Pd 1 pe dazzle 594

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).

Following blocking with FcR blocker, PBMCs, TILs, and NILs were stained with Fixable Viability Dye eFluor^® 660 (FVD 660; eBioscience) according to the manufacturer’s protocol to gate for live cells. Cells were then surface labeled with antibodies CD3-APC-H7 (BD Biosciences), CD4-Alexa Fluor 700 (BioLegend), LAP-PE (clone TW4-2F8; BioLegend), and PD-1- PE/Dazzle™ 594 (BioLegend) for 30 min at 4°C. After washing with staining solution, cells were fixed and permeabilized using fixation/permeabilization buffer (eBioscience) at 4°C for 45 min after thorough vortexing. Cells were then washed twice with permeabilization wash buffer (eBioscience) and blocked using mouse serum (Sigma-Aldrich) and rat serum (eBioscience) for 10 min. Cells were then stained with antibodies CTLA-4-PerCP-eFluor^® 710 (clone 14D3; eBioscience), FoxP3-PE-Cyanine7 (clone PCH101, eBioscience), and Helios-FITC (clone 22F6, BioLegend) for 30 min at 4°C. Cells were washed twice with permeabilization wash buffer (eBioscience) and were resuspended in flow cytometry staining buffer for flow cytometric analyses. Data were acquired with a BD FACSCanto II flow cytometer using BD FACSDiva software (BD Bioscience) and analyzed on BD FACSuite software (BD Biosciences). Out of 12 CRC samples, we did not consider intracellular staining data from 2 samples since no cell viability dye was used for these.