Cd56 apc

The percentages of cells producing IFN‐γ and granzyme‐B were measured by flowcytometry using various stimuli. For each condition, duplicate wells with 250 000 PBMCs were cultured in a 96 wells plate. Various combinations of the following stimuli were used as indicated; IL‐12 (0.25 ng/mL, Miltenyi), IL‐18 (50 ng/mL, MBL), CD28 monoclonal antibody (2 ug/mL, eBiosciences), R848 (1 μg/mL, Invivogen, San Diego, CA, USA), Escherichia coli ATCC 25922 (fixed for 20 minutes in 2% formaldehyde, 25 bacteria per lymphocyte), and E. coli K12 (fixed for 5 minutes in 1% formaldehyde, 25 bacteria per lymphocyte). For all conditions, cells were incubated for a total of 24 hours at 37°C at 5% CO₂. Brefeldin A (10 μg/mL, Sigma) was added after 6 or 21 hours of culture as indicated in the figure legend. Cells were stained with anti‐CD3‐PerCp‐Cy5.5(UCHT1), anti‐CD8‐APC‐H7(SK3), anti‐CD161‐eFluor450(HP‐3G10), anti‐TCR Vα7.2‐PE(3C10), CD56‐APC(N901, Beckman) and Live/dead Aqua, fixed, permeabilized and stained with anti‐IFN‐γ‐PE‐Cy7(4S.B3) and anti‐granzyme‐B‐FITC(GB11). Cytokine‐producing cells were detected by flowcytometry using a MACSQuant Analyser 10. Gating of cells was set on internal controls with low or absent expression on lineage negative cells. Only samples with more than 80 MAIT‐cell events were included for expression of surface markers, IFN‐γ and granzyme‐B.

To determine whether GCR and SIRT1 are associated with intracellular cytokine production in CD8+ and CD8– T and NKT-like cells, aliquots of blood were stimulated and treated as described above. Following washing of permeabilized cells, appropriately diluted GCR antibody (MCA2469, Bio-Rad, Sydney, Australia) was added to cells for 15 min. Cells were further washed and stained with anti-mouse IgG1 V450 secondary antibody for 15 min. Cells were then washed and stained with SIRT1 Alexa-Fluor 488, IFNγ PE (BD), TNFα PE (BD), CD3 perCP.CY5.5 (BD, Sydney, Australia), CD28 PECY7 (BD), CD56 APC (Beckman Coulter, Sydney, Australia), CD8 APC.CY7 (BD), and CD45 V500 (BD) for 15 min in the dark at room temperature. Appropriate controls and cells were analyzed as above.

Total blood counts, leucocyte, and monocyte counts were measured with a Sysmex XN-450 Hematology Analyzer (Sysmex Corporation, Kobe, Japan). Undiluted blood (50 µL) was incubated for 15 min in the dark at room temperature with the following antibodies: CD3-APC-750 (mouse, dilution: 1:25; Beckman Coulter, Woerden, The Netherlands), CD56-APC (mouse, 1:25; Beckman Coulter), CD16-FITC (mouse, 1:25; eBioscience Thermo Fisher Scientific, Breda, The Netherlands), CD14-PC7 (mouse, 1:25; eBioscience Thermo Fisher Scientific), CCR2-BV421 (mouse, 1:50 and 1:25; BD Biosciences), CD36-APC-700 (mouse, 1:10; Beckman Coulter), CD41-PC5.5 (mouse, 1:50; Biolegend, San Diego, CA, USA), CD11b-BV785 (mouse, 1:50 and 1:25; Biolegend). Followed by the addition of 1 mL 10× diluted lysis buffer (BD Pharm Lyse; BD Biosciences), samples were mixed and incubated for 10 min in the dark at room temperature. Samples were then measured on a Beckman Coulter FC500 flow cytometer, each sample was measured both stained and unstained. Flow cytometry data were analysed using Kaluza software (Beckman Coulter); for gating strategy, see Supplementary material online, Figure S1. Besides determining the different monocyte subsets (classical, intermediate, or non-classical). Atherogenic markers CCR2, CD36, CD41, and CD11b were determined per monocyte subset.