Carboxyfluorescein succinimidyl ester (cfse)

CD8+ OT1 cells were isolated from spleen of OT1 mice by CD8+ magnetic bead selection (Miltenyi), and OT1 cells were labeled with 2 μmol/L carboxyfluorescein succinimidyl ester (CFSE, Thermo Fisher), according to the manufacturer’s instructions. In a 3-day incubation, OT1 cells was co-cultured 1:1 with GM-CSF-primed neutrophils (100 ng/mL for 12 h) in cognate peptides SIINFEKL (10 ng/mL) and IL-2 (10 ng/mL), with or without a mouse PD-L1 neutralizing antibody (20 μg/mL, BioXCell). Proliferation of antigen activated CTLs was determined by flow cytometry. For cytotoxicity study, KPC cells were transfected with lentiviral vector (LV)-OVA or empty LV (GeneChem) at a MOI 50, and incubated with puromycin (5 μg/mL) for 5 days to select stable KPC-OVA or KPC-LV cell lines. OT1 cells were stimulated with SIINFEKL (10 ng/mL) and IL-2 (10 ng/mL) for 5 days to generate CTLs. CFSE-labeled GM-CSF-primed neutrophils and CTLs mixtures (1:1) were co-cultured with CFSE-free KPC-OVA or KPC-LV cells at a ratio of 20:1, with or without a mouse PD-1 (BioXCell, 20 μg/mL) or PD-L1 neutralizing antibody (BioXCell, 20 μg/mL) for 8 hr. Cytotoxicity was analyzed by calculating percentage of CFSE-PI+ cells using flow cytometry.

Mouse spleen CD3⁺ T cells were isolates by using MojoSort Mouse CD3 T Cell Isolation Kit (Biolegend, #480031) resulting in >95% purity. Isolated spleen CD3⁺ T cells were labeled with 5 μM carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, #C34554) for 10 min at room temperature (RT) and then blocked with 3 mL FBS for 10 min at 37°C. CFSE-labeled T cells were resuspended in culture medium (RPMI 1640 supplemented with 10% PBS, 1% L-Glutamine, 1% penicillin-streptomycin and 10% PBS, 1% L-Glutamine, 1% penicillin-streptomycin and 0.055mM 2-mercaptoethanol) and stimulated with 2.5 μg/mL coated anti-CD3 antibody (Bio X Cell, #BE0001-1) and 2.5 μg/mL soluble anti-CD28 (Bio X Cell, #BE0015-1) antibody in U-bottom 96-well plates for 2 days. For coculture studies, CFSE-labeled purified T cells (1 × 10⁶ cells/mL) were mixed with 5-day PBS or H-LPS treated WT or TRAM KO monocytes at a ratio of 1:1 in the presence of coated anti-CD3 and soluble anti-CD28 antibody for 2 days in a U-bottom 96-well plates (200 μL/well).