Anti pd 1 pe cy7

TILs were suspended in AIM-V (Thermo Fisher Scientific, Tokyo, Japan) supplemented with 10% AB serum (Biowest, Nuaillé, France) and then treated with Golgi STOP (1:1000), PMA (50 ng/mL), and ionomycin (1 µM) for 4 h. Harvested cells were stained with Zombie Yellow Fixable Viability kit (BioLegend), anti-CD45-BV785, anti-CD45RA-APC-Cy7 [HI100], anti-PD-1-PE-Cy7, and anti-Tim3-PE. After fixing with Foxp3/transcription factor staining buffer set (Thermo Fisher Scientific), cells were stained with anti-CD3-BV421 [UCTH1], anti-CD4-APC [RPA-T4], CD8-BV711 [RPA-T8], anti-IL-2 [PerCP-eFluor710], anti-TNFα-BV510 [Mab11], and anti-IFNγ-FITC [4 S.B3].

The cetuximab (Bristol-Myers Squibb, New York, NY), trastuzumab (Genentech, San Francisco, CA), pertuzumab (Genentech) and isotype (rituximab, Genentech) antibodies were purchased from the National Institutes of Health Pharmacy. Flow cytometry of haNK cells was performed on a BD LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed in FlowJo 9.9 (TreeStar Inc., Ashland, OR). Flow cytometry of tumor cells was performed on a BD FACSVerse flow cytometer (BD Biosciences) and analyzed in BD FACSuite (BD Biosciences). Staining of haNK cells was performed with four panels, and the antibodies used were: Anti-CD56-PErCP-Cy5.5, anti-CD16-APC-Cy7, anti-NKG2D-APC, anti-NKG2D-FITC, anti-CD107a-PE-Cy7, anti-GranzymeB-FITC, anti-perforin-APC, anti-NKp46-PE-Cy7, anti-NKp30-APC, anti-NKp44-PE, anti-2B4-FITC, anti-CD158a-PE, anti-CD158d-PE, anti-CD158j-APC, anti-PD-1-PE-Cy7, anti-PD-L1-APC, and anti-MICA-PE; all were obtained from BioLegend (San Diego, CA). Anti-CD56-PE, anti-CD16-PE-Cy7, CD11a-PE, Ki67-FITC, anti-HLA-ABC-FITC and anti-4-1BB-BV421 were obtained from BD Biosciences. In controlled experiments to detect CD16 on haNK cells, the anti-CD16 MAb was CD16-FITC (BD Biosciences) and the isotype control antibody was MIgG1k-FITC (BD Biosciences).

Expression of PD-1/PD-L1/PD-L2 was assessed retrospectively in prospectively collected blood samples. The following combination of human monoclonal antibodies were used according to the manufacturers’ instructions to identify different immune cell populations (CD3⁺, CD4⁺ and CD8⁺ T lymphocytes): anti-CD3-FITC, anti-PD-L1-PE, anti-CD8-APC Cy7, anti-PD-1-PE Cy7, anti-PD-L2-APC and anti-CD4-PerCp (BioLegend; San Diego, CA, US). The PBMC fraction was blocked for 5 minutes with a FAB anti-IgG. Samples were incubated for 45 minutes with the appropriate antibodies (2.5 μl) at room temperature and protected from light exposure. After incubation, 2 ml of a 1:1 solution of PBS-Fetal bovine serum (FBS) were added to each sample. Samples were then centrifuged at 1100 rpm for 5 minutes. The supernatant was decanted and cells were fixed in paraformaldehyde (1%).
Gating strategy were set using fluorescence minus one (FMO) for PD-1 /PD-L1 &PD-L2. The samples were acquired in a FACS Aria II Flow Cytometer (BD, Biosciences, San José, Cal, USA) and analyzed with FlowJo software 10.1 (Tree Star. Ashland, Or, USA). The leukocyte population was gated based on morphological parameters on a forward vs side scatter (FSC/SSC) plot.

Flow cytometry of NK cells was performed on a BD LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed in FlowJo 9.9 (TreeStar Inc., Ashland, OR). Staining of NK cells was performed with four panels, and the antibodies used were: anti-CD56-BV605, anti-NKG2D-APC, anti-CD107a-PE-Cy7, anti-GranzymeB-FITC, anti-perforin-APC, anti-NKp46-PE-Cy7, anti-NKp30-APC, anti-NKp44-PE, anti-2B4-FITC, anti-4-1BB-PerCP-Cy5.5, anti-CD95-BV421, anti-Tim3-BV421, anti-TRAIL-PE, anti-CD122-PerCP-Cy5.5, anti-CD122-BV510, anti-CD95L-PE, anti-Ki67-BV421, anti-CD40L-BV510, anti-PD-1-PE-Cy7; all were obtained from BioLegend (San Diego, CA). Anti-CD16-APC-H7, PD-L1-PE-Cy7, and CD11a-FITC, were obtained from BD Biosciences (San Jose, CA). Anti-NKG2A-PE was from R&D Systems (Minneapolis, MN), and anti-CD158a-PerCP-Cy5.5 from eBioscience.