Interleukin 4 (il 4)

Spleen cells were excised from mice and single-cell suspensions of splenocytes were prepared in RPMI 1640 by teasing the organ through a sterile nylon mesh. Naive CD4⁺ T cells were purified using an EasySep mouse naive CD4⁺ T cell isolation kit (Stemcell Technologies, Vancouver, BC, Canada) and cultured with plate-bound anti-CD3 (5 µg/ml; Bio X Cell), anti-CD28 (5 µg/ml; Bio X Cell), and cytokines used alone, including IL-12 (R&D Systems), IFN-γ (R&D Systems), IL-1 (R&D Systems), IL-4 (R&D Systems), IL-6 (Cell Signaling Technology), IL-23 (eBioscience), and TGF-β (Invitrogen), or in combination with Abs (Bio X Cell) for Th17-promoting (2.5 ng/ml TGF-β, 20 ng/ml IL-6, 10 µg/ml anti–IFN-γ, 10 µg/ml anti–IL-4, 10 µg/ml anti–IL-2), Th1-promoting (10 ng/ml IL-12, 5 µg/ml anti–IL-4), Th2-promoting (10 ng/ml IL-4, 5 µg/ml anti–IFN-γ), or regulatory T cell (Treg)–promoting (5 ng/ml TGF-β, 5 µg/ml anti–IFN-γ, 5 µg/ml anti–IL-4) conditions. In some experiments, naive CD4⁺ T cells were cultured in Th17-promoting conditions and IL-21 (20 ng/ml; eBioscience) or IL-23 (50 ng/ml) was added to cultures after 24 h. Cells were harvested after 48 h for real-time PCR analysis and after 72 h for cytokine expression by flow cytometry as previously described (23 (link)).

For infections, 500 third-stage larvae of N. brasiliensis were injected subcutaneously as described (Molofsky et al., 2015b (link)). Mice were killed at the indicated timepoints and tissues were harvested and analyzed. For cytokine injections, Interleukin-33 (R&D Systems) was given as 500 ng in 0.2 ml PBS i.p. every other day for three doses. Intranasal (i.n.) cytokine treatments was given for seven consecutive days, using 4μg of IL-13 and 2μg of IL-4 (both Peprotech). IL-4 was given in complex with anti-IL-4 mAb (BioXcell, clone 11B11, 10μg). 10ug of papain was given i.n. in a total volume of 40μL for three consecutive days and lungs were analyzed four or eight days from the last treatment.