Anti nkg2a apc clone z199

Flow cytometric analysis was performed on PBMCs and RB-derived cells according to standard procedures using a panel of mAbs that others and we have shown to be cross-reactive with RM immune cells (10 (link), 19 (link)) (Supplementary Table 2). The following Abs were used: anti–CD4-APCCy7 (clone OKT4), anti–HLA-DR-BV711 (clone L243), and anti-CD20 PerCpCy5.5 (clone 2H7) all from Biolegend, San Diego, CA, USA; anti–CD95-CF594 (clone DX2), anti–beta7-PECy5 (clone FIB504), anti–CCR7-PECy7 (clone 3D12), anti–Ki67-Alexa700 (clone B56), anti–CD3-BUV395 (clone SP34-2), anti–CD8-BUV496 (clone RPA-T8), anti–CD56-BV605 (clone B159), and anti–CD16-BV650 (clone 3G8) all from Becton–Dickinson, BD Biosciences, San Jose, CA, USA; anti–NKG2A-APC (clone Z199), from Beckman Coulter, Brea, CA, USA; Aqua Live/Dead amine dye-AmCyan from ThermoFisher Scientific, Invitrogen, Waltham, MA, USA; anti–CD38-FITC (clone AT-1) from STEMCELL Technologies, Vancouver, British Columbia, Canada; and anti–α4β7-PE (clone Act-1) obtained from the NIH Non-human Primate Reagent Resource, University of Massachusetts Medical School. Flow cytometric acquisition was performed on at least 100,000 CD3⁺ T cells on a BD LSRII Flow Cytometer driven by BD FACSDiva software. Analyses of the acquired data were performed by FlowJo software, Tree Star, Inc., Ashland, OR, USA.

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.