Newcastle University

Title:The Role of HLA Class II Antibody in Endothelial Cell Activation and Allograft Rejection

Gabriel Cantanhede, MD MSc¹, Vaughan Carter, PhD², Keith Giles, RegSci², John Kirby, PhD¹and Simi Ali, PhD¹.

¹Institute of Cellular Medicine, University of Newcastle, Newcastle, Tyne and Wear, United Kingdom

²Histocompatibility and Immunogenetics, National Health Service Blood and Transplant, Newcastle, Tyne and Wear, United Kingdom.

Background: Antibody-mediated rejection is one of the major causes of chronic rejection. This is mediated by endothelial cell activation, a process of endothelium microvascular inflammation and leukocyte recruitment. Despite evidence pointing to the relevance of HLA class II antibody in graft rejection, the role of antibody-endothelium interaction in the absence of complement is not fully understood. This project seeks to identify signalling cascades activated by HLA class II antibodies and mechanisms mediating endothelial cell – leukocyte interaction.

Methodology:Stably-transfected Ea.hy926 class II transactivator and HMEC-1 cells were stimulated with HB145, a pan-HLA class II mouse monoclonal antibody. Cell signalling pathways were examined using a R&D Human phosphokinase array, Western Blot and RT-qPCR. The expression of ICAM-1 and VCAM-1 was determined using flow cytometry. Wound healing assays were performed using μ-slide cell culture inserts. Eahy.926 CIITA and HMEC-1 cells were genotyped for HLA class I and II epitope expression using PCR-SSP. HB145 was characterised for class I and class II by Luminex.

Results:Interpreted HLA Types were Ea.hy926 CIITA DR11(5), 13(6); DQ6(1),7(3) and HMEC-1 DR12(5), 18(3); DQ4, 5(1). HB 145 was found a comprehensive pan-class II antibody.Eahy.926 cells stimulated with HB145 showed time-dependent phosphorylation of p-ERK, p-AKT, p-mTOR, and p-AMPK α1,α2. Phosphorylation of p-mTOR was abrogated by Rapamycin at 20nM. Stimulated Ea.hy 926 CIITA upregulated the expression of mRNA encoding Cyclin D1 (p<0.001) after 24h of treatment, while levels of c-myc remained unchanged. In HMEC-1 cells, HB145 induced a 3-fold expression of ICAM-1 after 72h of treatment (p<0.001). In Eahy.926 CIITA cells, HB145 treatment alone increased the overall wound closure rate, reaching faster wound closure (11h vs. 14h; p<0.005) when compared to W6/32.

Conclusions:Exposure of endothelial cells to HLA class II antibody induces an activation of signalling cascades characteristic of endothelial cell activation. This activation is further responsible for upregulation of adhesion molecules, which facilitate the interaction of donor endothelium and recipient leukocytes. Strategies to block endothelium-leukocyte interaction might reduce the incidence of allograft rejection and improve allograft survival.