Ifn γ pe

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).

ELISA kits for murine IL-6 was from R&D Systems (Minneapolis, MN). Fluorescein-conjugated mAbs including anti-CD3-PE-Cy7, CD4-PE-Cy5, CD8-PE, CD8-FITC, CD11b-APC, Ly6G-PE, Ly6C-FITC, CXCR2-Percp-Cy5.5, CD45-BV510, PD1-PE, PDL1-PE, LAG3-PE, CTLA4-PE, IFNγ-PE and isotype antibodies were purchased from BD biosciences. TIM3-PE was from Miltenyi Biotec (Bergisch-Gladbach, Germany). Antibody against STAT3 (79D7, 4904S), p-STAT3 (Tyr705, 9131S), ZEB1 (3396P), ZO-1(5406P), Snail(3879P), N-Cadherin(4061P) were obtained from Cell Signaling Technology (Beverly, MA, USA) and antibody against Actin was from Santa Cruz. IL6 inhibitor (501109) and IFN-γ inhibitor (505827) was from Biolegend. Luciferin substrate (K9909PE) for in vivo image was purchased from PerkinElmer Inc (Hopkinton, MA, USA). HE staining kit was from Beyotime Biotechnology. ShRNA for ZEB1 (TG513177) was purchased from OriGene.

PBMCs and other cells were stained with anti-human mAbs: anti-CD3 FITC, anti-CD4 BV605, anti-CD8 BV421, anti-CD14 BV711, anti-CD19 BV605, anti-CD1d APC, anti-HLA-DR BV421, IFN-γ PE, and IL-4 PE (all from BD Biosciences, Vienna, Austria). APC-labeled human CD1d tetramers loaded with the α-GalCer analogue PBS-57 were obtained from the MHC Tetramer Core Facility (Emory University Vaccine Center, Atlanta, GA). Anti-human ADRB2 (AbD Serotec, Oxford, UK) was coupled with alexa fluor647 fluorochrome by using a protein labeling kit (Life Technologies, Paisley, UK). Viability was assessed with Zombie Nir viability dye (BioLegend, London, UK) or with fixable viability dye eFluor506 (eBiosciences, Vienna, Austria). The corresponding isotype control mAbs were used to assess staining specificity.

Escherichia coli (DH5α, Invitrogen) was cultured overnight at 37°C in SOC broth. Bacteria were washed once in PBS and fixed in 2% paraformaldehyde for 20 min, then washed extensively before counting by Flow Cytometry (FACSVerse, BD Biosciences) and added to the THP1 (a human monocytic cell line derived from an acute monocytic leukemia patient) in a bacteria/cell ratio of 100:1 for 24 h. PBMCs were cultured for 1 h in round-bottom 96-well plates in the presence of E. coli-activated THP1 (1:2) or PMA (250 ng/μl) plus ionomycin (1 μg/μl) as positive control. Brefeldin A (10 μg/ml) was added and cells were incubated for further 4 h. Cells were harvested and stained as previously described. For the intracellular quantification of cytokine production, the following antibodies were used: IL-17 PE (Miltenyi Biotech), IFNγ PE and TNFα FITC (BD Biosciences).
The assay setup for both PMA/iono and bacterial stimulation was optimized on 2 HIV– and 1 HIV+ subjects. During each round of experiments, PBMCs from the same batch of the same HIV– subject were included. This allowed us to verify that the culture conditions were reliable and that the stimuli were working as expected.

Intracellular staining was carried out according to the manual of the BD Cytofix/Cytoperm™ kit (BD Biosciences). Briefly, PBMCs were harvested and adjusted to 1 × 10⁶ cells/mL. The cells were incubated with 0.1% GolgiStop (BD Biosciences) in an incubator for 4 h. The cells were incubated with mouse mAbs against human CD56 (FITC), CD3 (PerCP), and PD-1 (APC). These were followed by intracellular staining with mouse mAbs against human perforin (PE), granzyme B (PE), or IFN-γ (PE) (BD Pharmingen, San Jose, CA, USA), as well as the isotype control antibodies. After one wash with PBS, the cells were detected by the FACSCalibur cell analyzer. The data were analyzed as above.

An additional experiment was designed and performed to evaluate CD4+ T‐cell phenotypes. The mouse spleen was acquired under sterile condition, cut into small pieces and filtered with 70‐μm strainer to remove the capsule and connective tissue. The cell supernatant was collected and centrifuged at 1500 rpm for 10 min., with thrice washes atTris‐NH4 cl. FACSAria II flow cytometry (BD Biosciences, San Diego, CA, USA) was used to test spleen CD4+ T‐cell subgroups in animals mentioned above. The CD4+ T cells were isolated by flow cytometry and then were labelled with IFN‐γ‐PE, IL‐4‐PE and Foxp3‐PE antibodies (BD). The proportion of CD4 + IFN‐γ+ T cells, CD4 + IL‐4+ T cells and CD4 + Foxp3+ T cells was accounted. The mRNA expression of T‐bet, GATA‐3 and Foxp3 in lung tissues harvested from various groups was measured on basis of gene probes as listed in Table 1, using Rotor‐Gene 3000 fluorescence ration PCR instrument (Corbett Research, Sydney, Australia).