M5 114.15.2 anti mhcii

Freshly isolated or in vitro cultured ILC2s, from WT, MhcII^−/−, or QUAD-KO mice, were resupended in RPMI 1640 (GIBCO®) supplemented with 2 mM L-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin, and 10% Hyclone Fetal Bovine Serum (Thermoscientific). Where necessary, ILC2s were pulsed with OVA-peptide (323–339) for 2 hr at 37°C. Subsequently ILC2s were washed thoroughly and 1 × 10⁴ ILC2s were cultured with 1 × 10⁴ labeled CD3⁺CD4⁺ OTIITg or DO11.10Tg splenic T cells, which were sorted by flow cytometry, for 5 days at 37°C, 5% CO₂. Where indicated, the antibodies M5/114.15.2 (anti-MHCII, eBioscience), 16-10A1 (anti-CD80, BioLegend), and GL-1 (anti-CD86, BioLegend) were added to cultures at a concentration of 1 μg/ml. 11B11 (anti-IL-4, eBioscience) was incubated at a concentration of 10 μg/ml. Where appropriate, blockade of IL-2 was performed with 10 μg/ml of JES6-1A12 (eBioscience) and S4B6 (BD PharMingen). Cocultures containing in vivo-loaded OVA-DQ⁺ ILC2s were prepared by FACS-purifying ILC2s, based on Lin⁻ICOS⁺OVA-DQ⁺ or lineage⁻ICOS⁺OVA-DQ⁻, from the bronchoalveolar lavage of WT mice receiving an intranasal administration of OVA-DQ and IL-33 on 7 consecutive days. Cocultures were setup at a 1:1 ratio with 4 × 10⁴ cells per well. Human samples were taken under GCP guidance with ethical approval of the NRES Committee South Central.

To generate ILC2s in this experiment, mice were injected with IL-25Ig (5 μg/mouse) at days 0, 1, 3 and 5 after infection [17 (link), 22 (link)]. ILC2s (CD4^-IL-17RB⁺CD127⁺T1/ST2⁺GFP⁺) were sorted from pooled mesenteric lymph nodes using a cell sorter (FACSAria II, BD Biosciences). To study effector CD4+ T cells, mice were infected and effector CD4⁺ T cells (CD44⁺CD62L^-CD4⁺CD3⁺ cells) were sorted from the mesenteric lymph nodes from mice infected with T. spiralis on day 7 postinfection. Sorted ILC2s (5 x 10⁴ cells) were pulsed with 10 μg/ml T. spiralis extract or OVA antigen for 3 hours at 37°C. Subsequently ILC2s were washed and were then co-cultured with effector CD4⁺ T cells (1 x 10⁵ cells) for 3 days. The antibodies M5/114.15.2 (anti-MHCII, eBioscience) were added to cultures at a concentration of 1 μg/ml. The culture supernatants were collected after 3 days and were then analyzed for cytokine production using ELISA.