Bond rx platform

Syngeneic tumor tissues were fixed in 10% formalin, embedded in paraffin, and sectioned at a 4 μm thickness on a Leica RM2235 microtome. Sections were stained with rabbit monoclonal antibodies (Cell Signaling Technology) against murine CD8α (clone D4W2Z) and PD-L1 (clone D5V3B) using the automated Leica BOND Rx platform. Stained slides were imaged with a Leica Aperio VERSA scanner and analyzed with HALO software (Indica Labs). The percentage positivity for each marker was calculated as the density of positive cells per mm² divided by the total number of cells per mm².

Immunohistochemistry was done both manually on the bench or on the Leica bond Rx platform and described in the supplementary methods section. All antibodies, source and concentration used for both multiplex and single plex immunohistochemistry have been listed in Table S2.

Double immunostaining for CD8 and FOXP3 was performed on a Leica Bond RX platform, with antigen retrieval performed for 20 min at 97 °C Bond ER2 antigen retrieval solution. Primary antibodies were incubated for 30 min at room temperature (FoxP3 - Abcam: Clone 236A/E7 1:100 dilution; CD8 - DAKO: Clone C8/144B 1:50 dilution) and detected using the Leica Refine Polymer brown and red detection systems. Analysis was performed by two independent and blinded head and neck pathologists counting intratumoural CD8⁺ and FOXP3⁺ TIL in multiple random high-power fields at 200× magnification. Where possible, ten high-power fields were counted.

Tumor core biopsies specimens were obtained prior to and after treatment in order to evaluate the TILs in the tumor microenvironment. The core biopsies were formalin-fixed paraffin-embedded (FFPE) and stored in -20 °C. Immunohistochemistry (IHC) staining was performed on sequential 5 μm FFPE sections. Immunostaining for CD45RO was performed at the Johns Hopkins Hospital Immunopathology Laboratory using the Ventana autostainer with mouse monoclonal anti-CD45RO (MB-1) antibody (Ventana Medical Systems, Inc., Tucson, AZ, USA) according to the manufacturer’s protocols. CD8 IHC was completed at the Johns Hopkins University School of Medicine Tumor Microenvironment core utilizing mouse monoclonal anti-CD8 (4B11) antibody using the automated Leica Bond RX platform. All IHC was performed with 3–3′-diaminobenzidine detection and haematoxylin counterstaining. Manual multiplex IHC (mIHC) was performed as previously described [27 (link)].
Manual immunostaining for PD-L1 was completed as previously described28. The tissue specimens were deparrafinized, rehydrated, and immediately stained with the PD-L1 antibody clone SP142 (Spring Bioscience). Detection was performed by Tyramide Signal Amplification (TSA) system (PerkinElmer).

The staining was carried out on Bond RX platform (Leica System), following modified standard ‘IHC protocol F’ and Heat Induced Epitope Retrieval 1 (HIER1) for 20 min using the BOND Polymer Refine Detection kit. The ‘IHC protocol F’ was modified by 1) inserting three extra 5 min washing steps after HIER; 2) replacing the step ‘Mixed DAB Refine’ with ‘Bond Open Container’, which was supplied with freshly made 3-Amino-9-Ethylcarbazole (AEC); and 3) removing the step of ‘Hematoxylin’. One cycle of mIHC was carried out as one staining protocol, following the standard operation of the manufacturer’s protocol, except a) in the first cycle, the ‘Dewax’ step was selected, and b) in the last cycle, the ‘Hematoxylin’ step was added back as a sole staining step.
After each cycle of staining, slides were unloaded from the Bond machine and temporarily mounted in Tris-Buffered Saline with 0.1% Triton X-100 (TBST) and imaged using Zeiss AxioScan Z1 at 20x brightfield. After imaging, AEC was removed by ethanol by 2 brief washes in distilled water, 1 wash in 70% ethanol, and 1 wash in 100% ethanol for 3.5 min. The slides were then rehydrated through 2 min incubation in 70% ethanol, 1 min incubation in 30% ethanol, and then 4 washes in distilled water. The slides were then left to rest in TBST for the next cycle.

Automated RNAscope for PD-L1 was performed on sections from the adenocarcinoma and squamous cell carcinoma TMAs on a Leica Bond RX platform. Briefly, sections were cut at 4 μm, air dried overnight, baked at 60°C for 1 hour, dewaxed, and air-dried before pretreatments. For all tissue sections, a standard pretreatment protocol was used. Three RNAScope probes from Advanced Cell Diagnostics (ACD; Hayward, California) were used in this study: positive-control probe Hs-PPIB (313908 Accession # NM_000942.4-4 – 139 - 989); and probe to the immune pathway–associated biomarker PD-L1 – Hs-CD274 (600868 Accession # NM_014143.3 – sequence region 124 - 1122) were also used to stain the lung TMAs. Also a negative-control probe DapB (312038 Accession # NM_EF191515) was tested in a subset of adenocarcinoma and squamous cell carcinoma TMA cases with no expression observed on any cores. Detection of specific probe binding sites done was with the RNAScope 2.5 HD Reagent kit-brown from ACD (Cat. No. 322300).