小鼠异体皮移植创面使用细胞毒性T淋巴细胞抗原4-Ig重组腺病毒载体对其免疫功能的影响

目的　在小鼠背部移植异体皮后局部使用细胞毒性T淋巴细胞抗原 4 Ig(CTLA4 Ig)重组腺病毒载体 ,观察其对机体免疫功能的影响。　方法　将 6 0只BALB/c小鼠随机分为手术对照(A)组、CTLA4 Ig转染 (B)组及正常对照 (C)组 ,每组 2 0只。A、B组小鼠制作 1 .5cm× 1.5cm创面 ,移植同创面大小的C57BL小鼠皮肤后 ,A组小鼠创面涂抹不含腺病毒的交联聚丙烯酸树脂 (卡波姆霜 )0 .1g;B组小鼠创面涂抹含腺病毒的卡波姆霜 0 .1g,病毒滴度 5× 1 0 9/L。C组小鼠不作任何处理。术后 1d分别向 3组小鼠腹腔内注射体积分数 1 0 %绵羊红细胞 (SRBC)1ml。术后 7、1 4、2 1、2 8d收集血清进行凝集试验 ,检测SRBC抗体的效价 ,同时取BALB/c、C57BL及昆明小鼠的脾淋巴细胞作混合淋巴细胞培养 (MLC),观察CTLA4 Ig对脾淋巴细胞的抗原识别特异性及再次应答的影响。结果3组小鼠抗SRBC抗体效价组间比较 ,差异无显著性意义 (P >0.0 5 )。术后 1 4d内与A组比较 ,B组小鼠脾淋巴细胞与对C57BL小鼠脾淋巴细胞表现出特异性低反应 (P <0.0 5);1 4d后 ,A、B组的再次应答达到或超过C组 (P >0.0 5 )。结论　局部使用CTLA4 Ig重组腺病毒不影响小鼠的体液免疫功能 ,但可能引起系统性的特异性细胞免疫耐受。     

阻断第二信号诱导骨移植后对淋巴细胞亚群的抑制作用

目的　研究细胞毒性T淋巴细胞相关抗原(CTLA4Ig)对新鲜同种异体骨移植后血淋巴细胞亚群的影响。方法　在组织相容性抗原完全不同的两组小鼠间进行异位骨移植,于移植后第0、1天,腹腔注射CTLA4Ig100μg,分别于术后2、4、6周取受体外周血,利用流式细胞仪技术测定血淋巴细胞亚群CD     

H-2半相合小鼠非清髓性骨髓移植模型的建立

目的　建立H 2半相合小鼠非清髓性骨髓移植模型。方法　用氟达拉滨、阿糖胞苷、环磷酰胺联合60 Co 2Gy全身照射组成预处理方案 ,以C5 7BL/ 6J(H 2 b)为供鼠 ,以C5 7BL/ 6J×BALB/c杂交F1代小鼠为受鼠 ,进行H 2半相合骨髓移植 ,采用环孢素A联合甲氨蝶呤预防急性移植物抗宿主病 (aGVHD)。观察移植后小鼠造血恢复、aGVHD发生率及死亡率 ,5 0d内存活率。结果　移植小鼠平均在 (1 2 .0± 1 .6 )d造血恢复 ,仅接受预处理而不输注供鼠细胞的小鼠在 (1 3.5± 2 .0 )d恢复自身造血 ;移植小鼠移植后 7d到 45d内均可检测到供、受者混合造血细胞嵌合体 ;移植后 2 2d～ 2 5d小鼠出现典型的aGVHD症状。环孢素A联合甲氨蝶呤预防方案可有效降低aGVHD的发生率 ,并减轻其严重程度 ;aGVHD死亡率为 6 0 %,5 0d内存活率为 70 %。结论　以氟达拉滨、阿糖胞苷、环磷酰胺联合全身照射组成的非清髓性预处理方案 ,可在H 2半相合小鼠间进行成功的骨髓移植 ,并可产生稳定的供、受者混合造血细胞嵌合体。     

小鼠自体胰岛肌肉移植及疗效评价

目的探讨小鼠自体胰腺部分切除及自体胰岛的肌肉移植方法,并对术后小鼠进行疗效评价。方法选用C57BL/6小鼠,分为三组,胰腺部分切除后移植组为麻醉后切除十二指肠部沿胃大弯至脾部位的胰腺组织,并将胰岛纯化后移植至自体肌肉组织;以切除部分胰腺后未移植组和正常组作对照。结果小鼠自体胰腺部分切除和自体胰岛肌肉移植术后存活率较高,但在饲养过程中均出现持续体重下降和血糖不稳定,OGTT显示两组术后小鼠20、40 min的血糖均较正常组高,肌肉移植7 d后HE染色显示胰岛在肌肉中存活,但胰岛中心部位细胞有坏死。结论本研究建立了一种无免疫排斥下研究小鼠胰岛移植的方法,可为进一步探索改进胰岛移植研究提供参考。     

外用环孢素A联合CTLA4Ig延长异体移植鼠耳存活的研究

目的　探讨局部外用环孢素 A(Cs A)联合细胞毒性淋巴细胞相关抗原 4融合蛋白 (CTL A4 Ig)对异体复合组织移植的免疫抑制及诱导免疫耐受的作用。方法　建立吻合血管的同种异体大鼠耳廓移植模型 ,术后在移植耳皮肤表面外涂 Cs A并联合 CTL A4 Ig腹腔注射治疗 ,观察移植物的排斥反应及存活时间 ,检测移植后受体血清白细胞介素 - 2 (IL- 2 )含量变化。结果　对照组平均存活时间为 (7.8± 1.7)天 ;单纯用 Cs A治疗组为 (15 .2± 1.9)天 ,单纯CTL A4 Ig治疗组为 (16 .6± 2 .1)天 ;Cs A +CTL A4 Ig联合治疗组为 (2 8.8± 3.5 )天 ,与其它各组相比均有统计学意义 (P<0 .0 1) ;且联合治疗组的受体血清 IL - 2含量最低 ,尤以第 5、7天为著 ,与其它各组相比有统计学意义 (P<0 .0 1)。结论　局部外用 Cs A联合 CTL A4 Ig能有效抑制异体复合组织移植排斥反应 ,显著延长移植物存活时间。     

肿瘤浸润淋巴细胞与白介素2联用抑制Lewis肺癌生长和转移的研究

目的 研究Lewis肺癌(LLC）肿瘤浸润淋巴细胞(TIL）对荷瘤鼠体内的抗瘤作用。方法 从接种于C57BL/6小鼠体内的LLC瘤体中分离提取TIL，经1000u／ml白细胞介素-2(IL-2)的完全培养基中培养后，与LLC细胞以10：1接种于C57BL／6小鼠腋下，每口1000u IL-2瘤内注射，于接种后第18天处死小鼠。结果 经TIL和IL-2处理的小鼠肿瘤重量明显低于其它各组(P＜0．001)，且肺转移发生率也较其它各组低（P＜0.001）。结论 肿瘤浸润淋巴细胞与IL-2联用可明显抑制LLC的生长和转移。     

C57BL6近交系小鼠前列腺癌原位移植瘤模型的建立

目的建立C57BL6近交系小鼠前列腺癌原位移植瘤模型并探讨其应用价值.方法用微量注射器在C57BL6近交系小鼠的前列腺左右背外侧叶包膜下接种小鼠前列腺癌RM-1细胞2×106个建立原位移植瘤模型,以皮下移植瘤模型作为对照比较两者的肿瘤转移发生情况及生存期.结果原位模型小鼠全部发生肿瘤转移,其中盆腔淋巴结和肺的转移发生率分别为(10/10)、(8/10),对照组仅有肿瘤的局部生长和浸润,无转移发生;原位模型的小鼠平均生存期为(11.0±2.4)d较对照组(23.0±4.7)d明显缩短,差异有非常显著性(P＜0.01).结论小鼠的前列腺癌原位移植瘤模型较好地模拟了人癌体内的自然生长状况,是观察转移性前列腺癌较为适宜的模型.     

白细胞介素4在输注供体肝脏非实质细胞诱导免疫耐受中的作用

目的　探讨皮肤移植受体在输注供体肝脏非实质细胞 (NPC)诱导免疫耐受过程中 ,白细胞介素 4(IL 4)所起的作用。方法　将 2× 1 0 7个C3H/He (C3H)小鼠的肝脏非实质细胞通过尾静脉输入C57BL/ 6(B6)小鼠的体内 ,48h后B6小鼠腹腔注射环磷酰胺 2 0 0mg/kg ,1 8d后接受C3H小鼠皮片的移植 ,分别于NPC细胞输注前及输注后 7、1 8、30、60d采血监测IL 4水平的动态变化。结果　 1 5只接受皮片移植的小鼠 ,其皮片存活时间显著延长 ,皮片平均存活时间 (70± 1 7.2 )d ,正常对照组其移植皮肤的平均存活时间仅 (1 0± 0 .4)d ,用Elisa法检测NPC输注后不同时期IL 4水平的动态变化发现 ,B6小鼠体内血清IL 4水平逐渐升高 ,在长期耐受的小鼠中 ,其水平增加尤其显著 ,甚至成倍的提高。结论　IL 4水平的升高对于诱导和保持免疫耐受起着重要的作用     

门静脉输注供者脾细胞诱导的供者特异性免疫低反应性

目的 探讨经门静脉输注供者脾细胞能否诱导皮肤移植小鼠产生供者特异性的免疫低反应性及其可能机制.方法 取Balb/c小鼠,随机分为空白对照组(经小鼠门静脉输注RPMI 1640培养液)、受者脾细胞组(经小鼠门静脉输注Balb/c小鼠脾细胞)、供者脾细胞组(经小鼠门静脉输注C57BL/6小鼠脾细胞)、空白移植对照组(经小鼠门静脉输注RPMI 1640培养液,7 d后移植C57BL/6小鼠的皮肤)、实验对照组(经小鼠门静脉输注Balb/c小鼠脾细胞,7 d后移植C57BL/6小鼠的皮肤)、实验组(经小鼠门静脉输注C57BL/6小鼠脾细胞,7 d后移植C57BL/6小鼠的皮肤)以及第三方移植组(经小鼠门静脉输注C57BL/6小鼠脾细胞,7 d后移植C3H小鼠的皮肤).记录空白移植对照组、实验对照组、实验组和第三方移植组移植皮肤的存活时间,并观察移植皮肤的病理学变化;脾细胞输注后7 d,分别获取空白对照组、受者脾细胞组和供者脾细胞组小鼠的外周血、脾脏和肝脏,用流式细胞仪测定样本中CD4+CD25+Foxp3+调节性T淋巴细胞(CD4+CD25+Foxp3+Treg细胞)的比例.结果 实验组移植皮肤的存活时间为(19.8±4.6)d,明显长于空白移植对照组、实验对照组和第三方移植组,但仍未达到长期存活.皮肤移植后7 d,空白移植对照组和实验对照组的移植皮肤呈现重度急性排斥反应的病理学改变,而实验组移植皮肤呈现中度急性排斥反应的病理学改变.供者脾细胞组外周血、肝脏和脾脏中CD4+CD25+Foxp3+Treg细胞比例明显高于空白对照组和受者脾细胞组.结论 门静脉输注供者脾细胞可特异性地延长供者皮肤移植物的存活时间,减轻移植物的排斥反应,该效应可能与受者体内的CD4+CD25+Foxp3+Treg细胞增加有关.     

睾丸支持细胞与共刺激通路阻断剂对异体移植肾细胞的保护作用

目的　研究表达Fas配体 (FasL)的睾丸支持细胞与共刺激通路阻断剂细胞毒性T细胞相关抗原 4免疫球蛋白 (CTLA 4Ig)对异体移植肾细胞的保护作用 ,以提高移植肾的免疫耐受性。方法　酶消化法制备睾丸支持细胞及肾细胞。取 10 6个细胞混合移植于大鼠肾包膜下 ,实验大鼠分为 3组 :对照组 (10只 )、混合细胞移植组 (混合移植组 ,16只 )、联合CTLA 4Ig组 (CTLA组 ,10只 )。分别于术后 1、7、14、2 0d检测血清白细胞介素 2 (IL 2 )水平变化。术后 2 0d取出移植物 ,以亲和素 生物素 过氧化物酶复合物技术观察肾细胞存活状况 ;MD 2 0图像分析系统测定混合移植物灰度值 ;末端脱氧核糖核酸转移酶介导的X dUTP缺口末端标记 (TUNEL)法观察移植物中凋亡的细胞。　结果对照组无移植物存活 ;混合移植组 14只移植物存活 ,灰度值为 0 36 2± 0 0 17;CTLA组 10只移植物均存活 ,灰度值为 0 4 4 5± 0 0 2 1;混合移植组与CTLA组间灰度值差异有显著意义。术后CTLA组血清IL 2水平较混合移植组低 ,差异有显著意义 ;混合移植物中可见凋亡的淋巴细胞。　结论　异体肾细胞移植中 ,睾丸支持细胞与CTLA 4Ig对移植肾细胞具有协同保护作用。     

CTLA4-Ig对肝细胞移植大鼠免疫耐受的作用及其机制

目的 探讨细胞毒性淋巴细胞相关抗原4融合蛋白(CTLA4-Ig)诱导肝细胞移植大鼠免疫耐受的作用及机制.方法 10%D-氨基半乳糖(D-gal)一次性腹腔注射建立大鼠急性药物性肝衰竭模型;采用肝脏原位灌注法分离纯化肝细胞,经脾脏移植后随机分为两组.实验组腹腔一次性注射CTLA4-Ig,对照组不予处理.两组均分别于术后第1、3、5、7天采外周血观察白细胞介素(IL)-2、肿瘤坏死因子(TNF)及肝功能变化;术后1周测两组大鼠外周血T细胞亚群,处死大鼠后取脾脏苏木素-伊红(HE)染色.结果 实验组谷丙转氨酶(ALT)、血清总胆红素(TBil)于术后第7天分别为(6.5±7.3)IU/ml、(5.1±1.6)mmol/L,低于对照组.术后治疗组IL-2含量明显下降,第7天达到(1. 3138±0.8508)ng/L,两组差异有统计学意义(P＜0.05);术后TNF含量两组之间差异无统计学差异(P＞0.05).外周血CD4+T细胞、CD4+/CD8+T细胞实验组分别为(37.3±7.2)%、(1.5±0.1)%,低于对照组(P＜0.05),CD8+T细胞两组差异无统计学意义(P＞0.05).术后第7天治疗组脾内仍可见肝细胞或肝细胞团,对照组见大量淋巴细胞浸润,但很少见肝细胞.结论 CTLA4-Ig能诱导经脾同种异体肝细胞移植大鼠免疫耐受,使急性肝衰大鼠肝功能得到改善.可能是抑制T淋巴细胞亚群,且主要是抑制CD4+T细胞,使CD4+/CD8+T细胞比值下降;抑制IL-2的分泌.

Abstract：

Objective To investigate the immunosuppressive effect of cytotoxic T Iymphocyte associated antigen 4 Ig fusion protein (CTLA4-Ig) in rat allograft hepatocyte transplantation model and the mechanisms. Methods Acute liver failure (ALF) model was established by intraperitoneal injection of 10% D-gal solution to SD rates. Collagenase perfusion was performed on SD rats to separate liver cells. SD rats with ALF were subjected to intrasplenic hepatocyte transplantation and randomly divided into two groups. The experimental group received intraperitoneal injection of CTLA4-Ig. The concentrations of interleukin (IL)-2 and tumor necrosis factor (TNF), liver function and histologicy were observed at the 1st,3rd, 5th, 7th day after operation and the T lymphocyte subsets were detected by using immunohistochemistry at the 7th day after operation. Results The levels of ALT and TBil were respectively (6. 5 ±7.3) IU/ml and (5.1 ± 1.6) mmol/L at the 7th day after operation and significantly decreased after injection of CTLA4-Ig ( P ＜ 0. 01 ). IL-2 concentration in the experimental group was ( 1.3138 ± 0. 8508 ) ng/L at the 7th day after operation and significantly decreased (P ＜0. 05). TNF had no significant difference between two groups after operation ( P ＞ 0. 05 ). T lymphocyte subsets, mainly CD4 + , in the experimental group was significantly decreased as compared with control group ( P ＜ 0. 05 ), so did the CD4 +/CD8 +. Histological changes: At the 7th day after operation, there were some hepatocytes in the spleen of the experimental group. But in the control group, the changes in the spleen were characterized by severe lymphocyte infiltration. There were no hepatocytes both groups. Conclusion CTLA4-Ig can induce rat allogeneic hepatocytes intrasplenic transplantation immune tolerance. It may improve the liver function of rats with ALF.CTLA4-Ig can decrease T lymphocyte subsets, mainly CD4 + and concentrations of IL-2.     

Anti-CD154 or CTLA4Ig obviates the need for thymic irradiation in a non-myeloablative conditioning regimen for the induction of mixed hematopoietic chimerism and tolerance

BACKGROUND: Thymic irradiation (TI) or repeated administration of T cell-depleting monoclonal antibodies (TCD mAbs) is required in a previously described non-myeloablative regimen allowing allogeneic marrow engraftment with stable mixed chimerism and tolerance. As both treatments might be associated with toxicity in the clinical setting, we evaluated whether T-cell costimulatory blockade could be used to replace them. METHODS: C57BL/6 mice received depleting anti-CD4 and anti-CD8 mAbs on day -5, 3 Gy whole body irradiation (day 0), and 15x10(6) fully MHC-mismatched, B10.A bone marrow cells. In addition, hosts were injected with an anti-CD154 mAb (day 0) and/or CTLA4Ig (day +2). Chimerism in peripheral blood was followed by flow cytometric (FACS) analysis, and tolerance was assessed by skin grafting, and also by mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) assays. The frequency of certain Vbeta families was determined by FACS to assess deletion of donor-reactive T cells. RESULTS: Chimerism was transient and tolerance was not present in animals receiving TCD mAbs on day -5 without costimulatory blockade. The addition of anti-CD154 and CTLA4Ig, alone or in combination, reliably permitted induction of high levels of stable (>6 months) multi-lineage chimerism, with specific tolerance to skin grafts and donor antigens by MLR and CML assays. Long-term chimeras showed deletion of donor-reactive CD4+ peripheral blood lymphocytes, splenocytes, and mature thymocytes. Administration of TCD mAbs only 1 day before bone marrow transplantation plus anti-CD154 also allowed induction of permanent chimerism and tolerance. CONCLUSIONS: One injection of anti-CD154 or CTLA4Ig overcomes the need for TI or prolonged host TCD in a preclinical model for the induction of mixed chimerism and deletional tolerance and thus further decreases the toxicity of this protocol. Achievement of tolerance with conditioning given over 24 hr suggests applicability to cadaveric organ transplantation.     

CTLA4-Ig和IL-4诱导异种骨移植免疫耐受的体外研究

目的探讨CTLA4Ig和IL4在诱导异种骨移植免疫耐受中的作用。方法反应细胞为BALB/c小鼠脾淋巴细胞,刺激细胞为新西兰白兔血淋巴细胞,刺激抗原为兔骨上清液。采用经典的混合淋巴细胞培养法及骨上清液与淋巴细胞混合培养法作为异种骨移植的体外实验模型。在各培养液中分别加入CTLA4Ig、IL4及两者联合应用,通过测定其3HTdR掺入率,观察不同细胞因子对刺激淋巴细胞增殖的影响。结果1细胞刺激组CTLA4Ig和IL4均对淋巴细胞增殖有显著抑制作用P<0.001),CTLA4Ig与IL4联合应用并未显示出比CTLA4Ig单独应用更为明显的细胞增殖抑制作用(P>0.05)。2骨上清液刺激组:CTLA4Ig对细胞增殖无抑制作用(P>0.05),而IL4则有较为显著地细胞增殖抑制作用(P<0.05);CTLA4Ig与IL4联合应用也未产生比单独应用IL4更为明显的细胞增殖抑制作用(P>0.05)。结论CTLA4Ig对由细胞刺激产生的淋巴细胞增殖抑制效果较好,而IL4则对骨上清液刺激的细胞增殖抑制作用更好;CTLA4Ig与IL4联合应用并未产生协同抑制作用。     

In vitro study of immune tolerance induced by CTLA4-Ig in bone transplantation: The effect on cell proliferation stimulated by lymphocytes and bone supernatant

To clarify the effect of CTLA4-lg on immune rejection of bone grafts, we observed the effect of CTLA4-lg on lymphocyte proliferation of BALB/C mice stimulated by lymphocytes and bone supernatant of C57BL/6 mice. The splenic lymphocytes and bone supernatant of C57BL/6 mice, as the stimulator cells and stimulator antigens, were cultured in vitro with the splenic lymphocyte of BALB/C mice. At the same time, CTLA4-lg at a dose of 5,10 or 20 &mgr;g/ml and L6 (as control) at 20 &mgr;g/ml were added. Six days later, the incorporation of 3 H-TdR was determined. Results indicated that CTLA4-Ig at a dose of 5, 10 or 20 &mgr;g/ml significantly inhibited the cell proliferation stimulated by lymphocytes and bone supernatant of C57BL/6 mice. The effect was non-cytotoxic. L6 showed no significant inhibition of cell proliferation. CTLA4-Ig can efficiently block the proliferation of alloresponsive T cell stimulated by lymphocytes and bone supernatant of C57BL/6 mice. This study provides a basis for further study of CTLA4-induced immune tolerance of bone grafts.     

CTLA4Ig和西罗莫司短期联合使用延长异种胰岛移植物的存活

目的 观察细胞毒性T淋巴细胞相关抗原4融合蛋白(CTLA4Ig)与西罗莫司(SRL)联用阻断共刺激通路对异种胰岛移植物存活的影响.方法 取C57BL/6小鼠,腹腔注射链佐星,制成糖尿病模型.采用随机单位组设计分组法将糖尿病小鼠分为7组,各组均于小鼠左肾包膜下移植SD大鼠胰岛300胰岛当量.CTLA4Ig组分别于移植当天及移植后第2、4、6天腹腔注射CTLA4Ig 0.5 mg/d;SRL组分别于移植当天及移植后第1、2天给予SRL灌胃,0.2 mg·kg-1·d-1,其后隔天用药1次,共用2周;MRI组分别于移植当天及移植后第2、4天腹腔注射仓鼠抗小鼠CD154单克隆抗体(MR1)0.5 mg/d;CTLA4Ig和SRL联用组(SRL联用组)、CTLA4Ig和MRl联用组(MR1联用组)以及CTLA4Ig、MR1和SRL联用组(三药联用组)各药物的剂量与用法同上述各组;对照组仅行胰岛移植,不予以药物.观察至移植后200 d,通过监测受者血糖水平来判断排斥反应的发生情况.记录各组移植物的存活(即无排斥反应)时间.发生排斥反应者,或未发生排斥反应、移植物存活时间＞200 d者,取移植胰岛,行HE染色及免疫荧光染色,进行组织学观察.结果 对照组移植物存活时间中位数为17 d,该组最终均发生排斥反应.SRL组、MRl组和CTLA4Ig组移植物存活时间中位数分别为34 d、98 d和77 d.均明显长于对照组(P＜0.05),三组中分别有90%(9/10)、62.5%(5/8)和83.3%(5/6)的小鼠发生排斥反应.SRL联用组移植物存活时间中位数为130 d,明显长于上述4组(P＜0.01),有50%(3/6)的小鼠发生排斥反应.MR1联用组以及三药联用组移植物存活时间中位数均＞200 d,分别有42.9%(3/7)和25%(2/8)的小鼠发生排斥反应.组织学检查结果显示,对照组发生排斥反应时,其移植胰岛破坏严重,可见大量CD4+和CD8+淋巴细胞及巨噬细胞浸润,并可见IgG、IgM和补体C3沉积.其它组发生排斥反应者的组织学改变与对照组相似.SRL联用组存活200 d的小鼠,其移植胰岛组织中未见或仅有少量炎症细胞浸润,胰岛素和胰高血糖素染色阳性,未见IgG、IgM和补体C3沉积.结论 短期联合使用CTLA4Ig和SRL能显著延长小鼠体内大鼠来源的胰岛的存活时间.     

Combined Anti‐CD154/CTLA4Ig Costimulation Blockade‐Based Therapy Induces Donor‐Specific Tolerance to Vascularized Osteomyocutaneous Allografts

Tolerance induction by means of costimulation blockade has been successfully applied in solid organ transplantation; however, its efficacy in vascularized composite allotransplantation, containing a vascularized bone marrow component and thus a constant source of donor‐derived stem cells, remains poorly explored. In this study, osteomyocutaneous allografts (alloOMCs) from Balb/c (H2^d) mice were transplanted into C57BL/6 (H2^b) recipients. Immunosuppression consisted of 1 mg anti‐CD154 on day 0, 0.5 mg CTLA4Ig on day 2 and rapamycin (RPM; 3 mg/kg per day from days 0–7, then every other day for 3 weeks). Long‐term allograft survival, donor‐specific tolerance and donor–recipient cell trafficking were evaluated. Treatment with costimulation blockade plus RPM resulted in long‐term graft survival (>120 days) of alloOMC in 12 of 15 recipients compared with untreated controls (median survival time [MST] ≈10.2 ± 0.8 days), RPM alone (MST ≈33 ± 5.5 days) and costimulation blockade alone (MST ≈45.8 ± 7.1 days). Donor‐specific hyporesponsiveness in recipients with viable grafts was demonstrated in vitro. Evidence of donor‐specific tolerance was further assessed in vivo by secondary donor‐specific skin graft survival and third‐party graft rejection. A significant increase of Foxp3⁺ regulatory T cells was evident in tolerant animals. Donor cells populated peripheral blood, thymus, and both donor and recipient bone marrow. Consequently, combined anti‐CD154/CTLA4Ig costimulation blockade‐based therapy induces donor‐specific tolerance in a stringent murine alloOMC transplant model.     

Changes in expression of T-cell activation-related molecules and cytokines during tolerance induction in an allogeneic skin transplantation murine model

Bone marrow transplantation after treatment with busulfan and costimulatory blockade with monoclonal antibodies (mAb) cytotoxic T lymphocyte antigen 4 (CTLA4)-Ig and anti-CD154 mAb or two-signal blockade using anti-CD45RB and anti-CD154 mAb are nonmyeloablative treatment regimens for allogeneic transplantation. There may be differences in the mechanisms of donor cell engraftment and reactive cell deletion by which these regimens induce donor-specific tolerance. Therefore, this study was performed to investigate changes in T cells and cytokines during tolerance induction toward allogeneic skin grafts. BALB/c and C57BL/6 mice were used as donors and recipients, respectively. Skin and bone marrow transplantations were performed and busulfan was administered. Three groups were treated with mAb as follows: group 1, anti-CD154 mAb; group 2, anti-CD154 plus anti-CD45RB mAb; and group 3, anti-CD154 mAb plus CTLA4-Ig. The proportions of CD4+ or CD8+ T cells and the expression of CD45RB isoforms on splenocytes were measured using flow cytometry and the production of cytokines by CD4+ T cells using enzyme-linked immunosorbent assay. Group 2 showed a significant reduction in the proportions of CD8+ T cells and CD45RB high isoforms compared with groups 1 and 3. The levels of interleukin (IL)-2 and IL-4 in group 2 were lower and higher than those of groups 1 and 3, respectively. In conclusion, the combined use of anti-CD154 and anti-CD45RB mAb decreases the CD8+ T-cell population and the expression of CD45RB, resulting in a Th2 cytokine profile, which may be a characteristic mechanism leading to donor cell engraftment and reactive cell deletion for donor-specific tolerance.     

骨髓输注联合阻断共刺激通路延长大鼠皮肤移植物的存活时间

目的 探讨骨髓输注联合阻断共刺激通路对大鼠移植皮肤存活的影响及可能机制.方法 以Lewis大鼠为受者,皮肤移植前经尾静脉输注供者(BN大鼠)骨髓细胞2×108 个,并于骨髓输注当天、输注后第2、4、6及8天腹腔注射抗CD25单克隆抗体,同时按照分组要求腹腔注射细胞毒性T淋巴细胞相关抗原4融合蛋白(CTLA4Ig组)、抗CD154单克隆抗体(抗CD154单抗组)以及CTLA4Ig和抗CD154单克隆抗体(联合处理组),于骨髓输注后第8天移植BN大鼠的皮肤.另以仅行皮肤移植者为对照(对照组).观察各组移植物抗宿主病(GVHD)的发生情况、外周血中供者细胞嵌合率、T淋巴细胞凋亡率及移植皮肤的存活时间.结果 各组均未观察到GVHD的发生.骨髓输注后第7天即可在CTLA4Ig组、抗CD154单抗组及联合处理组观察到嵌合现象,至第21天时,嵌合率仍维持于一定水平,联合处理组明显高于其它三组(P＜0.01).骨髓输注后第7及21天,CTLA4Ig组、抗CD154单抗组及联合处理组间两两比较,T淋巴细胞凋亡率的差异无统计学意义,但均显著高于对照组(P＜0.05,P＜0.01).CTLA4Ig组、抗CD154单抗组及联合处理组移植皮肤的存活时间显著长于对照组(P＜0.01),联合处理组移植皮肤存活时间为(16.7±3.1)d,明显长于CTLA4Ig组和抗CD154单抗组(P＜0.05).结论 骨髓输注联合阻断共刺激通路能够延长移植皮肤存活时间,其机理可能与诱导嵌合和T淋巴细胞凋亡有关.     

Combined Coinhibitory and Costimulatory Modulation with Anti-BTLA and CTLA4Ig Facilitates Tolerance in Murine Islet Allografts

Complex interactions between positive and negative cosignaling receptors ultimately determine the fate of the immune response. The recently identified coinhibitory receptor, B and T lymphocyte attenuator (BTLA), contributes to regulation of autoimmune and potentially alloimmune responses. We investigated the role of BTLA in a fully major histocompatibility complex-mismatched mouse islet transplant model. We report that anti-BTLA mAb (6F7) alone does not accelerate graft rejection. Rather, while CTLA4Ig alone improved allograft survival, the addition of anti-BTLA mAb to CTLA4Ig led to indefinite (>100 days) allograft survival. Immediately after treatment with anti-BTLA mAb and CTLA4Ig, islet allografts showed intact islets and insulin production despite a host cellular response, with local accumulation of Foxp3+ cells. We clearly demonstrate that combined therapy with anti-BTLA mAb and CTLA4Ig mice induced donor-specific tolerance, since mice accepted a second donor-specific islet graft without further treatment and rejected third party grafts. CTLA4Ig and anti-BTLA mAb limited the initial in vivo proliferation of CFSE-labeled allogeneic lymphocytes, and anti-BTLA mAb enhanced the proportion of PD-1 expressing T cells while depleting pathogenic BTLA+ lymphocytes. We conclude that targeting the BTLA pathway in conjunction with CTLA4Ig costimulatory blockade may be a useful strategy for promoting immunological tolerance in murine islet allografts.