Purpose: This study aimed to evaluate the efficacy of an optimized immunosuppressive (IS) regimen, including a higher dose of anti-CD154mAb (TNX-1500), in prolonging graft survival in pig-to-baboon kidney xenotransplantation.
Methods: Nine baboons received kidneys from gene-edited pigs (n=7; either triple-knockout or double-knockout with growth hormone receptor knockout and six human transgenes - hCD46, hCD55, hTBM, hEPCR, hHO-1, and hCD47; Revivicor Inc). Group A (n=3) received induction therapy with anti-thymocyte globulin (ATG), anti-CD20mAb (rituximab), and C1-esterase inhibitor, followed by maintenance therapy with TNX-1500 (20mg/kg, Tonix Corp), rapamycin, methylprednisolone, and IL-6 receptor blockade (tocilizumab). Group B (n=6) received the identical regimen except for an increased TNX-1500 dose (30mg/kg). All animals were confirmed CMV-negative. Monitoring included blood counts, flow cytometry, serum and urine analyses, ultrasound examinations, and renal biopsies.
Results: In Group A, survival was 59, 86, and 120 days (median 86 days, mean 86 days), with AMR at the time of graft failure. In Group B, graft survival was significantly prolonged (median 214 days, mean 248 days,  p<0.05): >1 year (n=2), >6 months (n=2), and >3 months (n=2). One baboon developed antibody-mediated rejection (AMR) after IS reduction (day 439); the remaining five were euthanized with functioning grafts for complications believed to be unrelated to anti-CD154mAb therapy (on days 100, 117, 203, 225, and 405), though one had arterial thrombosis (day 117) and one had unexplained chronic diarrhea and weight loss (day 225).   
In Group B, five of six recipients developed mild focal thrombotic microangiopathy (TMA) between 3–6 months post-transplant, although serum creatinine levels remained normal or near-normal. No histopathological features of AMR were observed at 3 months post-transplant. All of the biopsies taken at >6 months demonstrated features of transplant glomerulopathy and/or progression of TMA. Despite these pathological changes, serum creatinine levels remained relatively stable in all four baboons surviving >6 months.
Flow cytometric analysis in Group B showed that both CD4+ and CD8+ T cell numbers remained suppressed <50% of baseline at 6 months. Notably, the number of regulatory T cells (Tregs) gradually recovered from ATG, and the percentage of Tregs increased compared to baseline for >3 months.
Conclusions: An anti-CD154mAb constitutes a key component of the IS regimen. Increasing its dose resulted in significantly prolonged graft survival with few associated complications. The combination of CD40/CD154 co-stimulation pathway blockade with rapamycin and tocilizumab may exert synergistic effects in suppressing effector T cells and expanding Tregs, offering a promising strategy to enhance long-term xenograft survival.