IL-21 production by TSLP-DC–polarized CD4 T cells is stable. (A) DCs were activated with TSLP (50 ng/ml, TSLP-DC, filled triangles) or in control medium (DC, circles). After 24 h, DCs were co-cultured with naive CD4 T cells and stimulated for 24 h with anti-CD3/CD28 beads. IL-21 concentration in the supernatants from seven independent experiments. (B) Quantification of IL-21 secretion by CD4 T cells polarized for 6 d with DCs, previously activated for 24 h with increasing doses of TSLP. SEM for four independent experiments; *, P < 0.05; **, P < 0.01; ***, P < 0.001, paired Student’s t test. (C) CD4 T cell fold expansion and IL-21 secretion from co-cultures with untreated CD1c⁺, TSLP-activated CD1c⁺ and TSLP-activated CD141⁺ DCs. SEM for 12 independent experiments; *, P < 0.05; ***, P < 0.001, paired Student’s t test. (D) Intracellular FACS staining of IL-21, TNF, and IL-4 by TSLP-DC–activated CD4 T cells at the indicated days of primary and secondary culture from a representative CD4 T cell donor. In primary culture CD4 T cells were activated by TSLP-DC. In secondary culture, cells from day 5 of primary culture were cultured for 6 d in medium alone (No DC), with TSLP-DC or LPS-DC. (E) Percentage of IL-21⁺/TNF⁺ and IL-21⁺/IL-4⁺ cells (among activated cells) and fold expansion in primary and secondary culture as indicated, in three independent experiments. NA, not applicable.