Anti tcrβ antibody h57 597

Naive (CD44^loCD62L^hi) CD4⁺ T cells were purified from the spleens of mice. After lysis of red blood cells, the CD4⁺ T cells were obtained using a CD4⁺ T cell isolation kit with anti-CD4 microbeads (Miltenyi Biotec), and naive CD44^loCD62L^hi cells were then further sorted to more than 99.5% purity using a FACS Aria cell sorter (BD Biosciences). Naive CD4 T cells were plated onto 24-well tissue culture plates (Costar) pre-coated with 10 μg ml⁻¹ agonistic anti-TCRβ antibody (H57-597) with 1 μg ml⁻¹ agonistic anti-CD28 antibody (clone 37.51, Biolegend). Non-polarized Th cell cultures contained IL-2 (15 ng ml⁻¹), anti-IL-4 antibody (BD Biosciences, BVD4-1D11) (1 μg ml⁻¹) and anti-IFNγ antibody (Biolegend, R4-6A2) (1 μg ml⁻¹). Oleic acid (Sigma-Aldrich) was dissolved in DMSO to a final concentration of 100 mM and was complexed to BSA. For lipid uptake experiments, naive CD4 T cells were cultured under indicated conditions in the presence of Bodipy FLC16, Bodipy LDL, or Bodipy 493/503, and mean fluorescence of Bodipy was measured by flow cytometry31 (link)32 (link)33 (link).

Splenic naïve CD4⁺ T cells were obtained by the negative selection using the Mojo Sort Mouse CD4 T Cell Isolation Kit (Biolegend #480006) and positive selection using CD62L MicroBeads, mouse (Miltenyi Biotec #130-049-701). Naïve CD4⁺ T cells were plated onto 24-well tissue culture plates (Costar #3526) pre-coated with 10 mg/ml anti-TCRβ antibody (H57-597, BioLegend) for 2 days. Culture medium contained 1 μg/ml anti-CD28 antibody (clone 37.51, BioLegend) and cytokine that induce differentiation of Th cell subsets. Th0 cell cultures contained 15 ng/ml IL-2 (WAKO), 1 μg/ml anti-IL-4 antibody (BioLegend) and 1 μg/ml anti-IFNγ antibody (BioLegend). Th1 cell cultures contained 15 ng/ml IL-2, 10 ng/ml recombinant mouse IL-12 (WAKO) and 1 μg/ml anti-IL-4 antibody. Th2 cell cultures contained 15 ng/ml IL-2, recombinant mouse 10 ng/ml IL-4 (WAKO) and 1 μg/ml anti-IFNγ antibody. Th17 cell cultures contained 10 ng/ml IL-6 (BD biosciences), 1 ng/ml TGFβ (BD biosciences), 1 μg/ml anti-IL-2 antibody (BioLegend), 1 μg/ml anti-IL-4 antibody and 1 μg/ml anti-IFNγ antibody. Regulatory T cell cultures contained 30 ng/ml IL-2, 10 ng/ml TGFβ, 1 μg/ml anti-IL-4 antibody and 1 μg/ml anti-IFNγ antibody.