Anti mouse cd3 145 2c11

Draining lymph nodes (dLNs) and spleen were triturated and dispersed through a 40 μm strainer and then washed with PBS. After washing, single cell suspension was prepared for flow cytometry analysis [6 (link),33 (link)]. For surface staining, cells were stained by using anti-mouse CD3 (145-2C11, BD Bioscience, San Jose, CA, USA), CD4 (GK1.5, BD Bioscience), CD8 (53-6.7, BD Bioscience), CD11b (M1/70, BD Bioscience), CD11c (N418, BD Bioscience), and Gr-1 (RB6-BC5, BD Bioscience). For intracellular cytokine staining, cells were incubated for 4 hours with PMA (200 ng/mL, Merck, NJ, USA) and ionomycin (500 mg/mL, Merck) in the presence of Golgistop (BD Bioscience). After stimulation, cells were surface stained as described above. Then, cells were re-suspended with 250 μL fixation solution (BD Bioscience) and washed by permeabilization buffer (BD Bioscience). Twenty minutes later, cells were stained with intracellular antibodies, i.e., anti-IFN-γ (XMG1.2, eBioscience, San Diego, CA, USA), anti-IL-17A (TC11-18H10, BD Bioscience), anti-GM-CSF (MPI-22E9, Biolegend, San Diego, CA, USA), and anti-IL-10 (JES5-16E3, Biolegend) antibodies. For Ki-67 (SoIA15, eBioscience) staining, cells were fixed and permeabilized by transcription factor staining buffer set (eBioscience) for nuclear protein staining. Results were analyzed by FlowJo (Tree star Inc., Oakland, CA, USA).

Single-cell suspensions were prepared as described above. Cells were stimulated for 4 h with 50 ng/mL of PMA (Sigma-Aldrich, St. Louis, MO, USA) and 500 mg/mL of ionomycin (Sigma-Aldrich, St. Louis, MO, USA) in the presence of GolgiStop (BD Biosciences, San Diego, CA, USA). After stimulation, the cells were washed and resuspended in a staining buffer, where they were stained with anti-mouse CD3 (145-2C11, BD Bioscience, San Jose, CA, USA), CD4 (GK1.5, Biolegend, San Diego, CA, USA), and CD8 (53-6.7, BD Bioscience, San Jose, CA, USA) antibodies and incubated for 30 min at 4 °C in PBS with 2% FBS. Cells were then fixed and permeabilized with the Cytofix/Cytoperm Solution Kit (BD Biosciences, San Jose, CA, USA) and stained with anti-mouse IFN-γ (XMG1.2, Biolegend, San Diego, CA, USA), IL-17A (TC11-18H10, Biolegend, San Diego, CA, USA), IL-17F (O79-289, Biolegend, San Diego, CA, USA), and GM-CSF (MP1-22E9, Biolegend, San Diego, CA, USA) antibodies for 1 h at room temperature. The results were analyzed using a BD LSRFortessa Cell Analyzer and BD FlowJo (Tree Star Inc., Oakland, CA, USA).

RPMI 1640 medium containing 10% FCS was used for CD4 naive T-cell culture. Antibodies used for cell culture were anti-mouse CD3 (145-2C11) and anti-mouse IL2 (300 u/ml) (BD BioSciences, San Jose, CA, USA).