Cytotoxic Degranulation of NK and CD8+ T Cells

Cytotoxic degranulation was assessed by measuring cell surface expression of CD107a as previously described (47 (link)). Resting or IL-2–activated NK cells isolated from splenocytes by negative selection using a mouse NK cell isolation kit (STEMCELL Technologies) were stimulated with YAC-1 or B16F10 cells for 4 hours. Lymphocytes were gated on forward scatter/side scatter, and the CD107a expression of CD3ε⁻NKp46⁺ NK cells was analyzed by flow cytometry. NK cell degranulation was determined by the percent increase of CD107a⁺ NK cells after stimulation with YAC-1 or B16F10 cells relative to CD107a⁺ NK cells without stimulation (ΔCD107a⁺ cells). For assessing CD8⁺ T cell-cytotoxic activity, IL-2–activated CD8⁺ T cells were isolated from splenocytes by negative selection using a mouse CD8⁺ T cell isolation kit (STEMCELL Technologies) and were stimulated with P815 cells preincubated with anti-CD3ε antibody (10 μg/ml, clone 145-2C11; BioLegend) or isotype control antibody (10 μg/ml, clone HTK888; BioLegend) for 4 hours. P815 cells are FcR⁺ and serve as a classical target for T cell cytotoxicity via their ability to bind antibodies to specific activating receptors (for example, CD3ε). In doing so, they can stimulate T cells through CD3ε. Cytotoxic degranulation was measured by surface expression of CD107a on CD3ε⁺CD8a⁺ T cells after lymphocyte gate.