Anti pe microbeads

PBMC were separated from whole blood by Pancoll (PAN-Biotech, Cat# P04-60500) density gradient centrifugation. Sorting was performed using autoMACS Pro System (Miltenyi Biotech) according to manufacturer's instructions. PBMC were stained with CD14-PE antibody (Immunotools, Cat# 21620144, clone 18D11) followed by incubation with anti-PE microbeads (Miltenyi Cat# 130-048-801). The depleted fraction was collected and resuspended in R10 medium – i.e., RPMI 1640 (Sigma-Aldrich) containing 10% heat inactivated FBS (Sigma-Aldrich), 100 U/ml penicillin, 100 U/ml streptomycin and 2 mM L-glutamine (Life Technologies), 1 mM sodium pyruvate, 100 U/ml non-essential amino-acids, 50 μM 2-mercaptoethanol (Invitrogen). 5 × 10⁵ cells/well were seeded in 96-well plates and stimulated with 20 µg/ml cGAMP or 1 µg/ml R848 (Invivogen France). For pDC depletion, PBMCs were stained with PE conjugated anti-BDCA4 (BioLegend Cat# 354504, RRID:AB_11219194) and anti-CD123 (BD Biosciences Cat# 555644, RRID:AB_396001) antibodies, and then sorted using anti-PE microbeads (Miltenyi) as above. Mock-depleted or pDC-depleted PBMCs were stimulated with R848 or ODN M362. Cell depletion was checked by flow cytometry on LSRII cytometer (BD Biosciences, RRID:SCR_002159). Supernatants were collected at 24 h and tested for IFNα by ELISA as described above.

We prepared single-cell suspensions from human lung tumors using the Tumor Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Macrophages from these single-cell suspensions were magnetically sorted using primary antibodies, human CD68-PE (phycoerythrin) (Miltenyi Biotec) and secondary antibody, anti-PE MicroBeads (Miltenyi Biotec) according to the instructions provided by the manufacturer. Patient characteristics are shown in table S3. We fragmented mouse tumor tissues into small pieces, followed by digestion with collagenase (5 μg/μl) supplemented with deoxyribonuclease (DNase; 10 μg/μl) for 30 min at 37°C. Further, we passed the tissue extract through a cell strainer and treated it with red blood cell lysis buffer. Subsequently, we centrifuged and suspended cells in MACS buffer (Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with 5% BSA. We magnetically sorted macrophages from single-cell suspensions using the primary antibody F4/80-PE mouse (Miltenyi Biotec, Bergisch Gladbach, Germany) and the secondary antibody anti-PE MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the instructions provided by the manufacturers.