Anti mouse il 12

Naïve CD4⁺ T cells were sorted from the spleens and lymph nodes with the MACS CD4⁺CD62L⁺ T Cell Isolation (30–093-227) Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were cultured under neutral (TH0) conditions in supplemented IMDM medium in the presence of activating antibodies (5 μg/ml plate-coated anti-CD3 and 1 μg/ml soluble anti-CD28) and iTreg polarizing cytokines: TGF-β [5 ng/ml], human IL-2 [20 ng/ml], αIL-4 [2 μg/ml], αIFN-γ [2 μg/ml] and αIL-12 [2 μg/ml].
Recombinant proteins (recombinant human IL-2 and TGF-β) and blocking antibodies (anti-mouse IL-4, anti-mouse IFN-γ, anti-mouse IL-12) for in vitro cell differentiation were purchased from eBioscience (San Diego, California,USA).

RPMI 1640 medium (Biological Industries) containing 10% FCS, antibiotics, sodium pyruvate and non-essential amino acids (all reagents from Sigma Aldrich) was used for all cultures. Various fluorochrome or biotin coupled antibodies to detect mouse CD4, CD25, CD44, CD45.1, CD45.2, CD62L, DO11.10-TCR clonotype (KJ1-26), IFNg, IL-4, IL-5, IL-13, T-bet, GATA-3, TCRValpha2, TCRVbeta5 and human CD4, CD45RO, CCR7, CD25, IFNg, IL-13, T-bet, GATA-3 along with appropriate isotype controls (BD Biosciences, eBioscience, BioLegend, Cell Signaling Technology) were used. Secreted IL-4, IL-13 and IFNg from mouse and human cell cultures were detected using ready enzyme-linked immunosorbent assays (eBiosciences). Cultures were supplemented with recombinant human IL-2 (Roche) where necessary. Dual color ELISpot kits (R&D Systems) were used to detect IFNg and IL-4 producing mouse CD4 cells. Brefeldin, Phorbol Myristate Acetate (PMA) and ionomycin were procured from Sigma. For human and mouse T cell activation in vitro culture grade anti-CD3 and anti-CD28 (eBiosciences) were used. For polarizing cultures recombinant cytokines IL-4, IFNg, IL-12 and antibodies anti-mouse IL-4, anti-mouse IFNg and anti-mouse IL-12 (all from eBiosciences) were used.