Antibody ligation of HLA class II induces YAP nuclear localization and formation of cytoplasmic YAP condensates in human endothelial cells

Antibody (Ab) crosslinking of HLA class II (HLA II) molecules on the surface of endothelial cells (ECs) triggers proliferative and prosurvival intracellular signaling, which are implicated in promoting chronic Ab-mediated rejection (cAMR). Despite the importance of cAMR in transplant medicine, the mechanisms involved remain incompletely understood. Here, we examined the regulation of yes-associated protein (YAP) nuclear cytoplasmic localization and phosphorylation in human ECs challenged with Abs that bind HLA II, which are strongly associated with cAMR. To examine changes in YAP localization in response to Ab-mediated engagement of HLA II, we used an adenoviral vector to express the class II transactivator or treatment with interferon γ. In unstimulated ECs expressing HLA II, YAP localized mainly in the cytoplasm. Stimulation with HLA II Ab (0.1-1 µg/mL) induced marked translocation of YAP to the nucleus. HLA II signaling triggered by high concentrations of HLA II Ab (1 µg/mL) also induced prominent YAP localization in cytoplasmic punctate structures that were disassembled by exposure to 1,6-hexanediol, suggesting that these structures are biomolecular condensates. Using multiple treatments, including stimulation with serum, thrombin or HLA I Ab and conditions (eg ECs plated at different densities) indicate that formation of YAP cytoplasmic puncta can be dissociated from YAP nuclear localization and phosphorylation at Ser127, a site in YAP targeted by the Hippo kinases LATS1/2. The results revealed that HLA II signaling regulates YAP subcellular distributions in ECs and demonstrate, for the first time, that HLA II Ab selectively stimulates YAP concentration in punctate structures.