Cd45.2 104

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).

Cell-surface staining was performed with fluorophore-conjugated antibodies against the following proteins: NK1.1 (PK136, Tonbo), CD11b (M1/70, Tonbo), CD27 (LG.3A10, BioLegend), KLRG1 (2F1, eBioscience), CD69 (H1.2F3, BioLegend), Ly49H (3D10, eBioscience), CD107a (1D4B, BioLegend), CD45.1 (A20, BioLegend), CD45.2 (104, Biolegend), TCRβ (H57–597, BioLegend), IFN-γ (XMG1.2, BioLegend), and Ly49D (4E5, BioLegend). Unless otherwise indicated, NK cells were defined as TCRβ-NK1.1⁺ cells. Intracellular cytokine staining was performed with the Cytofix/Cytoperm Plus Kit (BD). NK cells were enriched from spleens as mentioned above, stained with cell-surface antibodies, and then incubated with various dyes in Hank’s balanced salt solution plus Mg and Ca as follows: 100 nM Mitotracker Green (Life Technologies) for 30 min at 37°C to measure mitochondrial mass, 100 nM TMRE for 30 min at 37°C to measure mitochondrial membrane potential, 5 μm MitoSOX red (Invitrogen) for 15 min at 37°C to measure mitochondria-associated ROS, or 1:400 Cyto-ID autophagy detection reagent (Enzo Life Sciences) for 30 min at 37°C to measure autophagosomes. Flow cytometry and cell sorting were performed on the LSR II and Aria II cytometers (BD Biosciences), respectively. For experiments involving real-time PCR, cell populations were sorted to >95% purity. Data were analyzed with FlowJo software (Tree Star).