Fitc anti human granzyme b

Antibody were purchased from BioLegend, including FITC anti-human HLA-A2 antibody (Cat#343303), PE anti-human HLA-A2 antibody (Cat#343305), anti-human CD28 antibodies (Cat#302901), APC anti-human-CD69 (Cat#310909), APC anti-human CD8 (Cat#344721), PerCP anti-human IFN-γ (Cat#502524), and FITC anti-human granzyme B (Cat#515403). Flex-T monomer (Cat#280003) and PE conjugated streptavidin (Cat#405203) were purchased from BioLegend. Ficoll-Paque PLUS (Cat#17144003) was from GE and DMSO (Cat#D2650) was from Sigma-Aldrich. IMDM (Cat#SH30228.01) and penicillin-streptomycin (Cat#SV30010) were from HyClone, and FBS (Cat#S711-001S) was purchased from LONSERA. PBS (Cat#C10010500BT) was from Gibco (Waltham, Massachusetts, USA). D-biotin (Cat#2110450) was purchased from Invitrogen (Waltham, Massachusetts, USA), and Mitomycin C (Cat#50-07-7) was from Sinochem Holdings (Beijing, China). CFSE (Cat#T6802) was from Targetmol (Boston, Massachusetts, USA). EasySep Human CD8⁺ T Cell Isolation Kit (Cat#17953) was from Stem Cell Technologies (Vancouver, British Columbia, Canada), and IL-2 (recombinant human interleukin-2(¹²⁵Ala) injection) was from SL PHARM (Beijing, China). Leuko act cktl with GolgiPlug (Cat#550583) was purchased from BD.

HLA-A2 expressing T2A2 cells were loaded with peptides for subsequent T cell activation. Briefly, T2A2 cells were treated with 20 μg/mL mitomycin C (Sinochem) for 30 min to stop cell proliferation, and loaded with given epitope peptides. 0.5×10⁶ CD8⁺ T cells isolated from health donors were co-cultured with 0.5×10⁶ peptide-loaded T2A2 cells stained with 5 μmol/L CFSE (TargetMol), and stimulated with 1 μg/mL anti-human CD28 antibodies (BioLegend) and 50 IU/mL IL-2 (SL PHARM, Recombinant Human Interleukin-2(¹²⁵Ala) Injection). 50 IU/mL IL-2 and 20 μM mixed peptides were then supplemented every two days. The T cell activation marker CD69 (BioLegend), tetramer specific CD8⁺ T cells and apoptosis marker Annexin V-APC (BioLegend) on T2A2 cells were evaluated after 16 hours and 7 days, respectively. On day 7, stimulatory T cell-mediated T2A2 killing, we gated the total CD8⁺ (CFSE negative) and T2A2 cells (CFSE positive), then stained with LIVE/DEAD dye and then calculated the percentage of live T2A2 cells. On day 7, cells were re-stimulated with peptides for 6 hours in the presence of Leuko Act Cktl with GolgiPlug (BD) plus 50 IU/mL IL-2, and the production of IFN-γ and Granzyme B was checked with PerCP anti-human IFN-γ (BioLegend) and FITC anti-human Granzyme B (BioLegend) staining.