OR4 Development of an in vitro model of human antibody mediated rejection

Aim Antibody-mediated rejection (AbMR) remains a leading barrier to long term transplant survival. The immune mechanisms of AbMR are incompletely understood but recent evidence suggests that monocytes and NK cells are potential effectors against HLA ab-coated endothelium. We developed an in vitro model of human AbMR that incorporates the immune components currently known. Methods Human umbilical vein endothelial cells (HUVECs) served as endothelial targets and were pretreated with IFN-g for 48 h to maximize HLA expression. HLA abs were derived from sera pooled from highly sensitized patients on our waitlists. Primary NK cells and monocytes were isolated by immunomagnetic depletion from healthy controls and added to HLA ab-coated HUVECs. RNA was extracted for global gene expression analyses after 8 h. Analyses focused on 3 representative sets of transcripts: IFN-g response, chemokines, and endothelial activation (Table 1). Results NK cell/ HLA ab-mediated attack leads to increased chemokines that promote additional NK cell and monocyte recuitment (Fig. 1), many reflecting endothelial IFN-g effects. Adhesion molecules key to recruitment and leukocyte engagement were also increased, some of these being required for optimal NK cell activation (ICAM1). The presence of monocytes in the system (despite being separated from NK-HUVECs) increased all 3 responses observed with NK cells, particularly endothelial activation. Conclusions Our results suggest that we have a working model of human AbMR that incorporates 2 effector cell types, HLA abs, and HUVEC targets. This model argues for a key effector role by NK cells in AbMR, whose function is greatly enhanced by soluble mediators from monocytes in close proximity.