD25⁺CD127lo Treg, defined by robust expression of Foxp3. A further distinct population of CD4⁺CD39⁺ T lymphocytes can be identified, which do not express CD25 and FoxP3 and exhibit the memory effector cellular phenotype. Differential expression of CD25 and CD39 on circulating CD4⁺ T cells distinguishes between Treg and pathogenic cellular populations that secrete proinflammatory cytokines such as IFNγ and IL‐17. These latter cell populations are increased, with a concomitant decrease in the CD4⁺CD25⁺CD39⁺ Tregs, in the peripheral blood of patients with renal allograft rejection. We conclude that the ectonucleotidase CD39 is a useful and dynamic lymphocytes surface marker that can be used to identify different peripheral blood T cell‐populations to allow tracking of these in health and disease, as in renal allograft rejection.     

Absence of CD4CD25 regulatory T cell expansion in renal transplanted patients treated in vivo with Belatacept mediated CD28-CD80/86 blockade

AIMS: Belatacept is a new recombinant molecule (CTLA4-Ig) that interferes with the second activation signal of T lymphocytes. CTLA4-Ig induced T cell allograft tolerance in rodents but not in primates. We examined the changes in peripheral lymphocyte subsets, including regulatory T cells, in renal transplant patients treated with Belatacept. METHODS: A cross-sectional immunological study was carried out 6 months after transplantation in 28 patients enrolled in the Belatacept phase II study. Eighteen patients received Belatacept, mycophenolate mofetil and steroids (Belatacept group), while the control group of 10 patients received cyclosporine, mycophenolate mofetil and steroids (CsA group). Lymphocyte subsets were examined by flow cytometry. Foxp3 mRNA expression was measured by quantitative PCR. RESULTS: The number of T lymphocytes and the percentage of CD3+ T cells were similar in both groups. However, the percentage of CD3+ CD4+ T cells was lower in the Belatacept group than in the control CsA group (B=42.5%+/-13.7 vs CsA=52.9%+/-9, p<0.005), and the percentage of CD3+ CD8+ cells was higher in the Belatacept group than in the control (B=32.9%+/-6.7 vs CsA=19.5%+/-8.2, p<0.0002). The percentage of CD19+ cells was similar in both groups. Among CD56+cells, only the percentage of CD16+ cells was significantly higher in the Belatacept group than in the control (B=82%+/-12 vs CsA=59.7%+/-25, p=0.01). Among CD4 and CD8 T cells the percentage of activated lymphocytes expressing CTLA4, HLA-DR or CD40L was similar in both groups. The percentage of CD4+CD25+ T cells was higher in the CsA group. The percentage of regulatory CD4+CD25+ cells with bright CD25 staining was similar in both groups (B=3.6+/-2.3% vs CsA=4.7+/-1.9%, ns) as was the expression of FoxP3. CONCLUSION: Our results indicated that Belatacept did not induce regulatory T cell expansion in vivo. We suggest that Belatacept treatment should be maintained after transplantation to allow graft acceptance.     

Reduction of Foxp3-expressing regulatory T cell infiltrates during the progression of renal allograft rejection in a mouse model

BACKGROUND: Regulatory T (Treg) cells are the immune suppressors in the maintenance of immune homeostasis and tolerance to self and non-self antigens, and may have therapeutic potential in the treatment of transplant rejection in patients. However, Treg cell development and action are poorly understood in transplantation. In this study, the association of CD4(+)Foxp3(+) infiltrates within renal allograft tissue with graft survival was investigated in a mouse model. METHODS: Kidney donors from C57BL/6J mice (H-2(b)) were transplanted to bilaterally nephrectomized Balb/c recipient mice (H-2(d)). Treg cells were examined with FACS and immunohistochemical staining. RESULTS: Here we showed that without any immunosuppressive regimen, kidney allografts were mostly rejected from 20 to 60 days after transplantation. During the progression of allograft rejection Foxp3(+) Treg phenotype infiltrates were significantly diminished, while intragraft expression of TGF-beta1, IL-6, IL-17 and IL-23 was up-regulated. The regulatory function of CD4(+)CD25(+) infiltrates was confirmed by their suppressive activity in mixed lymphocyte reaction. Further in vitro studies indicated that primary renal tubular epithelial cell (TEC) cultures produced high levels of IL-6 in response to allogeneic lymphocyte or IL-17 stimulation, and neutralization of IL-6 increased CD4(+)CD25(+)Foxp3(+) cells in co-cultures with TEC. CONCLUSION: Diminution of Foxp3(+) Treg infiltrates associates with renal allograft rejection, and neutralization of IL-6 activity enhances Foxp3(+) Treg cell differentiation. Our findings suggest that increase in intragraft IL-6 may down-regulate infiltrating Foxp3(+) Treg cells.     

Everolimus and Basiliximab Permit Suppression by Human CD4+CD25+ Cells in vitro

Immunosuppressive drugs are essential for the prevention of acute transplant rejection but some may not promote long-term tolerance. Tolerance is dependent on the presence and regulatory function of CD4(+)CD25(+) T cells in a number of animal models. The direct effects of immunosuppressive drugs on CD4(+)CD25(+) cells, particularly those that interfere with IL-2 signaling are uncertain. We studied the effects of the rapamycin derivative everolimus and the anti-CD25 monoclonal antibody basiliximab on the regulatory capacity of human CD4(+)CD25(+) cells in vitro. Both drugs permitted the suppression of proliferation and IFN-gamma secretion by CD4(+)CD25(-) cells responding to allogeneic and other polyclonal stimuli; CTLA-4 expression was abolished on CD4(+)CD25(+) cells without compromising their suppressive ability. Everolimus reduced IFN-gamma secretion by CD4(+)CD25(-) cells before the anti-proliferative effect: this is a novel finding. Exogenous IL-2 and IL-15 could prevent the suppression of proliferation by CD4(+)CD25(+) cells and the drugs could not restore suppression. By contrast, suppression of IFN-gamma secretion was only slightly impeded with the exogenous cytokines. Finally, CD4(+)CD25(+) cells were more resistant than CD4(+)CD25(-) cells to the pro-apoptotic action of the drugs. Together these data suggest that CD4(+)CD25(+) cells may still exert their effects in transplant patients taking immunosuppression that interferes with IL-2 signaling.     

RATG对CD4+细胞和CD8+细胞共刺激分子基因表达和细胞因子分泌的影响

目的 探讨兔抗人胸腺细胞多克隆抗体(RATG)在体外对CD4+细胞和CD8+细胞共刺激分子基因表达和细胞因子分泌的影响.方法 从正常成人外周血单个核细胞中分离和纯化CD4+细胞和CD8+细胞,加入RATG,37℃下培养.分别于24、48和72 h收集培养上清液和细胞,以不处理的细胞为正常对照,正常兔Ig处理的细胞作为阴性对照.采用实时聚合酶链反应技术检测培养细胞的细胞毒性T淋巴细胞相关抗原4(CTLA4)、CD154、Foxp3、OX40、γ干扰素(IFN-γ)、白细胞介素2(IL-2)、IL-10和IL-2受体(CD25)的基因表达水平,应用Multiplex检测技术测定培养上清液中IFIN-γ、IL-2、IL_4和IL-10的水平.结果 与正常CD4+细胞比较,加入RATG的CD4+细胞培养24 h,其CTLA-4、CD154、Foxp3、OX40、IFN-γ、IL-2、IL-10和CD25基因转录表达均上调,阴性对照CD4+细胞则无这些基因转录表达的上调.处理48 h后,CD4+细胞的CD154和IL-2的基因转录表达呈现下调现象,而CTLA4、Foxp3、0X40、IFN-γ、IL-10和CD25的基因转录水平均较24 h时明显降低.处理72 h后,CD4+细胞的CTLA4、Foxp3、OX40和CD25的基因转录表达再次呈现高水平,CD154和IFN-γ的基因转录表达上调表达不明显,而IL-2和IL-10的基因转录表达则呈现明显的下调.RATG处理的CD4+细胞培养上清液中的IFN-γ、IL-2、IL-4和IL-10浓度显著增加,以处理24 h的水平最高,而阴性对照者未测出.与正常CD8+细胞相比较,加入RATG处理的CD8+细胞培养24 h,其CTLA4、Foxp3、OX40、IFN-γ,、IL-2、IL-10和CD25的基因转录表达呈现明显上调,而CD154基因转录表达稍有下调.RATG处理48 h,CD8+细胞的CTLA4、Foxp3、OX40、IFN-γ和CD25基因转录表达仍维持在高水平,CD154基因转录表达仅仅呈现低水平的上调,IL-10基因转录表达水平显著下降,而IL-2的基因转录表达则明显下调.处理72 h,CD8+细胞的CTLA4、Foxp3、OX40、IFN-γ、IL-10和CD25的基因转录表达仍维持在高水平,CD154基因转录表达则呈现下调,而IL-2基因转录表达的下调更为显著.阴性对照CD8+细胞则未呈现这些基因转录的上调现象.RATG处理的CD8+细胞培养上清液中IFN-γ、IL-2和IL-10显著增多,以处理24 h的浓度最高,IL-4浓度升高的幅度较小,而正常CD8+细胞和阴性对照CD8+细胞几乎检测不到IFN-γ、IL-2、IL-4和IL-10.结论 在体外,RATG可以刺激CD4+细胞和CD8+细胞上调多种促进免疫抑制的共刺激分子基因表达,促进其分泌与免疫调节相关的IFN-γ、IL-2、IL-4和IL-10.     

Influence of Immunosuppressive Drugs on the Development of CD4CD25 Foxp3 T Cells in Liver Transplant Recipients

Introduction

Many studies suggest that CD4⁺CD25^high T regulatory cells (Tregs) have a crucial role in downregulating the immune response to alloantigens. In this study, we investigated the possible influence of immunosuppressive therapy, including rapamycin and calcineurin inhibitors (CNIs; tacrolimus), on level of Tregs in liver allograft recipients.

Materials and Methods

We assessed 47 liver transplant recipients with stable liver function for ≥2 years, dividing them into 2 groups: Patients receiving rapamycin (n = 15), and those receiving tacrolimus (n=32). Thirty-eight, age-matched healthy subjects were used as normal controls. We examined the expression of CD4, CD25, and Foxp3 in peripheral blood T cells. Flow cytometry was performed with a FACSCalibur instrument with data analysis using Cell Quest software.

Results

Rapamycin significantly increased the prevalence of Tregs, including the percentage of CD4⁺CD25^high T cells in total lymphocytes and among total CD4⁺ T cells, compared with the healthy subjects and the CNI group. The prevalence of Tregs in the CNIs group was significantly lower than that of controls. Foxp3 was expressed in >95% of CD4⁺CD25^highT cells, whereas it was in <20% of CD4⁺CD25^low T cells and not expressed among CD4⁺CD25⁻ T cells.

Conclusions

Immunosuppressive therapy (rapamycin or CNIs) may have a different roles in tolerance induction among liver transplant recipients. Namely, rapamycin promoted the induction of a profile consistent with alloantigen tolerance; CNIs hampered this progression.     

Observational support for an immunoregulatory role of CD3+CD4+CD25+IFN-gamma+ blood lymphocytes in kidney transplant recipients with good long-term graft outcome.

There is evidence that interferon-gamma (IFN-gamma)-dependent interactions of dendritic cell (DC), T regulatory (Treg), and T suppressor (Ts) subpopulations contribute to allograft acceptance. We measured DC subsets, CD3+CD4+CD25+ (Treg phenotype) and CD3+CD8+CD28(-) (Ts phenotype) peripheral blood lymphocytes (PBL) expressing Foxp3, Th1 or Th2 cytokines, peripheral T- and B-cell counts, and plasma cytokines in 33 kidney transplant recipients with a serum creatinine of < or =1.8 mg/dl and 32 recipients with a serum creatinine of > or =2.0 mg/dl more than 100 days post-transplant. Cell subsets were measured in whole blood using four-color flow cytometry. Patients with increased creatinine had less frequently detectable CD3+CD4+CD25+IFN-gamma+ PBL than patients with good graft function (P = 0.017). In patients with good graft function, CD3+CD4+CD25+IFN-gamma+ PBL were associated with high Foxp3+, IL-2+, IL-12+, IL-4+, and IL-10+ CD3+CD4+CD25+ T PBL (P < 0.001), low CD3+CD8+CD28(-)Foxp3+ (P = 0.002), CD3+CD4+DR+ (P = 0.002), CD3+CD8+DR+ T (P = 0.005) and CD19+ B PBL (P = 0.005), and low lineage(-)HLA-DR+CD11c+CD123(-) DC1 (P = 0.006). Patients with impaired graft function did not show these associations. Additional flow cytometric analysis confirmed strong co-expression of IFN-gamma and Foxp3 by CD4+CD25+ PBL particularly in patients with good graft function. Our data support an immunoregulatory role of CD3+CD4+CD25+Foxp3+IFN-gamma+ cells in a subgroup of transplant recipients with good graft acceptance.     

CD154 on the surface of CD4+CD25+ regulatory T cells contributes to skin transplant tolerance

BACKGROUND: It is known that the infusion of whole blood from donors (donor-specific transfusion) into recipients combined with anti-CD154 therapy can prolong allograft survival. It has generally been agreed that the effectiveness of anti-CD154 therapy is caused by the inactivation of alloreactive CD4+ and CD8+ effector T cells. The recent literature has implicated CD4+CD25+ regulatory T cells in the suppression of autoimmunity and graft rejection, and we therefore examined whether CD154 blockade is effective because of its blockade of inflammatory T-cell activation or because of a direct impact on the regulatory T cells. METHODS: RAG(-/-) mice were adoptively transfused with CD4+ T cells or a subset of the population (CD4+CD25+ or CD4+CD25- T cells) alone or in combination with donor-specific transfusion and anti-CD154 and given an allo-skin transplant. The longevity of the transplant was determined over time. CD154(-/-)CD4+ T cells were used to assess the importance of CD154 in graft rejection and acceptance. RESULTS: CD154 blockade (or loss of CD154) on CD4+CD25+ regulatory T cells enhanced their immunosuppressive activities and was a contributing factor to anti-CD154-induced immune suppression in vivo. In a model of allograft tolerance, suppression was elicited by antigen and anti-CD154 or antigen alone if the CD4+CD25+ regulatory T cells were deficient in CD154 expression. CONCLUSIONS: Neutralizing the function of CD154 on regulatory T cells upon antigen exposure induces heightened levels of suppressive activities and is likely a contributing factor to the long-lived therapeutic effects of anti-CD154 treatment.     

Effect of mycophenolate mofetil regimen on peripheral blood lymphocyte subsets in kidney transplant recipients

BACKGROUND AND AIMS: There is growing evidence of the effects of immunosuppressive agents on "immune targets" in renal transplantation. Immunological monitoring could indirectly measure the suppressive effect of these drugs and guide early preventive interventions in transplant recipients. Due to the selective antiproliferative effect of mycophenolate mofetil (MMF) on lymphocytes, our goal was to determine whether MMF modulates peripheral blood lymphocyte subsets (PBLS) in kidney allograft patients. METHODS: We assessed absolute CD3(+), CD3(+)CD4(+), CD3(+)CD8(+), CD19(+), CD16(+)CD3(-) PBLS counts and CD4/CD8 ratios for 12 months in three groups of kidney allograft patients stratified according to maintenance immunosuppressive regimen: group A (n = 31), which started MMF with prednisone (P) + cyclosporine A (CyA), and two control groups, B (n = 19) and C (n = 15) on P + CyA + azathioprine (Aza) and P + CyA regimens, respectively. We compared intra- and intergroup lymphocyte counts and ratios. RESULTS: Intergroup comparisons showed a significant reduction in all PBLS in group A (CD19(+) from 3 months and other subsets from 6 months), whereas there were no significant changes in PBLS in the other group analyses or comparisons. CONCLUSIONS: Our findings suggest that (1) MMF modulates all PBLS in kidney allograft patients, causing a progressive reduction occurring earlier in CD19(+), and (2) we can rule out that these changes were caused by the "natural immunological evolution" of the transplantation. These results could offer a new method for immunological monitoring of transplant patients.     

A novel mechanism of action for anti-thymocyte globulin: induction of CD4+CD25+Foxp3+ regulatory T cells

T cell-depleting agents are being tested as part of clinical tolerance strategies in humans with autoimmunity and transplantation. The immunosuppressive activity of anti-thymocyte globulin (ATG) has been thought to result primarily from depletion of peripheral lymphocytes. Herein is reported for the first time that ATG but not anti-CD52 mAb (alemtuzumab) or the IL-2R antagonists causes rapid and sustained expansion of CD4+CD25+ T cells when cultured with human peripheral blood lymphocytes. These cells display enhanced expression of the regulatory markers glucocorticoid-induced TNF receptor, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), and forkhead box P3 and efficiently suppress a direct alloimmune response of the original responder lymphocytes. It is interesting that the cells do not suppress memory responses to the recall antigen mumps. Ex vivo expansion of regulatory T cells is due mainly to conversion of CD4+CD25- into CD4+CD25+ T cells and to a lesser degree to proliferation of natural CD4+CD25+ T cells. The induction of regulatory T cells depends on production of Th2 cytokines in the generating cultures. These novel data suggest that ATG not only may promote expansion/generation of regulatory T cells but also may be useful in future ex vivo expansion of these cells for cellular therapy in autoimmunity and clinical transplantation.     

Immunological evaluation of combination therapy with tacrolimus and sirolimus on long-term allograft survival in nonhuman primates

We reported that a 60-day course of combination therapy with tacrolimus and sirolimus induced long-term survival of renal allograft after withdrawal of immunosuppressants in Vervet monkeys. In the present study, the mechanism of drug-induced allograft survival was evaluated via Th1/Th2 cytokines, apoptosis and MLC activity in primates. MATERIALS AND METHODS: Cytokines were evaluated by ELISA. MLR and CTL assays were performed by incorporation of 72 hours (3)H-TdR and 4 hours (51)Cr release assay. RESULTS: A 60-day course of tacrolimus with sirolimus resulted in long-term survival of kidney allografts. (67% > 100 days) without intermittent acute rejection. Low sensitivity to MLR was seen in long-term renal allograft survival among monkeys treated with tacrolimus and sirolimus. Increased levels of CD3(+)CD8(+), CD3(+)/CD56(+) NKT cells and CD86(+)CD8(-)CD11(+) dendritic cells were observed. A population of high expression of CD4(+)FasL(+) was detected. In addition, the concentrations of IL-2 and IFN-gamma from long-term allograft surviving monkeys was not significantly increased, rather a late phase dominance of Th2, IL-4, IL-10, and TGF-beta was found correlated with long-term survival of recipients. In conclusion, the mechanism of tacrolimus and sirolimus induced long-term allograft survival in primates relates to up-regulated FasL expression, NKT cells and dendritic cells, with downregulation of MLR sensitivity. It is also associated with late-dominant expression of Th2 cytokines.