Yinchen Dong

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.

Primate renal transplants using immunotoxin.

BACKGROUND T-lymphocyte depletion 7 days before transplantation with immunotoxin FN 18-CRM9 has resulted in tolerance to subsequent renal allografts. We tested the effect of giving immunotoxin on the day of the transplantation and evaluated its effect on rhesus monkey and allograft survival, on antibody production, and on T-cell recovery. METHODS Major histocompatibility complex mismatched renal allografts were performed in rhesus monkeys. Immunotoxin was given starting on the day of transplantation, with and without prednisone and mycophenolate mofetil for 3 days. T-cell subsets and alloantibody levels were measured by flow cytometry. The ability of treated monkeys to develop antibody to tetanus, diphtheria, and xenoantibody was measured. Histology of renal transplants was read in a blinded manner. RESULTS Immunotoxin started on the day of transplantation resulted in prolonged allograft survival in all treatment groups. Graft loss between days 50 and 135 was most often due to interstitial nephritis. Later graft loss was due to chronic rejection. Monkeys had intact antibody responses to alloantigen, tetanus, diphtheria, and xenoantibody. Their CD4 cells recovered gradually over 6 months. CONCLUSIONS Immunotoxin reliably prolongs renal allograft survival when started on the day of transplantation, but interstitial nephritis and chronic rejection limit the development of long-term tolerance. T-cell-dependent B-cell responses remain intact after treatment.

Successful conversion from conventional immunosuppression to anti-CD154 monoclonal antibody costimulatory molecule blockade in rhesus renal allograft recipients.

BACKGROUND Several conventional forms of immunosuppression have been shown to antagonize the efficacy of anti-CD154 monoclonal antibody- (mAb) based costimulatory molecule blockade immunotherapy. Our objective was to determine if allograft recipients treated with a conventional immunosuppressive regimen could be sequentially converted to anti-CD154 mAb monotherapy without compromising graft survival. METHODS Outbred juvenile rhesus monkeys underwent renal allotransplantation from MHC-disparate donors. After a 60-day course of triple therapy immunosuppression with steroids, cyclosporine, and mycophenolate mofetil, monkeys were treated with: (1) cessation of all immunosuppression (control); (2) seven monthly doses of 20 mg/kg hu5C8 (maintenance), or; (3) 20 mg/kg hu5C8 on posttransplant days 60, 61, 64, 71, 79, and 88 followed by five monthly doses (induction+maintenance). Graft rejection was defined by elevation in serum creatinine>1.5 mg/dl combined with histologic evidence of rejection. RESULTS Graft survival for the three groups were as follows: group 1 (control): 70, 75, >279 days; group 2 (maintenance): 83, 349, >293 days, and; group 3 (induction+maintenance): 355, >377, >314 days. Acute rejection developing in two of four monkeys after treatment with conventional immunosuppression was successfully reversed with intensive hu5C8 monotherapy. CONCLUSIONS Renal allograft recipients can be successfully converted to CD154 blockade monotherapy after 60 days of conventional immunosuppression. An induction phase of anti-CD154 mAb appears to be necessary for optimal conversion. Therefore, although concurrent administration of conventional immunosuppressive agents including steroids and calcineurin inhibitors has been shown to inhibit the efficacy of CD154 blockade, sequential conversion from these agents to CD154 blockade appears to be effective.

Immunotoxin-treated rhesus monkeys: a model for renal allograft chronic rejection.

Background. Unlike acute and hyperacute rejection, chronic rejection (CR) still constitutes a poorly understood process. The onset is insidious, occurs in a period of months to years and, because the pathophysiology is not well understood, is untreatable. A reliable large-animal model for renal allograft CR is needed and has not been reported in the literature yet. Methods. CR biopsy changes were studied in major histocompatibility complex-mismatched renal allografts performed in nine rhesus monkeys that received CD3 T-lymphocyte depletion therapy with immunotoxin on the day of the transplantation (n=7) or 7 days before transplant (n=2). Results. Mean graft survival time was 613.77 days. Biopsy changes of CR were identified as soon as 84 days after transplant (mean, 336 days; range, 84–896 days). Most of the experimental animals had severe interstitial fibrosis, tubular atrophy, chronic transplant glomerulopathy, and chronic vascular rejection changes at the time of necropsy. A significant positive correlation between the severity of CR and the degree of CD68+ macrophage infiltrate of renal parenchyma and the degree of anemia and serum creatinine level elevations were also observed. Conclusions. Our findings are similar to those seen in human renal chronic allograft nephropathy, but in contrast, our model excludes all the nonimmune factors associated with chronic allograft nephropathy, including donor disease, injury from prolonged preservation, drug toxicity, and underlying recipient disease. Immunotoxin-treated rhesus monkeys emerge as an outstanding animal model for assisting us in understanding the pathophysiology of CR.

Surveillance of acute rejection in baboon renal transplantation by elevation of interferon-γ inducible protein-10 and monokine induced by interferon-γ in urine

Background. CXCR3 binding chemokines play a key role in recruitment of inflammatory cells into an organ transplant. This study addresses the question of whether urinary excretion of these chemokines correlates with acute rejection in a baboon kidney transplantation model. Methods. Seven outbred baboons underwent renal allotransplantation from major histocompatibility complex (MHC)-mismatched donors. The treatment of baboons consisted of anti-CD4 monoclonal antibody (mAb), anti-CD8 mAb, rapamycin, and mycophenolate mofetil (MMF). Urinary levels of interferon-&ggr; inducible protein-10 (IP-10) and monokine induced by interferon-&ggr; (Mig) were determined by ELISA. Renal biopsies were examined by immunohistochemical staining for CXCR3 and Mig. Results. Urinary levels of IP-10 and Mig increased significantly in all of the five baboons at the time of acute rejection of renal transplant. The IP-10 and Mig levels did not rise in two nonrejecting baboons. In two baboons, urinary levels of IP-10 and Mig rose before the elevation of the serum creatinine. In renal biopsies, expression of Mig was detected in glomeruli, tubules, and infiltrating cells, and the expression was significantly elevated in biopsies with acute rejection (P<0.01). CXCR3 was constitutively expressed in tubular cells in biopsies derived from both normal grafts and grafts with acute rejection. Whereas the infiltrating cells were increased in the biopsies with acute rejection, the expression of CXCR3 was also significantly higher (P<0.01) in these infiltrating cells compared with those in the normal controls. Conclusions. This study shows an important correlation between urinary excretion of IP-10 and Mig and acute rejection in baboon kidney transplantation.

Effect of immunosuppressants on T-cell subsets observed in vivo using carboxy-fluorescein diacetate succinimidyl ester labeling.

Background. The in vivo effects of immunosuppressants on T cells are classically determined using animal models of organ transplantation. These methods are technically difficult and time consuming. A simple in vivo method is needed for screening new immunosuppressants. Methods. Donor mouse spleen cells were labeled with a fluorescent dye, carboxy-fluorescein diacetate succinimidyl ester (CFSE), and then injected into the blood of recipient severe combined immunodeficiency mice. Three days after the injection, spleen cells of the recipient mice were isolated and the proliferating alloreactive T cells were analyzed by flow cytometry. Results. In control recipient mice, 50% of the T cells were proliferating, consisting of both CD4+ and CD8+ T cells. In cyclosporine- or FK506-treated mice, T-cell proliferation was suppressed in the CD4 subset but not in the CD8 subset. On the contrary, T-cell proliferation was significantly reduced in the CD8 subset but not in the CD4 subset in recipient mice treated with rapamycin. Conclusion. The present mouse model using carboxy-fluorescein diacetate succinimidyl ester labeling is simple and fast. It is useful for screening new immunosuppressants and for examining the effect on T-cell subsets.

Piceatannol in combination with low doses of cyclosporine A prolongs kidney allograft survival in a stringent rat transplantation model.

Background. The discovery of new immunosuppressive agents has enhanced short-term graft survival. However, current immunosuppressants often induce toxicities that limit their clinical use. Thus, there is a need for new immunosuppressants for use in clinical transplantation. Piceatannol blocks Syk and ZAP-70, tyrosine kinases involved in immune cell activation. We examined whether piceatannol prolongs kidney allograft survival in the stringent ACI-to-Lewis rat model. Methods. Kidney recipients were divided into four groups. Group 1 (n=8) received piceatannol 30 mg/kg per day intravenously and cyclosporine A (CsA) 2 mg/kg per day intramuscularly from day −3 to day 7 after transplantation. At day 8, piceatannol was reduced to 10 mg/kg per day and the combined treatment continued until day 60. Group 2 (n=9) received 2 mg/kg per day CsA alone from day −3 to day 60. Group 3 (n=4) received piceatannol alone as in group 1. Group 4 (n=2) received only the vehicle dimethyl sulfoxide from day −3 to day 60. Graft rejection was defined as either a serum creatinine level more than 2 mg/dL or animal death. Results. Group 1 animals survived for at least 115 days (n=8, P <0.05), with several animals maintaining their grafts for more than 200 days. In contrast, 8 of 9 animals in group 2 rejected their grafts within 10 days of transplantation; one animal survived for 71 days. Excellent graft function was maintained in group 1 animals despite withdrawal of immunosuppression. Conclusions. These results are the first to show that piceatannol, when combined with subtherapeutic dosages of CsA, prevents graft rejection, suggesting that targeting Syk and Zap could be useful for preventing graft rejection.

Graft survival in a rhesus renal transplant model after immunotoxin-mediated T-cell depletion is enhanced by mycophenolate and steroids

BACKGROUND Anti-CD3 immunotoxin (IT), a T-cell-depleting agent, prolongs survival of renal allografts in a rhesus monkey model without the need for long-term immunosuppression. In this study we sought to further prolong allograft survival by giving short-term conventional immunosuppression simultaneous with IT administration. METHODS MHC class II mismatched, juvenile rhesus monkeys were paired as donor and recipient for renal transplantation. Recipients received two to three daily doses of IT starting on the day of transplantation. Additional immunosuppression was given for no more than 60 days. Graft function was monitored by serum creatinine and renal biopsies. Flow cytometry was used to monitor T-cell recovery. RESULTS Graft survival time (GST) in animals receiving IT was prolonged compared with controls with 50% of IT-treated monkeys surviving >100 days. Animals treated with IT plus mycophenolate mofetil (MMF) and steroids had significantly enhanced GST (mean GST, 305 days) compared with those treated with IT alone (mean GST, 94 days). In contrast, addition of cyclosporine or 40-O-[2-Hydroxyethyl]rapamycin did not significantly increase graft survival time. A comparison among animals from all treatment groups with short (<100 days) and long (>100 days) GST demonstrated that those with the shorter GST had a higher blood T-cell count 2 weeks after transplantation. Full recovery of CD4+ T cells required longer than 6 months. CONCLUSIONS A combination with MMF and steroids given for 4 days after renal allograft transplantation significantly increases GST in IT-treated monkeys. We hypothesize that MMF and steroids suppress the initial T-cell activation mediated by IT.

Increased glomerular deposits of von willebrand factor in chronic, but not acute, rejection of primate renal allografts

BACKGROUND In our previously described primate renal allograft model, T cell ablation leads to long-term graft survival. The role of endothelial cell alteration in chronic rejection was examined in our model. METHODS Renal transplants were performed in rhesus monkeys using a T cell- depleting immunotoxin, FN18-CRM9. Sections from 10 rejected kidneys (5 acute and 7 chronic rejection) were examined after immunohistochemical staining for expression of endothelium-related proteins [von Willebrand factor (vWF), CD62P, and CD31], fibrinogen, and a macrophage marker (CD68). Glomerular staining for each antigen was graded on a semiquantitative scale. RESULTS Intense staining for vWF was consistently observed in glomerular endothelium, subendothelium, and mesangium in all kidneys removed due to chronic rejection. vWF staining was weak in kidneys showing acute rejection. The difference in glomerular staining was statistically significant. Staining for vWF in extraglomerular vessels was nearly identical in kidneys showing acute and chronic rejection. Expression of CD62P was increased in extraglomerular vessels in allografts with chronic rejection, but the glomeruli showed little or no staining. There was no significant difference in the glomerular staining for CD62P or CD31 in organs showing acute and chronic rejection. Fibrinogen staining of glomerular mesangium was seen in kidneys with chronic rejection. Macrophages (CD68+) infiltrating glomeruli were more numerous in kidneys showing chronic rejection. CONCLUSION Increased glomerular deposition of vWF in renal allografts showing chronic rejection, without increased staining for CD62P or CD31, suggests increased constitutive secretion of vWF from endothelial cells as a component of the mechanism of chronic rejection in our model.

Activation of T lymphocytes for adhesion and cytokine expression by toxin-conjugated anti-CD3 monoclonal antibodies

BACKGROUND Immunosuppressive drugs that target T cells are useful for prolonging allograft survival. The anti-CD3 immunotoxin FN18-CRM9 has been shown to effectively prolong renal allograft survival in a rhesus monkey model of transplantation. However, immunotoxin-treated monkeys showed increased levels of inflammatory cytokines and produced antibodies to donor proteins. To better understand the role of FN18-CRM9 in the production of cytokines and anti-donor antibodies in the monkey model, we examined whether this immunotoxin elicits functional responses in T cells. METHODS Purified normal rhesus monkey T cells (>98% purity) were incubated with immunotoxin FN18-CRM9 or the unconjugated anti-CD3 monoclonal antibodies and then examined for changes in protein tyrosine phosphorylation, adhesion to fibronectin, gene expression, and proliferation in the presence or absence of anti-CD28 monoclonal antibodies (mAb) and interleukin-2. RESULTS Immunotoxin treatment of T cells in vitro increased protein tyrosine phosphorylation, cell adhesion to the extracellular matrix, and expression of the inflammatory cytokines interferon-gamma and tumor necrosis factor-alpha. These immunotoxin effects were similar in magnitude to those induced by the unconjugated mAb. In contrast, immunotoxin-induced T cell proliferation was markedly less than that induced by the unconjugated mAb. Interestingly, the mitogenic molecules IL-2 and anti-CD28 mAb did not prevent immunotoxin-induced inhibition of cell proliferation. CONCLUSIONS The activation of T cells for protein phosphorylation, adhesion, and cytokine expression strongly suggests that the actions of FN18-CRM9 in vivo are not limited to the inhibition of protein synthesis.