Introduction: Costimulatory blockade is one of key immunosuppressants in kidney xenotransplantation. We investigated optimal combinations of double costimulatory blockade including two forms of anti-CD154 mAb (5C8, PG405), abatacept (CTLA-4Ig), and anti-ICAM-1 mAb (MD3) in pig-to-nonhuman primate kidney xenotransplantation.
Methods: Kidneys from six or eight genetically-engineered pigs (knock-out for GGTA1, CMAH, iGb3s, or B4GalNT2; knock-in for human CD39, CD46, CD55, or thrombomodulin) were transplanted into five cynomolgus monkeys. Common immunosuppressive regimen consisted of thymoglobulin, rituximab, anti-C5 inhibitor (crovalimab), etanercept, and triple maintenance immunosuppressants including prednisolone, tacrolimus, and mycophenolate mofetil. Additionally, three groups received different combinations of costimulatory blockade. Group A (n=3) received modified anti-CD154 mAb with Fc modification and IL-10-conjugation (PG405) and anti-ICAM-1 mAb (MD-3). Group B (n=1) received modified anti-CD154 mAb (PG405) and abatacept with anti-IL-6R mAb (tocilizumab). Group C (n=1) received original anti-CD154 mAb (5C8) and abatacept with tocilizumab. Contralateral native nephrectomy was performed at 4 weeks after transplantation. Kidney xenograft biopsies were performed at 4 weeks post-transplantation, and later timepoints.
Results: The serum levels of BUN and creatinine after native nephrectomy were higher in groups A and B compared to those in group C. Furthermore, serum levels of proinflammatory cytokines (IL-6, TNF-α) and complement activation (membrane attack complex [MAC]) were lower in group C compared to those in group A. Serum levels of TNF-α were markedly increased, while those of IL-6 and MAC were low in group B. Notably, de novo anti-pig-specific antibodies were detected in groups A and B, whereas they were not in group C. However, anti-pig-specific T cell responses were not significantly different among three groups. Number of memory T cells, monocyte, and NK cells in peripheral blood were kept low in group C. In parallel, kidney xenograft biopsies showed that antibody-mediated rejection occurred in all monkeys of groups A and B, whereas it did not in group C. Groups A and B exhibited mesangiolysis, thrombotic microangiopathy, microvascular inflammation, and vasculitis. Additionally, immunohistochemistry staining showed that groups A and B had significant intragraft infiltration of CD3+ T cells, CD68+ macrophages, and CD61+ platelets with deposition of IgG, C4d, or MAC, whereas group C show minimal intragraft infiltration of immune cells without significant IgG or complement deposition.
Conclusion: Combination of 5C8 clone of anti-CD154 mAb and abatacept seemed to suppress kidney xenograft rejection better than combination of a modified anti-CD154 mAb and anti-ICAM-1 mAb. Therefore, we need to development new double costimulatory blockade strategies including safer anti-CD154 mAb without thromboembolic complications.