Expanded Mouse CD4+CD25+FoxP3+ Regulatory T Cells Maintain a Normal Phenotype in vitro

Abstract

Purpose:: Regulatory T cells can inhibit a wide variety of autoimmune and inflammatory diseases, but their involvement in regulating ocular surface inflammation in dry eye induced immunopathology has recently started to be investigated (Niederkorn et al., 2006). CD4⁺CD25⁺FoxP3⁺ regulatory T cells make up 5-10% of the mouse CD4⁺ T cell population. Due to the limited number of regulatory T cells in vivo and the inherent loss of cells through the isolation procedure, it became necessary to expand regulatory T cells in vitro to begin to determine the mechanism of action used by these cells to inhibit ocular inflammation. The ability of in vitro expanded C57BL/6 and BALB/c CD4⁺CD25⁺FoxP3⁺ regulatory T cells to maintain a comparable phenotype to freshly isolated regulatory T cells was evaluated.

Methods:: Experimental dry eye was induced in C57BL/6 WT or BALB/c WT mice by exposing the mice to a dry, desiccating environment (DS) for 5 days (Dursun et al.; 2002) which induces decreased tear production, loss of goblet cells, and the presence of proinflammatory cytokines. Regulatory T cells were isolated using MACS isolation columns and expanded in vitro for up to 7 days in the presence of anti-CD3 and anti-CD28 antibody coated beads and IL-2 (20 ng/ml). In vitro cells were analyzed by flow cytometry for regulatory T cell markers. Cell supernatants were analyzed by Luminex analysis for TGF-ß and IL-10.

Results:: C57BL/6 WT and BALB/c WT in vitro regulatory T cells maintain a normal phenotype of CD4⁺, CD25⁺, GITR, CD62L, CTLA-4, and intracellular FoxP3⁺ as determined either by flow cytometry and/or immunohistochemistry following 7 days in vitro. The expression levels of these markers were comparable to the levels seen in freshly isolated regulatory T cells. Luminex analysis of the supernatants of C57BL/6 or BALB/c in vitro regulatory T cells revealed the presence of the inhibitory cytokines TGF-ß (8000+/-39.3 and 1333+/-27.3 pg/ml, respectively) and IL-10 (21.6+/-.086 ng/ml and 118+/-12 ng/ml, respectively) at day 7.

Conclusions:: Expanded regulatory T cells maintain the normal phenotype seen in freshly isolated cells for up to 7 days in vitro.

Keywords: cornea: tears/tear film/dry eye • inflammation • immunomodulation/immunoregulation