Tnf fitc

PD-1 FITC (1:300; J43; eBioscience), NK1.1 PE (1:300; PK136; eBioscience), CD3e PE-Dazzle594 (1:300; 145-2C11; BioLegend), CD3e PE (1:200; 145-2C11; eBioscience), FoxP3 PerCP-Cy5.5 (1:200; FJK-16s; eBioscience), CD8a PE-Cy7 (1:2000; 53-6.7; eBioscience), CD8a APC (1:600; 53-6.7; eBioscience), CD8a PerCP-Cy5.5 (1:400; 53-6.7; eBioscience), TCRb AF700 (1:100; H57-597; BioLegend), CD45 BV785 (1:1000; 30-F11; BioLegend), CD4 BV711 (1:400; GK1.5; BioLegend), CD19 BV650 (1:400; 6D5; BioLegend), CD19 PE (1:500; 1D3; eBioscience), CD11b BV605 (1:1000; M1/70; BioLegend), CD11b BV510 (1:400; M1/70; BioLegend), CD11c BV605 (1:400; N418; BioLegend), Siglec-F PE (1:300; E50-2440; BD Biosciences), F4/80 APC (1:200; BM8; BioLegend), Ly-6G AF 700 (1:300; 1A8; BioLegend), MHC II BV650 (1:4000; M5/114.15.2; BioLegend), CD64 BV421 (1:200; X54-5/7.1; BioLegend), TNF FITC (1:300; MP6-XT22; BioLegend), IFNg PE-Cy7 (1: 1000; XMG1.2; BD Biosciences), Eomes PE (1:200; W17001A; BioLegend), Ki-67 BV 650 (1:200, 11F6; Biolegend), GzmB FITC (1:100; GB11; BioLegend). Gating strategy is depicted in Supplementary Fig. 6d.

PBMCs were thawed, washed, counted and re-suspended in X-Vivo 15 medium supplemented with IL-4 (5 ng/ml) and IL-7 (5 ng/ml) on day 0. On d1, pooled 15-mer Her-2 peptides (PepMix, JPT Technologies, Berlin) were added at 1 µg/ml to 1 × 10⁶ cells per culture. IL-2 (40U/ml) was added on d3. T-cells were harvested on d12 and re-stimulated (0.4–0.5 × 10⁶ cells/well) with 1 µg/ml Her2 peptides or left un-stimulated as a negative control for 12 h. Pepmixes of influenza nucleoprotein and matrix protein were used as positive controls. Golgi-plug (BD-Biosciences) was added at 1 µl/ml to prevent cytokine secretion. The cells were harvested, washed, incubated with Gamunex and EMA, fixed and permeabilized with Cytofix/Cytoperm (BD-Biosciences) before staining with CD3-Pacific Orange (Invitrogen), CD4-Pacific Blue, TNF-FITC, IL-2-Alexa-Fluor-700, IL-5-PE (BioLegend), CD8-APC-Cy7, IFN-γ-PE-Cy7 (BD-Biosciences), IL-10-APC (Miltenyi-Biotec) and IL-17-PerCP-Cy5.5 (eBioscience). After washing, the cells were immediately measured using a BD-LSR-II flow cytometer with FACS-Diva software (BD-Biosciences).