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.     

Rat xenograft survival in mice treated with donor-specific transfusion and anti-CD154 antibody is enhanced by elimination of host CD4+ cells

BACKGROUND: Treatment with a donor-specific transfusion (DST) and a brief course of anti-mouse CD154 (anti-CD40-ligand) monoclonal antibody (mAb) prolongs the survival of both allografts and rat xenografts in mice. The mechanism by which allograft survival is prolonged is incompletely understood, but depends in part on the presence of CD4+ cells and the deletion of alloreactive CD8+ T cells. Less is known about the mechanism by which this protocol prolongs xenograft survival. METHODS: We measured rat islet and skin xenograft survival in euthymic and thymectomized mice treated with combinations of DST, anti-CD154 mAb, anti-CD4 mAb, and anti-CD8 mAb. Recipients included C57BL/6, C57BL/6-scid, C57BL/6-CD4null, and C57BL/6-CD8null mice. RESULTS: Pretreatment with a depleting anti-CD4 mAb markedly prolonged the survival of both skin and islet xenografts in mice given DST plus anti-CD154 mAb. Comparable prolongation of xenograft survival was obtained in C57BL/6-CD4null recipients treated with DST and anti-CD154 mAb. In contrast, anti-CD8 mAb did not prolong the survival of either islet or skin xenografts in mice treated with DST and anti-CD154 mAb. Thymectomy did not influence xenograft survival in any treatment group. Adoptive transfer of splenocytes from C57BL/6-CD4null recipients treated with DST and anti-CD154 mAb and bearing long-term skin xenografts revealed the presence of residual xenoreactive cells. CONCLUSIONS: These data suggest that treatment with DST and anti-CD154 mAb induces a state of "functional" transplantation tolerance. They also support the hypothesis that both the induction and maintenance of graft survival based on this protocol depend on different cellular mechanisms in allogeneic and xenogeneic model systems.     

Combination of anti-CD4 with anti-LFA-1 and anti-CD154 monoclonal antibodies promotes long-term survival and function of neonatal porcine islet xenografts in spontaneously diabetic NOD mice

Type 1 diabetes mellitus (T1DM) is caused by the autoimmune destruction of pancreatic islet beta-cells, which are required for the production of insulin. Islet transplantation has been shown to be an effective treatment option for TIDM; however, the current shortage of human islet donors limits the application of this treatment to patients with brittle T1DM. Xenotransplantation of pig islets is a potential solution to the shortage of human donor islets provided xenograft rejection is prevented. We demonstrated that a short-term administration of a combination of anti-LFA-1 and anti-CD154 monoclonal antibodies (mAbs) was highly effective in preventing rejection of neonatal porcine islet (NPI) xenografts in non-autoimmune-prone B6 mice. However, the efficacy of this therapy in preventing rejection of NPI xenografts in autoimmune-prone nonobese diabetic (NOD) mice is not known. Given that the current application of islet transplantation is for the treatment of T1DM, we set out to determine whether a combination of anti-LFA-1 and anti-CD154 mAbs could promote long-term survival of NPI xenografts in NOD mice. Short-term administration of a combination of anti-LFA-1 and anti-CD154 mAbs, which we found highly effective in preventing rejection of NPI xenografts in B6 mice, failed to promote long-term survival of NPI xenografts in NOD mice. However, addition of anti-CD4 mAb to short-term treatment of a combination of anti-LFA-1 and anti-CD154 mAbs resulted in xenograft function in 9/12 animals and long-term graft (>100 days) survival in 2/12 mice. Immunohistochemical analysis of islet grafts from these mice identified numerous insulin-producing beta-cells. Moreover, the anti-porcine antibody as well as autoreactive antibody responses in these mice was reduced similar to those observed in naive nontransplanted mice. These data demonstrate that simultaneous targeting of LFA-1, CD154, and CD4 molecules can be effective in inducing long-term islet xenograft survival and function in autoimmune-prone NOD mice.     

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.     

Long-term survival and function of intrahepatic islet allografts in baboons treated with humanized anti-CD154.

Clinical islet cell transplantation has resulted in insulin independence in a limited number of cases. Rejection, recurrence of autoimmunity, and impairment of normal islet function by conventional immunosuppressive drugs, e.g., steroids, tacrolimus, and cyclosporin A, may all contribute to islet allograft loss. Furthermore, intraportal infusion of allogeneic islets results in the activation of intrahepatic macrophages and endothelial cells, followed by production of proinflammatory mediators that can contribute to islet primary nonfunction. We reasoned that the beneficial effects of anti-CD154 treatment on autoimmunity, alloreactivity, and proinflammatory events mediated by macrophages and endothelial cells made it an ideal agent for the prevention of islet allograft failure. In this study, a nonhuman primate model (Papio hamadryas) was used to assess the effect of humanized anti-CD154 (hu5c8) on allogeneic islet engraftment and function. Nonimmunosuppressed and tacrolimus-treated recipients were insulin independent posttransplant, but rejected their islet allografts in 8 days. Engraftment and insulin independence were achieved in seven of seven baboon recipients of anti-CD154 induction therapy administered on days -1, 3, and 10 relative to the islet transplant. Three of three baboons treated with 20 mg/kg anti-CD154 induction therapy experienced delayed rejection episodes, first detected by elevations in postprandial blood glucose levels, on postoperative day (POD) 31 for one and on POD 58 for the other two. Re-treatment with three doses of anti-CD154 resulted in reversal of rejection in all three animals and in a return to normoglycemia and insulin independence in two of three baboons. It was possible to reverse multiple episodes of rejection with this approach. A loss of functional islet mass, as detected by reduced first-phase insulin release in response to intravenous glucose tolerance testing, was observed after each episode of rejection. One of two baboons treated with 10 mg/kg induction therapy became insulin independent post-transplant but rejected the islet graft on POD 10; the other animal experienced a reversible rejection episode on POD 58 and remained insulin independent and normoglycemic until POD 264. Two additional baboon recipients of allogeneic islets and donor bone marrow (infused on PODs 5 and 11) were treated with induction therapy (PODs -1, 3, 10), followed by initiation of monthly maintenance therapy (for a period of 6 months) on POD 28. Rejection-free graft survival and insulin independence was maintained for 114 and 238 days, with preservation of functional islet mass observed in the absence of rejection. Prevention and reversal of rejection, in the absence of the deleterious effects associated with the use of conventional immunosuppressive drugs, make anti-CD154 a unique agent for further study in islet cell transplantation.     

Evaluation of donor-specific transfusion sources: unique failure of bone marrow cells to induce prolonged skin allograft survival with anti-CD154 monoclonal antibody

BACKGROUND: Treatment with anti-CD154 monoclonal antibody (mAb) plus a donor-specific transfusion (DST) of spleen cells prolongs skin allograft survival in mice through a mechanism involving deletion of host alloreactive CD8(+) T cells. It is unknown if other lymphohematopoietic cell populations can be used as a DST. METHODS: Murine recipients of allogeneic skin grafts on day 0 were either untreated or given a DST on day -7 plus 4 doses of anti-CD154 mAb on days -7, -4, 0, and +4. Deletion of CD8(+) alloreactive cells was measured using "synchimeric" CBA recipients, which circulate trace populations of TCR transgenic alloreactive CD8(+) T cells. RESULTS: Transfusion of splenocytes, thymocytes, lymph node cells, or buffy coat cells led to prolonged skin allograft survival in recipients treated with anti-CD154 mAb. In contrast, bone marrow DST failed to delete host alloreactive CD8(+) T cells and was associated with brief skin allograft survival. Transfusions consisting of bone marrow-derived dendritic cells or a mixture of splenocytes and bone marrow cells were also ineffective. CONCLUSIONS: Donor-specific transfusions of splenocytes, thymocytes, lymph node cells, or buffy coat cells can prolong skin allograft survival in recipients treated with costimulation blockade. Bone marrow cells fail to serve this function, in part by failing to delete host alloreactive CD8(+) T cells, and they may actively interfere with the function of a spleen cell DST. The data suggest that transplantation tolerance induction protocols that incorporate bone marrow cells to serve as a DST may not be effective.     

Indefinite survival of neonatal porcine islet xenografts by simultaneous targeting of LFA-1 and CD154 or CD45RB

A variety of transient therapies directed against molecules involved in T-cell activation and function result in long-term islet allograft survival. However, there are relatively few examples of durable islet xenograft survival using similar short-term approaches, especially regarding highly phylogenetically disparate xenograft donors. Previous studies demonstrate that combined anti-lymphocyte function-associated antigen-1 (LFA-1) plus anti-CD154 therapy results in a robust form of islet allograft tolerance not observed with either individual monotherapy. Thus, the aim of this study was to determine whether the perturbation of anti-LFA-1, either alone or in combination with targeting CD154 or CD45RB, would promote neonatal porcine islet (NPI) xenograft survival in mice. NPI xenografts are rapidly rejected in wild-type C57BL/6 mice but reproducibly mature and restore durable euglycemia in diabetic, immune-deficient C57BL/6 rag-1(-/-) recipients. A short course of individual anti-LFA-1, anti-CD154, or anti-CD45RB therapy resulted in long-term (>100 days) survival in a moderate proportion of C57BL/6 recipients. However, simultaneous treatment with anti-LFA-1 plus either anti-CD154 or anti-CD45RB therapy could achieve indefinite xenograft function in the majority of recipient animals. Importantly, prolongation of islet xenograft survival using combined anti-LFA-1/anti-CD154 therapy was associated with little mononuclear cell infiltration and greatly reduced anti-porcine antibody levels. Taken together, results indicate that therapies simultaneously targeting differing pathways impacting T-cell function can show marked efficacy for inducing long-term xenograft survival and produce a prolonged state of host hyporeactivity in vivo.     

输注胰岛抗原特异性Treg细胞延长同系NOD小鼠移植胰岛的存活时间

目的 探讨输注胰岛抗原特异性调节性T淋巴细胞(Treg细胞)对非肥胖糖尿病(NOD)小鼠同系胰岛移植物存活时间的影响.方法·以未成熟树突状细胞(imDC)联合谷氨酸脱羧酶-65在体外诱导童贞T淋巴细胞分化成胰岛抗原特异性Treg细胞.以已发生糖尿病的NOD小鼠为受者,将分离得到的尚未进展为糖尿病的NOD小鼠的胰岛(500胰岛当量)移植至受者的肾包膜下,对照组不行移植,只观察血糖变化;单纯胰岛移植组只进行胰岛移植,不输注胰岛抗原特异性Treg细胞;实验组于术前1d静脉输注1×106个胰岛抗原特异性Treg细胞,然后进行胰岛移植.术后检测受者的血糖,以判断移植胰岛的存活时间,观察胰岛移植物的病理学变化.结果 对照组血糖持续高于11.1 mmol/L;单纯胰岛移植组小鼠的血糖于术后1～2 d降至正常,到7～17d时开始陆续升高,并维持在术前水平,移植物存活时间为(12.2±2.6)d;实验组小鼠的血糖于术后1～2 d降至正常,至第27天开始有小鼠血糖升高超过11.1 mmol/L,第43天时,所有小鼠的血糖均超过11.1mmol/L,移植物的存活时间为(35.2±4.3)d,明显长于单纯胰岛移植组(P＜0.01).单纯胰岛移植组的移植胰岛有明显的淋巴细胞浸润,并伴有胰岛细胞严重破坏,胰岛素染色未见完整的胰岛存在,仅有极少量残存的分泌胰岛素的胰岛细胞;实验组第15天时移植胰岛形态完整,仅有少量淋巴细胞浸润,分泌胰岛素的胰岛大量存在.结论 体外诱导产生的胰岛抗原特异性Treg细胞可以延缓自身免疫系统对移植胰岛的破坏,明显延长NOD小鼠移植胰岛的存活时间.     

Prolonged survival of rat islet xenografts in mice after CD45RB monotherapy

BACKGROUND: Pancreatic islet transplantation can correct the disordered glucose metabolism of type 1 diabetes, but the number of successful transplants has been low because of the need for long-term immunosuppression and the limited availability of human islets. New approaches, such as the use of tolerance-inducing treatment modalities and the use of islets of nonhuman sources, can possibly improve the success of islet transplantation. In the present study, the authors investigated the effect of anti-CD45RB treatment on the survival of islet xenografts. METHODS: Chemically induced diabetic mice underwent xenografting with rat islets and were treated with CD45RB antibodies on days -1, 0, and 5. Immunohistology and real-time polymerase chain reaction were used to study the effect of the treatment in the xenografts. The effect of anti-CD45RB treatment in peripheral blood of normal mice was measured with flow cytometry. RESULTS: In the treated mice, survival of the grafts was prolonged substantially. In the treated mice with functioning grafts, no lymphocytes were found infiltrating the transplanted islets on day 6; whereas in the untreated animals with functioning grafts, signs of rejection were evident. In the grafts of the treated animals, significantly less mRNA for interleukin (IL)-2, interferon-gamma, and IL-4 was found compared with the untreated mice. After CD45RB treatment, there was depletion or decrease of CD45RBbright cells from the peripheral blood. CONCLUSIONS: Our results show that a short course of anti-CD45RB monotherapy prolongs the survival of rat islet xenografts in C57BL/6 mice.     

Combination of donor-specific blood transfusion with anti-CD28 antibody synergizes to prolong graft survival in rat liver transplantation

Donor-specific blood transfusion (DST) has been shown to effectively induce tolerance to certain allografts. In addition, it is well known that blockade of costimulatory signals reduces the ability of T cells to respond to alloantigens, prolonging allograft survival in some transplant models. We assessed the effects of single or multiple DSTs in the absence or presence of anti-CD28 monoclonal antibodies (mAbs) on graft function and host survival in rat liver transplantation (LTx). Fully MHC-mismatched adult male Dark Agouti (DA) and Lewis (LEW) rats were used as donors and recipients, respectively. Heparinized DA blood was administered to na?ve LEW rats 7 days before LTx [DST(-7d)], 14 and 7 days before LTx [DST(1 x 2)], twice a week for 2 weeks prior to LTx [DST(2 x 2)] and once a week for 4 weeks prior to LTx [DST(1 x 4)]. For some experiments, two different monoclonal antibodies (mAb) to rat CD28 (JJ316 and JJ319) were administered in combination with some DST treatments. We found that DST administration induced a time- and dose-dependent increase in host survival. Treatment of LEW rats with JJ316 or JJ319 mAb alone failed to prolong graft survival over untreated rats; however, the combination of DST(1 x 2) with JJ316 or JJ319 mAb induced indefinite survival at 100 days following surgery. We found that this protective effect was associated with increased numbers of splenic CD4+ CD45RC- but not CD4+ CD25+ foxp3+ T-cells in long-term survivors. Our data suggest that the combination of suboptimal DST with CD28 mAb induces donor-specific tolerance that correlates with enhanced numbers of regulatory T-cells.     

基因转移CTLA4-Ig和抗CD154抗体在异种胰岛移植排斥反应中的作用

目的 探讨基因转移细胞毒性T细胞相关抗原4免疫球蛋白(CTLA4-Ig)和抗T细胞分化群154(CD154)抗体在异种胰岛移植排斥反应中的作用及机理.方法 建立人-大鼠异种胰岛移植模型,用携带CTLA4-Ig基因的重组腺病毒感染移植胰岛细胞,并用抗CD154抗体进行治疗,观察糖尿病大鼠胰岛移植后血糖变化、生存情况及移植物病理形态学改变,检测移植物CTLA4-Ig、胰岛素的表达和移植大鼠白细胞介素2(IL-2)、肿瘤坏死因子(TNF)-α的水平变化.结果 (1)糖尿病大鼠移植后2 d血糖降至正常,对照组血糖平均在移植后8 d升高,抗体治疗组、转染组和联合治疗组血糖分别在18、25和36 d升高.(2)对照组、抗体治疗组、转染组和联合治疗组的移植物存活时间分别为(10.0±2.1)d、(22.0±8.2)d、(28.0±6.5)d和(37.0±9.3)d,各组间比较差异有统计学意义(P＜0.05);移植大鼠生存时间分别为(21.0±5.7)d、(35.0±6.5)d、(48.0±8.5)d和(65.0 ±12.5)d,各组间比较差异有统计学意义(P＜0.05).(3)对照组在移植后1周内,IL-2、TNF-α的水平均急剧上升,较移植前显著升高(P＜0.01).(4)各治疗组移植物见成片的胰岛细胞团,未见淋巴细胞浸润,转染组和联合治疗组移植物可见CTLA4-Ig和胰岛素的表达.结论 基因转移CTLA4-Ig和抗CD154抗体均可抑制异种胰岛移植排斥反应,二者联合效果优于单独使用.     

Long-term survival and function of intraportal porcine and human islet xenografts in nondiabetic nude mice

Instant blood-mediated inflammatory reaction (IBMIR) is a serious obstacle to both clinical islet allotransplantation and future islet xenotransplantation via the portal vein. We have previously observed uniform long-term tilapia (fish) islet xenograft survival when islets were transplanted intraportally into nondiabetic nude mice (nDNM), but not in diabetic nude mice (DNM). In this study, we examined whether human islets (HI) and adult porcine islets (API) can tolerate intraportal transplantation into nDNM like tilapia islets. HI and API were transplanted intraportally into nDNM. Recipients were humanely killed either 14 or 28 days after transplantation and livers were processed for histology. Human insulin and human C-peptide were measured in the terminal serum samples of HI recipients. In six of seven HI and seven of seven API recipients, liver histology showed insulin-positive islet xenografts. In recipients with HI, the numbers of islets/ductal structures seen histologically correlated well with serum sample results. These results show that HI and API can survive and function long term after intraportal transplantation into nDNM recipients. Our previous and present data indicated that DNM and nDNM could be useful models to study "glucose toxicity" and the role of IBMIR in the fate of intraportal islet grafts.     

Prolonged survival of nonhuman primate renal allograft recipients treated only with anti-CD4 monoclonal antibody

The immunosuppressive efficacy of the monoclonal antibody OKT4A reactive with human and monkey CD4 cells was evaluated in cynomolgus renal allograft recipients. Low-dose (0.1 to 0.3 mg/kg/day) intact monoclonal antibodies (10 recipients) or F(ab')2 fragments (two recipients) were administered for 12 days. High-dose OKT4A (10 mg/kg) was administered on the day of transplantation as the only suppression in five animals. Four control animals received either no therapy or a monoclonal antibody nonreactive with monkey cells (OKT3). Maximum survival of the control animals and those treated with F(ab')2 was 11 days. Mean survival in the recipients of low-dose OKT4A was 25.4 +/- 4.3 days and in the group receiving high-dose OKT4A it was 39 +/- 6.4 days. All OKT4A-treated animals showed "coating" and CD4 modulation without depletion of circulating T cells. No modulation occurred in the F(ab')2-treated recipients. Serial allograft biopsy specimens showed reduced lymphocyte infiltration that was nearly complete in recipients of high-dose OKT4A. Biopsy-derived donor-reactive cytotoxic T-cell lines were generated regularly from recipients of low-dose, but not high-dose, OKT4A during periods of stable function. All animals treated with monoclonal antibodies developed an immunoglobulin G antimurine humoral response. Thus OKT4A is a potent immunosuppressive agent administered even as a single bolus, and depletion of CD4 cells is not required to suppress rejection. Anti-CD4 monoclonal antibodies may prove useful in patients, perhaps requiring only a limited number of higher-dose injections in the peritransplant period.     

Prolonged islet allograft survival in diabetic NOD mice by targeting CD45RB and CD154

Clinical islet transplantation is a successful procedure that can improve the quality of life in recipients with diabetes. A drawback of the procedure is the need for chronic administration of immunosuppressive drugs that, among other side effects, are potentially diabetogenic. Definition of immunosuppressive protocols that utilize nondiabetogenic compounds could further improve islet transplantation outcome. We used the NOD mouse to assess the effect of targeting the T-lymphocyte surface receptors CD45RB and CD154 in preventing loss of allogeneic islet grafts as a result of recurrence of autoimmunity and allorejection. Administration of the two antibodies led to significantly prolonged allograft survival, with a percentage of grafts surviving long-term. The therapeutic efficacy of the treatment was paralleled by a shift in CD45RB isoform expression on T-lymphocytes, increased in vitro responsiveness to interleukin-7, and increased in vitro gamma-interferon production after anti-CD3 antibody stimulation. Furthermore, graft infiltration by CD8+ T-cells was remarkably reduced. Recipient mice bearing functioning allografts were otherwise immunocompetent, as assessed in vivo and in vitro by numerous tests, including intragraft cytokine production, responsiveness to polyclonal stimulation and alloantigens, and analysis of cell subset phenotype. These data show that nondiabetogenic regimens of immunomodulation can lead to prolonged islet allograft survival in the challenging NOD mouse model.     

Long-term survival of hamster islet xenografts in mice under short-course treatment with nondepleting versus depleting anti-CD4 monoclonal antibodies

ABSTRACT: Xenogeneic grafts provide a potential alternative to the current shortage of human organs for transplantation. However, the prevention of rejection and tolerance induction of xenografts still remain to be further explored. Islet xenografts appear more promising than vascularized whole organ xenografts and additionally also more resistant to the recurrence of autoimmune disease than allografts. Recently, the nondepleting monoclonal antibody (mAb), which blocks the CD4 molecule on lymphocytes, was reported to be able to induce tolerance in allotransplantation and CD4 positive cells were further confirmed to be a major factor responsible for cellular xenograft rejection. Therefore, we hypothesize that anti-CD4 nondepleting mAb could also be effective in protecting cellular xenografts and inducing unresponsiveness of recipients. We studied the effect of the nondepleting anti-CD4 mAb YTS177.9 on islet xenograft survival by using the hamster-to-mouse islet transplantation model. Results were compared with that of the depleting anti-CD4 mAb GK1.5 that was shown to have similar binding sites on the CD4 molecule to mAb YTS 177.9. Our data show that mAb YTS 177.9 did effectively prolong the survival of islet xenografts and, in addition, also successfully did induce long-term acceptance of 40% grafts after only three penoperative injections of 0.5 mg mAb per mouse. The average survival of the graft was markedly prolonged to >66.8±37.1 days compared with controls (8.3±1.4 days) or with the depleting anti-CD4 mAb GK1.5 (25.7±5.5 days). However, the latter displayed a more profound inhibition in in vitro and ex vivo mixed lymphocyte xenoreaction than mAb YTS 177.9. Moreover, the activity of this nondepleting mAb was found to be dose-dependent and 80% of grafts survived permanently when the dose was increased to six injections of 0.5 mg mAb. Like mAb GK1.5, mAb YTS 177.9 also prevented rejection when given after a delay of two days posttransplant. In addition, we found that neither depleting nor nondepleting anti-CD8 mAb was effective in this model. Our results strongly suggest that an anti-CD4 nondepleting or blocking mAb alone is able to induce long-term acceptance of islet xenografts and that blocking the CD4 molecule is significantly superior to depleting CD4 positive cells for the protection of islet xenografts. This may indicate that CD4 cells play a major role in xenograft tolerance induction.