*Purpose: Transplant recipients developing donor specific 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 post transplant. We previously reported that ligation of HLA-I or II on endothelial cells (EC) with antibody (Ab) triggered activation of intracellular signal networks leading to EC activation and proliferation. However, the impact of combined Ab ligation of HLA-I and II 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 on primary human aortic EC was induced by adenoviral vector encoding class II transactivator (Ad-CIITA) or pretreatment with TNFα/IFNγ and confirmed by flow cytometry. Ad-CIITA infected or cytokine-treated EC were stimulated with HLA-I and II Ab and protein phosphorylation was detected by Western Blot and EC proliferation measured by BrdU incorporation.

*Results: Co-ligation of HLA-I and II on EC stimulated a significant increase in phosphorylation of PI3K, Akt, mTOR, S6K, S6RP, and ERK that was accompanied by EC proliferation. Pharmacological blockade of PI3K/mTOR with LY294002, but not with A66, a selective inhibitor of the p110α of PI3K, caused ERK hyper-phosphorylation. Accordingly, suppression of mTORC2 enhanced combined Ab-stimulated activation of ERK. Interestingly, inhibition of Akt with allosteric (MK-2206) or active site (GDC-0068) inhibitors reduced phosphorylation of c-Raf-1 Ser259. Importantly, the Akt allosteric inhibitor MK, but not GDC, caused over activation of ERK, suggesting that combined HLA-I and II Ab-mediated over activation of ERK is predominantly Akt-dependent. 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: Inhibition of the PI3K/Akt/mTORC2 signaling axis suppresses a novel negative mTORC2/Akt-dependent feedback loop, leading to enhanced MEK/ERK pathway activity following combined HLA-I/II Ab-activated signal networks in EC. This mechanism is different from the HLA-II antibody-mediated feedback loop, which is predominantly Akt-independent. Our data suggest 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.

« Back to 2019 American Transplant Congress