title: In vitro expanded CD4+CD25+Foxp3+ regulatory T cells maintain a normal phenotype and suppress immune-mediated ocular surface inflammation.
abstract: OBJECTIVE
  To determine whether in vitro expanded CD4(+)CD25(+)Foxp3(+) regulatory T cells can suppress immune-mediated ocular surface inflammation in a mouse model of dry eye.
  
  
  METHODS
  C57BL/6 or BALB/c mice were exposed to a dry, desiccating environment produced by maintaining low humidity (<40%), injections of scopolamine, and air flow produced by a fan. CD4(+)CD25(+) regulatory T cells were isolated and expanded in vitro in the presence of rmIL-2 and beads coated with anti-CD28 and anti-CD3. In vitro expanded regulatory T cells were phenotypically compared with freshly isolated regulatory T cells by flow cytometry and immunofluorescence. T-cell-deficient nude mice were reconstituted with CD4(+) T-effector cells from donor mice exposed to a desiccating environment for 5 days, in combination with or without freshly isolated or in vitro expanded regulatory T cells. Tear cytokine levels were determined by a multiplex bead-based immunoassay.
  
  
  RESULTS
  In vitro regulatory T cells maintained normal levels of CD4(+), CD25(+), and intracellular Foxp3(+), as determined by flow cytometry and immunohistochemistry. Freshly isolated and in vitro regulatory T cells were titrated in the presence of CD4(+) pathogenic T cells (CD4(+Path) T cells) in reconstitution experiments and most efficiently ablated tear cytokine levels and conjunctival cellular infiltration at a ratio of 1:1 (T Regs:CD4(+Path)).
  
  
  CONCLUSIONS
  Regulatory T cells expressed CD4(+), CD25(+), and intracellular Foxp3(+) at normal levels and retained their inhibitory function after in vitro expansion, providing a useful tool to determine the mechanism regulatory T cells use to sustain a homeostatic environment on the ocular surface.
authors.affiliation: Allergan, Inc., Irvine, California 92612, USA.
authors.forename: Karyn F
authors.initials: KF
authors.lastname: Siemasko
authors.forename: Jianping
authors.initials: J
authors.lastname: Gao
authors.forename: Virginia L
authors.initials: VL
authors.lastname: Calder
authors.forename: Rebecca
authors.initials: R
authors.lastname: Hanna
authors.forename: Margarita
authors.initials: M
authors.lastname: Calonge
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Pflugfelder
authors.forename: Jerry Y
authors.initials: JY
authors.lastname: Niederkorn
authors.forename: Michael E
authors.initials: ME
authors.lastname: Stern
mesh_terms: D019264:Adoptive Transfer; D000818:Animals; D015704:CD4 Antigens / Q000378:metabolism*; D015352:Dry Eye Syndromes / Q000139:chemically induced; D005260:Female; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D007150:Immunohistochemistry; D016130:Immunophenotyping; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism*; D007637:Keratoconjunctivitis / Q000276:immunology*; D008198:Lymph Nodes / Q000276:immunology; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D012601:Scopolamine; D013154:Spleen / Q000276:immunology; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*
chemical_list: D015704:CD4 Antigens; D051858:Forkhead Transcription Factors; C498833:Foxp3 protein, mouse; D053645:Interleukin-2 Receptor alpha Subunit; D012601:Scopolamine