(719) - Autoantibodies (Non-MHC Antibodies) in a Mouse Model of Chronic Rejection: Profiling By Antigen Microarrays

Abstract

Purpose: Several antibodies against non-MHC antigens have been linked to rejection and cardiac allograft vasculopathy. Our group previously identified several new non-HLA antibodies in patients with antibody-mediated rejec- tion with antigen microarrays technique. We hypothesized that non-MHC antibodies would increase in a mouse model of chronic rejection. Methods: As an experimental model of chronic rejection, C57BL/6 mice received a heterotopic heart transplant from Bm12 mice, which differ by only one MHC gene locus. As a control group, C57BL/6 mice received a syngeneic heart transplant. Serum samples from recipients were withdrawn pre and post-transplantation for analysis with antigen microarrays. For our custom antigen microarrays, 108 antigens were spotted onto nitrocellulose slides. After blocking the slides, they were probed with diluted mouse serum and then probed with fluorescently labeled secondary antibodies. Fluorescent reactivity was quantified with a scanner and significance analysis of micro- arrays was used to detect significant changes in reactivity between groups. Detection of anti-MHC antibodies was performed using flow cytometry. Results: In allogeneic transplant group, 40 non-MHC antibodies showed a signifi- cant increase in reactivity in post versus pre-transplant serum. In syngeneic trans- plant group, 8 non-MHC antibodies showed a significant decrease in reactivity in post versus pre-transplant serum. When comparing allogeneic with syngeneic post-transplant serum, 14 antigens, including nuclear and endothelial cell antigens, showed significantly increased reactivity in allogeneic group (figure 1). Flow cytometry failed to detect anti-MHC antibodies in either group. Conclusion: In this study, we found that multiple non-MHC antibodies but not anti-MHC antibodies correlated with rejection in this mouse transplant model. The appearance of these non-MHC antibodies may serve as a marker for rejection.