Journal: PLoS Biology

Article Title: MAIT cells launch a rapid, robust and distinct hyperinflammatory response to bacterial superantigens and quickly acquire an anergic phenotype that impedes their cognate antimicrobial function: Defining a novel mechanism of superantigen-induced immunopathology and immunosuppression

doi: 10.1371/journal.pbio.2001930

Figure Lengend Snippet: Human peripheral blood mononuclear cells (PBMCs) ( n = 8) were left untreated or stimulated for 24 h with SEB. They were then washed and rested for 3 days before they were exposed to K . pneumoniae lysate. Interferon (IFN)-γ + , tumor necrosis factor (TNF)-α + , interleukin (IL)-2 + , and IL-17A + events among MAIT cells were enumerated 24 h later by intracellular cytokine staining ( A ). PBMCs from a separate cohort were subjected to SEB stimulation, followed by 24 h of resting, before they were challenged with either K . pneumoniae or E . coli lysate. Twenty-four hours later, cells were interrogated for their intracellular IFN-γ content ( n = 13) and evaluated for staining with Annexin V or Fixable Viability Dye ( n = 4) ( B ). SEB-exposed CD3 + cells co-expressing LAG-3 were divided into 5 subpopulations based on CD161 and Vα7.2 staining. The relative contribution of each subpopulation to total CD3/LAG-3 double-expressors is depicted in a pie chart generated using PBMCs from 8 donors ( C ). Concomitant upregulation of CD69 and LAG-3 by SEB, or lack thereof, was also examined in conventional T (T conv ) and MAIT cell compartments. Representative FACS plots are shown ( n = 8) ( D ). In additional experiments, the frequencies of LAG-3 + MAIT cells were determined in PBMC cultures containing 20 μM SB203580 or PD98059 ( E ), 5 ng/mL recombinant human IL-12 (rIL-12) and/or recombinant human IL-18 (rIL-18) ( F ), 5 μg/mL anti-IL-12 and anti-IL-18 ± 200 ng/mL cyclosporine A (CsA) ( n = 3) ( G ). The proportions of LAG-3 + TIM-3 - and LAG-3/TIM-3 double-expressors among T conv and MAIT cells were also calculated at indicated time points after SEB stimulation of PBMCs ( n = 8) ( H ). In separate experiments, PBMC cultures were stimulated for 24 h with SEB. Cells were washed, and cultures were replenished with fresh medium containing 20 μg/mL of an anti-human LAG-3 monoclonal antibody (mAb) or a mouse IgG1 isotype control. Klebsiella lysate was added to cultures, followed, 24 h later, by enumeration of IFN-γ + MAIT cells ( n = 3) ( I ). Error bars represent SEM. The underlying data for this figure can be found in , and our gating strategies are provided in .

Article Snippet: A frozen stock of a K . pneumoniae clinical isolate, Parkwood-18, was a gift from Dr. Miguel Valvano (Queen’s University Belfast, Belfast, United Kingdom).

Techniques: Staining, Expressing, Generated, Recombinant, Control