Cd8a bv605

Flow cytometry was performed using fluorescence-conjugates or specific mAb and their controls followed by species-specific conjugate (Supplementary Table 2) using a FACS CantoII flow cytometer (Beckman Coulter) or a LSRFortessa (Becton Dickinson) from the flow cytometer facility Tuebingen. Positive cells in percentage (%) were calculated as follows: Surface expression in percent obtained with the specific antibody—surface expression in percent obtained with isotype control. Platelets were preselected by CD41a⁺ and CD62P⁻ (resting) or CD62P⁺ (activated). Data analysis was performed using FlowJo software (v.10). In order to verify the reproducibility of our flow cytometry system, we performed a Bland–Altman analysis (Supplementary Fig. 8f). For immunophenotyping of PBMC subsets of lung cancer patients and healthy control donors were identified by counterstaining with CD3-PECy5 (BD biosciences, San Diego, CA), CD19-APC/Fire750, CD4-Pacific Blue, CD8a-BV605, CD56-PECy7, CD14-BV785, HLA-DR-BV650 (Biolegend, San Diego, CA) and CD16-FITC (invitrogen). PD-1 and PDL-1 expression as well as activation levels were analyzed using a PD-1-APC or PDL-1-APC and a CD69-PE antibody (BD biosciences), respectively. Isotype controls were obtained from BD biosciences. Dead cells were excluded from analysis with LIVE/DEAD™ Fixable Aqua (Thermo Fisher Scientific, Waltham, MA).

Cells were stimulated for 4 hours at 37°C in RPMI complete containing 1X cell stimulation cocktail (eBioscience) and protein transport inhibitor cocktail (eBioscience). Stimulated cells were harvested and stained for surface markers and viability for 30 min at room temperature. Samples were stained with viability dye APCef780 (ThermoFisher), CD45 evolve655 (ThermoFisher), CD4 BV785 (BioLegend), CD3 eF450 (ThermoFisher), CD8a BV605 (BioLegend), CD335 BV510 (BioLegend) and Gr-1 FITC (eBioscience). Following overnight fixation using the Fixation/Permeabilization solution (eBioscience Foxp3 Staining Buffer Set), cells were permeabilized and stained for cytokines IL-17A AF488 (eBioscience), IL-22 PE (eBioscience) and IFNγ PE (eBioscience). Cells were analyzed using the Aria IIIu cytometer and data was analyzed using the FlowJo software.

Tumor samples were collected individually, and cell suspension was obtained after mechanical dissociation and incubation with collagenase D (Roche, Basiléia, Swiss) at a concentration of 0.22 U/mL per sample at 37 ºC for 40 min. Then, samples were washed twice with PBS 1X (pH 7.4), filtered in a 70 µm cell strainer (BD Biosciences), and resuspended in solutions containing anti-CD45-PerCpCy5.5 (BioLegend), anti-CD11c-PE (BD Biosciences), MHC-II-BV421 (BioLegend), F4/80-FITC (Thermo Fischer Scientific), anti-CD86-APC (BioLegend), anti-CD11b-Alexa-Fluor488 (BioLegend), anti-Gr-1-PE (BD Biosciences), CD8a-BV605 (BioLegend), and anti-IFN-γ-PE (BioLegend) mAbs for 30 min at 4ºC. For analysis of E7-specific intratumoral CD8 T cells, samples were stained with the APC-labeled H-2Db E7-specific dextramer (Immudex, Copenhage, Denmark), and subsequently stained with anti-CD8a-Pacific Blue (BioLegend). After two washes, cells were resuspended in PBS, acquired in a LSR Fortessa flow cytometer (BD Biosciences), and analyzed using the Flow Jo software (BD Biosciences).