Klrg1 2f1

To determine ex vivo T cell functionality from tumor-bearing mice, single cell suspensions from spleen and tumor were obtained and activated in vitro^{30 (link)}. Briefly, mononuclear cells were restimulated with 1X Cell Stimulation Cocktail (eBioscience) in the presence of Golgiplug and Golgistop (BD) according to manufacturer’s instructions in T cell media. 4–5 hours later, cells were stained with live/dead ghost dye at 1:500 (Tonbo) and the following antibodies at diluted in FACs buffer at 1:200 against CD45 (30F-11, BD), CD3 (17A2, Biolegend), CD4 (RM4.5, Tonbo), CD8 (53–6.7, Tonbo), Klrg1 (2F1, eBioscience), and CD44 (IM7, Tonbo) for 30 minutes at 4°C in the dark. Cells were washed 2X in FACs buffer, fixed/permeabilized using the BD cytofix/cytoperm kit (BD) and stained with anti-IFNγ (XMG1.2, Biolegend) diluted 1:100 in perm/wash buffer for 1 h at 4°C. Cells were washed 2X in perm/wash buffer, resuspended in FACs buffer and stored overnight at 4°C in the dark. Cells were acquired the following day on a Fortessa 1770 flow cytometer following the addition of counting beads (Sigma) and analyzed using FlowJo software (version 10).

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).