Anti mouse ifn γ

Naïve CD4+ T cells were isolated from WT and CerS2 null spleen using magnetic beads, using the naïve CD4+ T cell isolation kit for mice (Miltenyi Biotec) according to manufacturer’s instructions. Isolated cells were primed with plate-bound anti-CD3 (5 μg/mL; BD Biosciences, San Jose, CA, USA) and soluble anti-CD28 (1 μg/mL; BD Biosciences) in round bottomed 96-well plates with RPMI1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin (HyClone, Logan, UT, USA). For Th1 differentiation, the cells were cultured with recombinant murine IL-2 (20 ng/mL, R&D Systems, Minneapolis, MN, USA), recombinant murine IL-12 (10 ng/mL, PeproTech, Rocky Hill, NJ, USA) and 2 μg/mL anti-mouse IL-4 (2 μg/mL, R&D systems) for 48 h. For Th2 differentiation, the cells were stimulated with recombinant murine IL-2 (20 ng/mL; R&D Systems), recombinant murine IL-4 (10 ng/mL; BioLegend, San Diego, CA, USA), anti-mouse IFN-γ (2 μg/mL; R&D Systems), and anti-mouse IL-12 (2 μg/mL; R&D Systems). For Th17 differentiation, the cells were cultured with recombinant murine IL-2 (20 ng/mL; R&D Systems), recombinant murine TGF-β (5 ng/mL; BioLegend), recombinant murine IL-6 (25 ng/mL; PeproTech), anti-mouse IFN-γ (2 μg/mL; R&D Systems), and anti-mouse IL-4 (2 μg/mL; R&D Systems).

Cell culture supernatants collected at 48h post infection were aliquoted into 200 μL volumes and frozen at -80°C. An ELISA test was conducted for IFN-γ, IL-6 and TNF-α according to the manufacturer’s instructions (R&D Biosystems). Briefly, wells of Nunc Maxisorp 96-well plates were coated with anti-mouse IL-6 (2 μg/ml), anti-mouse IFN-γ (4 μg/ml) or anti-mouse TNF-α (0.8 μg/ml) (R&D BioSystems) overnight at room temperature. Before use, the plates were blocked with PBS containing 1% bovine serum albumin fraction V (BSA, Sigma) for 2 h at room temperature. Cell culture supernatant samples were added to the wells in a volume of 100 μl and the control samples were diluted in PBS 1% BSA. After the 2 h incubation at room temperature, wells were washed 4 times with PBS containing 0.05% Tween 20. The addition of the biotinylated monoclonal antibodies for each of the cytokines (IL-6 150ng/ml; IFN-γ 300ng/ml; TNF-α 50ng/ml) were added and incubated for 2 h at room temperature. Horseradish peroxidase-conjugated streptavidin (R&D Biosystems) was added according to the manufacturer’s recommendations and incubated for 30 min at room temperature. Standard curves were generated using purified recombinant IL-6, IFN–γ and TNF-α according to the manufacturer’s recommendations (R&D BioSystems) using MaxPro generated 4-parameter curve-fit for each cytokine.

CD4⁺ T cells were isolated from mouse splenocytes (SPL) using anti-mouse CD4 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The sorted CD4⁺ T cells were stimulated with plate-bound anti-mouse CD3 (BD Biosciences, CA, USA) and anti-mouse CD28 (BD) and cultured with anti-mouse IFN-γ (R&D Systems, MN, USA) and IL-4 (R&D Systems) to block Th1 and Th2 differentiation, respectively. Th17 differentiation was induced by culture with recombinant IL-6 (R&D Systems) and recombinant transforming growth factor-β1 (R&D Systems) for 3 days.