Graphical AbstractHighlights d RNA sequencing of MAIT cells following stimulation via T cell receptor or cytokines d TCR and cytokines induce distinct transcriptome with different modes of activation d TCR stimuli result in a rapid, polyfunctional, proinflammatory response d Cytokine stimulation results in a slower, more restricted response SUMMARY Mucosal-associated invariant T (MAIT) cells can be activated via either their T cell receptor (TCR), which recognizes MR1-bound pyrimidines derived from microbial riboflavin biosynthesis, or via cytokines. These two modes of activation may act in concert or independently, depending upon the stimulus. It is unknown, however, how MAIT cell responses differ with the mode of activation. Here, we define transcriptional and effector responses of human CD8 + MAIT cells to TCR and cytokine stimulation. We report that MAIT cells rapidly respond to TCR stimulation, producing multiple cytokines and chemokines, altering their cytotoxic granule content and transcription factor expression, and upregulating co-stimulatory proteins. In contrast, cytokine-mediated activation is slower and results in a more limited response. Therefore, we propose that, in infections by riboflavin-synthesizing bacteria, MAIT cells play a key early role in effecting and coordinating immune responses, while in the absence of TCR stimulation, their role is likely to differ.

Human mucosal-associated invariant T (MAIT) cells are an important T cell subset that are enriched in tissues and possess potent effector functions. Typically such cells are marked by their expression of Vα7.2-Jα33/Jα20/Jα12 T cell receptors, and functionally they are major histocompatibility complex class I-related protein 1 (MR1)-restricted, responding to bacterially derived riboflavin synthesis intermediates. MAIT cells are contained within the CD161++ Vα7.2+ T cell population, the majority of which express the CD8 receptor (CD8+), while a smaller fraction expresses neither CD8 or CD4 coreceptor (double negative; DN) and a further minority are CD4+. Whether these cells have distinct homing patterns, phenotype and functions have not been examined in detail. We used a combination of phenotypic staining and functional assays to address the similarities and differences between these CD161++ Vα7.2+ T cell subsets. We find that most features are shared between CD8+ and DN CD161++ Vα7.2+ T cells, with a small but detectable role evident for CD8 binding in tuning functional responsiveness. By contrast, the CD4+ CD161++ Vα7.2+ T cell population, although showing MR1-dependent responsiveness to bacterial stimuli, display reduced T helper 1 effector functions, including cytolytic machinery, while retaining the capacity to secrete interleukin-4 (IL-4) and IL-13. This was consistent with underlying changes in transcription factor (TF) expression. Although we found that only a proportion of CD4+ CD161++ Vα7.2+ T cells stained for the MR1-tetramer, explaining some of the heterogeneity of CD4+ CD161++ Vα7.2+ T cells, these differences in TF expression were shared with CD4+ CD161++ MR1-tetramer+ cells. These data reveal the functional diversity of human CD161++ Vα7.2+ T cells and indicate potentially distinct roles for the different subsets in vivo.

Mucosal‐associated invariant T (MAIT) cells are an abundant innate‐like T lymphocyte population that are enriched in liver and mucosal tissues. They are restricted by MR1, which presents antigens derived from a metabolic precursor of riboflavin synthesis, a pathway present in many microbial species, including commensals. Therefore, MR1‐mediated MAIT cell activation must be tightly regulated to prevent inappropriate activation and immunopathology. Using an in vitro model of MR1‐mediated activation of primary human MAIT cells, we investigated the mechanisms by which it is regulated. Uptake of intact bacteria by antigen presenting cells (APCs) into acidified endolysosomal compartments was required for efficient MR1‐mediated MAIT cell activation, while stimulation with soluble ligand was inefficient. Consistent with this, little MR1 was seen at the surface of human monocytic (THP1) and B‐cell lines. Activation with a TLR ligand increased the amount of MR1 at the surface of THP1 but not B‐cell lines, suggesting differential regulation in different cell types. APC activation and NF‐κB signaling were critical for MR1‐mediated MAIT cell activation. In primary cells, however, prolonged TLR signaling led to downregulation of MR1‐mediated MAIT cell activation. Overall, MR1‐mediated MAIT cell activation is a tightly regulated process, dependent on integration of innate signals by APCs.

Background A proportion of tuberculosis (TB) case contacts do not become infected, even when heavily exposed. We studied the innate immune responses of TB case contacts to understand their role in protection against infection with Mycobacterium tuberculosis, termed “early clearance.” Methods Indonesian household contacts of TB cases were tested for interferon-γ release assay (IGRA) conversion between baseline and 14 weeks post recruitment. Blood cell populations and ex vivo innate whole blood cytokine responses were measured at baseline and, in a subgroup, flow cytometry was performed at weeks 2 and 14. Immunological characteristics were measured for early clearers, defined as a persistently negative IGRA at 3 months, and converters, whose IGRA converted from negative to positive. Results Among 1347 case contacts, 317 were early clearers and 116 were converters. Flow cytometry showed a resolving innate cellular response from 2 to 14 weeks in persistently IGRA-negative contacts but not converters. There were no differences in cytokine responses to mycobacterial stimuli, but compared to converters, persistently IGRA-negative contacts produced more proinflammatory cytokines following heterologous stimulation with Escherichia coli and Streptococcus pneumoniae. Conclusions Early clearance of M. tuberculosis is associated with enhanced heterologous innate immune responses similar to those activated during induction of trained immunity.

Mucosal associated invariant T (MAIT) cells are abundant unconventional T cells thatcan be stimulated either via their TCR or by innate cytokines. The MAIT cell TCR recognises a pyrimidine ligand, derived from riboflavin synthesising bacteria, bound to MR1. In infection, bacteria not only provide the pyrimidine ligand but also co-stimulatory signals, such as TLR agonists, that can modulate TCR-mediated activation. Recently, type I interferons (T1-IFNs) have been identified as contributing to cytokine-mediated MAIT cell activation. However, it is unknown whether T1-IFNs also have a role during TCRmediated MAIT cell activation. In this study, we investigated the co-stimulatory role of T1-IFNs during TCR-mediated activation of MAIT cells by the MR1 ligand 5-amino-6-Dribitylaminouracil/methylglyoxal. We found that T1-IFNs were able to boost interferon-γ and granzyme B production in 5-amino-6-D-ribitylaminouracil/methylglyoxal-stimulated MAIT cells. Similarly, influenza virus-induced T1-IFNs enhanced TCR-mediated MAIT cell activation. An essential role of T1-IFNs in regulating MAIT cell activation by riboflavin synthesising bacteria was also demonstrated. The co-stimulatory role of T1-IFNs was also evident in liver-derived MAIT cells. T1-IFNs acted directly on MAIT cells to enhance their response to TCR stimulation. Overall, our findings establish an important immunomodulatory role of T1-IFNs during TCR-mediated MAIT cell activation.Keywords: co-stimulation r influenza virus r mucosal associated invariant T cells r TCR activation r type I interferons Additional supporting information may be found online in the Supporting Information section at the end of the article.

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Among 42 gram-negative bloodstream isolates from inpatients in 3 hospitals in Yangon, Myanmar, admitted during July–December 2014, 16 (38%) were extended-spectrum β-lactamase–producing Enterobacteriaceae and 6 (14%) produced carbapenemase. The high prevalence of multidrug-resistant gram-negative bacteria raises concerns about the empiric treatment of patients with sepsis in Yangon.

8 9 3 1human MAIT cells to TCR and cytokine stimulation. We report that MAIT cells rapidly respond 3 2 to TCR stimulation through the production of multiple effector cytokines and chemokines, 3 3 alteration of their cytotoxic granule content and transcription factor expression, and upregulation 3 4 of co-stimulatory proteins CD40L and 4-1BB. In contrast, cytokine-mediated activation is slower 3 5and results in more limited production of cytokines, chemokines, and co-stimulatory proteins; 3 6 differences in granule content and transcription factor expression are also evident. Therefore, we 3 7 propose that in infections by riboflavin-synthesizing bacteria, MAIT cells play a key early role in 3 8 effecting and coordinating the immune response, while in the absence of TCR stimulation (e.g. 3 9 viral infection) their role is likely to differ. 4 0 4 1