7 aad viability staining solution

Cytotoxic activity of CD4⁺ HTL lines was measured by flow cytometry using the BD Accuri C6 flow cytometer and software (BD Biosciences). HSC-4, Lu65 (matched HLA-DR with PD-L1-specific HTLs), and SAS (unmatched HLA-DR with PD-L1-specific HTLs) were labeled by using the CellTrace™ CFSE Cell Proliferation Kit (Life Technologies) after pretreatment with or without IFN-γ (500 U/ml) for 24 h. After co-culture with PD-L1-specific HTLs for 6 h, tumor cells were collected, and dead cells were detected with 7-AAD viability staining solution (BioLegend). Cytotoxicity of PD-L1-specific HTLs was assessed in various effector/target cell (E:T) ratios (0:1, 10:1, and 30:1).

1 × 10⁵ cells were used for each staining, including a non-stained sample for each set of measurements. Briefly, cells were washed twice in PBS (Sigma-Aldrich, Munich, Germany) and incubated for 10 min at 4 °C in 40 μL blocking buffer, composed of 39 μL 1% Bovine Serum Albumin (BSA) (Sigma-Aldrich, Munich, Germany) in PBS (Sigma-Aldrich, Munich, Germany) and 1 μL FcR human blocking reagent (Miltenyi, Bergisch Gladbach, Germany). Cells were then incubated with conjugated antibodies (PE anti-human CD235a (Glycophorin A) Antibody (clone HIR2), FITC anti-human CD71 Antibody (clone CY1G4), FITC anti-mouse/human CD44 Antibody (clone IM7), FITC anti-human CD49d Antibody (clone 9F10), all from Biolegend, San Diego, CA, USA, and at concentrations suggested by the manufacturer) for 30 min at 4 °C in the dark and analyzed using a CyFlow Cube 8 6-channel instrument (Sysmex Partec, Münster, Germany). For cell death assessment, cells were incubated for 10 min at 4 °C in the dark with 7-AAD Viability Staining Solution (Biolegend, San Diego, CA, USA) according to the manufacturer’s instructions. A minimum of 0.5 × 10⁵ cellular events was recorded and data were analyzed and visualized using FCS Express 4 (DeNovo Software).

Phorbol 12-myristate 13-acetate (PMA), ionomycin, and Brefeldin A (BFA) were obtained from Multisciences (China). Recombinant human IL-21 (rhIL-21) was purchased from PeoproTech GmbH (Rocky Hill, CT, USA). Anti-GrB antibody and isotype control were purchased from R&D Systems (Minneapolis, MN, USA). CpG ODN 2006 was purchased from InvivoGen (San Diego, CA, USA). Anti-human IgM + IgG for B-cell receptor (BCR) stimulation and APC-Cyanine7-anti-human CD19 antibody, FITC-anti-human CD4 antibody, PerCP-Cyanine5.5-anti-human CD4 antibody, FITC-anti-human CD14 antibody, PE-anti-human-IL-21 receptor (IL-21R) antibody, PE-Cyanine7-anti-human CD25 antibody, PE-anti-human GrB antibody, FITC-anti-human IFN-gamma antibody, and eFluor^® 660-anti-human IL-17A antibody for immunostaining were purchased from eBioscience (San Diego, CA, USA). Alexa Fluor^® 700-anti-human CD3 antibody, Brilliant Violet 570-anti-human CD56 antibody, APC-annexin V, and 7-AAD viability staining solution were purchased from Biolegend (San Diego, CA, USA).

Cells were washed with cold fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline (PBS) containing 1% sodium azide and 2% FBS) and incubated with the indicated mAbs at 4 °C for 30 min, after blocking with purified mouse IgG (BioLegend, clone MG1-45, 0.5 μg/mL, 4 °C, 15 min). After several washes, the cells were stained with 7-AAD Viability Staining Solution (BioLegend) and analyzed using a BD FACSAria III Cell Sorter (BD, Franklin Lakes, NJ, USA) and FlowJo software version 10 (BD).

EL4 cells that do not express VEGFR2 were labeled with Tag-It Violet Proliferation Cell Tracking Dye (Biolegend), and VEGFR2⁺ EL4 cells were labeled with Cell Proliferation Dye eFluor 670 (Thermo Fisher Scientific). Mouse CAR-T cells 4 days after Rv transduction, EL4 cells, and VEGFR2⁺ EL4 cells were co-cultured at the indicated E/T ratios. After 18 h, CountBright Absolute Cell Counting Beads (Thermo Fisher Scientific) and 7-AAD Viability Staining Solution (Biolegend) were added to the reaction wells, and the number of each target cell was analyzed using flow cytometry until 1000 beads were detected. Cytotoxicity was calculated using the following formula: Percentage of antigen-specific lysis = [(VEGFR2⁺ EL4 cells/ EL4 cells ratio in non-effector cell’s well) − (VEGFR2⁺ EL4 cells/ EL4 cells ratio in effector cell’s well)] / [VEGFR2⁺ EL4 cells / VEGFR2⁻ EL4 cells ratio in non-effector cell’s well] × 100.