Cyto x cytoperm kit

To perform FACS analyses, single-cell suspensions were prepared in PBS with 2% BSA. For the detection of PD-L1 and MHC I in A549 and H522, the suspension cells were stained with PD-L1-PE Cy7 and MHC I-APC (ebioscience). For the evaluation of CD8 + T cells and Tregs, the cells were stained with the following anti-human/mouse antibodies targeting surface markers at 4 °C for 30 minutes: CD8-FITC, 7-AAD, CD4-FITC, CD25-PE, CD107-PE and Foxp3-PE (ebioscience, Biolegend). The expression of IFN-γ and Foxp3 were detected by using BD Cyto x/Cytoperm kit (BD Biosciences) according to the manufacturer's instructions. The stained cells were analyzed using a FACSAria II ow cytometer (BD Biosciences). The data was analyzed with FlowJo software.

BMDCs were seeded in 6-well plates at 10 6 cell/well in 450 μL antibiotic-free RPMI. Where appropriate, cells were preincubated with anti-LTA antibody (5 μg/mL) for 1 h, then 50 μL sample were added to BMDCs and incubated at 37°C for 18 h. Cells were suspended by cell scraper, washed, Fc blocked for 15 min, then incubated for 30 min with anti-CD11c-PerCP-Cy5.5 (1:200; Invitrogen). After washing, cells were xed and permeabilized with BD Cyto x/Cytoperm kit (BD Biosciences) per manufacturer's directions. Finally, BMDCs were incubated for 30 min with anti-IL-10-PE (1:200; Invitrogen) to label intracellular cytokines and analysed by ow cytometry.

Immuno uorescence surface staining was performed by adding a panel of directly conjugated Abs to freshly prepared BMMCs and PBMCs. Dead cells were excluded from the analysis using a viability dye (Zombie AquaFixable viability dye or 7-AAD). After surface staining, cells were permeabilized using the Cyto x/Cytoperm kit (BD Pharmingen), and incubated with intracellular Abs. Cells were washed and measured using a FACSCanto II (BD Biosciences). Flow cytometry data were analysed using FlowJo v10 software.
To analyze IFNγ and TNF production, both BMMCs and PBMCs were stimulated for 4h at 37°C with 30 ng/ml PMA and 500 ng/ml ionomycin in the presence of 10 mg/ml brefeldin A (BFA; Sigma Aldrich). The production of IL-15 and IL-6 was assessed as previously described (14) . In summary, BMMCs were incubated for 12h in the presence of 10 mg/ml brefeldin A. IL-15 and IL-6 mean uorescence intensity (MFI) was measured with intracellular staining in the whole BMMC population.
The complete list of antibodies used for the experiments is shown in Suppl. Table 1.