Cd69 h1.2f3

The following antibodies were used in this study: CD4 (RM4–5; Biolegend), CD8⍺ (53–6.7; Biolegend), Thy1.1 (OX-7; Biolegend), CD44 (1M7; Biolegend), CD69 (H1.2F3; Biolegend), CD103 (2E7; Biolegend), V⍺2 (B20.1; Biolegend), IFNγ (XMG1.2; Biolegend), TNF⍺ (MP6-XT22; Biolegend), IL-2 (JES6–5H4; Biolegend). MHC-II tetramers were obtained from the National Institutes of Health tetramer core facility. Tetramer staining was performed for 1 hour at 37° C and a tetramer loaded with a human CLIP peptide was used as a negative staining control. All other staining was performed for 15–30 minutes at 4° C. Intravascular labeling was performed as described previously (25 (link)). Briefly, 3 μg of anti-V⍺2 was injected i.v. in 200 μl of PBS, and tissues were harvested 3 minutes later. Data were acquired using a LSRII Flow Cytometer (BD) in the OHSU Flow Cytometry Core Facility. Flow cytometry data were analyzed using FlowJo software (BD) version 9.9.6 or 10.5.3.

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).

Anti-mouse antibodies used for flow cytometry include the following: CD95(Jo2), CD138(281–2), CD4(RM4–5), CD38(90), MHC-II(2G9), CD23(B3B4), CD80(B7–1), CD44(IM7), B220(RA3–6B2), CD11b(M1/70), IgM(R6–60.2), IgD(11–26c.2a), CD19(1D3) and Mouse BD Fc block (2.4G2) from BD Biosciences. CD8α(53–6.7), GL7(GL7), CD86 (GL1), CD21(7E9), CD69(H1.2F3), CD25(PC61) and CD73 (Ty/11.8) were from Biolegend. Anti-NFκ-B (D14E12), anti-LSD1 (C69G12) and horseradish peroxidase conjugated anti-rabbit IgG were from Cell Signaling Technology. Alkaline phosphatase conjugated anti-mouse IgG-AP, alkaline phosphatase conjugated anti-mouse IgG2c-AP and anti-mouse IgG(H+L) were from Southern Biotech. PNA-FITC was from Vector laboratories and R848 was from Invivogen.