Anti t bet apc

Cells were labelled with flow antibodies for 15 minutes in the dark in PBS containing 2% BSA. Labelled cells were washed twice and resuspended in PBS containing 2% BSA. The prepared samples were analyzed using an LSR-II flow cytometer (BD Bioscience) or sorted using an Aria II cell sorter (BD Bioscience). Anti-CD19-PeCy7 (561739), anti-CD3-PeCy7 (552774), anti-NK1.1-BV421 (562921), anti-NKp46-FITC (560756), anti-NKp46-AF647 (560755), anti-CD49b-PE (553858), anti-CD49a-PerCP-Cy5.5 (564862), anti-CD62L-APC (553152), anti-T-bet-APC (561264), anti-CD45.2-AF700 (560693), anti-CD45.2-FITC (553772), anti-Gata3-AF647 (560068), anti-RORγt-PE (562607), anti-CD127-V450 (561205), anti-CD117-PE (553869), anti-LPAM-1-APC (562376), anti-Flt3-BV421 (566292), anti-Ly-6A/E-APC (565355), and anti-CD122-PE (553362) were purchased from BD Bioscience. Anti-IFN-γ-AF-700 (505823) and anti-CD25-Pacific Blue (102022) were purchased from Biolegend. Anti-CD253-APC (17-5951-82), anti-Eomes-PE (12-4875-82), and anti-CD127-PerCP-Cy5.5 (45-1271-80) were purchased from eBioscience.

PBMCs (2 × 10⁵ cells) were stimulated with M. avium bacilli and M. intracellulare bacilli individually for 48 h and incubated with GolgiPlug (1 μL/mL; BD Pharmingen, San Diego, CA, USA) for the final 5 h of culture. Cells were measured by flow cytometry using a fluorescence activated cell sorter (FACS) (FACSVerse, BD Biosciences, San Jose, CA, USA) and anti-CD3-APC-Cy7, anti-CD4-FITC, anti-IFN-γ-PE, anti-IL-4-PE, anti-IL-17A-APC, anti-CD25-PE, anti-Foxp3-APC, anti-T-bet-APC, anti-GATA3-APC, and anti-RORγT-APC antibodies (BD Pharmingen, San Diego, CA, USA). The PD-1, CTLA-4, and TIM-3 were also stained with anti-PD-1-PE, anti-CTLA-4-APC, and anti-TIM-3- APC (BD Biosciences, San Diego, CA, USA). Data were analyzed using BD FACSuite software (BD Biosciences, San Jose, CA, USA). A gate was set on the lymphocytes using characteristic forward scatter and side scatter parameters followed by CD3⁺CD4⁺ T cells (Supplementary Figure S1).