title: Humoral Reactivity of Renal Transplant-Waitlisted Patients to Cells From GGTA1/CMAH/B4GalNT2, and SLA Class I Knockout Pigs.
abstract: Antipig antibodies are a barrier to clinical xenotransplantation. We evaluated antibody binding of waitlisted renal transplant patients to 3 glycan knockout (KO) pig cells and class I swine leukocyte antigens (SLA).
  
  
  
  Peripheral blood mononuclear cells from SLA identical wild type (WT), α1, 3-galactosyltransferase (GGTA1) KO, GGTA1/ cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) KO, and GGTA1/ CMAH /b1,4 N-acetylgalactosaminyl transferase (B4GalNT2) KO pigs were screened for human antibody binding using flow cytometric crossmatch (FCXM). Sera from 820 patients were screened on GGTA1/CMAH/B4GalNT2 KO cells and a subset with elevated binding was evaluated further. FCXM was performed on SLA intact cells and GGTA1/SLA class I KO cells after depletion with WT pig RBCs to remove cell surface reactive antibodies, but leave SLA antibodies. Lastly, human and pig reactive antibodies were eluted and tested for cross-species binding and reactivity to single-antigen HLA beads.
  
  
  
  Sequential glycan KO modifications significantly reduce antibody binding of waitlisted patients. Sera exhibiting elevated binding without reduction after depletion with WT RBCs demonstrate reduced binding to SLA class I KO cells. Human IgG, eluted from human and pig peripheral blood mononuclear cells, interacted across species and bound single-antigen HLA beads in common epitope-restricted patterns.
  
  
  
  Many waitlisted patients have minimal xenoreactive antibody binding to the triple KO pig, but some HLA antibodies in sensitized patients cross-react with class I SLA. SLA class I is a target for genome editing in xenotransplantation.
authors.affiliation: 1 Department of Surgery, University of Alabama at Birmingham, Birmingham, AL. 2 Department of Surgery, Indiana University, School of Medicine, Indianapolis, IN. 3 Transplant Surgery, University of Alabama at Birmingham, Birmingham, AL.
authors.forename: Gregory R
authors.initials: GR
authors.lastname: Martens
authors.forename: Luz M
authors.initials: LM
authors.lastname: Reyes
authors.forename: Ping
authors.initials: P
authors.lastname: Li
authors.forename: James R
authors.initials: JR
authors.lastname: Butler
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Ladowski
authors.forename: Jose L
authors.initials: JL
authors.lastname: Estrada
authors.forename: Richard A
authors.initials: RA
authors.lastname: Sidner
authors.forename: Devin E
authors.initials: DE
authors.lastname: Eckhoff
authors.forename: Matt
authors.initials: M
authors.lastname: Tector
authors.forename: A Joseph
authors.initials: AJ
authors.lastname: Tector
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000910:Antibodies, Heterophile / Q000097:blood*; D015478:Antigens, Heterophile / Q000235:genetics / Q000276:immunology*; D003429:Cross Reactions; D005434:Flow Cytometry; D005700:Galactosyltransferases / Q000172:deficiency / Q000235:genetics / Q000276:immunology*; D055786:Gene Knockout Techniques; D005838:Genotype; D006680:HLA Antigens / Q000276:immunology; D006648:Histocompatibility; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II / Q000235:genetics / Q000276:immunology*; D006650:Histocompatibility Testing / Q000379:methods; D006801:Humans; D056724:Immunity, Humoral; D007074:Immunoglobulin G / Q000097:blood*; D016030:Kidney Transplantation; D006899:Mixed Function Oxygenases / Q000172:deficiency / Q000235:genetics / Q000276:immunology*; D017350:N-Acetylgalactosaminyltransferases / Q000172:deficiency / Q000235:genetics / Q000276:immunology*; D010641:Phenotype; D011485:Protein Binding; D013552:Swine; D014183:Transplantation, Heterologous; D014850:Waiting Lists
chemical_list: D000910:Antibodies, Heterophile; D015478:Antigens, Heterophile; D006680:HLA Antigens; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D007074:Immunoglobulin G; C438668:swine leukocyte antigen; D006899:Mixed Function Oxygenases; C074383:CMPacetylneuraminate monooxygenase; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C530267:alpha-1,3-galactosyltransferase 1, porcine; C071413:beta-1,4-N-acetyl-galactosaminyl transferase 2