Anti gzmb

Tumoral tissue was embedded in Tissue-Tek (Sakura) and sections (5 μm) were cut onto charged glass slides (Superfrost Plus Yellow) and rehydrated. Antigen retrieval was performed in citrate buffer pH 6.0 (sodium citrate 10 μM) at 90°C for 20 min. The sections were permeabilized (bovine serum albumin 10 mg/ml, horse serum 5%, Triton 0.5%, and sodium azide 0.02%) for 2 h and incubated with anti-CD8-FITC (BioLegend Cat# 100705, RRID:AB_312744), anti-CD103 (BioLegend Cat# 121426, RRID:AB_2563691), anti-PD-1-APC (BioLegend Cat# 135209, RRID:AB_2251944), anti-TIM-3-PE/Dazzle (BioLegend Cat# 134014, RRID:AB_2632738), anti-GZMB (BioLegend Cat# 372222, RRID:AB_2728389), anti-IFNγ-PE and anti-TCF-1 primary antibodies at room temperature (RT) for 18 h. The anti-CD103, anti-GZMB and anti-TCF-1/7 mAbs were revealed with a secondary antibody, either AF-647 (Jackson ImmunoResearch Labs Cat# 711-585-152, RRID:AB_2340621) or AF-594 (Jackson ImmunoResearch Labs Cat# 711-605-152, RRID:AB_2492288). The nuclei were counterstained with Hoechst (Invitrogen) for 10 min. The sections were mounted with Vectashield (Vector Laboratories).

Single‐cell suspensions were washed with FACS buffer (PBS, 5% FBS, 2 mM EDTA) and resuspended in Fc block for 15 min. hMDMs were stained with the following antibodies during 45 min at 4°C: Fixable Viability Due eFluor 506 (eBioscience, 65‐0866‐18); anti‐CD14 (BD Pharmingen, 555397); anti‐CD11b (eBioscience, 48‐0112‐82); anti‐CD206(BD Biosciences, 740309); anti‐CD163 (BioLegend, 333622); anti‐HLA‐DR (Thermo Fisher Scientific, 17‐9956‐42); anti‐CD80 (BD Bioscience, 557227); anti‐CD115 (Sony Biotechnology, RT2336540).
CD8+ T cells were stained with the following surface markers: Fixable Viability efluor780 (Thermo Fisher Scientific, 65‐0866‐18) and anti‐CD8 (BioLegend, 980908). Subsequently, cells were incubated for 30 min with Fix/Perm buffer (eBioscience, 00−5523). Cells were washed with permeabilisation buffer (eBioscience, 00−5523) and stained ON (4°C) in permeabilisation buffer with anti‐IFNg (BioLegend, 502542); anti‐TNFa (BioLegend, 986802); anti‐IL‐2 (BioLegend, 500328) and anti‐GZMB (BioLegend, 515406). Cells were subsequently washed and resuspended in FACS buffer. FACS data were acquired using a FACS Fortessa or FACS Symphony (BD Biosciences) and data were analysed using the FlowJo (TreeStar) program.