Lympholyte mammal medium

PBMCs were isolated by density gradient centrifugation on Lympholyte-Mammal medium (1.086 g cm⁻³ at 22 °C, Cedarlane Labs, Skanderborg, Denmark) and cultured in RPMI-1640 with 5 × 10⁻⁵M 2-mercaptoethanol, 1 mM glutamine, 1% pyruvate, 1% penicillin-streptomycin, 1% HEPES and 10% fetal calf serum (Invitrogen, Taastrup, Denmark), with 2 × 10⁵ cells/well were added in Nunclon microtiter plates (NUNC). The cells were stimulated in triplicates with UV-SvD, Hirep1, CTH93, CT043, CT414_aa605-840 and MOMP in 10 μg ml⁻¹. Synthetic peptides (20-mer) with 10 aa overlap covering CT043, CT414_aa605-840, MOMP and Hirep1 were purchased from (Genscript, Piscataway, NJ, USA) and used for stimulation as pools (5 μg ml⁻¹ of each peptide in the peptide pools) or as individual peptides (5 μg ml⁻¹). The peptides are listed in Supplementary Tables 1. Wells with medium alone and with Staphylococcal enterotoxin B (1 μg ml⁻¹) were included as negative and positive control, respectively. After 3 days of incubation at 37 °C with 5% CO₂, cell culture supernatants were harvested and stored at −20 °C.

Based on their phenotypic characteristics, Breg^IL-33 cells were enriched and isolated from peripheral blood of IL-33-injected WT and IL-10^−/− mice for further functional characterization both in vitro and in vivo. Briefly, the mice were sacrificed at the peak of Breg^IL-33 appearing in the blood, i.e. week-2 after the 1st IL-33 injection (a total of 5 injections) (Fig. 3). Blood was harvested by heart puncture and diluted in PBS +5% EDTA. PBMC were first isolated by density separation using Lympholyte-Mammal medium (Cedarlane Laboratories Ltd, Canada). Total B cells (CD43^-) were then negatively selected using the MACS CD43 microbeads (Miltenyi Biotec, Germany), followed by depletion of CD23⁺ cells using an anti-CD23-PE antibody (Clone B3B4, Becton Dickinson, UK) and anti-PE microbeads (Miltenyi Biotec, Germany). All procedures were performed according to the manufacturers' instructions. Purities obtained were: total B cells (CD19⁺) >95%; of which the negatively selected population (CD23⁻ B cells) were >80%, and the remaining positively selected (CD23⁺ B cells) >90%. The flow through obtained after both depletion steps was enriched in CD23⁻ B cells with IL-10 producing capacity (∼3 folds), majority of which were also of CD25⁺ B cells (data not shown).