ABSTRACT
BACKGROUND: This study investigates anti-nonGal antibodies (Abs) in baboons after alpha1,3-galactosyltransferase gene-knockout (GalT-KO) pig heart transplantation (Tx). METHODS: Four baboons underwent pig heart Tx under chronic immunosuppression, which was discontinued after graftectomy. During follow-up, one baboon also received a pig splenocyte infusion. Hearts and splenocytes were from GalT-KO pigs (n = 3) or pigs with low Gal expression (Gal-low, n = 2), all of swine leukocyte antigen (SLA) dd haplotype. Several weeks after graftectomy, sera were tested by flow cytometry and cytotoxicity assay on porcine peripheral blood mononuclear cells (PBMC) for elicited anti-nonGal Abs. Sera were adsorbed on a Gal immunoaffinity matrix, and tested for SLA haplotype specificity using PBMC from SLA aa, cc, and dd haplotypes. RESULTS: Before heart Tx, no baboon had anti-nonGal Abs demonstrable by binding or cytotoxicity to GalT-KO PBMC. All four baboons developed anti-nonGal Abs after Tx, demonstrable by flow cytometry, and three sera from baboons showed cytotoxicity to GalT-KO PBMC of SLA(dd) haplotype. After adsorption of anti-Gal Abs, the elicited anti-nonGal Abs showed similar binding to PBMCs from pigs of all three haplotypes (SLA(dd), SLA(aa), SLA(cc)). CONCLUSIONS: Anti-nonGal Abs developed after GalT-KO pig heart Tx into baboons. The most potent of these antibodies appeared to detect antigens shared by the three pig haplotypes tested. It remains unclear whether these antibodies are directed towards shared SLA determinants or other pig antigens, and whether antibodies with specificity for allelic SLA determinants are also present, but at lower titer.
Subject(s)
Antibodies, Heterophile/blood , Galactose/immunology , Galactosyltransferases/genetics , Heart Transplantation/immunology , Transplantation, Heterologous/immunology , Alleles , Animals , Animals, Genetically Modified , Antibodies, Heterophile/immunology , Cytotoxicity Tests, Immunologic , Haplotypes , Histocompatibility Antigens/immunology , Leukocytes, Mononuclear/immunology , Papio , Swine , Swine, MiniatureABSTRACT
Hearts from alpha1,3-galactosyltransferase knockout pigs (GalT-KO, n = 8) were transplanted heterotopically into baboons using an anti-CD154 monoclonal antibody-based regimen. The elimination of the galactose-alpha1,3-galactose epitope prevented hyperacute rejection and extended survival of pig hearts in baboons for 2-6 months (median, 78 d); the predominant lesion associated with graft failure was a thrombotic microangiopathy, with resulting ischemic injury. There were no infectious complications directly related to the immunosuppressive regimen. The transplantation of hearts from GalT-KO pigs increased graft survival over previous studies.
Subject(s)
Disaccharides/immunology , Galactosyltransferases/genetics , Heart Transplantation , Transplantation, Heterologous , Animals , Animals, Genetically Modified , Disaccharides/metabolism , Fluorescent Antibody Technique , Galactosyltransferases/metabolism , Myocardium/pathology , Papio , Swine , Transplantation, Heterologous/immunologyABSTRACT
BACKGROUND: The expression of galactose alpha 1,3 galactose (Gal) in pigs has proved a barrier to xenotransplantation. Miniature swine lacking Gal (Gal pigs) have been produced by nuclear transfer/embryo transfer. METHODS: The tissues of five Gal pigs of SLA dd haplotype (SLA) were tested for the presence of Gal epitopes by staining with the Griffonia simplicifolia IB4 lectin. Their sera were tested by flow cytometry for binding of IgM and IgG to peripheral blood mononuclear cells (PBMC) from wild-type (Gal) SLA-matched pigs; serum cytotoxicity was also assessed. The cellular responses of PBMC from Gal swine toward Gal SLA-matched PBMC were tested by mixed leukocyte reaction and cell-mediated lympholysis assays. RESULTS: None of the tissues tested showed Gal expression. Sera from all five Gal pigs manifested IgM binding to Gal pig PBMC, and sera from three showed IgG binding. In all five cases, cytotoxicity to Gal cells could be demonstrated, which was lost after treatment of the sera with dithiothreitol, indicating IgM antibody-mediated cytotoxicity. PBMC from Gal swine had no proliferative or cytolytic T-cell response toward Gal SLA-matched PBMC. CONCLUSIONS: Gal pigs do not express Gal epitopes and develop anti-Gal antibodies that are cytotoxic to Gal pig cells. The absence of an in vitro cellular immune response between Gal and Gal pigs is related to their identical SLA haplotype and indicates the absence of immunogenicity of Gal in T-cell responses. The model of Gal organ transplantation into a Gal SLA-matched recipient would be a valuable large animal model in the study of accommodation or B-cell tolerance.
