Monensin

For the detection of degranulation, freshly isolated lymphocytes were activated with 100 U/ml IL-2 (R&D Systems) overnight, and then cocultured with K562 cells at an effector to target ratio (E:T) of 10:1 for 2 hours. Anti-CD107a and monensin (10μg/mL; Sigma) were added directly into the medium and incubated for 4 hours. For the detection of cytokine production, isolated lymphocytes were directly stimulated with 50 ng/mL PMA (Sigma) plus 1 μg/mL ionomycin (Calbiochem, Darmstadt, Germany) for 2 hours and followed by the addition of monensin (10μg/mL; Sigma) (BD Sciences, SanJose, CA) for another 4 hours. Cells were then collected and stained with surface antibodies and stained intracellularly with anti-IFN-γ (eBioscience, San Diego, CA) and anti-TNF-α (Biolegend) in the dark. After incubation, the cells were washed, resuspended and finally detected by FACSCalibur (BD Bioscience).

For intracellular staining for IFN-γ production, PBMCs were incubated with 50 ng/ml of rhIL-12 (Miltenyi Biotec) and 50 ng/ml of rh-IL18 (R&D Systems) for 21 h at 37°C. One mM monensin (Sigma-Aldrich, Gillingham, UK) was added for the final 3 h. Cells were stained with antibodies to CD3-PE-Cy7 (eBioscience), CD16-APC-Cy7 (BD Biosciences) and CD56-ECD (Beckman Coulter) and subsequently fixed and permeabilized, followed by intracellular staining for IFN-γ-V450 (BD Biosciences). Dead cells were excluded by live/dead stain (Invitrogen).
For TNF-α production, PBMCs were stimulated with phorbol myristate acetate (PMA) (3 ng/ml) and ionomycin (100 ng/ml) (Sigma-Aldrich) for 3 h in the presence of 1 mM of monensin (Sigma-Aldrich). Cells were stained with antibodies to CD3-PE-Cy7 (eBioscience), CD16-APC-Cy7 (BD Biosciences) and CD56-ECD (Beckman Coulter) in the presence of live/dead stain followed by fixing, permeabilization and intracellular staining for TNF-α-FITC (BD Biosciences).

Purified CD45⁺ TIL were co-cultured for 4 h with B16F10 tumour cells, pulsed with Trp2 (1 µg) and gp100 (1 µg) peptides, in the presence of Brefeldin A (Invitrogen 00-4506-51, 1/1000), monensin (Merck M5273, 2 µm) and anti-CD107a mAb (clone 1D4B, Biolegend 121619, 1/50). TIL were stained with mAb specific for surface proteins prior to fixation and permeabilization. Permeabilized cells were then stained with anti-IFNγ (clone XMG1.2, Biolegend 505808, 1/200), anti-TNF (clone MP6-XT22, Biolegend 506321, 1/100) and anti-perforin (clone eBioOMAK-D, eBioscience 17-9392-80, 1/50) mAb.
For cytotoxicity experiments, freshly isolated CD8⁺ TIL were either kept in medium or pretreated with neutralising anti-Nrp-1 (clone 761704, R&D systems MAB59941, 10 µg/ml), anti-PD-1 (clone RMP1-14, Bio-X-Cell BE0146, 10 µg/ml) or isotype controls (clone 2A3, Bio-X-Cell BE0089, 10 µg/ml). Cytotoxic activity toward the B16F10 cell line, pulsed with Trp2 and gp100 peptides, or the MC-38 cell line was evaluated using a conventional 4 h and overnight chromium (⁵¹Cr) release assay.
For experiments with human freshly isolated lung TIL and CTL clone P62, activated T cells were incubated for 30 min to 1 h either with bovine serum albumin (BSA, Merck) or Sema-3A-Fc (R&D systems 1250-S3-025, 100 ng/ml), and their cytotoxic activity toward the autologous tumour cell was evaluated.

Unless otherwise specifed, the drug applications in these experiments began after the TNR blocking step and lasted until fixation. To enhance culture activity, the neurons/slices were treated with 40 μM bicuculline (#485-49-4; Merck, Germany) or 0.1% volume/volume DMSO (#67-68-5; Merck, Germany) as a control. To reduce culture activity by inhibiting AMPA and NMDA receptors, neurons were treated with 10 μM CNQX (#0190; Tocris Bioscience, Germany) and 50 μM AP5 (#0106; Tocris Bioscience, Germany). To block the activity of matrix metalloproteinases, neurons were treated with 10 μM GM6001 (#CC1010, Merck, Germany). To digest glycosaminoglycans, neurons were treated with 0.5 units/mL Chondroitinase ABC from Proteus vulgaris (#C3667, Merck, Germany) for 30 min following the blocking step. To perturb dynamin-dependent endocytosis, neurons were treated with 30 μM Dyngo^® 4a (#ab120689; Abcam, United Kingdom) for 2 h following the labeling step (for the experiment shown in Supplementary Fig. 15a) or throughout the experiment (for the experiment shown in Supplementary Fig. 15c). To perturb Golgi trafficking, neurons were treated with 5 μg/mL brefeldin (#B7651; Merck, Germany) or 1 μM monensin (#M5273; Merck, Germany) for 4 h, added from the onset of blocking.

In total, 5000 to 7500 PDAC cells in 50 µL complete medium in 96-well plates (Nunc) were cultured either with 50 µL of 1 mM NaOH solvent control, 1 mM 1-methyl-levo-trypthophan (1-l-MT, Sigma Aldrich) or 1 mM 1-methyl-dextro-tryptophan (1-d-MT, Sigma Aldrich), as shown in Figure 3, or with medium, as shown in Figure 5, overnight. After 24 h, short-term activated Vγ9Vδ2 γδ T cells with 12.5 IU/mL rIL-2 and 300 nM BrHPP were added at an E/T ratio of 25:1 to the indicated PDAC cells or cultured alone. In Figure 5, short-term activated Vγ9Vδ2 γδ T cells were cultured in medium, with 1 mM of previously titrated l-kynurenine (200 to 1000 µM, R&D System) or with 1 mM of previously titrated picolinic acid (Sigma Aldrich) for 24 h before coculturing these cells with different PDAC cells. For CD107a-assay, 10 µL FITC-labeled anti-human CD107a mAb clone H4A3 (50 µg/mL, BioLegend) was added directly, whereas 3 µM monensin (Merck) was added 1 h after coculturing the cells. After an additional 3 h, Vγ9Vδ2 γδ T cells were stained with PE-labeled anti-TCRγδ mAb (clone 11F2, BD Biosciences), and analyzed by flow cytometry.