Induction of Fas or FasL expressions on CD4⁺ T cells. (A) Naive or cultured CD4⁺ T cells prepared from BALB/c mice were stained with anti-Fas or -FasL mAb (green, blue, or red line) or isotype-matched control antibody (gray). Naive CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28 mAbs for 4 days in the absence (blue line) or presence of 100 ng/mL CCL19 and CCL21 (red line) in primary culture, and then restimulated with IL-2 alone or anti-CD3 mAb and IL-2 in secondary culture. Two days after, the cells were stained for Fas and FasL. Mean fluorescence intensity (MFI) is indicated in each panel. Similar results were obtained in another independent experiment. (B-C) Naive CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28 mAbs with or without CCL19 and CCL21 (100 ng/mL each) for 4 days, washed, and restimulated with anti-CD3 mAb and IL-2 for 6 hours. Then, CD4⁺ T cells were negatively purified using the mouse CD4⁺ T-cell isolation kit (Miltenyi Biotec, Auburn, CA; purity: 90%-95%). (B) Total cellular RNA was prepared from the T cells treated in the absence (▪) or presence (□) of CCL19 and CCL21 as described in “Materials and methods,” under “Real-time reverse transcription–PCR analysis.” Quantitative real-time PCR was carried out, and the results were expressed as 2^−ΔΔCt, which corresponded to the fold change between the T cells treated in the absence of CCL19 and CCL21 and those in the presence of these chemokines. (C) For determining the translocation of transcription factors indicated, nuclear extracts were prepared from CD4⁺ T cells untreated (□), treated in the absence of (⊡), or presence (▪) of CCL19 and CCL21 in the primary culture, and were assayed for the contents of these factors using BD TransFactor kits (BD Bioscience/Clontech). Results were expressed as OD values at 450 nm ± SD of triplicate tests.