Date: Sunday, June 2, 2019
Session Time: 4:30pm-6:00pm
Presentation Time: 5:06pm-5:18pm
Location: Room 309
*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.
To cite this abstract in AMA style:Jin Y, Sinnet-Smith J, Rozengurt E, Reed EF. Inhibition of mTORC2 Causes Hyper Phosphorylation of ERK Induced by Co-Ligation of HLA-I and II Antibody [abstract]. Am J Transplant. 2019; 19 (suppl 3). https://atcmeetingabstracts.com/abstract/inhibition-of-mtorc2-causes-hyper-phosphorylation-of-erk-induced-by-co-ligation-of-hla-i-and-ii-antibody/. Accessed June 23, 2021.
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