Phenotypic Analysis of MAIT Cells

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).