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.     

Combination induction therapy with monoclonal antibodies specific for CD80, CD86, and CD154 in nonhuman primate renal transplantation

BACKGROUND: Antibodies and fusion proteins specific for CD80, CD86, and CD154 have shown promise as agents capable of inducing donor-specific tolerance in rodents. These agents have also been shown to be synergistic with one another in many settings of counter-adaptive immunity. In the nonhuman primate, monoclonal antibodies specific for CD80 and CD86 have prolonged the time to rejection of renal allografts but have not resulted in tolerance. A monoclonal antibody specific for CD154 has resulted in markedly prolonged survival of kidney, islet, cardiac, and skin allografts, but again most animals have eventually developed rejection after prolonged periods of rejection-free survival off therapy. METHODS: A combination of monoclonal antibodies specific for CD80, CD86, and CD154 were used in a mismatched nonhuman primate renal-allograft model. Doses used were based on optimized treatment protocols for each agent individually. RESULTS: Treatment of four rhesus macaques with this combination yielded a mean rejection-free survival of 565 days (311-911 days), significantly greater than untreated controls (mean survival=7.0 days, P=0.001) and animals treated with only a combination of anti-CD80 and CD86 (mean survival=191 days, P=0.01). The survival of animals treated with this combination of monoclonal antibodies was not significantly greater than those treated with anti-CD154 alone, but the production of alloantibody was delayed compared with monotherapy anti-CD154. CONCLUSION: These data suggest that a synergy exists between these agents, particularly with regard to T-dependent B-cell responses, but that they fail to induce durable tolerance in nonhuman primates.     

CD80, but not CD86, express on cultured murine keratinocyte stem cells

Although cultured keratinocyte allografts are initially accepted, grafted donor cells are gradually replaced by recipient elements. The precise mechanisms underlying this process are not clear. Keratinocyte stem cells (KSCs) were isolated and purified from neonatal C57BL/6 (H-2(b)) and BALB/c (H-2(d)) mice. Cultured KSCs were negative for IA/IE, indicating the absence of any contamination with Langerhans cells. Immunostaining showed that over a 7-day culture period, KSCs expressed increasing amounts of CD80, while CD86 could not be detected. RT-PCR results were consistent with these data. Mixed lymphocyte reaction assays showed that KSCs cultured in 10% serum for 7 days stimulated allogeneic rather than syngeneic T-cell proliferation. This study demonstrates that the costimulatory molecule CD80, but not CD86, may be expressed on cultured KSCs. The data indicate KSCs could act as antigen-presenting cells and thus provide an alternative explanation for the ultimate rejection of allogeneic keratinocytes.     

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.     

Administration of dendritic cells transduced with antisense oligodeoxyribonucleotides targeting CD80 or CD86 prolongs allograft survival

BACKGROUND: The expression of costimulatory molecules on antigen-presenting cells is crucial in determining T-cell immune responses. We examined the effects of transduction with high-affinity antisense oligodeoxyribonucleotides (ODNs) designed to target the mRNA of CD80 or CD86 on the phenotype and function of dendritic cells (DCs). MATERIALS AND METHODS: DCs were propagated from C57BL/10 (B10; H2b) bone marrow cells in granulocyte macrophage-colony stimulating factor and interleukin (IL)-4, and transduced with anti-CD80 or anti-CD86 antisense ODNs (base-mismatched ODNs as controls). The effect of antisense ODN on phenotype was examined by flow cytometry. The allostimulatory function of DCs was assessed by mixed leukocyte reaction and cytotoxic activity in vitro, and the influence on allograft survival was assessed in vivo. RESULTS: ODNs were effectively incorporated by DCs, which were enhanced by the presence of lipofectamine. Antisense ODNs targeting CD80 or CD86 mRNA specifically suppressed the expression of CD80 or CD86 in DCs and inhibited their capacity to elicit the proliferative responses, donor-specific cytotoxic T-lymphocyte activity in C3H (H2k) spleen T cells. This was associated with decreased IL-2, but elevated IL-4 production, and an increase in T-cell apoptosis. In contrast with the administration of control DCs into C3H recipients that exacerbated rejection of B10 cardiac allografts, injection of DCs transduced with anti-CD80 or CD86 antisense ODN significantly prolonged the survival of heart allografts. CONCLUSION: Transduction with antisense ODN targeting CD80 or CD86mRNA is a feasible and effective approach to modify DCs that renders them tolerogenic by inducing T-cell hyporesponsiveness and apoptosis. This may lead to the development of new therapeutic strategies in transplantation.     

Role of donor macrophages after heart and lung transplantation

Since the 1960s, heart and lung transplantation has remained the optimal therapy for patients with end‐stage disease, extending and improving quality of life for thousands of individuals annually. Expanding donor organ availability and immunologic compatibility is a priority to help meet the clinical demand for organ transplant. While effective, current immunosuppression is imperfect as it lacks specificity and imposes unintended adverse effects such as opportunistic infections and malignancy that limit the health and longevity of transplant recipients. In this review, we focus on donor macrophages as a new target to achieve allograft tolerance. Donor organ‐directed therapies have the potential to improve allograft survival while minimizing patient harm related to global suppression of recipient immune responses. Topics highlighted include the role of ontogenically distinct donor macrophage populations in ischemia–reperfusion injury and rejection, including their interaction with allograft‐infiltrating recipient immune cells and potential therapeutic approaches. Ultimately, a better understanding of how donor intrinsic immunity influences allograft acceptance and survival will provide new opportunities to improve the outcomes of transplant recipients.     

Overview of tacrolimus-based immunosuppression after heart or lung transplantation.

Transplantation has evolved into an accepted treatment for end-stage heart or lung disease. Acute rejection, complications related to immunosuppressive protocols, and the development of chronic rejection continue to challenge the long-term success of heart and lung transplantations. Wide acceptance of tacrolimus as an important immunosuppressant in renal and hepatic transplantations has led subsequently to its investigation as primary immunosuppression in heart and lung transplant recipients, either combined with azathioprine or with the newer agents mycophenolate mofetil or rapamycin. Studies have shown that tacrolimus is an effective therapeutic alternative to cyclosporine for primary immunosuppression in heart or lung transplantation and demonstrates equivalent if not improved prophylaxis of acute rejection, and more recently demonstrates a potential influence on chronic rejection, particularly in lung transplant recipients. Of importance, the enhanced immunosuppressive activity of tacrolimus is achieved without increased risk of infection or malignancy. Differences in tolerability profiles and side effects between tacrolimus and cyclosporine may be used in selecting the optimal immunotherapy after thoracic transplantation. In particular, the lesser propensity of tacrolimus to cause hypertension and hyperlipidemia potentially offers decreased cardiovascular risk for heart and lung transplant recipients.     

Increased engraftment and GVHD after in utero transplantation of MHC-mismatched bone marrow cells and CD80low, CD86(-) dendritic cells in a fetal mouse model.

BACKGROUND: Bone marrow transplantation (BMT) is the only known cure for a variety of inherited diseases and requires the administration of high doses of immunosuppressive and myeloablative therapy. Because the fetus is immunoincompetent early in gestation, in utero stem cell transplantation (IUT) could avoid the need for this toxic conditioning. A major limitation to date of IUT is the low level of engraftment and failure to induce tolerance. Dendritic cells (DC) are considered very potent antigen-presenting cells, but DC progenitors (pDC) are strongly tolerogenic. METHODS: We examined the effect of donor pDC on the degree of engraftment and tolerance induction after IUT. Bone marrow-derived pDC (CD80low, CD86-) from male C57BL/6 mice (H2b) were injected with and without donor bone marrow (BM) intraperitoneally into 13 to 15-day BALB/c (H2d) fetuses. Engraftment was determined by flow cytometry and quantitative polymerase chain reaction and tolerance by skin grafts and the mixed lymphocyte reaction. RESULTS: At 1-month posttransplant, mice that received BM+pDC showed a higher degree of engraftment (29+/-46%) than mice that received pDC-enriched cells or BM cells alone (0.11+/-0.70% and 1.71+/-1.66%, respectively, P<0.001). However, 5/19 recipients of BM+pDC died within 6 weeks; 4/5 had significant donor cell engraftment in blood and/or tissues. Also, these mice had evidence of graft-versus-host disease (GVHD). Two mice out of 15 long-term survivors in the BM+pDC group had virtually complete replacement of host with donor hematopoietic cells. Skin grafts and mixed lymphocyte reaction studies showed no durable tolerance induction other than in the two fully engrafted recipients of BM+pDC. CONCLUSIONS: These results suggest that donor pDC, along with donor BM, can have a significant impact on engraftment of MHC-mismatched donor cells associated with an increased incidence of GVHD. However, marrow-derived pDC do not result in an increase in tolerance induction in utero even when microchimerism is present.     

Effect of calcitriol on bone loss after cardiac or lung transplantation.

Rapid bone loss after cardiac and lung transplantation results in an increased risk of osteoporotic fracture. This study examined the efficacy of treatment with calcitriol (1,25-dihydroxyvitamin D3) in preventing bone loss in patients undergoing cardiac or lung transplantation. In this 2-year double-blind, stratified study, 65 patients undergoing cardiac or single lung transplantation were randomly allocated to receive either placebo or calcitriol (0.5-0.75 microg/day), the latter for either 12 months or 24 months. All patients received 600 mg calcium/day. Bone mineral density (BMD) was measured every 6 months for 2 years by dual-energy X-ray absorptiometry. There was no significant difference between groups with respect to age or cumulative dose of prednis(ol)one or cyclosporine over the 2 years. Bone loss at the proximal femur was significantly reduced or prevented at all three sites by treatment with calcitriol for 2 years compared with treatment with calcium alone. Treatment with calcitriol for 12 months followed by calcium for 12 months resulted in similar proximal femoral bone loss to that seen in those patients treated with calcium for 24 months, suggesting calcitriol prophylaxis needs to be continued beyond 12 months. At the lumbar spine, there were no significant differences in BMD between groups. Over a period of 2 years, 22 new vertebral fractures/deformities occurred in 4 patients treated with calcium alone compared with one new vertebral fracture in 1 patient treated with calcitriol. Because the sample size was too low to provide reliable interpretation of vertebral fracture rates, this difference is likely a chance result. Mild hypercalcemia was common with calcitriol therapy, as was mild hypercalciuria (59% of patients vs. 10% controls), but there were no significant differences between groups in serum creatinine after 2 years. These data suggest calcitriol has a role in reducing proximal femur bone loss after cardiac or lung transplantation but treatment needs to be continued beyond 1 year.     

Cytopathology of pulmonary alveolar proteinosis complicating lung transplantation.

Pulmonary alveolar proteinosis is a disorder of unknown origin that occurs rarely after lung transplantation. We identified a patient with pulmonary alveolar proteinosis 66 days after undergoing single lung transplantation for idiopathic pulmonary fibrosis. We based the diagnosis on the presence of amorphous clumps or globules of acellular and finely granular material in bronchoalveolar lavage fluid (BALF). This material persisted for an 18.5-month period and was present in 9 of 14 lavage specimens. However, despite its presence in the native lung at autopsy, the material was seen in only 1 of 14 transbronchial lung biopsy specimens. Although uncommon, pulmonary alveolar proteinosis can be diagnosed readily in BALF by its distinctive cytopathologic features and should be considered in the differential diagnosis of pulmonary disease in lung transplant recipients.     

Pulmonary macrophages are involved in reperfusion injury after lung transplantation

BACKGROUND: Reperfusion injury is a perplexing cause of early graft failure after lung transplantation. Although recipient neutrophils are thought to have a role in the development of reperfusion injury, some researchers have shown that neutrophils are not involved in its earliest phase. Intrinsic donor pulmonary macrophages may be responsible for this early phase of injury. Using the macrophage inhibitor gadolinium chloride, we attempted to investigate the role of pulmonary macrophages in reperfusion injury after lung transplantation. METHODS: Using our isolated, ventilated, blood-perfused rabbit lung model, all groups underwent lung harvest followed by 18-hour storage (4 degrees C) and blood reperfusion for 30 minutes. Group I served as a control. Group II received gadolinium chloride at 7 mg/kg 24 hours before harvest. Group III received gadolinium chloride at 14 mg/kg 24 hours before harvest. RESULTS: Group III had significantly improved arterial oxygenation and pulmonary artery pressures compared with groups I and II after 30 minutes of reperfusion. CONCLUSIONS: The earliest phase of reperfusion injury after lung transplantation involves donor pulmonary macrophages.     

Protective effect of short-tem calcitriol or cyclical etidronate on bone loss after cardiac or lung transplantation.

Bone loss is most rapid in the immediate period after cardiac or lung transplantation. This randomized study compared the efficacy of 6 months of treatment with either calcitriol (1,25-dihydroxyvitamin D3; 0.5 microg/day) or two cycles of etidronate plus calcium in preventing bone loss in 41 patients undergoing cardiac or lung transplantation. Patients were followed for 18 months after cessation of treatment. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DXA). There were no significant differences between groups with respect to age or cumulative dose of prednis(ol)one or cyclosporin over the 2 years. Bone loss did not differ between groups after 6 months and, despite 6 months prophylaxis with either agent, bone loss was significant in both groups at 6 months and 12 months. However, compared with an untreated reference group, both therapies offered significant protection at 6 months and etidronate provided significant protective carryover after therapy had been discontinued. These data suggest short-term prophylaxis with calcitriol or cyclical etidronate is partially effective in reducing bone loss after cardiac or lung transplantation but treatment needs to be continued for a longer term.     

Clinical significance of costimulatory molecules CD80/CD86 expression in IgA nephropathy

BACKGROUND: IgA nephropathy (IgAN) is the most common form of human glomerulonephritis. Tubulointerstitial inflammation with infiltration of mononuclear cells plays an important role in the progression of IgAN. Activation of T cells requires costimulatory signals through binding of CD28 receptor with cognate ligands (CD80/CD86) located on antigen-presenting cells (APC). To assess the clinical significance of this regulatory pathway participation in the pathogenesis of IgAN, a comprehensive immunohistologic evaluation was conducted on renal tissue of IgAN in different phases of progressive injury. METHODS: Thirty-three cases of IgAN and ten cases of non-IgA mesangial proliferative glomerulonephritis (PGN) with minor tissue damage as controls were investigated. Monoclonal antibodies were used to assess the expression of CD80, CD86, CD68, CD14, CD45RO, human leukocyte antigen-DR (HLA-DR), and intercellular adhesion molecule-1 (ICAM-1) in renal tissues. Clinical and expression data were compared at the time of renal biopsy. RESULTS: CD80+ and CD86+ cells were observed more in IgAN patients with progressive renal injury than in mild cases and controls. CD80 was limited to tubular epithelial cells and was complemented by HLA-DR expression. CD86 was expressed in the glomerulus, periglomerular area, and peritubular interstitium. Activated T cells (CD45RO+), monocytes (CD14+), macrophages (CD68+), and CD86 showed similar distributions. Positive correlations were found between CD86+ cells and CD45RO, CD14, and CD68 positive cells and between CD80+ tubuli and peritubular interstitial CD45RO+ cells. The number of interstitial CD86 positive cells and the percentage of CD80+ tubuli were correlated with renal function. Most CD86+ cells were monocyte/macrophages. CONCLUSION: This study suggested that CD80 and CD86 activate T cells in IgAN, CD80/CD86 expressions correlated with renal function at the time of renal biopsy, and monocyte/macrophages and tubular epithelial cells act as APC.