The Study Of PD-L1 Expression In Lupus Nephritis And Its Gene Transfection

Objective: Lupus nephritis(LN) is the results of overactivation of autoreactivate T and B cells, characterized by various autoantibody production. T cell require two distinct signals for optimal T cell activation.First signal is delivered to T cells through the interaction of the T cell receptor (TCR) with its specific peptide bound to major histocompatibility complex .Second signal is provided by co-signaling expressed on antigen presenting cells (APC). T cell activation is affected by both stimulatory and inhibitory co-signaling. PD-L1(programmed death ligand-1) is one of the ligands of a novel immunoinhibitory molecule PD-1 (programmed death-1) that is inducibly expressed on activated T, B and myeloid cells. PD-L1-PD-1 have the immunoinhibitory function on effective T cell activation . Inducible costimulator (ICOS) -B7 homologous protein (B7h, ICOSL) is a new member of the CD28-B7 family of costimulatory molecules that regulates T cell-dependent humoral immune responses. ICOS-ICOSL have the immunostimulatory function on effective T cell activation. Blockade of ICOS-ICOSL costimulatory pathway inhibits the lupus nephritis in NZB/W F1 mice but not all. BXSB mice spontaneously develop an autoimmune syndrome closely resembling lupus nephritis in humans.We want to first examine the expression of PD-L1 on diseased and normal human LN kidney samples as well as in human renal tubular epithelial cells (RTEC ) line HK-2 cells. Second, construct a recombinant adenovirus that expresses PD-L1 for gene therapy in vivo. Last,we test the potential protective effects of PD-L1 gene and ICOSLmAb delivery to BXSB mice on the development of LN. Methods and results: MHC class II-expressing renal tubular epithelial cells can function as APC for T cells. To study the influence of inhibitory ligands on TEC-mediated T cell activation, we fist examined the expression of PD-L1 on diseased and normal human LN kidney samples as well as in RTEC line HK-2 cells. In situ hybridization and immunohistochemical staining revealed constitutive low expression of PD-L1 on proximal tubules at both mRNA and protein levels in normal kidneys, but much higher expression in kidneys with type IV lupus nephritis.The PD-L1 expression had a positive corelation with PD-1 expression and a negative corelation with the tubulo interstitial injury. RT-PCR, FACS and immunocytochemistry showed that PD-L1 is constitutively expressed on HK-2 cells, and is dramatically upregulated by IFN-γ. In vitro, pretreatment of IFN-γ-stimulated HK-2 cells with anti-PD-L1 significantly enhanced IL-2 secretion from cocultured mitogen-activated Jurkat cells. These results suggest that the PD-L1-PD-1 pathway negatively regulates T cell activation by TEC, and may play an inhibitory role in TEC-mediated immune activation and immunopathology in the kidney. Next,we construct a recombinant adenovirus that expresses PD-L1 for gene therapy in vivo.Full-length mouse PD-L1 cDNA linked with an IRES(internal ribosome entry site)-EGFP cassette was subcloned into pAdTrack-CMV shuttle plasmid. The product was linearized to mediate homologous recombination with AdEasy-1 vector in BJ5183 host bacteria. The positive clone was identified by restriction endonuclease digestion and further confirmed by sequencing. The recombined adenovirus DNA was transfected into 293 cells for packaging and amplification of AdPD-L1 virus. The expression of PD-L1 is monitored by EGFP fluorescence in infected cells. After transfection with adenovirus DNA, infectious virus was only produced to cause cytopathic effect in the permissive cell line 293 but not in the non-permissive cell line HeLa, confirming only replication-defective but not wild type virus was generated. The specific expression of mouse PD-L1 was verified by PCR and Western-blot in 293 cell after infection with AdPD-L1, but not Ad.EGFP, a similarly constructed control virus. AdPD-L1, but not AdEGFP, significantly inhibited mouse mixed leukocyte reaction. We have successfully constructed a recombinant adenovirus AdPD-L1 that suppresses T cell response in vitro. The virus will be useful to enforce negative co-stimulation via membrane-anchored PD-L1 to the unwanted T cell response in animal models of disease in vivo. The last,we test the potential protective effects of PD-L1 gene and ICOSLmAb delivery to BXSB mice on the development of LN. It was demonstrated that a single administration of intravenous injection of AdPD-L1 into BXSB mice not only had the PD-L1 transfected on renal tubular epithelial cells,but also effectively inhibited proteinuria and IgG subclasses dsDNA antibody production . Hypercellularity and deposition of IgG inglomeruli were also reduced.The administration of a single AdPD-L1 together with ICOSLmAb twice per week by tail vein injection resulted in almost complete amelioration of LN and the more better renal pathology improvement compared with only AdPD-L1 treatment.Our results demonstrated the involvement of the ICOS-ICOSL and PD-L1-PD-1 costimulatory pathway in the pathogenesis of lupus nephritis.The blockade of ICOS-ICOSL and the enhancement of PD-L1-PD-1 may be beneficial for the treatment of human LN. Conclusions: All the results suggest: 1. the PD-L1-PD-1 pathway negatively regulates T cell activation by TEC, and may play an inhibitory role in TEC-mediated immune activation and immunopathology in the kidney. 2.the therapeutic potential of simultaneous stimulation of the PD-L1-PD-1 mediated pathway by adenovirus-mediated PD-L1 gene transfer and blockade of ICOS-ICOSL costimulation by ICOSLmAb administration in the prevention of LN.