ox40 ligand expression abrogates the immunosuppressive function of retinal pigment epitheliumox40配体表达废除视网膜色素上皮细胞的免疫抑制作用.pdfVIP

Cunningham et al. Journal of Opthalmic Inflammation and Infection 2013, 3:12 /content/3/1/12 ORIGINAL RESEARCH Open Access OX40 ligand expression abrogates the immunosuppressive function of retinal pigment epithelium 1* 2 3 4 3 Matthew A Cunningham , Zhuqing Li , Baoying Liu , Steven Yeh and Robert B Nussenblatt Abstract Background: This study aims to investigate the role of OX40 ligand (OX40L) in ocular inflammation via abrogation of retinal pigment epithelium (RPE)-mediated immunosuppression using an in vitro expression approach. OX40L cDNA was polymerase chain reaction-amplified and cloned into an eYFP fusion vector. Cultured retinal pigment epithelial cells (ARPE-19) were transfected with the vector. Total RNA from unstimulated or inflammatory cytokine-stimulated ARPE cells were isolated and analyzed for OX40L expression by reverse transcription-polymerase chain reaction. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donors. Human ARPE cells (±OX40L ± GITR ligand (GITRL) expression) and PBMCs were co-cultured for in vitro proliferation studies. Results: Polymerase chain reaction confirmed the insertion of the OX40L gene into the fusion vector. Flow cytometry and fluorescence microscopy further confirmed surface expression of OX40L on ARPE cells after transfection. OX40L expression was induced in the RPE cells stimulated with pro-inflammatory cytokines. In the co-culture studies, there was a significant reversal (20% to 30%) of the RPE-induced suppression of activated PBMCs when the ARPE cells were transfected with OX40L. When both OX40L and GITRL were concomitantly transfected into ARPE cells, there was an additive reversal of RPE-mediated T cell suppression, wh