Isolation and Characterization of Mamu-KIR3DL05 NK Cells

Expanded NK cell cultures were incubated with anti-CD3 Ab (clone 6G12) and T cells were depleted using pan-mouse IgG Dynabeads (Dynal Biotech). Mamu-KIR3DL05⁺ and -KIR3DL05^- subsets were separated by FACS using a Mamu-A1*002 tetramer folded with Gag_71-79 GY9 that binds Mamu-KIR3DL05 [27 (link)]. NK cells were stained with PE-conjugated Mamu-A1*002-GY9 tetramer for 30 minutes at 37°C followed by staining with anti-CD3-Pacific Blue (clone SP34-2; BD Biosciences) and anti-NKG2A-APC (clone Z199; Beckman Coulter) or anti-NKG2A-Pacific Blue and anti-CD3-FITC (clone SP34; BD Biosciences) for 20 minutes at 25°C. Tetramer⁺CD3^-NKG2A⁺ and Tetramer^-CD3^-NKG2A⁺ subsets were sorted using a FACSAria II (BD Biosciences). After sorting, these NK cells subsets were stimulated with γ-irradiated K562 Clone 9.mbIL21 cells and maintained as described above. Mamu-KIR3DL05⁺ and -KIR3DL05^- NK cell subsets were sorted from PBMC by the same procedure for immediate use in viral suppression assays.