Analysis of PD-L1 on Kidney Vascular Endothelial Cells

Background: Programmed death ligand 1 (PD-L1), expressed on various types of cells including vascular endothelia, inhibits T cell activity via PD-1. Anti- PD-L1/ PD-1 antibodies block this interaction and enhance T cell cytotoxicity, which has been utilized as cancer immunotherapy. However, Anti-PD-L1/PD-1 antibodies sometimes induce interstitial nephritis with unclear etiology. This study examines PD-L1 expression on kidney endothelial cells (ECs) and its effects on T cells in mice. Methods: Female C57BL/6j mice were used. Kidney and lung cells were isolated by collagenase treatment and density gradient centrifugation, then analyzed by flow cytometry (FCM). Glomerular cells were separated using a mesh and collagenase treatment. Specific cell populations were separated using CD45 and CD31 microbeads. Mice received intraperitoneal injections of LPS, anti- PD-L1, or anti-CD3ε antibodies. Results: FCM analysis in healthy mice showed PD-L1 expression in ECs (CD31+CD45.2-) of both kidneys and lungs, with higher expression in kidneys. These cells also expressed other EC markers like ICAM-1 and VEGFR2. Immunofluorescence staining (IF) revealed PD-L1 co-staining in CD31+ glomerular ECs, peritubular capillary ECs, and interlobular artery ECs. Glomerular ECs had lower PD-L1 expression compared to peritubular capillary and arterial ECs. Co-culturing renal ECs with splenic cytotoxic T cells (CD8α+) induced IFN-γ secretion, significantly increased by anti-PD-L1 antibody. LPS administration increased PD-L1 expression in ECs of both kidneys and lungs. IF showed enhanced PD-L1 in glomerular and peritubular capillary ECs in kidneys with LPS injection. Anti-CD3ε antibody, inducing high IFN-γ levels, also increased PD-L1 expression in renal ECs, as shown by FCM. Blocking PD-L1 on ECs with anti-PD-L1 antibody before anti-CD3ε treatment led to increased leukocyte infiltration into the renal interstitium and elevated blood urea nitrogen levels. Conclusion: In healthy mice, PD-L1 is primarily expressed in peritubular capillaries of the kidney, and its expression increases during inflammation. PD-L1 on kidney ECs may inhibit T cell immune activation, suggesting a protective role for the kidneys.