In vitro expanded CD4+CD25+Foxp3+ regulatory T cells maintain a normal phenotype and suppress immune-mediated ocular surface inflammation

Karyn F. Siemasko, Jianping Gao, Virginia L. Calder, Rebecca Hanna, Margarita Calonge, Stephen C. Pflugfelder, Jerry Y. Niederkorn, Michael E. Stern

Research output: Contribution to journalArticlepeer-review

50 Scopus citations


PURPOSE. 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.

Original languageEnglish (US)
Pages (from-to)5434-5440
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Issue number12
StatePublished - 2008

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience


Dive into the research topics of 'In vitro expanded CD4+CD25+Foxp3+ regulatory T cells maintain a normal phenotype and suppress immune-mediated ocular surface inflammation'. Together they form a unique fingerprint.

Cite this