title: MicroRNA regulation in human CD8+ T cell subsets--cytokine exposure alone drives miR-146a expression.
abstract: BACKGROUND
  microRNAs (miRNAs) are emerging as key regulators of the immune system, but their role in CD8+ T cell differentiation is not well explored. Some evidence suggests that signals from cell surface receptors influence the expression of miRNAs in CD8+ T cells, and may have consequent effects on cell phenotype and function. We set out to investigate whether common gamma chain cytokines modulated human CD8+ T cell expression of miR-146a, which previous studies have associated with different stages of CD8+ differentiation. We also investigated how changes in miR-146a related to other miRNAs that alter with CD8+ differentiation status.
  
  
  METHODS
  We treated human CD8+ T cells with the cytokines IL-2, IL-7 or IL-15 either at rest or after stimulation with anti-CD3 and anti-CD28. For some experiments we also purified human CD8+ T cell subsets ex vivo. Flow cytometry was used in parallel to assess cell surface memory marker expression. Total RNA from these cells was subjected to microarray analysis and real-time PCR for miRNA expression. Nucleofection studies were performed to assess potential mRNA targets of miR-146a.
  
  
  RESULTS
  We find that miR-146a is up-regulated in naïve CD8+ T cells exposed to IL-2 or IL-15, even in the absence of an activating T cell receptor stimulus, but not when IL-7 is also present. miR-146a expression correlates with a memory phenotype in both ex vivo and in vitro cultured cells although in our hands overexpression of miR-146a was not sufficient alone to drive a full memory phenotype. In ex vivo analysis, miR-146a was one of a small number of miRNAs that was differentially expressed between naïve and memory CD8+ T cells.
  
  
  CONCLUSIONS
  miR-146a is emerging as a critical regulator of immune system. Our data shows that miR-146a expression is strongly influenced by the cytokine milieu even in the absence of a T cell receptor stimulus. Our results have implications for studies designed to assess the function of miR-146a, help to define a fingerprint of miRNA expression in CD8+ T cell subsets and may be useful when designing optimal protocols for T cell expansion as efficacy of T cell immunotherapy is correlated with an 'early' memory phenotype.
authors.affiliation: School of Biological Sciences, University of Auckland, Thomas Building, Auckland, NZ, New Zealand. h.sheppard@auckland.ac.nz.|Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, NZ, New Zealand. h.sheppard@auckland.ac.nz.
authors.forename: Hilary M
authors.initials: HM
authors.lastname: Sheppard
authors.affiliation: School of Biological Sciences, University of Auckland, Thomas Building, Auckland, NZ, New Zealand. dver013@aucklanduni.ac.nz.|Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, NZ, New Zealand. dver013@aucklanduni.ac.nz.
authors.forename: Daniel
authors.initials: D
authors.lastname: Verdon
authors.affiliation: School of Biological Sciences, University of Auckland, Thomas Building, Auckland, NZ, New Zealand. a.brooks@auckland.ac.nz.|Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, NZ, New Zealand. a.brooks@auckland.ac.nz.
authors.forename: Anna E S
authors.initials: AE
authors.lastname: Brooks
authors.affiliation: School of Biological Sciences, University of Auckland, Thomas Building, Auckland, NZ, New Zealand. v.feisst@auckland.ac.nz.|Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, NZ, New Zealand. v.feisst@auckland.ac.nz.
authors.forename: Vaughan
authors.initials: V
authors.lastname: Feisst
authors.affiliation: School of Biological Sciences, University of Auckland, Thomas Building, Auckland, NZ, New Zealand. yuyu.jh@gmail.com.|Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, NZ, New Zealand. yuyu.jh@gmail.com.
authors.forename: Yu-Yu Joyce
authors.initials: YY
authors.lastname: Ho
authors.affiliation: School of Biological Sciences, University of Auckland, Thomas Building, Auckland, NZ, New Zealand. n.lorenz@auckland.ac.nz.
authors.forename: Natalie
authors.initials: N
authors.lastname: Lorenz
authors.affiliation: Bioinformatics Institute, University of Auckland, Auckland, NZ, New Zealand. v.fan@auckland.ac.nz.
authors.forename: Vicky
authors.initials: V
authors.lastname: Fan
authors.affiliation: School of Biological Sciences, University of Auckland, Thomas Building, Auckland, NZ, New Zealand. n.birch@auckland.ac.nz.|Centre for Brain Research, University of Auckland, Auckland, NZ, New Zealand. n.birch@auckland.ac.nz.
authors.forename: Nigel P
authors.initials: NP
authors.lastname: Birch
authors.affiliation: School of Biological Sciences, University of Auckland, Thomas Building, Auckland, NZ, New Zealand. adid002@aucklanduni.ac.nz.
authors.forename: Alicia
authors.initials: A
authors.lastname: Didsbury
authors.affiliation: School of Biological Sciences, University of Auckland, Thomas Building, Auckland, NZ, New Zealand. r.dunbar@auckland.ac.nz.|Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, NZ, New Zealand. r.dunbar@auckland.ac.nz.
authors.forename: P Rod
authors.initials: PR
authors.lastname: Dunbar
mesh_terms: D000941:Antigens / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D016207:Cytokines / Q000494:pharmacology*; D053200:Fas-Associated Death Domain Protein / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007156:Immunologic Memory; D019409:Interleukin-15 / Q000494:pharmacology; D007376:Interleukin-2 / Q000494:pharmacology; D015851:Interleukin-7 / Q000494:pharmacology; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D054400:Receptors, CCR7 / Q000378:metabolism; D016176:T-Lymphocyte Subsets / Q000187:drug effects / Q000378:metabolism*; D048029:TNF Receptor-Associated Factor 6 / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D000941:Antigens; C515370:CCR7 protein, human; D016207:Cytokines; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D019409:Interleukin-15; D007376:Interleukin-2; D015851:Interleukin-7; C542542:MIRN146 microRNA, human; D035683:MicroRNAs; D011948:Receptors, Antigen, T-Cell; D054400:Receptors, CCR7; D048029:TNF Receptor-Associated Factor 6