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This Article Full Text Full Text (PDF) All Versions of this Article: iovs.08-2075v1 49/12/5434    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Siemasko, K. F. Articles by Stern, M. E. PubMed PubMed Citation Articles by Siemasko, K. F. Articles by Stern, M. E.

In Vitro Expanded CD4⁺CD25⁺Foxp3⁺ Regulatory T Cells Maintain a Normal Phenotype and Suppress Immune-Mediated Ocular Surface Inflammation

From ¹Allergan, Inc., Irvine, California; the ²University College London, London, United Kingdom; ³IOBA (Institute of Applied Ophthalmobiology), University of Valladolid, Valladolid, Spain; the ⁴Baylor College of Medicine, Houston, Texas; and the ⁵Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas.

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.