CRISPR/Cas9-Engineered HLA-Deleted Glomerular Endothelial Cells as a Tool to Predict Pathogenic Non-HLA Antibodies in Kidney Transplant Recipients

Significance Statement Evidence for the deleterious role of non-HLA antibodies after kidney transplantation is growing. Still, given the high heterogeneity and the number of potential targets, a candidate-based strategy to detect these antibodies can be misleading. We introduce a cell-based assay using human glomerular endothelial cells deleted for all HLA antigens as targets and recapitulate a large array of potential non-HLA antibodies in a single test. This approach confirms that the global burden of non-HLA antibodies targeting the endothelium is associated with microvascular inflammation and worse graft outcome, independent of HLA donor-specific antibodies. Our results demonstrate the clinical utility of the test for improving the pretransplant evaluation of immunologic risk and for designing mechanism-driven therapeutic approaches targeting non-HLA antibodies. Background After kidney transplantation, donor-specific antibodies against human leukocyte antigen donor-specific antibodies (HLA-DSAs) drive antibody-mediated rejection (ABMR) and are associated with poor transplant outcomes. However, ABMR histology (ABMRh) is increasingly reported in kidney transplant recipients (KTRs) without HLA-DSAs, highlighting the emerging role of non-HLA antibodies (Abs). Methods W e designed a non-HLA Ab detection immunoassay (NHADIA) using HLA class I and II?deficient glomerular endothelial cells (CiGEnC?HLA) that had been previously generated through CRISPR/Cas9-induced B2M and CIITA gene disruption. Flow cytometry assessed the reactivity to non-HLA antigens of pretransplantation serum samples from 389 consecutive KTRs. The intensity of the signal observed with the NHADIA was associated with post-transplant graft histology assessed in 951 adequate biopsy specimens. Results W e sequentially applied CRISPR/Cas9 to delete the B2M and CIITA genes to obtain a CiGEnC?HLA clone. CiGEnC?HLA cells remained indistinguishable from the parental cell line, CiGEnC, in terms of morphology and phenotype. Previous transplantation was the main determinant of the pretransplantation NHADIA result (P<0.001). Stratification of 3-month allograft biopsy specimens (n=298) according to pretransplantation NHADIA tertiles demonstrated that higher levels of non-HLA Abs positively correlated with increased glomerulitis (P=0.002), microvascular inflammation (P=0.003), and ABMRh (P=0.03). A pretransplantation NHADIA threshold of 1.87 strongly discriminated the KTRs with the highest risk of ABMRh (P=0.005, log-rank test). A multivariate Cox model confirmed that NHADIA status and HLA-DSAs were independent, yet synergistic, predictors of ABMRh. Conclusion The NHADIA identifies non-HLA Abs and strongly predicts graft endothelial injury independent of HLA-DSAs.