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A comparison of the in vitro human immune response to corneal cells from humans and genetically-modified pigs

Hidetaka Hara¹, Naoko Koike¹, Cassandra Long¹, Minoru Fujita¹, Danny S. Roh², James L. Funderburgh², Thomas E. Starzl¹, Yifan Dai³, David L. Ayares³, David K.C. Cooper¹

¹Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA; ²Eye & Ear Institute, University of Pittsburgh, Pittsburgh, PA; ³Revivicor Inc., Blacksburg, VA; United States

Purpose: To investigate in vitro the human (h) humoral and cellular responses to wild-type (WT) pig corneal endothelial cells (pCECs) and compare with CECs from h and genetically-engineered pigs – (i) α1,3-galactosyltransferase gene-knockout [GTKO], (ii) GTKO transgenic for hCD46 [GTKO/CD46], (iii) GTKO/CD46/CD55, (iv) dominant-negative class II transactivator knockdown [CIITA-DN].

Methods: Lysis of activated pCECs was determined by a h complement-dependent cytotoxicity (CDC) assay. The hCD4+T cell response to CECs from WT, GTKO, and GTKO/CD46 pigs and humans was measured by MLR. The expression of SLA class II on CIITA-DN pCECs with/without activation by IFN-γ was measured. MLR was also performed using PBMCs from WT, CIITA-DN pigs, and h.

Results: On CDC, when activated pCECs were used, there was no significant difference in lysis of WT and GTKO pCECs. However, GTKO/CD46 pCECs demonstrated significant resistance to lysis (p<0.05 vs WT and GTKO), and there was no lysis of GTKO/CD46/CD55 pCECs (p<0.05 vs GTKO/CD46). hCD4+T cell responses to GTKO and GTKO/CD46 pCECs and hCECs were significantly less than to WT pCECs before and after activation (p<0.01). Although the hCD4+T cell response to GTKO and GTKO/CD46 pCEC was greater than to hCECs before activation, there was no significant difference after activation. The expression of SLA class II on pCEC from CIITA-DN pigs was significantly down-regulated, even after activation, and the hCD4+T cell response to CIITA-DN cells was significantly lower than to h and WT cells (P<0.01).

Conclusions: Corneas from GTKO/CD46/CD55/CIITA-DN pigs (when available) should be significantly protected against h humoral and cellular immunity, and may be comparable to h corneas.