Retransplantation of discordant xenogeneic islets with costimulatory blockade

BACKGROUND: The aim of the study was to analyze the possibility of xenogeneic islet retransplantation using costimulatory blockade. METHODS: Streptozotocin-induced diabetic mice were transplanted under the kidney capsule with human islets. Mice were nephrectomized and retransplanted with 1000 human islets under the contralateral kidney capsule 14 days later. Four groups were performed group I, first and second Tx without MR1; group II, first Tx without MR1, second Tx with MR1; group III, first Tx with MR1, second Tx without MR1; group IV, first and second Tx with MR1. A control group was transplanted only once without MR1 with human islets. After second Tx, cross-matches between recipient, serum and human lymphocyte were done for detection of antidonor antibodies. RESULTS: In the control group, mean graft survival was 13 (+/-7) days. In group I, mean graft survival was 5 +/- 3 days. In group II, mean graft survival was 16 +/- 13 days. In group III, mean graft survival was 81 +/- 22 days. In group IV, no rejection were recorded and all graft survived more than 120 days. Pretransplant cross-matches were negative. In groups I and II all cross-matches were positive, while none were positive in group IV. CONCLUSION: Retransplantation of xenogeneic islets was associated with accelerated rejection. After presensitization, MR1 was unable to induce tolerance to a second Tx. MR1 given at the first Tx only allowed prolonged survival of the second Tx, but rejection still occurred. MR1 given at first and second Tx allowed long-term survival of retransplanted xenoislets and prevented occurrence of antidonor antibodies.     

Role of CD40-CD154 pathway in the rejection of concordant and discordant xenogeneic islets

BACKGROUND: Costimulatory blockade has been shown to allow long-term survival of xenogeneic islets. The aim of the present study was to evaluate the role of recipient CD40 and CD154 in the rejection process of concordant and discordant islet xenotransplantation (Tx). METHODS: Diabetic C57BL/6 mice, CD40- or CD154 knockout (KO) mice were transplanted with either concordant rat or discordant human islets. Experimental design: group 1, control (ie, C57BL/6 mice received islet Tx without therapy); group 2, C57BL/6 mice received islet Tx with anti-CD154 monoclonal Ab (mAb) therapy; group 3, CD40 KO mice; and group 4, CD154 KO mice were used as recipients without therapy. Mouse anti-rat mixed lymphocyte reactions (MLR) were performed using mouse splenocytes obtained from animals transplanted with rat islets in groups 1 to 4. RESULTS: In group 2, short-term anti-CD154 mAb therapy significantly prolonged rat-to-mouse and human-to-mouse xenograft survival, compared to controls. In CD40-KO and CD154-KO recipients, survival of concordant or discordant islets was not prolonged significantly compared to control groups. Mouse anti-donor rat cellular responses were reduced approximately 50% in group 2 but remained unmodified in groups 3 and 4, when compared to group 1. CONCLUSIONS: Improved graft survival and reduced MLR responses against donor cells in vitro among the anti-CD154 mAb-treated mice could be explained by specific targeting of activated T cells with subsequent inactivation by anergy and/or elimination by apoptosis, or complement- or cellular-mediated mechanisms. Rejection of xenografts and strong MLR responses against donor cells in vitro in CD40 or CD154 KO animals is possible through efficient activation of alternate pathways of costimulation.     

Regulation of xenogeneic porcine pancreatic islets

The use of xenogeneic porcine pancreatic islets has been shown to be a potentially promising alternative to using human allogeneic islets to treat insulin-dependent type 1 diabetes (T1D). This article provides an overview of the existing FDA regulatory framework that would be applied to the regulation of clinical trials utilizing xenogeneic porcine pancreatic islets to treat T1D.     

Combined donor specific transfusion and anti-CD154 therapy achieves airway allograft tolerance

BACKGROUND: The state of tolerance allows long term graft survival without immunosuppressants. Lung transplantation tolerance has not been consistently achieved in either small or large animal models. METHODS: The mechanisms and effectiveness of a tolerance induction protocol consisting of donor specific transfusion (DST; day 0) and a short course of co-stimulatory blockade (anti-CD154 antibody; days -7, -4, 0 and +4) were studied in the mouse heterotopic tracheal transplant model of chronic lung rejection. C57BL/6 mice received BALB/c tracheal grafts (day 0) and were treated with DST alone, anti-CD154 alone, the combination (DST/anti-CD154), or no treatment. No non-specific immunosuppressants were used. RESULTS: DST/anti-CD154 in combination, but neither treatment alone, markedly prolonged the lumen patency and survival (>100 days) of fully histo-incompatible allografts (p<0.05 versus control allografts at every time point studied up to 16 weeks) without immunosuppression. This protocol was donor antigen specific as third party grafts (C3H) were promptly rejected. In addition, DST/anti-CD154 did not result in mixed chimerism but induced transplantation tolerance via a peripheral mechanism(s), which included significantly reduced cytotoxic T cell activity (p<0.001) and a significantly increased percentage of CD4+CD25+ cells (p = 0.03). CONCLUSIONS: The DST/anti-CD154 protocol successfully induced and maintained long term, donor specific tolerance in the mouse heterotopic airway graft model of chronic lung rejection. This finding may lead us closer to successful tolerance induction in lung transplantation.     

供体脾细胞联合抗CD154输注诱导肺移植免疫耐受

目的 探讨供体特异性输注(DST)联合共刺激阻断(DST/抗CD154)诱导肺移植后闭塞性细支气管炎(OB)免疫耐受机制.方法 建立小鼠DST/抗CD154方案诱导的免疫耐受模型术后15、25、100 d取出移植气道检测形态学改变,利用CSME-80鼠cDNA芯片检测15d耐受组和同种异体移植组移植物内基因表达差异,分析差异有统计学意义的基因表达与排斥和耐受之间的关系.选择部分差异表达基因行实时荧光定量逆转录-聚合酶链反应( RT-qPCR),鉴定验证基因芯片的可靠性.结果 免疫耐受组OB前期存在大量显著表达差异基因,Rapl信号通路、CD40/CD40L相关及其他T细胞表面分子相关基因、细胞周期、细胞生长等促进纤维增殖的部分基因显著下调表达.部分促炎因子同同种异体移植组一样上调表达.结论 Rap1信号通路、CD40/CD40L相关及其他T细胞表面分子相关基因、细胞周期、细胞生长等促进纤维增殖下调表达的部分基因等可能与肺移植慢性排斥反应耐受相关.     

Prolonged survival of microencapsulated neonatal porcine islets in mice treated with a combination of anti-CD154 and anti-LFA-1 monoclonal antibodies

BACKGROUND: The aim of this study was to determine whether short-term administration of a combination of anti-CD154 and anti-LFA-1 monoclonal antibodies can prolong the survival of microencapsulated neonatal porcine islets (NPI) in immunocompetent mice. METHODS: Microencapsulated NPI were transplanted into the peritoneal cavity of streptozotocin-induced diabetic B6 mice that received a short-term treatment of a combination of anti-CD154 and anti-LFA-1 monoclonal antibodies. Blood glucose levels of each recipient were measured for more than 100 days posttransplantation or until graft rejection. Microcapsules were recovered to determine the presence of immune cells using immunoperoxidase staining. In addition, the levels of mouse anti-porcine immunoglobulin (Ig) G antibodies in the serum of each recipient were measured by flow cytometry. RESULTS: Short-term administration of a combination of monoclonal antibodies resulted in significant prolongation of microencapsulated NPI xenograft survival. All treated mice (n = 20) achieved normoglycemia within 10-35 days posttransplantation and 11/20 mice remained normoglycemic for more than 100 days posttransplantation. In contrast, only 1/20 of the untreated mice achieved normoglycemia and this mouse became diabetic at 17 days posttransplantation. Histological examination of the recovered microcapsules from long-term surviving treated mice revealed minimal cellular overgrowth containing intact viable islets, whereas several layers of immune cells surrounding the capsules containing nonviable islets were observed in untreated mice. The levels of mouse anti-porcine IgG was also reduced in treated recipients compared to untreated mice. CONCLUSIONS: These data demonstrate that short-term administration of anti-CD154 and anti-LFA-1 monoclonal antibodies can be effective in promoting long-term survival of microencapsulated NPI in immune-competent mice.     

Humanized anti-CD154 antibody therapy for the treatment of allograft rejection in nonhuman primates

The anti-CD154 antibody hu5C8 prevents acute allograft rejection and prolongs allograft survival after withdrawal of therapy in nonhuman primates. This study describes the use of hu5C8 as a rescue agent for rejection developing after the withdrawal of hu5C8. Twelve rhesus monkeys that had received renal allografts under hu5C8 induction and subsequently rejected were studied. Rescue with hu5C8 was analyzed based on the histological character of the rejection (acute versus chronic) and whether conventional therapy was received at the time of rescue or induction. The diagnosis of rejection and response to therapy was based on allograft function and histology. Four monkeys that had acute rejection associated with conventional immunosuppression and hu5C8 were not reversed by hu5C8 rescue. Four animals with isolated chronic rejection following prolonged rejection-free survival after the withdrawal of hu5C8 did not respond to hu5C8 rescue therapy. Hu5C8 rescue therapy effectively reversed acute rejection occurring in two monkeys after hu5C8 withdrawal. One of two animals with combined acute on chronic rejection responded to hu5C8 rescue therapy. Hu5C8 effectively reverses acute but not chronic allograft rejection and appears to have no synergistic effect with conventional rescue agents.     

Transplantation of islets of Langerhans

The present study attempts to evaluate the possibility of using cryopreserved pancreatic islets in rats. Insulin-releasing activity was studied for the purpose of clarifying the function of cryopreserved islets. Cryopreserved pancreatic islets were also transplanted into the portal veins of recipient rats. Rats recovered from the diabetic state, and the normalized condition was maintained for up to 20 weeks, although 4 weeks were needed before blood and urine glucose reached normal levels after transplantation. Intact B cells were found in the transplanted islet cell masses in the liver of the recipients, but B cells of the recipient's pancreases (streptozotocin-treated rats) showed a marked decrease, as well as degenerative changes.     

Engraftment of discordant xenogeneic swine bone marrow cells in immunodeficient mice

Abstract: We have recently demonstrated that swine bone marrow cells (BMC) can engraft in C.B-17 scid mice. While engraftment is enhanced by providing donor-specific porcine cytokines, the level of swine hematopoiesis declines between 3 and 6 weeks post-transplant. In the present study, the utility of several strains of immunodeficient mice as recipients of swine hematopoietic cells has been determined by comparing levels of swine bone marrow engraftment at 3 weeks after bone marrow transplant. Irradiated recipients were injected with lxlO8 swine BMC and were treated daily with porcine cytokines. The presence of swine cells in BMT recipients was detected by flow cytometry and marrow colony-forming assays. Recombination activating gene-1 (RAG-1)-deficient mice were not permissive for the engraftment of swine BMC, even with administration of increased doses of whole body irradiation, or with depleting anti-NK cell antibody. In contrast, NOD-scid mice showed improved swine BMC engraftment compared to C.B-17 scid mice. Levels of swine class I+, myeloid, and CD2+ cells in bone marrow, spleen, and peripheral blood, and the number of porcine myeloid progenitor cells was significantly higher in NOD-scid recipients than in simultaneous C.B-17 scid recipients. In addition, the sera from C.B-17 scid mice markedly inhibited the proliferation of swine BMC in vitro. A weaker inhibitory effect was also mediated by sera from RAG-1-deficient mice, but not by sera from NOD-scid mice. Together, our results indicate that multiple host elements resist xenogeneic hematopoietic engraftment and function, some of which are clearly independent of host T, B, or NK cells. Understanding the basis for the advantage of NODscid mice as recipients of discordant xenogeneic porcine BMC will help to identify these elements.     

Anti-CD25 mAb, anti-IL2 mAb, and IL2 block tolerance induction through anti-CD154 mAb and rapamycin in xenogeneic islet transplantation

BACKGROUND: We have used anti-CD154 monoclonal antibody (mAb; MR1) and rapamycin (rapa) to induce tolerance to islet xenografts. The aim of this study was to investigate whether classical anergy and/or regulation by interleukin (IL)2-dependent CD25+ T regulatory cells played roles in the induction and maintenance of tolerance in this model. METHODS: Streptozotocin-induced diabetic mice were transplanted with rat islets. We performed the following groups: control group, islet transplantation without therapy; rapamycin group, 0.2 mg/kg by oral gavage on days 0, 1, 2, and every other day to day 14; anti-CD154 mAb (MR1) group, 0.5 mg intraperitoneally on days 0, 2, and 4; combination therapy group with rapa and MR1. We then administered in addition to the combination therapy with early (from days 0 to 14 [for IL2] or to 28 [for anti-IL2 mAb and anti-CD25 mAb] post-transplantation) or late (from days 100 to 114 [for IL2] or to 128 [for anti-IL2 mAb and anti-CD25 mAb] posttransplantation) recombinant IL2 (2000 U, intraperitoneally twice a day), a neutralizing anti-IL2 mAb (S4B6-1, 0.3 mg intraperitoneally twice weekly), and a depleting anti-CD25 mAb (PC61, 0.3 mg intraperitoneally twice weekly), respectively. Histology was performed at time of rejection. RESULTS: Rapa and MR1 therapy alone significantly prolonged xenograft survival compared to the control group: median graft survival was 34 days versus 17 days (P<.05) and 98 days versus 17 days (P<.05), respectively, but rejection still occurred. Combination therapy with MR1 and rapa allowed indefinite graft survival (median graft survival [MGS]>200 days, P<.001). When exogenous IL2 was administered early with MR1 and rapa, rapid rejection developed in 18 of 18 mice (MGS 7 days), whereas when IL2 was given late, only 3 of 10 developed rejection. Early administration of anti-IL2 mAb led to rejection in 10 of 10 mice (MGS 42 days), whereas late administration led to rejection in only one of four mice. Early administration of anti-CD25 mAb led to rejection in eight of nine mice (MGS 49 days), whereas late administration led to rejection in only three of seven mice. CONCLUSIONS: Rapa and MR1 allowed indefinite graft survival of islet xenografts. Classical anergy and regulation by IL2-dependent CD25+ T regulatory cells were critical in the induction of tolerance in the immediate posttransplantation period and less important for maintenance of tolerance.     

Transplantation of islets transduced with CTLA4-Ig and TGFbeta using adenovirus and lentivirus vectors

BACKGROUND: A major problem facing islet transplantation is immune destruction of grafts by transplant rejection and autoimmunity. Some success in prolonging graft rejection has been obtained by transducing islets prior to transplantation with adenoviral (Ad) vectors containing CTLA4-Ig and TGFbeta. The purpose of this study was to see if lentiviral (LV) vectors would provide superior results compared with adenoviral vectors. METHODS: Islets were isolated from Sprague-Dawley rats and transduced with Ad or LV vectors containing LacZ, CTLA4-Ig, CTLA4, and TGFbeta1 using various MOIs. Islets transduced with LV were healthy as judged by DNA and insulin content, and insulin secretion. Using the kidney capsule transplant site, 500 transduced rat islets were transplanted into streptozotocin diabetic B6AF1 mice. RESULTS: Maintenance of normoglycemia was prolonged in recipient mice carrying islets transduced with Ad vectors containing CTLA4-Ig, CTLA4, and TGFbeta1. Return of hyperglycemia in controls was 17-18 days while loss of function for the experimental groups occurred at 20-27 days. For the lentivirus transduced islets, rejection of controls was 20+/-1.6 days, for CTLA4-Ig was 42+/-21 days and for TGFbeta was 28+/-3.2 days. CONCLUSIONS: Although islets transduced with either adenovirus or lentivirus containing CTLA4-Ig, CTLA4, and TGFbeta1 could prolong graft survival in a rat to mouse transplantation model, with the conditions of this study lentivirus provided no advantage over adenovirus vectors.     

Treatment with anti-CD154 antibody and donor-specific transfusion prevents acute rejection of myoblast transplantation

BACKGROUND: Achieving immunological tolerance to transplanted myoblasts would reduce the adverse effects associated with the sustained immunosuppression required for this experimental therapeutic approach in Duchenne muscular dystrophic patients. METHODS: Mdx mice were transplanted with fully allogeneic BALB/c myoblasts in the tibialis anterior muscles. Seven days before transplantation (-7), host mice received 107 total donor spleen cells i.v. (donor-specific transfusion, DST) with 500 microg of anti-CD154 mAb i.p. on days -7, -4, 0, +4. RESULTS: Results showed a high level of dystrophin expression in 83, 60, and 20% of the mice 1, 3, and 6 months, respectively, after transplantation of myoblasts. No antibodies against the donor cells were produced up to 3 months after transplantation. However, abundant activated cytotoxic cells were present in muscles still expressing high percentage of dystrophin positive fibers. CONCLUSIONS: In conclusion, the DST + anti-CD154 mAb treatments effectively prolonged myoblast survival, but this treatment could not develop tolerance to complete allogeneic myoblast transplantation.