Klrg1 2f1 klrg1

Cells were incubated with αCD16/32 (clone 2.4G2; hybridoma supernatant) and plated on U-bottom 96-well plates at ≤ 3 × 10⁶ cells/well in complete RPMI. Where indicated, cells were stained with Kb-SIINFEKL tetramer APC (NIH tetramer core) at 37°C for 30 min in the presence of αCD8α (53–6.7; Biolegend). For live-dead and surface staining, cells were washed with media, and stained in media for 20 min at RT with Fixable Viability Dye 780 (eBioscience) and surface antibodies for CD19 (6D5, Biolegend), CD8α (53–6.7, Biolegend), CD44 (IM-7; Tonbo), CD127 (A7R34, Tonbo), KLRG1 (2F1/KLRG1, Biolegend), CD45.1 (A20, Biolegend), CD45.2 (104, Biolegend), CD122 (TM-b1, Biolegend). After wash with RPMI, cells were fixed and permeabilized for 45 min at RT in Foxp3/Transcription Factor 1× Fix/Perm solution (Tonbo), followed by wash with 1× Flow Cytometry Perm Buffer (Tonbo) and intracellular staining for 45 min at RT with intracellular antibodies, including TCF1 (C63D9; Cell Signaling Technology), FOXO1 (C29H4, Cell Signaling Technology), T-bet (4B10, Biolegend), EOMES (Dan11mag; eBioscience). The cells were washed twice and resuspended in 1× Flow Cytometry Perm Buffer (Tonbo). Flow cytometry data was acquired on a four-laser (405, 488, 561, 638 nm) CytoFlex S (Beckman Coulter) and analyzed using FlowJo software (BD Biosciences).