Anti cd25 antibody

C57BL/6 mice were purchased from Clea (Clea Japan, Inc., Tokyo, Japan). Total CD4⁺ T cells isolated from mouse spleen were prepared using a FITC anti-mouse CD4 antibody (Cat#100510, RM4–5, BioLegend) and anti-FITC-microbeads (Cat#130-097-050, Miltenyi Biotec). Naive CD4⁺ T cells (CD44^low, CD62L^high) were prepared using a MojoSort Mouse CD4 T cell Isolation Kit (Cat#480033), BioLegend). Biotinylated anti-CD44 (Cat#103004, IM7) and anti-CD25 antibodies (Cat#102004, PC61) were purchased from BioLegend. CD4⁺ T cells (7.5 × 10⁵) were stimulated using immobilized anti-TCR-β mAb (3 µg/mL, H57–597, BioLegend) plus an anti-CD28 mAb (1.0 µg/mL, cat#40–0281, TONBO Biosciences) for 2 days, and then, the cells were further expanded under similar conditions for an additional 3 days. IL-2 conditions: IL-2 (10 ng/mL, Pepro Tech); Th2 conditions: IL-2 (10 ng/mL, Pepro Tech), IL-4 (1 ng/mL, Pepro Tech), and anti-IFN-γ mAb (2.5 µg/mL, cat#40–731, TONBO Biosciences). Th1 conditions: IL-2 (10 ng/mL, Pepro Tech), IL-12 (1 ng/mL, Pepro Tech), and anti-IL-4 mAb (2.5 µg/mL, cat#40–731, TONBO Biosciences). The cultured cells were subjected to intracellular staining, ELISA assay, and RT-PCR. All animal experiments received approval from the Ehime University Administrative Panel for Animal Care. All animal care was conducted in accordance with the guidelines of Ehime University.

PBMCs were isolated from AIH patients and healthy volunteers as mentioned above. In addition, single-cell suspensions were obtained by mechanical disruption of mouse spleens through 70-μm cell strainers. Erythrocytes were lysed using lysis buffer. Then, the freshly collected cells were resuspended in 100 μl of PBS and incubated with anti-CD3, anti-CD4, and anti-CD8a antibodies (BioLegend, San Diego, CA, United States ) at 4°C for 30 min. To detect Treg subpopulations, intracellular staining was performed using a transcription factor buffer set (BioLegend, San Diego, CA, United States ). After staining with anti-CD4 and anti-CD25 antibodies, the cells were fixed, permeabilized, and incubated with an anti-FOXP3 antibody (BioLegend, San Diego, CA, United States ). The frequencies of CD4+ CD25+ FOXP3+ Tregs, CD3+ CD4+ T cells, and CD3+ CD8a+ T cells were examined using a flow cytometer (Beckman Coulter, Brea, CA, United States).