Interleukin 2 (il 2)

Lymph nodes from naïve male BALB/c mice were collected and CD4+ T cells were enriched using anti-CD4-coated magnetic beads on an autoMACS (Miltenyi Biotec, Auburn, CA). Cells were flow-sorted to separate effector T cells (CD4+CD25−) and Tregs (CD4+CD25^bright). The CD4+CD25− cells were CFSE-labeled and plated at 2.5×10⁴–5.0×10⁴ cells per well (96–well round bottom dish) in the presence of 2.5×10⁴–5.0×10⁴ anti-CD3/anti-CD28-coated silicon beads (1:1 ratio) or magnetic beads (Dynabeads Mouse T-Activator, Invitrogen). Increasing amounts of CD4+CD25^bright cells were incubated with effector T cells in the presence or absence of recombinant mouse IL-21 (Peprotech). Alternatively, sorted effector T cells were rested (1×10⁶ cells/ml), and Tregs (0.5×10⁶ cells/ml) were incubated overnight with 500 IU rhIL-2 (BD Biosciences) with or without rIL-21 (150 ng/ml). The following day, cells were washed and co-incubated as above. Day 5 supernatants were analysed for cytokines by ELISA (IL-4: BD Pharmingen, IL-2, IL-13: RnD Systems), and day 3 cells harvested for flow cytometric analysis of proliferation by CFSE dilution. The frequency of cell division was determined using FlowJo software (Tree Star, Ashland, OR).

Lung ILC2 were sorted as lineage^- (CD3, CD4, CD8, CD11b, Gr1, CD11c, TCRb, TCRgd, FceR1, CD49b, CD19) CD45⁺ICOS⁺IL-33R⁺ from naïve male wildytipe of C3ar1^−/− mice. 1×10³ ILC2 were seeded at the bottom of round-bottom 96-well dishes and cultured in in RPMI containing 10 % FBS, L-glutamine, penicillin/streptomycin and 55 uM 2-mercaptoethanol, 10 ng/ml IL-2 (RnD Systems) combined with IL-33 (RnD Systems), with or without 1–2 ug/ml C3a (RnD Systems). Cultures were maintained for 3–5 days, and supernatants were harvested for ELISA.