T Cell Cytokine and Phospho-S6 Analysis

The cells were differentiated in vitro and stimulated with an immobilized anti-TCR-β mAb (3 μg/ml, H57-597; BioLegend) for 6 h with monensin (2 μM, cat# M5273; Sigma-Aldrich, St. Louis, MO, USA) for the intracellular staining of cytokines. Intracellular staining was then performed as described previously^{27 (link),52 (link)}. For the intracellular staining of phosphorylated S6 ribosomal proteins, the CD8 T cells were fixed and permeabilized with BD Phosflow Lyse/Fix Buffer (cat# 558049; BD Biosciences) and BD Phosflow Perm III (cat# 558050; BD Biosciences) in accordance with the manufacturer’s instructions. Flow cytometry (FACS) was performed using a FACS Caliber instrument (BD Biosciences) and Gallios instrument (Beckman Coulter, CA, USA), and the results were analyzed using the FlowJo software program (Tree Star, Ashland, OR, USA).