A Mixed-chimerism Protocol Utilizing Thymoglobulin and Belatacept Did Not Induce Lung Allograft Tolerance, Despite Previous Success in Renal Allotransplantation

Abstract

Background: In kidney transplantation, long-term allograft acceptance in cynomolgus macaques was achieved using a mixed-chimerism protocol based on the clinically available reagents, rabbit anti-thymocyte globulin (ATG), and belatacept. Here, we have tested the same protocol in cynomolgus macaques transplanted with fully allogeneic lung grafts.

Methods: Five cynomolgus macaques underwent left orthotopic lung transplantation. Initial immunosuppression included equine ATG and anti-IL6RmAb induction, followed by triple-drug immunosuppression for 4 mo. Post-transplant, a nonmyeloablative conditioning regimen was applied, including total body and thymic irradiation. Rabbit ATG, belatacept, anti-IL6RmAb, and donor bone marrow transplantation (DBMT) were given, in addition to a 28-d course of cyclosporine. All immunosuppressant drugs were stopped on day 29 after DBMT.

Results: One monkey rejected its lung before DBMT due to AMR, after developing donor-specific antibodies. Two monkeys developed fatal post-transplant lymphoproliferative disorder, and both monkeys had signs of cellular rejection in their allografts upon autopsy. The remaining 2 monkeys showed severe cellular rejection on days 42 and 70 post-DBMT. Cytokine analysis suggested higher levels of pro-inflammatory markers in the lung transplant cohort, as compared to kidney recipients.

Conclusion: Although the clinically applicable protocol showed success in kidney transplantation, the study did not show long-term survival in a lung transplant model, highlighting the organ-specific differences in tolerance induction.

FIGURE 1.

Schematic for protocol of delayed tolerance induction following organ transplantation. Animals received equine ATG for induction therapy before transplantation and were kept on triple-drug immunosuppression for 4 mo. Thereafter, a nonmyeloablative conditioning regimen including total body irradiation (TBI), thymic irradiation (TI), and thymoglobulin (rabbit ATG) was performed. After donor bone marrow transplantation, belatacept was administered as well as a 28-d course of cyclosporine. Lung animals received an anti-IL6 receptor blocker following initial organ transplantation and DBMT. All immunosuppressive medication was stopped thereafter (kidney protocol redrawn from Hotta et al). ATG, anti-thymocyte globulin; DBMT, donor bone marrow transplantation.

FIGURE 2.

A, Kaplan–Meier analysis of graft survival following lung (black curve) and kidney (gray curve) transplantation. Allograft survival following kidney transplantation was significantly superior as compared to animals undergoing lung transplantation (P = 0.01). B, Pathology of lung allografts. (1) Hematoxylin and eosin staining of an allograft lung at the time of euthanasia (M8216) on day 78 after lung transplantation before DBMT. The histology shows signs of the severe antibody as well as mild acute cellular rejection (ISHLT grading: A1, B1R, AMR, C4d pos) with thickening of alveolars as well as lymphocyte infiltration. (II) and (III): Histology of the allograft lung of M8216 at time of euthanasia showing positive C4d staining, suggesting severe antibody-mediated rejection. C, Pathology of lung allografts sampled pre-DBMT (I and II) of M7416 showing no signs of cellular or antibody-mediated rejection. Upon euthanasia on day 70 post-DBMT III–V), histology showed severe acute cellular as well as antibody-mediated rejection with positive C4d staining (ISHLT grading: A4, B2R, AMR, C4d pos). D, Chest radiographs of M7416 of the postoperative course showing a well-aerated left allograft lung pre-DBMT, but diffuse infiltration after DBMT on day 70 after BMT indicating graft rejection. DBMT, donor bone marrow transplantation; ISHLT, International Society for Heart and Lung Transplantation.

FIGURE 3.

Cellular lymphocyte reconstitution in allograft recipients. A, Lung recipients showed effective depletion of lymphocytes during conditioning and DBMT; reconstitution of lymphocytes was similar between lung and kidney allograft recipients (E). Both groups showed an overspilling reconstitution of CD3+CD8+ lymphocytes, with overall numbers being higher in the kidney group (B, F). Similarly, CD3+CD4+ lymphocytes underwent effective depletion, the kidney group showing higher absolute numbers upon reconstitution as compared with lung recipients (C, G). Lung recipients failed to reconstitute regulatory T cells to pre-DBMT levels throughout the entire postoperative monitoring phase (D). Further differentiation of CD8+ T-lymphocytes revealed a high percentage of effector memory T cells in both groups upon reconstitution. In addition, kidney recipients expressed a larger proportion of central memory T cells, whereas lung animals failed to reconstitute central memory cells (H, I). DBMT, donor bone marrow transplantation.

FIGURE 4.

Serum cytokine levels before DBMT (day 30) and after DBMT (d30pBMT). Lung recipients showed higher levels of proinflammatory cytokines (IL-1RA, IL-6, and INF-g) after DBMT as compared with kidney recipients (A, B, C). Also, lung recipients had slightly higher levels of IL-10 after DBMT as compared with pre-DBMT, whereas kidney recipients showed lower levels of IL-10 after DBMT as compared with preBMT (D). (E, F) Donor-specific antibodies in lung transplant recipients. Animals showing signs of humoral rejection upon euthanasia also showed detectable anti-B-cell antibodies (M8216 and M7416) as well as anti-T-cell donor-specific antibodies (M7416 and M4116). DBMT, donor bone marrow transplantation.