title: RPE cells resist bystander killing by CTLs, but are highly susceptible to antigen-dependent CTL killing.
abstract: OBJECTIVE
  Retinal pigmented epithelial (RPE) cells maintain the blood-retinal barrier, sustain retinal photoreceptor cell health and function, and may play a role in ocular immune privilege. If RPE immunomodulatory activities were antigen specific, their expression would require antigen presentation. In a study of antigen processing and major histocompatibility complex (MHC) class I-restricted presentation by RPE cells, the cells' sensitivity to the activity of cytotoxic T lymphocytes (CTLs) was determined.
  
  
  METHODS
  RPE was cultured, with and without proinflammatory cytokines and antigen, followed by the addition of beta-galactosidase (beta-gal)-specific CTLs. Cytotoxic activity was measured by the CTL-dependent activation of caspase-3 in the RPE. Sensitivity to the CTLs was used to evaluate the activity of pathways of antigen processing and presentation using an antigen (beta-gal) that was either applied to or expressed in RPE.
  
  
  RESULTS
  RPE cells were sensitive targets for activated CTL-mediated killing in vitro only if prepulsed with cognate peptide, or if beta-gal-expressing RPE was pretreated to induce upregulation of immunoproteasome. Activated CTLs induced apoptosis in RPE within 3 hours of coculture with antigen-positive RPE monolayers. Application of CTLs in a resting state to antigen-positive RPE led to their activation in the absence of exogenous antigen-presenting cells (APCs). This antigen-dependent activation and killing required 24 hours of co-incubation of RPE with resting CD8 T cells specific for beta-gal. Although RPE cells are highly phagocytic, functional evidence for processing that allowed phagocytosed antigens to load into class I MHC was not detected. RPE was minimally sensitive to bystander killing by activated CTLs.
  
  
  CONCLUSIONS
  Although there are many reports of T-cell inhibition by RPE, we found that CTLs efficiently killed RPE cells by induction of apoptosis in an antigen-dependent manner. The survival of RPE in the face of extensive CTL destruction of adjacent photoreceptor cells in vivo appears to be based on their insensitivity to injury via bystander mechanisms.
authors.affiliation: Department of Ophthalmology, University of Minnesota, Minneapolis, Minnesota 55455, USA. grege001@umn.edu
authors.forename: Dale S
authors.initials: DS
authors.lastname: Gregerson
authors.forename: Kathleen L
authors.initials: KL
authors.lastname: Lew
authors.forename: Scott W
authors.initials: SW
authors.lastname: McPherson
authors.forename: Neal D
authors.initials: ND
authors.lastname: Heuss
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Ferrington
mesh_terms: D000818:Animals; D017951:Antigen Presentation / Q000276:immunology; D000938:Antigen-Presenting Cells / Q000276:immunology*; D017209:Apoptosis; D015153:Blotting, Western; D024201:Bystander Effect; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D018920:Coculture Techniques; D003602:Cytotoxicity, Immunologic; D005434:Flow Cytometry; D007371:Interferon-gamma / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000276:immunology*; D051379:Mice; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000276:immunology* / Q000473:pathology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D001616:beta-Galactosidase; D053148:Caspase 3