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

2021 ◽  
Vol 32 (12) ◽  
pp. 3231-3251
Author(s):  
Baptiste Lamarthée ◽  
Carole Burger ◽  
Charlotte Leclaire ◽  
Emilie Lebraud ◽  
Aniela Zablocki ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Kidney Transplant ◽  
Transplant Recipients ◽  
Kidney Transplant Recipients ◽  
Hla Antibodies ◽  
Specific Antibodies ◽  
Antibody Mediated Rejection ◽  
Biopsy Specimens ◽  
Glomerular Endothelial Cells ◽  
Donor Specific Antibodies

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.

scholarly journals Non-HLA Antibodies and Epitope Mismatches in Kidney Transplant Recipients With Histological Antibody-Mediated Rejection

2021 ◽  
Vol 12 ◽  
Author(s):  
Marta Crespo ◽  
Laura Llinàs-Mallol ◽  
Dolores Redondo-Pachón ◽  
Carrie Butler ◽  
Javier Gimeno ◽  
...  
Keyword(s):  
Kidney Transplant ◽  
De Novo ◽  
Transplant Recipients ◽  
Kidney Transplant Recipients ◽  
Serum Samples ◽  
Hla Antibodies ◽  
Aortic Endothelial Cells ◽  
Antibody Mediated Rejection ◽  
Donor Specific Antibodies

BackgroundCorrelation between antibody-mediated rejection (ABMR) and circulating HLA donor-specific antibodies (HLA-DSA) is strong but imperfect in kidney transplant (KT) recipients, raising the possibility of undetected HLA-DSA or non-HLA antibodies contributing to ABMR. Detailed evaluation of the degree of HLA matching together with the identification of non-HLA antibodies in KT may help to decipher the antibody involved.MethodsWe retrospectively assessed patients with transplant biopsies scored following Banff’15 classification. Pre- and post-transplant serum samples were checked for HLA and non-HLA antibodies [MICA-Ab, angiotensin-II type-1-receptor (AT1R)-Ab, endothelin-1 type-A-receptor (ETAR)-Ab and crossmatches with primary aortic endothelial cells (EC-XM)]. We also analyzed HLA epitope mismatches (HLA-EM) between donors and recipients to explore their role in ABMR histology (ABMRh) with and without HLA-DSA.ResultsOne-hundred eighteen patients with normal histology (n = 19), ABMRh (n = 52) or IFTA (n = 47) were studied. ABMRh patients were HLA-DSApos (n = 38, 73%) or HLA-DSAneg (n = 14, 27%). Pre-transplant HLA-DSA and AT1R-Ab were more frequent in ABMRh compared with IFTA and normal histology cases (p = 0.006 and 0.003), without differences in other non-HLA antibodies. Only three ABMRhDSAneg cases showed non-HLA antibodies. ABMRhDSAneg and ABMRhDSApos cases showed similar biopsy changes and graft-survival. Both total class II and DRB1 HLA-EM were associated with ABMRhDSApos but not with ABMRhDSAneg. Multivariate analysis showed that pre-transplant HLA-DSA (OR: 3.69 [1.31–10.37], p = 0.013) and AT1R-Ab (OR: 5.47 [1.78–16.76], p = 0.003) were independent predictors of ABMRhDSApos.ConclusionsIn conclusion, pre-transplant AT1R-Ab is frequently found in ABMRhDSApos patients. However, AT1R-Ab, MICA-Ab, ETAR-Ab or EC-XM+ are rarely found among ABMRhDSAneg patients. Pre-transplant AT1R-Ab may act synergistically with preformed or de novo HLA-DSA to produce ABMRhDSApos but not ABMRhDSAneg. HLA epitope mismatch associates with ABMRhDSApos compared with ABMRhDSAneg, suggesting factors other than HLA are responsible for the damage.

scholarly journals Transcriptional Changes in Kidney Allografts with Histology of Antibody-Mediated Rejection without Anti-HLA Donor-Specific Antibodies

2020 ◽  
Vol 31 (9) ◽  
pp. 2168-2183 ◽  
Author(s):  
Jasper Callemeyn ◽  
Evelyne Lerut ◽  
Henriette de Loor ◽  
Ingrid Arijs ◽  
Olivier Thaunat ◽  
...  
Keyword(s):  
Etiologic Factor ◽  
Transplant Recipients ◽  
Kidney Transplant Recipients ◽  
Multicenter Cohort Study ◽  
Hla Antibodies ◽  
Transcriptional Signature ◽  
Antibody Mediated Rejection ◽  
Biopsy Specimens ◽  
Transcriptional Changes ◽  
Donor Specific Antibodies

BackgroundCirculating donor-specific anti-HLA antibodies (HLA-DSAs) are often absent in serum of kidney allograft recipients whose biopsy specimens demonstrate histology of antibody-mediated rejection (ABMR). It is unclear whether cases involving ABMR histology without detectable HLA-DSAs represent a distinct clinical and molecular phenotype.MethodsIn this multicenter cohort study, we integrated allograft microarray analysis with extensive clinical and histologic phenotyping from 224 kidney transplant recipients between 2011 and 2017. We used the term ABMR histology for biopsy specimens that fulfill the first two Banff 2017 criteria for ABMR, irrespective of HLA-DSA status.ResultsOf 224 biopsy specimens, 56 had ABMR histology; 26 of these (46.4%) lacked detectable serum HLA-DSAs. Biopsy specimens with ABMR histology showed overexpression of transcripts mostly related to IFNγ-induced pathways and activation of natural killer cells and endothelial cells. HLA-DSA–positive and HLA-DSA–negative biopsy specimens with ABMR histology displayed similar upregulation of pathways and enrichment of infiltrating leukocytes. Transcriptional heterogeneity observed in biopsy specimens with ABMR histology was not associated with HLA-DSA status but was caused by concomitant T cell–mediated rejection. Compared with cases lacking ABMR histology, those with ABMR histology and HLA-DSA had higher allograft failure risk (hazard ratio [HR], 7.24; 95% confidence interval [95% CI], 3.04 to 17.20) than cases without HLA-DSA (HR, 2.33; 95% CI, 0.85 to 6.33), despite the absence of transcriptional differences.ConclusionsABMR histology corresponds to a robust intragraft transcriptional signature, irrespective of HLA-DSA status. Outcome after ABMR histology is not solely determined by the histomolecular presentation but is predicted by the underlying etiologic factor. It is important to consider this heterogeneity in further research and in treatment decisions for patients with ABMR histology.

FC 128NON-HLA ANTIBODIES AND EPLET MISMATCHES IN KIDNEY TRANSPLANT RECIPIENTS WITH A HISTOLOGICAL PICTURE OF ANTIBODY-MEDIATED REJECTION WITH AND WITHOUT HLA DONOR-SPECIFIC ANTIBODIES

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Marta Crespo ◽  
Laura Llinas ◽  
Dolores Redondo Pachon ◽  
Carrie L Butler ◽  
Javier Gimeno ◽  
...  
Keyword(s):  
Kidney Transplant ◽  
Graft Survival ◽  
Kidney Transplant Recipients ◽  
Serum Samples ◽  
Hla Antibodies ◽  
Aortic Endothelial Cells ◽  
Specific Antibodies ◽  
Antibody Mediated Rejection ◽  
Before And After ◽  
Donor Specific Antibodies

Abstract Background and Aims Correlation between antibody-mediated rejection (ABMR) and HLA donor-specific antibodies (DSA) is strong but imperfect in kidney transplant (KT) recipients, raising the possibility of other detrimental antibodies contributing to ABMR. The role of non-HLA antibodies on outcomes is not well known. Method We retrospectively assessed KT biopsies scored according to Banff’15 classification. Pre- and post-KT serum samples were checked for HLA and non-HLA antibodies (MICA-Ab, angiotensin II type 1 receptor (AT1R)-Ab, endothelin-1 type A receptor (ETAR)-Ab and crossmatches with primary aortic endothelial cells (EC-XM)). We also analyzed HLA epitope mismatches between donors and recipients. Results One-hundred eighteen patients with normal (n=19), ABMR histology (n=52) or IFTA (n=47) in their biopsy were studied. Graft survival was worse in ABMR patients (p=0.003). Pre-KT HLA-DSA were more frequent in ABMR cases (p=0.006). At biopsy, 73% ABMR patients had HLA-DSA (p&lt;0.001). Pre-KT AT1R-Ab were more frequent in ABMR compared with IFTA and normal cases (p=0.003), without differences in other non-HLA antibodies. Fourteen patients with histological ABMR (27%) had no detectable HLA-DSA post-KT and only 3 had non-HLA Ab. However, these ABMR-DSA- cases showed similar biopsy changes and graft survival compared with ABMR-DSA+. Pre- or post-KT non-HLA antibodies other than AT1R-Ab were detected similarly in ABMR and in normal or IFTA cases. Both total class II and DRB1 epitope mismatches were associated with postransplant DSA and ABMR-DSA+. Multivariate analysis showed that both pre-KT HLA-DSA and AT1R-Ab (DSA: OR: 3.39 [1.20-9.59], p=0.021; AT1R-Ab: OR: 5.31 [1.75-16.10], p=0.003) were strong independent predictors of postransplant ABMR-DSA+ (Table 1). Conclusion Despite highly prevalent HLA-DSA before and after transplantation in KT with histological ABMR, 27% of cases did not show circulating HLA-DSA. Pre-KT AT1R-Ab associated with ABMR-DSA+, but not MICA-Ab, ETAR-Ab or EC-XM+. Any of them associated significantly with ABMR-DSA-. Epitope mismatch predicted both postransplant DRB-DSA and ABMR-DSA+. Detection of pre-KT HLA-DSA and/or AT1R-Ab, together with HLA epitope mismatch assessment, are valuable tools for better DSA and ABMR prediction in KT patients.

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