Canto 2 system

Two million cells were suspended in 100 μL PBS and incubated with APC‐labeled anti‐human CD133 antibody (Beckton‐Dickinson, Flanklin Lakes, NJ, USA), anti‐human CD326 (EpCAM) antibody (Miltenyi Biotec, K.K., Tokyo, Japan) for 60 min on ice in the dark. After washing with PBS, cells were resuspended in 1 mL PBS and acquired on CANTO II system (Beckton‐Dickinson). Data were analyzed using FlowJo v10.1r5 software (FlowJo, LLC, Ashland, OR, USA).

A549P/A549CisR and H157P/H157CisR cells were stained with PE-PD-L1 Ab (BioLegend, 358103) (5 μl/10⁶ cells) (unstained cells as control) and the fluorescence was detected using the Canto II system (Becton-Dickinson, San Antonio, TX). In experiments with IL-6 addition, A549P and H157P cells were cultured and passaged in the presence of IL-6 (10 ng/ml), and P6 cells were used in analyses.

A549P/A549CisR and H157P/H157CisR cells were stained with APC-PD-L1 Ab (BioLegend, 329707) (5 μl/10⁶ cells) (unstained cells as control) while NK cells were stained with PE-NKG2D Ab (BioLegend, 320805) or APC-PD-1 Ab (BioLegend, 329907), and the fluorescence was detected using the Canto II system (Becton-Dickinson).

Two million cells were suspended in 100 µL PBS and incubated with antibodies for 60 minutes on ice in the dark. Antibodies used were; APC anti-human CD133 (Miltenyi Biotec, Bergisch gladbach, Germany; 1:100), APC anti-mouse CD133 (Prominin-1) (Miltenyi Biotec; 1:50), APC anti-CD326 (EpCAM) (Miltenyi Biotec; 1:100). After washing with PBS, cells were re-suspended in 1 mL PBS and measured by a CANTO II system (Beckton-Dickinson). All Data were analyzed using FlowJo v10.1r5 software (Ashland, OR, USA).

One million cells were suspended in 100 μL PBS and incubated with antibodies for 60 min on ice in the dark. The antibodies used were PerCP anti-human CD3, FITC anti-human CD4, PE anti-human CD8, PE anti-mouse CD4, and FITC anti-mouse CD8a (BioLegend, San Diego, CA, USA). After washing with PBS, the cells were resuspended in 500 μL PBS and measured using a CANTO II system (Beckton-Dickinson, Franklin Lakes, NJ, USA). All data were analyzed using FlowJo v10.7.1 software (Ashland, OR, USA).

Isolated T cells were stained with 2.5 µM Carboxyfluorescein succinimidyl ester (CFSE; Thermo Fisher Scientific) in PBS for 4 min at room temperature and reaction was stopped with FCS. For the assessment of MDSC suppressive capacity, MDSC were co-cultured with anti-CD3/anti-CD28 stimulated CFSE-labeled T cells. For this, 5 × 10⁴ T cells (per well) were seeded into 96-well plates and cocultured with 0.31 × 10⁴, 0.63 × 10⁴, 1.25 × 10⁴, 2.5 × 10⁴ or 5 × 10⁴ MDSC. Each well was supplemented with 1 µl beads (Dynabeads™ Mouse T-Activator CD3/CD28, gibco®, Thermo Fisher Scientific, Karlsruhe, Germany). After 72 h, CFSE dilution of CD4⁺ and CD8⁺ T cells was analyzed by flow cytometry (BD Canto II system, BD Bioscience, Heidelberg, Germany). Unstimulated CFSE-labeled T cells only were used to set the threshold of proliferated T cells (CFSE^low).

Fresh blood samples were collected from patients and volunteers. Six-color flow cytometric analysis was performed to determine cell phenotypes. Nine specific monoclonal antibodies (BD Biosciences, San Jose, CA, USA) against CD3 (FITC), CD45 (Per CP), CD4 (FITC and APC), CD45RO (PE), CD8 (PE-CY7), CD19 (APC), CD28 (PE), CD56 (PE), and HLA-DR (APC) were used to differentiate lymphocyte subsets. Lymphocytes were gated by CD45. The lymphocyte subsets analyzed included T cells (CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, CD4⁺CD45RO⁺, CD8⁺CD28⁺, CD8⁺CD28⁻, and CD3⁺HLA-DR⁺), natural killer cells (CD3⁻CD56⁺), and B cells (CD3⁻CD19⁺). Flow cytometry was performed using a BD Canto II system, and BD Diva software was used for data analysis.