Anti tim 3 apc

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).

Expression of the various CD19-CARs on T cells was detected using either biotinylated anti-mouse FMC63 scFv monoclonal antibody (Biowan) followed by staining with streptavidin-phycoerythrin (PE) (BD Biosciences), or with an allophycocyanin (APC)-labeled anti-mouse FMC63 scFv monoclonal antibody (Biowan). 1 × 10⁶ cells were stained for cell surface markers to analyze T cell differentiation and exhaust status. The following antibodies were used: anti-CD3-FITC (BD Biosciences), anti-human CD3-APC (BD Biosciences), anti-CCR7-BV421 (BD Biosciences), anti-CD 45RO-APC (BD Biosciences), anti-CD4-BB515, anti-CD8-BV510 (BD Biosciences), anti-CD45RA-PE- Cy7 (BD Biosciences), anti-CD62L-PE (BD Biosciences), anti-PD-1-APC (BD Biosciences), anti-TIM-3APC (BD Biosciences), and anti-LAG-3-APC (BD Biosciences). Samples were analyzed on either FACSCanto II flow cytometers (BD Biosciences) or Novocyte3110 (AECE).