Mice were exposed for 10 successive days to PBS (mice subsequently develop airway inflammation after OVA/alum immunization followed by OVA aerosol exposure) or to 1% OVA (to develop tolerance). Both groups were immunized with OVA/alum on days 21 and 27, and spleens were isolated (3). Spleens from 3 animals were pooled in each group, and CD4⁺ T cells were isolated and subjected to stimulation with OVA/APCs in vitro. The cells were stained for the expression of CD4, CD25, and Notch1. Since no staining was detected in the top left quadrants of the “Airway inflammation” and “Tolerance” panels, the total fluorescence value in the FL1 channel for the isotype control stain (top left and top right quadrants) was subtracted from the experimental stains to arrive at net percent-positive values of 5% and 15% for the airway inflammation and tolerance conditions, respectively. Expression of CD25 and Notch1 was determined on equal numbers of CD4⁺ T cells for each condition. Numbers in the dot plots denote percentages. Results shown are representative of 4 independent experiments.