Selective CD28 inhibition modulates alloimmunity and cardiac allograft vasculopathy in anti-CD154-treated monkeys

AbstractBackgroundSelective CD28 inhibition is actively pursued as an alternative to B7 blockade using CTLA4-Ig based on the hypothesis that the checkpoint immune regulators CTLA-4 and PD-L1 will induce tolerogenic immune signals. We previously showed that blocking CD28 using a monovalent nonactivating reagent (single chain anti-CD28 Fv fragment linked to alpha-1 anti-trypsin: sc28AT) synergizes with calcineurin inhibitors in nonhuman primate (NHP) kidney and heart transplantation. Here, we explored the efficacy of combining a 3-week 'induction" sc28AT treatment with prolonged CD154 blockade.MethodsCynomolgus monkey heterotopic cardiac allograft recipients received sc28AT (10 mg/kg, d0-20, n=3), hu5C8 (10-30 mg/kg, d0-84, n=4), or combination (n=6). Graft survival was monitored by telemetry. Protocol biopsies and graft explants were graded according to ISHLT AR and CAV scores. Alloantibody, T cell phenotype and Tregs were analyzed by flow cytometry. Immunochemistry and gene expression (Nanostring) characterized intra-graft cellular infiltration.ResultsRelative to modest prolongation of median graft survival time with sc28AT alone (34 days), hu5C8 (133 days) and sc28AT+hu5C8 (141 days) prolonged survival to a similar extent. CD28 blockade at induction, added to hu5C8, significantly attenuated the severity of acute rejection and cardiac allograft vasculopathy (CAV) during the first 3 months after transplantation relative to hu5C8 alone. These findings were associated with decreased proportions of circulating CD8+ and CD3+CD28- T cells, and modulation of inflammatory gene expression within allografts.ConclusionsInduction with sc28AT promotes early cardiac allograft protection in hu5C8-treated NHPs. These results support further investigation of prolonged selective CD28 inhibition with CD40/CD154 blockade in NHP transplants. Background Selective CD28 inhibition is actively pursued as an alternative to B7 blockade using CTLA4-Ig based on the hypothesis that the checkpoint immune regulators CTLA-4 and PD-L1 will induce tolerogenic immune signals. We previously showed that blocking CD28 using a monovalent nonactivating reagent (single chain anti-CD28 Fv fragment linked to alpha-1 anti-trypsin: sc28AT) synergizes with calcineurin inhibitors in nonhuman primate (NHP) kidney and heart transplantation. Here, we explored the efficacy of combining a 3-week 'induction" sc28AT treatment with prolonged CD154 blockade. Methods Cynomolgus monkey heterotopic cardiac allograft recipients received sc28AT (10 mg/kg, d0-20, n=3), hu5C8 (10-30 mg/kg, d0-84, n=4), or combination (n=6). Graft survival was monitored by telemetry. Protocol biopsies and graft explants were graded according to ISHLT AR and CAV scores. Alloantibody, T cell phenotype and Tregs were analyzed by flow cytometry. Immunochemistry and gene expression (Nanostring) characterized intra-graft cellular infiltration. Results Relative to modest prolongation of median graft survival time with sc28AT alone (34 days), hu5C8 (133 days) and sc28AT+hu5C8 (141 days) prolonged survival to a similar extent. CD28 blockade at induction, added to hu5C8, significantly attenuated the severity of acute rejection and cardiac allograft vasculopathy (CAV) during the first 3 months after transplantation relative to hu5C8 alone. These findings were associated with decreased proportions of circulating CD8+ and CD3+CD28- T cells, and modulation of inflammatory gene expression within allografts. Conclusions Induction with sc28AT promotes early cardiac allograft protection in hu5C8-treated NHPs. These results support further investigation of prolonged selective CD28 inhibition with CD40/CD154 blockade in NHP transplants. * T.Z. and A.M.A. contributed equally to the first authorship of this study. #Correspondence: Richard N. Pierson III, MD, Division of Cardiac Surgery, Department of Surgery, University of Maryland School of Medicine, 110 South Paca Street 7N-134, Baltimore, MD 21201. E-mail: rpierson@som.umaryland.edu. Phone: 410.328.5842, Fax: 410.328.2750 Authorship Contributions: T.Z., A.M.A., and R.N.P.III designed the experiments, analysed the data, and wrote the article. W.S., N.A.O., E.B., and G.B. performed experiments and analysed data. E.S., L.B., X.C., T.M., S.D., D.H., E.R., E.W., A.K., and C.A. conducted all experiments. Disclosure : The authors declare no conflict of interest. Funding: This work was supported by the NIH (UO1 AI 066719), an ASTS Mid-Career Award, a contract from the DOD ORD (N00014-04-1-0821), and an AHA Grant-in-Aid, all to RNP; by the Other Tobacco Related Diseases research grant from the Maryland Restitution Fund Program, to AA and RNP; and by the NIH Nonhuman Primate Reagent Resource (OD010976 and AI126683). Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.

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