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Probable failure of chimerism induction in orthotopically transplanted monkey hearts in baboons
Probable Failure of Chimerism Induction in Orthotopically Transplanted Monkey Hearts in Baboons H. Izutani, S.R. Gundry, M. Asano, O. Fagoaga, C.W. Zuppan and L.L. Bailey
A
SHORTAGE of donor organs is a serious problem for infants with incurable heart defects, who cannot be reliably supported by a mechanical assist device. Cardiac xenografts represent a possible solution to this problem. Concordant xenografts, ie, organ engraftment between closely related species, induce a relatively weak immune reaction and extend the survival period compared to that involving discordant xenografts between widely distant species. Therefore, concordant xenotransplantation may provide permanent engraftment. In our previous study, six rhesus monkey hearts were orthotopically transplanted into baboons using an immunosuppressive protocol of cyclosporine (CyA), methotrexate (MTX), and antilymphocyte globulin. Average survival time was 272 days.1 Deaths were related to opportunistic infections and drug side effects. To extend survival, we proposed to induce tolerance that would allow graft acceptance while maintaining otherwise adequate immune responses. Investigators have previously demonstrated that mixed lymphohematopoietic chimerism provides an effective means of inducing long-term tolerance in allograft models.2– 4 Efficacy of a mixed chimerism approach for producing long-term survival of xenografts in concordant rat-to-mouse combinations has been established.2,5 In recent studies, significant lymphocyte chimerism induced by donor bone marrow transplantation (BMT) and irradiation seemed to be important in achieving concordant kidney xenograft tolerance in cynomolgus monkeys.6 In this study, we tried to induce mixed chimerism by nonlethal total lymphoid irradiation (TLI) of recipient animals and BMT in a rhesus monkey-to-baboon concordant cardiac xenotransplant model.
MATERIALS AND METHODS Animals, Experimental Design Eleven juvenile Olive baboons weighing 2.5 to 4.1 kg received heart transplants from ABH-compatible rhesus monkeys weighing 2.3 to 6.3 kg. Among a panel of five to seven rhesus monkeys, the one whose lymphocytes were the lowest stimulators against the recipient’s lymphocytes in xenogeneic mixed lymphocyte reaction was selected as the donor. Cardiac xenografts were transplanted orthotopically as previously described.7 Five recipients (group I) were given 80 cGy TLI biweekly for 5 weeks (10 times, total 800 cGy) before transplant. They also received 3 ⫻ 108/kg of donor bone marrow cell infusion intraoperatively. The donor bone marrow
cells were harvested from bilateral femurs and washed for injection. Six recipients (group II) were not given TLI or BMT.
Immunosuppressive Protocol Pretransplant treatment consisted of 15 mg/kg/d subcutaneous (SC) doses of CyA and 0.5 to 2 mg/kg/wk, twice a week, intravenous (IV) doses of MTX given for 2 weeks before transplant. Perioperative IV antithymocyte globulin (ATGAM, Upjohn, 15 mg/kg/d) was administered from day ⫺3 through ⫹4. Postoperatively, baboons were treated with daily SC doses of CyA (10 to 15 mg/kg/d) and twice a week IV doses of MTX (0.5 to 2 mg/kg/wk). The CyA dose was controlled to obtain a blood trough level of 200 to 300 mg/dL. The MTX dose was adjusted while monitoring white blood cell counts in the recipient’s peripheral blood. A 4-day course of postoperative methylprednisolone (250 mg/d) was also included. Ganciclovir (5 mg/kg/d) was given IV for 2 weeks after transplant to prevent cytomegalovirus (CMV) infection. Xenograft rejections were detected mainly by echocardiography as previously described in pediatric heart transplant patients.8 Findings indicating acute rejections were newly formed pericardial effusion, increased wall thickness, and/or decreased wall motion. Also, acute rejection was diagnosed by detecting an increase of peripheral blood lymphocytes and/or spontaneous blastogenesis (SB). SB was measured by thymidine uptake in peripheral blood lymphocyte culture as a marker of lymphocyte activity. Acute rejections were treated with a 4-day course of methylprednisolone. Histologic examination of the grafts explanted at the time of animal death was performed with hematoxylin and eosin staining.
Chimerism Detection To detect mixed lymphohematopoietic chimerism in the transplanted recipients in group I, flow cytometric analysis of peripheral blood lymphocytes and groin lymph node biopsy specimen was administered using anti-human monoclonal antibodies cross-reacting to the donor rhesus monkey or recipient baboon peripheral blood lymphocytes. Immunohistochemical staining was also performed on the frozen specimens of groin lymph nodes, spleen, liver, lung, and cardiac xenograft from recipient baboons. From the Division of Cardiothoracic Surgery (H.I., S.R.G., M.A., L.L.B.), Immunology center (O.F.), and Department of Pathology (C.W.Z.), Loma Linda University Medical Center, Loma Linda, California. Address reprint requests to Dr Steven R. Gundry, Division of Cardiothoracic Surgery, Loma Linda University of Medical Center, 11175 Campus St, Loma Linda, CA 92354. E-mail: [email protected].
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/00/$–see front matter PII S0041-1345(00)01114-3
Transplantation Proceedings, 32, 1049–1051 (2000)
1049
1050
IZUTANI, GUNDRY, ASANO ET AL
Fig 1. Actuarial survival curve of the transplanted recipients.
RESULTS Xenograft Survival
Actuarial survival curves of the xenograft-transplanted recipient baboons in groups I and II are shown in Fig 1. There was no significant difference in the actuarial survival rate between groups I and II. In group I, one recipient died 10 days after transplant of gastroenterocolitis due to radiation injury. The recipient suffered from severe diarrhea and appetite loss, which caused dehydration and malnutrition. Two recipients died of acute rejection on days 35 and 88, respectively. One of them developed grade II graft-vs-host disease (GVHD) and died of acute vascular rejection causing myocardial infarction on the 88th postoperative day. The other two recipients are still alive in healthy condition on days 495 and 319 with three and two subclinical acute rejection episodes detected by echocardiography, respectively. In group II, six recipients survived for 50 to 515 days after transplant. Three recipients died of moderate to severe acute rejection that did not respond to rescue
therapy. The other three recipients died of a noncardiac event. Two of them had aspiration pneumonia causing lethal infection. One of them suffered from CMV infection requiring long-term ganciclovir therapy causing renal failure. Graft survival days and pathologic findings of the transplant recipients in groups I and II are summarized in Table 1. Representative Clinical Course of Group I Recipients
Body weight, peripheral blood lymphocyte count, and SB were monitored in group I recipients treated with pretransplant TLI and intraoperative donor BMT. Representative clinical course of group I recipients is shown in Fig 2. One recipient was alive on day 495 postoperatively with three subclinical rejection episodes detected by an increased SB and peripheral blood lymphocyte count and treated by methylprednisolone rescue therapy (arrow shows timing of the therapy). The number of lymphocytes in the peripheral blood decreased gradually during TLI treatment before
Table 1. Pathologic Findings of Transplanted Recipients Animal No.
Survival (d)
Cardiac Xenografts
Group I 1 2 3
495 319 88
Alive Alive Focal moderate acute rejection, moderate acute vascular rejection old infarction, acute infarction
Group II 4 5 6 7 8 9 10 11
10 35 413 515 371 50 59 223
Multifocal recent subendocardial infarction Diffuse moderately severe acute rejection Patchy myocardial fibrosis Hypertrophy, mild rejection Hypertrophy, minimal rejection Diffuse, marked acute rejection Moderate acute rejection Focal moderate rejection, hypertrophy
Other Organs
Focal viral pneumonia
Lymphoid depletion of spleen Herpes hepatitis and nephritis, pneumonia Pleural effusion Viral nephritis Interstitial nephritis Interstitial nephritis
GVHD
— — ⫹
— —
PROBABLE FAILURE OF CHIMERISM
1051
Fig 2. Representative clinical course of group I recipients. Arrows show the timing of rescue therapy (methylprednisolone 250 mg/d ⫻ 4 days, IV injection) for acute rejection.
transplant. Also, MTX administration preoperatively caused lymphocytopenia. The number of lymphocytes increased and gradually approached the level prior to TLI, but these costs were maintained below 3000/mm3 by adjusting the dose of MTX. Animal body weight increased normally during the observation period. Chimerism Detection in Group I
One recipient developed grade II GVHD and died due to acute rejection causing myocardial infarction on the 88th postoperative day. The recipient had facial skin rash and mild diarrhea postoperatively. However, this recipient did not show any evidence of chimerism in peripheral blood lymphocytes or organ specimens. The other four recipients did not have any symptoms of GVHD. 0.5% of mixed chimerism was detected by flow cytometric analysis in the groin lymph node cells from one recipient 8 months after transplant, although chimerism was not detected in peripheral blood lymphocytes. The other four animals had no chimerism detected in any examinations. DISCUSSION
Mixed lymphohematopoietic chimerism provides an effective means of inducing long-term tolerance in allograft models.2– 4 The efficacy of the chimerism approach for producing long-term survival of xenografts in concordant rat-to-mouse combinations has been established.2,5 BMT from human to baboon with TLI on the recipient induced chimerism for up to 18 months.9 In this study, we tried to induce long-term survival and mixed chimerism by nonlethal TLI of recipient animals and donor BMT in a rhesus monkey-to-baboon concordant cardiac xenotransplant model. This strategy has not shown improvement in the overall survival in this study. We were able to detect chimerism in one recipient, in which 0.5% of mixed chimerism was detected by flow cytometric analysis in the groin
lymph node 8 months after transplant. The other four recipients did not show any evidence of chimerism induction. Fortunately, the recipient that showed the possibility of chimerism induction is still alive and can be studied further. In this study, we used anti-human monoclonal antibodies cross-reacting to the donor rhesus monkey or recipient baboon peripheral blood lymphocyte for flow cytometry and immunohistochemical staining to examine chimerism induction. The antibodies that reacted strongly to the target cells were chosen from the panel of HLA class I. When the antibodies were used for detection of donor rhesus monkey or recipient baboon cells, the sensitivity of reaction to the target antigen was a problem. Because the monoclonal antibody was not specific to the target antigen exactly, we had to prepare specific antibodies for this kind of study. In conclusion, pretreatment with TLI, intraoperative BMT, and immunosuppressive regimen does not seem to be effective in producing mixed lymphohematopoietic chimerism in concordant primate cardiac xenograft recipients. REFERENCES 1. Matsumiya G, Gundry SR, Nehlsen-Cannarella S, et al: J Heart Lung Transplant 15(1 Pt 2):93, 1996 2. Ildstad ST, Sachs DH: Nature 307:168, 1984 3. Sharabi Y, Sachs DH: J Exp Med 169:493, 1989 4. Kawai T, Cosimi AB, Colvin RB, et al: Transplantation 59:256, 1995 5. Sharabi Y, Aksentijevich I, Sundt III TM, et al: J Exp Med 172:195, 1990 6. Kimikawa M, Sachs DH, Colvin RB, et al: Transplantation 64:709, 1997 7. Kawauchi M, Gundry SR, Alonso de Begona J, et al: J Thorac Cardiovasc Surg 106:779, 1993 8. Boucek MM, Mathis CM, Boucek Jr RJ, et al: J Heart Lung Transplant 13:66, 1994 9. Fontes P, Rogers J, Rao AS, et al: Transplantation 11:1595, 1997
A
SHORTAGE of donor organs is a serious problem for infants with incurable heart defects, who cannot be reliably supported by a mechanical assist device. Cardiac xenografts represent a possible solution to this problem. Concordant xenografts, ie, organ engraftment between closely related species, induce a relatively weak immune reaction and extend the survival period compared to that involving discordant xenografts between widely distant species. Therefore, concordant xenotransplantation may provide permanent engraftment. In our previous study, six rhesus monkey hearts were orthotopically transplanted into baboons using an immunosuppressive protocol of cyclosporine (CyA), methotrexate (MTX), and antilymphocyte globulin. Average survival time was 272 days.1 Deaths were related to opportunistic infections and drug side effects. To extend survival, we proposed to induce tolerance that would allow graft acceptance while maintaining otherwise adequate immune responses. Investigators have previously demonstrated that mixed lymphohematopoietic chimerism provides an effective means of inducing long-term tolerance in allograft models.2– 4 Efficacy of a mixed chimerism approach for producing long-term survival of xenografts in concordant rat-to-mouse combinations has been established.2,5 In recent studies, significant lymphocyte chimerism induced by donor bone marrow transplantation (BMT) and irradiation seemed to be important in achieving concordant kidney xenograft tolerance in cynomolgus monkeys.6 In this study, we tried to induce mixed chimerism by nonlethal total lymphoid irradiation (TLI) of recipient animals and BMT in a rhesus monkey-to-baboon concordant cardiac xenotransplant model.
MATERIALS AND METHODS Animals, Experimental Design Eleven juvenile Olive baboons weighing 2.5 to 4.1 kg received heart transplants from ABH-compatible rhesus monkeys weighing 2.3 to 6.3 kg. Among a panel of five to seven rhesus monkeys, the one whose lymphocytes were the lowest stimulators against the recipient’s lymphocytes in xenogeneic mixed lymphocyte reaction was selected as the donor. Cardiac xenografts were transplanted orthotopically as previously described.7 Five recipients (group I) were given 80 cGy TLI biweekly for 5 weeks (10 times, total 800 cGy) before transplant. They also received 3 ⫻ 108/kg of donor bone marrow cell infusion intraoperatively. The donor bone marrow
cells were harvested from bilateral femurs and washed for injection. Six recipients (group II) were not given TLI or BMT.
Immunosuppressive Protocol Pretransplant treatment consisted of 15 mg/kg/d subcutaneous (SC) doses of CyA and 0.5 to 2 mg/kg/wk, twice a week, intravenous (IV) doses of MTX given for 2 weeks before transplant. Perioperative IV antithymocyte globulin (ATGAM, Upjohn, 15 mg/kg/d) was administered from day ⫺3 through ⫹4. Postoperatively, baboons were treated with daily SC doses of CyA (10 to 15 mg/kg/d) and twice a week IV doses of MTX (0.5 to 2 mg/kg/wk). The CyA dose was controlled to obtain a blood trough level of 200 to 300 mg/dL. The MTX dose was adjusted while monitoring white blood cell counts in the recipient’s peripheral blood. A 4-day course of postoperative methylprednisolone (250 mg/d) was also included. Ganciclovir (5 mg/kg/d) was given IV for 2 weeks after transplant to prevent cytomegalovirus (CMV) infection. Xenograft rejections were detected mainly by echocardiography as previously described in pediatric heart transplant patients.8 Findings indicating acute rejections were newly formed pericardial effusion, increased wall thickness, and/or decreased wall motion. Also, acute rejection was diagnosed by detecting an increase of peripheral blood lymphocytes and/or spontaneous blastogenesis (SB). SB was measured by thymidine uptake in peripheral blood lymphocyte culture as a marker of lymphocyte activity. Acute rejections were treated with a 4-day course of methylprednisolone. Histologic examination of the grafts explanted at the time of animal death was performed with hematoxylin and eosin staining.
Chimerism Detection To detect mixed lymphohematopoietic chimerism in the transplanted recipients in group I, flow cytometric analysis of peripheral blood lymphocytes and groin lymph node biopsy specimen was administered using anti-human monoclonal antibodies cross-reacting to the donor rhesus monkey or recipient baboon peripheral blood lymphocytes. Immunohistochemical staining was also performed on the frozen specimens of groin lymph nodes, spleen, liver, lung, and cardiac xenograft from recipient baboons. From the Division of Cardiothoracic Surgery (H.I., S.R.G., M.A., L.L.B.), Immunology center (O.F.), and Department of Pathology (C.W.Z.), Loma Linda University Medical Center, Loma Linda, California. Address reprint requests to Dr Steven R. Gundry, Division of Cardiothoracic Surgery, Loma Linda University of Medical Center, 11175 Campus St, Loma Linda, CA 92354. E-mail: [email protected].
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/00/$–see front matter PII S0041-1345(00)01114-3
Transplantation Proceedings, 32, 1049–1051 (2000)
1049
1050
IZUTANI, GUNDRY, ASANO ET AL
Fig 1. Actuarial survival curve of the transplanted recipients.
RESULTS Xenograft Survival
Actuarial survival curves of the xenograft-transplanted recipient baboons in groups I and II are shown in Fig 1. There was no significant difference in the actuarial survival rate between groups I and II. In group I, one recipient died 10 days after transplant of gastroenterocolitis due to radiation injury. The recipient suffered from severe diarrhea and appetite loss, which caused dehydration and malnutrition. Two recipients died of acute rejection on days 35 and 88, respectively. One of them developed grade II graft-vs-host disease (GVHD) and died of acute vascular rejection causing myocardial infarction on the 88th postoperative day. The other two recipients are still alive in healthy condition on days 495 and 319 with three and two subclinical acute rejection episodes detected by echocardiography, respectively. In group II, six recipients survived for 50 to 515 days after transplant. Three recipients died of moderate to severe acute rejection that did not respond to rescue
therapy. The other three recipients died of a noncardiac event. Two of them had aspiration pneumonia causing lethal infection. One of them suffered from CMV infection requiring long-term ganciclovir therapy causing renal failure. Graft survival days and pathologic findings of the transplant recipients in groups I and II are summarized in Table 1. Representative Clinical Course of Group I Recipients
Body weight, peripheral blood lymphocyte count, and SB were monitored in group I recipients treated with pretransplant TLI and intraoperative donor BMT. Representative clinical course of group I recipients is shown in Fig 2. One recipient was alive on day 495 postoperatively with three subclinical rejection episodes detected by an increased SB and peripheral blood lymphocyte count and treated by methylprednisolone rescue therapy (arrow shows timing of the therapy). The number of lymphocytes in the peripheral blood decreased gradually during TLI treatment before
Table 1. Pathologic Findings of Transplanted Recipients Animal No.
Survival (d)
Cardiac Xenografts
Group I 1 2 3
495 319 88
Alive Alive Focal moderate acute rejection, moderate acute vascular rejection old infarction, acute infarction
Group II 4 5 6 7 8 9 10 11
10 35 413 515 371 50 59 223
Multifocal recent subendocardial infarction Diffuse moderately severe acute rejection Patchy myocardial fibrosis Hypertrophy, mild rejection Hypertrophy, minimal rejection Diffuse, marked acute rejection Moderate acute rejection Focal moderate rejection, hypertrophy
Other Organs
Focal viral pneumonia
Lymphoid depletion of spleen Herpes hepatitis and nephritis, pneumonia Pleural effusion Viral nephritis Interstitial nephritis Interstitial nephritis
GVHD
— — ⫹
— —
PROBABLE FAILURE OF CHIMERISM
1051
Fig 2. Representative clinical course of group I recipients. Arrows show the timing of rescue therapy (methylprednisolone 250 mg/d ⫻ 4 days, IV injection) for acute rejection.
transplant. Also, MTX administration preoperatively caused lymphocytopenia. The number of lymphocytes increased and gradually approached the level prior to TLI, but these costs were maintained below 3000/mm3 by adjusting the dose of MTX. Animal body weight increased normally during the observation period. Chimerism Detection in Group I
One recipient developed grade II GVHD and died due to acute rejection causing myocardial infarction on the 88th postoperative day. The recipient had facial skin rash and mild diarrhea postoperatively. However, this recipient did not show any evidence of chimerism in peripheral blood lymphocytes or organ specimens. The other four recipients did not have any symptoms of GVHD. 0.5% of mixed chimerism was detected by flow cytometric analysis in the groin lymph node cells from one recipient 8 months after transplant, although chimerism was not detected in peripheral blood lymphocytes. The other four animals had no chimerism detected in any examinations. DISCUSSION
Mixed lymphohematopoietic chimerism provides an effective means of inducing long-term tolerance in allograft models.2– 4 The efficacy of the chimerism approach for producing long-term survival of xenografts in concordant rat-to-mouse combinations has been established.2,5 BMT from human to baboon with TLI on the recipient induced chimerism for up to 18 months.9 In this study, we tried to induce long-term survival and mixed chimerism by nonlethal TLI of recipient animals and donor BMT in a rhesus monkey-to-baboon concordant cardiac xenotransplant model. This strategy has not shown improvement in the overall survival in this study. We were able to detect chimerism in one recipient, in which 0.5% of mixed chimerism was detected by flow cytometric analysis in the groin
lymph node 8 months after transplant. The other four recipients did not show any evidence of chimerism induction. Fortunately, the recipient that showed the possibility of chimerism induction is still alive and can be studied further. In this study, we used anti-human monoclonal antibodies cross-reacting to the donor rhesus monkey or recipient baboon peripheral blood lymphocyte for flow cytometry and immunohistochemical staining to examine chimerism induction. The antibodies that reacted strongly to the target cells were chosen from the panel of HLA class I. When the antibodies were used for detection of donor rhesus monkey or recipient baboon cells, the sensitivity of reaction to the target antigen was a problem. Because the monoclonal antibody was not specific to the target antigen exactly, we had to prepare specific antibodies for this kind of study. In conclusion, pretreatment with TLI, intraoperative BMT, and immunosuppressive regimen does not seem to be effective in producing mixed lymphohematopoietic chimerism in concordant primate cardiac xenograft recipients. REFERENCES 1. Matsumiya G, Gundry SR, Nehlsen-Cannarella S, et al: J Heart Lung Transplant 15(1 Pt 2):93, 1996 2. Ildstad ST, Sachs DH: Nature 307:168, 1984 3. Sharabi Y, Sachs DH: J Exp Med 169:493, 1989 4. Kawai T, Cosimi AB, Colvin RB, et al: Transplantation 59:256, 1995 5. Sharabi Y, Aksentijevich I, Sundt III TM, et al: J Exp Med 172:195, 1990 6. Kimikawa M, Sachs DH, Colvin RB, et al: Transplantation 64:709, 1997 7. Kawauchi M, Gundry SR, Alonso de Begona J, et al: J Thorac Cardiovasc Surg 106:779, 1993 8. Boucek MM, Mathis CM, Boucek Jr RJ, et al: J Heart Lung Transplant 13:66, 1994 9. Fontes P, Rogers J, Rao AS, et al: Transplantation 11:1595, 1997