Background: Human natural killer (NK) cell-mediated cytotoxicity represents a hurdle in pig-to-human xenotransplantation. It was previously reported that the expression of human major histocompatibility complex class I molecules, including HLA-B27, -Cw3, -E, and -G, partially protects porcine endothelial cells (pEC) from human NK-mediated cytotoxicity and that HLA-G inhibits NK adhesion to pEC. Here, we tested if HLA-Cw4 expression on pEC alone, or concurrently with HLA-Cw3, prevents human NK adhesion and cytotoxicity against pEC via recognition of the killer-cell immunoglobulin-like receptors (KIR) CD158a (KIR2DL1) and CD158b (KIR2DL2/3), respectively.

Methods: Two pEC lines (2A2 and PEDSV.15) were transfected with HLA-Cw3 and HLA-Cw4. HLA and KIR expression on porcine and human cells were analyzed by flow cytometry. The effect of HLA expression on pEC on human NK-mediated cytotoxicity and adhesion was tested by (51)Cr-release and dynamic adhesion assays, respectively.

Results: HLA-Cw4 expression on pEC reduced cytotoxicity mediated by CD158a(+) polyclonal human NK cells by an average of 58%, and by CD158a(bright) NK cell clones by 68%, but not by NK cells expressing low levels of CD158. Co-expression of HLA-Cw3 and HLA-Cw4 on pEC did not mediate further protection against NK cytotoxicity. The expression of HLA-Cw4 reduced the adhesion of human NK cells on pEC by a mean of 53%.

Conclusions: While transgenic expression of HLA-Cw4 on pEC reduces NK cell adhesion and cytotoxicity, co-expression with HLA-Cw3 is not sufficient to completely overcome human NK-mediated cytotoxicity in vitro.