Immunophenotyping
Mononuclear cells were prepared as described above for two immunophenotyping panels. Panel 1 was designed to study intracellular cytokine production following stimulation. Cells were stained with FITC-conjugated CD107a (Biolegend, San Diego, CA, USA) at the time of stimulation. Stimulation was performed with phorbol myristate acetate (PMA, 50 ng/mL, Sigma-Aldrich) and ionomycin (1 μg/mL, Sigma-Aldrich) for a total of 3.5 hours at 37 degrees centigrade. After 30 minutes, 1.25 μg/mL of monensin (Sigma-Aldrich) was added to stimulated cells for the remaining 3 hours. Cells were then washed with phosphate-buffered saline (PBS, Sigma-Aldrich) and re-suspended. Cell surface staining was performed for 30 minutes on ice in the dark as follows: BV510-conjugated CD4, PerCP-Cy5.5-conjugated CD8, ECD-conjugated CD14/CD19/CD56, APC-Cy7-conjugated CD3 (all from Biolegend) and Live/Dead (red, 488nm, Invitrogen, Carlsbad, CA, USA). After washing, cells were fixed with fixation/permeabilization solution (eBioscience, San Diego, CA, USA) for 30 minutes at room temperature in the dark. Cells were washed with permeabilization buffer (eBioscience). Following centrifugation and re-suspension, the following intracellular dyes were added for 30 minutes at room temperature in the dark: PE-Cy7-conjugated IL-6, AF700-conjugated IFN-γ, AF647-conjugated IL-4, BV421-conjugated IL-17A and PE-conjugated IL-8 (all from Biolegend). Cells were then washed and run on an LSRII flow cytometer equipped with blue, red and violet lasers (BD Biosciences, San Jose, CA, USA). The staining procedure was identical for unstimulated cells. At least 50,000 cells were recorded per sample.
Panel 2 assessed the immunophenotype of cells without mitogenic stimulation. Cells were surface stained with PE-Cy7-conjugated HLA-DR, BV510-conjugated CD4, PerCP-conjugated CD69, ECD-conjugated CD14/CD19/CD56, AF700-conjugated CD45RA, APC-Cy7-conjugated CD3, BV421-conjugated CD25, BV605-conjugated CD31 (all from Biolegend) and Live/Dead (red, 488nm, Invitrogen). After washing, cells were fixed with fixation/permeabilization solution (eBioscience) for 30 minutes at room temperature in the dark. Cells were washed with permeabilization buffer (eBioscience). Following centrifugation and re-suspension, AF647-conjugated FoxP3 (Biolegend) antibodies were added for 30 minutes in the dark. Cells were then washed and run on an LSRII flow cytometer equipped with blue, red and violet lasers (BD Biosciences). At least 50,000 cells were recorded per sample.
In both panels during the gating process, doublets were first excluded based on FSC-A and FSC-H characteristics. Lymphocytes were then identified based on FSC-A and SSC-A characteristics. Dead, as well as CD14+, CD19+ and CD56+ cells were excluded based on positivity in the ECD channel. Further gating was performed within CD3+ cells. Flow cytometry data was analyzed using the FlowJo software package.