Pig-to-baboon heterotopic heart transplantation--exploratory preliminary experience with pigs transgenic for human thrombomodulin and comparison of three costimulation blockade-based regimens

Abstract

Background: Three costimulation blockade-based regimens have been explored after transplantation of hearts from pigs of varying genetic backgrounds to determine whether CTLA4-Ig (abatacept) or anti-CD40mAb+CTLA4-Ig (belatacept) can successfully replace anti-CD154mAb.

Methods: All pigs were on an α1,3-galactosyltransferase gene-knockout/CD46 transgenic (GTKO.CD46) background. Hearts transplanted into Group A baboons (n=4) expressed additional CD55, and those into Group B (n=3) expressed human thrombomodulin (TBM). Immunosuppression included anti-thymocyte globulin with anti-CD154mAb (Regimen 1: n=2) or abatacept (Regimen 2: n=2) or anti-CD40mAb+belatacept (Regimen 3: n=2). Regimens 1 and 2 included induction anti-CD20mAb and continuous heparin. One further baboon in Group B (B16311) received a modified Regimen 1. Baboons were followed by clinical/laboratory monitoring of immune/coagulation parameters. At biopsy, graft failure, or euthanasia, the graft was examined by microscopy.

Results: Group A baboons survived 15 to 33 days, whereas Group B survived 52, 99, and 130 days, respectively. Thrombocytopenia and reduction in fibrinogen occurred within 21 days in Group A, suggesting thrombotic microangiopathy (TM), confirmed by histopathology. In Group B, with follow-up for >4 m, areas of myofiber degeneration and scarring were seen in two hearts at necropsy. A T-cell response was documented only in baboons receiving Regimen 2.

Conclusions: The combination of anti-CD40mAb+belatacept proved effective in preventing a T-cell response. The expression of TBM prevented thrombocytopenia and may possibly delay the development of TM and/or consumptive coagulopathy.

Keywords: baboon; complement-regulatory proteins; costimulation blockade; heart; pig; thrombomodulin; thrombotic microangiopathy; xenotransplantation; α1,3-galactosyltransferase gene-knockout.

Figure 1. Xenoreactive IgM and IgG binding to GTKO pAECs in baboons receiving Regimens 1, 2, and 3, and modified Regimen 1 (B16311)

No increase in IgM was observed in any of the baboons treated with any of the regimens (A). The baboons treated with Regimen 2 (abatacept-based) developed an elicited anti-pig IgG antibody response (B). An elicited IgG antibody response was not seen in baboons receiving Regimens 1 or 3 or modified Regimen 1 (B). Relative mean fluorescence intensity (MFI) = MFI of anti-nonGal antibody / MFI of isotype control.

Figure 2. Mean (+/− SE) platelet counts (A), fibrinogen (B), and D-dimer (C) in baboons of Groups A and B

(A) In Group A, thrombocytopenia developed within 21 days (95×10³ /µl) post-transplant. In contrast, there was no decrease in platelet count in Group B. (B) Fibrinogen level was dramatically decreased in Group A by day 21 (105 mg/dL), whereas no reduction of fibrinogen was observed in Group B. (C) In both groups, there was an immediate rise in the D-dimer levels by day 3 (>2 ug/ml). This increased D-dimer level was sustained in Group A, although it slightly recovered in day 14 (1.3 ug/ml), and then gradually increased throughout the post-transplant course in Group B.

Figure 2. Mean (+/− SE) platelet counts (A), fibrinogen (B), and D-dimer (C) in baboons of Groups A and B

(A) In Group A, thrombocytopenia developed within 21 days (95×10³ /µl) post-transplant. In contrast, there was no decrease in platelet count in Group B. (B) Fibrinogen level was dramatically decreased in Group A by day 21 (105 mg/dL), whereas no reduction of fibrinogen was observed in Group B. (C) In both groups, there was an immediate rise in the D-dimer levels by day 3 (>2 ug/ml). This increased D-dimer level was sustained in Group A, although it slightly recovered in day 14 (1.3 ug/ml), and then gradually increased throughout the post-transplant course in Group B.

Figure 3. Histopathology and immunohistopathology in baboons of Groups A and B

(A) GTKO.CD46.CD55 pig heart in a baboon that received Regimen 1 (B19110) 33 days after transplantation. (Left) Areas of fibrosis/scar adjacent to infarcted myocardium. (Right) Vacuolated and attenuated myofibers adjacent to areas of frank infarction and focal small thrombus. (B) GTKO.CD46.CD55 pig heart in a baboon that received Regimen 1 (B19010) 18 days after transplantation. (Left) large thrombus in ventricular lumen with evidence of re-endothelialization. (Right) Focal area of eosinophilic myofiber degeneration. (C) Minimal features of myocardial ischemia or injury, with no cellular infiltrate in a GTKO.CD46.TBM pig heart in a baboon that received Regimen 1 (B16311) 52 days after transplantation. (TBM expression on the pig aortic endothelial cells was 96%). (D) Patchy focally extensive areas of fibrosis (scarring) in a GTKO.CD46.TBM pig heart in a baboon that received Regimen 3 (B5512) 99 days after transplantation. (TBM expression on the pig aortic endothelial cells was 8%).

Figure 3. Histopathology and immunohistopathology in baboons of Groups A and B

(A) GTKO.CD46.CD55 pig heart in a baboon that received Regimen 1 (B19110) 33 days after transplantation. (Left) Areas of fibrosis/scar adjacent to infarcted myocardium. (Right) Vacuolated and attenuated myofibers adjacent to areas of frank infarction and focal small thrombus. (B) GTKO.CD46.CD55 pig heart in a baboon that received Regimen 1 (B19010) 18 days after transplantation. (Left) large thrombus in ventricular lumen with evidence of re-endothelialization. (Right) Focal area of eosinophilic myofiber degeneration. (C) Minimal features of myocardial ischemia or injury, with no cellular infiltrate in a GTKO.CD46.TBM pig heart in a baboon that received Regimen 1 (B16311) 52 days after transplantation. (TBM expression on the pig aortic endothelial cells was 96%). (D) Patchy focally extensive areas of fibrosis (scarring) in a GTKO.CD46.TBM pig heart in a baboon that received Regimen 3 (B5512) 99 days after transplantation. (TBM expression on the pig aortic endothelial cells was 8%).