CD8+ TIL Cells were divided into the control, SOX-treated, anti-TIGIT-treated (#71340, 5 ug/mL, BPS Biosciences), and SOX + anti-TIGIT-treated groups. For the proliferation assay, CD8+ TIL Cells were seeded on 12-well plates and stimulated with anti-CD3 (#16–0038-81,Thermo)/CD28 (#16–0289-81,Thermo) at 37°C exposed to 5% CO2 to approximately 50% proliferation. Immunofluorescence staining was then conducted to measure KI-67 expression to detect the proliferation of CD8+T cell.
For intracellular cytokine stimulation assays, CD8+ TIL Cells were treated with SOX or anti-TIGIT at 37°C with 5% CO2 for 24 h. Cells were harvested and stimulated with 4 µL/mL PMA/ionomycin/BFA/monensin mixture (250×, #70-CS1003, MultiSciences Biotech Co., Ltd, China) for 5 h at 37°C with 5% CO2. The cells were fixed and permeabilized using the FOXP3/transcription factor staining buffer set (#00–5523-00, Thermo) for 30 min, and were then collected and stained with APC-Cy7 mouse anti-human CD8 (#555369, BD Biosciences) and PE anti-interferon (IFN)-γ antibody (#502509, Biolegend).
For intracellular cytokine stimulation assays, CD8+ TIL Cells were treated with SOX or anti-TIGIT at 37°C with 5% CO2 for 24 h. Cells were harvested and stimulated with 4 µL/mL PMA/ionomycin/BFA/monensin mixture (250×, #70-CS1003, MultiSciences Biotech Co., Ltd, China) for 5 h at 37°C with 5% CO2. The cells were fixed and permeabilized using the FOXP3/transcription factor staining buffer set (#00–5523-00, Thermo) for 30 min, and were then collected and stained with APC-Cy7 mouse anti-human CD8 (#555369, BD Biosciences) and PE anti-interferon (IFN)-γ antibody (#502509, Biolegend).