Macsquant 10

Immediately following the stimulation protocols above, supernatants were gently removed and stored at -20°C. Then, the cells were immediately fixed with 4% paraformaldehyde and incubated for 15 minutes at 37°C before incubating with 1ug/mL of FITC anti-human CD63, FITC anti-human CD35, or APC anti-human CD107a antibody or isotype control (Biolegend) for 30 minutes at 4°C. Cells were then run through either a FACS Calibur (Becton-Dickenson) or a MACS Quant 10 (Miltenyi Biotech) flow cytometer and analyzed using FlowJo (Becton-Dickenson). The isotype control showed a lower signal at all conditions than the anti-CD63, anti-CD35, and anti CD107a antibody conditions. For detection of myeloperoxidase (MPO), supernatants stored overnight or several days were thawed slowly on ice, and tested using the BioLegend LEGENDplex^TM Human MPO Capture Bead kit according to the manufacturer’s instructions. Samples were then run through either a FACS Calibur (Becton-Dickenson) or a MACS Quant 10 (Miltenyi Biotech) flow cytometer and analyzed using FlowJo (Becton-Dickenson).

γδ T-cells were expanded using methods described previously (10, 34 (link)). Briefly, PBMCs were thawed, enumerated, and seeded at a concentration of 1 × 10⁶ cells/mL in a 24 well-plate with culture media (RPMI1640 media +10% FBS and 10% penicillin-streptomycin) containing 300 IU/mL of IL2 (Miltenyi Biotec) and 5 µmol/L of ZOL (Sigma-Aldrich). Culture media was changed on days 3, 7, and 10, with fresh culture media culture containing IL2 (300 IU/mL) only. After 14 days, expanded Vγ9Vδ2+ T-cells were harvested to determine number, phenotype, and function by flow cytometry (MACSQuant 10; Miltenyi Biotec). Expanded Vγ9Vδ2+ T-cell phenotypes were assessed in a similar manner as previously described in the aforementioned section but with a slightly altered phenotype panel by replacing, Vδ1-APC-Vio770 with TCRγδ-APC-Vio770 and NKG2C-PE-Vio770 with FasL-PE-Vio770. The expanded Vγ9Vδ2+ T-cells were cryopreserved in liquid nitrogen at a concentration of 10 × 10⁶ cells/mL in freezing media (90% FBS, 10% DMSO) until use in the in vitro cytotoxicity assays, adoptive transfer experiments in xenogeneic mice, and bulk RNA-seq analysis.

Flow cytometric analysis was performed on FAPs and SCs using PDGFRα-Alexa Fluor (AF) 647 (1:50; Abcam, ab270085), ITGA5-APC (1:50; Miltenyi Biotec, 130-110-591), CD14-APC (1:50; Miltenyi Biotec, 130-110-578), CD61-APC (1:50; Miltenyi Biotec, 130-110-887), combined with CD56-PE (1:10; BD Biosciences, 345812) antibodies on a MACSQuant10 flow cytometer (Miltenyi Biotec). Unstained cells were used routinely as negative controls and to define gating parameters. Flow cytometric data was analysed using FlowJo (FlowJo LLC, version 10.7.1).