Il 23

PBMCs [1x10 (6) cells/mL] were cultured in RPMI-1640 containing 20% FBS and 1% PSG with CD3/CD28 DynaBeads for 48 hrs in the presence or absence of PLX51107 (250nM) or PLX2853 (10nM). On day 3, CD3/CD28 DynaBeads were removed, cells pelleted by centrifugation, resuspended and incubated in RPMI-1640 containing 1% FBS for 4-hour starvation at 37°C. Cells were then stimulated for 15 min with IL-23 (10 ng/mL, Sigma Aldrich) for STAT3 phosphorylation. For baseline level assessment, cells were left unstimulated for 15 min at 37°C. Cells were subsequently fixed with paraformaldehyde (1.5%) for 15 min and stained for the cell surface markers CD3 and CD4. After washing, cells were permeabilized with 90% ice-cold methanol for 30 min in 4°C and stained for the total and phosphorylated STAT3. Cells were analyzed on the LSRII (BD Biosciences) within 24 hours. Data analysis was performed using Flow Jo software (Tree Star).

Naïve CD4⁺ T cells were separated from PBMC samples using naïve CD4⁺ T cell Isolation kit (Miltenyi, Germany) according to the kit’s protocol. The cells were activated with anti-CD28 (2 μg/mL, eBioscience, USA) and anti-CD3 (5 μg/mL, eBioscience, USA). The polarization of Th1/Th2/Th17 was performed for 3 days in the presence of different polarizing conditions (25 (link), 26 (link)). For Th1 polarization, anti–IL-4 (10 μg/ml, eBioscience, USA) and IL-12 (10 ng/mL, Sigma, USA) were applied. For Th2 polarization, IL-4 (2.5 ng/mL, Sigma, USA) and anti–IFN-γ (10 μg/mL, eBioscience, USA) were applied. For Th17 polarization, TGF-β (5 ng/mL, Sigma, USA), IL-6 (10 ng/mL, Sigma, USA), IL-1β (10 ng/mL, Sigma, USA), IL-23 (20 ng/ml, Sigma, USA), anti–IFN-γ (10 μg/ml), and anti–IL-4 (10 μg/ml) were applied. Naïve CD4⁺ T cells were maintained in RPMI 1640 medium (HyClone, USA) supplemented with 10% FBS (HyClone, USA) at 37°C and 5% CO₂ for all experiments. The expression of MALT1 in polarized cells was analyzed, and the naïve CD4⁺ T cells being cultured normally for 3 days were utilized as control.

The polarization of Th1, T-helper 2 (Th2), and Th17 cells was performed as described previously (21 (link), 22 (link)). Briefly, transfected naïve CD4⁺ T cells were stimulated with the following polarizing conditions for 3 days: IL-12 (10 ng/ml, Sigma, USA) and anti–IL-4 (5 μg/ml, Affinity, USA) for Th1 polarization; IL-4 (2.5 ng/ml, Sigma, USA) and anti-IFN-γ (5 μg/ml, Affinity, USA) for Th2 polarization; TGF-β (10 ng/ml, Sigma, USA), IL-6 (10 ng/ml, Sigma, USA), IL-1β (10 ng/ml, Sigma, USA), IL-23 (10 ng/ml, Sigma, USA), anti–IL-4 (5 μg/ml), and anti-IFN-γ (5 μg/ml) for Th17 polarization. After polarization, the cells and supernatants were collected for flow cytometry and ELISA, respectively.