title: Identification of human preformed antibody targets in GTKO pigs.
abstract: BACKGROUND
  Human preformed antibodies continue to recognize porcine xenografts, despite the advent of α-galactosyltransferase knockout (GTKO) pigs. This study examined the potential reactivity of human preformed IgG and IgM antibodies toward antigens in the GTKO pig liver.
  
  
  METHODS
  Human serum was analyzed for the concentration of IgG, IgM, anti-αgal antibody, anti-non-αgal antibody and cytotoxicity toward domestic and GTKO fibroblasts and liver sinusoidal endothelial cells (LSEC). We detected preformed antibodies in human serum directed toward GTKO pig liver cells and tissue samples using advanced proteomic techniques. The targets of preformed antibodies were identified by MALDI TOF TOF mass spectrometry and validated by confocal microscopy, immunoblot, and immunoprecipitation.
  
  
  RESULTS
  Human serum used in this study contained 2.06 μg/ml IgG and 0.013 μg/ml IgM directed toward GTKO fibroblasts. Human IgG and IgM bound to GTKO LSEC in a dose-dependent manner and were cytotoxic. We detected 357 protein spots recognized by human IgG and 233 by human IgM. Two hundred and nineteen proteins were common to both human IgG and IgM. Mass spectrometry identified numerous immunoreactive proteins, of which 19 were membrane proteins on liver cells. The most significant to this study were α-enolase, CFTR, and E-cadherin, which were abundant in GTKO pig tissues and expressed on the surface of GTKO LSEC. Human IgG captured α-enolase, CFTR, and E-cadherin by immunoprecipitation validating the proteomic identification.
  
  
  CONCLUSIONS
  These experiments indicate that several membrane antigens in GTKO pigs could be recognized directly by human IgG or IgM. Further studies on the contribution of these antigens to antibody-mediated xenograft rejection are necessary.
authors.affiliation: Department of Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA. cburlak@iupui.edu
authors.forename: Christopher
authors.initials: C
authors.lastname: Burlak
authors.forename: Zheng-Yu
authors.initials: ZY
authors.lastname: Wang
authors.forename: Ray K
authors.initials: RK
authors.lastname: Chihara
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Lutz
authors.forename: Yueren
authors.initials: Y
authors.lastname: Wang
authors.forename: Jose L
authors.initials: JL
authors.lastname: Estrada
authors.forename: A Joseph
authors.initials: AJ
authors.lastname: Tector
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000910:Antibodies, Heterophile / Q000097:blood*; D000920:Antibody-Dependent Cell Cytotoxicity; D015478:Antigens, Heterophile; D003165:Complement System Proteins / Q000378:metabolism; D042783:Endothelial Cells / Q000276:immunology; D005700:Galactosyltransferases / Q000172:deficiency* / Q000235:genetics* / Q000276:immunology; D055786:Gene Knockout Techniques; D006084:Graft Rejection / Q000209:etiology / Q000276:immunology; D022781:Hepatocytes / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D007075:Immunoglobulin M / Q000097:blood; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D034421:Sus scrofa / Q000235:genetics* / Q000276:immunology*; D014183:Transplantation, Heterologous / Q000009:adverse effects / Q000276:immunology; D014312:Trisaccharides / Q000276:immunology
chemical_list: D000910:Antibodies, Heterophile; D015478:Antigens, Heterophile; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D014312:Trisaccharides; C076569:alpha-galactosyl epitope; D003165:Complement System Proteins; D005700:Galactosyltransferases