Background: Endothelial cells (EC) line the inner surface of blood vessels and serve as the interface between recipient circulating blood and the donor allograft. Transplant recipients developing donor specific HLA class II (HLA-II) antibodies (Ab) are at risk of acute and chronic antibody mediated rejection (AMR) and transplant vasculopathy (TV). We postulate that HLA-II Ab contribute to the process of AMR/TV by binding to HLA-II and triggering signal transduction pathways leading to EC activation, proliferation and migration. Herein, we characterize the signaling pathways triggered by Ab ligation of HLA-II on EC and explore the use of pharmacologic kinase and siRNA inhibitors as potential therapies to interrupt pathogenic signaling pathways.

Method: HLA-II expression inhuman aortic EC was induced by adenoviral vector expression of CIITA or by pretreatment with TNFa/IFNg and confirmed by flow cytometry. Ad-CIITA infected or cytokine-treated EC were stimulated with HLA-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 HLA-II on EC stimulated a significant increase in phosphorylation of Src, FAK, PI3K, Akt, mTOR, S6K, S6RP, MEK, and ERK that was accompanied by EC proliferation and migration. Pharmacological inhibitors targeting Src, FAK, PI3K/Akt, and MEK/ERK blocked HLA-II stimulated EC proliferation and migration. PI3K inhibitors, LY294002 and A66, blocked HLA-II mediated activation of Akt, mTOR, S6K, and S6RP, but had no effect on c-Raf-1 or MEK. Pharmacologic blockade of PI3K with LY294002 mediated hyperphosphorylation of ERK. Similarly, suppression of mTORC2 enhanced HLA II-stimulated phosphorylation of ERK. Furthermore, siRNA knockdown of Rictor caused over-activation of ERK while abolishing phosphorylation of Akt Ser473. Combined treatment of EC with MEK inhibitor UO126 and rapamycin abrogated HLA-II 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 MEK/ERK pathway activity in HLA-II Ab-activated signal networks in EC. This data suggests that combined ERK and mTORC2 inhibitors will be required to achieve optimal efficacy in controlling HLA II Ab-mediated AMR.

CITATION INFORMATION: Jin Y-.P., Reed E. Dual Inhibition of PI3K and mTORC2 Causes ERK Hyperactivation Induced by HLA-II Antibody in Human Endothelium Am J Transplant. 2017;17 (suppl 3).

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