In animal-based transplantation research, the measurement of anti-donor antibodies in transplant recipients is limited by lack of an appropriate technique. We have developed a novel immunoassay capable of quantifying antibody bound to cell-surface major histo- compatability complex (MHC) and non-MHC antigens, using splenocytes from wild-type and MHC-deficient mice as antigen-bearing target cells. We utilized our "cellular ELISA" (CELISA) technique to study the development of tolerance versus immunity in the B-cell compartment in response to neonatal exposure to allogeneic fetal liver cells (FLC). This neonatal tolerance protocol typically induces permanent acceptance of donor-type and third-party cardiac allografts, but rejection of both donor-type and third-party skin grafts occurs. C3H/He (C3H; H-2(k)) mice were injected as neonates with BALB/c (BALB; H-2(d)) FLC and transplanted as adults with C57BL/6 (B6; H-2(b)) cardiac grafts. Despite long-term acceptance of third-party B6 cardiac grafts, serum contained increased anti-B6 IgG and IgM levels as measured by CELISA; IgM production was elevated by 2 weeks posttransplant and remained stable, while IgG production increased rapidly between 2 and 5 weeks posttransplant. In another experimental setting, CELISA assays were able to detect that neonatal injection of C3H mice with FLC from wild-type B6 mice or from MHC class II-deficient or class I/II-deficient (B6 background) mice (CI(+)CII(+), CI(+)CII(-), CI(-)CII(-), respectively) prevented sensitization to B6 antigens by subsequent skin transplants but did not induce graft acceptance, whereas FLC from MHC class I-deficient-only (CI(-)CII(+)) did not prevent B6 sensitization. The CELISA technique is a simple and sensitive means for quantifying alloantibodies in mice and will assist in further delineating the role of the B-cell compartment in neonatally induced cardiac allograft acceptance.

Animals↗

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Induction of human histo-blood group A antigen expression in mouse cells by gene therapy using lentiviral vectors harbouring human ABH-related glycosyltransferase genes.

We recently discovered that ABO incompatibility, which evokes a rapid humoral immune response in adult heart transplantation, is not a barrier in infant heart transplantation, and that infant recipients of ABO-incompatible hearts develop specific B-cell tolerance to donor A/B antigens. An animal model of ABO-incompatible heart transplantation would allow detailed investigation of the mechanism(s) of acceptance of ABO-incompatible grafts, using experimental methods that would not be possible in humans. To determine the feasibility of such a model, the human alpha-1,2-fucosyltransferase (H-transferase; for H antigen expression) gene was cloned into a lentiviral vector, and the human alpha-1,3-N-acetylgalactosaminyltransferase (A-transferase; for A antigen expression) gene was cloned into a bicistronic lentiviral vector also containing the green fluorescent protein (GFP) gene; these replication-deficient vectors were denoted H-trs and A-GFP, respectively. Synthesis of the human histo-blood group A antigen in humans is dependent on expression of these two glycosyltransferases. HeLa cells, a human cell line known to be of blood group O origin, expressed cell surface A antigen as measured by cellular ELISA when transfected with A-GFP alone, and the level of A antigen expression was enhanced by transfection with H-trs in addition to A-GFP. Cell surface H antigen expression was observed on both mouse fibroblast and mouse endothelial cells only when infected with H-trs lentiviral particles. Expression of A antigen was dependent on infection with both H-trs and A-GFP lentiviral particles, approaching levels on human group A cells. These collective results indicate that expression of human histo-blood group A antigen at the cell surface can be induced in mouse cells by infection with H-trs and A-GFP, and that such A antigen expression is dependent on H-trs expression.

ABO Blood-Group System↗