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Difference in signal transduction between anti-A/B and anti-HLA antibody binding to endothelial cells

Kenta Iwasaki, Yuko Miwa, Masataka Haneda, Takaaki Kobayashi

Department of Applied Immunology, Nagoya University School of Medicine, Nagoya, Aichi, Japan

Background: As even αGal knock-out pigs could not suppress antibody-mediated rejection, much attention has been given to non-Gal antigens including both carbohydrate and protein. Accommodation, no graft injury even in the presence of anti-donor antibody, is a key factor for successful transplantation with anti-donor antibody. However the mechanisms of accommodation are still under debate. Considering that clinical outcome in ABO-incompatible renal transplantation is considerably better than that in HLA-incompatible transplantation, mechanisms of accommodation after antibody binding might also differ between carbohydrate and protein antigens. The purpose of this study is to compare signal transduction between anti-A/B and anti-HLA antibody reaction and to elucidate the mechanisms underlying accommodation.

Method: Blood type A- or B-transferase gene was transfected into human EA.hy926 endothelial cells. After cell sorting, A- or B-expressing cells at high levels were obtained.The expression of CD55/CD59 and HO-1/ferritin H were confirmed by flow-cytometry and quantitative RT-PCR, respectively. Western blots were performed using whole cell lysates with the antibodies against AKT, phosphor-AKT (Ser473), ERK, and phospho-ERK. Cell toxicity was estimated by MTT assay.

Result: Pre-incubation with low levels of anti-HLA or high levels of anti-A/B antibodies for 24 h exhibited resistance against complement-mediated cytotoxicity. Anti-A/B antibody ligation inactivated ERK1/2 pathway and increased complement regulatory proteins such as CD55 and CD59, whereas low titer of anti-HLA ligation activated PI3K/AKT pathway and increased cytoprotective genes such as HO-1 and ferritin H.

Discussion: Complement inhibition by up-regulation of CD55 and CD59 through ERK1/2 inactivation might be a substantial role in accommodation after ABO-incompatible transplantationMechanisms underlying accommodation development seemed to be different between anti-A/B and anti-HLA antibody. Since ERK and PI3K/AKT signaling pathway are widely conserved among several species, analysis of those signal transduction pathways would provide useful information on potential strategy against anti-nonGal antibody.