Figure 2: IL-21-induced pro-IL-1β expression is linage-restricted.

(a) Top panel: cDCs were rested 16 h and stimulated as indicated for 15 min. pSTAT3 was evaluated by flow cytometry. cDCs were gated as CD11c^hi cells. Bottom panel: cDCs were rested 1 h, treated with 20 ng ml⁻¹ IL-21, IL-6, IL-10 or Flt3L for 4 h, and intracellular pro-IL-1β analysed by flow cytometry. Shown are data representative of three experiments. (b) Summary of three experiments from lower panel of a. **P values of the untreated sample compared with IL-21, IL-6 and IL-10 treated samples are 0.0002, 0.0017 and 0.0049, respectively; NS, P=0.4; error bars are means±s.e.m. (c–e) cDCs were stimulated with 100 ng ml⁻¹ IL-21 or IL-10 for 1 h, then stimulated with 100 ng ml⁻¹ LPS for 4 h, and the expression of Il1b (c), Il6 (d), and Tnf (e) mRNA were determined. Shown are combined results of 3 independent experiments; error bars are means±s.e.m. (f) Top panel: BMMs were rested without M-CSF for 16 h, treated with IL-21 or LPS for 4 h, and intracellular pro-IL-1β assessed by flow cytometry. Bottom panel: BMMs (gated as CD11c⁺F4/80⁺ cells) were rested and treated with IL-21 or LPS for 15 min. pSTAT3 was evaluated by flow cytometry. Data are representative of two experiments (total of 6 individual samples). (g) Top panel: CD4⁺ T cells were pre-activated with 5 μg ml⁻¹ plate-bound anti-CD3+2 μg ml⁻¹ soluble anti-CD28 for 3 days, washed, rested 16 h, treated with IL-21 for 4 h, and intracellular pro-IL-1β assessed by flow cytometry. Bottom panel: Rested CD4⁺ T cells were treated with IL-21 for 15 min. pSTAT3 was evaluated by flow cytometry. Data are representative of three experiments. Statistical analysis was performed by Student’s t-test.