Anti mouse cd3 mab

CD4+ T cells were isolated as described above according to the manufacturer’s instructions, then, 4 × 10⁵ of the purified T cells were seeded in 96-well round-bottomed microtiter plates precoated overnight with 5 µg/ml of anti-mouse CD3 mAb (Biolegend, CA, USA) in the presence of 1 µg/ml of anti-mouse CD28 mAb and 1,000 U/ml of mouse IL-2 (Peprotech, Rocky Hill, NJ, USA).
For the activation assay, T cells were collected and stained with a PE-labeled anti-CD69 mAb for 5 h. For the T cell proliferation assay, 10 nM BrdU was added to the plates for 48 h, then incorporated BrdU was detected using a BrdU ELISA kit (BD Biosciences, San Jose, CA, USA) after DNase treatment according to the manufacturer’s protocol. For the apoptosis assay, cells were collected and stained with Annexin V and PI as recommended by the manufacturer (BD Biosciences, San Jose, CA, USA).

Total CD4⁺ naïve T cells were isolated from spleen and lymph nodes by using Magnisort™ Mouse CD4 naïve T cells Enrichment Kit (Invitrogen, CA, USA) according to the manufacturer’s instructions. Twenty-four-well plates were pre-coated with anti-mouse CD3 mAb (5µg/ml; BioLegend, CA, USA) and anti-mouse CD28 mAb (2 µg/ml; BioLegend, CA, USA) at 4°C overnight. For Th17 cell differentiation, 5×10⁵ CD4⁺ T cells were seeded in the 24-well plates and cultured at Th17 polarizing condition medium supplemented with IL-6 (20 ng/ml), TGF-b1 (2 ng/ml), anti-IL4 (10 µg/ml), and anti-IFN-γ (10 µg/ml) for 4 days. For Th1 cell differentiation, equal amount of CD4⁺ T cells were seeded in the 24-well plate and cultured at Th1 polarizing condition medium supplemented with IL-12 (20 ng/ml), anti-IL4 (10 µg/ml) for 4 days. For Treg cell differentiation, equal amounts of CD4⁺ T cells were seeded in the 24-well plates and cultured at Treg polarizing condition medium supplemented with TGF-b1(5 ng/ml) and IL-2 (2 ng/ml) for 4 days. During the differentiation, cells were treated with PTL (3 μM). After the differentiation, cells were collected and detected by FACS. All cytokines were purchased from R&D Systems and BD Biosciences. mRNA expression of Th1-, Treg-, and Th17-related cytokines, transcription factors, and surface receptors was measured by RT-qPCR.

Purified CD4 + T-cells isolated from lymph nodes and spleens of naive mice using a negative CD4 + T-cell isolation kit (Miltenyi Biotec, California, USA). The purified CD4 + T-cells were then labeled with 1 μM CFSE (Invitrogen) for 10 min, and seeded in 96-well plates precoated with 10 μg/mL anti-mouse CD3 mAb and 2 μg/mL anti-mouse CD28 mAb (BioLegend) at a density of 5 × 10⁵ cells per well. Cell growths were analyzed by FACS at 72 h post-culture.