Anti il 4 neutralizing antibody

Pooled spleen and lymph nodes from C57BL/6 mice were homogenized through a 40 µm filter and subjected to magnetic enrichment of naive CD4+ T cells using the eBioscience MagniSort mouse naive T cell kit (eBioscience/Invitrogen/ThermoFisher). Cells were grown in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with glutamine, penicillin, streptomycin, gentamicin, 2-mercaptoethanol, and 10% FBS. Purified naive T cells were cultured on anti-hamster IgG-coated plates in the presence of hamster anti-CD3 epsilon and anti-CD28 antibodies (eBioscience), neutralizing anti-IL-4 antibody (eBioscience), recombinant IL-12 (10 ng/mL), and recombinant IL-2 (50 U/mL) for 72 h. Cells were cultured in the presence of serially diluted FK506 or APX879 suspended in DMSO during these 72 h. During the last 4 h of culture, phorbol 12-myristate 13-acetate (PMA; Sigma), ionomycin (Sigma), and GolgiStop (BD Biosciences) were added to the culture to facilitate detection of intracellular cytokines.

Naïve CD4⁺ T cells were sorted from spleen and lymph nodes using the MACS CD4⁺CD62L⁺ T Cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were cultured in supplemented IMDM medium in the presence of 5 µg/ml plate-coated anti-CD3 (produced in house) and 1 µg/ml soluble anti-CD28 antibodies (BD Bioscience, San Jose, California USA).The following conditions were used: Th0 – no further cytokines or antibodies; Th17 -TGF-β [5 ng/ml], IL-6 [20 ng/ml], IL-23 [10 ng/ml], neutralizing anti-IL-4 [2 µg/ml] and anti-IFN-γ [2 µg/ml] antibodies; Th1 - IL-12 [10 ng/ml], neutralizing anti-IL-4 antibody [5 µg/ml] (eBioscience, San Diego, California, USA). Where indicated, concentration of secreted cytokines in culture supernatants was measured by Bioplex System (Bio-Rad, Hercules, California, USA), according to the manufacturer's instructions.