Clone 29e 2a3

H820 lung cancer cells which were used as a positive control for PD-L1 analysis were obtained from the American Type Culture Condition (ATCC, Manassas, USA). Three different clones of anti-PD-L1 antibodies were tested: (1) clone 29E.2A3 (BioLegend, San Diego, USA), (2) clone 130021 (R&D Systems, Minneapolis, USA), (3) clone MIH1 (eBioscience, San Diego, USA) (Figure 1). SW620 colorectal cancer cells, MCF7 and Sk-Br-3 breast cancer cells were used as a negative control and were obtained from the CLS cell lines service (Eppenheim, Germany) (Figure 2). H820 cells were grown in RPMI-1640 medium with 5% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, USA), SW620 and Sk-Br-3 cells were grown in Dulbecco's modified Eagle's medium with 4,5g/L glucose, 2mM L-glutamine (Gibco, Thermo Fisher Scientific, Waltham, USA) and 10% FBS. Cells were maintained at 37°C in 5% CO₂. MCF-7 cells were grown in Minimum Essential Eagle ready-to-use medium (CLS cell lines service (Eppenheim, Germany). For immunofluorescence analysis cells were detached from cell culture flasks using StemPro^® Accutase^® Cell Dissociation Reagent (Gibco, Thermo Fisher Scientific, Waltham, USA) washed and stained for PD-L1 with the same protocol like a patient sample.

To confirm specificity of commercially acquired anti-PD-1 mAb, clone J43 (eBioscience, San Diego, CA, United States) and clone EH12.2H7 (BioLegend, San Diego, CA, United States), as well as anti-PD-L1 mAb, clone MIH5 (eBioscience) and clone 29E.2A3 (BioLegend), HEK293T cells transiently transfected with recombinant woodchuck PD-1 (wcPD-1) and woodchuck PD-L1 (wcPD-L1) were examined as additional test targets. For this purpose, sequences encoding wcPD-1 and wcPD-L1 were determined, expression vectors constructed using pCDNA 3.1(+), and HEK293T cells transfected using PolyJet DNA transfection reagent (SignaGen Laboratories LLC, Gaithersburg, MD, United States). Surface and intracellular expression of the transfected protein was determined by flow cytometry and compared to non-transfected cells and an isotype control.

Peripheral blood mononuclear cells or expanded NK cells were surface stained with panels of monoclonal antibodies used at the manufacturer’s recommended concentration. Antibodies from BioLegend (San Diego, CA, USA) included anti-CD3 (clone OKT3), anti-CD14 (clone M5E2), anti-CD56 (clone HCD56), anti-NKG2D (clone 1D11), anti-CD57 (clone HCD57), anti-CD11b (clone M1/70), anti-CD69 (clone FN50), anti-Tim3 (clone F38-2E2), anti-PD-1 (clone EH12.2H7), and anti-CD95 (clone DX2). Antibodies from BD Biosciences (San Jose, CA, USA) included anti-CD16 (clone 3G8) and anti-CD94 (clone HP-3D9). Antibodies from R&D Systems included anti-NKG2A (clone 131411) and anti-KIR2DL2/DL3/DS2 (clone 180704). Antibodies from Beckman Coulter (Indianapolis, IN, USA) included anti-KIR3DL1/DS1 (clone Z27.3.7) and anti-KIR2DL1/DS1 (clone EB6B). All panels also included the Fixable Blue Dead Stain (Life Technologies, Carlsbad, CA, USA) as a viability dye. K562 cells were stained with antibodies to PD-L1 (BioLegend, clone 29E.2A3) and Gal-9 (BioLegend, clone 9M1-3) with DAPI (BioLegend) as a viability dye. After staining, cells were fixed with 2% paraformaldehyde and analyzed using the BD Fortessa instrument (BD, Franklin Lakes, NJ, USA) and FlowJo software (TreeStar, Ashland, OR, USA).