Prolonged survival of neonatal porcine islet xenografts in mice treated with a donor-specific transfusion and anti-CD154 antibody

BACKGROUND: Combined treatment with a single donor-specific transfusion (DST) and a brief course of anti-mouse CD154 monoclonal antibody (mAb) to induce co-stimulation blockade leads to long-term murine islet allograft survival. The authors hypothesized that this protocol could also induce long-term survival of neonatal porcine islet cell clusters (NPCC) in chemically diabetic immunocompetent mice and allow their differentiation into functional insulin-producing cells. METHODS: Pancreata from 1- to 3-day-old pigs were collagenase digested and cultured for 8 days. NPCC were recovered and transplanted into the renal subcapsular space. Recipients included chemically diabetic nonobese diabetic (NOD)-scid and C57BL/6 mice that were otherwise untreated, treated with anti-CD154 mAb alone, or treated with DST plus anti-CD154 mAb. Plasma glucose concentration and body weight were measured, and xenografts were examined histologically. RESULTS: NPCC fully differentiated and restored normoglycemia in four of five diabetic NOD-scid recipients but were uniformly rejected by diabetic C57BL/6 recipients. Anti-CD154 mAb monotherapy restored normoglycemia in 4 of 10 (40%) NPCC-engrafted, chemically diabetic C57BL/6 mice, but combined treatment with DST and anti-CD154 mAb restored normoglycemia in 12 of 13 (92%) recipients. Reversal of diabetes required 5 to 12 weeks. Surviving grafts were essentially free of inflammatory infiltrates 15 weeks after transplantation. CONCLUSIONS: Combination therapy with a single DST and a brief course of anti-mouse CD154 mAb without maintenance immunosuppression permits survival and differentiation of NPCC in diabetic C57BL/6 mice. Successful grafts were associated with durable restoration of normoglycemia and the absence of graft inflammation.     

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.     

CD154 blockade, sirolimus, and donor-specific transfusion prevents renal allograft rejection in cynomolgus monkeys despite homeostatic T-cell activation

BACKGROUND: CD154-specific antibodies have been shown to prevent acute rejection in many preclinical models including nonhuman primates (NHPs). However, they have been ineffective in pilot clinical trials, suggesting a need for more robust preclinical analysis. One factor affecting the disparate results may be related to the recipient's immune activation state. Specifically, adult humans have a high percentage of memory-phenotype T cells compared to young animals. Postdepletional homeostatic repopulation has been shown to enrich for memory-phenotype T cells and interfere with CD154-based therapies in rodents. METHODS: We developed a NHP model nonspecifically enriched for peripheral memory-phenotype T cells. Thymectomized cynomolgus macaques underwent depletion with polyclonal anti-thymocyte globulin followed by repopulation. Peripheral phenotype was serially determined using polychromatic flow cytometry. In vitro response to donor and environmental antigens was also confirmed before and after manipulation. We then tested a regimen previously successful in rhesus monkeys combining anti-CD154, sirolimus, and donor-specific blood transfusion (DST), in a second primate species with and without the provocation of increased peripheral homeostatic T-cell activation. RESULTS: Monkeys that were thymectomized (n=3) and depleted recovered via homeostatic repopulation with a repertoire enriched for cells with a memory surface phenotype compared to unmanipulated controls (n=3). Despite a repertoire markedly enriched for memory-phenotype cells, the regimen effectively prevented acute rejection for the duration of therapy. CONCLUSIONS: Cynomolgus monkeys can be rendered memory phenotype enriched using homeostatic repopulation. Despite a generally activated T-cell repertoire, anti-CD154, sirolimus, and DST effectively prevents rejection in cynomolgus monkeys.     

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.     

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.     

Renal transplantation: cyclosporin A and antibody development after donor-specific transfusion

The survival of a one haplotype, mismatched living-related renal allograft is improved by donor specific transfusion (DST) before transplantation although the mechanism is unclear. The major risk of DST is sensitization of the recipient to donor lymphocytes precluding transplantation. Fifty prospective recipients of a living related transplant received either DST with cyclosporin A (group I) or DST alone (group II). Persistent donor sensitization precluding transplantation occurred in no patients in group I but in six in group II (P less than 0.05). Ten of 14 of those who developed donor cytotoxicity had previously been pregnant or received greater than or equal to 10 third party transfusions compared with 11 of 36 without such a history (P less than 0.05). Alloantibodies detected by a cellular ELISA developed following DST in 29% patients and antiidiotypic antibodies detected by the short antiidiotypic assay (SAA) in 36%; antiidiotypic activity occurred more frequently in those given cyclosporin A (P less than 0.02). Potentiating activity in the SAA which occurred in sera from six patients after DST had no influence on transplant outcome. Persistent sensitization, particularly in potential transplant recipients who have been pregnant or received many transfusions, can be prevented by giving cyclosporin A with DST; the mechanisms of this effect may be the induction of antiidiotypic antibodies. Both alloantibodies and antiidiotypic antibodies are induced by DST and may protect a subsequent renal allograft from the specific donor.     

Follicular T cells mediate donor-specific antibody and rejection after solid organ transplantation

Following solid organ transplantation, a substantial proportion of chronic allograft loss is attributed to the formation of donor-specific antibodies (DSAs) and antibody-mediated rejection (AbMR). The frequency and phenotype of T follicular helper (Tfh) and T follicular regulatory (Tfr) cells is altered in the setting of kidney transplantation, particularly in patients who develop AbMR. However, the roles of Tfh and Tfr cells in AbMR after solid organ transplantation is unclear. We developed mouse models to inducibly and potently perturb Tfh and Tfr cells to assess the roles of these cells in the development of DSA and AbMR. We found that Tfh cells are required for both de novo DSA responses as well as augmentation of DSA following presensitization. Using orthotopic allogeneic kidney transplantation models, we found that deletion of Tfh cells at the time of transplantation resulted in less severe transplant rejection. Furthermore, using inducible Tfr cell deletion strategies we found that Tfr cells inhibit de novo DSA formation but only have a minor role in controlling kidney transplant rejection. These studies demonstrate that Tfh cells promote, whereas Tfr cells inhibit, DSA to control rejection after kidney transplantation. Therefore, targeting these cells represent a new therapeutic strategy to prevent and treat AbMR.     

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.     

CD4+CD25+T细胞联合CD154单抗抑制大鼠肝移植急性排斥反应的研究

目的探讨CD4 CD25 T细胞联合应用CD154单抗在抑制大鼠肝移植急性排斥反应中的作用。方法分离Lewis大鼠脾脏CD4 _CD25 T细胞后与DA大鼠脾细胞单向混合淋巴细胞反应行体外激活。用"二袖套法"行DA到Lewis的原位肝移植48例。A组为对照组;B、C组单独术前回输体外激活的CD4 CD25 T细胞或术后腹腔注射抗CD154单抗;D组联合应用CD4 CD25 T细胞和CD154单抗。每组大鼠12对。术后7 d各组处死6只受体,检测移植肝内T细胞亚群和细胞因子水平。余大鼠观察生存情况,死亡大鼠观察移植肝病理变化。结果D组受体生存期(52.00±10.64)d明显长于B、C组(P<0.01);移植肝内CD4 CD25 T细胞比例(16.43±4.28)%明显高于B、C组(P<0.05、P<0.01),而淋巴细胞浸润数量[(3.47±1.21)%×106]和(CD8 T细胞百分比(14.19±3.02)%明显低于B、C组(P<0.05、P<0.01);移植肝内白细胞介素- 2(IL-2)水平(6.44±1.83)ng/L低于B、C组(P<0.05),IL-10(43.72±7.55)ng/L和转化生长因子-β1(TGF-β1)(270.06±46.91)ng/L明显高于B、C组(P<0.05、P<0.01)。结论联合应用CD154单抗能明显增强CD4 CD25 调节性T细胞对大鼠肝移植急性排斥反应的抑制作用。     

Treatment with the humanized CD154-specific monoclonal antibody, hu5C8, prevents acute rejection of primary skin allografts in nonhuman primates.

BACKGROUND: Allogeneic skin transplantation remains a rigorous test of any immune intervention designed to prevent allograft rejection. To date, no single, clinically available immunosuppressant has been reported to induce long-term primary skin allograft survival in primates. We have previously shown that treatment with the humanized CD154-specific monoclonal antibody, humanized 5C8 (hu5C8), induces long-term renal allograft survival in nonhuman primates. In this study, we evaluated the efficacy of hu5C8 in preventing primary skin allograft rejection in rhesus monkeys. METHODS: Ten rhesus monkeys were transplanted with full-thickness skin allografts mismatched at both class I and class II major histocompatibility loci. Of these, two were given no treatment, five were treated with hu5C8 alone, and three received hu5C8 combined with whole blood donor-specific transfusion (DST). All recipients also received skin autografts for comparison. Animals were followed by inspection, serial biopsy, mixed lymphocyte culture, and alloantibody determination. RESULTS: Treatment with hu5C8 alone or hu5C8 plus DST greatly prolonged allograft survival. Rejection occurred in the untreated group within 7 days. Mean allograft survival in the monotherapy hu5C8 group was >236 days and in the DST group was >202 days; these differences were not significant. Rejection eventually occurred in most animals. Allograft survival was not correlated with the development of T cell hyporesponsiveness in mixed lymphocyte culture. Rejection was not predicted by the development of donor-specific alloantibody. CONCLUSION: These results show that treatment with the CD154-specific monoclonal antibody, hu5C8, greatly delays the onset of acute skin allograft rejection.     

输血诱导免疫耐受对大鼠小肠移植急性排斥反应的影响

目的 观察供体特异性输血(DST)诱导免疫耐受对大鼠小肠移植后急性排斥反应的影响.方法 采用SD至Wistar大鼠异位小肠移植模型,实验组分别予以DST,环孢素(CsA)及DST联合CsA干预,Wistar大鼠同系间移植作为对照,于3、5、7 d观察移植肠管病理变化及受体血清中肿瘤坏死因子(TNF)-α和干扰素(IFN)-γ表达程度.结果 病理显示CsA干预组在7 d出现轻度移植排斥反应;DST联合CsA干预组与对照组结果相似,在3、5、7 d均无明显的移植排斥反应发生.并且DST联合CsA干预组TNF-α表达程度低于单独CsA干预组,在第7天时与对照组比较差异无统计学意义(P＞0.05);IFN-γ表达程度低于单独CsA干预组,在第5、7天时与对照组比较差异无统计学意义(P＞0.05).结论 异基因大鼠间小肠移植在CsA配合应用的情况下,进行DST可诱导其产生一定的免疫耐受,预防及减轻大鼠小肠移植急性排斥反应的发生及反应程度.     

Significance of donor-specific antibodies in acute rejection

Significant advances in recent years in the diagnosis of antibody-mediated graft rejection have led to the re-evaluation of humoral alloreactivity in organ transplantation. By introducing the "C4d-test" into the work-up of transplant biopsies, donor-specific antibodies were claimed to be directly involved in about 30% of acute rejection episodes. The diagnostic criteria for antibody-mediated rejections of renal grafts are now incorporated in the "Banff classification" as refined at a recent consensus conference. Capillary C4d is not always concordant with circulating anti-HLA-antibodies, even if these are assayed with improved techniques. Antibody absorption within the graft and antigens other than HLA, therefore, have to be considered. Effective therapy of humoral rejection is now available. Serial assessment of humoral alloreactivity also in the posttransplantation period is now mandatory to identify at-risk patients.     

Monotherapy with the novel human anti-CD154 monoclonal antibody ABI793 in rhesus monkey renal transplantation model

BACKGROUND: This study assesses the safety and efficacy of the novel human anti-human CD154 monoclonal antibody ABI793 in rhesus monkeys. METHODS: Outbred rhesus monkeys were used for renal transplantation from major histocompatibility complex-mismatched donors. Seven recipients were treated with ABI793, and six untreated recipients were used as controls. Graft function was monitored by urine output, serum creatinine, and renal biopsy. Phenotypic analysis of peripheral blood lymphocytes and mixed lymphocyte reaction were performed before transplantation and periodically after transplantation. Anti-donor major histocompatibility complex class I antibody levels were measured at the time of sacrifice. RESULTS: Monkeys in the treated group demonstrated prolonged graft survival compared with controls. One monkey was sacrificed because of a urine leak on postoperative day 13. Three monkeys were sacrificed because of acute rejection (days 44, 149, and 158). Two monkeys were sacrificed because of chronic active rejection (days 154 and 221). One monkey was sacrificed on day 139 without rejection to observe the effects of ABI793 in the absence of rejection. There were no obvious clinical side effects of ABI793, but microscopic thromboembolic changes were observed in two monkeys. Lymphocyte subsets remained unaltered in all monkeys. Mixed lymphocyte reaction showed nonspecific suppression 6 weeks after transplantation. The monkeys with chronic active rejection showed relatively strong alloantibody responses. CONCLUSIONS: ABI793 induces prolonged renal allograft survival in rhesus monkeys. Nevertheless, thromboembolic complications may occur and chronic allograft nephropathy may develop after anti-CD154 treatment is discontinued.     

The course of untreated acute rejection and effect of repeated anti-CD3 monoclonal antibody treatment in rhesus monkey liver transplantation

The effect of single and repeated treatment of liver allograft rejection using an anti-CD3 monoclonal antibody (FN18) was studied in a rhesus monkey model. Eight RhLA-mismatched monkeys received initial postoperative immunosuppression with CsA/prednisolone for 28 days. After cessation, acute rejection occurred in all animals (days 28-50). Control animals (n = 3) receiving no rejection treatment developed a chronic progressive rejection and died at days 112-160. In the animals treated with FN18 (n = 5), the first acute rejection was successfully reversed. T lymphocytes were cleared from the peripheral blood and the graft. Increased class I and class II MHC-antigens on hepatocytes were reduced to normal levels within 5 days of treatment. The second rejection treatment remained ineffective in two animals with antiidiotypic antibodies to FN18 but was successful in two animals with a low antimouse response. These four animals survived 160-509 days. The results have a number of implications regarding the course of untreated rejection in human liver transplant recipients and repetitive rejection treatment with monoclonal antibodies.     

阻断共刺激通络抑制大鼠肝移植急性排斥反应的研究

目的研究CD40Ig阻断共刺激通路在大鼠肝移植急性排斥反应中的作用.方法用"二袖套法"行Lewis到Brown Norway(BN)大鼠肝移植32例.A组10例为对照组;B组11例,术中肝脏冷保存前门静脉注射Lipofectamine2000-pcDNA3.1空载体复合物,行肝移植;C组11例,肝脏冷保存前门静脉注射Lipofectamine2000-pcDNA3.1 CD40Ig复合物.术后第5天每组杀死BN 3只,取病理并用细胞凋亡的原位检测法检测肝脏凋亡情况,免疫组织化学检测CD40Ig表达,取脾脏分离淋巴细胞,混合淋巴细胞反应观察刺激指数,流式细胞仪检测与CD40Ig结合的T淋巴细胞比例.余大鼠观察生存情况.死亡大鼠观察病理变化.结果 A组平均存活时间(11.00±4.28) d,B组平均存活时间(12.75±5.57) d,C组平均存活时间(41.25±13.70) d(P<0.01).A组、B组病理示中、重度急性排斥反应,凋亡指数分别为33.67±5.69、39.00±5.29,C组免疫组织化学示CD40Ig表达,第5天病理示轻度急性排斥反应,凋亡指数为0.27±0.21(P<0.01),死亡大鼠病理示部分呈中度急性排斥反应,大部分示慢性排斥反应.混合淋巴细胞反应示C组Lewis大鼠对BN大鼠产生特异性耐受.流式细胞仪示与CD40Ig结合的T细胞占总T细胞11.57%.结论供体肝脏转染CD40Ig能有效阻断T细胞共刺激通路,特异性抑制急性排斥反应,延长受体存活时间,但对慢性排斥反应无效.     

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.