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

Abstract

BackgroundAfter 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).MethodsW e designed a non-HLA Ab detection immunoassay (NHADIA) using HLA class I and II–deficient glomerular endothelial cells (CiGEnCΔHLA) 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.ResultsW e sequentially applied CRISPR/Cas9 to delete the B2M and CIITA genes to obtain a CiGEnCΔHLA clone. CiGEnCΔHLA 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.ConclusionThe NHADIA identifies non-HLA Abs and strongly predicts graft endothelial injury independent of HLA-DSAs.

Renal Endothelial Cytotoxicity Assay to Diagnose and Monitor Renal Transplant Recipients for Anti-Endothelial Antibodies

“…The discovery of new targets for non-HLA antibodies is an ongoing process ( 4 , 19 , 43 – 49 ). Although several non-HLA antibody specificities have been identified, the currently available solid-phase assays are limited to the known non-HLA antigens ( 23 , 50 ). Only antibodies directed against TUBB4B were found to be present in the serum of the patient described here; however, this antibody has never been described to correlate with rejection or graft failure ( 20 ).…”

“…Lamarthée et al. recently described the relevance of a tool to identify pathogenic non-HLA antibodies in kidney transplant recipients by utilizing CRISPR/Cas9-engineered HLA-silenced glomerular endothelial cells ( 50 ). Their endothelial crossmatch test identifies non-HLA Abs and strongly predicts graft endothelial injury, independent of HLA-DSAs.…”

Microvascular Inflammation of the Renal Allograft: A Reappraisal of the Underlying Mechanisms

“…The evaluation of the NHADIA in an unselected cohort of 389 kidney transplant recipients revealed that preformed non-HLA antibodies were increased in the patients who had undergone previous kidney transplantation, supporting the role of allosensitization to minor histocompatibility antigens. The pretransplant NHADIA value correlated with MVI lesions on the kidney graft at 3 months and 12 months and was associated with the risk of developing ABMRh ( 102 ).…”

“…Finally, it is still unclear whether a single non-HLA antibody can have a significant impact on graft outcome or whether a combination of several non-HLA antibodies is needed, perhaps with a set of specific antibodies for each individual, therefore requiring potential full sequencing of every donor/recipient pair ( 105 ). One approach may be to develop assays allowing us to detect the burden of non-HLA antibody binding to target cells, as we did with our NHADIA ( 102 ).…”

Novel approaches in complement profiling; application in kidney transplantation

“…Salp20 has been shown to inhibit the AP via the displacement of properdin causing an accelerated decay of the C3bBb complex and subsequent inhibition of the AP up t0 70%. 49,50 However, no experiments have been performed yet in inhibiting the AP activation on a renal cellular level.…”