Basophil-derived RA acts in a paracrine fashion on the phenotype and function of T-helper cells. Purified naive CD4⁺ T cells cocultured with autologous basophils in a 1:1 ratio were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies (1 μg/mL) in the absence or presence of 10 ng/mL IL-3 and/or the RAR antagonist AGN (100 nM) and cultured for 10 days until analysis. (A) Expression of β7 and α4 integrins, CD38, CD25, and CCR9 in T-helper cells was assessed by flow cytometry. Data are given as delta mean fluorescence intensities (ΔMFI). Mean values of triplicates (± SD) of representative experiments of at least 3 are shown. The different panels represent data from independent experiments with cells from different donors. (B,C) Basophil-derived RA promotes Th2 polarization. Flow cytometric analysis of IL-4 and IFN-γ expression in CD4⁺ T cells. After coincubation with basophil for 10 days, T cells were restimulated for 4 hours with PMA and ionomycin (20 nM and 1 μM, respectively) in the presence of brefeldin A and double stained for intracellular IL-4 and IFN-γ. (B) (Left panels) Representative 2-parameter dot plots of CD4⁺ T cells with IFN-γ in the abscissa and IL-4 in the ordinate; percentage of cells in each quadrant is indicated. (Right panel) Ratios of the percentages of IL-4 and IFN-γ single-positive T cells (IL-4/IFN-γ). The mean values (± SD) of duplicates are shown. (C) Pairwise analysis of the different experimental conditions of 4 independent experiments with cells isolated from different donors is shown. Statistical significance was calculated using the one-tailed paired t test for each parameter and each pair of experimental conditions. Statistical significance is shown in the top of each panel. The data (mean of duplicates) from each individual experiment are connected by lines.