T cells were enriched with mouse CD4+ cells using beads (MACS cell isolation kits; Miltenyi Biotec, Auburn, CA; >95% of cells expressed the relevant surface marker). Purified CD4+ T cells were added (2 × 105 cells/well) to culture wells with RPE cells (1 × 105 cells/well). The cultures were maintained for 4 days (for evaluation of cytokine production) or 5 days (for evaluation of cell proliferation). The cultures were then assayed for BrdU uptake (the last 8 hours of culture, BrdU cell proliferation ELISA; Roche Diagnostic, Mannheim, Germany), as a measure of cell proliferation. Serum-free medium was used in cultures and assays involving T cells stimulated by anti-CD3 antibodies to mimic, as close as possible, the intraocular microenvironment outside the blood ocular barrier. Serum-free medium was composed of RPMI 1640 medium without the addition of FBS, and supplemented with 0.1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO) and 0.2% insulin, transferrin, selenium culture supplement (ITS+; Collaborative Biochemical Products, Bedford, MA).
Mouse Th22 cells were induced with a similar method that has been described in a previous report.
17 Splenic CD4
+ T cells were cocultured with anti-mouse CD3 antibody (2 μg/mL; BD Pharmingen, San Diego, CA); anti-mouse CD28 antibody (2 μg/mL; BD Pharmingen); anti-mouse IFN-γ antibody (5 μg/mL, R&D Systems, Minneapolis, MN); anti-mouse IL-4 antibody (5 μg/mL, R&D Systems); recombinant mouse TNF-α (50 ng/mL, R&D Systems); and recombinant mouse IL-6 (20 ng/mL, R&D Systems). The concentration of IL-22 in the supernatants of the T cell cultures was measured by ELISA (R&D Systems).