Discovery of non-HLA antibodies associated with cardiac allograft rejection and development and validation of a non-HLA antigen multiplex panel: From bench to bedside.

Abstract

We analyzed humoral immune responses to nonhuman leukocyte antigen (HLA) after cardiac transplantation to identify antibodies associated with allograft rejection. Protein microarray identified 366 non-HLA antibodies (>1.5 fold, P<.5) from a discovery cohort of HLA antibody-negative, endothelial cell crossmatch-positive sera obtained from 12 cardiac allograft recipients at the time of biopsy-proven rejection. From these, 19 plasma membrane proteins and 10 autoantigens identified from gene ontology analysis were combined with 48 proteins identified through literature search to generate a multiplex bead array. Longitudinal sera from a multicenter cohort of adult cardiac allograft recipients (samples: n=477 no rejection; n=69 rejection) identified 18 non-HLA antibodies associated with rejection (P<.1) including 4 newly identified non-HLA antigenic targets (DEXI, EMCN, LPHN1, and SSB). CART analysis showed 5/18 non-HLA antibodies distinguished rejection vs nonrejection. Antibodies to 4/18 non-HLA antigens synergize with HLA donor-specific antibodies and significantly increase the odds of rejection (P<.1). The non-HLA panel was validated using an independent adult cardiac transplant cohort (n=21 no rejection; n=42 rejection, >1R) with an area under the curve of 0.87 (P<.05) with 92.86% sensitivity and 66.67% specificity. We conclude that multiplex bead array assessment of non-HLA antibodies identifies cardiac transplant recipients at risk of rejection.