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

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 (CiGEnCHLA) that had been previously generated through CRISPR/Cas9-induced and 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 and genes to obtain a CiGEnCHLA clone. CiGEnCHLA 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 (<0.001). Stratification of 3-month allograft biopsy specimens (=298) according to pretransplantation NHADIA tertiles demonstrated that higher levels of non-HLA Abs positively correlated with increased glomerulitis (=0.002), microvascular inflammation (=0.003), and ABMRh (=0.03). A pretransplantation NHADIA threshold of 1.87 strongly discriminated the KTRs with the highest risk of ABMRh (=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.