CTLA4Ig promotes the induction of hematopoietic chimerism and tolerance independently of Indoleamine-2,3-dioxygenase

Bone marrow transplantation (BMT) under costimulation blockade induces mixed chimerism and tolerance in rodent models. Recent data, predominantly from in vitro studies, suggest that in addition to blocking the CD28 costimulation pathway CTLA4Ig also acts through upregulating the tryptophan-catabolizing enzyme indoleamine-2,3-dioxygenase (IDO). Here we demonstrate that even though CTLA4Ig is critically required for the induction of chimerism and tolerance in a murine model of nonmyeloablative BMT, IDO activity is not. No significant differences were detectable in the kynurenine to tryptophan ratios (indicative of IDO activity) in sera of BMT recipients treated with CTLA4Ig (tolerant group) versus BMT recipients treated without CTLA4Ig (nontolerant group) versus na?ve controls. In vivo inhibition of IDO immediately after BMT with CTLA4Ig or several months thereafter did not block achievement of chimerism and tolerance. Thus, IDO does not play a critical role in the induction or maintenance of chimerism and tolerance in a CTLA4Ig-based BMT model.     

供体脾细胞联合抗CD154单抗对非清髓性骨髓移植方案建立异基因嵌合体的影响

.06±3.77)%、胸腺(24.52±3.22)%]均可形成稳定的高水平嵌合,并持续超过100 d.结论 单纯2×108 Se门静脉输注只能诱导轻度免疫耐受,建立短暂(＜20 d)、低水平(＜20%)的外周性嵌合体;在此基础上联合1×108个BMc输注可延长嵌合体水平至40 d,但仍不稳定;而加入AH.F5后可显著增强BMc植入率,三者具有协同作用.外周嵌合体＞30%、中枢嵌合体＞20%是保证骨髓细胞发挥强大致耐受作用的基础.     

Minimal conditioning required in a murine model of T cell depletion,thymic irradiation and high-dose bone marrow transplantation for the induction of mixed chimerism and tolerance

In a recently developed murine model for the induction of mixed chimerism and tolerance, hosts are treated with T cell depleting monoclonal antibodies (TCD mAbs; days -5, -1 and +7), thymic irradiation (TI) (7 Gy), and a high dose of fully allogeneic bone marrow cells (BMC, 200 x 10(6)). To find the minimum amount of each treatment required for success with this approach, we treated groups with (1) a lower dose of TI (3.5 Gy), (2) fewer BMC (100 x 10(6)), (3) no TI, (4) no TI plus additional TCD mAbs on day +14, or (5) fewer injections of TCD mAbs (day -5 only). Chimerism was followed by flow cytometry (FCM), and tolerance was assessed by skin grafting. Without TI, no long-term chimerism or tolerance could be induced, even when an additional dose of TCD mAbs was administered on day +14. A reduction in the dose of either BMC or TI led to substantially reduced effectiveness, as demonstrated by lower levels of chimerism and poorer donor skin graft survival. However, the dose of TCD mAbs and hence the duration of recipient T cell depletion could be safely reduced and thus the potential toxicity of the conditioning regimen lowered.     

非清髓性方案在诱导大鼠后肢移植免疫耐受中的应用

目的 探讨基于淋巴细胞毒性相关抗原4-抗体重组腺病毒(AdCTLA4-Ig)的非清髓性方案在造血干细胞嵌合体诱导复合组织异体移植免疫耐受中的作用.方法 以近交系Brown Norway(RT1n)大鼠为供体,Lewis(RT11)大鼠为受体.以后肢移植当天记为day 0.实验分4组,A组:受体直接给予同种异体后肢移植,移植前不进行非清髓件预处理,移植后连续100 d,每天仅给予低剂量环胞素A(CsA),8 mg/kg腹腔注射.B组:受体先给予非清髓性预处理,移植前第33天至移植后第100天,每日用免疫抑制剂三联方案腹腔注射雷帕霉素(RAPA,0.2 mg/kg)+麦考(MMF,20 mg/kg)+甲泼尼龙(MP,10 mg/kg),在移植当日及移植前及移植后第30天分3次尾静脉注射AdCTLA4-Ig(5×109 PFU/d),后肢移植前30 d接受单次3 Gy(照射率0.5 Gy/min)低强度全身照射,不予骨髓移植(BMT).C组:受体预处理方案同B组,移植前30 d,在低强度全身照射后4 h内给予单次尾静脉注射供体骨髓细胞(100×106 cells).D组:受体预处理方案及BMT方法同C组,但大鼠后肢移植供体为第三方动物WF大鼠.在后肢移植后第100天开始,B、C及D组均停止免疫抑制剂三联方案,每日仅给予低剂量CsA(8 mg/kg),连续100 d,直至大鼠后肢移植物发生排异反应而坏死.通过外周血嵌合率检测、移植物抗宿主病检测、后肢移植物存活情况观察、移植物组织病理学检查与评价及混合淋巴细胞反应对免疫耐受状态进行分析评价.结果 C组外周血嵌合率移植当口为(38.8±10.6)%,并长期保持稳定,移植后第300天为(29.3±11.9)%,均未发生移植物抗宿主病,停止免疫抑制剂三联方案后移植物存活>200 d,A、B、D组均发生免疫排异,后肢移植物分别存活(8±2)、(18±3)及(20±2)d,与C组相比,差异有统计学意义(P<0.01).C组移植物病理学检查显示无毛囊炎及血管周围炎等慢性免疫排异现象,混合淋巴细胞反应显示为供体特异性免疫耐受状态.结论 基于AdCTLA4-Ig的非清髓性BMT方案可以诱导长期稳定的造血干细胞嵌合体状态,并可以诱导受体对大鼠后肢移植物的供体特异性部分性免疫耐受.     

Long-term outcome and alloantibody production in a non-myeloablative regimen for induction of renal allograft tolerance

BACKGROUND: Multilineage chimerism and long-term acceptance of renal allografts has been produced in non-human primates conditioned with a nonmyeloablative regimen. Our study was undertaken to evaluate the immunological and pathological status of long-term survivors and to define the role of splenectomy and of the primarily vascularized kidney in the regimen. METHOD: Monkeys were treated with the basic regimen, including: total body irradiation, thymic irradiation, antithymocyte globulin, donor bone marrow transplantation, and a 4-week course of cyclosporine after which no further immunosuppression was given. They were divided into four groups according to the timing of kidney transplantation (KTx) and splenectomy as follows; group A (n=13): KTx and splenectomy on the day of donor bone marrow transplantation (day 0); group B (n=3): KTx on day 0 without splenectomy; group C (n=7): splenectomy on day 0 but delayed KTx until 3 to 16 weeks post-donor bone marrow transplantation; group D (n=3): both splenectomy and KTx delayed until day 120 post-donor bone marrow transplantation. RESULTS: In group A, 11 of 13 monkeys developed chimerism and 9 monkeys achieved long-term survival of 4 to 70 months without evidence of chronic vascular rejection. Alloantibodies were detected in only one long-term survivor. In contrast, all three monkeys in group B developed alloantibodies and rejected their allografts. In group C, long-term survival without alloantibody production was observed in two of three monkeys that had developed chimerism. In group D, all three recipients were sensitized and rejected the kidney allografts rapidly after transplantation. CONCLUSIONS: 1) Production of anti-donor antibody was prevented in most recipients that developed mixed chimerism in the regimens with splenectomy at the time of donor bone marrow transplantation. 2) If splenectomy is not included in the initial conditioning regimen, induction of B cell tolerance is less likely and the result is late onset of alloantibody production and allograft rejection. 3) Immediate transplantation of the kidney at the time of recipient conditioning is not essential for induction of donor specific hyporesponsiveness by bone marrow transplantation.     

Permanent acceptance of both cardiac and skin allografts using a mild conditioning regimen for the induction of stable mixed chimerism in mice

Patients who are receiving an organ transplant nowadays are sentenced to the life-long administration of immunosuppressive drugs, which have serious side effects. The reliable induction of donor-specific tolerance therefore remains a major goal in organ transplantation. Previously, we have developed a sublethal, non-myeloablative murine model in which permanent mixed, multilineage chimerism and donor-specific tolerance are established. Our model involves engraftment of fully allogeneic T cell depleted donor bone marrow cells in low dose irradiated and anti-CD3 treated major histocompatibility complex (MHC)-disparate recipient mice. To investigate whether vascularized organ grafts are accepted in our model, we performed heterotopic heart transplantations in our mixed chimeric mice. Chimeric mice permanently accepted hearts from the bone marrow donor (>130 days) and rapidly rejected third party-type allografts (median survival time 9 days). Untreated control recipient mice rejected both donor- and third party-type allografts. In addition, mice that accepted their cardiac grafts, donor-specific acceptance of skin grafts was observed. In conclusion, the establishment of stable mixed chimerism with this low-toxicity regimen resulted in permanent donor-specific acceptance of vascularized organ as well as skin grafts across a full MHC barrier.     

Pig hematopoietic cell chimerism in baboons conditioned with a nonmyeloablative regimen and CD154 blockade.

BACKGROUND: In an attempt to induce mixed hematopoietic chimerism and transplantation tolerance in the pig-to-primate model, we have infused high-dose porcine peripheral blood progenitor cells (PBPC) into baboons pretreated with a nonmyeloablative regimen and anti-CD154 monoclonal antibody (mAb). METHODS: Group 1 baboons (n=2) received a nonmyeloablative regimen including whole body irradiation, pharmacological immunosuppression, porcine hematopoietic growth factors, and immunoadsorption of anti-Galalpha1,3Gal (Gal) antibody before infusion of high doses of PBPC (2.7-4.6x10(10) cells/kg). In group 2 (n=5), cyclosporine was replaced by anti-CD154 mAb. Group 3 (n=3) received the group 1 regimen plus anti-CD154 mAb. RESULTS: In group 1, pig chimerism was detected in the blood by flow cytometry (FACS) for 5 days (with a maximum of 14%), and continuously up to 13 days by polymerase chain reaction (PCR). In group 2, pig chimerism was detectable for 5 days by FACS (maximum 33%) and continuously up to 28 days by PCR. In group 3, initial pig chimerism was detectable for 5 days by FACS (maximum 73%). Two of three baboons showed reappearance of pig cells on days 11 and 16, respectively. In one, in which no anti-Gal IgG could be detected for 30 days, pig cells were documented in the blood by FACS on days 16-22 (maximum 6% on day 19) and pig colony-forming cells were present in the blood on days 19-33, which we interpreted as evidence of engraftment. Microchimerism was continuous by PCR up to 33 days. CONCLUSIONS: These results suggest that there is no absolute barrier to pig hematopoietic cell engraftment in primates, and that this may be facilitated if the return of anti-Gal IgG can be prevented.     

Nonlethal conditioning for the induction of allogeneic chimerism and tolerance to islet allografts

BACKGROUND: We reported that tolerance to skin grafts can be achieved by chimerism induction by way of nonlethal conditioning. In the present study, we evaluated the outcome of islet allografts implanted either simultaneously or after donor bone marrow cell (BMC) infusion when nonlethal conditioning was used. METHODS AND RESULTS: B10 (H-2b) mice were conditioned with antilymphocyte serum (ALS), 100 cGy total body irradiation (TBI), and given 30 x 10(6) allogeneic (B10.BR, H-2k) BMC on day 0. On day 2, cyclophosphamide was given intraperitoneally (IP), followed by a second BMC infusion on day 3. After chimeras were typed for allogeneic BMC engraftment on day 28, animals were rendered diabetic chemically and transplanted under the kidney capsule with islet allografts genetically matched or disparate to the BM. Donor-specific islet grafts were accepted (median survival time [MST] > 180 days, n=6), whereas all major histocompatibility complex (MHC)-disparate third-party BALB/c (H-2d) islet grafts were rejected (MST=13.8 days, n=4). When B10.BR BMC and islets were given simultaneously, graft acceptance (MST >140 days, n=4) was observed. Surprisingly, when MHC-disparate third-party islets (BALB/c) were given together with B10.BR BMC, long-term survival was also observed (MST >100 days, n=3). These findings suggested that conditioning alone at the time of islet implant might induce long-term engraftment without further treatment. However, only chimeric animals accepted a second-set donor-specific graft, whereas all other groups rejected it. CONCLUSION: Our data indicates that stable allogeneic chimerism and islet indefinite survival can be achieved by the use of a nonmyeloablative protocol. The results of the conditioning-only experiments are consistent with the possibility of graft accommodation.     

Short-term immunosuppression facilitates induction of mixed chimerism and tolerance after bone marrow transplantation without cytoreductive conditioning

BACKGROUND: Induction of mixed chimerism and tolerance usually requires cytoreduction or transplantation of high numbers of bone marrow cells (BMC). However, such protocols have only a suboptimal success rate and, more importantly, equivalent numbers of BMC cannot be routinely obtained in the clinical setting. The authors therefore evaluated whether a short-course of immunosuppression (IS) given in addition to co-stimulation blockade would facilitate chimerism induction and allow reduction of the minimally required number of BMC without cytoreduction. METHODS: B6 mice received 200, 100, or 50 x 10 unseparated BMC from Balb/c donors plus an anti-CD40L monoclonal antibody (mAb) and CTLA4Ig (without irradiation or cytotoxic drugs). Some groups were treated additionally with IS (rapamycin, methylprednisolone, and mycophenolate mofetil for 4 weeks after bone marrow transplantation), donor-specific transfusion (DST), or anti-OX40L mAb, as indicated. RESULTS: IS led to long-term multilineage chimerism in 9 of 10 mice receiving 200 x 10 BMC (without IS, 1 of 4; P<0.05), in all mice (n=10) receiving 100 x 10 (without IS, 6 of 9; P<0.05), and notably in 9 of 10 mice treated with 50 x 10 BMC (without IS, 4 of 10; P<0.05). With transient IS, donor skin grafts were accepted longer than 170 days in 9 of 10 mice receiving 200 x 10 (without IS, 0 of 5 mice; P<0.05), all mice receiving 100 x 10 (without IS, 6 of 9; P<0.05), and 6 of 11 mice receiving 50 x 10 BMC (without IS, 4 of 10). The use of DST or anti-OX40L mAb had no beneficial effect. CONCLUSIONS: Transient IS significantly improves rates of chimerism and donor skin graft survival, and allows lasting mixed chimerism after transplantation of only 50 x 10 BMC. Thus, IS might help in the further development of noncytoreductive chimerism protocols.     

Anti-CD40L monoclonal antibodies can replace anti-CD4 monoclonal antibodies for the nonmyeloablative induction of mixed xenogeneic chimerism

BACKGROUND: We have previously demonstrated that xenogeneic bone marrow engraftment and donor-specific tolerance can be induced in mice receiving anti-CD4, -CD8, -Thy-1.2, and -NK1.1 monoclonal antibodies (mAbs) on Days -6 and -1, 3 Gy total body irradiation (TBI), and 7 Gy thymic irradiation on Day 0, followed by injection of T-cell depleted (TCD) rat bone marrow cells. We have recently demonstrated that anti-CD40L mAb treatment is sufficient to completely overcome CD4 cell-mediated resistance to allogeneic marrow engraftment and rapidly induce CD4 cell tolerance in an allogeneic combination. METHODS: We investigated the ability of anti-CD40L mAb to promote mixed xenogeneic chimerism and donor-specific tolerance in B6 mice receiving anti-CD8, -Thy1.2 and -NK1.1 mAbs and 3 Gy TBI followed by TCD bone marrow transplantation (BMT) from F344 rats. RESULTS: Administration of anti-CD4 mAb in this model could be completely replaced by one injection of anti-CD40L mAb. Evidence for deletional tolerance was obtained in mixed chimeras prepared with this anti-CD40L-based regimen. However, anti-NK1.1 and anti-Thy1.2 mAb could not be replaced by anti-CD40L mAb. CONCLUSIONS: These results demonstrate that anti-CD40L in combination with xenogeneic BMT can tolerize preexisting peripheral and intrathymic CD4 cells to xenoantigens. However, anti-CD40L does not prevent NK cell and/or gammaDelta cell-mediated rejection of xenogeneic bone marrow.     

CD40 blockade combines with CTLA4Ig and sirolimus to produce mixed chimerism in an MHC-defined rhesus macaque transplant model

In murine models, T-cell costimulation blockade of the CD28:B7 and CD154:CD40 pathways synergistically promotes immune tolerance after transplantation. While CD28 blockade has been successfully translated to the clinic, translation of blockade of the CD154:CD40 pathway has been less successful, in large part due to thromboembolic complications associated with anti-CD154 antibodies. Translation of CD40 blockade has also been slow, in part due to the fact that synergy between CD40 blockade and CD28 blockade had not yet been demonstrated in either primate models or humans. Here we show that a novel, nondepleting CD40 monoclonal antibody, 3A8, can combine with combined CTLA4Ig and sirolimus in a well-established primate bone marrow chimerism-induction model. Prolonged engraftment required the presence of all three agents during maintenance therapy, and resulted in graft acceptance for the duration of immunosuppressive treatment, with rejection resulting upon immunosuppression withdrawal. Flow cytometric analysis revealed that upregulation of CD95 expression on both CD4+ and CD8+ T cells correlated with rejection, suggesting that CD95 may be a robust biomarker of graft loss. These results are the first to demonstrate prolonged chimerism in primates treated with CD28/mTOR blockade and nondepletional CD40 blockade, and support further investigation of combined costimulation blockade targeting the CD28 and CD40 pathways.     

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。     

Pattern of alloimmune response in second same donor allografts after induction of tolerance using CTLA4Ig.

BACKGROUND: The pattern of allograft acceptance in the presence of costimulatory blockade is manifested by the sequential appearance of Th1 cells, followed by Th2 cells. The aim of this study was to examine whether this phenomenon repeats itself after second same donor allotransplantation, hoping to determine whether acceptance in this setting provokes a predominance of the Th2 response. METHODS: Tolerance was achieved by transplantation of CTLA4Ig-transduced ACI liver allografts in Lewis recipients. Recipient long-term survivors received a second transplant, consisting of a cervical heterotopic heart from the same ACI donor strain. Animals were sacrificed at predetermined intervals following the second transplant and the heart and liver were processed for histology and cytokine mRNA expression. RESULTS: Recipients of CTLA4Ig-transduced livers survived indefinitely. Rechallenge with same donor strain second allograft was manifested by an anergic immune response in the second cardiac allograft, and a very mild transient infiltrate within the first accepted liver graft. Cardiac function was maintained with resolution of all infiltrates. The cytokine cascade was activated within the allografts; however, the pattern of acceptance was not associated with predominance of a specific Th subtype. CONCLUSIONS: The pattern of acceptance of an allograft following CTLA4Ig-mediated costimulatory blockade is not related to long-term predominance of Th2 cells, a phenomenon that may be unique to the setting of a tolerant liver. It may be likely that the infiltrating lymphocytes that are dominant in the second graft are suppressed by other memory mechanisms.     

Adult porcine islet transplantation in baboons treated with conventional immunosuppression or a non-myeloablative regimen and CD154 blockade

The aim of the present study was to assess the survival of adult porcine islets transplanted into baboons receiving either (I) conventional triple drug immunosuppressive therapy or (2) a non-myeloablative regimen and an anti-CD154 monoclonal antibody (mAb) aimed at tolerance-induction. Group 1 baboons (n = 3) were pancreatectomized prior to intraportal injection of 10,000 porcine islet equivalents (IE)/kg and immunosuppressed with anti-thymocyte globulin (ATG), cyclosporine and azathioprine. In Group 2 (n = 2), non-pancreatectomized baboons underwent induction therapy with whole body and thymic irradiation, and ATG. Extracorporeal immunoadsorption (EIA) of anti-Galalpha1,3Gal (Gal) antibody was carried out. Maintenance therapy was with cobra venom factor, cyclosporine. mycophenolate mofetil, methylprednisolone and anti-CD154 mAb. Porcine islets were injected intraportally (14,000 and 32,000 IE/kg, respectively) and high-dose pig mobilized peripheral blood progenitor cells (3 x 10(10) cells/kg) were infused into a systemic vein. Porcine islets were also implanted in the sternomastoid muscle to facilitate subsequent biopsies. In both groups. porcine C-peptide was measured, and histological examination of liver or sternomastoid muscle biopsies was performed at regular intervals. In Group 1, total pancreatectomy reduccd human C-peptide to < 0.1 ng/ml and induced insulin-requiring diabetes. The transplantation of porcine islets was followed by normalization of glycemia for 15-24 h. Porcine C-peptide was detected only transiently immediately after porcine islet injection (maximum 0.12 ng/ml). Histological examination of liver biopsies taken between days 2 and 19 did not reveal viable islets, but necrotic cell structures with mononuclear cell infiltrates were identified in portal venules. In Group 2, injection of porcine islets into non-pancreatectomized recipients induced a transient hypoglycemia (2-4 h) requiring concentrated intravenous dextrose administration. Porcine C-peptide was detectable for 5 and 3 days (maximum 2.8 and 1.0 ng/ml), respectively. Baboon #4 died on day 12 from small bowel intussusception. Liver and sternomastoid muscle biopsies showed well-preserved porcine islets, staining positive for insulin and glucacon, without signs of rejection. In baboon #5, viable islets were detected in the sternomastoid muscle biopsy on day 14, but not on day 28 or thereafter. A progressive mononuclear cell and macrophage infiltration was seen in the biopsies. In conclusion, conventional immunosuppression allowed survival of porcine islets in baboons for < 24 h. The non-myeloablative regimen prolonged survival of porcine islets for > 14 days. However, despite depletion of T cells, anti-Gal antibody and complement, and CD154-hlockade, porcine islets were rejected by day 28. These results suggest that powerful innate immune responses are involved in rejection of discordant xenogencic islets.     

Antithymocyte globulin affects the occurrence of acute and chronic graft-versus-host disease after a reduced-intensity conditioning regimen by modulating mixed chimerism induction and immune reconstitution

BACKGROUND: There have been no detailed analyses of the induction of donor cell-type chimerism, the onset and incidence of acute and chronic graft-versus-host disease (GVHD), and the immune recovery kinetics after reduced-intensity stem cell transplantation (RIST). METHODS: To address these, with particular emphasis on the impact of the use of antithymocyte globulin (ATG) in RIST, we compared 39 consecutively registered patients who underwent RIST from an HLA-matched related donor and 33 patients who underwent conventional marrow-ablative transplantation. RESULTS: The incidences of grades II to IV acute and chronic GVHD tended to be less in RIST with ATG than in either RIST without ATG or conventional marrow-ablative transplantation. In a multivariate analysis, the predictive factors for acute and chronic GVHD included, respectively, ATG and grades II to IV acute GVHD. In a chimerism analysis, the achievement of complete donor chimera in T-cell lineage was delayed in RIST without ATG compared with RIST with ATG (P=0.038), which might explain the observed delayed onset of acute GVHD in RIST with ATG compared with the other two regimens. The ratio of type 1 and 2 dendritic cells did not affect the development of GVHD, whereas the number of naive CD4+ T cells did. No difference was observed in the incidence of clinically definitive infection, including cytomegalovirus, among the three cohorts, regardless of the use of ATG. CONCLUSIONS: We suggest that the conditioning regimen and immunosuppressive strategy after RIST should be carefully balanced against the risk of GVHD and of relapse of the basic disorder caused by the lack of a graft-versus-leukemia benefit.     

CTLA4Ig gene transfer prolongs survival and induces donor-specific tolerance in a rat renal allograft

Organ transplantation requires lifelong antirejection therapy, which carries the risk of infection and cancer. A revolutionary approach is to transduce the organ graft with immunomodulatory genes to render them tolerated with no need of systemic immunosuppression. Prolonged allograft survival was achieved by adenovirus-mediated transduction of the cold-preserved kidney with sequences encoding CTLA4Ig, a recombinant fusion protein that blocks T cell activation. Organ expression of the transgene was achieved associated with mild infiltration of mononuclear cells in the transfected kidney. Mixed lymphocyte reaction as well as the production of both Thl and Th2 cytokines were reduced. Thus, the gene transfer technique to prolong graft survival is indeed effective and safe and can induce donor-specific unresponsiveness. Pending appropriate large animal testing, ex vivo genetic manipulation of the organ before surgery may hopefully represent a major step forward in human transplant medicine.     

Low-dose donor bone marrow cells and splenocytes plus adenovirus encoding for CTLA4Ig gene promote stable mixed chimerism and long-term survival of rat cardiac allografts

Co-stimulatory blockade combined with donor bone marrow transfusion engenders stable mixed chimerism and robust tolerance to various organ and cell transplants. However, repeated administration of costly agents to block the co-stimulatory pathway and the high doses of donor bone marrow cells (BMCs) used in most protocols are impeding clinical development of this strategy. To circumvent these shortcomings, we developed a plan in which repeated administration of costly agents was replaced by a single injection of adenovirus containing the gene of interest, and the high dose of donor BMCs replaced by a mixture of low-dose donor BMCs and splenocytes (SPLCs). Cardiac allografts from DA(RT-1(a)) rats were transplanted heterotopically into the abdomens of LEW(RT-1(1)) rats. A cocktail of adenovirus containing CTLA4Ig gene (AdCTLA4Ig), donor BMCs (100 x 10(6)), and SPLCs (50 x 10(6)) was administered to recipients via the portal vein immediately after grafting (n = 6). Treatment with regimens, including AdCTLA4Ig only, AdCTLA4Ig plus donor BMCs, and AdCTLA4Ig plus donor SPLCs, significantly prolonged cardiac allograft survival in recipient rats, while animals that received no treatment or treatment with control adenovirus (AdLacZ) promptly rejected their allografts. Nevertheless, LEW recipients treated with AdCTLA4Ig and the mixture of a low dose of donor BMCs and SPLCs developed stable mixed chimerism, rendering them long-term survivors of cardiac allografts that also accepted skin grafts from the donor but not the third-party strain. We conclude that blockade of CD28-B7 pathway with AdCTLA4Ig plus a mixture of low doses of donor BMCs and SPLCs is a feasible strategy to induce long-term mixed chimerism with a potential application for clinical development.     

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.     

Targeted T-cell depletion or CD154 blockade generates mixed hemopoietic chimerism and donor-specific tolerance in mice treated with sirolimus and donor bone marrow

BACKGROUND: The administration of donor specific bone marrow (DSBM) to mice conditioned with antilymphocyte serum (ALS) and sirolimus can result in stable multilineage mixed chimerism and long-term graft survival. This study seeks to determine if either the targeted depletion of CD4 and/or CD8 pos T cells or costimulation blockade can substitute for ALS and preserve the efficacy of this regimen. METHODS: C57BL/6 recipients of BALB/c skin allografts were treated with DSBM (150 x 10(6) cells), sirolimus (24 mg/kg intraperitonealy), and either ALS or various monoclonal antibodies (alphaCD4, alphaCD8, alphaCD154 alone or in combination). Recipient peripheral blood mononuclear cell (PBMC) depletion, donor chimerism, and deletion of donor reactive T cells were assessed using flow cytometry. The specificity of immunologic nonreactivity and the presence of immunoregulatory activity were assessed through a mixed lymphocyte reaction assay. RESULTS: The administration of ALS, sirolimus, and DSBM resulted in sustained recipient PBMC depletion, transient chimerism, and prolonged graft survival. The substitution of an equivalent degree and duration of targeted depletion of either CD4 or CD8 pos T cells alone for ALS failed to produce chimerism or prolonged graft survival. In contrast, depletion of both CD4 and CD8 pos T cells resulted in durable multilineage chimerism, indefinite allograft acceptance (>350 days), and donor-specific tolerance to secondary skin grafts. Substitution of alphaCD154 monoclonal antibody for ALS also resulted in a state of mixed chimerism and donor specific tolerance. This tolerant state appears to be maintained at least partially through clonal deletion and suppression. CONCLUSION: Either combined CD4 and CD8 T-cell depletion or alphaCD154 blockade can effectively substitute for ALS in producing chimerism and tolerance in this model.