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.     

Induction of transplantation tolerance by allogeneic donor-derived CD4(+)CD25(+)Foxp3(+) regulatory T cells

Several studies have shown that recipient-derived CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are involved in transplantation tolerance. However, it is not clear whether allogeneic donor-derived Tregs are able to regulate T cell alloreactivity after solid organ allograft transplantation. Related studies in experimental bone marrow transplantation have shown that allogeneic donor-derived Tregs are capable of promoting early and long-term allogeneic hematopoietic engraftment, accompanied by tolerance to donor and recipient antigens. However, in these models, donor-derived Tregs are syngeneic with respect to the T responder cells. The role of Tregs in solid organ transplantation models where recipient-derived T responder and donor-derived Tregs are allogeneic has been scarcely studied. In order to determine whether allogeneic Tregs were able to regulate T cell alloreactivity, CD4(+)CD25(-) and CD8(+) T responder cells were cultured with stimulator dendritic cells in several responder-stimulator strain combinations (C57BL/6-->BALB/c, BALB/c-->C57BL/6 and C3H-->BALB/c) in the presence of responder-derived, stimulator-derived or 3rd-party-derived Tregs. Then, the frequency of IFN-gamma+ alloreactive T cells was determined by means of ELISPOT assay. The results of this study demonstrate that, regardless of the responder-stimulator strain combination, both responder-derived and stimulator-derived Tregs, but not 3rd-party-derived Tregs, significantly inhibited CD4(+) and CD8(+) T cell alloreactivity. The effect of allogeneic stimulator-derived Tregs was dependent on IL-10 and TGF-beta and reversed by exogenous IL-2. In vivo experiments in nu/nu recipients reconstituted with CD4(+)CD25(-) T responder and Tregs showed that recipient and donor-derived, but not 3rd-party-derived Tregs, significantly enhanced skin allograft survival. Importantly, T cells from both recipient-derived and donor-derived Treg-reconstituted nu/nu recipients exhibited donor-specific unresponsiveness in vitro. These results show that allogeneic donor-derived Tregs significantly inhibit T cell alloreactivity and suggest their potential use in the induction of transplantation tolerance.     

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.     

Adoptive transfusion of ex vivo donor alloantigen-stimulated CD4(+)CD25(+) regulatory T cells ameliorates rejection of DA-to-Lewis rat liver transplantation

BACKGROUND: Adoptive transfusion of splenocytes from long-term survivors of a tolerance model of rat orthotopic liver transplantation can induce acceptance of liver allografts in a rejection model preconditioned with donor gamma-irradiation before liver transplantation. Recent studies suggest that the regulatory T cells (Treg cells) in splenocytes from long-term survivors play an important role in the induction of liver graft tolerance, but this observation was made from a rejection model preconditioned with donor gamma-irradiation; little is known about the role of Treg cells in liver graft rejection using a naive rejection model. In this study, we examined the therapeutic potential of CD4(+)CD25(+) Treg cells in a naive rejection model of rat liver transplantation. METHODS: Freshly isolated or ex vivo alloantigen-stimulated CD4(+)CD25(+) Treg cells (1 x 10(6) cells) from naive Lewis RT(1) (LEW) rats were adoptively transferred into another LEW rat on days 1 and 7 after liver transplantation from a Dark Agouti RT1(a) (DA) rat. Recipients were treated with or without oral tacrolimus (FK506) (0.1 mg/kg/day) from days 1 to 7 after transplantation. For ex vivo alloantigen-stimulation, CD4(+)CD25(+) Treg cells from LEW rats were cocultured with mitomycin C-treated DA (donor alloantigen specific) or Brown Norway (BN)(RT1(n), third party) splenocytes for 72 hours. Ex vivo alloantigen-specific CD4(+)CD25(-) T-cell proliferation responses were assessed with fresh and stimulated CD4(+)CD25(+) Treg cells. RESULTS: Freshly isolated, donor alloantigen-stimulated and third-party alloantigen- stimulated CD4(+)CD25(+) Treg cells suppressed antigen-specific CD4(+)CD25(-) T-cell proliferation ex vivo, and adoptive transfusion of these 3 kinds of CD4(+)CD25(+) Treg cells prolonged survival of the liver allografts. The group transfused with the donor alloantigen-stimulated CD4(+)CD25(+) Treg cells had the greatest mean survival among the 3 groups (fresh Treg cells, 21 +/- 2 days, n = 6; third-party alloantigen-stimulated Treg cells, 20 +/- 2 days, n = 6; donor alloantigen-stimulated Treg cells, 30 +/- 2 days, n = 6). When combined with short-term tacrolimus administration, adoptive transfusion of donor antigen-stimulated Treg cells induced the greatest survival time in recipients (greater than 60 days; n = 6). CONCLUSION: Adoptive transfusion of ex vivo donor alloantigen-stimulated CD4(+)CD25(+) Treg cells combined with short-term tacrolimus treatment may represent a new strategy for preventing rejection after liver transplantation.     

Efficient peripheral construction of functional human regulatory CD4(+)CD25(high)Foxp3(+) T cells in NOD/SCID mice grafted with fetal human thymus/liver tissues and CD34(+) cells

Regulatory T cells, especially CD4(+)CD25(+) regulatory T cells are critical regulators of immune tolerance in humans and mice. Mice with humanized immunity have been developed by various transplantation strategies of human tissues or cells related to immunity, which are being extensively applied in biomedical research. However, it is unclear whether human CD4(+)CD25(+) regulatory T cells can normally develop in human thymic grafts and efficiently populate in the periphery in NOD/SCID mouse recipients. In human thymic grafts, high percentage of mature human CD4(+)CD25(high) regulatory T cells was detected. Human CD4(+)CD25(+) regulatory T cells maturing in fetal human thymus grafts could subsequently output to the periphery of NOD/SCID mouse recipients. Importantly, these cells exhibited Foxp3(+)CD45RO(+)CTLA4(+)CD127(-) phenotype, similarly to those in healthy individuals. In addition, human CD4(+)CD25(+) regulatory T cells maturing in human thymic grafts suppressed proliferative response of CD4(+)CD25(-) T cells to allogeneic antigens, though the peripheral CD4(+)CD25(+) regulatory T cells in fetal human thymus-grafted NOD/SCID mice showed somewhat decreased immunosuppressive ability compared with normal CD4(+)CD25(+) regulatory T cells. Thus, this humanized animal model is suitable for examining development and function of human CD4(+)CD25(+) regulatory T cells in vivo.     

A potential side effect of cyclosporin A: inhibition of CD4(+)CD25(+) regulatory T cells in mice

BACKGROUND: CD4CD25 regulatory T (Treg) cells are essential for the induction and maintenance of immunologic self-tolerance as well as transplant tolerance. The effects of cyclosporin A (CsA), a widely used immunosuppressive agent, on CD4CD25Treg cells in mice were investigated. METHODS: Balb/c mice were injected with CsA or control solution for one month. The levels, phenotype, and function of CD4CD25Treg cells in these mice were then assayed. RESULTS: The percentages and total cell numbers of CD4CD25Treg cells in the peripheral blood and spleen were significantly reduced after the treatment with CsA. The total numbers of CD4CD25Treg cells in the thymus of CsA-treated mice were markedly reduced as compared to the control mice. However, the percentage of CD4CD25Treg cells in the thymus of CsA-treated mice was markedly enhanced. More CD4CD25Treg cells expressing high levels of CD44 and CD45RB, and less CD4CD25Treg cells expressing CD62L were observed in CsA-treated mice, compared with the control mice. CD4CD25Treg cells expressed slightly lower levels of Foxp3 in CsA-treated mice. Furthermore, CsA markedly impaired the immunosuppressive function of CD4CD25Treg cells. CONCLUSIONS: CsA significantly impaired the development and function of CD4CD25Treg cells. The present studies suggest that CsA may block the potential induction of immune tolerance and increase the susceptibility to develop autoimmune diseases while preventing graft rejection.     

Immune regulation by CD4+CD25+ regulatory T cells: implications for transplantation tolerance

In transplantation research, the achievement of life-long tolerance for the graft without the need for immunosuppressive drugs, is a major goal. In the immune system various mechanisms are in place that help to prevent unwanted immunity. These mechanisms of peripheral tolerance include deletion, anergy, ignorance and suppression. In the last decade it has been demonstrated convincingly that a naturally occurring subset of CD4+ T cells, the so-called CD4+CD25+ regulatory T cells, play a key role in the suppression/regulation of immune responses. These cells have been shown to exist in mice, rats and humans, and can be found in thymus, peripheral blood, lymphoid organs and at sites of inflammation. CD4+CD25+ regulatory T cells can down-regulate the immune response by affecting T cell responses, antibody production, cytokine secretion and antigen-presenting cells. CD4+CD25+ regulatory T cells are generated in the thymus, but importantly recent evidence suggests that they can also be generated in the periphery. This latter finding is of particular importance for transplantation immunology, since it suggests that specific manipulation or induction of these cells is achievable in vivo. Here we review the recent developments on the CD4+CD25+ regulatory T cells and we discuss the potential use of these cells in transplantation immunology.     

B7-1 and B7-2 differentially control peripheral homeostasis of CD4(+)CD25(+)Foxp3(+) regulatory T cells

Targeting the CD28/B7 interaction remains among the most promising approaches to treat transplant rejection. A drawback to this approach is however the observations of decreased numbers of naturally occurring regulatory T cells (Tregs) in CD28(-) or B7-deficient non-obese diabetic (NOD) mice, cells that maintain immunological self tolerance, prevent autoimmunity and control immune responses to transplants. In this study, therefore, we investigated the relative contributions of B7-1 and B7-2, the only known ligands of CD28, on the thymic development and peripheral homeostasis of Tregs. Our data indicates that the absence of both B7-1 and B7-2 result in a dramatic reduction in the frequencies of Tregs in thymus and peripheral tissues of B7-1/B7-2-deficient mice with no apparent changes in the percentage and distribution of conventional T-cell subsets. In addition, neither B7-1 nor B7-2 expression alone is sufficient for the development and peripheral homeostasis of Tregs. Interestingly, while B7-1 and B7-2 equally contribute to thymic development of Tregs, the significant loss in peripheral homeostasis of Tregs is more evident in the absence of B7-2 as compared to B7-1. Consistent with these results we found that B7-2-deficient DCs are less effective than B7-1-deficient DCs in maintaining Tregs in vitro due to their inability to induce IL-2 production by conventional T cells and sustain Tregs expression of CD25. This study provides the first demonstration of relative roles of B7-1 and B7-2 in Tregs homeostasis and suggests that therapeutic approaches designed to selectively interrupt CD28/B7-2 interaction could indeed have measurable impact on sustaining Tregs homeostasis.     

抗CD25单抗联合血浆分离在高敏肾移植受体应用的临床研究

目的 探讨抗CD25单抗联合血浆分离在群体反应性抗体(PRA)阳性的高敏肾移植受体中的应用价值。方法 在41例PRA平均为52．2％的高敏肾移植受体中，24例仅用双滤过法血浆分离(DFPP)预处理(对照组)，17例在DFPP处理的基础上加用两个剂量的抗CD25单抗诱导治疗(治疗组)，比较两组急性排斥反应的发生率、严重程度和逆转情况、1年人／肾存活率以及并发症的发生。结果 治疗组仅2例(11．8％)发生急排，分别为IA和ⅡJ3级，对照组1例(4．2％)发生超排，10例(41．7％)发生急排，ⅡA级以上有6例，2组差异有显著性意义(P=0．039)。一年人／肾存活率治疗组为100％／94．1％，对照组为100％／91．7％，两组并发症无明显差异。结论PRA阳性的高敏肾移植受体在DFPP基础上应用抗CD25单抗能显著降低急性排斥的发生，减轻排斥反应的严重程度，获得满意的1年人／肾存活率。     

The efficacy and safety of high-dose mizoribine in ABO-incompatible kidney transplantation using anti-CD20 and anti-CD25 antibody without splenectomy treatment

Background

Mizoribine (MZR) has been developed as an immunosuppressive agent in Japan, but it shows less potent immunosuppressive effects at doses up to 3 mg/kg/d. In this study, we investigated whether high-dose MZR (6 mg/kg/d) was effective for ABO-incompatible (ABO-i) living donor kidney transplantation (LKT) using treatment with anti-CD25 and anti-CD20 monoclonal antibodies without splenectomy.

Methods

Since 2007, we encountered 24 cases of ABO-i LKT using anti-CD20 and anti-CD25 monoclonal antibody without splenectomy. The pretransplant immunosuppressive regimen consisted of two doses of anti-CD20 antibody, mycophenolate mofetil (MMF), prednisolone, a calcineurin inhibitor (cyclosporine [7 mg/kg] or tacrolimus [0.2 mg/kg] and two doses of anti-CD25 antibody. Antibody removal by plasmapheresis was performed before LKT up to several times according to the antibody titer. The posttransplant regimen consisted of high-dose mizoribine (6 mg/kg/d) instead of MMF (MZR group, n = 12).

Results

The 1-year graft survival rates for the MZR and MMF groups were both 100%. The rejection rate in the MZR group (eight %) was not significantly higher than that in the MMF group (seventeen %) Serum creatinine level was not significantly different between the two groups. In the MZR group 6 (50%) patients developed CMV antigenemia-positivity versus 11 (92%) in the MMF group (P < .05). The number of patients who developed CMV disease was 0 in the MZR group and 1 (8%) in the MMF group. The number of patients treated with ganciclovir was 0% and 8%, respectively (not significant).

Conclusions

We obtain good clinical results with high-dose MZR in ABO-i LKT using anti-CD20 and anti-CD25 antibody treatment without splenectomy.