Anti-CD25 treatment and FOXP3-positive regulatory T cells in heart transplantation

The interleukin-2 receptor alpha chain (IL-2Ra, CD25) plays a major part in shaping the dynamics of T cell populations following immune activation, due to its role in T cell proliferation and survival. Strategies to blunt the effector responses in transplantation have been developed by devising pharmaceutical agents to block the IL-2 pathways. However, such strategies could adversely affect the CD25(+)FOXP3(+)T regulatory (T reg) populations which also rely on intereukin-2 signaling for survival. The present study shows that a cohort of heart allograft recipients treated with Daclizumab (a humanized anti-CD25 antibody) display FOXP3 expression patterns consistent with functional T regulatory cell populations. High levels of FOXP3 were observed to correlate with lower incidence of and recovery from acute rejection, as well as lower levels of anti-donor HLA antibody production. Therefore, T reg populations appear fully functional in patients treated with Daclizumab, even when 5 doses were administered. By comparison, patients treated with fewer doses or no Daclizumab had a higher incidence of acute rejection, antibody production and graft failure. Therefore, our data indicates that Daclizumab treatment does not interfere with the generation of regulatory T cells and has a beneficial effect on heart allograft survival.     

T regulatory cells and transplantation tolerance

Despite the development of successful immunosuppression protocols and tremendous improvement in short-term graft survival rates, the problem of chronic graft loss remains the bane of clinical transplantation. The induction and maintenance of transplantation tolerance is the “Holy Grail” of transplantation.The recent identification and characterization of regulatory T cells has opened up exciting opportunities for tolerance induction, immunotherapy, and immunomodulation in transplantation. This review focuses on current understanding of regulatory T cells and their role in transplantation tolerance.     

Role and mechanisms of CD4+CD25+ regulatory T cells in the induction and maintenance of transplantation tolerance

To gain transplantation tolerance between donor organs and hosts is the ultimate goal of all sorts of organ transplantations. Induction of regulatory T cells has been demonstrated to lead to transplantation tolerance. This paper will review subsets of regulatory T cells, the role and mechanisms of CD4(+)CD25(+) regulatory T cells (Tregs) in graft rejection and tolerance, pathway used by Tregs to recognized alloantigens, pathways of Tregs homing into the graft and effects of immunosuppression on Tregs. It was well known that Tregs play a pivotal role in transplantation tolerance. The mechanisms by which Tregs exert their regulatory effect in the induction and maintenance of transplantation tolerance, anthropogenically, consist of physical cell-to-cell contact with potential target cells, autocrine and paracrine properties. ICAM-1, TGF-beta, CTLA-4, GITR and OX40 (CD134), etc. are involved in the regulatory function of Tregs through cell-to-cell contact mechanism. IL-10 and TGF-beta are two important soluble mediators involved in the autocrine mechanism by which Tregs exert their regulatory function. Paracrine properties refer to re-educate potentially destructive alloresponsive T cells to gain regulatory function. All that discussed above could illustrate, at least partially, how naturally occurring Tregs exert their regulatory function in vivo as they constitute only 5-10% of peripheral CD4(+) T cells.     

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.     

CD4+CD25+ regulatory T cells mediate acquired transplant tolerance

The Holy Grail of clinical organ transplantation is the safe induction of allograft tolerance. Transplant tolerance has been successfully induced in animal models. Since T cells play a pivotal role in graft rejection, modulating T cell function has been the primary focus of studies aimed at inducing transplant tolerance. Rodent models of transplant tolerance induction include central deletion and peripheral mechanisms involving activation-induced cell death (AICD), anergy, immune deviation, and production of regulatory T cells. These mechanisms are not mutually exclusive. Although clonal deletion and anergy limit self-reactive T cells in the thymus, these mechanisms alone are not sufficient for controlling self-reactive T cells in the periphery. There is now evidence that the adult animal harbors two functionally distinct populations of CD4(+) T cells; one mediates autoimmune disease and the other dominantly inhibits it. The latter cells express CD4, CD25 and CTLA-4. These thymus-derived T cells have recently been shown to mediate the induction and maintenance of transplant tolerance. These CD4(+)CD25(+) T cells are similar in origin, phenotype, and function to those that maintain natural self-tolerance and T cell homeostasis in the periphery. Against this background, is it possible that alloantigen specific regulatory T cells might be generated and expanded ex vivo before organ transplantation and then infused to induce long-term tolerance, perhaps without the need for chronic immunosuppression?     

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.     

树突状细胞和CD4+CD25+调节性T细胞在免疫耐受中的作用及关系

诱导免疫耐受具有高效、低毒、经济等优点,在器官移植中有着极其重要的意义.在建立免疫耐受中,树突状细胞(DC)起到克隆清除、克隆无能、表达T细胞抑制因子、选择性激活辅助T细胞和诱导调节性T细胞尤其是CD4+CD25+调节性T细胞的产生等作用.而CD4+CD25+调节性T细胞主要以DC为作用靶点,影响其分化成熟,抑制DC向...     

Tolerance to rat heart grafts induced by intrathymic immunomodulation is mediated by indirect recognition primed CD4+CD25+ Treg cells

BACKGROUND: In a rat model (PVG.R8-to-PVG.1U) disparate for one class I antigen, RT.1Aa, we previously demonstrated that intrathymic immunomodulation with donor antigens resulted in prolonged survival of cardiac allografts that underwent chronic rejection. However, long-term survivors developed a regulatory cell population that prevented both acute and chronic rejection when adoptively transferred into secondary graft recipients. The purpose of this study was to characterize these regulatory cells with particular emphasis on CD4+CD25+ Treg cells. METHODS: Spleens, lymph nodes, and peripheral blood lymphocytes of secondary tolerant recipients were characterized using antibodies to various T cell markers in flow cytometry. In vitro MLR and in vivo adoptive transfer experiments were conducted to investigate the involvement of CD4+CD25+ T cells in the observed tolerance. The presence of various cytokines in the sera of graft recipients and MLR culture supernatants was tested using ELISA. RESULTS: Tolerant recipients compared with naive rats had substantially higher percentages of CD4+CD25+ T cells in the spleen (28+/-3% vs. 11+/-5%) and blood (23+/-6% vs. 9+/-4%). Tolerant animals also had higher levels of serum IL-10 than naive and rejecting animals. CD4+CD25+ T cells from secondary long-term graft survivors inhibited donor-specific proliferative responses in vitro that was associated with high IL-10 production. Importantly, depletion of CD4+CD25+ T cells from splenocytes of tolerant rats abrogated their ability to transfer tolerance to tertiary graft recipients. CONCLUSIONS: Our data demonstrate that cardiac allograft tolerance in this model is mediated by CD4+CD25+ Treg cells primed by indirect recognition and is associated with high levels of IL-10.     

大鼠CD4^＋CD25^＋调节性T细胞的分离及功能鉴定

目的：研究利用免疫磁珠分选法稳定分离正常大鼠脾脏CD4^＋CD25^＋调节性T细胞的方法。方法：采用免疫磁珠两步法分离大鼠脾组织CD4^＋CD25^＋T细胞。首先采用藻红蛋白（PE）标记的抗CD25抗体和抗PE多功能磁珠试剂盒阳性分选CD25^＋T细胞,再用抗异硫氰酸荧光素（FITC）标记抗体和抗IgG磁珠阳性分选获得CD4^＋CD25^＋T细胞。分离后的细胞经流式细胞仪检测分离纯度,台盼蓝染色检测细胞存活率,体外增殖实验检测其对CD4^＋CD25^-T细胞的免疫抑制作用。结果：两次阳性分选后获得的CD4^＋CD25^＋T细胞纯度为（90.4±1.6）%,细胞存活率为（92.6±2.4）%。体外增殖实验表明,CD4^＋CD25^＋T细胞能明显抑制CD4^＋CD25^-T细胞的增殖（P〈0.01）。结论：采用免疫磁珠法两次阳性分选,可稳定地获得纯度理想并有免疫抑制功能的大鼠CD4^＋CD25^＋T细胞。     

The imbalance between regulatory and IL-17-secreting CD4T cells in multiple-trauma rat

Background

It has been well recognised that a deficit of numbers and function of CD4⁺CD25⁺Foxp3⁺cells (Treg) is attributed to the development of auto-immune diseases, inflammatory diseases, tumour and rejection of transplanted tissue; however, there are controversial data regarding the suppressive effect of Treg cells on the T-cell response in auto-immune diseases. Additionally, interleukin-17 (IL-17)-producing cells (Th17) have a pro-inflammatory role. The balance between Th17 and Treg may be essential for maintaining immune homeostasis and has long been thought as one of the important factors in the development/prevention of auto-immune diseases, inflammatory diseases, tumour and rejection of transplanted tissue, but their role in multiple trauma remains unclear.

Objective

This study aims to investigate whether an imbalance of Treg and Th17 effector cells is characteristic of rats suffering from multiple trauma.

Methods and subjective

Sixty Sprague-Dawley (SD) rats were randomly divided into three groups. The control group (n = 20, group I) no received procedures (normal). The sham group (n = 20, group II) only received anaesthesia, cannulation and observation. The bilateral femoral shaft fractures with haemorrhagic shock groups (n = 20, group III). Rats in groups II and III were killed at the end of 4 h after models were established. Peripheral blood samples were collected for assessment of Treg cells, Th17 cells and cytokines (IL-17, IL-6, IL-2, transforming growth factor beta (TGF-β)) and intestine tissue was collected for intestine histological analysis.

Results

We observed decreased Treg/Th17 ratios in CD4⁺T cells in rats with multiple trauma and a strong inverse correlation with disease activity (intestinal histological scores).

Conclusion

We suggest a role for immune imbalance in the pathogenesis and development of multiple trauma. The alteration of the index of Treg/Th17 cells likely indicates the therapeutic response and progress in the clinic.     

肝移植术后潜在免疫耐受者外周血自然杀伤细胞、调节性T细胞和记忆性T细胞的表达及其临床意义

目的 探讨肝移植术后潜在免疫耐受者免疫状态,为临床指导抗排斥治疗提供依据.方法 纳入1999年1月至2007年12月在本中心行肝移植存活至今潜在免疫耐受者29例,分为3～5年组(3Y组)10例,5～11年组(5Y组)19例,另纳入肝移植术后反复排斥反应者(RJ)10例,年龄匹配健康对照组(HC)17例;流式检测外周血单个核细胞中自然杀伤(NK)细胞、记忆性T细胞、调节性T细胞(Treg)3个亚群的表达.结果 3Y、5Y组CD4+ CD25+T细胞(3Y:27.56±4.63;5Y:30.07±3.91)、静息性Treg占CD4+T细胞比例(3Y:0.22±0.05;5Y:0.17±0.03)比R J组明显升高(CD4+ CD25+ T:11.78±4.28;静息性Treg:0.05±0.02)(P＜0.05);5Y组NK细胞占淋巴细胞比例(29.11±3.31)比RJ组(13.92±3.11)明显升高(P＜0.05);效应性记忆性CD8+T细胞比例3Y组(12.92±2.05)比RJ组(8.74±1.69)明显升高(P＜0.05).结论 肝移植术后潜在免疫耐受者外周血NK细胞、静息性调节性T细胞和效应性记忆性CD8+T细胞比例相对排斥反应者明显升高,可作为潜在的耐受标志物.     

Anti-CD28 antibody-induced kidney allograft tolerance related to tryptophan degradation and TCR class II B7 regulatory cells.

B7/CTLA-4 interactions negatively regulate T-cell responses and are necessary for transplant tolerance induction. Tolerance induction may therefore be facilitated by selectively inhibiting the B7/CD28 pathway without blocking that of B7/CTLA-4. In this study, we selectively inhibited CD28/B7 interactions using a monoclonal antibody modulating CD28 in a rat model of acute kidney graft rejection. A short-term treatment abrogated both acute and chronic rejection. Tolerant recipients presented few alloantibodies against donor MHC class II molecules, whereas untreated rejecting controls developed anti-MHC class I and II alloantibodies. PBMC from tolerant animals were unable to proliferate against donor cells but could proliferate against third-party cells. The depletion of B7+, non-T cells fully restored this reactivity whereas purified T cells were fully reactive. Also, NK cells depletion restored PBMC reactivity in 60% of tolerant recipients. Conversely, NK cells from tolerant recipients dose-dependently inhibited alloreactivity. PBMC anti-donor reactivity could be partially restored in vitro by blocking indoleamine-2,3-dioxygenase (IDO) and iNOS. In vivo, pharmacologic inhibition of these enzymes led to the rejection of the otherwise tolerated transplants. This study demonstrates that an initial selective blockade of CD28 generates B7+ non-T regulatory cells and a kidney transplant tolerance sustained by the activity of IDO and iNOS.     

Cytokines affecting CD4+ T regulatory cells in transplant tolerance. III. Interleukin-5 (IL-5) promotes survival of alloantigen-specific CD4+ T regulatory cells

CD4⁺ T cells mediate antigen-specific allograft tolerance, but die in culture without activated lymphocyte derived cytokines. Supplementation of the media with cytokine rich supernatant, from ConA activated spleen cells, preserves the capacity of tolerant cells to transfer tolerance and suppress rejection. rIL-2 or rIL-4 alone are insufficient to maintain these cells, however. We observed that activation of naïve CD4⁺ CD25⁺ FOXP3⁺ Treg with alloantigen and the Th2 cytokine rIL-4 induces them to express interleukin-5 specific receptor alpha (IL-5Rα) suggesting that IL-5, a Th2 cytokine that is produced later in the immune response may promote tolerance mediating Treg.This study examined if recombinant IL-5(rIL-5) promoted survival of tolerant CD4⁺, especially CD4⁺ CD25⁺ T cells. CD4⁺ T cells, from DA rats tolerant to fully allogeneic PVG heart allografts surviving over 100 days without on-going immunosuppression, were cultured with PVG alloantigen and rIL-5. The ability of these cells to adoptively transfer tolerance to specific-donor allograft and suppress normal CD4⁺ T cell mediated rejection in adoptive DA hosts was examined. Tolerant CD4⁺ CD25⁺ T cells' response to rIL-5 and expression of IL-5Rα was also assessed.rIL-5 was sufficient to promote transplant tolerance mediating CD4⁺ T cells' survival in culture with specific-donor alloantigen. Tolerant CD4⁺ T cells cultured with rIL-5 retained the capacity to transfer alloantigen-specific tolerance and inhibited naïve CD4⁺ T cells' capacity to effect specific-donor graft rejection. rIL-5 promoted tolerant CD4⁺ CD25⁺ T cells' proliferation in vitro when stimulated with specific-donor but not third-party stimulator cells. Tolerant CD4⁺ CD25⁺ T cells expressed IL-5Rα.This study demonstrated that IL-5 promoted the survival of alloantigen-specific CD4⁺ CD25⁺ T cells that mediate transplant tolerance.