Cd27 lg 3a10

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).

Lungs were perfused with PBS, diced into ~2-cm pieces and incubated with Liberase TM (42.4 μg/ml) and DNAse I (10 U/ml; both from Roche) for 45 min at 37 °C before being mashed through a 70-μM cell strainer and washed with complete RPMI. Remaining blood cells were lysed with ACK cell lysing buffer (Invitrogen) and single cell suspensions were incubated with biotinylated antibodies to CD3ε (clone 145-2C11), CD19 (1D3), B220 (RA3-6B2), CD5 (53-7.3), TCRβ (H57-597), TCRγδ (GL3), CD11c (N418), F4/80 (BM8), Gr-1 (RB6-8C5), Ter119 (TER-119), CD49b (DX5; all eBioscience) and CD27 (LG3A10, BioLegend) all at 25 μg/ml per 2 × 10⁸ cells. Cells were then incubated with anti-biotin microbeads (Miltenyi; 1:5 dilution at 10⁸ cells per ml) and depleted following manufacturer’s protocol. For each ILC enrichment, the depletion was repeated twice and yielded >95% pure ILC populations (Supplementary Fig. 3a).