Tigit buv395

PBMC was stained using Zombie Live/dead fixable cell stain (Thermo Fisher Scientific, Waltham, MA, USA) prior to staining with specific antibodies. Antibodies used were CD3 BUV496 (BD Bioscience), CD19 PeCy7 (BD Bioscience), CD56 BB700 (BD Bioscience), CD16 BV650 (BioLegend), CD96 BV421 (BD Bioscience), TIGIT BUV395 (BD Bioscience), DNAM1 (CD226) BUV805 (BD Bioscience). PBMC was stained in Live/Dead cell stain for 15 min in PBS at RT, washed in FACS buffer (PBS + 2% FCS), and blocked in Human Fc block (BD Bioscience) for five minutes at RT. Cell staining was performed in FACS buffer for 30 min at 4 °C, followed by two washes in FACS buffer, and fixation in 2% paraformaldehyde (PFA, Electron Microscopy Sciences). Samples were acquired on a LSR Fortessa (BD Bioscience) flow cytometer and analysed using FlowJo software (BD Bioscience). Flow cytometry gating is shown in Additional file 1: Fig. S1. NK cells were defined as CD3-CD19-CD56 + viable single lymphocytes. NK cell subsets were defined as mature (CD16 + CD56mid), regulatory (CD16-CD56mid) and immature (CD16-CD56hi). Expression of DNAM1, TIGIT and CD96 was calculated using the geometric mean of the mature, regulatory and immature NK cell subsets.

PBLs and cancer cells from direct and indirect co‐cultures were used for multi‐marker flow cytometry to detect the expression of immune checkpoint markers. Cells were washed and resuspended in 100 μl of staining buffer containing Human Fc Block™ (564219, BD Biosciences). The expression of immune checkpoint receptors was determined using the following antibodies: PD‐1 PE‐Dazzle 594 (329940, Biolegend), VISTA BV421 (566750, BD Biosciences), CTLA‐4 BV786 (563931, BD Biosciences), TIM‐3 BV650 (565565, BD Biosciences), LAG‐3 PE (565617, BD Biosciences), TIGIT BUV395 (747845, BD Biosciences) and CD8 BV510 (563919, BD Biosciences). In parallel, we determined the expression of respective ligands: CD80 BV510 (740150, BD Biosciences), CD86 Alexa700 (564544, BD Biosciences), PD‐L1 PE‐Cy7 (558017, BD Biosciences), PD‐L2 BV786 (563843, BD Biosciences), VISTA BV421 (566750, BD Biosciences), MHC‐II BV650 (564231, BD Biosciences), GAL‐9 PE (565890, BD Biosciences) and PVR BUV395 (748272, BD Biosciences). Flow cytometry was performed by recording 50 000 events/sample using the LSRFortessa X‐20 instrument and FlowJo V10.7.1 software (BD Biosciences).