阻断ICOS/B7h信号的供体特异性输血对移植受体CD4+CD25+调节性T细胞的影响

目的 观察阻断ICOS/B7h信号的供体特异性输血(DST)对异基因小鼠心脏移植术后体内CD4+CD25+调节性T细胞(Treg)的影响.方法 按陈氏方法建立小鼠颈部异位心脏移植模型,实验分3组,异基因组及同基因组:供心分别来源于BALB/C和C57BL/6小鼠,受体均为C57BL/6小鼠,未予治疗.治疗组:移植当天给予受体鼠(C57BL/6)尾静脉注射5×106 ICOS-Fc靶定的供体(BALB/C)脾B淋巴细胞,d0～6连续给予受体鼠尾静脉注射ICOS-Fc 200 μg/d.术后统计各组移植物的存活时间,通过流式细胞术检测受体鼠外周血中CD4+CD25+Treg的亚群比例,利用逆转录-聚合酶链反应(RT-PCR)检测移植物中FOXP3的mRNA表达,在混合淋巴细胞反应中检测CD4+CD25+Treg对CD4+CD25-效应T细胞(Teff)的增殖抑制效率.结果 与异基因组比较,治疗组心脏移植物存活时间明显延长[(84.38±29.14)d比(7.00±0.76)d,P＜0.01].各组中,治疗组受体外周血中CD4+CD25+Treg亚群比例显著上调[(15.60±5.69)%,P＜0.01].与其他两组比较,治疗组心脏移植物中FOXP3 mRNA表达显著上调.以正常鼠为对照,耐受鼠脾脏中获取的CD4+CD25+Treg能够更高效地抑制CD4+CD25-Teff在混合淋巴细胞培养中的增殖效应.结论 通过阻断ICOS/B7h信号的DST可以诱导异基因小鼠心脏移植耐受,CD4+CD25+Treg在耐受的形成与维持中均起着重要作用.     

Long‐term small bowel allograft function induced by short‐term FK 506 application is associated with split tolerance

Abstract Functional long‐term allograft survival after experimental small bowel transplantation (SBT) is limited by chronic rejection. Initial application of high‐dose FK 506 has been shown to induce stable long‐term graft function. In order to examine whether this long‐term function is associated with donor‐specific tolerance, we analyzed the functional status of recipient T cells in vivo and in vitro. One‐step orthotopic SBT was performed in the allogeneic Brown Norway (BN)‐to‐Lewis rat strain combination. FK 506 was given daily at a dose of 2 mg/kg from days 0‐5 in the rejection model and from days 0‐9 in the long‐term functional model. Mean survival time in the rejection model was 98 ± 2.8 days. Histological examination of these small bowel allografts disclosed signs of chronic rejection. In contrast, all animals of the long‐term functional model survived long term (> 250 days) without clinical signs of chronic rejection. The latter model, furthermore, produced evidence of donor‐specific tolerance. Whereas heterotopic Dark Agouti (DA) hearts were rejected regularly within 7 days, BN hearts survived indefinitely (> 70 days). In vitro, mixed leukocyte reactivity of CD4 + T cells was similarly strong against donor (BN) antigens as against third‐party (DA) antigens. The split tolerance revealed by our in vivo and in vitro results enabled acceptance of both the small bowel allograft without signs of chronic rejection and of donor‐specific heart allografts.     

Analysis of cellular events in hepatic allografts: Donor progenitors induce intragraft chimerism

Abstract Long‐term graft acceptance and tolerance induction after allogeneic rat liver transplantation are well described. However, the underlying mechanisms remain unclear. In this study we investigated the cellular events within the liver graft during initial immunosuppression and long‐term acceptance. Orthotopic liver transplantation was performed in the Dark Agouti (DA)‐to‐Lewis (LEW) and LEW‐to‐DA rat strain combination. In order to achieve long‐term acceptance, LEW recipients of DA livers were treated with two different short‐term therapies. Non‐parenchymal cells (NPC) were isolated from liver allografts on days + 10 and + 100 after transplantation and donor‐specific leukocytes were immunophenotyped by flow cytometry. Both the monotherapy and triple therapy prolonged graft survival (> 100 days). Liver allografts from LEW donors into DA recipients were spontaneously accepted across a complete MHC mismatch without immunosuppression. Liver allograft rejection was induced by infiltrating alloreactive immunocompetent cells. But the intensities of cell infiltration in the early and late phases after transplantation did not correlate with eventual outcome. Donor‐specific NPC decreased to 18‐25% on day + 10 in both therapeutic groups, but had rebounded to up to 40% by day + 100. Recurrence of donor‐specific cells was caused almost exclusively by rising T cell counts. The persistence of dendritic cells in the late phase after transplantation could be clearly demonstrated. Repopulation by donor‐specific T lymphocytes was observed in long‐term accepted liver grafts. This recurrence may be based on the differentiation of liver‐derived progenitor cells. The persistent coexistence of donor and recipient cells within the liver allograft (intrahepatic chimerism) appears to be characteristic and may be important for long‐term acceptance.     

Organ transplant specificity of tolerance to skin grafts with heart or kidney grafts plus nondepleting anti-CD4 monoclonal antibody (RIB 5/2) and intravenous donor alloantigen administration

BACKGROUND: CD4+ T cells play an essential role in allograft rejection. Monoclonal anti-rat CD4 antibody, RIB 5/2, has been shown to modulate the CD4 glycoprotein without eliminating recipient T cells. A single dose of monoclonal anti-rat CD4 antibody RIB 5/2 plus donor splenocytes results in donor-specific unresponsiveness to heart and kidney allografts, but not skin allografts. This study examined whether tolerance to the more resistant skin graft could also be achieved with RIB 5/2. METHODS: Buffalo (RT1(b)) recipients were given a single dose (20 mg/kg) of monoclonal antibody RIB 5/2 IP plus IV Lewis (RT1(l)) splenocytes (25 x 10(6)) 21 days before Lewis heart, kidney, or skin grafts. In addition, Lewis skin was grafted either simultaneously with or after long- term Lewis heart or kidney allograft acceptance (>50 days). RESULTS: While IV alloantigen plus RIB 5/2 results in long-term acceptance of both heart and kidney, skin allografts are rejected when transplanted alone. Simultaneous transplantation with a Lewis kidney, but not with a Lewis heart, resulted in long-term Lewis skin graft acceptance. However, recipients tolerant to Lewis kidney or heart alone will not accept subsequent Lewis skin grafts, while recipients of simultaneous Lewis skin and kidney grafts subsequently accept a second Lewis, but not third-party Brown Norway (RT1(n)), skin graft. CONCLUSION: RIB 5/2 plus Lewis donor splenocytes tolerize for donor-specific heart and kidney but not skin grafts. However, Lewis skin grafted simultaneously with a Lewis kidney, but not Lewis heart, is accepted and protects a subsequent donor-specific Lewis skin graft.     

AdCTLA-4Ig combined with donor splenocytes,bone marrow cells and anti-ICOS antibody treatment induce tolerance in a rat heart transplantation model

It is difficult to induce rat cardiac allograft tolerance by co-stimulator blockade of the B7-CD28 pathway with CTLA-4Ig monotherapy alone. However, combined therapies of AdCTLA-4Ig plus donor-specific spleen-cell infusion, bone marrow cell infusion, and anti–ICOS antibody have been demonstrated to effectively induce indefinite acceptance of rat cardiac allografts. In this report, we compared the tolerance of cardiac allograft tolerant recipients induced by the above three strategies. The results show that treating Lewis recipients of a DA cardiac allograft with a combination of AdCTLA4-Ig and anti-ICOS antibody, donor splenocytes or bone marrow cells produced indefinite graft survival. Second transplantation of donor type skin or heart grafts could not affect the survival of primary heart graft in anti-ICOS treated groups, but results in rejection of primary heart grafts in other two groups, and that co-stimulator blockade, CD28 and ICOS simultaneously with CTLA-4Ig and anti-ICOS antibody, facilitates the development of CD25+ CD4+ regulatory T cells and induces stable transplantation tolerance in the rat cardiac allograft model. This also provides an effective therapy in clinical transplantation for promoting permanent graft survival by generating regulatory T cells.Abbreviations BMC bone marrow cells - ICOS inducible co-stimulator - pfu plaque-forming units - SPC spleen cell     

Endogenous Memory CD8 T Cells Are Activated Within Cardiac Allografts Without Mediating Rejection

Endogenous memory CD8 T cells infiltrate MHC‐mismatched cardiac allografts within 12–24 h posttransplant in mice and are activated to proliferate and produce IFN‐γ. To more accurately assess the graft injury directly imposed by these endogenous memory CD8 T cells, we took advantage of the ability of anti‐LFA‐1 mAb given to allograft recipients on days 3 and 4 posttransplant to inhibit the generation of primary effector T cells. When compared to grafts from IgG‐treated recipients on day 7 posttransplant, allografts from anti‐LFA‐1 mAb‐treated recipients had increased numbers of CD8 T cells but these grafts had marked decreases in expression levels of mRNA encoding effector mediators associated with graft injury and decreases in donor‐reactive CD8 T cells producing IFN‐γ. Despite this decreased activity within the allograft, CD8 T cells in allografts from recipients treated with anti‐LFA‐1 mAb continued to proliferate up to day 7 posttransplant and did not upregulate expression of the exhaustion marker LAG‐3 but did have decreased expression of ICOS. These results indicate that endogenous memory CD8 T cells infiltrate and proliferate in cardiac allografts in mice but do not express sufficient levels of functions to mediate overt graft injury and acute rejection.     

协同信号途径阻断和供体脾细胞输注诱导预存同种记忆性T淋巴细胞大鼠心脏移植物的耐受

目的 联合阻断OX40/OX40L和CD28/B7双协同信号途径和供体特异性脾细胞输注,诱导预存同种反应性记忆T淋巴细胞大鼠对同种心脏移植物的耐受.方法 建立大鼠预存同种反应性记忆T淋巴细胞的移植模型,采用免疫磁珠法分选CD8+CD44-记忆性T细胞;对过继转移供体特异性CD8+记忆性T细胞3 d的Lewis大鼠分别/合并输注AdCTLA4Ig、AdOX40Ig、供体脾细胞(DST),同时移植来至DA大鼠的心脏;48 h后取出心脏进行组织学分析、细胞因子表达分析,同时观察不同处理移植心脏存活时间.结果 AdCTLA4Ig+AdOX40Ig+DST组分别与AdCT-LA4Ig,AdOX40Ig和DST比较,心脏组织病理级别较低,白细胞介素(IL)-2及干扰素(IFN)-γ的表达水平也明显比其他组低很多,而心脏存活时间显著延长.结论 联合阻断OX40/OX40L和CD28/B7和供体特异性脾细胞输注能较好地诱导大鼠心脏移植物耐受.     

Induction of Tolerance by Exosomes and Short-Term Immunosuppression in a Fully MHC-Mismatched Rat Cardiac Allograft Model

Exosomes are MHC‐bearing vesicles secreted by a wide array of cells. We have previously shown that donor‐haplotype exosomes from bone marrow dendritic cells (DCs) injected before transplantation significantly prolong heart allograft survival in congenic and fully MHC‐mismatched Lewis rats. Here we show that donor exosomes administered after transplantation are similarly able to prolong allograft survival, however, without inducing tolerance. We therefore tested the effect of exosomes combined with short‐term LF 15‐0195 (LF) treatment, which blocks the maturation of DCs, so that donor‐MHC antigens from exosomes could be presented in a more tolerogenic environment. LF treatment does not preclude the development of a strong antidonor cellular response, and while LF, but not exosome, treatment inhibits the antidonor humoral response and decreases leukocyte graft infiltration, allografts from LF‐treated recipients were either acutely or strongly chronically rejected. Interestingly, when combined with LF treatment, exosomes induced a donor‐specific allograft tolerance characterized by a strong inhibition of the antidonor proliferative response. This donor‐specific tolerance was transferable to naïve allograft recipients. Moreover, exosomes/LF treatment prevented or considerably delayed the appearance of chronic rejection. These results suggest that under LF treatment, presentation of donor‐MHC antigens (from exosomes) can induce regulatory responses that are able to modulate allograft rejection and to induce donor‐specific allograft tolerance.     

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.     

The role of Foxp3+ regulatory T cells in liver transplant tolerance

The liver has long been considered a tolerogenic organ that favors the induction of peripheral tolerance. The mechanisms underlying liver tolerogenicity remain largely undefined. In this study, we characterized Foxp3-expressing CD4+ CD25+ regulatory T cells (Treg) in liver allograft recipients and examined the role of Treg in inherent liver tolerogenicity by employing the mouse spontaneous liver transplant tolerance model. Orthotopic liver transplantation was performed from C57BL/10 (H2b) to C3H/HeJ (H2k) mice. The percentage of CD4+ CD25+ Treg was expanded in the liver grafts and recipient spleens from day 5 up to day 100 posttransplantation, associated with high intracellular Foxp3 and CTLA4 expression. Immunohistochemistry further demonstrated significant numbers of Foxp3+ cells in the liver grafts and recipient spleens and increased transforming growth factor beta expression in the recipient spleens throughout the time courses. Adoptive transfer of spleen cells from the long-term liver allograft survivors significantly prolonged donor heart graft survival. Depletion of recipient CD4+ CD25+ Treg using anti-CD25 monoclonal antibody (250 microg/d) induced acute liver allograft rejection, associated with elevated anti-donor T-cell proliferative responses, CTL and natural killer activities, enhanced interleukin (IL)-2, interferon-gamma, IL-10, and decreased IL-4 production, and decreased T-cell apoptotic activity in anti-CD25-treated recipients. Moreover, CTLA4 blockade by anti-CTLA4 monoclonal antibody administration exacerbated liver graft rejection when combined with anti-CD25 monoclonal antibody. Thus, Foxp3+ CD4+ CD25+ Treg appear to underpin spontaneous acceptance of major histocompatability complex- mismatched liver allografts in mice. CTLA4, IL-4, and apoptosis of alloreactive T cells appear to contribute to the function of Treg and regulation of graft outcome.     

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.     

The kinetics of tolerance induction by nondepleting anti-CD4 monoclonal antibody (RIB 5/2) plus intravenous donor alloantigen administration

BACKGROUND: CD4+ T cells play an essential role in allograft rejection. The monoclonal anti-rat CD4 antibody, RIB 5/2, has been shown to modulate the CD4 glycoprotein without eliminating the recipient T cells. We have successfully induced tolerance to rat heart allografts by recipient pretreatment with a single dose of RIB 5/2 plus intravenous administration of donor splenocytes. In this study, we explored whether this potent regimen could induce tolerance to the more resistant kidney and skin allografts. Furthermore, we examined the kinetics and requirements for tolerance to be met by a single dose of RIB 5/2 plus i.v. alloantigen. METHODS: The efficacy of a single i.p. dose of 20 mg/kg RIB 5/2 plus i.v. donor antigen (25x10(6) splenocyte) pretreatment 0, 21, or 40 days before receipt of an MHC-mismatched Lewis (RT1l) to Buffalo (RT1b) rat cardiac, renal, or skin allograft was studied. Another group of Buffalo recipients treated with RIB 5/2 plus an i.v. alloantigen +/-thymectomy received kidney transplants after 40 days. Attempts to prevent tolerance used interleukin-2 or prior sensitization. Mixed lymphocyte cultures, cytotoxic assays, and precursor frequencies of helper and cytotoxic cells, by limiting dilution analysis, serially measured in vitro cell-mediated immunity. RESULTS: RIB 5/2 administration combined with i.v. alloantigen 21 days before induced tolerance to heart and kidney allografts but did not prolong skin graft survival. In contrast, kidney allografts delayed for 40 days after pretreatment were acutely rejected and survival was not affected by the thymectomy. MLC, CTL, and pTH, and pCTL precursor frequencies from recipients of long-term grafts were specifically suppressed to donor, but not third party, alloantigen. CONCLUSION: A single dose of the nondepleting anti-CD4 monoclonal antibody, RIB 5/2, plus i.v. alloantigen is a potent inducer of tolerance to heart and kidney, but not skin, allografts. The RIB 5/2-induced donor unresponsiveness to a delayed kidney or cardiac allograft is time dependent but can be prolonged if specific alloantigen is present. Suppression of cell-mediated allo-immune responsiveness correlates with allograft acceptance.     

CD25+CD4+ regulatory T cells develop in mice not only during spontaneous acceptance of liver allografts but also after acute allograft rejection

BACKGROUND: Liver grafts transplanted across a major histocompatibility barrier are accepted spontaneously and induce donor specific tolerance in some species. Here, we investigated whether liver allograft acceptance is characterized by, and depends upon, the presence of donor reactive CD25CD4 regulatory T cells. METHODS: CD25 and CD25CD4 T cells, isolated from CBA. Ca (H2) recipients of C57BL/10 (B10; H2) liver and heart allografts 10 days after transplantation, were transferred into CBA. Rag1 mice to investigate their influence on skin allograft rejection mediated by CD45RBCD4 effector T Cells. RESULTS: Fully allogeneic B10 liver allografts were spontaneously accepted by naive CBA.Ca recipient mice, whereas B10 cardiac allografts were acutely rejected (mean survival time=7 days). Strikingly, however, CD25CD4 T cells isolated from both liver and cardiac allograft recipients were able to prevent skin allograft rejection in this adoptive transfer model. Interestingly, CD25CD4 T cells isolated from liver graft recipients also showed suppressive potency upon adoptive transfer. Furthermore, depletion of CD25CD4 T cells in primary liver allograft recipients did not prevent the acceptance of a secondary donor-specific skin graft. CONCLUSIONS: Our data provide evidence that the presence of CD25CD4 regulatory T cells is not a unique feature of allograft acceptance and is more likely the result of sustained exposure to donor alloantigens in vivo.     

Infectious tolerance mediated by CD8+ T-suppresor cells after UV-B-irradiated donor-specific transfusion and rat heart transplantation

AIMS: CD8+CD28- human T-suppressor cells (Ts), which can be generated in vitro, act directly on APC rendering them tolerogenic to unprimed and primed CD4+ T cells. The aim of this study was to investigate the possibility that CD8+ T cells mediate the induction of tolerance in a heart transplantation model in rodents. MATERIALS AND METHODS: Blood from Lewis rats was UV-B-irradiated and transfused into ACI recipients on days -21, -14, and -7 before heart allograft transplantation on day 0. CD4(+) and CD8(+) T cells were positively selected from ACI rats, which had tolerated Lewis heart allografts for more than 100 days and were adoptively transferred to naive ACI rats pretreated (day -1) with gamma irradiation. These ACI rats underwent transplantation with Lewis hearts 24 hours after adoptive transfer of putative T-suppressor cells. RESULTS: Adoptive transfer of CD8(+) T cells from tolerant ACI to naive ACI rats significantly prolonged Lewis heart mean allograft survival time (MST +/- SD) to 69 +/- 13 days as compared with 15 +/- 1 and 14 +/- 1 days in animals adoptively transferred with CD4+ T cells or untreated controls, respectively (P < .001). Similarly, adoptive transfer of CD8(+) T cells from secondary ACI recipients to naive syngeneic animals also significantly prolonged survival of heart allografts to MST +/- SD of 72 +/- 4 for CD8(+) and 15 +/- 4 days for CD4(+) T cells (P < .001). CONCLUSIONS: These data demonstrate that allogeneic tolerance induced in ACI recipients by treatment with UV-B-irradiated blood from Lewis donors is mediated by CD8+ T-suppressor cells.     

Donor apoptotic cell–based therapy for effective inhibition of donor‐specific memory T and B cells to promote long‐term allograft survival in allosensitized recipients

Allosensitization constitutes a major barrier in transplantation. Preexisting donor‐reactive memory T and B cells and preformed donor‐specific antibodies (DSAs) have all been implicated in accelerated allograft rejection in sensitized recipients. Here, we employ a sensitized murine model of islet transplantation to test strategies that promote long‐term immunosuppression‐free allograft survival. We demonstrate that donor‐specific memory T and B cells can be effectively inhibited by peritransplant infusions of donor apoptotic cells in combination with anti‐CD40L and rapamycin, and this treatment leads to significant prolongation of islet allograft survival in allosensitized recipients. We further demonstrate that late graft rejection in recipients treated with this regimen is associated with a breakthrough of B cells and their aggressive graft infiltration. Consequently, additional posttransplant B cell depletion effectively prevents late rejection and promotes permanent acceptance of islet allografts. In contrast, persistent low levels of DSAs do not seem to impair graft outcome in these recipients. We propose that B cells contribute to late rejection as antigen‐presenting cells for intragraft memory T cell expansion but not to alloantibody production and that a therapeutic strategy combining donor apoptotic cells, anti‐CD40L, and rapamycin effectively inhibits proinflammatory B cells and promotes long‐term islet allograft survival in such recipients.     

The Immunobiology of Inductive Anti-CD40L Therapy in Transplantation: Allograft Acceptance is Not Dependent Upon the Deletion of Graft-Reactive T Cells

CD40–CD40L costimulatory interactions are crucial for allograft rejection, in that treatment with anti‐CD40L mAb markedly prolongs allograft survival in several systems. Recent reports indicate that costimulatory blockade results in deletion of graft‐reactive cells, which leads to allograft tolerance. To assess immunologic parameters that were influenced by inductive CD40–CD40L blockade, cardiac allograft recipients were treated with multiple doses of the anti‐CD40L mAb MR1, which was remarkably effective at prolonging allograft survival. Acute allograft rejection responses such as IL‐2 producing helper cell priming, Th1 priming, and alloantibody production were abrogated by anti‐CD40L treatment. Interestingly, the spleens of mice bearing long‐term cardiac allografts following inductive anti‐CD40L treatment retained precursor donor alloantigen‐reactive CTL, IL‐2 producing helper cells, and Th1 in numbers comparable to those observed in naïve mice. These mice retained the ability to reject donor‐strain skin allografts, but were incapable of rejecting the original cardiac allograft, or a second donor‐strain cardiac allograft. Further, differentiated effector cells were incapable of mediating rejection following adoptive transfer into mice bearing long‐term allografts, suggesting that regulatory cell function, rather than effector cell deletion was responsible for long‐term graft acceptance. Collectively, these data demonstrate that inductive CD40–CD40L blockade does not result in the deletion of graft‐reactive T cells, but induces the maintenance of these cells in a quiescent precursor state. They further point to a tissue specificity of this hyporesponsiveness, suggesting that not all donor alloantigen‐reactive cells are subject to this regulation.     

Anti-CD25 mAb administration prevents spontaneous liver transplant tolerance

Liver allografts are accepted spontaneously in all mouse strain combinations without immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we examined the effect of CD4+ CD25+ T regulatory cells (Treg) on the induction of mouse liver transplant tolerance. Orthotopic liver transplantation was performed from B10 (H2b) to C3H (H2k) mice. Depleting rat anti-mouse CD25 mAb (PC61) was given to the donors or recipients (250 microg/d IP) pretransplant or to the recipients postoperatively. At day 5 posttransplantation, both effector T cells (mainly CD8) and CD4+ CD25+ Treg were increased in the liver allografts and host spleens compared to na?ve mice. Anti-CD25 mAb administration, either pretransplantation or posttransplantation, reduced the ratio of CD4+ CD25+ Treg to the CD3 T cells of liver grafts and recipient spleens and induced liver allograft acute rejection compared to IgG treatment. Anti-CD25 mAb administration elevated anti-donor T-cell proliferative responses and CTL and NK activities of graft infiltrates and host splenocytes; reduced CTLA4, Foxp3, and IDO mRNA levels; increased IL-10 and IFN-gamma; and decreased IL-4 mRNA levels in the livers or host spleens. The number of apoptotic T cells was reduced significantly in the liver grafts and treated host spleens. Therefore, anti-CD25 mAb administration changed the balance of CD4+ CD25+ Treg to activated T cells of liver graft recipients, preventing liver transplant tolerance. This was associated with enhanced anti-donor immune reactivity, downregulated Treg gene expression, and reduced T cell apoptosis in the grafts and host spleens.     

Intragraft CD11b+IDO+ Cells Mediate Cardiac Allograft Tolerance by ECDI‐Fixed Donor Splenocyte Infusions

We have previously shown that pre‐ and post‐transplant infusions of donor splenocytes treated with 1‐ethyl‐3‐(3’‐dimethylaminopropyl)‐carbodiimide (ECDI‐SPs) provide permanent donor‐specific protection of islet allografts. The efficacy of donor ECDI‐SPs in protecting vascularized cardiac allografts and mechanism(s) of protection are unknown. In this study, we show that infusions of ECDI‐SPs significantly prolong cardiac allograft survival concomitant with an impressive accumulation of CD11b⁺IDO⁺ cells in the cardiac allograft, and that the presence of this population is dependent on Gr1⁺ cells. Consequently, depletion of Gr1⁺ cells or inhibition of indoleamine 2,3 dioxygenase (IDO) activity abrogates graft protection by ECDI‐SPs infusions. In addition, T cells from ECDI‐SPs treated recipients secrete high levels of interleukin 10 and interleukin 13 upon in vitro restimulation, which are also dampened in recipients treated with the IDO inhibitor. Furthermore, combination of donor ECDI‐SPs with a short course of rapamycin provides indefinite cardiac allograft survival in 100% of the recipients. These findings reveal a novel mechanism of donor ECDI‐SPs in inducing cardiac transplant tolerance and provide several targets that are amenable to therapeutic manipulations for tolerance induction for cardiac transplantation.     

Prevention of allograft heart valve failure in a rat model

OBJECTIVE: Allograft heart valves are commonly used in cardiac surgery. Despite mounting evidence that these valves are immunogenic, leading to premature failure, current clinical practice does not attempt to minimize or control such a response. The objective of this study was to evaluate immune modulatory approaches to ameliorate allograft valve failure in a rat model. METHOD: Aortic valve grafts were implanted infrarenally into Lewis rat recipients (n = 32). There were 4 transplant groups: syngeneic grafts (Lewis to Lewis), untreated allografts (Brown Norway to Lewis), allograft recipients treated with cyclosporine (INN: ciclosporin) (10 mg/kg per day for 7 or 28 days), and allograft recipients treated with anti-alpha4 integrin and anti-beta2 integrin monoclonal antibodies for 7 days. At 7 and 28 days the valves were examined for structural integrity and cellular infiltration. RESULTS: Both cyclosporine and anti-alpha4/beta2 integrin treatment resulted in significant reduction in leaflet infiltration by macrophages (ED1(+)), T cells (CD3(+)), and CD8(+) T cells at 7 days with preservation of structural integrity when compared with control allografts. Twenty-eight days after implantation, daily treatment with cyclosporine preserved leaflet structural integrity and inhibited cellular infiltration. However, a short course of cyclosporine (7 days) failed to prevent destruction of the valves at 28 days. CONCLUSIONS: Immune modulatory approaches aimed at T-cell activation or trafficking decrease leaflet cellular infiltration and prevent allograft valve structural failure. However, short-course therapy does not appear to be sufficient and must be maintained to allow long-term preservation of leaflet structural integrity (28 days).