免疫毒素及可溶性抗原诱导胰岛素移植耐受

目的 通过静脉注入抗ＣＤ４、ＣＤ８免疫毒素及供体可溶性抗原诱导胰岛植物免疫耐受。方法 供、受体分别为Ｗｉｓｔａｒ大鼠和ＳＤ大鼠,移植前１４天,７天分别将免疫毒素各２００μｇ,供体可溶性抗原５００μｇ经静脉注入受体,然后将供体５００个胰岛移植于受体（糖尿病大鼠）左侧肾包膜下。结果 用免疫毒素及供体可溶性抗原联合处理组胰岛移植物存活时间显著延长（Ｐ〈０．０１）。而单独应用抗ＣＤ４、ＣＤ８免疫毒素或供体     

免疫毒素及可溶性抗原内诱导特异性淋巴细胞无反应的研究

通过诱导特异性淋巴细胞凋亡产生免疫耐受。方法 将抗ＣＤ４，ＣＤ８免疫毒素各２００μｇ／ｋｇ，抗体可溶性抗原５００μｇ经静脉注入受体，混合淋巴细胞培养测定处理后受体淋巴细胞的反应性，ＴＵＮＥＬ法标记凋亡的淋巴细胞。结果 免疫毒素及供体可溶性抗原联合应用可产生持续性免疫无反应，单用免疫毒素仅产生７－１４天的免疫无反应性。     

免疫缺陷树突状细胞诱导异种胰岛细胞移植耐受

目的　研究受体来源免疫缺陷树突状细胞(dendriticcell, DC)诱导异种胰岛细胞移植的免疫耐受作用及其机制。方法　从BALB/C小鼠骨髓干细胞诱导分化免疫缺陷DC,负载Wistar大鼠MHC抗原。将上述DC通过尾静脉回输糖尿病小鼠体内(预处理组), 7d后分别将Wistar或SD大鼠胰岛细胞移植于受体鼠肾包膜下。观察移植物存活时间,检测T细胞增殖及TH1 /TH2细胞因子的表达。结果　与对照组相比,预处理组胰岛细胞存活时间明显延长(P < 0. 0 5 ),而以SD大鼠胰岛细胞作为供体的移植物存活时间无明显改变。免疫缺陷DC预处理受体鼠T细胞增殖反应微弱,且TH1 /TH2细胞因子表达明显下降。结论　负载异种MHC抗原的免疫缺陷型DC预处理受体可诱导抗原特异性T细胞无能,以及TH1 /TH2细胞因子的低表达,从而有效地延长异种胰岛细胞存活时间。     

供体抗原经门静脉处理及移植物从门静脉回流对大鼠胰腺移植…

以３５只雄性ＳＤ大鼠为受体，制成糖尿病模型，以同龄Ｗｉｓｔａｒ雄性大鼠为供体，行全县腺移植。供体抗原分别经门静灌周围静脉处理，处理时间分别为移植前预处理或移植时处理，移植物静脉分别从门静脉或下腔静脉回流，结果表明供体抗原经门静脉预处理及移植物从门静脉加流对胰腺移植物功能存活的延长具有协同效应，移植时处理延长胰腺移植物效果不如移植前预处理，但可能具有一定的实用意义。     

诱导免疫耐受预防大鼠肾脏移植排斥反应的研究

目的 探讨具有临床应用前景的移植耐受诱导方法。方法 以Lewis大鼠和DA大鼠分别作为肾脏移植的受体和供体,采用注射抗淋巴细胞血清（ALS）、输入供体骨髓细胞（BMT）和环磷酰胺（Cp）注射方法进行移植耐受诱导,观察肾脏移植物的存活情况及受体对供体细胞抗原免疫应答改变。结果 经耐受诱导的大鼠肾脏移植物存活时间明显延长,7只鼠中5只移植物存活73－90 d时仍无排斥反应迹象,混合淋巴细胞反应及诱导迟发型超敏反应表现为供体特异性降低。结论 诱导免疫耐受预防肾脏移植排斥反应具有重要的临床意义,此方法有一定的临床应用前景。     

大鼠胸腺内注射同种抗原对甲状旁腺移植物存活的影响

目的　改善甲状旁腺移植物的存活时间。方法　用SDLewis及DA大鼠进行甲状旁腺移植实验。由供体Lewis大鼠的脾细胞提取抗原。按照不同的抗原注射途径（尾静脉、门静脉及胸腺内）,是否合用抗淋巴细胞血清及第3品系大鼠的甲状旁腺移植共分为9组。结果　胸腺内注射抗原结合抗淋巴细胞血清的应用，使甲状旁腺移植物的平均存活期达到（196.00±3.96）d，与其他各组相比差异有非常显著性（P＜0.01）。结论　大鼠胸腺内注射抗原结合抗淋巴细胞血清的应用成功地诱导受体产生了供体特异性免疫耐受。     

CTLA4Ig基因在糖尿病大鼠胰岛移植中的作用

为探讨融合蛋白（ＣＴＬＡ４Ｉｇ） 基因在糖尿病大鼠体内表达及其产物延长胰岛移植物存活的作用，利用脂质体（Ｌｉｐｏｆｅｃｔｉｎ） 载体包裹ＣＴＬＡ４ＩｇｃＤＮＡ 质粒后转染鼠胰岛和肌肉细胞，检测移植后ＣＴＬＡ４Ｉｇ 基因表达水平和Ｔ 淋巴细胞转化率，观察ＣＴＬＡ４Ｉｇ 基因表达在延长糖尿病鼠胰岛移植物和大鼠存活时间的作用。结果： 胰岛移植术后实验（ Ａ） 组、对照（Ｂ） 组Ｔ 淋巴细胞转化率有明显差异（ Ｐ＜ ０ ．０５） 。Ａ 组胰岛移植后第７ 天２ 只大鼠血清ＣＴＬＡ４Ｉｇ 呈阳性（ 阳性率为２０ ％ ） ，分别为１４ ｎｇ／ ｍｌ 和３１ ｎｇ／ ｍｌ。Ｂ 组胰岛移植后维持血糖正常时间平均３ ．６ ±５ ．１ 天，Ａ 组胰岛移植后维持血糖正常时间平均１４ ．８ ±１２ ．３ 天，两组比较差异有显著性意义（ Ｐ＜ ０ ．０５ ） 。Ａ 组大鼠平均存活２４ ．０ ±１０ ．８ 天，最长４５ 天，Ｂ 组大鼠平均存活１０ ．８ ±４ ．８ 天，最长２１ 天，两组大鼠生存时间差异有统计学意义（ Ｐ＜ ０ ．０１） 。本实验结果提示：ＣＴＬＡ４Ｉｇ 基因可以在受体大鼠胰岛细胞或肌肉组织表达，其表达产物可使胰岛移植物和受体鼠存活时间明显延长。     

胸腺内胰岛移植的实验研究

目的观察胸腺内和肾包膜下胰岛移植对移植物存活时间的影响。方法以Ｃ５７ＢＬ／６小鼠为受体,ＢＡＬＢ／ｃ小鼠为供体,肾包膜下胰岛移植分为单纯移植和移植的同时加腹腔内一次性注射兔抗小鼠胸腺细胞血清（ＡＴＳ）两组,胸腺内移植组亦分为单纯移植和移植加用ＡＴＳ两组。结果单纯胸腺内移植组其移植物的存活期为（１９．５±１０．１）天,长于单纯肾包膜下移植组（１４．０±２．１）天;移植的同时加用ＡＴＳ,则肾包膜下移植组移植物的存活期为（４３．０±１１．７）天,胸腺内移植组可达（９３．８±２５．５）天以上,其中６只（６／８）胸腺内移植物获长期存活,并且诱导了受体对供体的特异无反应性。结论胸腺可能为胰岛移植的理想部位,而且在诱导免疫耐受中具有重要作用。     

供体抗原注入胸腺处理对大鼠胰腺移植功能存活的影响

在大鼠胰腺移植模型上将供体Ｗｉｓｔａｒ大鼠的脾细胞于移植前或移植时注入受体ＳＤ大鼠胸腺内，辅以短期免疫抑制剂，结果显示能明显延长胰腺移植物功能存活。崦单纯短期免疫抑制剂或单纯供体脾细胞注入胸腺均未能延长胰腺功能存活。提示供体脾细胞注入胸腺预处理延长胰腺功能存活效果优于移植时处理，但移植时处理可能更有实用价值。     

供体抗原经门静脉处理及移植物从门静脉回流对大鼠胰腺移植物功能存活的影响

以35只雄性SD大鼠为受体，制成糖尿病模型，以同龄Wistar雄性大鼠为供体，行全胰腺移植。供体抗原分别经门静脉或周围静脉处理，处理时间分别为移植前预处理或移植时处理，移植物静脉分别从门静脉或下腔静脉回流，结果表明供体抗原经门静脉预处理及移植物从门静脉回流对胰腺移植物功能存活的延长具有协同效应，移植时处理延长胰腺移植物效果不如移植前预处理，但可能具有一定的实用意义。     

Operational tolerance induced by pretreatment with donor dendritic cells under blockade of CD40 pathway.

BACKGROUND: Dendritic cells can mount immune response as competent antigen presenting cells. Recently, it has been reported that immature dendritic cells induce prolongation of allograft survival. However, the ability of mature dendritic cells to induce operational tolerance is unclear. Therefore, in this study, we examined the ability of splenic mature dendritic cells to induce operational tolerance to fully allogeneic antigens using mouse heterotopic heart transplantation model. METHODS: CBA (H2k) mice received i.v. injections with donor splenic dendritic cells or B cells in the absence or presence of monoclonal antibody (mAb) specific for CD40 ligand or CD80/CD86 2 weeks before transplantation of a C57BL/10 (H2b) heart. RESULTS: When donor dendritic cells were injected i.v. 2 weeks before transplantation, rejection response was accelerated compared with that of naive mice [median survival time (MST) = 7 and 8 days, respectively]. However, when CD40 pathway was blocked by anti-CD40 ligand mAb, i.v. injection of donor dendritic cells but not B cells induced indefinite graft survival (MST >100 and 20 days, respectively). Mice treated with anti-CD40 ligand mAb alone rejected their grafts with a MST of 18 days. Intravenous injection of donor dendritic cells and B cells in combination with anti-CD80/CD86 mAbs was less effective to induce graft prolongation (MST = 9.5 and 13 days, respectively). CONCLUSIONS: Therefore, under blockade of CD40 pathway, mature dendritic cells were tolerogens in vivo independent of CD80/86 pathways.     

Long-term survival of hamster islet xenografts in mice under short-course treatment with nondepleting versus depleting anti-CD4 monoclonal antibodies

ABSTRACT: Xenogeneic grafts provide a potential alternative to the current shortage of human organs for transplantation. However, the prevention of rejection and tolerance induction of xenografts still remain to be further explored. Islet xenografts appear more promising than vascularized whole organ xenografts and additionally also more resistant to the recurrence of autoimmune disease than allografts. Recently, the nondepleting monoclonal antibody (mAb), which blocks the CD4 molecule on lymphocytes, was reported to be able to induce tolerance in allotransplantation and CD4 positive cells were further confirmed to be a major factor responsible for cellular xenograft rejection. Therefore, we hypothesize that anti-CD4 nondepleting mAb could also be effective in protecting cellular xenografts and inducing unresponsiveness of recipients. We studied the effect of the nondepleting anti-CD4 mAb YTS177.9 on islet xenograft survival by using the hamster-to-mouse islet transplantation model. Results were compared with that of the depleting anti-CD4 mAb GK1.5 that was shown to have similar binding sites on the CD4 molecule to mAb YTS 177.9. Our data show that mAb YTS 177.9 did effectively prolong the survival of islet xenografts and, in addition, also successfully did induce long-term acceptance of 40% grafts after only three penoperative injections of 0.5 mg mAb per mouse. The average survival of the graft was markedly prolonged to >66.8±37.1 days compared with controls (8.3±1.4 days) or with the depleting anti-CD4 mAb GK1.5 (25.7±5.5 days). However, the latter displayed a more profound inhibition in in vitro and ex vivo mixed lymphocyte xenoreaction than mAb YTS 177.9. Moreover, the activity of this nondepleting mAb was found to be dose-dependent and 80% of grafts survived permanently when the dose was increased to six injections of 0.5 mg mAb. Like mAb GK1.5, mAb YTS 177.9 also prevented rejection when given after a delay of two days posttransplant. In addition, we found that neither depleting nor nondepleting anti-CD8 mAb was effective in this model. Our results strongly suggest that an anti-CD4 nondepleting or blocking mAb alone is able to induce long-term acceptance of islet xenografts and that blocking the CD4 molecule is significantly superior to depleting CD4 positive cells for the protection of islet xenografts. This may indicate that CD4 cells play a major role in xenograft tolerance induction.     

Infusion of Lin- bone marrow cells results in multilineage macrochimerism and skin allograft tolerance in minimally conditioned recipient mice

Donor-specific immunological tolerance using high doses of donor bone marrow cells (BMC) has been demonstrated in mixed chimerism-based tolerance induction protocols; however, the development of graft versus host disease (GVHD) remains a risk. In the present study, we demonstrate that the infusion of low numbers of donor Lin(-) bone marrow cells (Lin(-) BMC) 7 days post allograft transplantation facilitates high level macrochimerism induction and graft tolerance. Full-thickness BALB/c skin allografts were transplanted onto C57BL/6 mice. Mice were treated with anti-CD4 and anti-CD8 mAbs on day 0, +2, +5, +7 and +14 along with low dose busulfan on day +5. A low dose of highly purified Lin(-) BMC from BALB/c donor mice was infused on day +7. Chimerism and clonal cell deletion were evaluated using flow cytometry. Donor-specific tolerance was tested by donor and third-party skin grafting and mixed leukocyte reaction (MLR). Lin(-) BMC infusion with minimal immunosuppression led to stable, mixed, multilineage macrochimerism and long-term allograft survival (>300 days). Mixed donor-recipient macrochimerism was observed. Donor-reactive T cells were clonally deleted and a 130% increase in CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) was observed in the spleen. Tolerant mice subsequently accepted second donor, but not third-party (C3H), skin grafts and recipient splenocytes failed to react with allogeneic donor cells indicating donor-specific immunological tolerance was achieved. We conclude that the infusion of donor Lin(-) BMC without cytoreductive recipient conditioning can induce indefinite survival of skin allografts via mechanisms involving the establishment of a multilineage macrochimeric state principally through clonal deletion of alloreactive T cells and peripherally induced CD4(+)Foxp3(+) Tregs.     

Robust tolerance to fully allogeneic islet transplants achieved by chimerism with minimal conditioning

BACKGROUND: Whether mixed chimeras induced by nonmyeloablative conditioning are tolerant to challenge with donor allogeneic islet grafts is unknown. Here we investigate whether our nonmyeloablative, costimulation blockade-free and sirolimus (SRL)-based protocol could facilitate mixed chimerism via bone marrow transplantation (BMT) and induce islet allograft tolerance. METHODS: After low dose (1-3 Gy) total body irradiation (TBI, day -1), with or without prior lymphocyte depletion, C57BL/6 mice were transfused with 40 x 10(6) BALB/c bone marrow cells (day 0) and received SRL (3 mg/kg/day) for 4 weeks. Chimerism was monitored by flow cytometry and the recipients were rendered diabetic chemically and challenged with donor islets. RESULTS: Mixed chimerism was achieved in mice treated with TBI 3 Gy/SRL but it declined over time in 60% (9/15) of them. Long-term stable chimerism was established in 100% of recipients over 50 weeks with either antilymphocyte serum (ALS, 9/9), anti-CD4 (4/4), or anti-CD4 plus anti-CD8 (5/5) prior to BMT. TBI conditioning could be reduced to 1 Gy, with 90% (9/10) maintaining chimerism in the long-term. When TBI was substituted with cyclophosphamide (CTX) or busulfan (BUS), all mice remained chimeric in the long-term. The chimeras showed no proliferative response to donor antigen and accepted both first and second donor-specific islet grafts indefinitely while rejecting third-party grafts. CONCLUSIONS: This data provides the first evidence that stable fully allogeneic chimeras induced with BMT after nonmyeloablative conditioning with SRL and lymphocyte-depleting antibodies exhibit robust donor-specific tolerance to islet grafts.     

Importance of thymus to maintain operational tolerance to fully allogeneic cardiac grafts

BACKGROUND: The effectiveness of donor-specific blood transfusion (DST) before transplantation has been established. Anti-CD4 monoclonal antibody (anti-CD4) augments the ability of DST to induce indefinite prolongation. Therefore, we investigated the importance of thymus to maintain the unresponsiveness to alloantigens. METHODS: CBA mice were pretreated with 0.25 mL of DST or two doses of anti-CD4 before transplantation of a C57BL/10 heart. Some mice were thymectomized. RESULTS: Naive CBA mice rejected C57BL/10 grafts acutely with a median survival time of 7 days. When mice were pretreated with anti-CD4 plus DST 4 weeks before transplantation, all grafts survived indefinitely (>100 days), whereas mice treated with DST alone or anti-CD4 alone rejected acutely (median survival time, 7 and 12 days, respectively). To investigate the importance of thymus, mice pretreated with anti-CD4 plus DST 4 weeks before transplantation were thymectomized or underwent a sham operation 1 day before grafting. Mice with the sham operation accepted grafts indefinitely, whereas thymectomized mice rejected the majority of the grafts (median survival time, 20 days). CONCLUSIONS: The thymus is important in maintaining the operational tolerance induced by anti-CD4 plus DST 4 weeks before grafting.     

Combined donor specific transfusion and anti-CD154 therapy achieves airway allograft tolerance

BACKGROUND: The state of tolerance allows long term graft survival without immunosuppressants. Lung transplantation tolerance has not been consistently achieved in either small or large animal models. METHODS: The mechanisms and effectiveness of a tolerance induction protocol consisting of donor specific transfusion (DST; day 0) and a short course of co-stimulatory blockade (anti-CD154 antibody; days -7, -4, 0 and +4) were studied in the mouse heterotopic tracheal transplant model of chronic lung rejection. C57BL/6 mice received BALB/c tracheal grafts (day 0) and were treated with DST alone, anti-CD154 alone, the combination (DST/anti-CD154), or no treatment. No non-specific immunosuppressants were used. RESULTS: DST/anti-CD154 in combination, but neither treatment alone, markedly prolonged the lumen patency and survival (>100 days) of fully histo-incompatible allografts (p<0.05 versus control allografts at every time point studied up to 16 weeks) without immunosuppression. This protocol was donor antigen specific as third party grafts (C3H) were promptly rejected. In addition, DST/anti-CD154 did not result in mixed chimerism but induced transplantation tolerance via a peripheral mechanism(s), which included significantly reduced cytotoxic T cell activity (p<0.001) and a significantly increased percentage of CD4+CD25+ cells (p = 0.03). CONCLUSIONS: The DST/anti-CD154 protocol successfully induced and maintained long term, donor specific tolerance in the mouse heterotopic airway graft model of chronic lung rejection. This finding may lead us closer to successful tolerance induction in lung transplantation.     

Anti-CD8 abrogates effect of anti-CD4-mediated islet allograft survival in rat model.

We studied the effects of anti-CD4 treatment of diabetic ACI rats on the induction of tolerance to allogeneic (Lewis) islet allografts. When given as a 4-day treatment regimen, OX38, a mouse anti-rat CD4 antibody, caused depletion of greater than 80% of CD4+ cells from the peripheral blood of treated rats. After induction of diabetes (a single high-dose bolus of streptozocin) and 3 days after the initiation of anti-CD4 immunotherapy, recipient ACI rats were transplanted with fully allogeneic (Lewis) islets of Langerhans via the portal circulation. These transplanted islets were capable of returning the anti-CD4-treated ACI recipients to normoglycemia, which was maintained indefinitely in the absence of further immunosuppression. In contrast, treatment of recipient rats with OX8, an anti-CD8 monoclonal antibody (MoAb), induced only a slight prolongation of graft survival (less than or equal to 30 days). Further characterization of the cellular requirements for the induction of long-term transplantation survival revealed that successful pretransplantation anti-CD4 therapy could be ablated by the coincident treatment of recipient rats with depleting levels of anti-CD8 MoAb. These data point to the necessity of a regulator CD8+ cell in the induction of anti-CD4-mediated transplantation survival in this rat model of islet transplantation.     

Regulation of skin and islet allograft survival in mice treated with costimulation blockade is mediated by different CD4+ cell subsets and different mechanisms

BACKGROUND: Donor-specific transfusion (DST) and a brief course of anti-CD154 monoclonal antibody (mAb) induces permanent islet and prolonged skin allograft survival in mice. Induction of skin allograft survival requires the presence of CD4 cells and deletion of alloreactive CD8 cells. The specific roles of CD4 and CD4CD25 cells and the mechanism(s) by which they act are not fully understood. METHODS: We used skin and islet allografts, a CD8 T cell receptor (TCR) transgenic model system, and in vivo depleting antibodies to analyze the role of CD4 cell subsets in regulating allograft survival in mice treated with DST and anti-CD154 mAb. RESULTS: Deletion of CD4 or CD25 cells during costimulation blockade induced rapid rejection of skin but only minimally shortened islet allograft survival. Deletion of CD4 or CD25 cells had no effect upon survival of healed-in islet allografts, and CD25 cell deletion had no effect upon healed-in skin allograft survival. In the TCR transgenic model, DST plus anti-CD154 mAb treatment deleted alloreactive CD8 T cells, and anti-CD4 mAb treatment prevented that deletion. In contrast, injection of anti-CD25 mAb did not prevent alloreactive CD8 T cell deletion. CONCLUSIONS: These data document that (1) both CD4CD25 and CD4CD25 cells are required for induction of skin allograft survival, (2) CD4CD25 T cells are not required for alloreactive CD8 T cell deletion, and (3) CD4CD25 regulatory cells are not critical for islet allograft tolerance. It appears that skin and islet transplantation tolerance are mediated by different CD4 cell subsets and different mechanisms.