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.