Anti mouse il 4

MultiScreen filter 96-well-plates (Millipore) were coated with the anti-mouse IL4 or anti-mouse IFNγ capture antibody (R&D Systems) and incubated overnight at 4°C. The plates were then blocked with 200 μL of RPMI-10 medium (RPMI 1640 supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μM 2-mercaptoethanol, and 2 mM L-glutamine) and incubated for 2 h at 37°C. In parallel, spleens were harvested from mice at day 5 post-A2 challenge and prepared to a single cell suspension. The cell suspensions were collected by centrifugation for 10 min at 200 × g and suspended in RPMI-10 at 10⁷ cells/mL. Spleen cell suspensions were added to the wells, and cells were stimulated with either 10 μg/mL RSV M2 _(82−90) peptide, 10 μg/mL RSV F _(51−66) peptide, 10 μg/mL RSV G _(183−198) peptide, or 10 μg/mL eGFP _(200−208) (irrelevant peptide control) for 24 h at 37°C and 5% CO₂. Plates were washed 4 times with wash buffer (0.05% Tween-20 in PBS), anti-mouse IL4 or anti-mouse IFNγ detection antibody (R&D Systems) was added, and plates were incubated overnight at 4°C. Detection antibody was removed, plates were washed, and cytokine spots were developed using NBT/BCIP substrate (R&D Systems). Spots were enumerated using an ELISPOT reader (AID, San Diego).

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).

Mouse spleens were harvested and dissociated into single-cell suspensions. The CD4+ T cells were selected positively using anti-mouse CD4 microbeads (Miltenyi Biotec Inc., Bergisch Gladbach, Germany). The sorted CD4+ T cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Gibco) and stimulated with 1 μg/ml plate-bound anti-mouse CD3 (BD Biosciences), 2 μg/ml anti-mouse CD28 (BD Biosciences), 2 μg/ml anti-mouse IL-4 (R&D Systems, MN, USA), 2 μg/ml anti-mouse interferon-γ (IFN-γ) (R&D Systems), 20 ng/ml recombinant IL-6 (R&D Systems), and 2 ng/ml recombinant transforming growth factor-β1 (TGF-β1) (R&D Systems) for 3 days.