Introduction: Pig-to-human islet xenotransplantation is one attractive approach with potential to alleviate the shortage of islet donors for beta cell replacement in diabetes. For islet xenotransplantation to play a vital role in diabetes care, safe and effective strategies for prevention of islet xenograft rejection must be developed. Blocking CD40L:CD40 costimulation with anti-CD40L antibodies has allowed several investigators to achieve long-term porcine islet xenotransplant survival in nonhuman primates. Antagonistic anti-CD40 antibodies are in clinical development in autoimmunity and transplantation to block CD40L:CD40 costimulation without thromboembolic risks. Herein, we investigate the efficacy and mechanisms of action of maintenance immunosuppression with anti-CD40 Ab 2C10.R4 or CTLA4-Ig in combination with rapamycin in pig-to-nonhuman primate (NHP) islet xenotransplant.
Methods: Cultured pig islets (25,000 IE/kg) were transplanted intraportally into diabetic cynomolgus macaques. Induction immunosuppression included basiliximab and CTLA4-Ig and then maintenance with rapamycin and either CTLA4Ig (cohort A, n=4) or 2C10R4 (cohort B, n=3). B and T cell phenotypes and functional profiles were assessed by flowcytometry using cryopreserved blood and spleen, lymph node, and liver samples. The development of donor-specific antibodies (DSAs), levels of BAFF, APRIL, and cytokines were monitored in serum. 
Results: Islet xenograft survival was ≥306 days in 1 of 4 recipients in cohort A (Fig.1). Conversely, the cohort B regimen facilitated long-term (>1 yr) functional islet xenograft survival in 3 of 3 recipients. The examination of infiltrating cells in tissues revealed few CXCR3+ and CXCR4+ B cells and a high abundance of PD-1+ exhaust-like B and T cell subsets in cohort B recipients. We noted, a significantly higher presence of CD8 TEM cells in blood and tissue that express cytokine and cytolytic effectors in cohort A (Fig.1). Prolonged suppression of DSAs, B cells expressing BAFFR and TACI, lower serum levels of BAFF/APRIL, and preponderance of naïve and immature B cells post-tx were noted only in cohort B (Fig.1). Furthermore, only cohort B showed expansion of Breg and Treg cells post-tx, both subsets displaying higher IL-10/TNF-A (alpha) ratios in Cohort B vs A (Fig.2).
Conclusion:  Maintenance with anti-CD40 plus rapamycin was associated with suppression of early B cell sensitization, posttransplant expansion of TEM cells, and B/ T cell activation and proliferation. Blocking CD40 signaling regulated cellular immunity by increasing the frequency of functional regulatory B and T cells. The long-term functional and histologic survival of islet xenografts indicates the robust effectiveness of the anti-CD40 plus rapamycin maintenance regimen in the stringent pig-to-NHP xenoislet transplant model.