title: Modulation of the leader peptide sequence of the HLA-E gene up-regulates its expression and down-regulates natural killer cell-mediated swine endothelial cell lysis.
abstract: BACKGROUND
  The inhibitory function of HLA class I molecules, HLA-G1 and HLA-E, on natural killer (NK) cell-mediated cytolysis has previously been reported. In this study, we report on a study of the effects of the co-expression of these molecules on the inhibition of NK cell-mediated cytolysis, using a newly constructed gene.
  
  
  METHODS
  Complementary DNA (cDNA) of HLA-G (G1 and G3), HLA-E, and human beta2-microglobulin (hbeta2m) were prepared and transfected into swine endothelial cell (SEC) and Chinese hamster ovarian tumor (CHO) cell. The leader peptide sequences of HLA-G1 and HLA -E genes were changed to VMAPRTLFL or VMAPRTLVL, which corresponds to the original HLA-G1 and HLA-A2. The cell surface expression of the modified genes was evaluated by flow cytometry, and NK cell-mediated cytolysis by human peripheral blood mononuclear cells (PBMC) was assessed.
  
  
  RESULTS
  The transfectant with the hbeta2m and HLA-G1 genes showed a clear expression of the HLA-G1 molecule and had an inhibitory effect on NK cell-mediated SEC lysis. Whereas neither the transfectant with the hbeta2m and HLA-E genes, nor that with the hbeta2m and HLA-G3 genes, expressed the HLA molecule on SEC, the transfectant with triple genes, hbeta2m, HLA-E, and HLA-G3, expressed the HLA-E molecule and also inhibited NK-mediated SEC lysis. Conversely, the modification of the leader sequence of the HLA-E gene successfully induced the expression of the HLA-E molecule on the SEC surface. Furthermore, the transfectant expressed both HLA-G1 and HLA-E molecules, thus efficiently enhancing the inhibition of NK-mediated SEC lysis.
  
  
  CONCLUSIONS
  The co-expression of HLA-G1 and HLA-E molecules with the modified genes has potential for use in preventing xenograft rejection, as mediated by human NK cells.
authors.affiliation: Division of Organ Transplantation, Department of Regenerative Medicine, Osaka University Graduate School of Medicine, Japan.
authors.forename: Katsuyoshi
authors.initials: K
authors.lastname: Matsunami
authors.forename: Shuji
authors.initials: S
authors.lastname: Miyagawa
authors.forename: Rie
authors.initials: R
authors.lastname: Nakai
authors.forename: Mako
authors.initials: M
authors.lastname: Yamada
authors.forename: Ryota
authors.initials: R
authors.lastname: Shirakura
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003602:Cytotoxicity, Immunologic / Q000276:immunology; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000276:immunology; D006680:HLA Antigens / Q000235:genetics*; D015395:Histocompatibility Antigens Class I / Q000235:genetics*; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology*; D010051:Ovarian Neoplasms; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals / Q000235:genetics*; D011994:Recombinant Proteins / Q000276:immunology; D013552:Swine; D014162:Transfection; D001613:beta 2-Microglobulin / Q000235:genetics; D000097625:HLA-E Antigens
chemical_list: D000911:Antibodies, Monoclonal; D017931:DNA Primers; D006680:HLA Antigens; D015395:Histocompatibility Antigens Class I; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D001613:beta 2-Microglobulin; D000097625:HLA-E Antigens