Study of MAIT Cell Activation in Viral Infections In Vivo v1

MAIT cells are abundant, highly evolutionarily conserved innate-like lymphocytes expressing a semi-invariant T cell receptor (TCR), which recognizes microbially derived small intermediate molecules from the riboflavin biosynthetic pathway. However, in addition to their TCR-mediated functions they can also be activated in a TCR-independent manner via cytokines including IL-12, -15, -18, and type I interferon. Emerging data suggest that they are expanded and activated by a range of viral infections, and significantly that they can contribute to a protective anti-viral response. Here we describe methods used to investigate these anti-viral functions in vivo in murine models. To overcome the technical challenge that MAIT cells are rare in specific pathogen-free laboratory mice, we describe how pulmonary MAIT cells can be expanded using intranasal bacterial infection or a combination of synthetic MAIT cell antigen and TLR agonists. We also describe protocols for adoptive transfer of MAIT cells, methods for lung homogenization for plaque assays, and surface and intracellular cytokine staining to determine MAIT cell activation.

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Lung Homogenization v1

10.17504/protocols.io.bmgzk3x6 ◽

2020 ◽

Author(s):

Timothy S C Hinks ◽

Bonnie van Wilgenburg ◽

Huimeng Wang ◽

Liyen Loh ◽

Marios Koutsakos ◽

...

Keyword(s):

Cell Activation ◽

Viral Infections ◽

Intracellular Cytokine Staining ◽

Cell Receptor ◽

Type I ◽

Independent Manner ◽

Mait Cells ◽

Specific Pathogen ◽

Mait Cell

This is part 3.4 of the "Study of MAIT Cell Activation in Viral Infections In Vivo" collection of protocols. Collection Abstract: MAIT cells are abundant, highly evolutionarily conserved innate-like lymphocytes expressing a semi-invariant T cell receptor (TCR), which recognizes microbially derived small intermediate molecules from the riboflavin biosynthetic pathway. However, in addition to their TCR-mediated functions they can also be activated in a TCR-independent manner via cytokines including IL-12, -15, -18, and type I interferon. Emerging data suggest that they are expanded and activated by a range of viral infections, and significantly that they can contribute to a protective anti-viral response. Here we describe methods used to investigate these anti-viral functions in vivo in murine models. To overcome the technical challenge that MAIT cells are rare in specific pathogen-free laboratory mice, we describe how pulmonary MAIT cells can be expanded using intranasal bacterial infection or a combination of synthetic MAIT cell antigen and TLR agonists. We also describe protocols for adoptive transfer of MAIT cells, methods for lung homogenization for plaque assays, and surface and intracellular cytokine staining to determine MAIT cell activation.

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Influenza A Virus Infection v1

10.17504/protocols.io.bmgyk3xw ◽

2020 ◽

Author(s):

Timothy S C Hinks ◽

Bonnie van Wilgenburg ◽

Huimeng Wang ◽

Liyen Loh ◽

Marios Koutsakos ◽

...

Keyword(s):

Influenza A ◽

Cell Activation ◽

Viral Infections ◽

Intracellular Cytokine Staining ◽

Cell Receptor ◽

Type I ◽

Independent Manner ◽

Mait Cells ◽

Mait Cell

This is part 3.3 of the "Study of MAIT Cell Activation in Viral Infections In Vivo" collection of protocols. Collection Abstract: MAIT cells are abundant, highly evolutionarily conserved innate-like lymphocytes expressing a semi-invariant T cell receptor (TCR), which recognizes microbially derived small intermediate molecules from the riboflavin biosynthetic pathway. However, in addition to their TCR-mediated functions they can also be activated in a TCR-independent manner via cytokines including IL-12, -15, -18, and type I interferon. Emerging data suggest that they are expanded and activated by a range of viral infections, and significantly that they can contribute to a protective anti-viral response. Here we describe methods used to investigate these anti-viral functions in vivo in murine models. To overcome the technical challenge that MAIT cells are rare in specific pathogen-free laboratory mice, we describe how pulmonary MAIT cells can be expanded using intranasal bacterial infection or a combination of synthetic MAIT cell antigen and TLR agonists. We also describe protocols for adoptive transfer of MAIT cells, methods for lung homogenization for plaque assays, and surface and intracellular cytokine staining to determine MAIT cell activation.

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MAIT Cell Adoptive Transfer v1

10.17504/protocols.io.bmgxk3xn ◽

2020 ◽

Author(s):

Timothy S C Hinks ◽

Bonnie van Wilgenburg ◽

Huimeng Wang ◽

Liyen Loh ◽

Marios Koutsakos ◽

...

Keyword(s):

Adoptive Transfer ◽

Cell Activation ◽

Viral Infections ◽

Intracellular Cytokine Staining ◽

Cell Receptor ◽

Type I ◽

Independent Manner ◽

Mait Cells ◽

Mait Cell

This is part 3.2 of the "Study of MAIT Cell Activation in Viral Infections In Vivo" collection of protocols. Collection Abstract: MAIT cells are abundant, highly evolutionarily conserved innate-like lymphocytes expressing a semi-invariant T cell receptor (TCR), which recognizes microbially derived small intermediate molecules from the riboflavin biosynthetic pathway. However, in addition to their TCR-mediated functions they can also be activated in a TCR-independent manner via cytokines including IL-12, -15, -18, and type I interferon. Emerging data suggest that they are expanded and activated by a range of viral infections, and significantly that they can contribute to a protective anti-viral response. Here we describe methods used to investigate these anti-viral functions in vivo in murine models. To overcome the technical challenge that MAIT cells are rare in specific pathogen-free laboratory mice, we describe how pulmonary MAIT cells can be expanded using intranasal bacterial infection or a combination of synthetic MAIT cell antigen and TLR agonists. We also describe protocols for adoptive transfer of MAIT cells, methods for lung homogenization for plaque assays, and surface and intracellular cytokine staining to determine MAIT cell activation.

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MAIT Cell Intracellular Cytokine Staining v1

10.17504/protocols.io.bmg2k3ye ◽

2020 ◽

Author(s):

Timothy S C Hinks ◽

Bonnie van Wilgenburg ◽

Huimeng Wang ◽

Liyen Loh ◽

Marios Koutsakos ◽

...

Keyword(s):

Cell Activation ◽

Viral Infections ◽

Intracellular Cytokine Staining ◽

Cell Receptor ◽

Type I ◽

Intracellular Cytokine ◽

Independent Manner ◽

Mait Cells ◽

Mait Cell

This is part 3.5 of the "Study of MAIT Cell Activation in Viral Infections In Vivo" collection of protocols. Collection Abstract: MAIT cells are abundant, highly evolutionarily conserved innate-like lymphocytes expressing a semi-invariant T cell receptor (TCR), which recognizes microbially derived small intermediate molecules from the riboflavin biosynthetic pathway. However, in addition to their TCR-mediated functions they can also be activated in a TCR-independent manner via cytokines including IL-12, -15, -18, and type I interferon. Emerging data suggest that they are expanded and activated by a range of viral infections, and significantly that they can contribute to a protective anti-viral response. Here we describe methods used to investigate these anti-viral functions in vivo in murine models. To overcome the technical challenge that MAIT cells are rare in specific pathogen-free laboratory mice, we describe how pulmonary MAIT cells can be expanded using intranasal bacterial infection or a combination of synthetic MAIT cell antigen and TLR agonists. We also describe protocols for adoptive transfer of MAIT cells, methods for lung homogenization for plaque assays, and surface and intracellular cytokine staining to determine MAIT cell activation.

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Viral Plaque Assay v1

10.17504/protocols.io.bmg3k3yn ◽

2020 ◽

Author(s):

Timothy S C Hinks ◽

Bonnie van Wilgenburg ◽

Huimeng Wang ◽

Liyen Loh ◽

Marios Koutsakos ◽

...

Keyword(s):

Cell Activation ◽

Viral Infections ◽

Mdck Cells ◽

Intracellular Cytokine Staining ◽

Type I ◽

Tissue Culture Dish ◽

Mait Cells ◽

Viral Plaque ◽

Mait Cell

This is part 3.6 of the "Study of MAIT Cell Activation in Viral Infections In Vivo" collection of protocols. Collection Abstract: MAIT cells are abundant, highly evolutionarily conserved innate-like lymphocytes expressing a semi-invariant T cell receptor (TCR), which recognizes microbially derived small intermediate molecules from the riboflavin biosynthetic pathway. However, in addition to their TCR-mediated functions they can also be activated in a TCR-independent manner via cytokines including IL-12, -15, -18, and type I interferon. Emerging data suggest that they are expanded and activated by a range of viral infections, and significantly that they can contribute to a protective anti-viral response. Here we describe methods used to investigate these anti-viral functions in vivo in murine models. To overcome the technical challenge that MAIT cells are rare in specific pathogen-free laboratory mice, we describe how pulmonary MAIT cells can be expanded using intranasal bacterial infection or a combination of synthetic MAIT cell antigen and TLR agonists. We also describe protocols for adoptive transfer of MAIT cells, methods for lung homogenization for plaque assays, and surface and intracellular cytokine staining to determine MAIT cell activation. Abstract: Viral plaque assays are used to determine influenza viral titers. A diluted solution of egg-adapted Influenza A viruses/lung-infected tissue homogenates are applied to a six-well tissue culture dish containing a monolayer of Madin-Darby canine kidney (MDCK) cells. The infected MDCK cells grow under a semisolid overlay medium (agar) containing trypsin. A plaque is produced when a virus particle infects a cell, replicates, and then kills the cell. This process can be repeated several times as surrounding cells can be infected by newly replicated virus and killed. When visualized by eye, plaques appear as white spots. The assay is measured in PFU/mL.

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MAIT Cell Expansion in Donor Mice v1

10.17504/protocols.io.bmeqk3dw ◽

2020 ◽

Author(s):

Timothy S C Hinks ◽

Bonnie van Wilgenburg ◽

Huimeng Wang ◽

Liyen Loh ◽

Marios Koutsakos ◽

...

Keyword(s):

Adoptive Transfer ◽

Cell Activation ◽

Viral Infections ◽

Type I ◽

Specific Pathogen Free ◽

Tlr Agonists ◽

Mait Cells ◽

Specific Pathogen ◽

Mait Cell

This is part 3.1 of the "Study of MAIT Cell Activation in Viral Infections In Vivo" collection of protocols. Collection Abstract: MAIT cells are abundant, highly evolutionarily conserved innate-like lymphocytes expressing a semi-invariant T cell receptor (TCR), which recognizes microbially derived small intermediate molecules from the riboflavin biosynthetic pathway. However, in addition to their TCR-mediated functions they can also be activated in a TCR-independent manner via cytokines including IL-12, -15, -18, and type I interferon. Emerging data suggest that they are expanded and activated by a range of viral infections, and significantly that they can contribute to a protective anti-viral response. Here we describe methods used to investigate these anti-viral functions in vivo in murine models. To overcome the technical challenge that MAIT cells are rare in specific pathogen-free laboratory mice, we describe how pulmonary MAIT cells can be expanded using intranasal bacterial infection or a combination of synthetic MAIT cell antigen and TLR agonists. We also describe protocols for adoptive transfer of MAIT cells, methods for lung homogenization for plaque assays, and surface and intracellular cytokine staining to determine MAIT cell activation. Abstract: MAIT cells are rare in specific pathogen-free mice [6], typically comprising about 1 x 104 recoverable pulmonary MAIT cells in an infection-naive adult C57BL/6 mouse. Therefore, for adoptive transfer experiments, the MAIT cell population should first be expanded using intranasal infection [15] or immunization (5-OP-RU with TLR agonists) [3, 15] (see Note 5). When planning the adoptive transfer experiment, estimate that one S. Typhimurium BRD509-infected mouse will yield 1–2 x 106 sorted MAITcells, which are enough for 10–20 recipient mice (105 MAIT cells/RAG2–/–γC–/– mouse in this case). Infect donor mice 7 days earlier than the adoptive transfer.

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Type I interferons are important co-stimulatory signals during T cell receptor mediated MAIT cell activation

10.1101/686170 ◽

2019 ◽

Cited By ~ 1

Author(s):

Rajesh Lamichhane ◽

Henry Galvin ◽

Rachel F Hannaway ◽

Sara M de la Harpe ◽

Fran Munro ◽

...

Keyword(s):

T Cell ◽

T Cell Receptor ◽

Cell Activation ◽

Cell Receptor ◽

Interferon Γ ◽

Type I Interferons ◽

Type I ◽

Mait Cells ◽

Mait Cell

AbstractMucosal associated invariant T (MAIT) cells are abundant unconventional T cells which can be stimulated either via their T cell receptor (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 Toll-like receptor 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 (5-A-RU/MG). We found that T1-IFNs were able to boost interferon-γ and granzyme B production in 5-A-RU/MG-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 confirmed using 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.

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Review for "Type I interferons are important co‐stimulatory signals during T cell receptor mediated human MAIT cell activation"

10.1002/eji.201948279/v2/review2 ◽

2019 ◽

Keyword(s):

T Cell ◽

T Cell Receptor ◽

Cell Activation ◽

Cell Receptor ◽

Type I Interferons ◽

Type I ◽

Mait Cell

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Outcome of SARS-CoV-2 infection linked to MAIT cell activation and cytotoxicity: evidence for an IL-18 dependent mechanism

10.1101/2020.08.31.20185082 ◽

2020 ◽

Cited By ~ 4

Author(s):

Héloïse Flament ◽

Matthieu Rouland ◽

Lucie Beaudoin ◽

Amine Toubal ◽

Léo Bertrand ◽

...

Keyword(s):

Disease Severity ◽

Cell Activation ◽

Viral Infections ◽

Immune Cell ◽

Dependent Manner ◽

Mait Cells ◽

Inflammatory Phase ◽

Immune System Dysfunction ◽

Cell Counts ◽

Mait Cell

Immune system dysfunction is paramount in Coronavirus disease 2019 (COVID-19) severity and fatality rate. Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells involved in mucosal immunity and protection against viral infections. Here, we studied the immune cell landscape, with emphasis on MAIT cells, in a cohort of 182 patients including patients at various stages of disease activity. A profound decrease of MAIT cell counts in blood of critically ill patients was observed. These cells showed a strongly activated and cytotoxic phenotype that positively correlated with circulating pro-inflammatory cytokines, notably IL-18. MAIT cell alterations markedly correlated with disease severity and patient mortality. SARS-CoV-2-infected macrophages activated MAIT cells in a cytokine-dependent manner involving an IFNα-dependent early phase and an IL-18-induced later phase. Therefore, altered MAIT cell phenotypes represent valuable biomarkers of disease severity and their therapeutic manipulation might prevent the inflammatory phase involved in COVID-19 aggravation.

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