Effect of adenovirus-mediated transfer of the CTLA4IG gene in hamster-to-rat xenotransplantation

BACKGROUND: Costimulatory molecules such as CD28 and B7 are essential for T cell activation, as well as playing a role in the T cell receptor and major histocompatibility complex pathway. It is well known that rejection in allotransplantation is diminished by treatment with CTLA4Ig, but whether a similar effect occurs in xenotransplantation remains to be determined. METHODS: In this study, we investigated whether adenovirus-mediated gene transfer with CTLA4Ig cDNA by intravenous injection to the recipient is effective in suppression in hamster-to-rat cardiac xenotransplantation. RESULTS: With CTLA4Ig gene transfer, the duration of gene expression was clearly prolonged, based on reduced production of antiadenovirus antibody and shrinkage of the spleen. The survival of cardiac xenografts was significantly prolonged with CTLA4Ig gene transfer compared to the control graft, and survival with combination use of FK506 and CTLA4Ig gene transfer in xenotransplantation was also significantly prolonged compared to that with CTLA4Ig gene transfer alone. Cessation of the cardiac graft in the combination treatment occurred in parallel with the elevation of antihamster IgM antibodies in rat sera. CONCLUSIONS: These results suggest that adenovirus-mediated CTLA4Ig gene transfer is effective for immunosuppression in hamster-to-rat xenotransplantation.     

Long-term acceptance of rat cardiac allografts on the basis of adenovirus mediated CD40Ig plus CTLA4Ig gene therapies

BACKGROUND: We have previously demonstrated that blockade of either CD80/86-CD28 or CD40-CD154 costimulatory pathways by using adenovirus vector coding CTLA4Ig (AdCTLA4Ig) or CD40Ig (AdCD40Ig) genes induced donor-specific tolerance in rat liver transplantation. In this study, we asked whether these gene-therapy-based costimulation blockade would induce tolerance in cardiac transplantation. METHODS: Heterotopic heart transplantation was performed in a full major histocompatibility complex (MHC) barrier combination of ACI (RT1avl) to Lewis (LEW, RT1l) rats. Vector (1 x 10(9) plaque forming unit [PFU]), AdLacZ, AdCTLA4Ig, or AdCD40Ig, was administered intravenously to recipient animals immediately after grafting, and graft survival, serum CTLA4Ig/CD40Ig levels, and graft histology were assessed. Tolerance was determined by secondary skin-graft challenging. RESULTS: Allografts of both untreated and AdLacZ controls were promptly rejected within 7 days, whereas a single treatment with AdCTLA4Ig or AdCD40Ig significantly prolonged median graft survival to 55.5 and 28.5 days, respectively. In contrast, the combined AdCTLA4Ig and AdCD40Ig gene therapy maintained high CTLA4Ig and CD40Ig levels through the posttransplant period and allowed long-term cardiac allograft survival for more than 270 days. However, both donor and third-party skin grafts were rejected in the animals who harbored cardiac grafts over 150 days. Also, typical features of chronic rejection were evident in the long-term surviving grafts. CONCLUSION: Simultaneous blockade of CD28 and CD154 pathways by AdCTLA4Ig plus AdCD40Ig induces a strong immunosuppression that allows long-term acceptance of full MHC mismatched cardiac graft in rats. This strategy, however, was not enough to induce tolerance to skin grafts and to avoid chronic rejection, as shown in the liver-transplantation model.     

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.     

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。     

Adenovirus‐mediated gene transfer of CTLA4lg gene results in prolonged survival of heart allograft

Abstract It has been demonstrated that the administration of CTLA4Ig protein can induce the suppression of allograft and xenograft rejection. The purpose of this study is to determine the effect of adenovirus‐mediated gene transfer with CTLA4Ig gene on the transgene expression and suppression of alloimmune response in allogeneic cardiac transplantation of rats. Adenoviral vectors with β galactosidase or human CTLA4Ig cDNA (Adex/LacZ, Adex/hCTLA4Ig) were constructed. These vectors were transduced to the liver of Lewis (LEW) rats by intravenous injection. Then LEW rats received heterotopic cardiac transplantation from Dark Agouti rats. Experiments were performed using both CTLA4Ig gene transduction and/or immunosuppressant FK506. The transgene expression after adenovirus‐mediated transfer with CTLA4Ig cDNA lasted for several months, compared with several weeks after that in controls, which was proportional to the blood concentration of CTLA4Ig protein. By the administration of 1 × 10⁹ PFU of Adex/hCTLA4Ig, the survival of cardiac grafts was significantly prolonged, compared with controls or the use of 1 × 10⁸ PFU of Adex/hCTLA4Ig. In the rats with beating grafts over 100 days, the blood concentrations of CTLA4Ig were undetectable. The combination therapy using a low titer Adex/hCTLA4Ig and low‐dose of FK506 was synergistically effective on this cardiac transplantation model. In conclusion, adenovirus‐mediated gene transfer with CTLA4Ig gene was efficient for the prolongation of both transgene expression and allograft survival.     

Gene therapy-mediated CD40L and CD28 co-stimulatory signaling blockade plus transient anti-xenograft antibody suppression induces long-term acceptance of cardiac xenografts

BACKGROUND: The authors have previously demonstrated that inhibition of CD28 and CD40 ligand (CD40L) co-stimulatory signals by adenovirus-mediated cytotoxic T-lymphocyte-associated (CTL) antigen 4 (A4) immunoglobulin (Ig) and CD40Ig gene therapies induces tolerance or long-term acceptance in rat liver and heart allograft transplantation. In this study, the authors examined whether co-stimulation blockade with a brief course treatment of FK779, a novel leflunomide derivative, would be an ideal strategy for controlling xenograft rejection. METHODS: Hamster hearts were transplanted into Lewis rats. Adenovirus vector coding (Ad) CD40Ig, CTLA4Ig, or LacZ gene (1 x 10(9) plaque-forming units) was administered intravenously to recipient rats 2 days before or immediately after xenografting. FK779 (10 mg/kg/day) was administered orally to recipients for 7 days beginning on day -1. Graft survival, graft histology, and xenoreactive antibodies were examined. RESULTS.: Both untreated and AdLacZ-treated control rats rejected cardiac xenografts, with a median survival time (MST) of 3 days. Co-stimulatory blockade alone by AdCTLA4Ig, AdCD40Ig, or both could not overcome such delayed xenograft rejection (DXR) (MST, 3-4 days). Under a short-course FK779 treatment that suppressed T-cell-independent xenoreactive antibodies, administration of AdCD40Ig (MST, 30.5 days) but not AdCTLA4Ig (MST, 9 days) significantly prolonged xenograft survival as compared with the FK779 monotherapy (MST, 7 days). In contrast, DXR and cellular rejection were controlled successfully and all xenografts were accepted for over 100 days when AdCTLA4Ig and AdCD40Ig were administered under FK779 induction therapy. However, chronic rejection was present in all long-term surviving xenografts. CONCLUSIONS.: Gene therapy-based co-stimulation blockade with FK779 induction treatment seems to be an attractive strategy with which to control xenograft rejection.     

CTLA4IgG treatment induces long-term acceptance of rat small bowel allografts

BACKGROUND: CTLA4 immunoglobulin (Ig)G that binds to B7 effectively inhibits the signaling of CD28/CTLA4-B7 pathway and induces antigen specific T cell unresponsiveness in vitro and in vivo. Using CTLA4IgG, we examined induction of long-term graft survival and the mechanism of maintenance of tolerance in rat allogeneic small bowel transplantation. METHODS: Small bowels of Brown-Norway rats (RT1n) were heterotopically transplanted into Lewis rats (RT1l). Recipients were treated with an i.p. injection of either CTLA4IgG or control IgG for 7 days. RESULTS: Long-term survival was observed in rats treated with CTLA4IgG, whereas control rats died within 16 days after transplantation. To examine whether a tolerant state was established in long-term survival rats, secondary transplantation was performed using small bowels of Brown-Norway rats or ACI (RT1b) rats. It was demonstrated that small bowels of Brown-Norway rats were accepted; however, those of ACI rats were rejected within 10 days. Serum concentrations of interleukin (IL)-4 were maintained at >50 microg/ml for 7 days after transplantation in rats treated with CTLA4IgG but <15 microg/ml in control rats. IL-2 concentration was reduced to half in CTLA4IgG-treated rats compared with that in control recipients. Serum IFN-gamma in CTLA4IgG-treated recipients increased after transplantation and was not distinguishable from that of control recipients during the first 7 days after transplantation. Conclusion. We demonstrated that CTLA4IgG treatment alone for 7 days induced a long-term donor specific tolerance in rat allogeneic small bowel transplantation. The induction of long-term acceptance of small bowel allografts by CTLA4IgG is not caused by simply the shift of anti-alloimmune responses from Thl to Th2 cytokine production.     

环孢素对共刺激阻断剂延长移植肾存活效应的影响

目的:探讨环孢素(CsA)对共刺激阻断剂CTLA4Ig延长移植肾存活效应的影响。方法:肾移植大鼠分为对照组(第1组)、CTLA4Ig组(第2组)、CsA CTLA4Ig组(第3组)、CTLA4Ig IL-2组(第4组)和CsA CTLA4Ig IL-2组(第5组),观察术后血肌酐(Scr)、移植肾病理改变、移植肾存活时间。结果:与第1组、第4组相比,第2组、第3组、第5组移植肾存活时间显著延长(P<0.01),其中,第3组移植肾存活时间最长(66.1±10.6)d;术后15天,第2组Scr显著低于第3组、第5组(P<0.05);术后30天,第3组、第5组Scr显著低于第2组(P<0.01);术后30天,第3组、第5组移植肾淋巴细胞浸润明显少于第2组。结论:CsA可增强CTLA4Ig延长移植肾存活的效应,对外源性IL-2逆转CTLA4Ig的效应具有抵抗作用。     

Immunosuppressive activity of the Chinese medicinal plant Tripterygium wilfordii. II. Prolongation of hamster-to-rat cardiac xenograft survival by combination therapy with the PG27 extract and cyclosporine

BACKGROUND: PG27 is an immunosuppressive fraction purified from an extract of a Chinese medicinal plant Tripterygium wilfordii, which we investigated alone and in combination with cyclosporine (CsA) in a concordant, hamster-to-rat cardiac xenotransplantation model. METHODS: Golden Syrian hamster hearts were heterotopically transplanted into the abdomen of Lewis rat recipients, which were treated intraperitoneally or orally with PG27, CsA, or both. RESULTS: Combination therapy with 30 mg/kg(day of PG27 and CsA at 10 mg/kg/day successfully suppressed acute hamster-to-rat cardiac xenograft rejection. Treatment with PG27 or CsA alone was ineffective. Among several effective combinations, the best regimen involved PG27 at 30 mg/kg/day and CsA at 5 mg/ kg/day from days 8 to 35 and then CsA at 5 mg/kg/day from days 36 to 100, which produced 100% survival beyond 100 days. CsA suppressed the heterospecific lymphocytotoxic antibody response and inhibited IgG but not IgM xenoantibody production (which led to xenograft rejection), whereas PG27 alone did not prevent antibody production. The PG27/CsA combination blocked the lymphocytotoxic antibody response and IgG and IgM xenoantibody production induced by cardiac xenotransplantation. CONCLUSIONS: PG27 combined with CsA substantially prolonged hamster-to-rat cardiac xenograft survival, as well as completely inhibiting xenoantibody production.     

小剂量环孢素A联用细胞毒T淋巴细胞A4-Ig治疗器官移植排斥反应

目的　研究小剂量环孢素A(CsA)联用细胞毒T淋巴细胞A4 Ig(CTLA4 Ig)治疗器官移植排斥反应的效果。　方法　采用Ono′s方法建立大鼠心脏移植排斥反应动物模型 ,将实验动物分为 4组。A组 :对照组 ,未给予任何治疗 ;B组 :腹腔内注射CsA ,10mg·kg-1·d-1,连续 7d ;C组 :术后第 2天 1次性注射CTLA4 Ig 10 0 μg ;D组 :术后 1～ 7d连续腹腔内注射CsA ,2mg .kg-1.d-1,术后第 2天加用CTLA4 Ig 5 0 μg。观察移植心脏存活天数及术后IL 2含量和组织学变化。　 结果　A、B、C、D 4组大鼠移植心脏存活时间分别为 ( 7 2± 0 7)、( 19 4± 2 1)、( 3 1 6± 1 8)和 ( 2 4 6± 2 1)d ,与对照组相比 ,各治疗组大鼠心脏存活时间明显延长 ,差异有显著性意义 (q =3 2 7 83 ,P <0 0 5 ) ,D组与C组相比差异无显著性意义 (q =1 86,P >0 0 5 ) ;各治疗组术后IL 2含量明显减低 ,与对照组相比差异有非常显著性意义 (q=9 82 ,P <0 0 1) ;A、B、C和D组排斥反应分级分别为Ⅳ、Ⅲ、Ⅰ和Ⅰ级。　结论　CTLA4 Ig抗排斥反应能力比CsA强 ,小剂量CsA联合使用CTLA4 Ig能增强治疗排斥反应疗效 ,两者具有正协同作用。     

Development of donor-specific immunoregulatory T-cells after local CTLA4Ig gene transfer to pancreatic allograft

BACKGROUND: CTLA4Ig gene transfer directly to graft tissue might have the potential to avoid the need for systemic immunosuppression. In our previous studies of bio-breeding (BB) rats, local adenovirus-mediated CTLA4Ig gene transfer protected the pancreas from autoimmune and alloimmune responses. This study investigated the potency of local CD28/B7 costimulatory blockade for induction of donor-specific tolerance and further examined the existing mechanisms. METHODS: Brown Norway (BN; RT1)-pancreaticoduodenal grafts transfected with Ad.CTLA4Ig via intraarterial ex vivo perfusion were transplanted into streptozotocin-induced diabetic Lewis (LEW; RT1) rats. RESULTS: Ad.CTLA4Ig transduced grafts combined with a short course of FK506 resulted in indefinitely prolonged survival (>156 days vs. 19.5 days with FK506 alone). CTLA4Ig was predominantly expressed in grafts on day 4. The expression was gradually diminished and was only slightly detectable at day >100. The proliferative responses against BN antigen were remarkably enhanced among recipients with rejected grafts, but the T-cells from tolerant recipients (>100 days) showed poor cytotoxic responses. On adoptive transfer assay, the splenic T-cells of tolerant recipients were able to suppress the rejection of BN, but not third-party Wistar Furth (WF; RT1) hearts in irradiated (480 cGy) LEW recipients. The percentage of CD4CD25 splenic T-cells was significantly increased in tolerant recipients (13.53 +/- 4.06% vs. 6.06 +/- 0.56% in naive rats). CONCLUSION: CTLA4Ig gene transfer to the pancreaticoduodenal allograft combined with a short course of FK506 induces donor-specific tolerance. The mechanism of maintaining tolerance could be explained by development of splenic T suppressor cells.     

Combined gene therapy with adenovirus vectors containing CTLA4Ig and CD40Ig prolongs survival of composite tissue allografts in rat model

BACKGROUND: The blockade of costimulatory signal pathway by anti-CD40 ligand antibody or cytotoxic T lymphocyte antigen 4 immunoglobulin (CTLA4Ig) prolongs allograft survival in various vascularized organ transplantations. Because of the short half life of these agents, repeated administration of proteins is required to achieve significant graft survival. Furthermore, there is limited information regarding the effect of cosimulatory blockade on the survival of composite tissue allografts. Therefore, we examined the effect of adenovirus-mediated gene transfer of CTLA4Ig or CD40Ig gene or both in composite tissue allotransplantation. METHODS: The hind limbs removed from male ACI rats (RT1 ) were transplanted into female Lewis rats (RT1 ) heterotopically. The recombinant adenovirus carrying CTLA4Ig (AxCTLA4Ig) or CD40Ig (AxCD40Ig) was intravenously administered after limb transplantation. RESULTS: Limb allograft survival was significantly prolonged by either AxCTLA4Ig or AxCD40Ig treatment at 1 x 10 plaque forming unit (mean survival time [MST] of 39.4+/-6.0 and 13.0+/-2.9, respectively) compared with the adenovirus vector containing beta-galactosidase-treated group (MST of 4.8+/-0.8). Combination of AxCTLA4Ig and AxCD40Ig led to significant prolongation of graft survival (MST of 49.2+/-6.6). Serum levels of CD40Ig were higher in rats treated with combination therapy than those treated with AxCD40Ig alone, whereas the serum levels of CTLA4Ig in rats treated with AxCTLA4Ig alone and AxCTLA4Ig and AxCD40Ig combined were very similar. CONCLUSION: This study indicates that an adenovirus-mediated gene therapy of CTLA4Ig or CD40Ig has a therapeutic potential for preventing rejection in composite tissue transplantation. Furthermore, a combination therapy of AxCTLA4Ig and AxCD40Ig was even more effective in preventing acute rejection and prolonging the survival of allografted limbs without apparent complication.     

Prevention of Acute Lung Allograft Rejection in Rat by CTLA4Ig

CTLA4 immunoglobulin (CTLA4Ig), which binds with a high affinity to B7-1 and B7-2, interrupts T-cell activation by inhibiting costimulatory signal. CTLA4Ig has been used in hopes of achieving antigen-specific tolerance induction in several solid organ transplants. In lung allograft rejection, however, its use has been controversial in terms of its effect on prevention of rejection. In the present study, the effect of murine CTLA4Ig on rat-lung allograft rejection was investigated. Rat left-lung transplantation was performed in an RT1 incompatible donor (Brown Norway; BN)-recipient (F344) combination. All allografts (n = 12) without any treatment were rejected within 7 days after transplantation. A single injection of murine form CTLA41g at a dose of 100 microg intraperitoneally (ip) or intravenously (iv) on day 1 post-transplantation achieved long-term graft survival (>90days) in 2/5 (40%) and 3/8 (38%), respectively. Moreover, 6/7 (86%) allografts in rats that received iv injection of 500 microg CTLA4Ig survived more than 90days. Allograft survival in the CTLA4Ig 500 microg iv recipient group was significantly longer than that in the no-treatment control or control immunoglobulin group (p <0.01). Four out of seven recipients bearing functional allografts for more than 90 days with the CTLA4Ig treatment accepted donor-specific skin grafts, whereas all third-party skin grafts (n=3) were rejected. Prevention of rat-lung allograft rejection could be achieved by intravenous administration of CTLA4Ig, resulting in long-term allograft survival with acceptance of donor-specific skin grafts.     

Importance of the Gal alpha1-3 Gal antigen in discordant islet xenotransplantation: immunosuppression, which inhibits porcine islet xenograft rejection in ordinary mice, is equally effective in Gal-knockout mice

BACKGROUND: Islet xenotransplantation will most likely be performed in diabetic patients treated with immunosuppressive drugs. The importance of the galactosyl alpha(1-3) galactose (Galalpha1-3Gal) antigen in immunosuppressed islet xenograft recipients has not been studied. METHODS: Fetal porcine islet-like cell clusters (ICCs) were transplanted into the renal subcapsular space of both Gal-knockout mice and ordinary mice. Transplantations were performed in untreated mice and mice immunosuppressed with cyclosporine A (CsA) plus 15-deoxyspergualin (DSG). Studies were also performed in immunosuppressed Gal-knockout mice that had been actively immunized against Galalpha1-3Gal. Evaluation was performed 12 days after transplantation using morphologic techniques. The levels of serum immunoglobulin (Ig)G and IgM to the Galalpha1-3Gal antigen or to the ICCs were determined. RESULTS: No difference in the morphologic appearance could be seen between ordinary mice and Gal-knockout mice. No deposits of IgG, IgM, or C3 could be detected. Almost no difference could be seen between immunosuppressed Gal-knockout mice and immunosuppressed ordinary mice. In immunosuppressed, immunized Gal-knockout mice, the results were similar. In ordinary mice treated with CsA+DSG, the levels of anti-Gal IgM were lower than they were in untreated mice, whereas the levels of anti-Gal IgG were similar. In Gal-knockout mice (including immunized animals) treated with CsA+DSG, the levels of anti-Gal IgG and IgM were lower than they were in untreated Gal-knockout mice. CONCLUSIONS: After renal subcapsular transplantation, antibodies against Galalpha1-3Gal have no major influence on islet xenograft rejection in the pig-to-mouse model. Immunosuppression, which inhibits rejection in the pig-to-mouse model, is equally effective when transplantation is performed across the Galalpha1-3Gal barrier.     

Blockade of both CD28/B7 and OX40/OX40L co-stimulatory signal pathways prolongs the survival of islet xenografts

CTLA4Ig, a recombinant fusion protein composed of the extracellular domain of human CTLA4 and the constant region of human IgG1, inhibits the interaction of CD28/B7 pathway by binding the B7 molecule. OX40Ig, a recombinant fusion protein composed of the extracellular domain of human OX40 and the constant region of human IgG1, abrogates the interaction of OX40/OX40L pathway by binding the OX40L on APCs. So blockade of CD28/B7 or OX40/OX40L co-stimulatory pathways alone in mice with CTLA4Ig or OX40Ig can result in finitely prolonging the survival of islet grafts (43.2 +/- 4.81 and 67.7 +/- 7.74 days, respectively). In this study, a novel replication-defective adenovirus containing both of the CTLA4Ig and OX40Ig genes, AdCTLA4Ig-IRES-OX40Ig, was constructed by homologous recombination and injected into the streptozocin-rendered diabetic BalB/c mouse recipients (H-2d) through the tail vein, at the same day, the freshly isolated islets from Lewis rats (RT-1) were transplanted under the left kidney capsule of the recipients. The results showed that the mean survival time of the islet xenografts in the AdCTLA4Ig-IRES-OX40Ig-treated diabetic mice was significantly prolonged (100.3 +/- 14.94 days), while those in the untreated or AdEGFP-treated mice were rejected in normal fashion (6.7 +/- 0.94 and 7.0 +/- 1.0 days, respectively). In conclusion, utilizing AdCTLA4Ig-IRES-OX40Ig in vivo which can simultaneously express CTLA4Ig and OX40Ig proteins can improve the survival of Lewis-->BalB/c islet xenografts.     

Effects of Gene Transfer CTLA4Ig and Anti-CD40L Monoclonal Antibody on Islet Xenograft Rejection in Mice

Blockade of a costimulatory pathway by adenovirus-mediated cytotoxic T lymphocyte associated antigen 4 immunoglobulin (CTLA4-Ig) gene transfer and anti-CD40L mAb(MR1) have been reported to enhance graft survival in several experimental transplantation models. In this study, we investigated the effects of gene transfer of CTLA4Ig and MR1 on islet xenograft rejection in mice. Recombinant adenovirus AdCTLA4Ig was constructed to express CTLA4Ig. Islet grafts from adult male DA rats transferred with AdCTLA4Ig were transplanted to streptozocin-induced diabetic Balb/c mice. The diabetic mice were treated with MR1 after transplantation. We evaluated the islet xenograft mean survival time as well as changes in interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) levels in transplanted mice. The mean survival of islet xenografts in the MR1 treatment group was 34.9 ± 5.62 days, in the AdCTLA4Ig treatment group it was 56.5 ± 10.64 days, and in the AdCTLA4Ig plus MR1 treatment group it was 112.9 ± 19.26 days, all significantly prolonged compared with an untreated group (8.1 ± 0.83 days). Within 1 week after transplantation the levels of IL-2 and TNF-α showed sharp increases in the untreated group, being significantly higher than those observed prior to transplantation. In conclusion, using both AdCTLA4Ig and MR1 can improve the islet xenograft survival. The beneficial effects of the combined use of the 2 reagents were superior to either 1 alone, possibly related to down-regulated expression of Th1 cell-related cytokines.     

Induction of regulatory cells and control of cellular but not vascular rejection by costimulation blockade in hamster-to-rat heart xenotransplantation

BACKGROUND: In heart allograft in the rat, a sustained costimulation blockade with CTLA4Ig prevents alloreactive T-cell activation and promotes a long-term graft survival through the action of tolerogeneic dendritic cells. It is unclear whether similar mechanisms might occur after xenotransplantation. To test that hypothesis, we have analyzed the action of CTLA4Ig in a model of CD4(+)T cell-mediated xenograft rejection. METHODS: Hamster hearts were transplanted into LEW.1A rats receiving an accommodation-inducing treatment consisting of a short course administration of LF15-0195 and a daily administration of cyclosporine A (CSA). To achieve long-term delivery of CTLA4Ig, an intravenous administration of an adenovirus vector coding for mouse CTLA4Ig (Ad-CTLA4Ig) was added to the accommodation induction protocol. On day 40 post-transplantation, rejection was induced by CSA withdrawal. In other xenograft recipients, CD28/B7 costimulation was inhibited at that time only by injections of CTLA4Ig or anti-CD28 antibodies. Graft survival, immunohistology, as well as development of antibodies and regulatory cells were examined. RESULTS: Xenografts survived 6 days after CSA withdrawal in controls and were rejected, as previously described, through the action of CD4(+) xenoreactive T cells. Interfering with CD28/B7 costimulation inhibited this xenoreactive T cell response and delayed rejection to day 10. In recipients that had received Ad-CTLA4Ig, survival was prolonged to day 19 and this was accompanied by the appearance of regulatory cells exhibiting non-donor-specific suppressive activity dependent on IL-2, NO, and IDO. These regulatory cells were different from those previously identified after Ad-CTLA4Ig administration in heart allograft in the rat. In these recipients, rejection occurred as a consequence of an evoked anti-donor IgM response and complement activation and not of a cellular rejection as complement inhibition with cobra venom factor further prolonged xenograft survival. CONCLUSION: CD28/B7 blockade delays CD4(+) T cell-mediated rejection after CSA withdrawal in accommodated recipients of hamster heart xenografts. In addition, a sustained expression of CTLA4Ig has the potential of inducing cellular regulatory mechanisms. However, such treatment does not prevent the development of xenoreactive IgM antibodies that participate in vascular rejection processes in a complement-dependent manner.