Fitc clone eh12.2h7

Flow cytometry was performed on PBMCs from baseline, after Cycle 1, after Cycle 3, and after surgery using an antibody panel designed to identify tumor-related T cells, Effector cytotoxic T lymphocytes (CTLs), and pro-apoptotic T cells. To perform flow cytometry analysis of the phenotype and functional potential of immune cells, the following antibodies were used as per published protocols^{43 (link),44 (link)}: CD8-PE-Cy7 (clone RPA-T8, catalog 304006, BD Pharmingen), CD11a-APC (clone HI111, catalog 301212, BioLegend), PD-1 fluorescein isothiocyanate (FITC) (clone EH12.2H7, catalog 32990, BioLegend), CX3CR1-APC/Cy7 (clone 2A9-1, catalog 341616, BioLegend), Bim-PE (clone C34C5, catalog 12186S, Cell Signaling Technology), NKG7 monoclonal antibody (AG1490 Rb mAb 8H3/8K3)-FITC conjugated (Fusion antibodies with a contract with Dong lab). T cells were stained for surface markers before intracellular staining. Data was collected on a CytoFLEX LX (Beckman Coulter, Atlanta, GA) and analysis was performed with the R software version 4.1.1.

Human primary peripheral blood NK cells were purchased from STEMCELL Technologies (70036) and stimulated with 1 μg ml⁻¹ human IL-2 (PeproTech) overnight in RPMI 1640 plus 10% FCS hi and 1% penicillin–streptomycin. Cells were stained and sorted using anti-CD56 (PerCP/Cy5.5, clone MEM-188, 304625, 1:20 dilution, BioLegend with IgG2a, PerCP/Cy5.5, clone MOPC-173, 400251, 1:20 dilution, BioLegend), anti-SIRPα (APC, clone 15-414, 372105, 1:20 dilution, BioLegend with IgG2a, APC, clone MOPC-173, 400219, 1:20 dilution, BioLegend), anti-CD94 (FITC, clone DX22, 305504, 1:20 dilution, BioLegend with IgG1, FITC, clone MOPC-21, 400107, 1:20 dilution, BioLegend), anti-CD3 (AF488, clone UCHT1, 300415, 1:20 dilution, BioLegend with IgG1, AF488, clone MOPC-21, 400129, 1:20 dilution, BioLegend), anti-ILT2 (APC, clone GHI/75, 333719, 1:20 dilution, BioLegend with IgG2b, APC, clone MPC-11, 400321, 1:20 dilution, BioLegend) and anti-PD-1 (FITC, clone EH12.2H7, 329903, 1:20 dilution, BioLegend with IgG1, FITC, clone MOPC-21, 400107, 1:20 dilution, BioLegend). The sorted population (FACSAria Fusion with FACSDiva 8.0.3, BD Biosciences) was used as effector cells.