Cytometer setup tracking beads

We isolated peripheral blood mononuclear cells (PBMCs) and plasma from heparinized blood by density gradient centrifugation on Ficoll-Isopaque (Pharmacia, Piscataway, NJ). We stored plasma at −20°C for further immunological analysis. We immediately washed and stained the freshly isolated PBMCs with the following fluorochrome-conjugated antibodies purchased from BioLegend, BD, or Invitrogen: Vα7.2-PE, CD3-PE-Texas Red, CD4-Amcyan, CD8-FITC, CD161-APC, CD38-PE-Cy7, and DAPI. After 45 minutes incubation at 4°C, we analyzed at least 10⁵ lymphocytes on a FACSAria III flow cytometer (BD Biosciences, San Jose, CA) and analyzed data using FlowJo 10 software (TreeStar Inc, Ashland, OR). We used Cytometer Setup & Tracking beads (BD Biosciences) to check for inter-day variability, and Fluorescence Minus One (FMO) controls. The gating strategy is shown in Figure 1A. We defined MAIT cells as live (DAPI⁻) CD3⁺CD4⁻CD161^hiVα7.2⁺ cells, expressed as a percentage of total CD3⁺ lymphocytes, and used CD38 as a marker of cell activation. We also assessed the frequency of circulating live CD3⁺CD4⁻CD161^loVα7.2⁺ cells, which have been suggested to be MAIT-derived cells associated with absent or reduced cytokine secretion in vitro[18] (link), [20] (link), [26] (link), [27] , and also associated with MAIT cell loss and functional exhaustion in HIV patients [20] (link).

Data acquisition was performed on a FACS Aria^TM SORP cytometer (BD Biosciences) fitted with a 640 nm (30 mW) red laser, a 355 nm (60 mW) UV laser, a 405 nm (50 mW) violet laser, a 488 nm (100 mW) blue laser, and a 561 nm (50 mW) yellow/green laser. The Hoechst dye was excited by the UV laser and fluorescence was collected in two channels: UV-1 450/50 band-pass (BP) filter and UV-2′ 660/40 long-pass (LP) filter. An LP 635 nm dichroic mirror was used to split the emission wavelengths. The instrument was calibrated each time with Cytometer Setup&Tracking Beads (BD Bioscience). The Coefficient of Variation of the instrument (% CV) was routinely examined before each experiment. A 100 µm nozzle and window extension (WE) 3 were used for data acquisition and sorting. Imaging flow cytometry was performed with an ImageStream imaging cytometer (Amnis) fitted with a 375 UV laser, a 488 blue laser, a 561 yellow-green laser, a 642 red laser, and a 785 nm infrared laser. Acquisition was performed with the INSPIRE^® software and analysis was performed using IDEAS^® image analysis software. Pictures were taken at ×60 magnification at low-speed high-sensitivity mode.

Cytokines levels of IL-2, IL-4, IL-6, IL-10, IL-17, TNF-α, and IFN-γ were measured from peripheral blood samples collected by standard methods; specifically, they were obtained by venipuncture in 5-mL tubes without an anticoagulant. The tubes were centrifuged and the serum was removed and stored at -80°C until cytokine levels were examined by flow cytometry. Measurements of Th1 (IL-2, TNF, and IFN-γ), Th2 (IL4, IL-6, and IL-10) and Th17 (IL-17) cytokines were obtained by flow cytometry (FACSCanto II; BD Biosciences, San Jose, CA, USA) using the BD Cytometric Bead Array Human Th1/Th2/Th17 Cytokine Kit (CBA; BD Biosciences), according to manufacturer's instructions. The limits of detection for each cytokine were as follows: 0.1 pg/mL (IL-2), 0.03 pg/mL (IL-4), 1.4 pg/mL (IL-6), 0.5 pg/mL (IFN-ɣ), 0.9 pg/mL (TNF-α), 0.8 pg/mL (IL-17A), and 16.8 pg/mL (IL-10). For data analysis, FCAP array software was used (Soft Flow Inc., Pecs, Hungary). The sensitivity and instrument performance were evaluated using Cytometer Setup &Tracking Beads (BD Biosciences) prior to the sample analysis.