Figure 5: NA-mediated costimulation induces Th2 differentiation.

(a) Naive CD4⁺ T cells were stimulated with immobilized anti-CD3∈ (10 μg ml⁻¹) plus anti-CD28 (10 μg ml⁻¹) and the indicated ligands for 3 days, followed by culture with IL-2 (10 ng ml⁻¹) for an additional 3 days. Cells were subjected to real-time PCR analysis (b), or restimulated with immobilized anti-CD3∈ plus anti-CD28 for 6 h for staining of intracellular cytokines IL-4 and IL-13. (b,c) Naive CD4⁺ T cells from WT and IL-4Rα^−/− mice were activated similarly as in (a) and cells were subjected to real-time PCR analysis (c) or restimulated with immobilized anti-CD3∈ plus anti-CD28 for 6 h for staining of intracellular cytokines IL-4 and IL-13. *P<0.05, Student’s t-test (compared with anti-CD3/CD28 plus non-CpG in WT cells). (d) Naive CD4⁺ T cells were stimulated for 48 h with anti-CD3∈ plus anti-CD28 and the indicated ligands and cytokine production was analysed by ELISA. *P<0.05, Student’s t-test (compared with anti-CD3/CD28 alone). (e) Naive CD4⁺ T cells with or without γ-irradiated naive CD4⁺ T cells (Dead cells) at a 1:1 ratio were stimulated with anti-CD3∈ plus anti-CD28 in the presence or absence of DNase I or RNase A, and analysed similarly as in (a). (f) Naive CD4⁺ T cells from OT-II Tg mice were co-cultured with irradiated T cell-depleted splenocytes from C57BL/6 mice as APCs, together with OVA peptide (10 μM) in the presence or absence of non-CpG for 6 days. CD4⁺ T cells were restimulated with immobilized anti-CD3∈ plus anti-CD28 for 6 h for staining of intracellular cytokines. Error bars indicate s.d. Data are representative of at least three independent experiments.