Cd19 apc fire 750

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).

After 2–4 days, B cells were gently harvested and incubated with fluorochrome-labeled detection antibodies for 30 min at 4 °C in the dark, washed two times, and resuspended in 400 µL washing buffer (PBS containing 0.05% bovine serum albumin). Fluorescence was detected with a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA) and analyzed with Kaluza software from Beckman Coulter. The following antibodies were used for detection: BTLA Brilliant Violet 421 (344512), CD19 APC-Fire 750 (302257), GITR Brilliant Violet 421 (371207), OX40 PE-Cy5 (350009) and TIM-3 Pacific Blue all from Biolegend (San Diego, CA, US), CD137 PE (12-1379-42), CD27 PE-Cy5 (15-0279-42), CD39 PE-Cy7 (25-0399-42), CD73 FITC (11-0739-42), LAG3 PE (12-2239-42) and PD-1 PE (12-2799-41) from eBioscience (San Diego, CA, US), CD86 Alexa Fluor 700 (561124) and CTLA4 PE-Cy5 (561717) from BD Biosciences (Franklin Lakes, NJ, USA).