CRISPR/Cas9-Engineered HLA-Deleted Glomerular Endothelial Cells as a Tool to Predict Pathogenic Non-HLA Antibodies in Kidney Transplant Recipients
Authors: Baptiste Lamarthée, Carole Burger, Charlotte Leclaire, Emilie Lebraud, Aniela Zablocki, Lise Morin, Xavier Lebreton, Béatrice Charreau, Renaud Snanoudj, Soëli Charbonnier, Tifanie Blein, Mélanie Hardy, Julien Zuber, Simon Satchell, Morgan Gallazzini, Fabiola Terzi, Christophe Legendre, Jean Luc Taupin, Marion Rabant, Claire Tinel, Dany Anglicheau
Journal: Journal of the American Society of Nephrology (2021)

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 Δ 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 Δ HLA clone. CiGEnC Δ 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 &lt;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.