Baptiste Lamarthée1, Carole Burger1, Charlotte Leclaire1, Emilie Lebraud1, Aniela Zablocki1, Lise Morin1, Xavier Lebreton2, Béatrice Charreau3, Renaud Snanoudj4, Soëli Charbonnier5, Tifanie Blein5, Mélanie Hardy6, Julien Zuber2,5, Simon Satchell7, Morgan Gallazzini1, Fabiola Terzi1, Christophe Legendre2, Jean Luc Taupin6, Marion Rabant1,8, Claire Tinel1, Dany Anglicheau9,2. 1. Necker-Enfants Malades Institute, Institut National de la Santé et de la Recherche Médicale (INSERM) U1151, University of Paris, Paris, France. 2. Department of Nephrology and Kidney Transplantation, Necker Hospital, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris, France. 3. Center for Research in Transplantation and Immunology, INSERM UMR1064, IHU CESTI, LabEx IGO and LabEx Transplantex, Nantes University, Nantes, France. 4. Immunology and Histocompatibility Laboratory, Saint-Louis Hospital, AP-HP, LabEx Transplantex, INSERM U1160, University Paris Diderot, Paris, France. 5. Laboratory of Human Lymphohematopoiesis, Imagine Institute, INSERM U1163, University of Paris, Paris, France. 6. Immunology and Histocompatibility Laboratory, Saint-Louis Hospital, AP-HP, INSERM U976, IRSL, University of Paris, Paris, France. 7. Bristol Renal, Bristol Heart Institute, Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom. 8. Department of Pathology, Necker Hospital, AP-HP, Paris, France. 9. Necker-Enfants Malades Institute, Institut National de la Santé et de la Recherche Médicale (INSERM) U1151, University of Paris, Paris, France dany.anglicheau@aphp.fr.
Abstract
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 (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. RESULTS: W 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. CONCLUSION: The NHADIA identifies non-HLA Abs and strongly predicts graft endothelial injury independent of HLA-DSAs.
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 (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. RESULTS: W 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. CONCLUSION: The NHADIA identifies non-HLA Abs and strongly predicts graft endothelial injury independent of HLA-DSAs.
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