Clone c8 144b

TMA blocks were sectioned at 2.5 μm. Sections were mounted on glass slides, dried and baked at 60°C for 30 min. H&E staining and double immunohistochemistry for Pan-CK and CD8 were performed. Double immunohistochemistry was performed using Bond RX (Leica Biosystems). Slides were dewaxed using Bond dewax solution (product code AR9222, Leica Biosystems). Heat-induced epitope retrieval in citrate buffer based (code AR9640, Leica Biosystems) at pH6 for 20 min at 100°C was followed by incubation with primary mouse pancytokeratin antibody (Dako, clone AE1/AE3, Ref M351501-2); dilution 1:400; for 30 min. Slides were incubated with HRP (horseradish peroxidase)-polymer for 15 min. Visualization was accomplished using 3,3-Diaminobenzidine (DAB) for 10 min, leading to a brown staining signal (Bond polymer refine detection, Leica Biosystems, Ref DS9800). As a second step, mouse CD8 antibody was used (Dako-Agilent, clone C8/144B, Ref M7103); dilution 1:100; incubation time 30 min. Alkaline Phosphatase (AP)-polymer was used as secondary antibody; incubation time 15 min. Visualization was accomplished using fast red resulting in a red chromogen (Red polymer refine Detection, Leica Biosystems, Ref DS9390). Samples were counterstained with hematoxylin and mounted with Aquatex (Merck).

For immunohistochemistry (IHC) staining, the ALI PDOs were processed as described above. IHC staining of the various antibodies was performed according to established staining protocols of the routine laboratory. The following antibodies were used: PAX8 (dilution 1:100, clone MRQ-50, Cell Marque), vimentin (dilution 1:5000, clone V9, Agilent), PD-L1 (dilution 1:50, clone E1L3N, Cell Signaling Technology), CA9 (dilution 1:8000, clone EPR 23055-5, Abcam), CD8 (dilution 1:50, clone C8/144B, Agilent), LCA (dilution 1:2000, clone 2B11 + PD7/26, Agilnet), and Granzyme B (dilution 1:50, clone 11F1, Leica). Stainings were performed on an Autostainer 480S (Fa Medac).

Samples of lung tissue of all patients were collected in buffer (formaldehyde 4%), subsequently excised in paraffin embedding, and sectioned after fixation. In the accredited routine laboratory of Karolinska Hospital, the blocks were dehydrated and paraffin-embedded using automated tissue processors. Sections (5 μm) were then cut and stained with H&E using automated staining machines. Immunostaining was conducted in Ventana Ultra Benchmark (Ventana Medical Systems, USA), following the manufacturer’s instructions. Immunohistochemistry for PD-L1 and CD8⁺ T-cells was performed using (Roche Cat# 790-4905, RRID: AB_2819099, clone SP263 catalogue # 07494190001, Ventana) and (Santa Cruz Biotechnology Cat# sc-53212, RRID: AB_1120718, clone C8/144B catalogue # M7103, Dako, Agilent), respectively.

The formalin-fixed, paraffin-embedded (FFPE) samples were stained for both PD-L1 and CD8. Immunohistochemistry of the FFPE tumor samples was performed on a BenchMark Ultra autostainer (Ventana Medical Systems). Briefly, paraffin sections were cut at 3 µm, heated at 75 °C for 28 min and deparaffinized 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 min at 95 °C (CD8) or 48 min at 95 °C (PD-L1). CD8 was detected using clone C8/144B (1/100 dilution, 32 min at 37 °C, Agilent/DAKO) and PD-L1 was detected using clone 22C3 (1/40 dilution, 1 h at RT, 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). A Pannoramic® 1000 scanner from 3DHISTECH was used to scan the slides at a 40 × magnification. The stained FFPE slides were scored by a blinded pathologist using Slidescore (www.slidescore.com). Of each biopsy, five representative areas of 0.2mm² were selected to assess the number of CD8⁺ cells/mm².

Where p16 testing was not performed as standard of care, formalin-fixed paraffin-embedded tissue blocks were obtained and retrospectively tested for p16 as described elsewhere.^{4 (link)} The formalin-fixed paraffin-embedded blocks received (168/791 from the Manchester cohort) were also analyzed for TILs using multiplex immunohistochemistry. Primary antibodies against pan-cytokeratin (clone AE1/AE3/PCK26) and CD4 (clone SP35) were obtained from Roche Diagnostics, anti-CD68 (1 in 3,000, clone KP1) from Abcam, and anti-CD8 (1 in 300, clone C8/144B) from Agilent DAKO (Santa Clara, CA). Fluorescent immunohistochemistry was conducted using the Ventana Ultra Discovery Autostainer. Multiplex images were acquired using the Olympus VS120-L100-W-12 (Olympus Corporation, Tokyo, Japan). Data analysis was performed using HALO v3.1.1076.429 (Indica Labs, Albuquerque, NM) software. REporting recommendations for tumor MARKer prognostic studies (REMARK) guidelines were followed throughout.^{26 (link)}