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Blocking of costimulation prevents kidney graft rejection in rhesus monkeys
Blocking of Costimulation Prevents Kidney Graft Rejection in Rhesus Monkeys M.A. Ossevoort, J. Ringers, L. Boon, K. Lorre´, Y. van den Hout, E.-M. Kuhn, M. de Boer, M. Jonker, and P. de Waele
I
MMUNOLOGIC rejection of an allograft through T-cell dependent mechanisms is an inevitable result after organ transplantation between genetically nonidentical individuals. Therefore, administration of drugs suppressing the recipient T-cell function is required for successful transplantation of allogeneic organs. Nowadays, calcineurin inhibitors and glucocorticosteroids are used clinically. Cessation of immunosuppression generally leads to graft loss. Induction of antigen-specific nonresponsiveness to the donor tissue by a short-term therapy would be optimal to prevent graft loss. There is accumulating evidence that blocking the costimulatory pathways, such as CD80/CD86CD28/CTLA4, and CD40L-CD40, offers a valid approach for immune suppression after solid organ transplantation. Blocking both CD80 and CD86 using CTLA4-Ig or a combination of anti-CD80 and anti-CD86 monoclonal antibodies (MAbs) results in prolongation of allograft survival in rodent transplantation models.1,2 The additional blockade of the CD40-CD40L pathway has been described to result in prolongation of allograft survival and even to induce allograft tolerance.3,4 In a previous study,5 it was demonstrated that treatment with anti-CD80 MAb in combination with cyclosporine (CyA) resulted in a significant prolongation of skin graft survival time in rhesus monkeys. These promising data initiated the present study exploring the use of anti-CD80 and anti-CD86 MAbs in combination with anti-CD40 MAb or CyA as a prophylactic treatment to prevent rejection in a preclinical kidney transplant model in rhesus monkeys. MATERIALS AND METHODS In this study, the efficacy of the prophylactic treatment of antiCD80 MAb (B7-24, Innogenetics, Gent, Belgium) and anti-CD86 MAb (1G10, Innogenetics) together or in combination with antiCD40 MAb (5D12, Pangenetics, Amsterdam, The Netherlands) or CyA was tested in a life-supporting kidney transplant model in rhesus monkeys. The rhesus monkeys received one partly MHCmatched kidney graft on day 0. The mixed lymphocyte reaction (MLR) from the recipient against the donor antigens was positive. Kidney function was monitored by serum creatinine level. The survival time represents the first rejection episode. The MAbs were given intravenously (IV) daily for 14 days starting at day 21. The dose of anti-CD80 and anti-CD86 MAbs was 0.5 mg/kg and that of anti-CD40 MAb was 1 mg/kg on day 21. The dose of all three
antibodies was 0.25 mg/kg from day 0 to day 12. The CyA (10 mg/kg) was given intramuscularly [IM] starting on day 0 until day 35 (animals treated with CyA alone) or on day 21 to day 35 (animals treated with MAbs in combination with CyA). At several time points during the experiment, blood samples were taken to determine the blood levels of CyA, serum levels of the MAbs, and the rhesus monkey anti-mouse antibody response (ELISA). For the determination of the lymphocyte subsets by FACScan analysis, blood samples were taken prior, during, and after treatment with the MAbs.
RESULTS
Since many anti-human leukocyte MAbs crossreact with nonhuman primates, the rhesus monkey is a useful animal model in which to test the immunosuppressive efficacy of these reagents. During the treatment period with the MAbs, no signs of side effects or rejection were noted. Serum concentrations (trough level) of the anti-CD80 and anti-CD86 MAbs were 6 and 4 mg/mL serum, respectively. Anti-CD40 MAb was not detectable in the serum. As long as the MAbs were detectable in the serum, anti-mouse Ig (GAM)-coated cells could be found in the circulation representing the binding of the injected mouse MAbs to the peripheral blood mononuclear cells (PBMC; Fig 1). The serum concentration of the anti-CD80 or anti-CD86 MAbs decreased to an undetectable level at day 21 or 15, respectively. At day 21 still anti-mouse Ig-coated cells could be detected in the circulation, whereas at day 35 no anti-mouse Ig-coated cells were present. Treatment with anti-CD80 and anti-CD86 MAbs alone had no effect on the amount of CD31 and CD201 cells in the circulation (Fig 1). Nevertheless, during treatment with anti-CD80, anti-CD86, and anti-CD40, the amount of CD201 cells tends to decrease whereas the amount of CD31 cells were unaffected (Fig 1). Induction of the anti-antibody response was delayed using anti-CD80, anti-CD86, and anti-CD40 MAbs compared From the Biomedical Primate Research Centre, Rijswijk, and University Hospital, Leiden, and Pangenetics, Amsterdam, The Netherlands; and Innogenetics, Gent, Belgium. Supported by BIOMED II grant BMH4-CT96-0127. Address reprint requests to Dr M.A. Ossevoort, BRRC, PO Box 3306, 2280 HV Rijswijk, the Netherlands.
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/98/$19.00 PII S0041-1345(98)00576-4
Transplantation Proceedings, 30, 2165–2166 (1998)
2165
2166
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tionally, in vitro the CD40/CD40L-mediated rhesus monkey B-cell proliferation could strongly be inhibited by adding the anti-CD40 MAb (data not shown). The mean survival time of untreated kidney transplanted rhesus monkeys is 6 days (n 5 4). Prophylactic short-term treatment (14 days) with anti-CD80 plus CD86 MAbs alone or in combination with anti-CD40 MAb resulted in a prolonged graft survival of 28 6 7 days (n 5 4) and 24 6 7 days (n 5 4), respectively. Kidney graft rejection started after the antibody therapy was stopped. The survival time of animals treated with the anti-CD80 and anti-CD86 MAbs in combination with CyA (35 days, 10 mg/kg IM) was similar to that of animals treated with CyA alone. However, histologic examination of the rejected kidney showed less signs of vascular rejection in the animals treated with anti-CD80 and anti-CD86 MAbs alone compared to the combined treatment with CyA or anti-CD40 MAb. CONCLUSIONS
Fig 1. Representative example of the effects of daily injection with anti-CD80 and anti-CD86 MAbs (A) or anti-CD80, antiCD86, and anti-CD40 MAbs (B) on rhesus monkey peripheral blood lymphocyte subsets. MAbs were treated from day 21 to day 12. PBMCs were stained with anti-mouse Ig-FITC (MAbcoated cells, closed squares), anti-CD3-FITC (T cells, open squares), and anti-CD20-PE (B cells, open circles).
with anti-CD80 and anti-CD86 MAb alone and other immunosuppressive murine MAbs such as anti-CD4.6 This demonstrates an additional inhibitory effect of the antiCD40 MAb therapy on the B-cell response, which coincides with the decrease of CD201 cells in the circulation. Addi-
Thus, prolonged graft survival can be achieved by blocking the CD80/CD86-CD28/CTLA4 and CD40-CD40L pathways in a preclinical vascularized transplant model in rhesus monkeys. No rejection occurs as long as the MAb concentration in the serum remained high. Additionally, treatment with the anti-CD80 and anti-CD86 MAbs shows a beneficial effect on the type of rejection and the presence of antiCD40 MAb has a suppressive effect on the B-cell response. These promising results justify further investigation to achieve the final goal of induction of donor-specific tolerance. REFERENCES 1. Lenschow DJ, Zeng Y, Hatchcock KS, et al: Transplantation 60:1171, 1995 2. Bashuda H, Seino K, Kano M, et al: Transplant Proc 28:1039, 1996 3. Larsen CP, Elwood ET, Alexander DZ, et al: Nature 381:434, 1996 4. Kirk AD, Harlan DM, Amstrong NN, et al: Proc Natl Acad Sci USA 94:8789, 1997 5. Ossevoort MA, Lorre´ K, Boon L, et al: submitted 6. Jonker M, Neuhaus P, Zurcher C, et al: Transplantation 39:247, 1985
I
MMUNOLOGIC rejection of an allograft through T-cell dependent mechanisms is an inevitable result after organ transplantation between genetically nonidentical individuals. Therefore, administration of drugs suppressing the recipient T-cell function is required for successful transplantation of allogeneic organs. Nowadays, calcineurin inhibitors and glucocorticosteroids are used clinically. Cessation of immunosuppression generally leads to graft loss. Induction of antigen-specific nonresponsiveness to the donor tissue by a short-term therapy would be optimal to prevent graft loss. There is accumulating evidence that blocking the costimulatory pathways, such as CD80/CD86CD28/CTLA4, and CD40L-CD40, offers a valid approach for immune suppression after solid organ transplantation. Blocking both CD80 and CD86 using CTLA4-Ig or a combination of anti-CD80 and anti-CD86 monoclonal antibodies (MAbs) results in prolongation of allograft survival in rodent transplantation models.1,2 The additional blockade of the CD40-CD40L pathway has been described to result in prolongation of allograft survival and even to induce allograft tolerance.3,4 In a previous study,5 it was demonstrated that treatment with anti-CD80 MAb in combination with cyclosporine (CyA) resulted in a significant prolongation of skin graft survival time in rhesus monkeys. These promising data initiated the present study exploring the use of anti-CD80 and anti-CD86 MAbs in combination with anti-CD40 MAb or CyA as a prophylactic treatment to prevent rejection in a preclinical kidney transplant model in rhesus monkeys. MATERIALS AND METHODS In this study, the efficacy of the prophylactic treatment of antiCD80 MAb (B7-24, Innogenetics, Gent, Belgium) and anti-CD86 MAb (1G10, Innogenetics) together or in combination with antiCD40 MAb (5D12, Pangenetics, Amsterdam, The Netherlands) or CyA was tested in a life-supporting kidney transplant model in rhesus monkeys. The rhesus monkeys received one partly MHCmatched kidney graft on day 0. The mixed lymphocyte reaction (MLR) from the recipient against the donor antigens was positive. Kidney function was monitored by serum creatinine level. The survival time represents the first rejection episode. The MAbs were given intravenously (IV) daily for 14 days starting at day 21. The dose of anti-CD80 and anti-CD86 MAbs was 0.5 mg/kg and that of anti-CD40 MAb was 1 mg/kg on day 21. The dose of all three
antibodies was 0.25 mg/kg from day 0 to day 12. The CyA (10 mg/kg) was given intramuscularly [IM] starting on day 0 until day 35 (animals treated with CyA alone) or on day 21 to day 35 (animals treated with MAbs in combination with CyA). At several time points during the experiment, blood samples were taken to determine the blood levels of CyA, serum levels of the MAbs, and the rhesus monkey anti-mouse antibody response (ELISA). For the determination of the lymphocyte subsets by FACScan analysis, blood samples were taken prior, during, and after treatment with the MAbs.
RESULTS
Since many anti-human leukocyte MAbs crossreact with nonhuman primates, the rhesus monkey is a useful animal model in which to test the immunosuppressive efficacy of these reagents. During the treatment period with the MAbs, no signs of side effects or rejection were noted. Serum concentrations (trough level) of the anti-CD80 and anti-CD86 MAbs were 6 and 4 mg/mL serum, respectively. Anti-CD40 MAb was not detectable in the serum. As long as the MAbs were detectable in the serum, anti-mouse Ig (GAM)-coated cells could be found in the circulation representing the binding of the injected mouse MAbs to the peripheral blood mononuclear cells (PBMC; Fig 1). The serum concentration of the anti-CD80 or anti-CD86 MAbs decreased to an undetectable level at day 21 or 15, respectively. At day 21 still anti-mouse Ig-coated cells could be detected in the circulation, whereas at day 35 no anti-mouse Ig-coated cells were present. Treatment with anti-CD80 and anti-CD86 MAbs alone had no effect on the amount of CD31 and CD201 cells in the circulation (Fig 1). Nevertheless, during treatment with anti-CD80, anti-CD86, and anti-CD40, the amount of CD201 cells tends to decrease whereas the amount of CD31 cells were unaffected (Fig 1). Induction of the anti-antibody response was delayed using anti-CD80, anti-CD86, and anti-CD40 MAbs compared From the Biomedical Primate Research Centre, Rijswijk, and University Hospital, Leiden, and Pangenetics, Amsterdam, The Netherlands; and Innogenetics, Gent, Belgium. Supported by BIOMED II grant BMH4-CT96-0127. Address reprint requests to Dr M.A. Ossevoort, BRRC, PO Box 3306, 2280 HV Rijswijk, the Netherlands.
© 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/98/$19.00 PII S0041-1345(98)00576-4
Transplantation Proceedings, 30, 2165–2166 (1998)
2165
2166
OSSEVOORT, RINGERS, BOON ET AL
tionally, in vitro the CD40/CD40L-mediated rhesus monkey B-cell proliferation could strongly be inhibited by adding the anti-CD40 MAb (data not shown). The mean survival time of untreated kidney transplanted rhesus monkeys is 6 days (n 5 4). Prophylactic short-term treatment (14 days) with anti-CD80 plus CD86 MAbs alone or in combination with anti-CD40 MAb resulted in a prolonged graft survival of 28 6 7 days (n 5 4) and 24 6 7 days (n 5 4), respectively. Kidney graft rejection started after the antibody therapy was stopped. The survival time of animals treated with the anti-CD80 and anti-CD86 MAbs in combination with CyA (35 days, 10 mg/kg IM) was similar to that of animals treated with CyA alone. However, histologic examination of the rejected kidney showed less signs of vascular rejection in the animals treated with anti-CD80 and anti-CD86 MAbs alone compared to the combined treatment with CyA or anti-CD40 MAb. CONCLUSIONS
Fig 1. Representative example of the effects of daily injection with anti-CD80 and anti-CD86 MAbs (A) or anti-CD80, antiCD86, and anti-CD40 MAbs (B) on rhesus monkey peripheral blood lymphocyte subsets. MAbs were treated from day 21 to day 12. PBMCs were stained with anti-mouse Ig-FITC (MAbcoated cells, closed squares), anti-CD3-FITC (T cells, open squares), and anti-CD20-PE (B cells, open circles).
with anti-CD80 and anti-CD86 MAb alone and other immunosuppressive murine MAbs such as anti-CD4.6 This demonstrates an additional inhibitory effect of the antiCD40 MAb therapy on the B-cell response, which coincides with the decrease of CD201 cells in the circulation. Addi-
Thus, prolonged graft survival can be achieved by blocking the CD80/CD86-CD28/CTLA4 and CD40-CD40L pathways in a preclinical vascularized transplant model in rhesus monkeys. No rejection occurs as long as the MAb concentration in the serum remained high. Additionally, treatment with the anti-CD80 and anti-CD86 MAbs shows a beneficial effect on the type of rejection and the presence of antiCD40 MAb has a suppressive effect on the B-cell response. These promising results justify further investigation to achieve the final goal of induction of donor-specific tolerance. REFERENCES 1. Lenschow DJ, Zeng Y, Hatchcock KS, et al: Transplantation 60:1171, 1995 2. Bashuda H, Seino K, Kano M, et al: Transplant Proc 28:1039, 1996 3. Larsen CP, Elwood ET, Alexander DZ, et al: Nature 381:434, 1996 4. Kirk AD, Harlan DM, Amstrong NN, et al: Proc Natl Acad Sci USA 94:8789, 1997 5. Ossevoort MA, Lorre´ K, Boon L, et al: submitted 6. Jonker M, Neuhaus P, Zurcher C, et al: Transplantation 39:247, 1985