PD-1T TILs as precision biomarker in NSCLC 31 2 tissue (n=3), showed fixation and/or staining artefacts (n=3), or were non-NSCLC histology (n=1) (Fig. 1A, Table S1). We excluded bronchial biopsies as they frequently showed unspecific antibody staining due to mechanical damage, and lymph node resections due to the presence of PD-1 bright T cells in normal lymphoid tissue, which could potentially lead to false positive results. Prespecified subgroup analyses were performed to compare (i) samples derived from tumor resections and biopsies, (ii) samples from primary and metastatic sites, and (iii) samples that were obtained either directly before the start of nivolumab or pembrolizumab or before any prior line of systemic treatment. Fresh tumor samples were collected from 16 patients with NSCLC undergoing primary surgical treatment between July 2017 and February 2019 at NKI-AVL. The study was approved by the Institutional Research Board of NKI-AVL (CFMPB484). All patients consented to research usage of material not required for diagnostic use either by opt-out procedure or via prior informed consent (after May 23, 2018). Representative tumor tissue samples were procured from surgical resection specimens by a pathologist. Half of each sample was formalin fixed and embedded in paraffin for further histological analysis, the other half was immediately processed into tumor fragments that were cryopreserved until further usage (see sample processing and flow cytometry analysis). Sample processing and flow cytometry analysis For flow cytometry analysis, cryopreserved tissue fragments were thawed and processed into single-cell suspensions by enzymatic digestion using RPMI1640 medium (Thermo Fisher) supplemented with 1% Penicillin-Streptomycin (Roche), 12.6µg/ml Pulmozyme (Roche) and 1mg/ml Collagenase type IV (Sigma), as described previously14. Samples were then washed in PBS (Sigma), filtered over a 150µM filter mesh, resuspended in 50µL PBS, and incubated with Fc receptor blocking agent (eBioscience) and with live/dead Zombie UV (Biolegend) for 20 min at 4°C. Cells were washed, resuspended in 50µl of staining buffer (PBS (Sigma), 0.5% bovine serum albumin (Sigma), 0.1% NaN3 (Invitrogen)) containing the below-described antibodies, and incubated for 20 min at 4°C. After washing twice, cells were taken up in 200µl IC Fixation Buffer (eBioscience) and incubated for 20 min. Subsequently, samples were washed twice before data acquisition. For staining the following antibodies were used: anti-CD45 PerCP Cy5.5 (2D1, RRID:AB_1548697) from Invitrogen; anti-CD8 BUV563 (RPA-T8, RRID:AB_2870199), -PD-1 PE-Cy7 (EH12.1, RRID:AB_10611585), all from BD Biosciences; anti-CD3 FITC (SK7, RRID_ AB2043993), -CD4 BV421 (SK3 RRID:AB_2566015), all from Biolegend. PD-1T lymphocytes
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