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Hidetaka Hara, Naoko Koike, Cassandra Long, Danny S. Roh, James L. Funderburgh, Thomas E. Starzl, Yifan Dai, David Ayares, David K.C. Cooper; A Comparison Of The In Vitro Human Immune Response To Corneal Cells From Humans And Genetically-Modified Pigs. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1151.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate in vitro the human humoral and cellular responses to wild-type (WT) pig corneal endothelial cells (pCEC) and compare with CEC from humans and genetically-engineered pigs - (i) α1,3-galactosyltransferase gene-knockout [GTKO], (ii) GTKO+transgenic for a human complement-regulatory protein [GTKO/CD46], (iii) dominant-negative class II transactivator transgenic [CIITA-DN].
Pooled human serum was used to measure IgM/IgG binding to pCEC from WT, GTKO, and GTKO/CD46 pigs by flow cytometry. Lysis of pCEC was determined by a human complement-dependent cytotoxicity (CDC) assay. The human CD4+T cell response to CEC from all 3 pigs and humans was measured by mixed lymphocyte reaction (MLR). The expression of swine leukocyte antigen (SLA) class II on CIITA-DN pCEC with/without activation by IFN-γ was measured. MLR was performed using peripheral blood mononuclear cells (PBMC) from WT, CIITA-DN pigs, and humans.
There was greater binding of IgM and IgG to WT than to GTKO and GTKO/CD46 pCEC (p<0.01). CDC of quiescent WT pCEC was greater than of GTKO and GTKO/CD46 pCEC (p<0.05); after activation, CDC was increased and there was no significant difference in lysis of WT and GTKO pCECs, but GTKO/CD46 pCECs demonstrated significant resistance to lysis (p<0.01, p<0.05, respectively). Human CD4+T cell responses to GTKO and GTKO/CD46 pCEC and human CEC were significantly less than to WT pCEC (p<0.05) before and after activation. Although the human CD4+T cell response to GTKO and GTKO/CD46 pCEC was greater than to human CEC 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. The human CD4+T cell response to CIITA-DN cells was significantly lower than to human and WT cells (P<0.01).
Corneas from GTKO/CD46/CIITA-DN pigs should be significantly protected against human humoral and cellular immunity, and may be comparable to human corneas.
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