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M. E. Stern, K. F. Siemasko, J. Gao, V. L. Calder, R. Hanna, M. Calonge, S. C. Pflugfelder, J. Y. Niederkorn; In vitro Expanded CD4+CD25+FoxP3+ Regulatory T Cells Inhibit Ocular Surface Inflammation in a Mouse Model of Dry Eye. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1924. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Experimental dry eye is induced by exposing mice to a dry, desiccating environment (DS) for 5 days resulting in decreased tear production, loss of conjunctival goblet cells, and the presence of proinflammatory cytokines in the tear film. Adoptive transfer of CD4+ T cells from mice exposed to a DS environment to a T cell-deficient nude mouse recipient results in transfer of dry eye disease to the recipient mouse, proving that dry eye is mediated by CD4+ T cells. The ability of in vitro expanded CD4+CD25+FoxP3+ regulatory T cells to suppress immune-mediated ocular surface inflammation in this mouse model of adoptive transfer of dry eye was investigated.
Regulatory T cells were isolated using MACS isolation columns and expanded in vitro for 7 days in the presence of anti-CD3 and anti-CD28 antibody coated beads and IL-2 (20 ng/ml). T cell-deficient nude recipients received donor DS treated CD4+ T cells (5 X106) and different ratios of in vitro regulatory T cells. Tears were analyzed for cytokines by Luminex analysis.
In vitro regulatory T cells were titrated in the presence of CD4+ T cells in reconstitution experiments of nude mice. Seven day in vitro regulatory T cells on either the C57BL/6 or BALB/c background work most efficiently in ablating disease transfer by CD4+ T cells at 1:1 (in vitro T Regs: CD4+) based on tear cytokine level analysis of IL-12 (C57BL/6 background: 83.65% inhibition, *P<0.03; BALB/c background: 97.87% inhibition, *P<0.008), IFN-γ (C57BL/6 background: 49.26% inhibition, *P<0.04; BALB/c background: 77.45% inhibition, *P<0.03) and TNF-α (C57BL/6 background: 48.38% inhibition, *P<0.03; BALB/c background: 86.4% inhibition, *P<0.03). The 1:4 and 1:2 (in vitro T Reg:CD4+) ratios had diminishing, but not statistically significant, effects on tear cytokine levels in the BALB/c WT background.
Expanded regulatory T cells maintain their inhibitory function, providing a tool to further determine the mechanisms by which regulatory T cells function.
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