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.

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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Mucosal associated invariant T (MAIT) cells are anti‐microbial innate‐like T cells that are abundant in blood and liver. MAIT cells express a semi‐invariant T‐cell receptor (TCR) that recognizes a pyrimidine ligand, derived from microbial riboflavin synthesis, bound to MR1. Both blood and liver derived (ld)‐MAIT cells can be robustly stimulated via TCR or by cytokines produced during bacterial or viral infection. In this study, we compared the functional and transcriptomic response of human blood and ld‐MAIT cells to TCR signals (Escherichia coli or the pyrimidine ligand) and cytokines (IL‐12 + IL‐18). While the response of blood and ld‐MAIT cells to TCR signals were comparable, following cytokine stimulation ld‐MAIT cells were more polyfunctional than blood MAIT cells. Transcriptomic analysis demonstrated different effector programmes of ld‐MAIT cells with the two modes of activation, including the enrichment of a tissue repair signature in TCR‐stimulated MAIT cells. Interestingly, we observed enhancement of IL‐12 signaling and fatty acid metabolism in untreated ld‐MAIT cells compared with blood MAIT cells. Additionally, MAIT cells from blood and liver were modulated similarly by TCR and cytokine signals. Therefore, we report that blood and ld‐MAIT cells are fundamentally different but undergo conserved changes following activation via TCR or by cytokines.

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

Cannabinoid receptor 2 (CB2) is predominantly distributed in immune tissues and cells and is a promising therapeutic target for modulating inflammation. In this study we designed and synthesised a series of 2,4,6-trisubstituted 1,3,5-triazines with piperazinylalkyl or 1,2-diethoxyethane (PEG2) chains as CB2 agonists, all of which were predicted to be considerably more polar than typical cannabinoid ligands. In this series, we found that triazines containing an adamantanyl group were conducive to CB2 binding whereas those with a cyclopentyl group were not. Although the covalent attachment of a PEG2 linker to the adamantyl triazines resulted in a decrease in binding affinity, some of the ligands produced very interesting hCB2 signalling profiles. Six compounds with notable hCB2 orthosteric binding were functionally characterised in three pathways; internalisation, cyclic adenosine monophosphate (cAMP) and ERK phosphorylation (pERK). These were predominantly confirmed to be hCB2 agonists, and upon comparison to a reference ligand (CP 55,940), four compounds exhibited signalling bias. Triazines 14 (UOSD017) and 15 were biased towards internalisation over cAMP and pERK, and 7 was biased away from pERK activation relative to cAMP and internalisation. Intriguingly, the triazine with an amino-PEG2-piperazinyl linker (13 [UOSD008]) was identified to be a mixed agonist/inverse agonist, exhibiting apparent neutral antagonism in the internalisation pathway, transient inverse agonism in the cAMP pathway and weak partial agonism in the pERK pathway. Both the cAMP and pERK signalling were pertussis toxin (PTX) sensitive, implying that 13 is acting as both a weak agonist and inverse agonist at CB2 via Gαi/o. Compound 10 (UOSD015) acted as a balanced high intrinsic efficacy agonist with the potential to produce greater hCB2-mediated efficacy than reference ligand CP 55,940. As 10 includes a Boc-protected PEG2 moiety it is also a promising candidate for further modification, for example with a secondary reporter or fluorophore. The highest affinity compound in this set of relatively polar hCB2 ligands was compound 16, which acted as a slightly partial balanced agonist in comparison with CP 55,940. The ligands characterised here may therefore exhibit unique functional properties in vivo and have the potential to be valuable in the future development of CB2-directed therapeutics.

Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes that are abundant in mucosal tissues and the liver where they can respond rapidly to a broad range of riboflavin producing bacterial and fungal pathogens. Neutrophils, which are recruited early to sites of infection, play a nonredundant role in pathogen clearance and are crucial for controlling infection. The interaction of these two cell types is poorly studied. Here, we investigated both the effect of neutrophils on MAIT cell activation and the effect of activated MAIT cells on neutrophils. We show that neutrophils suppress the activation of MAIT cells by a cell-contact and hydrogen peroxide dependent mechanism. Moreover, highly activated MAIT cells were able to produce high levels of TNF-α that induced neutrophil death. We therefore provide evidence for a negative regulatory feedback mechanism in which neutrophils prevent overactivation of MAIT cells and, in turn, MAIT cells limit neutrophil survival.Keywords: MAIT cells r Mucosal immunity r Neutrophils r TNF-α Additional supporting information may be found online in the Supporting Information section at the end of the article.

Mucosal associated invariant T (MAIT) cells are abundant unconventional T cells that can 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 TCR‐mediated 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‐d‐ribitylaminouracil/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.