OR18. Composite impact of co-ligation of HLA-I and HLA-II antibody-mediated activation of human aortic endothelial cells

Aim Solid organ transplant recipients developing donor specific HLA antibodies (DSA) are at risk for antibody-mediated rejection (AMR). It has been reported that as many as 30% of transplanted recipients develop HLA class I (HLA-I) and/or class II DSA during the first year posttransplant. We previously reported that ligation of HLA I or HLA II on the surface of endothelial cells (EC) with antibody (Ab) triggered intracellular signal networks leading to EC proliferation. However, the impact of combined HLA-I and II Ab ligation on endothelial function remains unknown. We hypothesized that combined HLA-I and II ligation cooperate to potentiate EC activation and proliferation signal networks. Methods HLA-II expression in human aortic EC was induced by adenoviral vector expression of CIITA or by pretreatment with TNF α /IFN γ and confirmed by flow cytometry. Ad-CIITA infected or cytokine-treated EC were stimulated with combined HLA-I and II Ab, protein expression and phosphorylation were detected by Western Blot, EC proliferation was measured by BrdU incorporation and analyzed by flow cytometry. Results Ligation of both HLA-I and II on EC with Ab stimulated a significant increase in phosphorylation of Src, FAK, PI3K, Akt, mTOR, S6K, S6RP, and ERK that was accompanied by EC proliferation. Pharmacological inhibitors targeting Src, PI3K/Akt, and MEK/ERK blocked HLA-II stimulated EC proliferation. Inhibition of PI3K with LY294002 caused ERK overactivation, but no effects on Src, FAK, nor c-Raf. Similarly, suppression of mTORC2 enhanced combined Ab-stimulated ERK activation. Interestingly, 24 h treatment with rapamycin caused hyperphosphorylation of Akt at Thr308, which is different from either HLA-I or HLA-II Ab-mediated signal events. Furthermore, combined treatment of EC with MEK inhibitor UO126 and rapamycin abrogated combined Ab-stimulated activation of Akt and ERK, and further blocked EC proliferation. Conclusions Treatment with dual PI3K/mTOR inhibitors suppresses a novel negative feedback loop mediated by mTORC2, leading to enhanced PI3K/Akt and MEK/ERK pathway activity in combined HLA-I and II Ab-activated signal networks in EC. This data suggests that combined ERK and dual PI3K/mTORC2 inhibitors will be required to achieve optimal efficacy in controlling combined HLA I and II Ab-mediated AMR.