受体血诱导大鼠肝移植免疫耐受的机制探讨

目的将受体血预先经门静脉输入供体，探讨肝移植后免疫排斥反应的变化。方法将LEW大鼠和ACI大鼠作为受体和供体。移植前7d将受体血经门静脉输入供体，移植后检测移植肝内供体源性树突状细胞，检测移植肝组织内IFN-γmRNA的表达，检测移植肝内浸润细胞的凋亡。结果移植前7d受体血经门静脉输入供体组的生存时间显著延长，为（33．7±2．6）d，无处置组为（10．1±0．7）d。移植肝组织内IFN-γmRNA有明显表达，移植肝内检测到大量供体源性树突状细胞。移植肝的浸润淋巴细胞凋亡显著多于无处置组。结论移植肝内浸润淋巴细胞凋亡抑制了肝移植排斥反应。     

预输注RelB shRNA慢病毒修饰树突细胞诱导大鼠肝移植免疫耐受

背景:肝移植后的排斥反应是威胁患者和移植物长期存活的主要原因。诱导受者产生特异性免疫耐受是解决排斥反应的理想措施。目的:探讨RNAi RelB树突状细胞预输注诱导大鼠肝移植特异性免疫耐受的可能性。方法:将近交系雄性清洁级DA(RT1a)大鼠和近交系雄性SPF级Lewis(RT11)大鼠分别作为供、受体,行原位肝移植手术。术前随机配对分为4组:①对照组,受体鼠移植前不做预输注。②治疗组:受体鼠移植前7d静脉输注供体大鼠RNAi RelB树突状细胞(5×106)。③未成熟树突状细胞组:受体鼠移植前7d静脉输注供体大鼠未成熟树突状细胞(5×106)。④成熟树突状细胞组:受体鼠移植前7d静脉输注供体大鼠成熟树突状细胞(5×106)。结果与结论:与对照组、未成熟树突状细胞组以及成熟树突状细胞组相比,治疗组移植肝的平均生存时间显著延长。移植后第7天,治疗组天冬氨酸转氨酶,总胆红素水平低于对照组、成熟树突状细胞组、未成熟树突状细胞组(P<0.01),移植后第14天治疗组、未成熟树突状细胞组天冬氨酸转氨酶,总胆红素均有轻微下降,两组比较差异仍有显著性意义(P<0.01)。移植后第7天,与治疗组比较,对照组、成熟树突状细胞组、未成熟树突状细胞组γ-干扰素、白细胞介素2水平升高(P<0.01),而白细胞介素4、白细胞介素10下降(P<0.01);移植后第14天治疗组、未成熟树突状细胞组γ-干扰素、白细胞介素2水平均有下降,白细胞介素4、白细胞介素10水平均有升高,两组比较差异仍有显著性意义(P<0.01)。对照组、成熟树突状细胞组、未成熟树突状细胞组移植后第7天排斥活动指数为8.0-9.0。未成熟树突状细胞组第14天肝细胞、内皮细胞坏死及汇管区炎性细胞浸润进一步增多。治疗组移植后第7天排斥活动指数为6.0-8.0,第14天时排斥活动指数为4.0-5.0。结果提示RNAi RelB树突状细胞预输注可以减轻移植肝排斥程度,延长移植肝生存时间,这是通过间接途径实现的,其机制可能与T细胞的调节和无能有关。     

肝移植受体免疫耐受的诱导及CD4^＋CD25^＋T细胞的重要作用

背景:解决肝移植排斥反应的惟一方法是诱导免疫耐受,而环孢素A对诱导鼠肝移植免疫耐受及T细胞都存在一定影响,可为各种免疫抑制剂的合理使用提供依据.目的:探讨环孢素A诱导肝移植后免疫耐受的可行性及方法,分析CD4+CD25+调节性T细胞在诱导耐受中的作用.设计、时间及地点:随机分组,动物实验观察,2007-01/2008-04在中山大学医学实验动物中心和深圳市第三人民医院肝病研究所完成.材料:健康LEW大鼠66只,BN大鼠78只,CD4、CD25抗体和Foxp3抗体(克隆号PCH101)由美国eBioscience公司生产,环孢素A针剂由北京诺华制药提供.方法:根据用药情况建立6组肝移植模型:同基因对照组:BN大鼠进行同种异体肝移植,移植后不用药.急性排斥组:LEW大鼠作为供体,BN大鼠作为受体,移植后不用药.环孢素A小、中、大剂量组:LEW大鼠作为供体,BN大鼠作为受体,移植后分别给予环孢素A1.0,1.5,2.0 mg/(kg·d),疗程均为5 d.环孢素A中剂量延期组:LEW大鼠作为供体,BN大鼠作为受体,移植后给予环孢素A1.5 mg/(kg·d),疗程7 d.除急性排斥组和环孢素A小剂量组各24只外,其他6只/组.主要观察指标:观察各组大鼠移植后生存时间及外周血变化,并检测急性排斥组和组肝、环孢素A小剂量组脾组织中CD4+CD25+Foxp3+Treg的含量.结果:同基因对照组存活时间>100 d,而不发生排斥反应.环孢素A小、中、大剂量组存活时间分别为(51.5±2.4),(53.6±3.6).(23.2±2.1)d,环孢素A中剂量延期组和急性排斥组分别为(57.8±7.2),(20.6±3.2)d,除同基因对照组外,各组间差异均有统计性意义(F=114.82,P<0.01).各组外周血CD4+CD25+Foxp3+Treg的比例无明显差别及增加(P>0.05).环孢素A小剂量组肝组织中CD4+CD25+Foxp3+Treg的含量明显增加,且高于急性排斥组(P<0.05).结论:小剂量环孢素A可以诱导免疫耐受,大剂量环孢素A突然停药会导致加速性排斥.CD4+CD25+Foxp3+Treg能促进移植肝受体的免疫耐受,但其增加只存在于移植的肝内,外周血、脾脏未见增加.     

浸润淋巴细胞凋亡诱导大鼠肝移植耐受

目的观察移植术后大鼠肝脏内不同细胞成分的凋亡现象，寻找耐受过程中的关键细胞成分，探讨大鼠肝移植耐受机制。方法分离肝脏间质细胞，流式细胞仪检测移植术后大鼠肝脏内不同细胞成分的凋亡现象。结果BN→LEw组大鼠移植肝脏在术后早期发生急性排斥反应，并且移植物内间质细胞存在大量的凋亡，急性排斥反应评分及细胞凋亡指数逐渐升高，术后7d达到高峰随后下降，前者术后28d与术前无明显差异（P〉0.05）；细胞凋亡指数至术后28d较术前仍有明显差异，明显高于LEw→LEw组（P〈0.05）。移植物内发生凋亡的细胞为来自于受体的浸润T淋巴细胞。结论移植物排斥反应评分下降与移植肝脏内CTL清除有关，来源于受体的浸润T淋巴细胞凋亡导致移植肝脏免疫耐受。     

低剂量西罗莫司协同CD4~+CD25~+ T-reg诱导大鼠肝移植免疫耐受的实验研究

目的我们通过体外诱导、扩增并分选出CD4+CD25+T-reg回输入受体大鼠,并协同低剂量西罗莫司去诱导大鼠肝移植免疫耐受,研究探讨CD4+CD25+T-reg细胞亚群在移植免疫耐受过程中扮演的角色。方法将实验动物分为急性排斥组(DA→LEW)、免疫耐受组(LEW→DA)、低剂量西罗莫司(0.1 mg·kg-1·d-1)和CD4+CD25+T-reg协同作用组(实验组)。每组8只实验动物。各组肝移植大鼠的存活率比较,应用HE染色法观察移植肝术后7 d组织病理学变化,检测CD4+CD25+Foxp3+T-reg细胞亚群在各组大鼠移植肝脏、外周血总单核细胞数中所占的百分比。RT-PCR检测术后7 d移植肝脏Foxp3 m RNA的表达水平。用ELISA检测血浆中细胞因子IL-10、TGF-β的水平。结果实验组对比排斥组,能够长期存活,获得免疫耐受(P<0.05)。实验组移植肝脏中CD4+CD25+Foxp3+T-reg细胞亚群所占的比例明显高于排斥组(P<0.001),且在移植肝脏中,实验组Foxp3 m RNA表达含量明显增加(P<0.001)。实验组血浆中细胞因子IL-10、TGF-β的表达水平高于排斥组(P<0.001)。结论我们通过流式细胞分选技术将体外扩增诱导成熟的受体大鼠CD4+CD25+T-reg细胞分离收集,然后回输受体协同低剂量的西罗莫司能够成功地诱导了长期肝移植免疫耐受。从而为临床应用CD4+CD25+T-reg细胞抑制免疫排斥反应,诱导移植免疫耐受提供了一条新思路和理论实验依据。     

抗ICOS免疫疗法抑制鼠心脏移植的加速性和慢性排斥反应

背景:在DA鼠移植给路易鼠模型中,用AdCTLA-41g免疫球蛋白阻断B7-CD28共刺激信号可以诱导心脏同种异体移植的长期免疫耐受.然而这种耐受是不完全的,并最终发生排斥.尤其是在心脏移植后功能良好时将供体皮肤移植给受体可以引发加速性移植心脏的排斥反应.目的:重新评价抗 ICOS 抗体疗法与AdCTLA-41g疗法相结合在同种异体移植中的作用.验证抗ICOS疗法无论是否应用AdCTLA-41g疗法都起到阻断心脏加速性排斥的作用.方法:在DA(供体)→LEW(受体)鼠之间进行心脏移植手术.心脏移植给予受体鼠一次静脉注射109 pfu AdCTLA-41g.生存期50 d的受体鼠接受全厚皮片移植到侧胸壁,第50天开始静脉注射抗ICOS抗体(1 mg/kg)或者IgG,隔天注射1次,共2周.完全移植排斥被定义为心脏停止跳动,或者组织学上通过单核细胞浸润和心肌细胞坏死的苏木精一伊红染色证实.结果与结论:AdCTLA-41g处置受体生存大于100 d的心脏切片可见典型的ICOS+单核细胞浸润,并表现出血管病理性改变.AdCTLA-41g疗法与抗ICOS抗体结合可介导出对慢性排斥稳定的耐受.抗ICOS疗法明显减少了ICOS阳性炎性细胞浸润,并阻断了皮肤二次移植引发的心脏加速性免疫捧斥反应.实验结果表明了ICOS在慢性及急性心脏移植免疫排斥中的重要作用,并揭示出阻断血管化器官同种异体移植慢性排斥的革命性观点.     

受体血预先经门静脉输入供体诱导移植肝浸润细胞凋亡

背景:肝移植后排斥反应发生时,肝细胞被浸润的T淋巴细胞破坏,肝细胞减少,肝功能进行性恶化.诱导肝移植免疫耐受仍然是目前面临的重大课题.目的:观察受体血预先经门静脉输入供体对大鼠移植肝内浸润淋巴细胞凋亡的影响.设计、时间及地点:随机对照动物实验,于2002-05/2004-05在中国医科大学器官移植实验室完成.材料:选用纯系大鼠制作大鼠原位肝移植模型.24只供体为雄性ACI大鼠(RT1a),24只受体为雄性LEW大鼠(RT1I).方法:大鼠肝移植采用改良的两袖套法进行原位移植.受体大鼠按随机数字表法分为4组,每组6只.对照组未作处理;受体血经阴茎静脉输入组于移植前7 d将受体血1 mL.经阴茎静脉输入供体;非受体血经门静脉输入组于移植前7 d将非受体大鼠(BN大鼠)血1 mL经门静脉输入供体;受体血经门静脉输入组于移植前7 d将受体血1 mL经门静脉输入供体.主要观察指标:肝移植后观察大鼠生存时间;移植后检测各组大鼠血清γ-干扰素质量浓度;移植后观察移植肝组织学变化;移植后7 d检测移植肝内树突状细胞数量;移植后3,5,7,14 d检测移植肝内浸润淋巴细胞的凋亡情况.结果:受体血经门静脉输入组大鼠的生存时间显著长于对照组(P＜0.01).肝移植后3,5 d,受体血经门静脉输入组大鼠的血清γ-干扰素质量浓度显著高于对照组(P＜0.05),受体血经阴茎静脉输入组、非受体血经门静脉输入组大鼠的生存时间及血清γ-干扰素质量浓度与对照组无显著差异(P＞0.05).受体血经门静脉输入组大鼠移植肝汇管区内的淋巴细胞浸润显著减少,移植肝内检测到大量供体来源的树突状细胞.移植后受体血经门静脉输入组大鼠肝组织切片每平方毫米的凋亡细胞数显著多于对照组(P＜0.01).结论:受体血预先经门静脉输入供体,可以延长异系肝移植受体大鼠生存时间,促进移植肝内浸润淋巴细胞的凋亡.     

MAdCAM-1在大鼠小肠移植肠管中的表达及其意义

【目的】通过建立大鼠小肠移植模型,观察粘附素细胞粘附分子-1（MAdCAM-1）在大鼠小肠移植肠管中的表达及移植肠管的改变。【方法】实验分三组,第Ⅰ组为同系同基因移植（从LEW鼠到LEW鼠）;第Ⅱ组为异系同基因移植（从DA鼠到LEW鼠）;第Ⅲ组为使用抗-MAdCAM-1抗体的异系同基因移植。将DarkAgouti（DA）鼠的小肠异位移植到Lewis（LEW）鼠中。观察各组移植肠管的形态和组织学变化;并用免疫染色检测MAdCAM-1和整合素a4137在移植肠管中的表达。通过荧光激活细胞分类术来分析移植肠管中肠系膜淋巴结和肠管Peyer＇s淋巴结的淋巴细胞浸润情况。【结果】实验组大鼠移植肠管存活期为（7．0±3．3）d,对照组为（24．6±8．4）d（P〈0．05）。并且,在实验组的移植肠管中MAdCAM-1的表达被抑制。而对照组在Peyefs淋巴结中CD4^＋T细胞和CD8^＋T细胞无显著差别,实验组的肠系膜淋巴结中CD4^＋T细胞有显著降低。【结论】在小肠移植中,通过阻断抗-MAdCAM-1抗体可以用来预防急性排斥反应。     

白细胞分化抗原74和CXC趋化因子配体9阳性巨噬细胞亚群在大鼠肝移植排斥反应中的作用

目的 探讨在肝移植排斥反应中巨噬细胞亚群分类及变化。方法 建立大鼠肝移植模型分为：免疫耐受组（B-B），将BN大鼠供体的肝脏移植至BN大鼠受体；免疫排斥组（L-B），将Lewis大鼠供体的肝脏移植至BN大鼠受体，使用单细胞RNA测序和高通量RNA测序区分大鼠移植肝巨噬细胞亚群，发现排斥反应高度差异的基因，免疫组化确定蛋白表达和细胞亚群的变化和分布。结果 CD68阳性巨噬细胞在排斥组多于耐受组（P <0.05），巨噬细胞可分为9个亚群，排斥反应中巨噬细胞第8群的CXC趋化因子配体9(CXCL9)明显升高，第5群的白细胞分化抗原74(CD74)基因明显升高（P <0.05）。排斥反应中巨噬细胞差异基因综合第1位的是CD74，第2位是CXCL9。与耐受组比较，排斥组肝脏汇管区可见大量CD74阳性巨噬细胞浸润，并且在肝血窦CD74阳性巨噬细胞浸润也明显增加（P <0.05），在排斥肝脏汇管区和肝血窦可见大量CXCL9阳性巨噬细胞浸润以汇管区为著（P <0.05），在排斥肝脏汇管区CD14阳性细胞明显增加（P <0.05）。结论 在肝移植排斥反应中CD74阳性巨噬细...     

受体骨髓干细胞于大鼠移植肝内向血管内皮细胞分化的实验研究

目的:研究受体骨髓干细胞在大鼠移植肝中向血管内皮细胞(VECs)的分化情况及其对大鼠原位肝脏移植术后早期排斥反应的影响。方法:用双袖套法建立大鼠原位肝移植模型,供体为雌性Wistar大鼠,受体为雌性SD大鼠。受鼠随机分为对照组(n=6)和实验组(n=12)。其中对照组仅行原位肝移植,实验组于术中经门静脉注射雄性SD大鼠骨髓干细胞0.5mL(1×107/mL)。实验组再随机分为3组,每组4只,分别在术后第7、14、21天切取肝脏,并行Sry原位杂交结合vonWillebrand因子(vWF)免疫组化的双标染色,观察受体骨髓干细胞向VECs转化的情况,同时观察术后早期的病理变化。结果:实验组术后一般情况明显优于对照组。实验组于术后第7天在肝组织内发现Sry与vWF双染阳性细胞,该细胞于第14和21天在血管壁出现。对照组肝脏病理学检查为急性重度排斥反应,实验组为急性轻到中度排斥反应。结论:受体骨髓干细胞能在移植肝的环境中诱导分化为VECs,表达vWF,并可部分替代移植肝本身的VECs;经门静脉输注受体骨髓干细胞可减轻移植肝的急性排斥反应。     

Suppression of hepatic allograft rejection in the rat by mitomycin C-treated donor splenocytes: analysis of the immune status

A single intraperitoneal injection of 3 x 10(6) donor splenocytes treated with mitomycin c (MMC) seven days before hepatic transplantation prolongs survival of hepatic allografts in the ACI (RT1a) to LEW (RT1l) rat combination. This effect is donor specific. An intravenous injection of the same dose of splenocytes treated with MMC seven days before transplantation also tends to prolong hepatic allograft survival. Furthermore, lymphocytotoxic antibody can be detected in rats 30 days after transplantation. Adoptive transfer of 5 x 10(7) splenocytes taken from long-term surviving hepatic allograft recipients pretreated with MMC-treated donor ACI splenocytes into irradiated (750 rads) LEW rats prolongs the survival of donor-type skin grafts, whereas third-party strain (BN) grafts are rejected. Similarly, prolonged survival of ACI cardiac allografts in irradiated (450 rads) LEW recipients is achieved following the transfer of spleen cells taken from longterm surviving hepatic allograft recipients pretreated with MMC-treated donor ACI splenocytes, whereas third-party (BN) cardiac allografts show rejection. These findings suggest the presence of donor-specific suppressor cells and indicate that a single injection of donor splenocytes treated with MMC to the recipient seven days before transplantation can induce specific suppression of rejection in a rat hepatic allograft model.     

Transfer of specific unresponsiveness to organ allografts by thymocytes. Specific unresponsiveness by thymocyte transfer

Prolonged survival of vascularized organ allografts has been produced in unmodified inbred rats by transfer of thymocytes from enhanced, engrafted, syngeneic animals. For these thymocytes to increase significantly the survival of test allografts they must be harvested 6-9 d after transplantation. Thymectomy of the enhanced, engrafted animals during the same critical period causes acute rejection of othewise long surviving grafts. For optimal effect, the enhanced thymocyte donor must be actively and passively immunized and receive a cardiac allograft. The necessity for erythrocytes in the initial active immunization regimen is noted. Additionally, the antigenic specificity of the suppressor effect has been established with two histoincompatible donor rat strains. Cellular and humoral host responses mounted by test graft recipients after thymocyte transfer from enhanced, engrafted donors are different from those mounted either by unmodifed animals acutely rejecting their grafts or by enhanced rats bearing well-functioning grafts. Numbers of T lymphocytes are reduced in the grafted hearts and in the spleens of test graft recipients, a finding paralleled by the complete absence of specific direct lymphocyte-mediated cytotoxicity. In contrast, cytotoxic antibody production, although delayed, is increased in magnitude, peaking around the time of graft rejection. These studies provide evidence that different biological manipulations can modify separate pathways in the complex cellular and humoral responses towards organ allografts. They demonstrate that cellular immunity is critically involved in immunological enhancement of vascularized organ allografts, a phenomenon hitherto considered primarily humoral. It seems clear that cells with suppressor activity are present within the thymus during the early phases of immunological enhancement.     

共刺激阻断剂细胞毒性T淋巴细胞相关抗原4Ig基因修饰树突状细胞对移植肾存活的影响

背景：前期的研究表明，通过受体全身注射T细胞活化所需的B7／CD28共刺激信号阻断剂细胞毒性T淋巴细胞相关抗原4Ig可以明显延长大鼠移植肾存活，但所需剂量大、影响范围广、特异性差，可能造成全身性不良反应。 目的：为了提高细胞毒性T淋巴细胞相关抗原4Ig治疗的靶向性，采用细胞毒性T淋巴细胞相关抗原4Ig基因对肾移植供、受体树突状细胞进行体外修饰，观察两种树突状细胞单独以及联合应用对移植肾存活的影响。 设计、时间及地点：随机对照动物实验，于2003—04／2004—07在解放军全军肾病中心实验室完成。 材料：肾移植供体为近交系Brown-Norway（BN）大鼠，受体为近交系Lewis大鼠，Wistar大鼠作为无关淋巴细胞供体。 方法：分离培养供、受体大鼠骨髓源性树突状细胞，以细胞毒性T淋巴细胞相关抗原4Ig基因重组腺病毒转染供、受体大鼠骨髓源性树突状细胞。构建大鼠肾移植模型，将单独的供、受体树突状细胞，混合的供、受体树突状细胞于肾移植前24h经股静脉注入受体，以未注射树突状细胞的受体为对照。 主要观察指标：①移植肾存活时间。②术后20d应用MTT法检测受体脾细胞对供体及无关抗原刺激反应程度。 结果：与对照组比较，受体树突状细胞未能延长移植肾存活，但与供体树突状细胞联合应用时可使移植肾存活时间较单独应用供体树突状细胞时明显延长（P〈0．01）。注入供体树突状细胞与混合的供、受体树突状细胞大鼠的脾细胞对供体抗原刺激的反应均明显低于对照组（P〈0.01），而对无关抗原刺激的反应与对照组无差异。 结论：细胞毒性T淋巴细胞相关抗原4Ig基因修饰的供体树突状细胞可以明显延长大鼠移植肾存活时间，受体树突状细胞无此作用，但两种树突状细胞联合应用可以使移植肾获得更长的存活时间。     

Mapping of the Epstein-Barr virus and C3dg binding sites to a common domain on complement receptor type 2

This study has examined the ability of adoptively transferred CD4+ and CD8+ T cells to mediate rejection of a fully allogeneic DA renal graft in the PVG nude rat. Transfer, at the time of transplantation, of naive CD4+ T cells caused rapid graft rejection and primed CD4+ cells were several times more potent. In contrast, naive or specifically sensitized CD8+ cells were entirely ineffective at mediating renal allograft rejection. Whereas nonrejecting grafts showed only a mild cellular infiltrate, rejecting grafts in CD4+ reconstituted animals showed a substantial infiltrate and many of the infiltrating cells had a phenotype (MRC OX8+, MRC OX19-), consistent with NK cells. Experiments using a mAb (HIS 41) against an allotypic determinant of the leukocyte common antigen confirmed that the majority (greater than 80%) of the cellular infiltrate in rejecting grafts derived from the host rather than from the CD4+ inoculum. Infiltrating mononuclear cells, obtained from rejecting allografts 7 d after transplantation in CD4+-injected PVG nude hosts, showed high levels of in vitro cytotoxicity against not only kidney donor strain Con A blasts but also third-party allogeneic Con A blasts, as well as against both NK and LAK susceptible targets. When splenocytes from nontransplanted nude PVG rats were tested in vitro they also demonstrated high levels of lytic activity against both NK and LAK susceptible targets as well as allogeneic Con A blasts, which were not susceptible to lysis by spleen cells from euthymic rats. These findings suggest that injected CD4+ cells may cause renal allograft rejection by the recruitment of extrathymically derived, widely alloreactive cells into the kidney in this model of graft rejection.     

Anti-interleukin 2 receptor monoclonal antibodies spare phenotypically distinct T suppressor cells in vivo and exert synergistic biological effects

The therapeutic efficacies of ART-18, ART-65, and OX-39, mouse antibodies of IgG1 isotype recognizing distinct epitopes of the p55 beta chain of the rat IL-2-R molecule, were probed in LEW rat recipients of (LEW X BN)F1 heterotopic cardiac allografts (acute rejection in untreated hosts occurs within 8 d). A 10-d course with ART-18 prolongs graft survival to approximately 21 d (p less than 0.001). Therapy with ART-65, but not with OX-39, was effective (graft survival approximately 16 and 8 d, respectively). Anti-IL-2-R mAb treatment selectively spared T cells with donor-specific suppressor functions; the CD8+ (OX8+ W3/25-) fraction from ART-18-modified recipients, and primarily the CD4+ (W3/25+ OX8-) subset from ART-65-treated hosts conferred unresponsiveness to naive syngeneic rats after adoptive transfer, increasing test graft survival to approximately 16 and 45 d, respectively. Concomitant administration of ART-18 and ART-65 to recipient animals in relatively low doses exerted a strikingly synergistic effect, with 30% of the transplants surviving indefinitely and 50% undergoing late rejection over 50 d. These studies provide evidence that anti-IL-2-R mAbs selectively spare phenotypically distinct T cells with suppressor functions. The data also suggest that in vivo targeting of functionally different IL-2-R epitopes may produce synergistic biological effects.     

Failure of long surviving, passively enhanced kidney allografts to provoke T-dependent alloimmunity. I. Retransplantation of (AS X AUG)F1 kidneys into secondary AS recipients

Long survival of (AS X AUG)F1 rat kidney allografts in AS recipients was induced by passive enhancement with AS anti-AUG antiserum at the time of grafting. After 1-3 mo, the kidney allografts were transferred to second AS recipients, either naive or sensitized against AUG tissue. Naive second recipients did not reject the grafts acutely and failed to mount T-dependent immunity against AUG targets. When later challenged with spleen cells carrying the AUG haplotype, the naive second AS recipients showed strong IgM, IgG, and cytotoxic T-cell responses after grafting, and the kidneys were rapidly destroyed by immune rejection in all but one rat. It is concluded that long-surviving kidney allografts fail to activate helper T cells and induce in naive second recipients the same state of unresponsiveness observed in the first recipient.     

T cell requirements for the rejection of renal allografts bearing an isolated class I MHC disparity

This study has examined the cellular and humoral responses underlying the rejection of rat renal allografts bearing an isolated RT1Aa class I MHC disparity. RT1Aa disparate kidneys were rejected promptly by high responder RT1u but not by low responder RT1c recipients (median survival time 10 d and greater than 100 d, respectively). The magnitude and phenotype of the cellular infiltrate were similar in rejecting and nonrejecting RT1Aa disparate kidneys. Paradoxically, graft infiltrating cells and spleen cells from RT1u recipients showed minimal ability to lyse donor strain lymphoblasts in vitro, whereas effector cells from RT1c recipients showed modest levels of cytotoxicity. Injection of RT1u rats with MRC OX8 mAb was highly effective at selectively depleting CD8+ cells from graft recipients but had no effect in prolonging the survival of RT1Aa disparate grafts despite the complete absence of CD8+ cells from the graft infiltrate, which included numerous CD4+ T cells and macrophages. RT1u, but not RT1c, recipients mounted a strong alloantibody response against RT1Aa disparate kidneys. Immune serum obtained from RT1u recipients that had rejected a RT1Aa disparate graft was able, when injected into cyclosporin-treated RT1u recipients, to restore their ability to reject a RT1Aa, but not a third-party RT1c, kidney. These results suggest that CD8+ cells in general and CD8+ cytotoxic effector cells in particular are unnecessary for the rapid rejection of RT1Aa class I disparate kidney grafts by high responder RT1u recipients. By implication, CD4+ T cells alone are sufficient to cause prompt rejection of such grafts and they may do so by providing T cell help for the generation of alloantibody.     

Induction of myocardial nitric oxide synthase by cardiac allograft rejection.

Cardiac transplantation, effective therapy for end-stage heart failure, is frequently complicated by allograft rejection, the mechanisms of which remain incompletely understood. Nitric oxide (NO), a vasodilator which is cytotoxic and negatively inotropic, can be produced in large amounts by an inducible NO synthase (iNOS) in response to cytokines. To investigate whether iNOS is induced during cardiac allograft rejection, hearts from Lewis or Wistar-Furth rats were transplanted into Lewis recipients. At day 5, allogeneic grafts manifested reduced contractility and histologic evidence of rejection (inflammatory infiltrate, edema, necrosis of myocytes). The mRNA for iNOS and iNOS protein were detected in ventricular homogenates and in isolated cardiac myocytes from rejecting allogeneic grafts but not in tissue and myocytes from syngeneic control grafts. Immunocytochemistry showed increased iNOS staining in infiltrating macrophages and in microvascular endothelial cells and cardiac muscle fibers and also in isolated purified cardiac myocytes from the rejecting allografts. Using a myocardial cytosolic iNOS preparation, nitrite formation from L-arginine and [3H] citrulline formation from [3H]L-arginine were increased significantly in the rejecting allogeneic grafts (P < 0.01). Myocardial cyclic GMP was also increased significantly (P < 0.05). The data indicate myocardial iNOS mRNA, protein and enzyme activity are induced in infiltrating macrophages and cardiac myocytes of the rejecting allogeneic grafts. Synthesis of NO by iNOS may contribute to myocyte necrosis and ventricular failure during cardiac allograft rejection.     

CD28-B7 blockade after alloantigenic challenge in vivo inhibits Th1 cytokines but spares Th2

Blocking the CD28-B7 T cell costimulatory pathway with the fusion protein CTLA4Ig inhibits alloimmune responses in vitro and in vivo and induces tolerance to cardiac allografts in mice and rats, but the mechanisms mediating the tolerant state in vivo are unknown. Here, we report the effects and potential mechanisms of CTLA4Ig in the rat renal allograft model. LEW rats were nephrectomized and received renal allografts from major histocompatibility complex-incompatible WF rats. While all untreated and control immunoglobulin (Ig)-treated animals acutely rejected their allografts and died, 86% of rats that received a single injection of CTLA4Ig on day 2 after transplantation had prolonged survival (> 60-100 days) with preserved renal function. By contrast, only 29% of animals that received CTLA4Ig on the day of engraftment had prolonged survival. Long-term survivors (> 100 days) exhibited donor-specific tolerance, accepting donor-matched WF but acutely rejecting third-party BN cardiac allografts. Immunohistological analysis of grafts sampled at 1 week after transplantation showed that both control and CTLA4Ig-treated animals had mononuclear cell infiltrates, with a higher percentage of CD4+ cells in the CTLA4Ig- treated group. However, while this was associated with vasculitis and tubulitis in control grafts, there was no evidence of tissue injury in CTLA4Ig-treated animals. The immune response leading to graft rejection in control animals was characterized by expression of the T helper (Th) type 1 cytokines interleukin (IL)-2 and interferon-gamma. In contrast, the persistent CD4+ infiltrate without graft rejection in CTLA4Ig- treated animals was associated with increased staining for the Th2- related cytokines IL-4 and IL-10. Furthermore, grafts from CTLA4Ig- treated animals had marked upregulation of intragraft staining for IgG1, but not IgG2a or IgG2b. Administration of rIL-2 to CTLA4Ig- treated animals restored allograft rejection in 50% of animals tested. These results confirm that blockade of the CD28-B7 pathway after alloantigenic challenge induces donor-specific acceptance of vascularized organ allografts, and indicates in this model that CTLA4Ig inhibits Th1 but spares Th2 cytokines in vivo.