Cd3 pacific blue

ICS assays were performed as previously described^{5 (link)}. In brief, freshly isolated mouse splenocytes or monkey PBMCs were seeded into 96-well plates (2 × 10⁶ cells per well) and incubated with an EBOV GP peptide pool (2 µg/ml). One hour later, brefeldin A (BD Phamingen, CA, USA) was added and incubated for another 10 h. The cells were then harvested, stained with surface antibodies (CD3-Pacific blue, CD8-APC-Cy7, CD4-FITC; BD Biosciences, CA, USA) for 1 h, then permeabilized and stained with intracellular antibodies (IFN-γ-PE, IL-2-APC, TNF-α-PE-Cy7; BD Biosciences, CA, USA). Finally, the cells were detected with an LSR Fortessa SORP instrument (BD Biosciences, CA, USA).

Whole blood and mononuclear cells isolated from intestinal biopsies were stained for flow cytometry using a six-color technique as described previously to assess changes in the levels of major T cell populations and their immune activation status. The mAb combination used was: CD3-Pacific Blue, CD4-allophycocyanin, CD8-Texas Red, HLA-DR-allophycocyanin-Cy7, CD38-PE (BD Biosciences) and Ki-67–FITC (BD Pharmingen). All Abs were validated and titrated using PBMCs from PTMs [17 (link),75 (link)]. Samples were stained for Ki-67 using the Ki-67/FITC–conjugated mouse anti–human mAb set (BD Pharmingen) as per the manufacturer’s instructions. Stained cells were analyzed with an LSRII flow cytometer (BD Biosciences) and FlowJo Version 7.6 software (TreeStar). CD4⁺ and CD8⁺ T cell percentages were obtained by first gating on lymphocytes, then on CD3⁺ T cells. Activation markers were determined by gating on lymphocytes, then on CD3⁺ T cells, and finally on CD4⁺CD3⁺ or CD8⁺CD3⁺ T cells.

Flow cytometry was performed on cryopreserved cells. As per the gating strategy in figure 1A, NK cells were identified using a combination of Fixable blue dead cell stain (Life technologies), CD3-Pacific Blue, CD56-PE-Cy7 and CD16- APC-Cy7 (BD Biosciences). NK cell receptor expression was assessed using combinations of CD158a-PerCP-Cy5.5 (eBioscience), CD158b-FITC, KIR3DL1-Alexafluor700 (both Biolegend), KIR2DL3-PE, KIR2DL1-APC (both R&D Systems), NKG2A-PE (Beckman Coulter), NKG2D-APC, CD94-FITC, 2B4-FITC, NKp46-PE, NKp30-APC (all BD Biosciences), TRAIL-PE (Biolegend), CD161-FITC (Miltenyi) and CD160-Alexafluor647 (Biolegend). For intracellular staining, cells were fixed and permeabilized (PermA/B solution, Caltag) according to the manufacturers' instructions, prior to incubation with Perforin-PerCP-Cy5.5 (eBioscience). At least 1500 NK cells were acquired for all samples on either a five laser BD LSRFortessa or a four laser BD LSRII system, equipped with FACSDiva Version 8.8.3 (BD biosciences). Rainbow beads ensured a consistent, comparable level of fluorescence across all samples on different days of acquisition. Gates were set using fluorescence minus one or unstimulated samples where appropriate. The data were analysed using FlowJo version 9.5.3 (Treestar, OR, USA).

The predicted peptides were synthesized (Genscript Corporation). PBMCs were cultured in complete RPMI (Core Media Preparation Facility MSKCC) with peptides at 1 μg/mL, peptide vehicle (DMSO, Sigma-Aldrich) and CEF peptide pool (2 μg/ml, C.T.L) for 21 days with peptide restimulation at day 7 and day 14. IL-2 (Proleukin, Chiron) and IL-15 (Peprotech, cat#200-15) were added every 3 days at 10 IU/mL and 10 ng/mL respectively. Intracellular Cell Staining (ICS) was performed at day 14, and day 21 after 6 hr re-stimulation in the presence of monensin for 5 hr (GolgiStop, BD). Cells were then stained for 15 min with viability dye (LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, ThermoFisher) at 4°C followed by 30 min incubation with CD45-APC-H7 (BD PharMingen, clone 2D1), CD3-Pacific Blue (BD PharMingen, clone UCHT1), CD4-PerCP-Cy5.5 (eBioscience, clone OKT4), CD8-PE (BD Biosciences, clone SK1). Cells were then fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) for 20 min at 4°C and washed with BD Perm/Wash (BD Biosciences). The ICS was performed in BD Perm/Wash with IFN-γ-FITC (eBioscience, clone GZ-4) and TNF-α-PE-Cy (eBioscience, clone MAb11) at 4°C for 30 min. Samples were acquired on a BD LSRII flow cytometer (BD Biosciences) and the analysis was performed on FlowJo software (FlowJo, LLC).

PBMCs (2 × 10⁵) were immunostained on ice for 20 min with combinations of the following fluorochrome-conjugated mouse monoclonal antibodies or their concentration-matched isotype controls: 1 µg/mL CD3-Pacific Blue (Clone UCHT1; BD Biosciences), 1 µg/mL CD3-PeCy7 (Clone UCHT1; eBioscience, Hatfield, UK), 1 µg/mL CD56-PE (Clone AF12-7H3; Miltenyi Biotec), 5 µg/mL NKp46-Pacific Blue (Clone 9E2; BioLegend, Cambridge, UK) or 10 µg/mL NKG2D-PeCy7 (Clone 1D11; BioLegend). After incubation, samples were washed in PBS prior to flow cytometric analysis on a CyAN_ADP cytometer (Dako, Cambridgeshire, UK) where receptor expression was studied on 5000 CD3⁻ CD56^DIM NK cells.