R M Gorczynski1, Z Chen, X M Fu, H Zeng. 1. The Toronto Hospital, Department of Surgery and Immunology, University of Toronto, Ontario, Canada.
Abstract
BACKGROUND: Portal venous (p.v.) peritransplant immunization with dendritic cells from bone marrow cultures, along with cyclosporine (10 mg/kg), produces antigen-specific increased renal allograft survival compared with recipients receiving intravenous (i.v.) immunization. Increased survival is associated with altered cytokine production from recipient T cells restimulated with donor antigen. We used a suppressive subtractive hybridization approach to explore a role in the regulation of transplant rejection for other genes differentially expressed after p.v. immunization. METHODS: Subtractive hybridization was performed using tissue from p.v. and i.v. immunized mice and a novel polymerase chain reaction-based approach. A gene-bank search was used to identify the source of the differentially expressed cDNAs. One product, the mouse homologue of rat OX-2, was further analyzed using Western gels and FACS analysis of dendritic cells (NLDC145+) isolated from p.v.-immunized mice. RESULTS: Eighty cDNA clones were obtained by suppressive subtractive hybridization. Differential expression was confirmed in Northern RNA blots. One clone showed sequence homology to a gene encoding a molecule on rat dendritic cells (MRC OX-2), with homology to genes encoding the costimulatory molecules CD80 (B7-1) and CD86 (B7-2). In p.v.-immunized mice, a monoclonal antibody to the rat OX-2 molecule identified, by Western blot analysis, increased expression of a molecule with molecular weight (43 kDa) analogous to rat MRC-OX-2; labels (by FACS analysis) indentified increased numbers of a population of cells staining with NLDC145; and blocks indentified increased graft survival. CONCLUSION: Our data suggest that OX-2 is functionally important in the increased graft survival seen in p.v.-immunized mice receiving renal allografts.
BACKGROUND: Portal venous (p.v.) peritransplant immunization with dendritic cells from bone marrow cultures, along with cyclosporine (10 mg/kg), produces antigen-specific increased renal allograft survival compared with recipients receiving intravenous (i.v.) immunization. Increased survival is associated with altered cytokine production from recipient T cells restimulated with donor antigen. We used a suppressive subtractive hybridization approach to explore a role in the regulation of transplant rejection for other genes differentially expressed after p.v. immunization. METHODS: Subtractive hybridization was performed using tissue from p.v. and i.v. immunized mice and a novel polymerase chain reaction-based approach. A gene-bank search was used to identify the source of the differentially expressed cDNAs. One product, the mouse homologue of ratOX-2, was further analyzed using Western gels and FACS analysis of dendritic cells (NLDC145+) isolated from p.v.-immunized mice. RESULTS: Eighty cDNA clones were obtained by suppressive subtractive hybridization. Differential expression was confirmed in Northern RNA blots. One clone showed sequence homology to a gene encoding a molecule on rat dendritic cells (MRC OX-2), with homology to genes encoding the costimulatory molecules CD80 (B7-1) and CD86 (B7-2). In p.v.-immunized mice, a monoclonal antibody to the ratOX-2 molecule identified, by Western blot analysis, increased expression of a molecule with molecular weight (43 kDa) analogous to rat MRC-OX-2; labels (by FACS analysis) indentified increased numbers of a population of cells staining with NLDC145; and blocks indentified increased graft survival. CONCLUSION: Our data suggest that OX-2 is functionally important in the increased graft survival seen in p.v.-immunized mice receiving renal allografts.