title: In vitro cytokine licensing induces persistent permissive chromatin at the Indoleamine 2,3-dioxygenase promoter.
abstract: Mesenchymal stromal cells (MSCs) are being investigated as therapies for inflammatory diseases due to their immunosuppressive capacity. Interferon (IFN)-γ treatment primes MSC immunosuppression partially through induction of Indoleamine 2,3-dioxygenase (IDO1), which depletes tryptophan necessary to support proliferation of activated T cells. We investigated the role of histone modifications in the timing and maintenance of induced IDO1 expression in MSCs under clinical manufacturing conditions, such as cryopreservation.
  
  
  
  We used chromatin immunoprecipitation and quantitative polymerase chain reaction (PCR) to assay levels of transcriptionally permissive acetylated H3K9 and repressive trimethylated H3K9 histone modifications surrounding the transcriptional start site for IDO1, and reverse transcriptase PCR and immunoblotting to detect messenger RNA (mRNA) and protein.
  
  
  
  MSCs derived from three donors approached maximum IDO1 mRNA levels following 24 hours of in vitro cytokine treatment. Induction of IDO1 expression correlated with increased acetylation of H3K9 concomitant with reduction of trimethylated H3K9 modifications at the promoter. Examination of two additional donors confirmed this result. While induced IDO1 levels decreased within 2 days after cytokine removal and freeze thawing, the activated chromatin state was maintained. Upon re-exposure to cytokines, previously primed MSCs accumulated near-maximum IDO1 mRNA levels within 4-8 h.
  
  
  
  Our data indicate that in vitro priming of MSCs causes chromatin remodeling at the IDO1 promoter, that this alteration is maintained during processing commonly used to prepare MSCs for clinical use and that, once primed, MSCs are poised for IDO1 expression even in the absence of cytokines.
authors.affiliation: Cellular and Tissue Therapies Branch, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Yazmin I
authors.initials: YI
authors.lastname: Rovira Gonzalez
authors.affiliation: Cellular and Tissue Therapies Branch, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, Maryland, USA. Electronic address: Patrick.Lynch@fda.hhs.gov.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Lynch
authors.affiliation: Cellular and Tissue Therapies Branch, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Elaine E
authors.initials: EE
authors.lastname: Thompson
authors.affiliation: Cellular and Tissue Therapies Branch, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Brian G
authors.initials: BG
authors.lastname: Stultz
authors.affiliation: Cellular and Tissue Therapies Branch, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, Maryland, USA. Electronic address: Deborah.Hursh@fda.hhs.gov.
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Hursh
mesh_terms: D000107:Acetylation; D002478:Cells, Cultured; D002843:Chromatin / Q000235:genetics* / Q000378:metabolism; D042002:Chromatin Assembly and Disassembly; D015925:Cryopreservation; D016207:Cytokines / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase / Q000235:genetics* / Q000378:metabolism; D007371:Interferon-gamma / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects; D059630:Mesenchymal Stem Cells / Q000187:drug effects / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger; D024363:Transcription Initiation Site; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D002843:Chromatin; D016207:Cytokines; C576710:IDO1 protein, human; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma
keywords: chromatin remodeling; cytokine licensing; histone modifications; immunomodulation; indoleamine 2,3-dioxygenase; mesenchymal stromal cells