ILC2 Transfer and Ovalbumin Immunization

ILC2 were isolated as recently described (21 (link)). In brief, donor mice were hydrodynamically injected with 4 µg each of IL-25 and IL-33 vectors to induce ILC2. Three days postinjection (dpi), ILC2 were sort-purified from spleen and mesenteric lymph nodes (MLNs) and in vitro expanded as indicated above. OTII transgenic CD4⁺ T cells were purified by negative selection (Stemcell Technologies) according to the manufacturer’s instructions and assessed at ≥95% purity. One day prior to cell transfer, recipient mice were intraperitoneally (i.p.) immunized with 20 µg Ovalbumin (Invivogen) in 200 µl Imject Alum (Thermo Scientific). 2 × 10⁶ CD4⁺ OTII T cells were co-transferred intravenously (i.v.) with 1 × 10⁶ WT or Btn2a2^–/–ILC2 into 8–14-week-old recipient hosts as indicated. Mice were sacrificed 3 days post adoptive cell transfer, and spleens were harvested for flow cytometric analyses.