*Purpose: In kidney transplantation recipients (KTRs), donor-specific antibodies directed against Human Leucocyte Antigens (HLA-DSA) are thought to drive antibody-mediated rejection (AMR) and poor transplant outcome. However, evidence of microvascular inflammation, the histological hallmark of AMR, is increasingly reported in patients without HLA-DSA, strongly supporting the implication of non-HLA antibodies (Abs). In KTRs displaying a severe vascular rejection in the absence of HLA-DSA, we previously showed that their pretransplant sera (D0) contain IgG Abs specifically targeting conditionally immortalized human glomerular endothelial cells (CiGEnC).

*Methods: As a human cell, the CiGEnC cells constitutively express HLA class I molecules and class II after activation. In order to develop a cell-based assay allowing the detection of non-HLA Abs reactivity to human glomerular endothelial cells even in patients with circulating HLA-DSA, we used the CRISPR-Cas9 technology to delete B2M and CIITA genes in CiGEnC cells to suppress the expression of class I and II HLA antigens, respectively. Phenotyping of the produced cells was assessed by flow cytometry, qPCR and confocal microscopy. The produced CiGEnCΔHLA clone was then used as target cell in a new cell-based assay to assess the presence of preformed non-HLA Abs in the sera of a large cohort of 389 consecutive KTRs.

*Results: A two-step process of gene-editing and selection of a CiGEnCΔHLA clone was performed. The engineered CiGEnCΔHLA clone remained undistinguishable from the parent cell line in terms of morphology and expression of various endothelial markers. The CiGEnCΔHLA clone was then used to assess the presence of preformed non-HLA Abs in pretransplant serum samples of 389 consecutive KTRs. Multivariate regression analysis revealed that pretransplant serum reactivity associated to retransplantation status (P<0.001). Addressing the prognosis significance of our cell-based assay, we demonstrated that the pretransplant serum reactivity associated with microvascular inflammation at 3 months and 12 months post-transplantation, independently of HLA-DSAs. In a time-to-event analysis, the pretransplant serum reactivity to CiGEnCΔHLA cells also associated with occurrence of histological lesions suggestive of AMR (i.e. microvascular inflammation >2 or C4d positivity).

*Conclusions: We have developed an engineered human glomerular endothelial cell line lacking HLA antigen that allowed us to identify preformed non-HLA Abs with significant consequences on allograft outcome. These results support the link between the presence of non-HLA Abs and endothelial allograft injury.

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