7 aad

A total of 4 × 10⁵ stimulated OVA-specific CD8⁺ T cells were added to 1 × 10⁵ B16F10-mCherry-OVA and treated with fresh condition medium (1:2 dilution). After 48 h, cells were stained using anti-Annexin V, 7-AAD (BioLegend, 640930) and anti-CD8 for 20 min at 4 °C, and were immunophenotyped by flow cytometry using an LSRFortessa or a FACSCanto II (Becton Dickinson). For CGRP, 4 × 10⁵ stimulated OVA-specific CD8⁺ T cells were added to 1 × 10⁵ B16F10- mCherry-OVA and treated with CGRP (100 nM). After 24 h, the cells were stained using anti-Annexin V, 7-AAD (BioLegend, 640930) and anti-CD8 for 20 min at 4 °C, and were immunophenotyped by flow cytometry using an LSRFortessa or a FACSCanto II (Becton Dickinson). Cytokine expression levels were analysed after in vitro stimulation (PMA–ionomycin; see ‘Intracellular cytokine staining’).

To detect the effect of therapy on cell cycle progression, cells were fixed with ice-cold 70% ethanol for at least 2 h, washed with PBS, and incubated with PI staining solution (BD, 556463). The cell cycle was analyzed using a Fortessa Flow Cytometer (BD Biosciences). For 7AAD staining, cells were collected with trypsinization, and washed with PBS, stained with 7AAD (Biolegend, 640922), and analyzed on a Fortessa Flow Cytometry. Results are from three independent experiments performed.

BM was extracted from the femur and tibia by centrifugation for 5 min at 6500× g. Spleen cells were prepared by gentle crushing in RPMI. Cells were counted and 5 × 10⁵ cells were stained in PBS. For analysis of the macrophage population, the following antibodies were used: anti-CD11b (clone M1/70, Biolegend, cat#101206) conjugated with fluorescein isothiocyanate (FITC), anti-F4/80 (clone BM8, StemCell Technologies, Vancouver, Canada, cat#60027PE) conjugated with phycoerythrin (PE), 7AAD (Biolegend, cat#420404). For analysis of the erythroblastic population and of its apoptosis, anti-CD71 FITC (clone R17217, Biolegend, cat#113806), anti-Ter119 PE (clone TER119, Biolegend, cat#116208), 7AAD and Annexin V (Biolegend, cat#640920 conjugated with Allophycocyanin (APC) were used. Cells were stained on ice and protected from light for 1 h. Data acquisition was made with a BD Accuri C6 (BD Biosciences, Franklin Lake, NJ, USA). A gate excluding debris was made with a FSC-SSC dot plot, and 10,000 events inside that gate were collected from each sample. Data was analyzed with the BD Accuri C6 Software. Gating strategies are available in the Supplementary Data (Figures S1 and S2).

Fluorochrome-conjugated isotype controls, anti-human CD3 (clone HIT3a), CD4 (clone OKT4), CD8 (clone HIT8a), CD45 (clone HI30), CD45RA (clone HI100), CD62L (clone DREG56), CD107a (clone H4A3), and CD279 (PD-1, clone EH12.2H7) were purchased from BD Biosciences (Franklin Lakes, NJ) or BioLegend. CARs were detected with AF647- or AF488-conjugated goat anti-human IgG-Fc Fab-fragments (Jackson ImmunoResearch, West Grove, PA). All antibody staining was performed in PBS containing 1% FBS after blocking with PBS containing either 10% human serum (Capricorn Scientific, Ebsdorfergrund, Germany) or 20 μg/mL mouse-IgG (Sigma-Aldrich). Samples were fixed with Cell Fix (BD Biosciences) before analysis, except when 7-aminoactinomycin D (7-AAD) staining was used. For 7-AAD staining, antibody stainings were performed as described and 7-AAD (BioLegend) was added 5 min before acquisition. Flow cytometric data were acquired using an LSR II cytometer (BD Biosciences) and analyzed using FlowJo (version 10; Tree Star, Ashland, OR).

HCT116 and RKO cells were seeded (0.5 × 10⁶ cells per well) and reverse transfected using Lipofectamine RNAiMax (Life Technologies, Waltham, MA, USA) with 15 nM mirVana™ miRNA mimics (miR-1 and cel-miR-67 as the negative control). After 48 h (HCT116) and 72 h (RKO), cells were harvested for apoptosis, cell proliferation and cell cycle analyses. The treatment duration for each cell line was equivalent to two doubling times (Doubling time: 23 h for HCT116 and 36 h for RKO) [64 (link)]. Dying/dead cells (AnnexinV+, AnnexinV+/7AAD+ and 7AAD+ only) were analyzed, following staining with APC AnnexinV and 7AAD (Cat #640930, Biolegend, San Diego, CA, USA), according to the manufacturer’s instructions. The cell cycle profile of each cell line was determined following fixation (70% ethanol overnight at 4 °C), using FXCyle PI/RNase (Cat #F10797, Invitrogen, Carlsbad, NM, USA) staining solution. Cells were sorted using BD FACSymphonyA5 and BD™ LSR II flow cytometers and data analyses were performed using Flowjo™ version 10.7.1 (BD, Ashland, OR, USA).

Cells (3 × 10⁵) in 1 ml culture medium were incubated with or without 2 μg/ml Gal-9. Two days later, cells were harvested and stained with APC-labeled anti-PD-1 and/or PE-labeled anti-TIM-3 antibodies (BioLegend, 1:50 each). After staining, cells were washed and resuspended in 0.5 ml staining/wash buffer containing 0.5 μg/ml 7-AAD (BioLegend). Counting beads (10000 BD Accudrop Beads) were added per sample and a fixed number of 3000 bead events were acquired for each sample. Data were analyzed with FlowJo as described above. Viable cells were gated according to normal scatter parameters (FSC/SSC) and 7-AAD exclusion, and cell survival was calculated using the following formula: Cell survival (%) = 100*(viable cell count in Gal-9-treated sample)/(viable cell count in control).
In some cases, cell death was determined by staining cells with FITC-labeled Annexin V (BioLegend, 1:20) and PI per the manufacturer’s instructions.