Whole blood was collected from healthy individuals at the Suzhou Blood Center
(Suzhou, China) and subjected to density gradient separation on Ficoll-Paque Plus (GE
Healthcare, USA). After centrifugation, the peripheral blood mononuclear cell (PBMC)
layer was collected, seeded onto a tissue culture plate, and incubated at 37°C in a
5%-CO2 incubator. After 2-h incubation, cells in suspension were
collected following gentle pipetting the medium, and these were predominantly T
cells. The isolated T cells were labeled with carboxyfluorescein succinimidyl ester
(CFSE; Biolegend) as previously described (14 (link)). Meanwhile, A549 cells were treated with cisplatin (25 mg/mL; Biolegend)
for 3 h. The CFSE-labeled T cells were then seeded into 96-well plates
(2×105 cells/well) that had been pre-coated overnight with anti-CD3 (5
µg/mL, Biolegend) and anti-CD28 (2.5 µg/mL, Biolegend) at 4°C. The cisplatin-treated
A549 cells with or without B7-H1 blocking antibody (50 µg/mL, Biolegend) were then
added to CFSE-labeled T cells at a T:A549 ratio of 1:2, 1:4, or 1:8. Each condition
was tested in triplicate. After 72 h, all cells were collected and T-cell
proliferation was examined by flow cytometry.
(Suzhou, China) and subjected to density gradient separation on Ficoll-Paque Plus (GE
Healthcare, USA). After centrifugation, the peripheral blood mononuclear cell (PBMC)
layer was collected, seeded onto a tissue culture plate, and incubated at 37°C in a
5%-CO2 incubator. After 2-h incubation, cells in suspension were
collected following gentle pipetting the medium, and these were predominantly T
cells. The isolated T cells were labeled with carboxyfluorescein succinimidyl ester
(CFSE; Biolegend) as previously described (14 (link)). Meanwhile, A549 cells were treated with cisplatin (25 mg/mL; Biolegend)
for 3 h. The CFSE-labeled T cells were then seeded into 96-well plates
(2×105 cells/well) that had been pre-coated overnight with anti-CD3 (5
µg/mL, Biolegend) and anti-CD28 (2.5 µg/mL, Biolegend) at 4°C. The cisplatin-treated
A549 cells with or without B7-H1 blocking antibody (50 µg/mL, Biolegend) were then
added to CFSE-labeled T cells at a T:A549 ratio of 1:2, 1:4, or 1:8. Each condition
was tested in triplicate. After 72 h, all cells were collected and T-cell
proliferation was examined by flow cytometry.