CTLA-4 Ig腺病毒基因治疗联合供体细胞输注诱导混合嵌合体和大鼠心脏移植耐受

目的　在心脏移植手术中实施CTLA 4Ig腺病毒基因治疗并联合术后输注供体骨髓细胞 ,诱导异基因大鼠心脏移植耐受 ,并对相关机制进行研究。方法　将异基因DA大鼠的心脏移植给受体LEW大鼠 ,同时经门静脉输注供体DA的脾细胞 (SC ,3× 10 8)、CTLA 4Ig腺病毒 [( 1～ 5 )× 10 9PFU ml],第 4天由舌静脉输注DA的骨髓细胞 (BMC ,3× 10 8)。观察、记录心脏移植物的存活时间。并对皮肤移植的受体作同样处理 ,观察皮肤移植存活情况。通过MLR、IL 2逆转实验及嵌合体的测定 ,探讨耐受机理 ,并检测了CTLA 4Ig的体内表达、TH1 TH2型细胞因子的表达。结果　单用CTLA 4Ig腺病毒基因治疗 ,或CTLA 4Ig腺病毒基因治疗联合单独的供体脾细胞或骨髓细胞能不同程度地延长异基因心脏移植物的存活 ,但不能延长皮肤移植物的存活。脾细胞、CTLA 4Ig腺病毒和骨髓细胞(SC Ad BMC)处理组的心脏移植物存活时间明显超过其它各耐受诱导组 ,并且能够诱导皮肤耐受。RT PCR实验证明 ,在受体内不同的组织CTLA 4Ig基因的表达量有所不同 ,并且随着时间的推移表达下降。TH1和TH2型细胞因子的检测显示 ,耐受大鼠体内未发现这两类细胞因子的偏移现象。MLR证明耐受大鼠的免疫应答表现为供体特异性降低 ,IL 2逆转实验、嵌合体检测表明 ,该耐受可能与     

人CTLA4Ig腺病毒载体的构建及其生物学功能研究

目的：通过同源重组构建CTLA4Ig腺病毒载体。研究其生物学活性，并用于基因治疗诱导心脏移植耐受。方法：通过基因重组技术，将CTLA4Ig基因克隆至腺病毒穿梭质粒pCA13中，然后将该质粒与腺病毒辅助质粒同源重组，经过293细胞包装，构建CTLA4Ig腺病毒载体，用RT-PCR，SDS-PAGE及Western blot等技术，检测包装的病毒感染293细胞后CTLA4Ig蛋白的表达及分泌，通过体外实验，观察病毒感染的293细胞上清对混合淋巴细胞反应（MLR）的抑制作用，通过大鼠体内实验，检测用CTLA4Ig腺病毒载体基因治疗后，CTLA4Ig在体内的表达及分泌，通过体外实验，观察病毒感染的293细胞上清对混合淋巴细胞反应（MLR）的抑制作用，通过大鼠体内实验，检测用CTLA4Ig腺病毒载体基因治疗后，CTLA4Ig在体内的表达。结果：CTLA4Ig腺病毒载体构建成功，体外实验证实，CTLA4Ig腺病毒感染的293细胞上清液，能够抑制异基因脾细胞的单向MLR，体内实验表明，经过静脉途径给予受体大鼠CTLA4Ig腺病毒载体，能够诱导移植耐受，延长心脏移植物的存活。结论：构建的CTLA4Ig腺病毒载体，体外感染293细胞能够分泌CTLA4Ig蛋白，该蛋白可抑制T细胞的活化，CTLA4Ig腺病毒载体可用于体内基因治疗诱导移植耐受。     

联体共生诱导异基因大鼠心脏移植耐受

目的通过异基因大鼠联体共生诱导心脏移植耐受并探讨其耐受机理.方法将DA和LEW的脾细胞(2×108)分别经舌静脉注射给对方,2d后均经腹腔注射环磷酰胺(80mg/kg),第3天实施联体手术,联体1w后分开,在DA→LEW之间进行异位心脏移植手术.观察、记录移植物的存活时间,通过胸腺和脾脏内嵌合体检测、MLR、体内细胞转移、IL-2逆转实验,探讨耐受机理.结果异基因心脏移植物的存活时间明显延长;嵌合体检测显示耐受的形成和嵌合体的存在成相关性,MLR和体内细胞转移实验证明,受体大鼠的免疫应答受抑制表现为供体特异性,并且受体存在抑制细胞;IL-2逆转实验表明该耐受与克隆失活(anergy)有关.结论通过联体共生诱导大鼠耐受、延长心脏移植存活,移植耐受的形成涉及多种机制.     

供体骨髓细胞促进大鼠心脏移植耐受及机制的研究

经门静脉给予受体LEW大鼠3×10~8个DA大鼠供体脾细胞,2d后腹腔注射80mg/kg的环磷酰胺,第4天由舌静脉输注1×10~8骨髓细胞。2周后实施心脏移植手术。观察、记录移植物的存活时间,通过过继性转移实验,MLR、CTL活性测定及嵌合体分析,探讨耐受机制。结果表明,异基因心脏移植物的存活时间进一步延长;该耐受状态可被过继性转移;MLR、CTL活性表明,受体大鼠的免疫应答被特异性抑制;嵌合体水平提高。结论:异基因骨髓细胞输注可加深由脾细胞和环磷酰胺诱导的大鼠心脏移植耐受,该耐受与嵌合体水平、抑制细胞的存在有关。输注骨髓细胞对于改善、维持耐受状态,延长大鼠心脏移植物存活时间是一有效的方法。     

门静脉输注供体脾细胞诱导基因大鼠心脏移植耐受

目的通过门静脉输注同种异基因脾细胞、腹腔给予环磷酰胺诱导同种心脏移植耐受 ,并探讨其耐受机理。方法经门静脉给予受体大鼠 3× 10 8个异基因供体脾细胞 ,2 d后腹腔注射 80 m g/kg的环磷酰胺 ,10 d后实施心脏移植手术。观察、记录移植物的存活时间 ,通过 ML R、DTH、IL- 2逆转实验及过继性转移实验 ,探讨耐受机理。结果异基因心脏移植物的存活时间明显延长 ;ML R和 DTH实验证明 :受体大鼠的免疫应答受抑制 ,且该耐受表现为供体特异性 ;IL- 2逆转实验、过继性转移实验表明 :该耐受与克隆失活、抑制细胞和“感染”耐受机理有关。结论本诱导方案对于大鼠心脏移植存活时间的延长是一有效的方法 ,移植耐受的形成涉及多种机制。     

诱导HO-1表达延长大鼠心脏移植物存活时间

目的了解血红素加氧酶-1(HO-1)在延长异基因大鼠心脏移植物存活中的作用及其机制。方法雄性LEW(RT11)和LEW.1W(RT1u)大鼠分别作为供体和受体,受体鼠分别应用CoPPIX 5 mg/(kg.d),SnPPIX5 mg/(kg.d)。对照组及实验组均6只鼠。由移植手术前1日开始至发生排斥,比较心脏移植物存活时间,分别在移植后第5天取心脏进行HE染色及CD3、CD25、ED1和K i-67染色;并测定HO-1活性和用W estern b lot定量HO-1蛋白;进行受体脾细胞接受供体脾细胞刺激的混合淋巴细胞培养。结果对照组心脏移植物平均7 d出现排斥;应用CoPPIX诱导HO-1表达后存活时间为13.5 d,显著长于对照组(P<0.05)。而应用SnPPIX治疗后存活时间为6.5 d。对照组与SnPPIX治疗组的心脏移植物中有中等量的细胞浸润,CoPPIX治疗组移植物组织中的CD3 T细胞、CD25 细胞、巨噬细胞和增殖细胞都明显少于SnPPIX及对照组。同对照组相比,CoPPIX诱导HO-1表达明显抑制体内脾细胞增生(P<0.05),而SnPPIX无抑制。结论诱导HO-1表达能够抑制大鼠淋巴细胞的免疫活性并延长异基因心脏移植物的存活。     

诱导HO-1表达延长大鼠心脏移植存活时间

目的 了解血红素加氧酶-1（HO-1）在延长异基因大鼠心脏移植物存活中的作用及其机制。 方法 雄性LEW (RT1l)和LEW.1W(RT1u)大鼠分别作为供体和受体，受体鼠分别应用CoPPIX (5mg·kg-1·day-1), SnPPIX (5mg·kg-1·day-1)对照组及实验组，各组均6只鼠。由移植手术前1日开始至发生排斥，比较心脏移植物存活时间，分别在移植后第5天取心脏进行HE染色及CD3、CD25、ED1和Ki-67染色；并测定HO-1活性和用Western blot定量HO-1蛋白；进行受体脾细胞接受供体脾细胞刺激的混合淋巴细胞培养。结果 对照组心脏移植物平均7d出现排斥；应用CoPPIX诱导HO-1表达后存活时间为13.5d，显著长于对照组(p<0.05)。而应用SnPPIX治疗后存活时间为6.5d。对照组与SnPPIX治疗组的心脏移植物中有中等量的细胞浸润，CoPPIX治疗组移植物组织中的CD3+T细胞、CD25+细胞、巨噬细胞和增殖细胞都明显少于SnPPIX及对照组。同对照组相比，CoPPIX诱导HO-1表达明显抑制体内脾细胞增生(p<0.05)，而SnPPIX无抑制。结论 诱导HO-1表达能够抑制大鼠淋巴细胞的免疫活性并延长异基因心脏移植物的存活。     

а-MSH基因转染的小鼠同种异体移植心脏存活期延长

目的 观察a-黑素细胞刺激素(a-MSH)基因原位转染供者小鼠心脏对小鼠同种异体心脏移植排斥反应的影响.方法 将携带a-MSH基因和绿色荧光蛋白(GFP)基因的重组腺相关病毒(rAAV-a-MSH)和单纯携带GFP的重组腺相关病毒(rAAV-GFP)分别经主动脉灌注原位转染供者小鼠心脏,再行同种异体心脏移植,观察心脏移植物的存活时间.测定移植心脏的GMSH表达情况,并于术后7 d测定移植心脏的细胞因子及趋化因子水平.结果 腺相关病毒载体能有效地将a-MSH基因导人小鼠心脏,与rAAV-GFP空载体组[(7.0±0.33)d]和PBS组[(7.0±2.23)d]相比,rAAV.a-MSH原位转染组[(16.3±2.21)d]的心脏移植物存活时间延长.术后7 d,rAAV-a-MSH原位转染组心脏移植物的Thl细胞因子(IL-2、IFN-a)水平较其它两组减低,而Th2细胞因子(IL4、IL-10)水平升高,趋化因子MCP-I、RANTES水平下降.结论 携带a-MSH基因的rAAV载体经原位转染供者小鼠心脏,能促进移植心脏局部的细胞因子格局由Th1型向Th2型偏移,降低趋化因子水平,从而延长同种异基因移植心脏的存活时间.     

抗原负载的免疫缺陷树突状细胞诱导异种胰岛移植耐受

目的:探讨负载异种MHC抗原的免疫缺陷树突状细胞(dendriticcell,DC)预处理受体对异种胰岛移植的耐受诱导作用及其机制。方法:从BALB/c小鼠骨髓干细胞分别诱导分化成熟DC及免疫缺陷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,7天后分别将Wistar或SD大鼠胰岛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细胞因子表达。结果:对照组胰岛存活时间为8.2±1.1天;成熟DC组胰岛存活时间缩短为6.1±1.1天(P<0.05);免疫缺陷DC组胰岛存活时间显著延长,为42.3±3.5天(P<0.05)。SD大鼠胰岛移植物平均存活时间与正常受体组无差异。与正常受体鼠相比,成熟DC预处理组的T细胞增殖反应强烈,而Th1/Th2细胞因子水平无明显差异。免疫缺陷DC预处理组的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,从而有效延长异种胰岛存活时间。     

FK506和CTLA4Ig转基因腺病毒在耐受诱导中的作用

目的　评价Tacrolimus(FK5 0 6 )和细胞毒性T淋巴相关抗原4免疫球蛋白(CTLA4Ig)转基因腺病毒(AdCTLA4Ig)在耐受诱导中的作用。方法　按照是否接受FK5 0 6处理、心脏移植后有无骨髓细胞输注(bonemarrowtransfusion ,BMT)、术后有无AdCTLA4Ig输注及移植心脏来源,将实验动物随机分为8组,观察各组心脏移植物的存活时间,于术后第30天测定其中4组的混合淋巴细胞反应的特异性和非特异性抑制率以及其中4组的异基因嵌合水平。结果　无处理对照组移植心脏存活时间为(6 .5±0 .5 5 )d ;AdCTLA4Ig单一处理组及联合BMT组移植心脏存活时间分别为(37.5±2 .88)d和(39.2±3.31)d ,与无处理组比较,差异有统计学意义(P <0 .0 5 ) ,当把FK5 0 6处理加入到上述2组的处理方案中时,移植心脏存活时间缩短(P <0 .0 5 )。术后30d混合淋巴细胞反应(MLR)结果显示,AdCTLA4Ig +BMT组和AdCTLA4Ig组的特异性抑制率明显高于FK5 0 6 +AdCTLA4Ig +BMT组和FK5 0 6+AdCTLA4Ig组(P <0 .0 5 ) ,而非特异性抑制率并没有差别。术后30d的异基因嵌合水平检测显示,BMT +AdCTLA4Ig组的胸腺和外周血嵌合水平高于AdlacZ腺病毒组、无处理对照组和AdCTLA4Ig组(P <0 .0 5 )。结论　在耐受诱导过程中,当FK5 0 6和AdCTLA4Ig联合应用时,前者会对后者的作用产     

Monoclonal Antibody Treatment to Prolong the Secondary Cardiac Allograft Survival in Alloantigen‐primed Mice

We have previously shown that costimulation blockade using a combination of monoclonal antibodies (mAbs) – CTLA4Ig, antibodies to CD154, LFA‐1, and OX40L – can induce tolerance of cardiac allografts in mice with adoptively transferred CD4⁺ memory T cells [ 1 ]. However, the effect of costimulatory blockade in secondary allograft rejection has not been studied. B6 mice that rejected BALB/c skin grafts for more than 4 weeks (defined as alloantigen‐primed mice) were used as recipients. The recipient mice were treated with the mAbs to CD154, LFA‐1, OX40L, and CD122 on days 0, 2, 4, and 6 after the secondary transplantation of BALB/c heart. The mean survival time (MST) of secondary cardiac allografts in rats treated with antibodies to CD154 and LFA‐1 (2‐antibodies approach) and those treated with antibodies to CD154, LFA‐1, OX40L, and CD122 (4‐antibodies approach) was greater than that of the controls (MST = 6.7 days, 22.2 days, and 3.2 days, respectively). The 4‐antibodies approach prevented lymphocytic infiltration in the grafts, inhibited memory T‐cells proliferation in the spleen, increased IL‐10 secretion in the serum, and enhanced the expression of CD4⁺ Foxp3⁺ regulatory T cells (Tregs) in spleen. Expression levels of alloreactive antibodies were high in the recipient mice of experimental and control groups. Inhibiting the memory T cells by costimulation blockade extended allograft survival in secondary transplant models but could not induce tolerance of graft. Alloreactive antibodies may participate in alloresponse and play an important role in secondary cardiac allograft rejection.     

Simultaneous administration of a low-dose mixture of donor bone marrow cells and splenocytes plus adenovirus containing the CTLA4Ig gene result in stable mixed chimerism and long-term survival of cardiac allograft in rats

T-cell costimulatory blockade combined with donor bone marrow transfusion may induce mixed chimerism, rendering robust tolerance in transplanted organs and cells. However, most protocols entail high doses of donor bone marrow cells (BMCs) or repeated administration of costly agents that block costimulatory pathways, thus delaying clinical development. To circumvent these shortcomings, we developed a strategy in which the dosage of donor BMCs was reduced but compensated by donor splenocytes (SPLCs). Furthermore, repeated administration of costly agents was replaced with a single injection of adenovirus expressing a gene of interest. In rat cardiac transplantation models, cardiac allografts from DA (RT-1a) rats were transplanted heterotopically into the abdomen of LEW (RT-11) recipient rats. Immediately after cardiac transplantation, an adenovirus vector (AdCTLA4Ig; 5 x 10(9) plaque-forming units) containing the gene for CTLA4Ig was administered to recipients (n = 6) simultaneously with a low dose of donor BMCs (1 x 10(8)/rat) and SPLCs (5 x 10(7)/rat) via the portal vein. The treated LEW recipient rats developed long-lasting mixed chimerism (>10% at >100 days) and exhibited long-term cardiac allografts (mean survival time of > 200 days) compared with control recipients. Moreover, recipients displaying long-lasting mixed chimerism accepted subsequent donor skin allografts while promptly rejecting third-party skin allografts. These results suggest that blockade of the CD28-B7 pathway, using adenovirus-mediated CTLA4Ig gene transfer, in concert with a low dosage of donor BMCs and SPLCs, may represent a feasible strategy to induce stable mixed chimerism and permit long-term survival of cardiac allografts.     

Anti-ICAM-1 antibody and CTLA-4Ig synergistically enhance immature dendritic cells to induce donor-specific immune tolerance in vivo

Immature dendritic cells (DC) have been demonstrated to induce T-cell hyporesponsiveness in vitro and immune tolerance in vivo. However, immature DC (iDC) may become mature once infused in vivo, thus limiting the prolongation of the allograft survival. Considering that mature DC express high level of B7, intercellular adhesion molecule-1 (ICAM-1), and T-cell activation needs costimulation signals provided by DC, we selected anti-ICAM-1 mAb and cytotoxic T lymphocyte antigen-4Ig fusion protein (CTLA-4Ig) for in vivo administration to block costimulation pathways in order to further improve the efficacy of iDC to induce immune tolerance. Seven days before allogeneic cardiac transplantations, the recipients were intravenously (i.v.) pretreated of donor-derived iDC with or without simultaneous injections of anti-ICAM-1 mAb and CTLA-4Ig. CTLA-4Ig or anti-ICAM-1 mAb administration alone resulted in significant prolongation of cardiac allograft survival induced by iDC. When used simultaneously, CTLA-4Ig and anti-ICAM-1 mAb induced permanent allografts acceptance even in 90% recipients. The recipients could keep the skin alive for a longer time in the donor-specific second transplantation, but no effect was observed on the skin from C3H third-party mice. The efficient induction of donor-specific tolerance observed above may be related to the more potent inhibition of donor-specific T-cell responses including cytotoxicity activity, Th1 cytokines production, and alloantibody production by the combined use of anti-ICAM-1 mAb and CTLA-4Ig. Our data suggest that anti-ICAM-1 antibody and CTLA-4Ig can synergistically enhance iDC to induce donor-specific immune tolerance in vivo.     

Effective induction of immune tolerance by portal venous infusion with IL-10 gene-modified immature dendritic cells leading to prolongation of allograft survival

Dendritic cells (DC) not only initiate T cell responses, but are also involved in the induction of tolerance. The functional properties of DC are strictly dependent on their state of maturation. It has been shown that immature DC can induce immune tolerance and prolong allograft survival. Interleukin-10 (IL-10) is an important immunosuppressive cytokine which inhibits maturation and function of DC. In order to improve the tolerogenicity of DC, we and others showed that adenovirus vectors can effectively mediate IL-10 genetic modification of DC, and IL-10 genetic modification can inhibit MHC II, B7.2, and CD40 expression, IL-12 secretion and the T cell stimulatory capacity of DC. The primary aim of this study is to examine the in vivo effects of this approach on allograft survival in a murine cardiac allograft transplantation model. To our surprise, we observed that infusion of immature DC genetically modified to express IL-10 (DC-IL-10) via the tail vein could not prolong allograft survival in the recipients, but shortened their survival. More interestingly, portal venous infusion of DC-IL-10 markedly prolonged allograft survival. The diverse effects of DC-IL-10 infusion through different routes may be due to the different immune responses to alloantigens in recipients that received DC-IL-10 via either the portal or the tail vein. Decreased cytotoxicity, polarization of Th2 response, poor T cell stimulating activity of liver DC and enhanced incidence of donor DC in the recipients may contribute to the more efficient prolongation of allograft survival observed after portal venous infusion of DC-IL-10. These results suggest that portal venous infusion may be an effective approach for immature DC to induce immune tolerance or hyporesponsiveness against donor antigens, and prolong allograft survival.Abbreviations APC Antigen-presenting cells - CTL Cytotoxic T lymphocytes - DC Dendritic cells - DC-IL-10 IL-10 gene-modified immature dendritic cells - iDC Immature dendritic cells - IL-10 Interleukin-10 - MLR Mixed leukocyte reaction - MOI Multiplicity of infection     

Fibronectin-mononuclear cell interactions regulate type 1 helper T cell cytokine network in tolerant transplant recipients

Fibronectin (FN), expressed primarily by macrophages, endothelial cells, and smooth muscle cells, represents an integral feature of the rejection response in transplant recipients. Here we demonstrate a unique pattern of cellular FN expression in rat recipients of cardiac allografts rendered tolerant in an infectious manner with either nondepleting CD4 mAb or regulatory spleen cells. Unlike in rejecting controls, cellular FN in tolerant hosts was restricted to the graft vessels and no vascular cell adhesion molecule-1 or intercellular adhesion molecule-1 expression could be found, supporting the role of FN in leukocyte sequestration at the graft site. The lack of myocardial FN in tolerant rats, despite dense macrophage infiltration, correlated with profound depression of Th1 (interleukin-2 and interferon-gamma) cytokines. Treatment with CD4-depleting mAb prevented tolerance induction and restored myocardial expression of FN in parallel with marked increase in the expression of interleukin-2 and interferon-gamma mRNA/protein. Furthermore, connective segment-1 peptide-facilitated adjunctive blockade of FN-alpha4beta1 interactions in recipients conditioned with CD4 depleting mAb, significantly depressed intragraft expression of interleukin-2 and interferon-gamma mRNA/protein. Hence, the lack of FN associated with infiltrating leukocytes plays an important role in the maintenance of tolerance in transplant recipients by depressing local expression of Th1 cytokines that otherwise facilitate acute graft rejection.     

TGF-beta(1) gene modified immature dendritic cells exhibit enhanced tolerogenicity but induce allograft fibrosis in vivo

Administration of donor-derived immature dendritic cells (DC) can prolong the survival of MHC-mismatched cardiac allografts. Genetic modification of DC by immunosuppressive molecules can enhance their potential tolerogenicity. In this study bone marrow derived immature DC were genetically modified by transforming growth factor (TGF) beta1 by recombinant Ad. TGF-beta(1) gene modified immature DC (TGF-beta-DC) displayed a characteristic phenotype of immature DC, decreased ability to secrete interleukin 12, and reduced allostimulatory ability. TGF-beta-DC induced alloantigen-specific T cell hyporesponsiveness in vitro and in vivo, and Th2 cytokine polarization. mRNA expression of donor MHC class II (Ia(b)) and human TGF-beta(1) was detected in spleen and lymph nodes of the allogeneic recipients for 3 weeks after TGF-beta-DC infusion, indicating that microchimerism of TGF-beta-DC is exhibited in allogeneic recipients. In this murine cervical heterotopic heart transplantation model, the survival of the allograft in recipients intravenously infused with TGF-beta-DC 7 days before transplantation was greatly prolonged, and about 67% of cardiac grafts survived more than 40 days. Histological analysis of the allografts showed that the normal myocardial architecture was well preserved, accompanied by very little necrotic cells, but interstitial fibrosis replaced myocytes, and moderate collagen suffused the whole cardiac allograft in the recipients infused with TGF-beta-DC. mRNA expression of type III procollagen was markedly increased in the allografts of the recipients infused with TGF-beta-DC. Our results suggest that infusion of TGF-beta(1) gene modified immature DC prolongs the survival of the allograft through the effective induction of donor-specific T cell hyporesponsiveness. However, TGF-beta(1) expressed by gene modified immature DC can cause the fibrosis of the allografts, which may limit the application of this approach in the allograft transplantation.     

TGF-beta1 and donor dendritic cells are common key components in donor-specific blood transfusion and anti-class II heart graft enhancement, whereas tolerance induction also required inflammatory cytokines down-regulation

Heart allograft tolerance in adult recipients can be induced in the LEW.1W to LEW.1A congeneic strain combination by pre-graft donor-specific blood transfusion (DST). Long-term survivors accept LEW.1W graft but reject third party skin grafts. As tolerant recipients of heart allografts showed an increase in anti-donor class II antibodies, we hypothesize that these antibodies could be instrumental in tolerance induction. However, anti-donor MHC class II alone prolonged graft survival but did not induce heart allograft tolerance in this combination. We analyzed the immune response patterns in heart allograft recipients following the injection of anti-donor class II antibodies (prolongation) or DST priming (tolerance). As suggested by the different phenomena, several immunological patterns were strikingly different between the two models. In strong contrast to DST-tolerant recipients, at 5 days after transplantation, neither Th1/Th2 nor inflammatory cytokines were inhibited in recipients treated with anti-donor class II antibodies, in which only prolongation of graft survival was induced. Nevertheless, in both models, depletion of resident dendritic cells (DC) from donor hearts inhibited tolerance induction (DST) or shortened allograft survival (anti-donor class II antibodies). Moreover, TGF-beta1 was not down-regulated and administration of neutralizing anti-TGF-beta1 antibody, which inhibited tolerance induction (DST), also shortened allograft survival (anti-donor class II antibodies). These results suggest that, in these two MHC class II-restricted models, both TGF-beta1 and donor DC have a pivotal role in prolonging graft survival. However, in the days following transplantation, further inhibition of inflammatory cytokine production, particularly Th1 and macrophage-derived cytokines is required for tolerance induction.