Anti-MHC class I antibody-mediated activation of cell proliferation and survival signaling pathways in endothelial cells

Anti-HLA antibodies (Ab) have been shown to contribute to the process of transplant vasculopathy by binding to HLA class I molecules expressed by the endothelial cells (EC) of the graft and transducing intracellular signals that elicit cell proliferation. Anti-HLA Ab mediate rejection by ligation of MHC class I molecules on the surface of endothelial cells and activate cell proliferation and survival pathways. The purpose of this thesis was to elucidate the role of MHC class I molecules to elicit cell proliferation and survival signaling pathways in endothelial cells. The first aim determined the role of mTOR in class I induced EC proliferation and explored the relationship between mTOR complexes and their downstream targets following ligation of HLA class I molecules. Small interfering RNA (siRNA) knockdown of mTOR inhibited MHC class I mediated cell proliferation and phosphorylation of proteins downstream of mTORC1 and mTORC2. Long-term pretreatment with the mTOR inhibitor rapamycin significantly blocked both mTOR-Raptor and mTOR-Rictor complex formation and class I induced Akt Ser473 phosphorylation and Bcl-2 expression. These results support the role of anti-HLA Ab in the process of transplant vasculopathy and suggest that exposure of the graft endothelium to anti-HLA Ab may promote proliferation through the mTOR pathway. The second aim characterized the interaction between mTOR and ERK in EC following MHC class I and integrin ligation. As a novel finding, MHC class I or intergin ligation can phosphorylate ERK Thr202/Tyr204 through an mTORC2 dependent mechanism. Lastly, the purpose of aim three was to characterize the role of the class I signaling pathway in the pathogenesis of antibody mediated rejection (AMR) by developing a mouse vascularized heterotopic cardiac allograft model in which B6.RAG1 KO hosts (H-2Kb/D b) received a fully MHC-incompatible Balb/c (H-2Kd/D d) allograft and were passively transfused with anti-donor class I antibody. Allografts treated with anti-H-2Kd antibody lead to features of AMR including microvascular changes accompanied by C4d deposition. MHC class I induced signaling molecules were elevated in anti-class I treated mice compared to isotype controls. These results provide the first analysis of the interrelationships between these signaling molecules in vivo that reflects the signaling pathway in vitro.