title: Treg cell function in rheumatoid arthritis is compromised by ctla-4 promoter methylation resulting in a failure to activate the indoleamine 2,3-dioxygenase pathway.
abstract: OBJECTIVE
  Functionally impaired Treg cells expressing abnormally low levels of CTLA-4 have been well documented in rheumatoid arthritis (RA). However, the molecular defect underlying this reduced expression is unknown. The aims of this study were to assess the role of DNA methylation in regulating CTLA-4 expression in Treg cells isolated from RA patients and to elucidate the mechanism of their reduced suppressor function.
  
  
  METHODS
  CTLA-4 expression in Treg cells from RA patients and healthy controls was measured by quantitative polymerase chain reaction (PCR) and flow cytometry. Methylation of the CTLA-4 gene promoter was analyzed by bisulfite-specific PCR, followed by sequencing. Methylation-dependent transcriptional activity of the CTLA-4 gene promoter was measured by luciferase assay, and NF-AT binding to the CTLA-4 gene promoter was determined by chromatin immunoprecipitation. The role of CTLA-4 expression in controlling Teff cells was analyzed using an autologous mixed lymphocyte reaction.
  
  
  RESULTS
  Down-regulation of CTLA-4 expression in Treg cells from RA patients was caused by methylation of a previously unidentified NF-AT binding site within the CTLA-4 gene promoter. As a consequence, Treg cells were unable to induce expression and activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO), which in turn resulted in a failure to activate the immunomodulatory kynurenine pathway.
  
  
  CONCLUSIONS
  We show for the first time that epigenetic modifications contribute to defective Treg cell function in RA through an inability to activate the IDO pathway. Therefore, this study sets a precedent for investigating potential therapeutic strategies aimed at reinforcing the IDO pathway in RA patients.
authors.affiliation: Kennedy Institute and University of Oxford, Oxford, UK.
authors.forename: Adam P
authors.initials: AP
authors.lastname: Cribbs
authors.forename: Alan
authors.initials: A
authors.lastname: Kennedy
authors.forename: Henry
authors.initials: H
authors.lastname: Penn
authors.forename: Jordan E
authors.initials: JE
authors.lastname: Read
authors.forename: Parisa
authors.initials: P
authors.lastname: Amjadi
authors.forename: Patricia
authors.initials: P
authors.lastname: Green
authors.forename: Khaja
authors.initials: K
authors.lastname: Syed
authors.forename: Szymon W
authors.initials: SW
authors.lastname: Manka
authors.forename: Fionula M
authors.initials: FM
authors.lastname: Brennan
authors.forename: Bernard
authors.initials: B
authors.lastname: Gregory
authors.forename: Richard O
authors.initials: RO
authors.lastname: Williams
mesh_terms: D001172:Arthritis, Rheumatoid / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D060908:CTLA-4 Antigen / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D015536:Down-Regulation; D006801:Humans; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D015398:Signal Transduction / Q000502:physiology*; D050378:T-Lymphocytes, Regulatory / Q000276:immunology / Q000378:metabolism*
chemical_list: D060908:CTLA-4 Antigen; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase