Freshly isolated cells (∼2 × 106) from draining lymph nodes and spleen of immunized mice were stained with a 5-μM solution of CFSE (carboxy fluorescein diacetate succinimidyl; Invitrogen Corp., Carlsbad, CA) at room temperature for 8 minutes. After staining, the cells were incubated with warm fetal bovine serum at 37°C for 10 minutes. The cells were washed twice in phosphate-buffered saline (PBS) and were cultured in HL-1 medium (Lonza Ltd., Allendale, NJ) for 4 days at 37°C in 7.5% CO2 in the presence or absence of the immunizing antigen (IRBP 10 μg/mL; peptides 25 μg/mL each). The cells were harvested after 4 days, washed, and stained with anti-mouse CD4 or CD8 antibody conjugated to APC (allophycocyanin; BD Biosciences, Franklin Lakes, NJ) according to standard protocol. After the cells were washed, they were resuspended in PBS for flow cytometry (FACSCalibur; BD Biosciences). Propidium iodide (PI; Roche Pharmaceuticals, Branchburg, NJ) was added to each sample 2 minutes before flow cytometry, to exclude dead cells from the analysis. Approximately 75,000 to 100,000 cells were collected from the lymphocyte gate and analyzed (FlowJo 8.5.1 software; Treestar, Inc., San Carlos, CA). The frequency of PI-negative CD4+ or CD8+ T cells that proliferated in response to stimulation with immunizing antigens were determined by subtracting the expression of CFSE in unstimulated CD4 or CD8 T cells from antigen-stimulated samples. Analyzed data are expressed as percentages of total PI-negative CD4+ or CD8+ cells.