采用经姜黄素处理的未成熟树突状细胞延长大鼠移植肾存活时间

目的 探讨姜黄素(Cur)处理的树突状细胞(DC)诱导同种T淋巴细胞低反应性的效果以及对大鼠移植肾存活时间的影响.方法 体外培养Wistar大鼠骨髓来源的DC,经Cur处理后,以流式细胞仪检测细胞CD11c、CD80、CD86及主要组织相容性复合物(MHC)Ⅱ类抗原的变化,酶联免疫吸附试验测定DC分泌白细胞介素12(IL-12)的水平,混合淋巴细胞反应(MLR)检测其刺激Lewis大鼠T淋巴细胞增殖的能力,二次MLR测定其诱导的T淋巴细胞抗原特异性低反应性.以Wistar大鼠为供者,Lewis大鼠为受者,进行肾移植.术前第7天,经尾静脉给受者输注用Cur处理的供者DC,分设不处理对照组和未成熟DC对照组(经尾静脉注射供者的未成熟DC),术后观察移植肾存活时间及组织学改变情况,第14天检测受者T淋巴细胞对供者成熟DC的反应性.结果 Cur能明显抑制DC共刺激分子CD11c、CD80、CD86及MHCⅡ类抗原的表达以及IL-12的分泌(P＜0.05).同种T淋巴细胞对经Cur处理过的DC刺激的增殖能力明显减低,且这种低反应性具有抗原特异性.对照组和未成熟DC对照组移植肾的存活时间分别为(8.6±2.1)d和(22.4±7.4)d,实验组为(31.5±6.9)d,实验组移植肾存活时间明显长于对照组和未成熟DC对照组(P＜0.05),且其移植肾组织的损伤程度最轻.实验组受者的T淋巴细胞对供者成熟DC刺激的反应性明显低于对照组(P＜0.05),而对第三方无关抗原的刺激保持较高增殖强度.结论 Cur能抑制DC成熟功能,诱导供者特异性的T淋巴细胞低反应性,移植前输注经Cur处理的未成熟DC能显著延长大鼠移植肾的存活时间.     

霉酚酸酯预处理供者未成熟树突状细胞回输受者延长移植物的存活

目的观察霉酚酸酯(MMF)处理的供者来源的树突状细胞(DC)回输受者延长移植物存活的作用。方法在DC前体的体外培养过程中加入MMF处理,利用同种小鼠异位心脏移植模型,设单纯移植组、供者未成熟DC回输受者组,以及MMF处理的供者未成熟DC回输受者组,观察MMF处理后DC的抗原提呈能力的变化、移植心脏存活时间并做微嵌合和病理学分析。结果MMF处理后DC的抗原提呈能力明显下降,单纯移植组移植心脏的存活期仅为8d,未成熟DC回输受者组心脏存活期为21d,而MMF处理的未成熟DC组移植物存活时间延长为30d,差异有统计学意义(P<0.01);MMF处理的供者未成熟DC在受者体内的嵌合期可达28d以上,且病理分析显示可以明显抑制炎症的产生。结论MMF处理的DC回输受者能够诱导针对移植供者的特异性免疫耐受,进而延长移植物的存活。     

未成熟树突状细胞诱导协调性异种胰岛移植耐受的研究

目的　研究未成熟树突状细胞 (DC)在协调性异种胰岛移植中的免疫耐受诱导作用。方法　分别应用 2 0 μg/L粒细胞集落刺激因子 (GM CSF)和 5 0 0 μg/LGM CSF 2 0 0 μg/L白细胞介素4 (IL 4) ,从BALB/c受鼠骨髓培养未成熟DC及成熟DC ,然后负载Wistar大鼠的主要组织相容性复合物 (MHC)抗原。将两种DC回输至糖尿病小鼠体内 ,7d后分别将Wistar大鼠和SD大鼠胰岛移植于糖尿病小鼠左肾包膜下 ,监测移植物存活情况 ,检测T细胞增殖反应 ,并进行病理切片检查。结果　负载MHC抗原成熟DC预处理的BALB/c小鼠体内胰岛迅速被排斥 ,负载MHC抗原未成熟DC预处理的BALB/c小鼠胰岛存活时间明显延长 (P <0 .0 1 ) ,但以SD大鼠作为供者的胰岛移植物迅速被排斥。耐受鼠的脾细胞对Wistar大鼠脾细胞的增殖反应微弱 ,而排斥鼠脾细胞则出现明显增殖反应。结论　未成熟DC预处理受者可诱导T细胞无能 ,并有效延长异种胰岛移植后的存活时间。     

西罗莫司与未成熟树突状细胞延长小鼠皮肤移植存活时间的协同作用

目的 研究西罗莫司（SRL）对小鼠骨髓源树突状细胞（DC）分化及成熟的影响，观察西罗莫司与未成熟树突状细胞在延长小鼠皮肤移植存活时间中的协同作用。方法 （1）在诱导C57BL／6小鼠骨髓细胞定向分化为DC时加入SRL，通过流式细胞仪检测CD11c、CD86及MHCⅡ类分子表达情况，经脂多糖（LPS）刺激后，再检测各分子表达的变化。（2）通过单向混合淋巴细胞反应（MLR）观察经SRL处理的DC刺激同种异基因小鼠T细胞增殖情况。（3）以C57BL／6小鼠为供者，BALB／c小鼠为受者建立皮肤移植模型。观察皮肤移植前7d经尾静脉注射供者未成熟DC及经胃管连续灌注SRL7d的受者移植皮片存活情况及组织学变化。结果 （1）经SRL处理的DC表面CD11c表达仅有轻度降低，但CD86和MHCⅡ类分子表达明显减少。（2）MLR显示经SRL处理的DC刺激同种异基因小鼠T细胞增殖的能力降低。（3）受者皮肤移植术前联合应用供者未成熟DC和SRL，可减轻移植皮片炎症反应并延长其存活时间。结论 SRL对DC分化的影响不明显，但可抑制DC发育成熟。受者术前应用SRL和未成熟DC可延长皮肤移植的存活时间。     

输注转染MyD88siRNA基因的供者树突状细胞延长小鼠移植心存活时间

目的 探讨输注供者来源的转染了髓样分化因子88(MyD88)siRNA基因的树突状细胞(DC)在延长同种小鼠移植心存活时间中的作用及机制.方法 以脂质体为载体,将化学合成的MyD88siRNA导入BALB/c小鼠(供者)骨髓来源的DC中,制备转染MyD88siRNA基因的DC(MyD88siRNA-DC).随机将27只C57BL/6小鼠(受者)平均分为磷酸盐缓冲液(PBS)对照组、培养8 d的DC(Day8-DC)组及MyD88siRNA-DC组,分别将PBS、Day8-DC及MyD88siRNA-DC输注至受者体内.于输注后第7、14和21天时应用免疫双荧光染色法观察供者DC在受者脾脏内的存活情况;混合淋巴细胞反应(MLR)测定受者脾脏内T淋巴细胞对供者同种抗原的反应性.另取27对供、受者(BALB/c小鼠和C57BL/6小鼠),通过袖套管技术建立颈部异位心脏移植模型,随机平均分为PBS对照移植组、Day8-DC移植组及MyD88siRNA-DC移植组,各组于移植前7 d分别经受者门静脉注射0.5 ml PBS、2.0× 106个Day8-DC及2.0× 106个MyD88siRNA-DC.于输注后第7天,观察各组移植心的存活时间;病理检查观察排斥反应程度;酶联免疫吸附试验测定受者血清巾Th1及Th2型细胞因子[γ干扰素(INF-γ)、白细胞介素(IL)-12、IL-4和IL-10]水平的变化.结果 MyD88siRNA-DC在受者脾脏内的存活时间明显延长,MyD88siRNA-DC组受者脾脏内T淋巴细胞对供者抗原的反应性最低(P＜0.01).PBS对照移植组、Day8-DC移植组及MyD88siRNA-DC移植组移植心的存活时间分别为:(6.67±1.37)d、(13.67±2.25)d和(24.50±4.42)d,与PBS对照移植组相比,Day8-DC移植组移植心存活时间延长(P＜0.01),而MyD88siRNA-DC移植组移植心存活时间较Dby8-DC移植组进一步延长(P＜0.01);MyD88siRNA-DC移植组移植心排斥反应病理分级最低,受者血清中INF-γ和IL-12水平显著降低(P＜0.01),而IL-4和IL-10水平明显升高(P＜0.01).结论 输注转染MyD88siRNA基因的供者DC能够延长同种小鼠移植心的存活时间;其机制可能与诱导受者Th1/Th2免疫偏移及形成供、受者微嵌合状态有关.     

输注负载胎盘滋养层细胞抗原的受者aaDC延长小鼠移植心的存活时间

目的 探讨输注负载供者胎盘滋养层细胞抗原的受者耐受性选择性激活树突状细胞(aaDC)对小鼠移植心存活时间的影响.方法 雌性Balb/c小鼠和雄性C57BL/6小鼠合笼后,获取雌鼠胎盘滋养层细胞,并提取其抗原.体外制备Balb/c小鼠的aaDC,分别与滋养层细胞抗原或者C57BL/6小鼠脾细胞抗原共培养,获得负载滋养层细胞抗原或者C57BL/6小鼠脾细胞抗原的aaDC,测定培养液中白细胞介素10(IL-10)和IL-12的含量以及aaDC表面CD40、CD80、CD86和主要组织相容性复合物(MHC)Ⅱ类分子的表达.取Balb/c小鼠,分别接受负载滋养层细胞抗原或者C57BL/6小鼠脾细胞抗原的aaDC输注(滋养层细胞抗原组和脾细胞抗原组),7 d后接受C57BL/6小鼠心脏异位移植,以注射生理盐水者为对照组,输注负载滋养层细胞抗原aaDC后接受昆明小鼠心脏移植者为第三供者对照组.术后记录移植心存活时间,并检测受者脾脏和移植心组织中IL-2、IL-10和γ干扰素(IFN-7)mRNA的表达.结果 负载脾细胞抗原的aaDC,CD86和MHCⅡ类分子的表达明显升高,而负载滋养层细胞抗原的aaDC,仅MHCⅡ类分子表达升高.滋养层细胞抗原aalDC的培养液中,IL-10为(122.6±15.4)ng/L,IL-12为(232.8±25.4)ng/L,空白对照aaDC的IL-10和IL-12分别为(35.4±8.5)ng/L和(267.3±12.5)ng/L,二者间IL-10的差异有统计学意义(P<0.05),而IL-12的差异无统计学意义(P>0.05).脾细胞抗原aaDC培养液中的IL-10和IL-12与空白对照aaDC相近,差异均无统计学意义(P>0.05).对照组移植心存活时间为(6.73±0.58)d,脾细胞抗原组为(12.28±0.76)d,滋养层细胞抗原组为(38.83±2.79)d,第三供者对照组为(6.68±0.62)d.滋养层细胞抗原组移植心存活时间明显长于脾细胞抗原组和对照组(P<0.01),而第三供者对照组和对照组间移植心存活时间的差异无统计学意义(P>0.05).结论 预先输注负载供者胎盘滋养层细胞抗原的受者aaDC,可延长小鼠移植心的存活时间.     

未成熟树突状细胞在异种移植中的免疫递呈及调节作用

目的　观察树突状细胞 (DC)对异种抗原的免疫递呈作用 ,并探讨未成熟DC对T细胞的免疫调节作用。方法　从BALB/c小鼠骨髓诱生未成熟DC及成熟DC。将DC与小鼠T细胞进行混合淋巴细胞培养 (MLC)作为异种移植体外模型 ,以胰岛细胞作为靶细胞检测细胞毒效应。结果　MLC结果表明保留DC组及体内、外致敏成熟DC组刺激指数 (SI =2 .0 9,3 .91,2 .81)与去除DC组 (SI=1.0 9)相比差异有显著性。体内、外致敏未成熟DC组 (SI =1.0 8,1.16)与体内、外对照组(SI =2 .98,2 .81)相比差异有显著性。细胞毒检测表明成熟DC诱导T细胞最大杀伤活性为 61% ,而未成熟DC诱导T细胞最大杀伤活性为 9%。结论　DC主要以间接方式递呈异种抗原 ,而未成熟型DC可诱导异种抗原特异性T淋巴细胞无能。     

转染IKK2dn的受者树突状细胞回输延长同种异体肾移植大鼠的存活时间

目的 探讨IKK2dn基因转染并负载供者抗原的受者未成熟树突状细胞(imDC)延长同种异体肾移植大鼠的存活时间及其机制.方法 获取和培养Lewis大鼠骨髓源性DC,转染IKK2dn并负载BN大鼠可溶性抗原进行体外实验,检测CD86和主要组织相容性复合物(MHC)Ⅱ的表达及DC刺激T淋巴细胞增殖的能力.肾移植受者为Lewis大鼠,用随即数字表法分DC组、空转染组、转染组、对照组,术前7d分别输注1×10~7个D、Adv-0-DC、负载BN抗原的Adv-IKK2dn-DC和等量生理盐水,供者均为BN大鼠.另设第三方供者组,术前处理同转染组,供者为Wistar大鼠.移植后检测各组受者T淋巴细胞的增殖能力及血清白细胞介素2(IL-2)和γ干扰素(IFN-γ)的表达水平,观察各组大鼠的存活时间和发生排斥反应情况.结果 DC的体外实验结果显示:与转染IKK2dn前相比,转染后DC仍能低水平表达CD86和MHC Ⅱ,负载供者抗原后CD86和MHCⅡ表达均增加,而转染IKK2dn后再负载供者抗原,CD86和MHC Ⅱ的表达未发生明显变化;DC负载供者抗原后,刺激T淋巴细胞增殖的能力明显增强(P<0.05),而转染IKK2dn并负载供者抗原后不能有效刺激T淋巴细胞增殖.肾移植术后的检测结果显示:转染组T淋巴细胞的增殖能力明显低于其他4组(P<0.05或P相似文献   

核因子κB诱骗剂处理的树突状细胞延长小鼠移植心脏存活时间

目的探讨核因子κB（NF-κB）诱骗剂处理供者树突状细胞（DC）对同种异体小鼠移植心脏存活时间的影响。方法在体外以NF-κB诱骗剂处理供者骨髓来源的DC，并于心脏移植术前7d经门静脉输注2×10^6个DC给受者，术后1～7d受者接受亚治疗量的环孢素A（10mg·kg^-1·d^-1）腹腔注射（联合方案组），并设不作任何处理（对照组）、单用环孢素A（CsA组）、单纯输注未经处理DC（DC对照组）和单纯输注经处理DC（DC实验组）的对照组，另设接受来自第三方供者的对照组（第三供者组，受者的处理同联合方案组），所有受者均接受腹腔心脏移植。观察各组移植心脏的存活时间；采用酶联免疫吸附法检测术后第7天受者血清白细胞介素2（IL-2）、IL-4、IL-10和γ干扰素（IFN-γ）的含量。结果移植心脏平均存活时间（MST），对照组为7d，CsA组为10．3d，DC对照组为7．6d，DC实验组为21．4d，联合方案组为53．6d，第三供者组为9d，DC实验组移植心脏MST明显长于对照组和DC对照组，差异有统计学意义（P〈0．01）；联合方案组移植心脏MST明显长于CsA组、DC实验组及第三供者组，差异有统计学意义（P〈0．01）。联合方案组IL-2和IFN-γ的含量最低，而IL-4和IL-10的含量最高，与其它各组比较，差异均有统计学意义（P〈0．05）；与对照组、DC对照组及CsA组相比，DC实验组IL-2及IFN-γ的含量也显著降低（P〈0．05），而IL-4和IL-10则升高（P〈0．05）。结论术前输注经NF-κB诱骗剂处理的供者DC可延长同种小鼠移植心脏的存活时间，若术后加用短程亚治疗量CsA，则移植心脏的存活时间得到进一步延长。     

第三方骨髓间充质干细胞对同种异体皮肤移植的影响

目的 探讨第三方骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)对同种异体皮肤移植的影响.方法 40只雌性C57BL/6和50只雄性BALB/C小鼠分别作为皮肤移植的供体和受体.将50只BALB/C小鼠随机分为5组,每组10只:空白对照组,直接进行皮肤移植;给予受体环磷酰胺组(Cyelophosphamide,CP组);给予受体SD大鼠BMSCs移植组(SD-BMSCs组);给予受体CP+SD-BMSCs移植组(CP+SD-BMSCs组);CM-DiI荧光标记组.CP+SD-BMSCs组给予大剂量CP腹腔注射,200 mg/kg,2 d,移植当天自受体小鼠尾静脉注射1×10~6个SD-BMSCs.检测项目包括:观察皮片存活时间、单向混合淋巴反应、组织HE染色、组织荧光镜检,流式细胞仪检测受体外周血单核细胞中SD-BMSCs的含量等.结果 CP+SD-BMSCs组皮肤移植物存活时间(14.5±1.9)d,空白对照组为(8.O±0.8)d,CP组为(11.1±1.4)d,SD-BMSCs组为(10.9±1.4)d,CP+SD-BMSCs组皮肤移植物存活时间明显比后3组延长(P<0.05).混合淋巴反应结果显示,BMSCs对淋巴细胞的增殖抑制作用存在量效关系.HE染色发现CP+SD-BMSCs组不但有血管生成,淋巴细胞浸润也较少.CM-DiI标记的SD-BMSCs能够在BALB/c小鼠体内存活30 d以上,SD-BMSCs组与CP+SD-BMSCs组外周血流式细胞仪均检测到SD-BMSCs.结论 BMSCs能够在异种受体体内存活较长时间;第三方BMSCs能够延长同种异体移植物的存活时间;BMSCs能够抑制异种T淋巴细胞的活化增殖.     

Antilymphocytic antibodies and marrow transplantation. V. Suppression of secondary disease by host-versus-theta-graft reaction.

An approach to block secondary disease was investigated in mice sensitized against the Th-1.1 (theta-AKR) alloantigen on the marrow donor's T cells. To avoid a concomitant sensitization against the donor's histocompatibility antigens, prospective marrow recipients were sensitized against thymocytes of a third-party strain sharing the donor's Th-1 alloantigen but not his histocompatibility antigens. Advantage was taken of the fact that rats carry a Th-1.1-like theta-antigen which induces anti-Th-1.1 antibodies in Th-1.2 mice. CBA/J and (C57BL/6 x CBA)F1 Th-1.2 mice were sensitized against rat thymocytes and tranfused with spleen and bone marrow of AKR/J Th-1.1 after irradiation with 800 to 900 R. Although unsensitized recipients died within 3 weeks of acute secondary disease, sensitized mice survived the observation period of 50 days as chimaeras. Sensitized recipients were killed by the transplantation of spleen cells from congenic AKR/Cum carrying the Th-1.2 antigen. The host-versus-theta-graft approach suppressed secondary disease following H-2-compatible and -incompatible marrow grafts. Its hemopoietic and Tcell chimaeras tolerated skin grafts of the donor strain while rejecting third-party skin grafts.     

Application of recipient-derived dendritic cells to induce donor-specific T-cell hyporesponsiveness

Administration of donor-derived immature dendritic cells (DC) treated with NF-kappaB oligodeoxyribonucleotides (ODN) prevents allograft rejection. We attempted to explore the use of recipient-derived DC pulsed with donor antigens, in which the donor antigens were presented to host T cells via an indirect pathway (cross-priming). Expression of CD40, CD80, and CD86 on DC was significantly inhibited by treatment with NF-kappaB ODN, whereas MHC class I and II were minimally affected. Normal C3H DC pulsed with B10 antigens stimulated proliferative responses and donor-specific CTL activity in C3H T cells, both of which were, however, markedly inhibited when DC were treated with NF-kappaB ODN. This manipulation was associated with reduced IFN-gamma and increased IL-10 production in the supernate, suggesting a Th2 bias. More frequent apoptotic T cells were observed in cultures with NF-kappaB ODN DC. In contrast to administration of normal DC pulsed with donor antigens that accelerated rejection of B10 cardiac allografts (median survival time [MST] 7 days versus 10 days in no-DC treatment control, P < .05), a single injection of 2 x 10(6) NF-kappaB ODN DC significantly prolonged allograft survival (MST 50 days, P < .05 compared with no-DC treatment control). The anti-donor CTL activity in infiltrating T cells isolated from cardiac grafts in recipients that received NF-kappaB ODN DC was significantly suppressed. These data indicate that vaccination with immature DC, propagated from recipient BM is an attractive approach to induce T-cell hyporesponsiveness.     

Participation of the liver in generation of a vigorous anti-donor response after inoculation of donor spleen cells.

BACKGROUND: There is a general agreement that a preferential accumulation of alloantigens within the liver could induce hyporesponsiveness to the inoculated antigens. Entrapment of antigens in the liver may evoke an unique immune response in the organ and play a key role in determination of the fate of the transplanted grafts. To understand the immune response in the liver after inoculation of allogeneic donor antigens, we examined the immune response to systemically inoculated alloantigen in rats whose sensitized liver was replaced with that of naive rats or in naive rats whose liver was replaced with that of sensitized rats. METHODS: Using implantation of syngeneic liver (alloantigen-accumulated/naive) in rats (naive/alloantigen-sensitized), we compared the immune responses to alloantigen between rats with hepatic/extrahepatic alloantigen at 24 hr after alloantigen inoculation. This was called sensitized-liver-grafted (SLG)/sensitized-liver-removed (SLR) treatment. The immune response to donor alloantigen in this model was evaluated by survival of skin or heart grafts, complement-dependent cytotoxicity (CDC) titer and delayed-type hypersensitivity (DTH) response. RESULTS: Compared with the mean survival time (MST) in donor spleen cell inoculated (DSI) rats (skin and heart, MST: 8.2+/-1.1 and 10.7+/-2.3 days), SLG rats rejected allografts in an accelerated fashion (skin and heart, MST: 5.5+/-0.5 and 4.2+/-0.8 days), associated with higher CDC titer and DTH response. In contrast, allograft survival was moderately prolonged in SLR (skin and heart, MST: 16.5+/-2.6 and 29.5+/-3.7 days) associated with suppressed CDC titer and DTH response. The survival of third-party allograft after SLG or SLR treatment (skin, MST: 9.3+/-1.5 or 9.7+/-0.6 days) indicated that immunological hyper/hyporesponsiveness was donor-specific. CONCLUSIONS: A strong anti-donor immune response was induced by the transfer of donor antigen-baring liver to naive rats 24 hr after alloantigen inoculation, whereas removal of the liver suppressed alloimmune response. Our results indicate that vigorous anti-alloimmune response occurred in the liver after systemic inoculation of donor spleen cells.     

Prevention of murine acute graft-versus-host disease by recipient-derived TGFbeta1-treated dendritic cells

Acute graft-versus-host disease (GVHD) remains the major barrier to allogeneic bone marrow transplantation (allo-BMT). Evidence has accumulated that transforming growth factor beta1-treated dendritic cells (TGFbeta-DC), deficient in surface costimulatory molecules, inhibit alloantigen-specific T-cell responses and induce graft hyporeactivity. To analyze the effect of TGFbeta-DC on GVHD after allo-BMT, 5.0 x 10(6) recipient-derived TGFbeta-DC were injected into C57BL/6 (H-2b) with bone marrow-splenocyte grafts from major histocompatibility complex (MHC) disparate BALB/c mice (H-2d). Survival analysis showed TGFbeta-DC cotransplantation resulted in significant prolongation of allograft survival, namely a mean survival time (MST) of 44.3 +/- 4.5 days, versus the untreated MST of 9.5 +/- 0.6 days (P < .01). However, mature DC aggravated the GVHD with an MST of 6.6 +/- 0.6 days (P < .01). In addition, the third-party C3H-derived TGFbeta-DC did not enhance the survival rate (MST = 9.7 +/- 0.5 days). Furthermore, serum IFN-gamma, IL-12, and IL-18 levels in TGFbeta-DC cotransplanted mice were reduced compared with untreated BMT hosts, while serum IL-10 levels were not changed. These results suggest that TGFbeta-DC cotransplantation may attenuate the severity of GVHD after BMT.     

Microchimerism does not induce tolerance and sustains immunity after in utero transplantation

BACKGROUND: To date, over 40 in utero transplants have been performed in humans; the only successes were documented in the treatment of severe combined immunodeficiency syndromes. Hemoglobinopathies and metabolic disorders are candidate diseases for this approach; however, when applied clinically, the results have been discouraging. To address the role of the fetal immune system in the outcome of in utero transplantation, we have developed a murine model of in utero transplantation in immunologically intact murine recipients and have studied chimerism and tolerance/immunity to allogeneic donor cells through the lives of the animals. METHODS: We have performed experiments in which purified murine sca-1+/lin- cells and c-kit+/lin- cells of C57BL/6 (H2b) mice were injected into Balb/c (H2d) fetal recipients at early gestational ages. Chimerism was tested by highly sensitive semiquantitative polymerase chain reaction assay and tolerance/immunity to donor cells was studied by in vivo (skin grafts, responses to postnatal boosts) and in vitro (mixed lymphocyte culture, cytotoxicity, and cytokine release) assays. RESULTS: One hundred percent (10/10) of mice transplanted with c-kit+ cells and 44% (4/9) of mice transplanted with sca+ cells showed circulating donor cells within the first 6 months of life (P=0.031). Mice in the sca+ group rejected donor skin grafts at a mean time of 9.1+/-0.2 days, whereas mice in the c-kit+ group rejected donor skin grafts at a mean time of 15.1+/-0.7 days (P=0.001). The difference between the transplanted groups and non-transplanted controls was also significant (P<0.05). All mice transplanted with sca+/lin- cells showed greater response to donor cells than to third-party cells at all effector to target ratios (P=0.002). Differences in response to donor alloantigen between sca+ and c-kit+ groups were significant (P=0.003). Cytokine quantification demonstrated higher TH1 than TH2 cytokine release in all groups, and the response to donor cells was higher in the sca+ compared with c-kit+ mice (P=0.031). CONCLUSION: These results demonstrate a low level of chimerism and tolerance in mice transplanted in utero with sca+/lin- and c-kit+/lin- cells. The possibility of active in utero immunization to donor cells is supported by accelerated skin graft rejection in mice transplanted with sca+ cells and enhanced in vitro immune responses in mice with persistent microchimerism.     

Induction of donor-specific unresponsiveness to rat cardiac allograft by donor leukocytes and cyclosporine

Many recent reports have emphasized the importance of donor antigens in the induction of allograft tolerance. This study examines the effect of pretransplant infusion of 10(8) donor leukocytes (DL) combined with peritransplant cyclosporine (CsA) on W/F cardiac allograft survival in Lewis rats. Peritransplant recipient treatment consisted of CsA 20 mg/kg given i.m. on days 0, +1, and +2 relative to heart transplantation. Lewis recipients, 5-8 per group, were pretreated with 10(8) DL with or without peritransplant CsA. A single DL transfusion on day -3 or day -7 prior to transplantation significantly prolonged the mean survival time (MST) of W/F hearts from 7.0 +/- 0.9 days in controls to 12.2 +/- 4.5 days and 12.4 +/- 1.0 days (P less than 0.01), respectively. Two DL infusions on days -7 and -3 or on days -14 and -7 prolonged the MST to 10.6 +/- 1.3 days (P less than 0.02) and 16.4 +/- 2.8 days (P less than 0.001), respectively. The administration of peritransplant CsA alone significantly prolonged W/F heart allograft survival to 43.1 +/- 2.7 days. When pretransplant DL transfusion on day -3 was combined with CsA treatment, 4/8 animals maintained their grafts indefinitely (greater than 100 days). Similarly, DL infusion on day -7 with peritransplant CsA led to indefinite graft survival in 3/5 animals. Administration of DL on days -7 and -3 combined with CsA resulted in indefinite graft survival (greater than 100 days) in 4/6 animals. Transfusion of DL on day -3 alone or in combination with peritransplant CsA, had no effect on a third-party (ACI) heart allograft survival prolongation compared with appropriate controls. To define the underlying mechanisms responsible for donor-specific unresponsiveness in this model, pooled sera and unseparated spleen cells were passively transferred from recipients of long-term cardiac allografts to syngeneic rats receiving donor-type (W/F) or third-party (ACI) cardiac allografts. Transfer of serum (1 ml on days 0, and 1, 0.5 ml on days +2, +3, and +4) from ungrafted recipients of DL on days -14 and -7 led to significant donor graft survival of 9.8 +/- 0.4 days (P less than 0.02) in unmodified hosts. Similarly, passive transfer of serum obtained at 20 and 100 days after transplantation significantly prolonged the MST of donor-type hearts in syngeneic untreated hosts to 11.3 +/- 0.8 and 10.0 +/- 1.1 days, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prolongation of adult skin allograft survival by cotransplantation of neonatal skin in antilymphocyte serum and donor bone marrow cell-treated mice

Adult male B6AF1 (H-2KIb/kDb/d) mice were given skin allografts from adult male C3H (H-2k) mice, with and without contralateral cotransplants. The cotransplants were of either adult or neonatal (less than 24 hr) C3H skin. In recipient mice treated peritransplantation with antilymphocyte serum and posttransplantation with an i.v. injection of donor-strain bone marrow cells, the presence of a neonatal cotransplant resulted in significantly longer survival of the adult grafts. Median survival time (MST) for adult grafts, for the group that received a neonatal cotransplant was greater than 100 days, in comparison to MSTs of 59 days and 55 days for the groups that received only single adult grafts or the adult graft with an adult cotransplant. These results suggest an active immunomodulatory contribution of neonatal tissue, and we term this phenomenon the "cotransplant effect." This prolongation of survival of the adult grafts by the neonatal cotransplants was statistically significant in animals treated with ALS and BMC, but not in recipients that were treated with ALS only (MST = 34 days, in comparison with 29 days for groups that received either a single adult graft or an adult graft with an adult cotransplant). The graft-prolonging effects of an infusion of donor BMC thus appear to potentiate the expression of the cotransplant effect. An understanding of this effect may lead to improved methods of controlling the allograft response to adult tissues in the clinical setting.     

Induction of indefinite survival of fully allogeneic cardiac grafts and generation of regulatory cells by intratracheal delivery of alloantigens under blockade of the CD40 pathway

BACKGROUND: The authors previously showed that intratracheal delivery (ITD) of donor splenocytes induced prolonged survival of fully allogeneic cardiac grafts in mice. In this study, this treatment protocol was combined with blockade of the CD40 pathway in an attempt to induce operational tolerance. METHODS: CBA mice were given donor splenocytes (1x107) or Kb peptide (100 microg) by ITD with or without antibody specific for mouse CD40 ligand (MR1, 200 microg) 7 days before transplantation of a C57BL/10 heart. Also, splenocyte (5 x 107) from primary recipient CBA mice given ITD of donor splenocytes or Kb peptide plus MR1 were adoptively transferred into naive CBA secondary recipients 7 days after the pretreatment and C57BL/10 hearts were transplanted into those recipients the same day. RESULTS: ITD of donor splenocytes and Kb peptide induced prolonged survival of cardiac grafts (median survival time [MST], 74 and 56 days, respectively), whereas naive control mice and mice pretreated with syngeneic splenocytes had acute graft rejection (MST in both groups, 7 days). When MR1 was included, all grafts survived indefinitely (>200 days), but mice pretreated with MR1 alone had graft rejection (MST, 54 days). Mice bearing cardiac grafts had acceptance of skin grafts from C57BL/10 but not BALB/c mice, demonstrating that operational tolerance was induced. Secondary recipients given adoptive transfer of splenocytes from primary recipients of the combined treatment had acceptance of C57BL/10 grafts, suggesting that regulatory cells were generated within 7 days of pretreatment. CONCLUSIONS: ITD of donor splenocytes or Kb peptide under blockade of the CD40 pathway induced operational tolerance and generated regulatory cells.     

Rapamycin-conditioned, alloantigen-pulsed dendritic cells promote indefinite survival of vascularized skin allografts in association with T regulatory cell expansion

Clinically-applicable protocols that promote tolerance to vascularized skin grafts may contribute to more widespread use of composite tissue transplantation. We compared the properties of alloantigen (Ag)-pulsed, rapamycin (Rapa)-conditioned and control bone marrow-derived host myeloid dendritic cells (DCs) and their potential, together with transient immunosuppression (anti-lymphocyte serum+cyclosporine), to promote long-term, vascularized skin graft survival in Lewis rats across a full MHC barrier. Both types of DCs expressed low levels of CD86, but Rapa DC expressed lower levels of MHC II and CD40 and were less stimulatory in MLR. While both Rapa and control DCs produced low levels of IL-12p70 and moderate levels of IL-6 and IL-10 following TLR ligation, Rapa DC secreted significantly lower levels of IL-6 and IL-10 in response to LPS. Donor Ag-pulsed Rapa DC, but not control DC, induced long-term skin graft survival (median survival time >133 days) when administered 7 and 14 days post-transplant. Circulating T cells in hosts with long-surviving grafts were hyporesponsive to donor alloAg stimulation, but proliferated in response to third-party stimulation and produced IFN-gamma and IL-10. When recipients of long-surviving grafts were challenged with skin grafts, donor but not third-party grafts were prolonged, suggesting underlying regulatory mechanisms. Both flow cytometry and immunohistochemical analysis revealed that donor Ag-pulsed Rapa DC infusion expanded CD4+ Foxp3+ Treg in recipients' spleens, graft-associated lymph nodes and the graft. These data demonstrate for the first time that pharmacologically-modified, donor Ag-pulsed host DC administered post-transplant can promote indefinite vascularized skin graft survival, associated with Treg expansion.     

Intragraft delivery of 16, 16-dimethyl PGE2 induces donor-specific tolerance in rat cardiac allograft recipients

The stable prostaglandin E2 analogue, 16,16-dimethyl PGE2 (di-M-PGE2) was continuously infused by osmotic pump directly into rat heterotopic cardiac allografts. Intragraft delivery of 20 micrograms/kg/day di-M-PGE2 for 2 weeks completely prevented graft rejection for more than 150 days (n = 10), while untreated Buffalo recipients rejected Lewis cardiac allografts within 8 days after transplantation (mean survival time = 7.4 +/- 0.5 days, n = 5). When given for only 1 week, 20 micrograms/kg/day had a partial effect, since 60% of recipients accepted grafts long-term and 40% experienced rejection by day 14 (n = 5). In contrast, systemic intravenous administration of 20 micrograms/kg/day di-M-PGE2 for 2 weeks could not prolong graft survival (MST = 7.0 +/- 0.0 days, n = 3), and the higher dose of 200 micrograms/kg/day resulted in death by day 2 (n = 5). Long-term BUF recipients of LEW cardiac allografts accepted LEW donor strain skin grafts for more than 35 days while rejecting third-party Wistar Furth skin grafts in a normal fashion (MST = 7.3 +/- 0.5 days, n = 3), indicating the induction of donor-specific tolerance. Long-surviving LEW cardiac allografts retransplanted into naive BUF recipients were rejected within 7 days (MST = 6.7 +/- 0.5 days, n = 3), indicating no change in graft immunogenicity. Therefore, a 14-day infusion of di-M-PGE2 directly into a strongly MHC-mismatched cardiac allograft uniformly has resulted in long-term engraftment and the development of recipient donor-specific tolerance.