CD4+CD25+免疫调节性T细胞对CD4+T细胞介导的移植物抗宿主病预防作用的研究

目的研究CD4+CD25+免疫调节性T(Treg)细胞在小鼠骨髓移植后,对移植物抗宿主病的预防作用及其作用机制.方法用C3H(H-2k)小鼠骨髓作为供体,提取C3H(H-2k)小鼠CD4+T及CD4+CD25+T细胞,C3H×B6(H-2k/b)F1小鼠为骨髓移植的受者.在受者接受致死量全身放射后,输注供者去除T细胞的骨髓(ATBM),使其造血功能重建(ATBM组).于不同的实验组给予CD4+(CD4组)T细胞,CD4+CD25+T(CD25组)或二者同时输注(CD4/CD25组).观察各组小鼠移植物抗宿主病(GVHD)的发生率.结果所有10只ATBM组小鼠至骨髓移植后60天仍全部存活,无GVHD发生;所有10只CD4组小鼠在骨髓移植10天内全部死于GVHD(P＜0.01);所有5只CD25组小鼠于骨髓移植后60天仍全部存活,无明显GVHD发生(P＞0.05);同样,所有6只CD4/CD25组小鼠至骨髓移植后仍全部存活,无明显GVHD发生(P＞0.05).结论在同种异基因小鼠的骨髓移植模型中,CD4+CD25+T不诱导GVHD的发生,并有预防CD4+T细胞介导的GVHD发生的作用.     

Host CD25+CD4+Foxp3+ regulatory T cells primed by anti-CD137 mAbs inhibit graft-versus-host disease

CD25(+)CD4(+)Foxp3(+) regulatory T cells (Tregs) play a pivotal role in the maintenance of self-tolerance and regulation of immune responses. Previous studies have demonstrated that CD137 signals can promote proliferation and survival of Tregs in vitro. Here, we show that in vivo CD137-induced expansion of Tregs in naive mice was dependent upon IL-2 secreted by memory T cells. Tregs primed by anti-CD137 mAbs had a higher immunosuppressive capacity. Preconditioning with anti-CD137 mAbs significantly inhibited graft-versus-host disease (GVHD) in the C57BL/6 → (C57BL/6 × DBA/2) F1 acute GVHD model. In this disease model, a high proportion of host Tregs remained long-term in the recipient spleen, whereas donor hematopoietic cells replaced other host bone marrow-derived cells. Transient depletion of Tregs before transfer of donor cells completely abrogated the inhibitory effect of anti-CD137 mAbs on GVHD. In addition, adoptive transfer of anti-CD137-primed Tregs ameliorated GVHD. Our results demonstrate that it is possible to enhance the survival and/or the immunosuppressive activity of host Tregs in nonmyeloablative GVHD, and that 1 way of accomplishing this is through the prophylactic use of anti-CD137 mAbs in nonmyeloablative GVHD.     

The role of CD4+CD25hi regulatory T cells in the physiopathogeny of graft-versus-host disease

Donor T cells present within hematopoietic stem cell transplants promote engraftment, contribute to T-cell reconstitution and provide an antileukemic effect. However, they are also responsible for the life-threatening graft-versus-host disease. The subpopulation of CD4+CD25hi regulatory T cells, initially identified as crucial players in the regulation of autoimmune processes, might also play a role in the control of alloreactivity. Experimental studies in mice indicate that donor regulatory T cells indeed control alloreactive responses and reduce graft-versus-host disease. Recent clinical reports also suggest that higher numbers of CD4+CD25hi cells within the transplant or in the blood of grafted patients might be associated with reduced graft-versus-host disease. Hence, these cells are attractive immunoregulatory candidates to prevent graft-versus-host disease in humans.     

Blockade of chronic graft-versus-host disease by alloantigen-induced CD4+CD25+Foxp3+ regulatory T cells in nonlymphopenic hosts

CD4(+)CD25(+) regulatory T cells (Tregs) are well known to suppress immunopathology induced in lymphopenic animals following T cell reconstitution, including acute graft-versus-host disease (GVHD) post-bone marrow transplantation. The regulatory potential of this subset in nonlymphopenic hosts and in chronic, Th2-mediated GVHD is less clear. We have generated alloantigen-specific cells from CD4(+)CD25(+) populations stimulated with MHC-disparate dendritic cells and found them to express a stable Treg forkhead box p3(+) phenotype with enhanced suppressive activity mediated by cell contact. When transferred into nonlymphopenic F1 hosts, nonspecific Tregs proliferated as rapidly as CD4(+)CD25(-) cells but displayed distinct growth kinetics in vitro. Tregs, expanded in response to alloantigen in vitro, displayed greatly enhanced suppressive activity, which was partially antigen-specific. They were effective inhibitors of chronic GVHD, blocking donor cell engraftment, splenomegaly, autoantibody production, and glomerulonephritis. CD25(+) and CD25(-) cells were equally susceptible to inhibition by immunosuppressive drugs targeting TCR signaling and rapamycin, but Tregs were resistant to inhibition by dexamethasone. The data indicate that alloantigen-driven expansion, rather than homeostatic proliferation, is key to the effectiveness of CD4(+)CD25(+) Tregs in GVHD and suggest that cellular therapy with alloantigen-induced Tregs in combination with glucocorticoid treatment would be effective in prevention of chronic GVHD after immune reconstitution.     

CD25+CD4+ regulatory T cells suppress CD4+ T cell-mediated pulmonary hyperinflammation driven by Pneumocystis carinii in immunodeficient mice

The CD4(+) T cell-mediated inflammatory response to Pneumocystis carinii (PC) critically contributes to the clinical severity of PC pneumonia. It has been suggested that lymphopenic conditions predispose individuals to this immunopathology, although the mechanisms remain poorly understood. Another set of evidence indicates that a subpopulation of CD4(+) T cells constitutively expressing the CD25 molecule prevent lymphopenia-induced autoimmunity and inflammatory bowel disease. We tested the ability of this CD25(+)CD4(+) population to regulate CD4(+) T cell-mediated inflammatory response to PC. Adoptive transfer of CD25(-)CD4(+) cells into PC-infected recombination-activating gene-2-deficient mice led to lethal pneumonia within 13 days post-transfer. PC infection appeared to trigger CD25(-)CD4(+) cells, since recipients with reduced PC load survived up to 5 weeks after transfer. In contrast, transfer of CD25(+)CD4(+) cells did not induce lethal pneumonia and prevented the development of the disease induced by CD25(-)CD4(+) cells. Furthermore, CD25(-)CD4(+) cells reduced the PC load in the lung, while CD25(+)CD4(+) cells suppressed this immune response. Our results indicate an essential role for CD25(+)CD4(+) T cells in the control of PC-driven immunopathology, and suggest that in immunocompromised hosts PC pneumonia may result from a deficiency in regulatory T cells.     

Foxp3+CD4+ T cell-mediated immunosuppression involves extracellular nucleotide catabolism

Foxp3(+)CD4(+) T cells represent a population of naturally arising suppressor T cells that are crucial for the control of autoimmune responses. The suppressive activity of this T cell subset relies on multiple mechanisms that include secretion of anti-inflammatory factors such as TGF-beta or IL-10. Novel studies now establish that, through the generation of the immunosuppressive factor adenosine, the ectoenzymes CD39 and CD73 are important contributors to the regulatory activity of Foxp3(+)CD4(+) T cells.     

Tracking ex vivo-expanded CD4+CD25+ and CD8+CD25+ regulatory T cells after infusion to prevent donor lymphocyte infusion-induced lethal acute graft-versus-host disease.

Donor bone marrow (BM)-derived CD4+ CD25+ regulatory T cells, maturing in the host thymus, are critical in inhibiting graft-versus-host disease (GVHD) after donor lymphocyte infusion (DLI) in murine BM chimeras. Data presented here demonstrate that fresh CD25+ cells isolated from donor-type mice can be expanded ex vivo by a variety of methods. Ex vivo-expanded CD4+ CD25+ and CD8+ CD25+ cells were potent suppressors of donor response to host alloantigens in mixed lymphocyte reaction assays. Both fresh and ex vivo-expanded CD4+ CD25+ cells persisted long-term in vivo and effectively prevented DLI-induced GVHD in CD25-/- BM chimeras. Importantly, co-infused CD4+ CD25+ cells with DLI cells migrated to peripheral lymphoid organs and survived long-term in DLI-treated CD25-/- chimeras, but not in DLI-treated CD25+/+ chimeras, indicating homeostatic control of CD25+ cells and an available niche required for their long-term persistence. Furthermore, maintenance of CD25 expression seemed necessary for suppressive function, because only the CD25+ cell fraction, but not the CD25- fraction isolated after adoptive transfer, was suppressive in vitro. Ex vivo-expanded CD8+ CD25+ cells weakly prevented GVHD, apparently because of a rapid disappearance of these cells after adoptive transfer. Taken together, these data suggest that the therapeutic use of ex vivo-expanded CD4+ CD25+ cells may be a feasible, nontoxic modality for controlling GVHD in the clinic. Because of strict homeostatic control, an available niche may be required for long-term persistence of infused regulatory T cells.     

Blockade of IL-6-signaling inhibits the pathogenesis of CD4+ T cell-mediated lethal graft-versus-host reaction against minor histocompatibility antigen

Graft-versus-host reaction (GVHR) is considered as a problem in hematopoietic cell transplantation. We found that CD45RB(high) CD62L(+) na?ve CD4(+) T cells from wild-type B10D2 (H-2d MMTV6(-)) mice immediately differentiated into effector T cells producing high-levels of various cytokines after the transfer into BALB/c RAG2(-/-) (H-2d MMTV6(+)) mice. The expanded CD4(+) T cells, which have almost TCR Vβ3 chain, recognized the minor antigen of recipient mice and brought typical severe GVHR symptoms such as eyelid irritation, diarrhea, and liver failure. Eventually, all of the recipient mice transferred CD4(+) T cells was dead within 10 days. We demonstrated here that blockade of IL-6 signaling by administration of anti-IL-6 receptor (IL-6R) monoclonal antibody (mAb) remarkably inhibited the CD4(+) T cell-mediated lethal GVHR. In addition, we confirmed that the in vivo injection of anti-IL-6R mAb prevented the generation of effector CD4(+) T cells which produce the inflammatory cytokines such as IFN-γ, TNF-α, and IL-17. These findings indicated that IL-6 was a critical factor in the CD4(+) T cell-dependent acute GVHR induced by a minor-antigen, suggesting that IL-6-mediated signaling pathway would be a strong therapeutic target in T cell-mediated GVHR as well as other diseases including autoimmune and inflammation.     

Differential upregulation of interleukin-18 receptor alpha chain between CD4+ and CD8+ T cells during acute graft-versus-host disease in mice.

Interleukin-18 (IL-18), a unique cytokine that stimulates both T helper 1 (Th1) and Th2 responses, is associated with acute graft-versus-host disease (aGVHD), the major limiting toxicity following allogeneic stem cell transplantation. Here, we investigated the mechanism underlying the upregulation of IL-18 receptor (IL-18R) expression on T cells in murine aGVHD models. The induction of aGVHD elevated host serum IL-12 levels and increased expression of IL-18Ralpha chain (IL-18Ralpha) on engrafted T cells, in particular on CD8+ T cells. However, IL-18Ralpha expression did not increase on the CD4+ T cells of an IL-12-deficient host, indicating the IL-12-dependent upregulation of IL-18Ralpha expression on CD4+ T cells during aGVHD. Purified donor CD8+ T cells transferred in the host increased IL-18Ralpha expression. In vitro experiments showed that IL-18Ralpha expression upregulated on CD8+ T cells but not on CD4+ T cells on stimulation through the T cell receptor (TCR). These results suggest that IL-18Ralpha expression is differentially upregulated between CD4+ and CD8+ T cells during aGVHD, depending on endogenous IL-12 and TCR engagement, respectively.     

CD4+CD25+调节性T细胞在肿瘤免疫中的作用

CD4+CD25+调节性T细胞(Tr)是体内自然发生的调节性T细胞的重要亚群,具有无反应性和免疫抑制两大特性,主要通过与靶细胞的直接接触而起作用,其在体内不仅参与自身免疫性疾病、移植排斥反应等,还在肿瘤的发生、发展及免疫治疗中发挥重要作用.近几年来,Tr在肿瘤免疫中的作用倍受关注.     

Patients suffering from acute graft-versus-host disease after bone-marrow transplantation have functional CD4+CD25hiFoxp3+ regulatory T cells

Acute Graft-Versus-Host Disease (aGVHD), mediated by CD4(+) and CD8(+) effector T cells, is a life-threatening complication in hematopoietic stem cell (HSC) transplantation. Naturally-occurring CD4(+)CD25(hi)(Foxp3(+)) regulatory T cells (T(reg)) have been shown to modulate tolerance to aGVHD in murine graft models. In this report, we investigated their role in the prevention of aGVHD in patients transplanted with bone-marrow-derived HSC. When CD4(+)CD25(hi)Foxp3(+) T cells were isolated from bone-marrow grafts, they showed no suppressive activity. The analysis of their function in patients suffering from aGVHD after transplantation revealed a gain of suppressive activity indicating their inability to control the aGVHD induction. Thus, our findings clearly demonstrate that CD4(+)CD25(+) and CD4(+)CD25(hi)Foxp3(+) T cells, when administered in steady-state physiological conditions, do not influence the outcome of aGVHD after bone-marrow transplantation.     

Distinct roles of CTLA-4 and TGF-beta in CD4+CD25+ regulatory T cell function

Both CTLA-4 and TGF-beta have been implicated in suppression by CD4+CD25+ regulatory T cells (Treg). In this study, the relationship between CTLA-4 and TGF-beta in Treg function was examined. Blocking CTLA-4 on wild-type Treg abrogated their suppressive activity in vitro, whereas neutralizing TGF-beta had no effect, supporting a TGF-beta-independent role for CTLA-4 in Treg-mediated suppression in vitro. In CTLA-4-deficient mice, Treg development and homeostasis was normal. Moreover, Treg from CTLA-4-deficient mice exhibited uncompromised suppressive activity in vitro. These CTLA-4-deficient Treg expressed increased levels of the suppressive cytokines IL-10 and TGF-beta, and in vitro suppression mediated by CTLA-4(-/-) Treg was markedly reduced by neutralizing TGF-beta, suggesting that CTLA-4-deficient Treg develop a compensatory suppressive mechanism through CTLA-4-independent production of TGF-beta. Together, these data suggest that CTLA-4 regulates Treg function by two distinct mechanisms, one during functional development of Treg and the other during the effector phase, when the CTLA-4 signaling pathway is required for suppression. These results help explain contradictions in the literature and support the existence of functionally distinct Treg.     

Neonatal autoimmune disease: influence of CD4+ CD25+ regulatory T cells

Although previous studies have emphasized the tolerogenic property of murine neonatal immune system, recent studies indicate that neonatal mice are prone to autoimmune disease. This chapter will summarize the evidence for neonatal propensity to autoimmune ovarian disease (AOD) and describe the new finding that autoantibody can trigger a T cell-dependent autoimmune disease in neonatal but not adult mice. Based on depletion or addition of the CD4+ CD25+ T cells, disease resistance of older mice is explicable by the emergence of CD4+ CD25+ regulatory T-cell function after day 5, whereas disease susceptibility is associated with resistance to regulation by CD4+ CD25+ T cells.     

供体凋亡淋巴细胞预输注对猪肝移植受体CD4+T、CD8+T、CD4+CD25+调节性T细胞的影响

背景：凋亡细胞能够主动调节机体的免疫功能,并能通过调节机体细胞免疫和体液免疫的途径诱导免疫耐受,但这些结果只在大鼠肝脏移植模型中证实。 目的：探讨通过⁶⁰Co γ射线体外处理后的供体淋巴细胞预输注诱导猪肝移植特异性免疫耐受的作用中,对淋巴细胞亚群的影响。 方法：建立非转流小型猪原位肝移植模型。将受体猪随机摸球法均分为2组：空白对照组,受体猪无特殊处理,行肝移植;淋巴细胞组：受体猪在肝移植前7 d经耳静脉注射⁶⁰Co γ射线处理过的5×10⁸个供体淋巴细胞。观察两组受体猪移植后的存活时间,移植后T淋巴细胞亚型CD4⁺T、CD8⁺T、CD4⁺CD25⁺Tr变化及病理。 结果与结论：移植后3 d,两组病理活检均呈急性中、重度排斥反应;移植后6 d,两组均呈急性重度排斥反应。移植后1,3,6 d CD4⁺T、CD8⁺T、CD4⁺CD25⁺Tr升降趋势,两组间差异无显著意义(P > 0.05)。提示,⁶⁰Co γ射线体外处理过的淋巴细胞预输注未能够诱导猪同种异体肝移植特异性免疫耐受,未能引起T淋巴细胞亚群变化有关。     

The roles of soluble osteopontin using osteopontin-transgenic mice in vivo: proliferation of CD4+ T lymphocytes and the enhancement of cell-mediated immune responses.

We generated transgenic mice expressing osteopontin (OPN) under the control of the alpha(1)-antitrypsin promoter. These mice (OPN-T mice) expressed OPN mRNA in liver and kidney, and released a large amount of plasma OPN, which increased after stimulation with turpentine oil. Before sensitization, the number of CD4+ T cells in lymph nodes was significantly higher in OPN-T than nontransgenic mice, and that in spleen was slightly higher, whereas that of CD8+ T cells was no different between OPN-T and nontransgenic mice. After sensitization, the CD4+ T cell numbers in spleen increased significantly, while there were almost no changes in the CD8+ T cells in lymph nodes and spleen. The intensity of contact hypersensitivity responses to 2,4-dinitrofluorobenzene (DNFB) was obviously enhanced in OPN-T mice. In the delayed-type hypersensitivity (DTH) model elicited by DNFB, the number of CD8+ T cells among DNFB-2,4,6-trinitrobenzenesulfonic acid (TNBS)-peritoneal exudate cells was significantly higher in OPN-T than nontransgenic mice, while there was almost no difference in that of CD4+ T cells. Adoptive transfer experiments revealed that the enhanced reactivity is carried by CD4+ and CD8+ T cells, respectively, although the ability of transferring DTH was significantly lower in CD8+ than in CD4+ T cells. The enhancement of CD8+ T cell migration was observed in OPN-T mice. These results suggest that OPN induces a proliferation of effector CD4+ and CD8+ cells in cell-mediated reactions and plays a role in the migration of CD8+ T cells.     

Opposing peripheral fates of tissue-restricted self antigen-specific conventional and regulatory CD4+ T cells

The development of self antigen-specific T cells is influenced by how the self antigen is expressed. Here, we created a mouse in which a model self antigen is conditionally expressed in different tissue environments. Using peptide:MHCII tetramer-based cell enrichment methods, we examined the development of corresponding endogenous self antigen-specific CD4⁺ T cell populations. While ubiquitous self antigen expression resulted in efficient deletion of self antigen-specific T cells in the thymus, some tissue-restricted expression patterns resulted in partial deletion of the population in peripheral lymphoid organs. Deletion specifically affected Foxp3⁻ conventional T cells (Tconv) with a bias towards high avidity TCR expressing cells in the case of thymic, but not peripheral deletion. In contrast, Foxp3⁺ Treg exhibited elevated frequencies with increased TCR avidity. T cells surviving deletion were functionally impaired, with Tconv cells exhibiting more impairment than Tregs. Collectively, our results illustrate how postthymic recognition of tissue-restricted self antigens results in opposing developmental fates for Tconv and Treg cell subsets.     

CD4+CD25+ regulatory T cells: II. Origin, disease models and clinical aspects

Autoimmune diseases afflict approximately 5% of the population and reflect a failure in the immune system to discriminate between self and non-self resulting in the breakdown of self-tolerance. Regulatory CD4+CD25+ T cells (Treg cells) have been shown to play an important role in the maintenance of immune homeostasis and self-tolerance by counteracting the development and effector functions of potentially autoreactive T cells. We have in the previous APMIS review described the phenotype and physiology of Treg cells. The present overview deals with the thymic origin of Treg cells and their role in disease models such as autoimmune gastritis and inflammatory bowel disease. Finally, we will consider some aspects of the therapeutic potential of Treg cells.