Anti human cd107a h4a3

The following antibodies were used: anti-human CD3 (HIT3a, Biolegend), anti-human CD8 (SK1, Biolegend), anti-human NKp30 (P30-15, Biolegend), anti-human HLA-A2 (BB7.2, Biolegend), anti-human HER2 (24D2, Biolegend), anti-mouse TCR-β chain (H57-597, Biolegend), anti-human CD107a (H4A3, Biolegend), anti-human IFN-γ (B27, Biolegend). Corresponding isotype controls mIgG1 (MOPC-21, Biolegend), mIgG2b (MG2b-57, Biolegend) and Armenian Hamster IgG (HTK888, Biolegend) were used. Anti-B7H6 clone 1.18 and corresponding isotype control were from in-house production.^{9 (link)} For CD8⁺ T cell activation, anti-human functional-grade purified anti-CD3 (UCHT1, Biolegend) and anti-CD28 (37.51, Biolegend) were used.

Activated CD8⁺ T cells were stimulated with PMA/ionomycin plus protein transport inhibitor cocktail by adding 2 µg mL⁻¹ antimouse CD107a (LAMP‐1) (1D4B, BioLegend, 1:200) for 3 h. Cells were washed once with the staining buffer (PBS with 2% FBS) and then analyzed by flow cytometry. For KP‐10 analysis, CD8⁺ T cells were pretreated with 10 × 10⁻⁶m of KP‐10 for 6 h and then labeled with the antimouse CD107a (LAMP‐1) (1D4B, BioLegend) or antihuman CD107a (H4A3, BioLegend, 1:200) antibody.

Cells were stained with the following antibodies from Biolegend: anti-human CD107a (H4A3), anti-human Granzyme B (GB11), and anti-human Perforin (#353312). Antibodies were coupled with APC and Pacific Blue fluorochromes. For intracellular cytokine production analysis, cells were either stimulated with PMA + Ionomycin + Brefeldin A for 4 h at 37 °C or incubated only with Brefeldin A and further stained with ebioscience IC kit according to the manufacturer’s instructions. Flow cytometry acquisition was performed on a LSR Fortessa (BD) and data was analyzed with FlowJo X software (Tree Star).