Nkg2a pe

The following antibodies were used for flow cytometry: CD3 Alexa 700 (SP34-2 BD Biosciences), CD4 AmCyan (L200 BD Biosciences), CD8 PerCP-Cy5.5 (SKI eBiosciences), CD8 AmCyan (SKI BD Biosciences), CD28 PE-Texas Red (CD28.2 Beckman Coulter, BD Biosciences), CD95 PE (DX2 BD Biosciences, eBiosciences), CCR5 Allophycocyanin (3A9 BD Biosciences), Ki-67 FITC (B56 BD Biosciences), CD56 PerCP-Cy5.5 (MEM-188 Invitrogen), CD16 Pacific Blue (3G8 BD Biosciences, Biolegend), CD20 Allophycocyanin-Cy7 (L27 BD Biosciences), HLA-DR PE-Texas Red (TU36 Invitrogen, Immu357 Beckman Coulter), NKG2A PE (Z199 Beckman Coulter), CD14 FITC (M5E2 BD Biosciences, R&D Systems), STAT5 PE (47/Stat5 (pY6) BD Biosciences), BrdU FITC (B44 BD Biosciences), BrdU Allophycocyanin (B44 BD Biosciences). Anti-CCR7 (150503) was purchased as purified immunoglobulin from R&D Systems, conjugated to biotin using a Pierce Chemical Co. biotinylation kit, and visualized with streptavidin–Pacific Blue (Invitrogen). Rhesus recombinant anti-IL-15 and rhesus recombinant control IgG1 mAb were provided through the National Institutes of Health’s Nonhuman Primate Reagent Resource Program. Simian recombinant IL-7 was provided by Cytheris SA (Issy-Les-Moulineaux, France). Rhesus recombinant IL-15 was provided by Francois Villinger (Emory University) through the Resource for Nonhuman Primate Immune Reagents.

KB tumor cells were treated with C-IgG– or F-IgG–FITC for 30 min at 37°C and collected at the designated time points. Cells were washed with flow buffer (5% FBS in PBS), fixed in 1% formalin in PBS, mounted on 25 × 75 × 1 mm slides (Erie Scientific, Portsmouth, NH) and stained with DAPI for nuclear identification (Vector Labs, Burlingame, CA) prior to analysis by immunofluorescence (IF) microscopy. Intracellular analysis of NK cell ERK phosphorylation was also assessed following coculture with KB tumor cells. NK cell activation was assessed 48 hours post coculture via FACS analysis with CD56‐phycoerythrin (PE), CD158-allophycocyanin (APC), CD57‐FITC, NKG2A-PE (Beckman Coulter, Brea, CA), CD56‐APC, CD3‐V450, CD16‐FITC, NKG2D-APC, CD69–phycoerythrin-cyanin 7 (Pe-Cy7) and KIR/NKAT2‐FITC, CD94‐APC, NKp46/CD335-APC antibodies (BD Biosciences, San Jose, CA). In murine studies, IF microscopy of tumor sections was performed (16 (link)). Sections were analyzed on an Olympus Fluoview 1000 laser scanning confocal microscope. In murine studies with C‐IgG– and F‐IgG–FITC, tissues were harvested in the dark at indicated times. Tumors were dissociated (17 (link)) and stained with FR‐α–APC (R&D Systems, Minneapolis, MN). FR⁺/FITC⁺ populations were assessed using a Becton Dickinson FACS Calibur flow cytometer (Becton‐Dickinson, San Jose, CA) (16 (link)).

Cryopreserved peripheral blood mononuclear cells (PBMC) were stained with combinations of antibodies that comprehensively interrogate the breadth of NK-cell receptors including the killer immunoglobulin receptors (KIRs), the c-type lectin receptors (NKG2), and the natural cytotoxicity receptors (NCRs). These included CD158a-PerCP-Cy5.5 (eBioscience), KIR3DL1-Alexafluor700 (Biolegend), NKG2A-PE (Beckman Coulter), NKG2D-APC, CD94-FITC, 2B4-FITC, NKp46-PE, NKp30-APC, CD158b-FITC (all BD Biosciences), TRAIL-PE (Biolegend), CD161-FITC (Miltenyi) and CD160-Alexafluor647 (Biolegend). Dead cells were excluded using the Fixable Blue Dead Cell Stain (Life Technologies) prior to surface staining and fixation. For perforin staining, samples were then permeabilized (Perm A/B, Caltag) and stained with anti-Perforin-PerCP-Cy5.5 (eBioscience). NK cells were identified as CD3 negative lymphocytes expressing CD56 and/or CD16. At least 1500 NK cells were acquired per sample. Fluorescence minus one was used to set gates. The data were analyzed with Flowjo v9.5.4 (Treestar).