B7-1 (CD80) and B7-2 (CD 86) expression in human tubular epithelial cells in vivo and in vitro

BACKGROUND: Tubulointerstitial inflammation with infiltration of mononuclear cells plays an important role in acute allograft rejection and in the progression of renal diseases. We therefore investigated in vivo the expression of the costimulatory molecules B7-1 and B7-2 on proximal tubular epithelial cells (PTEC) under normal and pathologic conditions and analyzed the regulation and functional role of these molecules after cytokine and CD40 activation in vitro. METHODS: Immunohistological staining for B7-1 and B7-2 on cryostat sections of core needle biopsies from patients with different renal diseases was examined. Patients were divided into three groups: group A: diffuse interstitial inflammation; group B: minor interstitial inflammation; group C: no interstitial inflammation. In addition, the expression of B7-1 and B7-2 protein and mRNA of cultured PTEC that had been stimulated with cytokine-combinations in absence or presence of a stimulatory anti-CD40 antibody was investigated by means of FACS analysis and RT-PCR. The functional role was analyzed in MKLCs with cytokine and anti-CD40 prestimulated PTEC by measuring IFN-gamma and IL-2 expression in absence or presence of CTLA4-Ig by ELISA. RESULTS: Group A patients showed intense tubular staining for B7-1 and B7-2, group B patients showed mild staining, whereas in group C patients B7-1 and B7-2 staining was negative or only weakly positive. In vitro, the presence of B7-1 and B7-2 on PTEC was increased after stimulation with combinations of IL-1alpha, IL-4, IFN-gamma or IL-13 instead of IL-4 and CD40 activation. B7-1 and B7-2 mRNA could be detected in PTEC as well. In MKLCs only cytokine and anti-CD40 prestimulated PTEC were able to stimulate IFN-gamma and IL-2 production by purified T cells, which could be blocked dose-dependently by CTLA4-Ig. CONCLUSIONS: This study clearly shows that B7-1 and B7-2 can be induced on PTEC in vivo and in vitro. After B7-1 and B7-2 induction, PTEC costimulate CD28 on T lymphocytes resulting in cytokine production. This might be of relevance in allograft rejection and in various kidney diseases.     

Graded function of CD80 and CD86 in initiation of T-cell immune response and cardiac allograft survival.

CD28 and cytotoxic T-lymphocyte antigen-4 (CTLA-4), which have opposing functions in T-cell responses, share the two ligands, CD80 (B7-1) and CD86 (B7-2). To understand the functions of CD80 and CD86, respectively, CD80(low) dendritic cells (DCs) and CD86(low) DCs were prepared by using RNA interference. Then CD80 and CD86 functions were analysed by in vitro mixed lymphocyte reaction and cytokine production assay. Effect on cardiac allograft survival was assayed in vivo. In this study, graded stimulatory function of CD80 and CD86, stronger inhibition of proliferation, and stronger prolongation of transplant survival were observed when CD80 and CD86 were blocked simultaneously.     

Spontaneous allograft tolerance in B7-deficient mice independent of preexisting endogenous CD4+CD25+ regulatory T-cells

BACKGROUND: Acute cardiac allograft rejection requires host, but not donor, expression of B7-1/B7-2 costimulatory molecules. However, acute cardiac rejection requires direct antigen presentation by donor-derived antigen presenting cells to CD4 T-cells and does not require indirect antigen presentation to CD4 T-cells. Given this discrepancy in the literature and that the consequence of allograft exposure in B7-deficient mice is unknown; the goal of the study was to examine the antidonor status of allografted B7-1/B7-2-deficient hosts. METHODS: C57Bl/6 B7-1/B7-2-/- mice were grafted with heterotopic BALB/c hearts. Recipients bearing long-term surviving allografts were used to examine the status of antidonor reactivity in vitro and in vivo. Tolerance was examined in vivo through adoptive transfer of splenocytes from graft-bearing animals to secondary immune-deficient Rag-1-/- hosts bearing donor-type or third-party cardiac allografts and by regulatory T-cell depletion with anti-CD25 antibody. RESULTS: When transferred to B7-replete Rag-1-/- recipients, cells from na?ve B7-1/B7-2-/- mice readily initiated cardiac allograft rejection. However, splenocytes transferred from long-term allograft acceptor B7-1/B7-2-/- hosts failed to reject donor-type hearts but acutely rejected third-party allografts. In addition, such cells did not reject (donorxthird-party) F1 allografts. Finally, in vivo depletion of regulatory T-cells did not prevent long-term acceptance. CONCLUSIONS: Results demonstrate that B7-deficient T-cells are capable of acute cardiac allograft rejection in a B7-replete environment. Importantly, results also show that B7-deficient hosts do not simply ignore cardiac allografts, but rather spontaneously develop transferable, donor-specific tolerance and linked suppression in vivo. Interestingly, this tolerant state does not require endogenous CD4+CD25+ regulatory T-cells.     

Treatment with humanized monoclonal antibodies against CD80 and CD86 combined with sirolimus prolongs renal allograft survival in cynomolgus monkeys

BACKGROUND: Co-stimulatory blockade has been shown to prolong allograft survival in different transplant models. We investigated the effect of combining humanized anti-CD80 and anti-CD86 monoclonal antibodies (mAb) with sirolimus in cynomolgus monkey renal transplant recipients. METHODS: After renal transplantation, groups of four animals were treated daily with sirolimus, sirolimus and nine weekly doses of mAb, two weekly doses of mAb, or sirolimus and two weekly doses of mAb. RESULTS: Survival was significantly better in monkeys treated with the combination of sirolimus and mAb when compared with treatment with either agent alone (P=0.0067 by log-rank analysis). When combined with sirolimus, nine weekly doses of mAb did not result in an additional survival benefit compared with only two mAb doses (P=0.74). None of the treatment regimens used in this study resulted in development of transplantation tolerance. CONCLUSIONS: Sirolimus can be successfully combined with humanized mAb against CD80 and CD86. Induction with a short course of mAb is effective in prolonging allograft survival in combination with sirolimus.     

Cross-species costimulation: relative contributions of CD80, CD86, and CD40

BACKGROUND: The response of human CD4+ T cells against porcine cells is of comparable magnitude to that induced by human leukocyte antigen-mismatched allogeneic cells. This reflects productive interactions between key costimulatory molecules across the species barrier. Inhibition of these molecular interactions will be crucial in overcoming CD4+ T-cell-mediated rejection of xenografts. We have performed a detailed investigation to determine the expression profiles and relative contributions of the three key costimulatory molecules in the porcine-human xenogeneic response. Whereas only porcine CD86 is constitutively expressed on resting endothelial cells, both CD40 and CD80 are rapidly expressed after activation. All three costimulatory molecules are expressed by professional antigen-presenting cells. METHODS: We have isolated full-length cDNA clones for human and porcine CD80, CD86, and CD40. Human fibroblast cell lines (M1) coexpressing DR1 were transfected with these cDNAs and used in mixed lymphocyte reactions and flow cytometric studies in vitro. RESULTS: These data provide the first characterization of the expression profile and functional role of porcine CD80. Functional assays demonstrate that pCD40, pCD80, and pCD86 are independently capable of costimulating human CD4+ T cells, albeit with differing kinetics. Proliferative responses were of comparable magnitude to those obtained when costimulation was provided by human CD40, CD80, and CD86. CONCLUSIONS: These data have implications for therapy targeting the direct pathway of xenorecognition; costimulatory molecule blockade must be directed against both the B7/CD28 and CD40/CD40L pathways.     

Induction therapy with monoclonal antibodies specific for CD80 and CD86 delays the onset of acute renal allograft rejection in non-human primates

CD80 and CD86 (also known as B7-1 and B7-2, respectively) are both ligands for the T cell costimulatory receptors CD28 and CD152. Both CD80 and CD86 mediate T cell costimulation, and as such, have been studied for their role in promoting allograft rejection. In this study we demonstrate that administering monoclonal antibodies specific for these B7 ligands can delay the onset of acute renal allograft rejection in rhesus monkeys. The most durable effect results from simultaneous administration of both anti-B7 antibodies. The mechanism of action does not involve global depletion of T or B cells. Despite in vitro and in vivo evidence demonstrating the effectiveness of the anti-B7 antibodies in suppressing T cell responsiveness to alloantigen, their use does not result in durable tolerance. Prolonged therapy with murine anti-B7 antibodies is limited by the development of neutralizing antibodies, but that problem was avoided when humanized anti-B7 reagents are used. Most animals develop rejection and an alloantibody response although still on antibody therapy and before the development of a neutralizing antibody response. Anti-B7 antibody therapy may have use as an adjunctive agent for clinical allotransplantation, but using the dosing regimens we used, is not a tolerizing therapy in this non-human primate model.     

CD80和CD86在IgA肾病肾小管间质中的表达及其临床意义

细胞免疫在IgA肾病的发生、发展过程中发挥着重要作用.在发病早期表现为外周血T细胞过度活化.其中,CD80和CD86是两个重要的共刺激分子.肾小管间质病变程度是影响IgA肾病进展和预后的重要因素之一~([1-3]).因此,我们探讨CD80与CD86在IgA肾病进展过程中的作用.     

监测大鼠心脏移植前后共刺激分子B7及其受体表达的意义

目的观察共刺激分子B7-1、B7-2及其受体CD28、OX40和CD11a在大鼠心脏移植排斥反应中的变化,探讨其作用及意义。方法用流式细胞仪动态监测大鼠异位心脏移植前后不同时间外周血白细胞中B7分子及OX40、CD11a、CD28受体的表达变化。结果术后淋巴细胞和单核细胞中B7-1、B7-2分子及OX40、CD11a受体的表达高峰值均出现在术后第7d或维持高峰平台至术后第7d,此时移植物排斥反应多表现为2~3级;CD28受体的表达在移植前后与排斥反应程度之间无明显相关。结论动态监测大鼠外周血淋巴、单核细胞中B7-1、B7-2分子及OX40、CD11a受体的表达有助于观察移植排斥反应的进程。     

The role of CD8+ and CD4+ cells in islet allograft rejection

The requirements of CD8+ and CD4+ cells for islet graft rejection in combinations with different histoincompatibilities were investigated by in vivo administration of anti-Lyt-2.2 (CD8) mAb, anti-L3T4 (CD4) mAb, or both to recipient mice. In B10.AQR----B10.A (H-2K-incompatible) and B10.A(5R)----B10.A (H-2K- and IA-incompatible) combinations, administration of either anti-Lyt-2.2 (CD8) or anti-L3T4 (CD4) mAb completely blocked islet graft rejection, indicating that neither CD8+ cells nor CD4+ cells alone were capable of mediating rejection, and that collaboration of CD8+ cells and CD4+ cells was necessary. On the other hand, in the BALB/c----B6 (H-2- and non-H-2-incompatible) combination, administration of anti-Lyt-2.2 (CD8) or anti-L3T4 (CD4) mAb resulted in rejection of most of the grafts, although survival was prolonged significantly, and administration of both anti-Lyt-2.2 (CD8) and anti-L3T4 (CD4) mAb together completely blocked rejection. These results suggested that either CD8+ or CD4+ cells were capable of mediating rejection, but that rejection was maximal in the presence of both T cell subsets. Immunohistochemical analyses showed marked depletion of CD8+ cells and CD4+ cells in grafted islets as well as spleens when anti-Lyt-2.2 (CD8) and anti-L3T4 (CD4) mAb, respectively, were injected.     

HLA-G-treated tolerogenic dendritic cells induce tolerogenic potential by increasing expression of B7-1 (CD80) molecules

BACKGROUND: By consensus, HLA-G has a role in the induction of tolerance via interaction with dendritic cells and manipulation of co-stimulatory molecule expression. The purpose of this study was to demonstrate that HLA-G modified dendritic cells exhibit a decreased ability to stimulate T-cells in vitro due to increased expression of B7-1, which provides regulatory signalling to T-cells as a consequence of binding CD28 and CD152 ligands. METHODS: Bone-marrow cells were cultured from Brown Norway (BN) rat femurs and sorted with anti-rat dendritic cell Ab (OX62) microbeads. Isolated dendritic cells (DCs) were treated with HLA-G tetramer for 3 days. Initially, cells were plated with media, alloantigenic splenocytes, and T-cells and then observed in mixed lymphocyte reaction for thymidine uptake. Also, the cells were analyzed by flow cytometry using antibodies for MHC-II (IA), CD80, and CD86. RESULTS: This study displays that HLA-G-modified DCs decrease induction of an alloproliferative response. In addition, this study demonstrated that HLA-G tetramer treatment decreases CD86 and permits expression of CD80 without altering MHC-II expression. DISCUSSION: This study specifically investigated the role of B7-1 (CD80), showing that HLA-G treatment of immature DCs allows expression of B7-1 (CD80) without altering MHC-II expression and simultaneously decreases B7-2 (CD86) expression. Therefore, a low level of B7-2 (CD86) allows attenuation of T-cell response after activation, both of which are beneficial to immune tolerance. The selective expression of B7-1 (CD80) is important and may serve as a guide for future therapies aimed at transplant tolerance.     

Defective expression of B7-2 (CD86) on monocytes of dialysis patients correlates to the uremia-associated immune defect

BACKGROUND: Specific cellular immune reactions in patients with chronic renal failure (CRF) are impaired by a defect of the antigen-presenting cells. To elucidate the molecular background for this defect, we determined the expression of human lymphocyte antigen (HLA)-DR and costimulatory molecules on monocytes of hemodialysis patients. METHODS: The expression of HLA-DR, B7-1 (CD80), and B7-2 (CD86) molecules was determined on CD14+ monocytes of chronic hemodialysis patients prior to a dialysis session. Mononuclear cells of these patients were cultured, and expression of the respective antigens was determined after in vitro activation by various stimuli. Results were correlated with in vitro proliferation of T cells in a phytohemagglutinin (PHA) assay and the clinical response to a hepatitis B vaccination. All data were compared with healthy controls and patients with CRF who were not on dialysis. RESULTS: Monocytes of chronic hemodialysis patients but not CRF patients expressed low levels of costimulatory B7-2, while HLA-DR expression was normal. B7-1 was only expressed on activated monocytes, and the expression reached normal levels in hemodialysis patients. Baseline expression of B7-2 highly correlated with the results of T-cell proliferation assays in hemodialysis patients and also with the clinical immune response. CONCLUSIONS: Impaired expression and up-regulation of B7-2 is an important feature of the cellular immune defect in chronic hemodialysis patients. It leads to reduced costimulation and effector activation of T cells and contributes to a molecular explanation for the impaired response of hemodialysis patients to the hepatitis B vaccination.     

The role of donor lymphoid cells in the transfer of allograft tolerance

Tolerance to murine skin allografts across a MHC disparity was induced by conditioning primary hosts with sublethal fractionated total-body irradiation (FTBI) and transfusion of allogeneic bone marrow (BM). Tolerance could be adoptively transferred to secondary hosts conditioned by FTBI with infusion of spleen cells from hosts bearing intact skin allografts greater than 60 days. Tolerance could not be transferred by tolerant host spleen (THS) preparations from which cells of the donor genotype had been deleted by cytotoxic alloantisera. Deletion of host genotype cells, however, did not diminish the capability of THS to transfer tolerance. All of the tolerizing activity of THS appeared to reside within cells of the donor genotype. Small numbers of normal donor spleen cells could induce tolerance in FTBI hosts but only at the expense of very high mortality, in contrast to the low mortality observed with tolerizing injections of allogeneic donor cells from THS or injections of normal semiallogeneic F1 hybrid spleen cells. If an active immune response is responsible for tolerance induction/transfer in this model, allogeneic donor lymphoid cells derived from BM, in contrast to donor spleen cells, must be capable of mounting this response without concomitant severe GVHD. In future experiments, cells of donor genotype can be isolated from THS and purified in sufficient numbers to compare their tolerizing efficiency vs. that of normal donor cells, detect possible suppression of normal host cell alloreactivity in vitro and identify the donor cell phenotypes involved.     

联合阻断CD28/B7与CD40/CD40L共刺激通路诱导抗原特异性免疫耐受

目的探讨阻断CD28／B7与CD40／CD40L共刺激通路对同种异体小鼠移植心脏存活时间的影响及其机理。方法实验分4组进行，均以C57BL／6小鼠为受者，BALB／c小鼠为供者，施行腹部异位心脏移植，根据分组要求，MR1组于移植当天静脉内注射抗CD40L单克隆抗体（MR1抗体）0．25mg／d，移植后第2、4天改为腹腔注射0．25mg／d；抗B7组于移植当天至术后第4天腹腔内注射抗B7—1和抗B7—2抗体各0．1nag／d；联合处理组术后联合使用MR1抗体和抗B7抗体，二者的用法同MR1组和抗B7组；对照组术后不使用任何抗体。记录各组移植心的存活时间；移植后60d时对移植心脏组织行病理学检查。联合处理组的受者于心脏移植后150d分别接受供者来源（BALWc小鼠）及无关供者来源（C3H小鼠）的皮肤移植，对照组的受者也同时接受两种皮肤移植，术后不进行处理，术后观察移植皮片的存活时间。结果对照组移植心脏存活时间为（7．86±1．57）d，与对照组比较，MR1组、抗B7组和联合处理组的移植心脏存活时间均得到显著延长，但联合处理组延长最为明显，均超过150d；MR1组和抗B7组移植心脏组织病理学检查均呈慢性排斥反应改变，而联合处理组未见明显慢性排斥反应征象。联合处理组移植的供者来源皮肤存活时间均超过50d，而无关供者来源的皮肤则被很快排斥；对照组两种来源的皮肤移植后存活时间均较短。结论联合阻断CD28／B7与CD40／CD40L共刺激通路可延长移植心脏存活时间，诱导出抗原特异性免疫耐受。     

Coadministration of either cyclosporine or steroids with humanized monoclonal antibodies against CD80 and CD86 successfully prolong allograft survival after life supporting renal transplantation in cynomolgus monkeys

BACKGROUND: Recent studies have shown some efficacy using monotherapy with monoclonal antibodies (mAb) against CD80 and CD86 receptors after life-supporting renal transplantation in non-human primates. Our study was designed to evaluate the efficacy of combinations of the same mAbs with either microemulsion cyclosporine (CsA) or steroids. METHODS: Unilateral renal transplantation was performed in 16 blood group-matched and MLR-mismatched cynomolgus monkeys that were assigned to four different treatment groups. All monkeys in groups I, II, and IV were treated with the combination of a CD80 (h1F1) and CD86 (h3D1) mAb given at 20 mg/kg each preoperatively, then 5 mg/kg at weekly intervals starting postoperative (po) day 0 until poday 56 (9 doses). In group I the animals (n=4) were treated with mAbs only. In group II (n=4) mAbs were combined with a CsA regimen adjusted daily to maintain target 24 hr trough levels of 150-300 ng/ml CsA for poday 0 to poday 56. In group III (n=4) the animals received CsA monotherapy according to the same regimen as group II. In group IV methylprednisone was administered at 2 mg/kg IV on poday 0-2, then at 0.5 mg/kg/day prednisone per gavage that was and tapered to 0.2 mg/kg/day on which they were maintained until poday 56. All animals were off all immunosuppressive treatment after poday 56 and were then followed until poday 119. RESULTS: The mean survival of groups I-IV was 74 (range 9-119 days), 113 (96-119 days), 39 (22-71 days), and 79 days (6 to 119), respectively. All animals in group I showed clinical evidence of acute severe rejection (fever, creatinine increase, anuria) within the first week posttransplant, including those that retained renal function until poday 119. Only one animal in group II had a moderate clinical rejection during the treatment period and three of four animals survived the intended follow-up period. All animals in group III had multiple biopsy proven or severe clinical rejection episodes within the first 21 days and only one animal survived beyond poday 40. Moderate or severe acute rejection was diagnosed in three of four animals of group IV within the first 28 days post transplant and only one animal survived until poday 119. CONCLUSION: Our data show that combining a calcineurin inhibitor or prednisone with mAbs designed to block costimulatory signals does not antagonize the immunosuppressive efficacy of these mAbs. In addition, combining CsA with mAbs directed against the CD80 and CD86 receptors significantly prolongs graft survival when compared to CsA monotherapy. Therefore clinical trials of humanized mAbs to CD80 and CD86 used in combination with conventional immunosuppression can be considered.