Rtk2071

PBMC were seeded with an IL-10 neutralizing monoclonal antibody (clone: JES3-9D7, Biolegend) or matched isotype control (clone: RTK2071, Biolegend) at a concentration of 2.5 μg/ml for 2 h. PBMC were then stimulated and incubated overnight. Alternatively, PBMC were seeded with an IL-12 neutralizing monoclonal antibody (clone: MT3279H, Mabtech) or matched isotype control (clone: MPC-11, Biolegend) at 1 μg/ml for 5 h. PBMC were then stimulated, together with an additional dose of αIL-12/isotype control, for 24 h.

LEAF purified anti-mouse TNF antibody (MP6- × T22, BioLegend) or LEAF purified rat IgG1 isotype control (RTK2071, BioLegend) was injected i.p. (5 mg kg^–1 body weight) twice weekly for the indicated times.

All cells were purified by flow cytometry using BD FACS ARIA III (BD biosciences, San Jose, CA) with a purity of >95%. The isolated ILC2s were cultured (at least 5 × 10³ cells/well) in 96-well round-bottom plates with Gibco^™ Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, MA) that was supplemented with 10% fetal bovine serum (FBS), 2% antibiotics (penicillin and streptomycin), and 0.05 mM b-mercaptoethanol. The cells were maintained in a 37 °C incubator with 5% CO₂. All mouse ILC2s were cultured in the presence of recombinant mouse (rm) IL-2 (10 ng/mL), rmIL-7 (10 ng/mL), and/or rmIL-33 (10 ng/mL). The mouse cells were stimulated via mouse CD200-Fc (2724-CD-050; R&D Systems) and the corresponding IgG1 isotypes (110-HG-100; R&D Systems). In some experiments, cells were treated with either anti-ST2 blocking antibody (DIH4; BioLegend) or IgG1, κ isotype control (RTK2071; BioLegend). Human ILC2s were cultured in the presence of rhIL-2 (10 ng/mL), rhIL-7 (10 ng/mL), and/or rhIL-33 (10 ng/mL). The human cells were stimulated via human CD200-Fc (2724-CD-050; R&D Systems) and the corresponding IgG1 isotypes (110-HG-100; R&D Systems).