Immunomagnetic separation beads

Naïve CD4 cells were isolated from C57BL/6J mouse spleens using immunomagnetic separation beads (Miltenyi Biotec, 130-106-643) according to the manufacturer's instructions and seeded into 96-well plates (5 × 10⁵/well) precoated with anti-CD3 antibody (5 μg/ml) (BioLegend, 100309) with the addition of soluble anti-CD28 antibody (2 μg/ml) (BioLegend, 102102). MSCs, sh-NC-MSCs, sh-Chi3l1-MSCs, or sh-Chi3l1-MSCs plus Stattic (20 μM, Selleck, S7024) were seeded into 96-well plates (5 × 10³/well) 6 h before CD4 cell seeding. Th17 differentiation medium contained TGF-β (1.0 ng/ml) (PeproTech, AF-100-21C), IL-6 (30 ng/ml) (PeproTech, 216-16), IL-1β (20 ng/ml) (PeproTech, 211-11B), IL-23 (20 ng/ml) (BioLegend, 589002), anti-IL-4 (10 μg/ml) (BioLegend, 504102), and anti-IFN-γ (10 μg/ml) (BioLegend, 505833).
Th17 cells were differentiated for 72 h and restimulated with Cell Stimulation Cocktail (Tonbo Biosciences, TNB-4975) for 6 h before further analysis for intracellular cytokines by flow cytometry.

CD3⁺ T cells were isolated from the spleen using immunomagnetic separation beads (Miltenyi Biotec, 130-095-130) according to the manufacturer's protocol. CD3⁺ T cells (10⁶/ml in PBS) were labeled with 5 μM CFSE (Invitrogen, C34554) for 10 min at 37°C with gentle vortexing every 5 min. Labeling was terminated by adding a 5-fold volume of RPMI-1640 medium supplemented with 10% FBS.

Naïve splenic CD8⁺ T cells were isolated by immunomagnetic separation beads (Miltenyi Biotec, Bergisch Gladbach, Germany), and stimulated with anti-CD3 and anti-CD28 in the presence or absence of IFNα (2000 U/ml) for 48 h. Total RNA was isolated using Trizol reagent (Ambion). NEBNext^® UltraTM RNA Library Prep Kit for Illumina^® (NEB, USA) was used for generating sequencing libraries, and samples were sequenced on the Illumina HiSeq platform. Sequencing reads were mapped to the mouse reference genome using Hisat2 v2.0.5. edgeR was used for the normalization and identification of differentially expressed genes. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P-value < 0.05 and │log2 (fold change) │> 1 were considered as differentially expressed.