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Presenter: Burcin, Ekser, Pittsburgh, United States
Authors: Burcin Ekser1,2, Cassandra Long1, Goutham Kumar1, Hidetaka Hara1, Eefje M. Dons1, Jnanesh Thacker3, Yoshiya Toyoda3, Massimiliano Veroux2, David Ayares4, David K.C. Cooper1, Mohamed Ezzelarab1
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Heart xenotransplantation using genetically-engineered pigs: can CTLA4-Ig replace anti-CD154mAb in the immunosuppressive regimen?
Burcin Ekser1,2, Cassandra Long1, Goutham Kumar1, Hidetaka Hara1, Eefje M. Dons1, Jnanesh Thacker3, Yoshiya Toyoda3, Massimiliano Veroux2, David Ayares4, David K.C. Cooper1, Mohamed Ezzelarab1
1Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, PA, United States; 2Department of Surgery, Transplantation and Advanced Technologies, Vascular Surgery and Organ Transplant Unit, University Hospital of Catania, Catania, Italy; 3Department of Cardiothoracic Surgery, Division of Cardiac Surgery, University of Pittsburgh, Pittsburgh, PA, United States; 4Revivicor, Inc., Blacksburg, VA, United States
Background: Heterotopic heart xenotransplantation (HTx) using genetically-engineered pigs can be achieved without sensitization to pig antigens with an anti-CD154mAb-based immunosuppressive regimen (IS). We investigated whether CTLA4-Ig could replace anti-CD154mAb.
Methods: Study 1: GTKO pig-to-baboon artery patch Tx was carried out (n=6). IS was anti-CD154mAb-based (n=4) or CTLA4-Ig-based (n=2) with ATG, MMF, and steroids in both regimens. Study 2: HTx was carried out in baboons using GTKO.hCD46.hCD55 pigs (n=4). IS was anti-CD154mAb-based (n=2) or CTLA4-Ig-based (n=2), but HTx recipients received additional anti-CD20mAb. CD3+, CD4+, CD8+ T cell and CD19+, CD20+, CD21+ B cell counts and IgG and IgM antibody (Ab) binding against WT and GTKO PBMCs were determined by flow cytometry. Follow-up in both studies was for one month.
Results: Study 1: Baboons with GTKO patch grafts did not develop anti-pig Abs if IS was anti-CD154mAb-based, but did develop sensitization (5.5-fold increase in anti-pig IgG, no increase in anti-pig IgM) if IS was CTLA4-Ig-based. Study 2: The addition of anti-CD20mAb to the regimen (compared to historical controls) depleted all B cells in the blood for >1m (mean pre-Tx CD19#, CD20# and CD21# were 142, 650, 395, while post-Tx were 2, 2, 4, respectively). Despite this additional IS, baboons receiving a CTLA4-Ig-based IS regimen developed anti-pig IgG (with a 2.3-fold increase against WT and 1.6-fold increase against GTKO PBMCs), whereas those receiving anti-CD154mAb did not. After ATG, repopulation of CD3+T cells occurred more rapidly in CTLA4-Ig-treated baboons (73% recovery) than in anti-CD154mAb-treated baboons (9% recovery).
Conclusions: When either GTKO artery patch grafts or GTKO.CD46.CD55 heart grafts were transplanted into baboons, an anti-CD154mAb-based IS regimen prevented sensitization to pig antigens, whereas a CTLA4-Ig-based regimen did not, even with additional B cell depletion, though less sensitization occurred despite increased antigen load. Potent clinically-applicable IS or additional pig genetic modifications will be required to prevent T cell-dependent Ab development. Alternative approaches to block CD40-CD154 signaling pathway might be necessary.
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