Blg420801

B16 cells-expressing ovalbumin (courtesy of Lea Eisenach, Weizmann institute of Science) were suspended in DMEM medium w/o phenol red, supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, 100 mg/ml of streptomycin, 2 mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate, and 50 mM β-mercaptoethanol (Biological Industries). Cells were seeded 50–100 × 10³ B16 cells per well in a 24-well plate; 150–300 × 10³ OT-I T-cells pre-activated and cultured for 7 days, were added on top of the B16 cells. After 24–48 h, cells were collected, and a single-cell suspension was prepared and stained with live/dead stain (L23105, Life Technologies, Carlsbad, CA, USA), CD8 (BLG100734, Biolegend), FasL (BLG106606, Biolegend), and PD-1 (BLG135225, Biolegend). Cells were then fixated (BLG420801, Biolegend), permeablized (BLG521002, Biolegend), and stained for intracellular granzyme B (BLG515406, BioLegend). The mean fluorescent intensity of Granzyme B, FasL, and PD-1 of live CD8⁺ cells was measured and analyzed using flow cytometry (Becton Dickinson; FlowJo software).

The following antibodies were used, in this study, for flow cytometry: Pacific blue-CD3 (clone SK7, BioLegend, San Diego, CA, United States), PECy7 or FITC-CD8 (clone HIT8a, BioLegend), FITC-PD-1 (clone EH12.2H7, BioLegend), FITC-TIM-3 (clone F38-2E2, BioLegend), FITC-LAG-3 (clone 11C3C65, BioLegend) APC-CD25 (clone BC96, BioLegend), APC-CD28 (clone CD28.2, BioLegend), APC-vio770-CD45RA (clone HI100, BioLegend) and PerCP-CCR7 (clone G043H7, BioLegend). TIL cultures were washed and re-suspended in cell-staining buffer (BioLegend). Cells were incubated for 30 min with the antibodies on ice, washed in buffer and measured using MACSQuant flow cytometer (Miltenyi Biotech).
For localization and quantification of granzyme B, cells were fixed (BLG420801, Biolegend), permeabilized (BLG421002, Biolegend), and stained with granzyme B-specific antibodies (BLG515406, BioLegend). For carboxy fluorescein succinimidyl ester (CFSE) cell proliferation assays, TIL cultures were stained before seeding at day 11 of the REP with 5 μM CFSE (Thermo Fisher Scientific) for 20 min at 37°C, according to the manufacturer’s instructions. Four days later cells were taken for flow cytometry analysis and their CFSE fluorescence intensity was determined. Samples were analyzed using FlowJo software (FlowJo LLC, Ashland, OR).