Mp4 25d2

MMCP-1 in serum samples was measured according to the manufacturer’s instructions (eBioscience). OVA-specific IgE and IgG1 were assessed as previously described, using clones TOε and TOSG1C6 (BioLegend, San Diego, CA) as standards^{24 (link)}. The sandwich ELISA for murine IL-4 was constructed using 11B11 to capture and biotinylated BVD6-24G2 to detect (BioLegend, San Diego, CA) with recombinant protein standard from Shenandoah Biotechnology (Warwick, PA). Murine IL-13 was detected using paired antibodies from Affymetrix/eBioscience (San Diego, CA): eBio13A and biotinylated eBio1316H. Human IL-4 was detected using the antibody pair 8D4-8 and biotinylated MP4-25D2 (BioLegend, San Diego, CA).
Detection of IL-4 production from murine mast cells was enabled by culture in the presence of azide-free anti-IL-4Rα (10μg/ml, clone mIL4R-M1, BD Biosciences, Franklin Lakes, NJ) to block cellular uptake of secreted IL-4. Similarly, IL-4 reuptake by human mast cells was inhibited with anti-IL-4Rα (clone 25463, R&D Systems/biotechne, Minneapolis, MN).

Human peripheral blood was obtained from healthy volunteers who are not taking any medications. PBMCs were prepared by using lymphocyte separation medium (MP Biomedicals), according to the manufacturer’s instructions. Subsequently, total CD4⁺ T cells were isolated by means of negative selection with CD14 microbeads (Miltenyi Biotec), followed by positive selection with CD4 microbeads (Miltenyi Biotec). Naive and effector memory CD4⁺ T cells were further sorted as CD3⁺CD4⁺ CD45RA⁺CD45RO⁻CD25⁻ and CD3⁺CD4⁺CD45RA⁻CD45RO⁺ cells, respectively, by using the FACSAria III cell sorter (BD Biosciences). For naive T-cell differentiation, naive T cells undergoing fluorescence-activated cell sorting (FACS) were stimulated with plate-coated anti-CD3 (10 μg/mL, OKT3; BioLegend) and soluble anti-CD28 (2 μg/mL, CD28.2; BioLegend) for 4 or 7 days. For T_H2 polarization, IL-2 (10 ng/mL), IL-4 (10 ng/mL), and anti–IFN-γ (5 μg/mL, B27; BioLegend) were added. For T_H17 polarization, IL-2 (10 ng/mL), IL-6 (20 ng/mL), TGF-β (10 ng/mL), IL-23 (20 ng/mL), IL-21 (20 ng/mL), IL-1β (20 ng/mL), anti–IFN-γ (5 μg/mL), and anti–IL-4 (5 μg/mL, MP4-25D2; BioLegend) were added. TGF-β was from PeproTech, and the rest of the cytokines were from eBioscience. Effector memory CD4⁺ T cells were stimulated with plate-coated anti-CD3 (10 μg/mL) for 48 hours in the presence of UA or DMSO.

Blood was obtained from volunteers (patients with asthma and healthy controls). UK HRA approval was granted following Research Ethics Committee (North West-Liverpool Central) review and written consent obtained from volunteers. Peripheral blood naïve CD4⁺ T cells were purified using the naive CD4+ T cell Isolation Kit II (Miltenyi Biotec, 130-094-131) according to the manufacturer’s instructions. 250,000 splenic naïve CD4⁺ T cells per well were cultured on anti-CD3 coated plates (clone OKT3, 5 μg ml^-1, BioLegend 317325) supplemented with anti-CD28 (clone CD28.2, 2 μg ml^-1, BioLegend 302933) and IL-2 (10 ng ml^-1, R&D 204-IL-010), and additionally IL-4 (12.5 ng ml^-1, R&D 202-IL-010) and anti-IFNγ neutralising antibody (clone B27, 1 μg ml^-1, BioLegend, 506531) in T_H2 conditions, or IL-12 (2.5 ng ml^-1, R&D 219-IL-005) and anti-IL-4 neutralising antibody (clone MP425D2, 1 μg ml^-1, BioLegend, 500837) in T_H1 conditions. Cells were cultured for 4 weeks with a weekly stimulation (4 days) and rest (3 days) schedule.