Tnf α pe cy7

Antibodies for surface markers staining of DCs and T cells were obtained from BD Biosciences, New York, USA (anti-human CD3-PE, CD3-FITC, CD8-PerCP, CD8-APC, CD56-PE, NKG2D-APC, CD4-FITC, CD4-PerCP, CD107a-FITC, CD25-APC, CD45RO-FITC, CD27-PerCPCY5.5, CD57-APC, CCR7-PE, CD14-APC, CD80-PE, CD83-APC, CD86-FITC, HLA-DR-FITC). Antibodies for intracellular proteins staining were also obtained from BD Biosciences (anti-human IFNγ-APC, TNFα-PECY7, granzyme B-FITC, FoxP3-PE). The intracellular staining was performed by fixing and permeabilizing cells with Cytofix/Cytoperm (BD Biosciences). The experiments were performed by using FACS CantoII (BD Biosciences) flow cytometer, and data were analyzed by using the Flowjo software.

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).

Batched stored samples were thawed quickly at 37°C and washed twice with 1X BD PermWash buffer. Cells were then incubated in 1X BD PermWash for 10 min before incubation with the antibody cocktail mix in 2% FCS at 4°C for 45 min. After incubation, cells were washed twice with PBS (2% FCS) and the resuspended in 0.3 mL PBS for cell acquisition using a Beckton Dickinson LSRII flow cytometer (SORF model). The following monoclonal antibody-fluorochrome conjugates were used: IL-2-R-phycoerythrin (PE), CD8-V500, IFN-γ-Alexa Fluor-700, TNF-α-PE-Cy7, Ki67-Fluorescein isothiocyanate (FITC), all from BD, CD27 PE-Cy5, HLA-DR- Allophycocyanin-Cy7 (APC-Cy7), CD3-BV650 (Biolegend), CD4 PE-Cy5.5 (Invitrogen), CD45RA PE-Texas Red-X (Beckman Coulter). A minimum of 50,000 ViViD negative (viable) CD3 events were collected using FACS DIVA v6 software. Post-acquisition compensation and analysis was performed in FlowJo version 9 (FlowJo, LLC). Supplementary Figure 1 shows the gating strategy employed.

Staining and flow cytometry were conducted as previously described (34 (link)). In brief, batched stored samples were thawed quickly and washed, incubated in BD PermWash, and then stained with the antibody cocktail mix. After incubation, cells were washed and then resuspended in PBS for cell acquisition using a BD LSRII flow cytometer (SORF model). The following monoclonal antibody–fluorochrome conjugates were used: IL-2-R–phycoerythrin (PE), CD8-V500, IFN-γ–Alexa Fluor 700, TNF-α–PE-Cy7, Ki67–fluorescein isothiocyanate (FITC), all from BD; CD27–PE-Cy5, HLA-DR–allophycocyanin-Cy7 (APC-Cy7), CD3-BV650 (BioLegend); CD4–PE-Cy5.5 (Invitrogen); and CD45RA–PE-Texas Red-X (Beckman Coulter). A minimum of 50,000 ViViD-negative (viable) CD3⁺ events were collected using BD FACSDiva v6 software. Postacquisition compensation and analysis was performed in FlowJo version 9 (FlowJo, LLC). Supplemental Figure 2 shows the gating strategy employed. Measurable response to BCG was characterized by the polyfunctionality of the CD3⁺CD4⁺ T cell response assessed by analyzing permutations of TNF-α, IL-2, and IFN-γ expression after stimulation using combinatorial polyfunctionality analysis of antigen-specific T cell subsets (COMPASS) (35 (link)). PFS and FS, which summarize the functional profile of each subject, were calculated from posterior probabilities as described previously (35 (link)).

PBMCs were cultured in 96-well flat-bottom plates in Basal Medium Eagle (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin (all from Thermo Fisher Scientific). PBMCs were stimulated with recombinant Human IL-3 (100 ng/mL; PEPROTECH, Rocky Hill, NJ, USA) and a TLR7 agonist, imiquimod (R837) (100 ng/mL; InvivoGen, San Diego, CA, USA) or a TLR9 agonist, CpG ODN 2216 (5 μg/mL; Miltenyi Biotec, Bergisch Gladbach, Germany) for 6 h at 37 °C in a 5% CO₂ incubator. GolgiPlug (100 ng/mL; BD Biosciences) was added during the final 3 h of stimulation to block cytokine secretion. After staining the cell-surface antigens, intracellular cytokines were stained using the BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences), anti-IFN-α-APC (Miltenyi Biotec), and anti-tumor necrosis factor α (TNF-α)-PE-Cy7 (BD Biosciences), or their isotype control antibodies.