Characteristics of human leukocyte antigen-E expression in transgenic porcine liver

Overexpression of human leukocyte antigen-E (HLA-E) in genetically modified pigs is a potential strategy to reduce immune responses generated by human natural killer (NK) cells after solid organ transplantation. Therefore, evaluating the effects of in vivo HLA-E expression is essential to clarify emerging issues of transgene efficiency in pig organs. This study aims to investigate the expression of HLA-E protein in the liver of transgenic pigs with reference to the hepatic lobular architecture and to determine whether the addition of the two protective transgenes (hFUT2xhGLA) in pig cells affects the HLA-E expression pattern in triple transgenic liver. Here, we used confocal immunofluorescence and Western blotting to study the distribution and quantification of HLA-E protein in the liver of transgenic pigs carrying only the HLA-E transgene and from triple transgenic pigs (hFUT2xhGLAxHLA-L) designed to express HLA-E and human alpha 1,2-fucosyltransferase (hFUT2) and alpha-galactosidase A (hGLA). In all transgenic animals the HLA-E protein was evenly distributed in hepatic lobules with no evidence of zonation pattern or gradient formation, and almost absent in interlobular connective tissue septa. Quantification of immunofluorescence and Western blot revealed a significantly higher expression of HLA-E protein in the liver of transgenic pigs compared to non-genetically modified pigs. However, no significant differences were observed in the level of HLA-E protein between the single and triple transgenic animals. This study demonstrates that expression of the HLA-E transgene in porcine liver is stable, not affected by hFUT2xhGL1 genetic background and likely independent of mechanisms maintaining hepatic zonation of the liver lobules.