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

Superantigens (SAgs) are potent exotoxins secreted by Staphylococcus aureus and Streptococcus pyogenes. They target a large fraction of T cell pools to set in motion a "cytokine storm" with severe and sometimes life-threatening consequences typically encountered in toxic shock syndrome (TSS). Given the rapidity with which TSS develops, designing timely and truly targeted therapies for this syndrome requires identification of key mediators of the cytokine storm's initial wave. Equally important, early host responses to SAgs can be accompanied or followed by a state of immunosuppression, which in turn jeopardizes the host's ability to combat and clear infections. Unlike in mouse models, the mechanisms underlying SAg-associated immunosuppression in humans are ill-defined. In this work, we have identified a population of innate-like T cells, called mucosa-associated invariant T (MAIT) cells, as the most powerful source of pro-inflammatory cytokines after exposure to SAgs. We have utilized primary human peripheral blood and hepatic mononuclear cells, mouse MAIT hybridoma lines, HLA-DR4-transgenic mice, MAIThighHLA-DR4+ bone marrow chimeras, and humanized NOD-scid IL-2Rγnull mice to demonstrate for the first time that: i) mouse and human MAIT cells are hyperresponsive to SAgs, typified by staphylococcal enterotoxin B (SEB); ii) the human MAIT cell response to SEB is rapid and far greater in magnitude than that launched by unfractionated conventional T, invariant natural killer T (iNKT) or γδ T cells, and is characterized by production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2, but not IL-17A; iii) high-affinity MHC class II interaction with SAgs, but not MHC-related protein 1 (MR1) participation, is required for MAIT cell activation; iv) MAIT cell responses to SEB can occur in a T cell receptor (TCR) Vβ-specific manner but are largely contributed by IL-12 and IL-18; v) as MAIT cells are primed by SAgs, they also begin to develop a molecular signature consistent with exhaustion and failure to participate in antimicrobial defense. Accordingly, they upregulate lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin-3 (TIM-3), and/or programmed cell death-1 (PD-1), and acquire an anergic phenotype that interferes with their cognate function against Klebsiella pneumoniae and Escherichia coli; vi) MAIT cell hyperactivation and anergy co-utilize a signaling pathway that is governed by p38 and MEK1/2. Collectively, our findings demonstrate a pathogenic, rather than protective, role for MAIT cells during infection. Furthermore, we propose a novel mechanism of SAg-associated immunosuppression in humans. MAIT cells may therefore provide an attractive therapeutic target for the management of both early and late phases of severe SAg-mediated illnesses

Broadband Multi-wavelength Properties of M87 during the 2017 Event Horizon Telescope Campaign

Abstract: In 2017, the Event Horizon Telescope (EHT) Collaboration succeeded in capturing the first direct image of the center of the M87 galaxy. The asymmetric ring morphology and size are consistent with theoretical expectations for a weakly accreting supermassive black hole of mass ∼6.5 × 109 M ⊙. The EHTC also partnered with several international facilities in space and on the ground, to arrange an extensive, quasi-simultaneous multi-wavelength campaign. This Letter presents the results and analysis of this campaign, as well as the multi-wavelength data as a legacy data repository. We captured M87 in a historically low state, and the core flux dominates over HST-1 at high energies, making it possible to combine core flux constraints with the more spatially precise very long baseline interferometry data. We present the most complete simultaneous multi-wavelength spectrum of the active nucleus to date, and discuss the complexity and caveats of combining data from different spatial scales into one broadband spectrum. We apply two heuristic, isotropic leptonic single-zone models to provide insight into the basic source properties, but conclude that a structured jet is necessary to explain M87’s spectrum. We can exclude that the simultaneous γ-ray emission is produced via inverse Compton emission in the same region producing the EHT mm-band emission, and further conclude that the γ-rays can only be produced in the inner jets (inward of HST-1) if there are strongly particle-dominated regions. Direct synchrotron emission from accelerated protons and secondaries cannot yet be excluded

Therapeutic control of leishmaniasis by inhibitors of the mammalian target of rapamycin.

Leishmaniasis is a serious global health problem affecting many people worldwide. While patients with leishmaniasis can be treated with several agents, drug toxicicty and the emergence of resistant strains render available treatments ineffective in the long run. Inhibitors of the mammalian target of rapamycin (mTOR) have been demonstrated to exert anti-pathogen properties. In this study, we tested the therapeutic efficacy of several mTOR inhibitors in controlling infection with Leishmania major. Rapamycin, GSK-2126458 and KU-0063794 were administered to BALB/c mice, which had received an intrafootpad injection of the parasite. Footpad swelling and parasite burden were assessed, and cytokine production by mouse splenocytes and phenotypic changes in draining lymph node cells were evaluated. Treatment with a clinically relevant dose of rapamycin or with GSK-2126458, but not with KU-0063794, dramatically lowered both the footpad swelling and the parasite load in the draining lymph node. Importantly, the employed dose of rapamycin did not kill the promastigotes in vitro as judged by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and electron microscopy. Moreover, the IL-4 production capacity of splenocytes harvested from infected mice that were treated with rapamycin was significantly reduced. Consequently, the IFN-γ:IL-4 production ratio was elevated, suggesting a T helper-type 1 (Th1)-skewed cytokine profile. Finally, the expression level of CD69, an early activation marker, on splenic and lymph node CD4+ and CD8+ T cells was enhanced in rapamycin-treated mice. Taken together, our findings suggest that select mTOR inhibitors may be used in therapeutic settings for the management of leishmaniasis. We propose that the beneficial effects of such inhibitors stem from their immunomodulatory properties. Therefore, the adjuvanticity of mTOR inhibitors may also be considered in vaccination strategies against Leishmania species

Human peripheral blood mucosa-associated invariant T (MAIT) cells are a predominant source of interferon (IFN)-γ after exposure to staphylococcal enterotoxin B (SEB).

<p>Human peripheral blood mononuclear cells (PBMCs) were left untreated or stimulated for 24 h with 100 ng/mL of SEB. Intracellular IFN-γ was detected by flow cytometry among bulk CD3⁺, CD3⁺CD161^-, CD3⁺CD161^low, and CD3⁺CD161^high cells (<b>A</b>). PBMCs were additionally stained with monoclonal antibodies (mAbs) to T cell receptor (TCR) γδ and TCR Vα7.2 and with PBS-57-loaded CD1d tetramer. Events corresponding to γδ T (green), invariant natural killer T (<i>i</i>NKT) (red), MAIT (blue), and conventional T (T_conv) (cyan) cells were superimposed to generate a dot plot (<b>B</b>). The frequency of IFN-γ⁺ cells and the mean fluorescence intensity (MFI) of IFN-γ staining were determined in 7 donors, each of whom is represented by a circle (<b>C</b>). Five subpopulations were defined among SEB-stimulated CD3⁺ cells based on their co-expression of CD161 and Vα7.2, or lack thereof. The proportion of IFN-γ⁺ cells for each subpopulation is demonstrated in representative FACS plots (<b>D</b>). Historical data from 24 donors were subjected to Spearman’s rank correlation analysis to test the association between MAIT cell and IFN-γ⁺ cell frequencies (<b>Fig 1E</b>). Mean ± SEM values are shown in <b>C</b> (<i>n</i> = 7) and <b>D</b> (<i>n</i> = 9). *, **, ***, and **** in panel C denote statistical differences with <i>p</i> < 0.05, <i>p</i> < 0.01, <i>p</i> < 0.001, and <i>p</i> < 0.0001, respectively. The underlying data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001930#pbio.2001930.s002" target="_blank">S1 Data</a>, and our gating strategies are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001930#pbio.2001930.s003" target="_blank">S2 Data</a>.</p

Staphylococcal enterotoxin B (SEB) induces rapid expression of LAG-3, TIM-3 and PD-1 by human mucosa-associated invariant T (MAIT) cells in vivo.

<p>Human peripheral blood mononuclear cell—reconstituted NOD-<i>scid</i> IL-2Rγ^null (hPBMC-NSG) mice (<i>n</i> = 3/group) were injected with PBS or 100 μg SEB. Twenty-four hours later, human interferon (IFN)-γ was measured in the serum by ELISA. Error bars represent SEM, and ** denotes <i>p</i> < 0.01 (<b>A</b>). Nine days after SEB injection, splenic cells were stained with anti-human CD45, CD3, CD161, and Vα7.2 monoclonal antibodies (mAbs) to confirm the presence and/or expansion of human hematopoietic cells, T cells and MAIT cells in hPBMC-NSG mice (<b>B</b>). In addition, MAIT cells were assessed for surface expression of human LAG-3, TIM-3 and PD-1 (open histograms). Filled histograms represent background staining with isotype controls (<b>C</b>). The underlying data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001930#pbio.2001930.s002" target="_blank">S1 Data</a>, and our gating strategies are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001930#pbio.2001930.s003" target="_blank">S2 Data</a>.</p

IL-12/IL-18-mediated transactivation of mucosa-associated invariant T (MAIT) cells following staphylococcal enterotoxin B (SEB) stimulation requires signaling through p38 and MEK1/2.

<p>Human peripheral blood mononuclear cells (PBMCs) (<i>n</i> = 6) were exposed to SEB in the absence or presence of 20 μM SB203580 and/or PD98059, and intracellular interferon (IFN)-γ accumulation in MAIT cells was detected after 24 h by flow cytometry (<b>A</b>). Intracellular phosphorylated p38 was traced in MAIT cells after stimulation with SEB in the absence or presence of anti-IL-12 and/or anti-IL-18 monoclonal antibodies (mAbs). Representative FACS plots are shown along with a bar graph summarizing data obtained from 3 individuals (*: <i>p</i> < 0.05; NS: non-significant) (<b>B</b>). In separate experiments, PBMCs were left untreated or exposed to SEB or to recombinant human IL-12 (rIL-12) and/or recombinant human IL-18 (rIL-18) for 24 h before intracellular p38 phosphorylation in MAIT cells was analyzed (<i>n</i> = 6). Error bars represent SEM (<b>C</b>). The underlying data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001930#pbio.2001930.s002" target="_blank">S1 Data</a>, and our gating strategies are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001930#pbio.2001930.s003" target="_blank">S2 Data</a>.</p

Staphylococcal enterotoxin B (SEB) stimulates human peripheral blood and hepatic mucosa-associated invariant T (MAIT) cells to produce classic pro-inflammatory cytokines except interleukin (IL)-17.

<p>Human peripheral blood mononuclear cells (PBMCs) were stimulated with 100 ng/mL of SEB, and culture supernatants were collected at indicated time points for cytokine analysis (<b>A</b>). Data from 4 healthy donors were averaged and used to generate a heat map illustrating interferon (IFN)-γ, interleukin (IL)-2, IL-17A, and tumor necrosis factor (TNF)-α levels. PBMCs (<i>n</i> = 8) were exposed to SEB, and the frequencies of IFN-γ⁺, IL-2⁺, TNF-α⁺, and IL-17⁺ events among conventional T (T_conv) and MAIT cells were determined at indicated time points (<b>B</b>). Freshly isolated and SEB-stimulated PBMCs (<i>n</i> = 3) were also examined to assess the intracellular T-bet and RORγT contents of MAIT cells relative to background staining with isotype controls (filled histograms in representative plots) (<b>C</b>). Non-parenchymal hepatic mononuclear cells (HMNCs) were isolated from tumor-free liver tissue samples of patients with colorectal carcinoma (<i>n</i> = 20), in which CD3⁺Vα7.2⁺CD161⁺ MAIT cell frequencies were calculated and compared with those determined in 20 PBMC samples (<b>D</b>). HMNCs were incubated for 12 h or 24 h with SEB, followed by cytofluorimetric analysis of IFN-γ and IL-17 production by hepatic MAIT cells (<b>E</b>). In a limited number of experiments, blood MAIT and T_conv cells were purified using a cell sorter and co-incubated with autologous CD14⁺ monocytes in the presence or absence of SEB. IFN-γ and IL-17A contents of culture supernatants were measured after 2 h, 6 h, or 12 h by ELISA (<b>F</b>). *, **, and **** denote <i>p</i> < 0.05, <i>p</i> < 0.01, and <i>p</i> < 0.0001, respectively. The underlying data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001930#pbio.2001930.s002" target="_blank">S1 Data</a>, and our gating strategies are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001930#pbio.2001930.s003" target="_blank">S2 Data</a>.</p

Staphylococcal enterotoxin B (SEB) administration to mucosa-associated invariant T (MAIT)^high DR4⁺ chimeric mice upregulates anergy markers on MAIT cells.

<p>Bone marrow cells from DR4-transgenic (DR4 tg) and B6.CAST mice were prepared and co-injected at a 1:1 ratio into γ-irradiated B6 mice. Control chimeras were generated by co-transferring B6 and DR4 tg marrow cells into B6 recipients. The expression of HLA-DR, or lack thereof, was assessed by flow cytometry in the above chimeras as well as in wild-type B6, DR4 tg, and B6.CAST mice. Filled histograms correspond to background staining with a mouse IgG2a isotype control (<b>A</b>). In addition, lung MAIT (CD3⁺ 5-OP-RU-MR1 tetramer⁺) cell frequencies in the above animals were determined (<b>upper panels in B</b>). 6-formylpterin (6-FP)-loaded MR1 tetramer was used as a negative staining control (<b>lower panels in B</b>). Chimeric mice (<i>n</i> = 3 per group) were injected i.p. with PBS or with 10 μg SEB, monitored for weight loss, and sacrificed on day 3, at which point total non-parenchymal lung mononuclear cells (MNCs) were enumerated (<b>C</b>) and PD-1⁺, LAG-3⁺ and TIM-3⁺ lung MAIT cell proportions were determined by flow cytometry (<b>D</b>). Error bars represent SEM, and ** denotes <i>p</i> < 0.01. The underlying data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001930#pbio.2001930.s002" target="_blank">S1 Data</a>, and our gating strategies are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001930#pbio.2001930.s003" target="_blank">S2 Data</a>.</p

Staphylococcal enterotoxin B (SEB) can activate human mucosa-associated invariant T (MAIT) cells by an interleukin (IL)-12/IL-18-dependent mechanism.

<p>Peripheral blood mononuclear cells (PBMCs) were exposed to SEB for 24 h, and interferon (IFN)-γ⁺ events were enumerated among Vβ13.2⁺ and Vβ2⁺ MAIT cells (<b>A</b>). Human PBMCs (<i>n</i> = 4) were stimulated with SEB before culture supernatants were harvested at indicated time points to assay for IL-18, IL-12p70, IFN-α2, IL-7, and IL-15. Data were averaged to generate a heat map (<b>B</b>). The relative frequencies of CD218a⁺ and CD218a^- cells within the CD3⁺IFN-γ⁺ gate was determined at indicated time points post-SEB stimulation (<i>n</i> = 3) (<b>C</b>). CD3⁺ cells exhibiting high, intermediate and low surface levels of CD218a were further analyzed for CD161 and Vα7.2 positivity (<b>D</b>). CD218a and CD212 expression by MAIT cells was assessed in untreated and SEB-stimulated PBMC cultures (<i>n</i> = 7) (<b>E</b>). Filled and open histograms correspond to staining with isotype controls and anti-CD218a/CD212, respectively (<b>E</b>). In several experiments, neutralizing monoclonal antibodies (mAbs) to IFN-γ, IL-12, and/or IL-18 (or isotype control[s]) were added to PBMC cultures prior to SEB stimulation. Twenty-four hours later, the percentages of CD69⁺ (<b>F</b>) and cytokine⁺ events (<b>G-H</b>) were determined among total (<b>F-H</b>) or fractionated (<b>H</b>) MAIT cells. In additional cultures, PBMCs were stimulated for 24 h with SEB or with recombinant human IL-12 (rIL-12) and/or recombinant human IL-18 (rIL-18) in parallel before cell-surface expression of CD69 (<i>n</i> = 8) and intracellular IFN-γ accumulation (<i>n</i> = 4) in MAIT cells were evaluated (<b>I</b>). Error bars represent SEM, and *** indicates a statistically significant difference with <i>p</i> < 0.001. The underlying data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001930#pbio.2001930.s002" target="_blank">S1 Data</a>, and our gating strategies are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001930#pbio.2001930.s003" target="_blank">S2 Data</a>.</p