Anti cd3 percp clone sk7

The following fluorochrome-conjugated monoclonal antibodies (mAbs) were used to evaluate NKA by flow cytometry: anti-CD3-PerCP (clone SK7, BD Bioscience, San Jose, CA, USA), anti-CD56-PE (clone NCAM16.2, BD Bioscience, San Jose, CA, USA), anti-CD107a/LAMP1-FITC (clone H4A3, BD Bioscience, San Jose, CA, USA), and anti-IFN-γ-FITC (clone 25723.11, BD Bioscience, San Jose, CA, USA). The following fluorochrome-conjugated mAbs were used for the analysis of NKG2D and 2B4 on NK cells: anti-CD3-PerCP (clone SK7, BD Bioscience, San Jose, CA, USA), anti-CD56-FITC (clone NCAM16.2, BD Bioscience, San Jose, CA, USA), anti-CD314/NKG2D-PE (clone 149810, R&D Systems, Minneapolis, MN, USA), anti-CD244/2B4-PE (clone C1.7, BD Bioscience), and mouse IgG1 isotype control (clone MOPC-21, BD Bioscience, San Jose, CA, USA).

HLA-A2 PE-labeled tetramers loaded with TcTLE peptide (TLEEFSAKL), derived from T. cruzi KMP-11 protein [22 (link)], and also HLA-A2 PE-labeled tetramers with a modified Flu-MP peptide (Flu-MP*; GILGFVTTL) derived from the influenza virus matrix protein [22 (link),26 (link)], and a modified CMV pp65 peptide (CMV*; DLSPMVATV), employed as controls, were produced by the National Institute of Health (NIH) Tetramer Facility (Atlanta, GA, USA). In total, 1 × 10⁶ PBMCs per tube were stained with 0.5 μg/mL tetramers and anti-CD3-PerCP (clone SK7) and anti-CD8-FITC (clone SK1) conjugates (BD Biosciences, San José, CA, USA) for 20 minutes in the dark at room temperature. After the cells were washed with staining buffer (1% fetal bovine serum in 1X PBS), they were resuspended in 500 μL of 1X PBS. At least 50,000 events gated for CD3⁺ CD8⁺ T cells were acquired and analyzed using a FACSAria II flow cytometer (BD Immunocytometry Systems) and FlowJo 9.3.2 software (Tree Star, Inc.). The gating strategy is shown in Fig 1A and 1B. The cut-off point for HLA-A2/TcTLE tetramer CD8⁺ T cells was established at 0.063% after determining the average background value of K1-specific CD8⁺ T cells from HD (0.027%) plus three standard deviations (0.012%). A cut-off point for HLA-A2/Flu-MP* and HLA-A2/CMV* tetramer CD8⁺ T cells was fixed at 0.1% [26 (link)].

Changes in TIGIT expression levels on CD226⁺ NK cells were detected by flow cytometry. For staining, PBMCs were seeded in 96-well round-bottom plates (50,000–60,000 per well) and stimulated with IL-10 (10 ng/mL; R&D Systems), IL-12 + IL-15 (10 ng/mL and 50 ng/mL, respectively), or transforming growth factor-beta (TGF-β) (50 ng/mL; R&D Systems) respectively. Unstimulated cells were used as negative controls. Cells were incubated in RPMI media containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% penicillin/streptomycin (PS) for 24 h at 37°C with 5% CO₂ in a total volume of 200 μL, then harvested, washed, and stained with anti-CD3-PerCP (clone SK7; BD Biosciences), anti-CD56-PE-Cy7 (clone B159; BD Biosciences), anti-CD16-APC-Cy7 (clone 3G8; BD Biosciences), and anti-TIGIT-APC (clone A15153G; Biolegend) in staining buffer on ice in the dark for 20 min. APC mouse IgG1, κ isotype control (clone MOPC-21, Biolegend) was used to define gates. Cells were then washed twice with PBS containing 2% FBS, re-suspended in staining buffer, and analyzed using a BD FACS Canto II cytometer (BD Biosciences).