IL-4与IL-10联合诱导异种骨移植免疫耐受的体外研究

目的　探讨 IL - 4和 IL - 10在诱导异种骨移植免疫耐受中的作用。方法　反应细胞为 BAL B/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 IL - 4、IL - 10及两者联合应用 ,通过测定其 3H- Td R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　无论在细胞刺激组还是骨上清液刺激组 ,IL- 4对淋巴细胞增殖均有显著抑制作用 (P<0 .0 0 1和 P<0 .0 5 ) ,IL- 10未表现出抑制作用 (P>0 .0 5 )。在两组 IL- 4和 IL - 10联合应用均产生比 IL - 4单独应用更为明显的细胞增殖抑制作用 (P<0 .0 0 1和 P<0 .0 5 )。结论　 IL - 4对由细胞或骨上清液刺激产生的淋巴细胞增殖均有很好的抑制作用 ,IL- 10没有表现出抑制作用 ;IL- 4与 IL- 10联合应用有协同抑制作用。     

CTLA4-Ig抑制异种混合淋巴细胞反应

目的：研究共刺激分子细胞毒性T细胞抗原4（CTLA4）对人－猪异种移植反应中，T细胞活化的抑制作用。方法：用转染CTLA4－Ig融合基因的猪血管内皮细胞系（PIEC）和CTLA4－Ig融合蛋白抑制T细胞增殖反应，结果：B7／CD28共刺激信号途径在异种细胞排斥反应中起重要作用，CTLA4－Ig的抑制效应没有对同种细胞反应的影响强。结论：上述结果的机制可能是因 种间分子不相容性降低协同分子的匹配性，或者异种移植反应中其它协同分子“替代性”参与了辅佐信号的传导。     

无能T淋巴细胞的建立及其免疫生物学特性的研究

目的探讨体外诱导T淋巴细胞无能的条件,并分析无能T淋巴细胞的免疫生物学特性。方法以Wistar大鼠的脾细胞为刺激细胞,SD大鼠的脾细胞为反应细胞,进行混合淋巴细胞反应(MLR),分别单独或联合加入剂量不等的抗B71单克隆抗体(抗B71单抗)、抗B72单克隆抗体(抗B72单抗),制备无能T淋巴细胞。将无能T淋巴细胞和刺激细胞混合,分别加入抗CD3单克隆抗体、刀豆蛋白(ConA)及白细胞介素2(IL2),分析各种情况下无能T淋巴细胞的增殖情况;将刺激细胞与同种异体SD大鼠脾细胞混合,分别加入不同数量的无能T淋巴细胞,分析SD大鼠淋巴细胞的增殖情况。结果单独应用抗B71单抗或抗B72单抗,对淋巴细胞增殖的抑制作用不明显,而将二者联用,能明显阻断T淋巴细胞增殖;ConA及IL2能使无能T淋巴细胞增殖,而抗CD3单克隆抗体不能使无能T淋巴细胞发生增殖;无能T淋巴细胞对同种异体T淋巴细胞的增殖也产生抑制作用,该效应具有抗原特异性。结论联合抗B71单抗和抗B72单抗可诱导T淋巴细胞无能;无能T淋巴细胞在体外呈免疫无反应性,并能抑制同种异体T淋巴细胞的增殖,该抑制作用具有抗原特异性。     

重组腺相关病毒载体介导CTLA4Ig转染延长同种大鼠心脏移植存活时间

目的　研究重组腺相关病毒载体介导 CTLA4Ig(rAAV CTLA4Ig)全身转染对大鼠同种心脏移植的影响。方法　以BN大鼠为供者,Lewis大鼠为受者,建立心脏移植模型。实验分为两组。对照组:供、受者不给予任何处理;转染组:受者于心脏移植前30 d,通过尾静脉全身注射 1×109斑点形成单位(PFU)的 rAAV CTLA4Ig。观察移植心存活时间;ELISA法检测血清 CTLA4Ig、干扰素 γ(IFN γ)和白细胞介素 4(IL 4)水平;免疫组织化学法(SABC法)检测移植心组织中 CD4 和CD8 T细胞的浸润。对移植心存活时间明显延长的受者,用其脾细胞与供者脾细胞进行混合淋巴细胞培养,观察受者对供者的免疫反应状态。结果　转染组移植心存活时间较对照组明显延长(P<0.05);转染组血清CTLA4Ig蛋白一直维持在26.67~35.47 mg/L,移植后转染组血清 IFN γ水平下调,血清 IL 4水平上调(P<0.05);对照组出现典型的急性排斥反应表现,转染组心肌组织基本正常,间质内无炎性细胞浸润或血管外周及心肌间质内有局灶性炎性细胞浸润,未见坏死;对照组移植心组织中浸润的CD4 和CD8 T细胞数量明显高于转染组(P<0.01);转染组受者对供者的脾细胞增殖反应明显低于对照组(P<0.05)。结论　重组腺相关病毒载体可以介导 CTLA4Ig基因的持续表达,通过全身途径转染受者可以明显延长     

冻干同种与异种骨移植免疫反应的比较研究

目的: 通过比较冻干同种与异种骨移植免疫反应, 探讨异种骨移植排斥机制。方法: 取C57BL/6小鼠 10只和新西兰兔 1只, 分别为同种和异种骨供体, 通过处理制成冻干骨。取 40只BALB/c小鼠为骨移植受体, 随机分为A、B两组 (每组 20只), 分别在其股后肌袋植入冻干同种和冻干异种骨。分别于术后 1、2、4、6周分批取材检测。通过观察术后受者的淋巴细胞刺激指数、淋巴细胞亚群分析、细胞因子产生及组织学表现, 比较冻干同种与异种骨移植免疫反应。结果: 冻干异种骨移植组的细胞刺激反应在各不同时期均比同种组强 (P<0. 05 ), 而且其CD4 和CD8 T细胞亚群及IL 2分泌均高于同种组 (P<0. 05)。组织学检查也表明其细胞浸润多、成骨少。结论: 冻干同种与异种骨移植排斥机制基本相同, 二者均以Th1反应为主, 但异种骨移植排斥反应较强。     

转染CTLA4Ig基因的树突状细胞抑制反应性T细胞增殖

目的 探讨转染人细胞毒性T淋巴细胞相关抗原4免疫球蛋白（CTLA4Ig）基因的树突状细胞（DCsRev）对反应性T细胞的作用。方法 通过重组逆转录病毒将目的基因CTLA4Ig转染到大鼠骨髓来源的树突状细胞（DCs）中。采用逆转录聚合酶链反应（RT-PCR）和斑点酶联免疫吸附试验（Dot-ELISA）检测CTLA4Ig在DCs中的表达；采用混合淋巴细胞反应（MLR）检测DCsRev对反应性T细胞的作用。结果 检测结果证实CTLA4Ig基因成功转染至DCs。DCsRev的数量以及用DCsRev预处理反应性T细胞的时间长短与抑制MLR中T细胞增殖有一定的效应关系。当DCsRev数量在10^3～10^4。之间时，其抑制率最高，抑制率可达到为69．12％；用DCsRev预处理反应性T细胞12 h时，其对MLR中T细胞增殖的抑制率最高，为98．3％，12 h以后，随着预处理时间的延长，抑制率却不断下降。经DCsRev诱导的大鼠体内脾淋巴细胞在MLR中增殖低下。结论 转染CT-LA4Ig基因的DCs不但丧失了刺激MLR的能力，并且能够抑制MLR中反应性T细胞的增殖，提示DCsRev可能诱导抗原特异性T细胞的免疫耐受。     

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。     

免疫耐受与移植

本文概述了通过主动诱导、阻断免疫应答和建立白细胞微嵌合体诱导耐受的最新进展。细胞毒性T细胞相关抗原4(CTLA4)可通过阻断共刺激使T细胞无能,诱导耐受。IL-4和IL-10是T辅助细胞(Th)2细胞因子,能诱使免疫向Th2方向偏离,促进耐受形成;IL-10也是细胞因子合成抑制因子,可通过抑制宿主抗原提呈细胞抑制免疫。重点阐述了CTLA4和IL-4、IL-10的作用机制和研究现状,各种诱导方法的联合应用及其在异种移植中的具体实施。在用CTLA4成功诱导同种异体骨移植耐受的基础上,探讨了异种骨移植的特点,以及通过诱导免疫耐受防止异种骨移植排斥的可行性。     

同种异体与异种骨移植免疫反应的比较研究

目的比较分析同种异体与异种骨移植后的免疫反应,观察IL-4和IL-10对免疫反应的影响。方法取C57BL/6小鼠和新西兰兔作为同种异体和异种骨供体,分别制成新鲜骨和冻干骨。于股后肌袋内分别植入新鲜同种异体、新鲜异种、新鲜异种(给予IL-4和IL-10治疗)、冻干同种异体及冻干异种骨。结果在植入新鲜骨的各组中,新鲜异种组早期细胞刺激反应较新鲜同种组强(P< 0.05)。1周时新鲜异种组CD4 T细胞亚群和CD4/CD8比值与新鲜同种组间差异无显著性,但2周时明显低于新鲜同种组。细胞因子检测显示,新鲜异种组的IL-2分泌较新鲜同种组多(P< 0.05),而且IFN-γ分泌少,IL-4分泌多(P<0.05)。组织学检查发现,新鲜异种组细胞浸润多,且以单核、巨噬细胞为主。与新鲜异种组相比,用IL-4和IL-10治疗后明显抑制了骨抗原二次刺激引起的细胞增殖(P<0.01);早期IFN-γ分泌增加,2周时IFN-γ和IL-4分泌均明显减少(P<0.05);组织学检查显示,注射IL-4和IL-10后植骨处局部炎细胞浸润减少,诱导成骨增多。而在植入冻干骨的各组中,冻干异种组的细胞刺激反应始终比冻干同种组强(P<0.05),而且其CD4 和CD8 T细胞亚群及IL-2分泌均高于同种异体组(P<0.05)。结论新鲜异种骨移植早期排斥反应强、后期弱,早期以Th2反应为主,而同种异体骨移植免疫排斥以Th1反     

CTLA4-Ig与器官移植免疫耐受的诱导

应用CTLA4Ig或导入CTLA4Ig基因在动物器官移植中诱导了免疫耐受。其主要作用机制是CTLA4Ig可以阻断B7:CD28/CTLA4共刺激通路,阻碍第二信号的传递,从而抑制T细胞活化。CTLA4Ig为诱导器官移植后的免疫耐受提供了新的方法。     

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.     

细胞毒性T淋巴细胞相关抗原4诱导新鲜异体骨移植免疫耐受的实验研究

目的　利用外源性细胞毒性T淋巴细胞相关抗原4 ( cytotoxic T lymphocyte- associated antigen 4immuno globlin,CTL A4 - Ig)阻断T细胞反应的第2信号,诱导新鲜同种异体骨移植免疫耐受。　方法　采用近交系小鼠BAL B/C 6 6只作为骨移植的受体,进行异位肌袋一次及第2次骨移植。一次移植分为3组,每组18只,一组移植C5 7BL/6小鼠骨骼,注射L6 (对照品) ,为AL组;一组移植C5 7BL/6小鼠骨骼,注射CTL A4 - Ig,为AC组;一组移植同系BAL B/C小鼠骨骼,注射PBS缓冲液,为AB组。在第2、4及6周,进行血淋巴细胞亚群分析,血清抗体测定,供体细胞及抗原二次刺激实验及组织学观察。另取12只BAL B/C小鼠,进行第2次移植实验,供体为C5 7BL/6小鼠的骨骼,注射CTL A4 - Ig后2周,分别移植C5 7BL/6 ( BC组)和C3H( BH组)小鼠的骨骼,检测产生免疫耐受的特异性。　结果　AL组与AB组比较,产生了强烈的免疫排斥反应,移植后2、4及6周,CD4 T细胞的增殖明显增加( P<0 .0 5 ) ,血清抗体明显增多( P<0 .0 5 ) ,供体细胞及骨抗原二次刺激的细胞增殖也明显增加( P<0 .0 5 ) ;AC组免疫排斥反应较轻,在CD4 T细胞的增殖、血清抗体及二次刺激的细胞增殖方面均与AB组相似( P>0 .0 5 ) ;组织学观察也显示,异体骨周围的淋巴细胞浸润消失,软骨诱导及新骨形     

Lentiviral expression of CTLA4Ig inhibits primed xenogeneic lymphocyte proliferation and cytokine responses

BACKGROUND: Co-stimulatory blockade is known to inhibit lymphocyte responses and to prolong allograft and xenograft survival. The present study examines the effect of transgenic expression of cytotoxic T lymphocyte-associated molecule-4 immunoglobulin (CTLA4Ig) by a porcine endothelial cell line (PIEC) transduced by a lentiviral vector, on primed xenogeneic T-cell proliferative and cytokine responses. METHODS: Splenocytes from mice primed with PIEC were used as responder cells in a secondary proliferative assay. CTLA4Ig transduced and wild-type PIEC were used as stimulator cells. Responder cells were assayed for proliferation and cytokine production. RESULTS: Proliferation was profoundly inhibited by CTLA4Ig transduced cells compared with control cells. Cytokine analysis by enzyme linked immunospot demonstrated that production of interferon-gamma, IL4 (interleukin 4) and IL10 was inhibited by CTLA4Ig transduced cells compared with control cells. CONCLUSION: CTLA4Ig inhibited primed indirect xenogeneic T-cell proliferative and cytokine responses in vitro. Expression of immunomodulatory molecules by xenogeneic tissues has potential therapeutic applications for future xenotransplantation.     

Local expression of IDO, either alone or in combination with CD40Ig, IL10 or CTLA4Ig, inhibits indirect xenorejection responses

Abstract: Background: To overcome cell‐mediated xenorejection by transgenic expression of immunomodulatory molecules by a graft, it is likely that expression of multiple molecules will be required. Previous studies support the use of the immunomodulatory agents indoleamine 2,3‐dioxygenase (IDO), CD40Ig, interleukin 10 (IL10), and CTLA4Ig for suppression of rejection responses. We examined the effects of local expression of these molecules by a porcine cell line (PIEC) on indirect murine xenorejection responses in vitro and in vivo. Methods: The PIEC stable lines expressing IDO, CD40Ig, and IL10 as single molecules were generated. In addition, PIEC lines expressing IDO with either CD40Ig, IL10 or CTLA4Ig were generated to produce cell lines expressing two molecules. BALB/c mice were primed with wild type PIEC, followed by harvesting splenocytes used as responder cells and PIEC expressing immunomodulatory molecules as stimulators, in proliferation and cytokine assays. In vivo effects of modified PIEC were examined by transplantation of PIEC lines expressing the immunomodulatory molecules under the renal capsule of naïve mice. PIEC grafts were harvested for histological evaluation at days 7 and 14. Results: Proliferation of primed BALB/c splenocytes was inhibited most significantly by IDO compared with control cells (49%, P = 0.02). In addition both Th1 (interferon‐gamma) and Th2 (IL4 and IL10) cytokines were markedly inhibited in vitro by IDO expression. IL10 expressing cells did not inhibit proliferation as potently (37%, P = 0.03) whilst CD40Ig lead to an increase in proliferative responses (59%, P = 0.02). Co‐expression of CD40Ig, IL10, and CTLA4Ig with IDO resulted in further modest reductions in proliferation compared with IDO expression alone. When transplanted under the renal capsule of BALB/c mice, those grafts expressing IDO demonstrated significantly lower levels of lymphocyte infiltration at days 7 and 14 than control grafts and those expressing CD40Ig, CTLA4Ig or IL10 alone. Grafts co‐expressing IDO and a second molecule were no better protected than those expressing IDO alone. Graft cell viability (PIECs) was reduced in some IDO expressing grafts suggesting high levels of IDO expression may inhibit PIEC viability, however, grafts co‐expressing IDO‐CTLA4Ig and IDO‐IL10 were not affected in this way. Conclusion: Indoleamine 2,3‐dioxygenase appears to be a potent molecule for protecting xenografts from cell‐mediated rejection responses activated via the indirect pathway. Co‐expression of IDO with both CTLA4Ig and IL10 warrants further investigation. Overall these findings support pursuing further studies, in larger animal models, to determine whether increased IDO activity within the graft itself can attenuate xenorejection responses.     

CTLA4Ig基因对大鼠胰岛移植后排斥反应的治疗作用

目的　研究CTLA4Ig基因在糖尿病大鼠体内表达及其产物对胰岛移植物存活的作用。方法　利用Lipofectin载体包裹CTLA4IgcDNA质粒后转染鼠胰岛和肌肉细胞 ,检测移植后CT LA4Ig表达和T淋巴细胞转化率。结果　胰岛移植术后 7dT淋巴细胞转化试验 ,实验组 (A组 )和对照组 (B组 )每分钟脉冲数 (cpm)分别为175 .7± 98.2 ,2 5 4.4± 116 .3 ,两组比较差异显著 (P <0 .0 5 )。A组胰岛移植第 7d ,2只大鼠血清CTLA4Ig呈阳性 (阳性率 2 0 % )。A、B两组胰岛移植后血糖维持正常时间分别为 (14.8± 12 .3)d和 (3 .6± 5 .1)d ,两组比较差异显著 (P <0 .0 5 )。A、B两组大鼠平均存活时间分别为 (2 4.0± 10 .8)d和 (10 .8± 4.8)d ,两组比较 ,差异有极显著性 (P <0 .0 1)。结论　脂质体包裹的CTLA4IgcDNA转染肌细胞和胰岛细胞 ,可以在受体大鼠胰岛细胞或肌肉组织中表达 ,其表达产物可使胰岛移植物和受体鼠存活时间明显延长 ,抑制细胞免疫活性 ,发挥其治疗排斥反应的作用     

Negative and positive co-signaling with anti-BTLA (PJ196) and CTLA4Ig prolongs islet allograft survival

The novel coinhibitory receptor B and T lymphocyte attenuator (BTLA) has been implicated in the regulation of autoimmune and may potentially play a role in alloimmune responses. An anti-BTLA monoclonal antibody has been reported to prolong fully major histocompatibility complex-mismatched cardiac allograft survival, and we test the hypothesis that anti-BTLA monoclonal antibody PJ196 may synergize with cytotoxic T lymphocyte antigen-4 immunoglobulin (CTLA4Ig) costimulatory blockade in islet transplantation. We investigated the potential of PJ196, and show that it did not deplete BTLA expressing cells, but it caused down-regulation of BTLA on the surface of lymphocytes and accumulation of cells with regulatory phenotype at the graft site, promoting islet allograft acceptance together with CTLA4Ig. The combination of BTLA coinhibitory modulation and CTLA4Ig costimulatory blockade may be an effective adjunctive strategy for inducing long-term allograft survival.     

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