title: OX40 ligand expression abrogates the immunosuppressive function of retinal pigment epithelium.
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, when compared to the reversal caused by RPE cells expressing either OX40L alone or GITRL alone.
  
  
  CONCLUSIONS
  Using an in vitro approach, we found that OX40L causes an abrogation of the RPE-mediated immunosuppression. OX40L appears to be regulated in the ARPE-19 cell line and may play an important role in the pathogenesis of various ocular inflammatory conditions.
authors.affiliation: Vitreoretinal Service, Department of Ophthalmology and Visual Sciences, The University of Iowa Hospitals & Clinics, Iowa City, IA, 52242, USA. macunning@gmail.com.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Cunningham
authors.forename: Zhuqing
authors.initials: Z
authors.lastname: Li
authors.forename: Baoying
authors.initials: B
authors.lastname: Liu
authors.forename: Steven
authors.initials: S
authors.lastname: Yeh
authors.forename: Robert B
authors.initials: RB
authors.lastname: Nussenblatt