Multiparameter flow cytometry
tumor
TUMOR, APC
Fresh uninvolved tumor-adjacent normal lung and tumor tissues collected at surgery were disaggregated using the BD Medimachine System (BD Biosciences) to make a single-cell suspension for flow cytometry staining. Disaggregated cells were Fc-blocked using 5% goat serum (G9023, Sigma-Aldrich) for 30 minutes on ice. Surface staining was performed in 1× DPBS with 1% BSA (A8577, Sigma-Aldrich) for 30 minutes on ice using fluorochrome-conjugated monoclonal antibodies against CD45 (BUV395, clone HI30, 563792, BD Biosciences, 5 µl per sample), CD3 (PerCP-Cy5.5, clone SK7, 340949, BD Biosciences, 10 µl per sample), CD8 (AF700, clone RPA-T8, 557945, BD Biosciences, 5 µl per sample), CD4 (BUV496, clone SK3, 612936, BD Biosciences, 5 µl per sample), PD-1 (SB645, clone MIH4, 64-9969-42, eBioscience, 4 µl per sample), TIM3 (BV605, clone F38-2E2, 345018, BioLegend, 4 µl per sample), CD103 (BV711, clone Ber-Act8, 563162, BD Biosciences, 5 µl per sample), CTLA-4 (BV786, clone BNI3, 563931, BD Biosciences, 3 µl per sample), GITR (AF488, clone eBioAITR, 53-5875-42, eBioscience, 5 µl per sample), LAG3 (PE, clone 3DS223H, 2-2239-42, eBioscience, 5 µl per sample), CD56 (PE-Cy7, clone B159, 557747, BD Biosciences, 5 µl per sample), ICOS (BV421, clone C398.A4, 313524, BioLegend, 5 µl per sample) and CD25 (APCFire/750, clone BC96, 302642, BioLegend, 5 µl per sample). After surface staining, cells were fixed and permeabilized using eBioscience Foxp3/Transcription Factor Staining Buffer Set (00-5523-00, Thermo Fisher Scientific) according to the manufacturer’s instructions and stained using FOXP3 (PE-eFluor610, clone PCH101, 61-4776-42, eBioscience, 5 µl per sample) and Ki67 (APC, clone 20Raj1, 17-5699-42, eBioscience, 5 µl per sample) anti-human antibodies. For the memory panel, Fc-blocked cells were surface stained for 30 minutes on ice using monoclonal antibodies against CD27 (FITC, clone M-T271, 555440, BD Biosciences, 20 µl per sample), CCR7 (PerCP-Cy5.5, clone 150503, 561144, BD Biosciences, 5 µl per sample), CD45RA (V450, clone HI100, 560362, BD Bioscience, 5 µl per sample), CD3 (APC, clone UCHT1, 555335, BD Biosciences, 20 µl per sample), CD4 (BUV496, clone SK3, 612936, BD Biosciences, 5 µl per sample), CD8 (AF700, clone RPA-T8, 557945, BD Biosciences, 5 µl per sample), CD45RO (APC-H7, clone UCHL1, 561137, BD Biosciences, 5 µl per sample), BTLA (PE, clone J168-540, 558485, BD Biosciences, 5 µl per sample) and CD28 (PE-Cy7, clone CD28.2, 560684, BD Biosciences, 5 µl per sample). For the functional panel, Fc-blocked cells were surface stained for 30 minutes on ice using monoclonal antibodies against PD-1 (PerCP-Cy5.5, clone EH12, 329914, BioLegend, 5 µl per sample), TIM3 (APC, clone F38-2E2, 17-3109-42, eBioscience, 5 µl per sample), CD8 (APC-Cy7, clone RPA-T8, 557760, BD Biosciences, 3 µl per sample) and CD3 (PE-Cy7, clone UCHT1, 563423 BD Biosciences, 5 µl per sample). After staining, cells were fixed using the BD Fix/Perm buffer solution from the Fixation/Permeabilization Kit (554714, BD Biosciences) by incubating them for 20 minutes in the dark at room temperatures. Cells were then washed and stained with monoclonal antibodies against perforin (FITC, clone DG9, 11-9994-42, eBiosciences, 5 µl per sample), granzyme B (V450, clone GB11, 561151, BD Biosciences, 5 µl per sample) and IFNγ (PE, clone B27, 559327, BD Biosciences, 10 µl per sample) anti-human antibodies using the BD Perm Buffer I solution that was diluted with water according to the manufacturer’s instructions. Cells were stained for 30 minutes on ice for intracellular markers. Dead cells were stained using LIVE/DEAD Fixable Yellow Dead Cell Stain dye (L-34968, Life Technologies, 1 µl per sample) and excluded from the analysis. Data were acquired using the BD Fortessa X20 or Canto II (BD Bioscience) with BD FACSDiva software version 8.0.1 and analyzed using FlowJo software version 10.5.3 (Tree Star). Experiments and gating related to the presented results were conducted once. Detailed information on flow cytometry antibody panels is provided in Supplementary Table 