Chapter 2 32 were identified by using peripheral blood T cells from healthy donors as external reference to establish the cut-off as described previously10. Data acquisition was carried out on a BD LSR II SORP cell analyzer (BD Biosciences). Data was collected using the BD FACS Diva Software version 8.0.1, and further analyzed with FlowJo v10.6.1 (Tree Star Inc.) and GraphPad Prism v8.0e (GraphPad Software Inc.). Immunohistochemistry Separate immunohistochemistry (IHC) stainings of consecutive FFPE tumor tissue sections were performed on a BenchMark Ultra autostainer Instrument (Ventana Medical Systems). Paraffin sections were cut at 3 µm. Sections for PD-1 staining were dried overnight at room temperature and stained within 48 hours to reduce background staining. Prior to staining, sections were initially baked at 75°C for 28 minutes and deparaffinised in the instrument with EZ prep solution (Ventana Medical Systems). Heat-induced antigen retrieval was carried out using Cell Conditioning 1 (CC1, Ventana Medical Systems) for 32 minutes (CD68 and CD20CD3 double staining) or 48 minutes (PD-1 and PD-L1) at 95°C. PD-1 was detected using clone NAT105 (Lot number V0002089, Ready-to-Use, 16 minutes at RT, Roche Diagnostics (Cat. # 7099029001). PD-L1 was detected using clone 22C3 (1/40 dilution, 1 hour at RT, Agilent/DAKO) and CD68 was detected using clone KP1 (1/10000 dilution, 32 minutes at 37°C, Agilent/DAKO). Bound antibody was detected using the OptiView DAB Detection Kit (Ventana Medical Systems). Slides were counterstained with Hematoxylin and Bluing Reagent (Ventana Medical Systems). For double staining of CD20 (Yellow) and CD3 (Purple), CD20 was detected in the first sequence using clone L26 (1/800 dilution, 32 minutes at 37°C, Agilent/DAKO). CD20 bound antibody was visualized using anti-Mouse NP (Ventana Medical systems) for 12 minutes at 37°C followed by anti-NP AP (Ventana Medical systems) for 12 minutes at 37°C, followed by the Discovery Yellow detection kit (Ventana Medical Systems). In the second sequence of the double staining procedure CD3 was detected using clone SP7 (1:100 dilution, 32 minutes at 37°C, Thermo Scientific). CD3 was visualized using anti-Rabbit HQ (Ventana Medical systems) for 12 minutes at 37°C followed by antiHQ HRP (Ventana Medical systems) for 12 minutes at 37°C, followed by the Discovery Purple Detection Kit (Ventana Medical Systems). Slides were counterstained with Hematoxylin and Bluing Reagent (Ventana Medical Systems). PD-1T, PD-L1 and CD68 immunostainings were scanned at x20 magnification with a resolution of 0.50 per µm2 using an Aperio slide AT2 scanner (Leica Biosystems). CD20-CD3 immunostaining was scanned at x20 magnification with a resolution of
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