Article in the TME correlate with PLA2G2D expression, Pearson’s R coefficients and P values were determined for a range of cytokines and PLA2G2D across various tumour types included in TCGA. In the case of type I (α and β subtypes were included) and type III (λ) interferons, and in the case of TGF-β isoforms, the gene with the highest R coefficient per cytokine was plotted. Analyses were performed using the GEPIA2 TCGA data browser 63 . Log 2 -transformed fold changes in the RNA expres- sion of genes in NSCLC biopsies before anti-PD-1 therapy (pre) and after (post) onset of acquired therapy resistance were obtained from a previous study 16 . IHC and multiplex immunofluorescence PLA2G2D was stained by automated IHC using the Ventana Benchmark ULTRA v.12.1 (Ventana Medical Systems). FFPE sections of 4- μ m thick- ness were stained for PLA2G2D (Supplementary Table 6) using OptiView (760-700, Ventana). In brief, after deparaffinization and heat-induced antigen retrieval with CC1 (950-500, Ventana) for 32 min, the tissue samples were incubated with PLA2G2D antibody for 32 min at 37 °C. Incubation was followed by Optiview detection and haematoxylin II counterstaining for 8 min followed by a blue colouring reagent for 8 min according to the manufacturer’s instructions (Ventana, Roche). Multiplex immunofluorescence analysis was performed by automated immunofluorescence using the Ventana Benchmark Discovery ULTRA (Ventana Medical Systems). Slides of 4- μ m-thick FFPE sections were stained for CD11c, CD8, CD68, LAMP3 and CXCL13 (Supplementary Table 6). In brief, after deparaffinization and heat-induced antigen retrieval with CC1 (950-224, Ventana) for 32 min, incubation with anti- CD8 was performed for 32 min at 37 °C, followed by Omnimap anti- mouse HRP (760-4310, Ventana) and detection with R6G (950-240, Ventana). An antibody denaturation step was performed with CC2 (950- 123, Ventana) at 100 °C for 20 min. Second, incubation with anti-CD68 was performed for 12 min at 37 °C, followed by Omnimap anti-mouse (760-3210 Ventana) and detection with DCC (760-244, Ventana). An anti- body denaturation step was performed with CC2 (950-123, Ventana) at 100 °C for 20 min. Third, incubation with anti-LAMP3 was performed for 60 min at 37 °C, followed by Omnimap anti-rabbit (760-3211 Ventana) and detection with Red610 (760-245, Ventana) for 8 min. An antibody denaturation step was performed with CC2 at 100 °C for 20 min. Fourth, incubation with anti-CD11c was performed for 12 min at 37 °C, followed by Omnimap anti-rabbit HRP (760-4311, Ventana) and detection with Cy5 (760-238, Ventana) for 8 min. An antibody denaturation step was performed with CC2 at 100 °C for 20 min. Finally, anti-CXCL13 was incubated for 20 min at 37 °C, followed by Omnimap anti-rabbit HRP (760-4311, Ventana) and detection with FAM (760-243, Ventana) for 8 min. Finally, slides were washed in PBS and mounted with Vectashield containing 4′,6-diamidino-2-phenylindole (Vector Laboratories). Slides were imaged with an Axioscan 7 (ZEISS). Automated quantification of PLA2G2D + cells PLA2G2D-stained whole slides were scanned using the NanoZoomer (20×, Hamamatsu, HT2.0) and analysed using the open-source digital pathology software QuPath (v.0.5.1) 64 . All slides were set as ‘Brightfield (H-DAB)’, and for each slide, stain vectors were set for haematoxylin and DAB. ROIs of 200 μ m by 200 μ m in TDLNs and primary tumour tissues were selected for positive cell detection. Interfollicular regions and the centre of the primary tumour or the LN metastasis were selected for ROIs, and germinal centres, tissue borders and anthracotic areas were excluded from ROI selection. The number of ROIs per tissue depended on the tissue size. Cell segmentation and positive cell detection was performed on the basis of nuclear detection and mean DAB stain inten- sity using the built-in QuPath positive cell detection command, with the following settings: detection image, haematoxylin OD; requested pixel size, 0.5 μ m; background radius, 8.0 μ m, performing background by reconstruction; median radius, 0.0 μ m; sigma, 1.5 μ m; minimum area, 10.0 μ m, maximum area, 200.0 μ m; threshold, 0.1; maximum background, 2.0; cell expansion, 4.0 μ m, including nuclei and smooth boundaries. Three staining intensity thresholds were set using the fol- lowing settings: threshold compartment: cytoplasm: DAB OD mean, threshold positive 1+, 0.08; threshold positive 2+, 0.16; threshold posi- tive 3+, 0.24. For each slide, ROI measurements were exported and used for analysis. Per TDLN or tumour, as well as per patient, the average of all ROIs per tissue was used to calculate the percentage of positive cells, positive cells per mm 2 and the number of different positive cell intensities (1+, 2+ or 3+). Collection of TDLN tissue from patients with melanoma or NSCLC for cell culture We collected fresh TDLN tissue from patients with NSCLC or melanoma who underwent surgery at the Erasmus MC. Viable cells were obtained using the scrape cytology method, as described previously 65,66 . In brief, excised LNs were bisected during the standard pathology workflow, and a scalpel blade was used to shave along the cut surface and rinsed in 3 ml RPMI culture medium to obtain cells. This way, the LN samples could still be used for further processing without interfering with diagnostic workflows. The cell suspensions were filtered through a 35-μm filter cap (Falcon) and centrifuged at 400 g for 7 min. The medium was discarded and the cells were cryopreserved at −196 °C in RPMI containing 40% fetal bovine serum (FBS) and 10% dimethyl sulfoxide. All TDLN tissue samples were stored after resection at 4 °C and processed within 18 h. Flow-cytometry analysis TDLN samples were thawed in RPMI + 10% FBS, centrifuged for 7 min at 400 g , resuspended in RPMI with 10% FBS and incubated for 60 min at 37 °C. Then, cells were washed and resuspended in FACS buffer (0.5% BSA, 10% NaN 3 sodium azide and 2 mM EDTA in PBS) and stained for cell-surface markers (Supplementary Table 7) in Brilliant Stain Buffer (BD) for 30 min at 4 °C. Cells were washed with PBS and labelled with Fixable Viability Stain 575V (BD) for 15 min at 4 °C. For intracellular stain - ing, the FoxP3 fixation/Permeabilization Kit (eBioscience) guidelines were followed. All cells were then washed and resuspended in FACS buffer before analysis on a BD FACSymphony A5 flow cytometer (FACS Diva software v.9; BD). TDLN T cell in vitro stimulation assay TDLN samples were thawed and stained for cell-surface molecules as described above. Cells were washed and resuspended in MACS buffer (0.5% BSA and 2 mM EDTA in PBS). Before cell sorting, Helix NP Blue was added according to the manufacturer’s guidelines, and sorting was performed using a BD FACSAria III. We sorted three CD8 + T cell populations: CD8 + T naive (alive lineage − CD5 + CD8 + CCR7 + CD45 + ), CD8 + T non-naive PD-1 int (alive lineage − CD5 + CD8 + non(CCR7 + CD45RA + )PD-1 int ) and CD8 + T non-naive PD-1 hi (alive lineage − CD5 + CD8 + non(CCR7 + CD45 RA + )PD-1 hi ). The lineage cocktail contained antibodies against CD19, CD20, CD11c, CD68, CD56, CD14, CD16 and CD123. CD5 was used instead of CD3 to allow stimulation with anti-CD3 after sorting. In the TDLN samples, the lineage-negative CD5 + cell population overlapped with the lineage-negative CD3 + cell population by more than 98%, as veri- fied by flow cytometry. Sorted cells were collected in 100% FBS. Then, cells were resuspended in culture medium consisting of RPMI with 10% FBS, 1% penicillin–streptomycin and primocin (100 μ g ml −1 ). Per patient, concentrations were determined on the basis of the condition with the lowest cell yield, so that the other conditions contained cells x times the lowest cell count. The antibodies and culture materials used are listed in Supplementary Table 7. To mimic chronic T cell activa- tion, stimulation was performed using plates coated with anti-CD3 at 5 μ g ml −1 24 h before culture. Cells were seeded in 100 μ l culture medium with the addition of 2 μ g ml −1 soluble anti-CD28 at day 0. Cells were centrifuged, resuspended in culture medium and passaged onto a fresh anti-CD3-coated plate on days 2, 3 and 4, and analysed on day 5. This assay was adapted from a previous study 67 . On day 5, cells were washed