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Relative efficiency of porcine and human CTLA4-Ig in inhibiting human CD4+Tcell responses costimulated by porcine and human B7 molecules

Tadatsura Koshika¹, Carol Phelps², Jason Fang¹, Seung Lee¹, Minoru Fujita¹, David Ayares², David K.C. Cooper¹, Hidetaka Hara¹

¹Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh, Pittsburgh, PA; ²Revivicor, Blacksburg, VA; United States

Purpose: GTKO pigs transgenic for porcine (p) CTLA4-Ig have been produced to reduce T cell-mediated rejection following xenotransplantation. The serum level of soluble pCTLA4-Ig greatly exceeded the clinical therapeutic level, rendering the pigs immune-incompetent. Our aim was (i) toevaluate whether soluble pCTLA4-Ig from pCTLA4-Ig transgenic pigs could bind to p and human (h) B7 molecules (CD80/CD86), (ii) inhibit the xenogeneic direct responses of hCD4⁺T cells to pAECs, and (iii) to determine the efficacy of pCTLA4-Ig transgenic cells to inhibit host T cell-mediated xenogeneic immune responses.

Methods: Soluble pCTLA4-Ig produced by transgenic pigs was evaluated for binding to p and h B7 molecules on p aortic endothelial cells (pAECs) and hPBMCs, and for its inhibitory effect on allogeneic/xenogeneic hT cell responses to these cells. Both pCTLA4-Ig-expressing PBMCs and pAECs were evaluated for their direct inhibitory effect on hCD4⁺T cell responses.

Results: Soluble pCTLA4-Ig and purified hCTLA4-Ig showed similar binding to pB7 molecules, but pCTLA4-Ig showed significantly less binding to hB7 molecules. In mixed lymphocyte reaction, pCTLA4-Ig and hCTLA4-Ig equally inhibited the response of hCD4⁺T cells to pAECs, but pCTLA4-Ig was less effective in inhibiting the h allogeneic response. The hCD4⁺T cell response to PBMCs from pCTLA4-Ig pigs was significantly lower than that of non-pCTLA4-Ig pigs. Although pCTLA4-Ig was detected in the cytoplasm of pCTLA4-Ig-expressing pAECs, no soluble pCTLA4-Ig was detected in the supernatant during culture, and pCTLA4-Ig-expressing pAECs did not inhibit the xenogeneic direct hT cell response.

Conclusions: (i) Soluble pCTLA4-Ig only inhibits the h direct T cell xenogeneic response. (ii) High-level tissue-specific production of pCTLA4-Ig will be required to suppress the direct T cell response. (iii) hCTLA4-Ig will need to be administered to suppress the indirect response. (iv) The discrepancy between the high levels of soluble CTLA4-Ig in the pigs and its absence in the cell supernatant requires study.