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MAIT cells are licensed through granzyme exchange to kill bacterially sensitized targets.

Kurioka A, Ussher JE, Cosgrove C, Clough C, Fergusson JR, Smith K, Kang YH, Walker LJ, Hansen TH, Willberg CB, Klenerman P - Mucosal Immunol (2014)

Bottom Line: Mucosal-associated invariant T (MAIT) cells are an innate-like T-cell population restricted by the non-polymorphic, major histocompatibility complex class I-related protein 1, MR1.We show that resting human MAIT cells are uniquely characterized by a lack of granzyme (Gr) B and low perforin expression, key granule proteins required for efficient cytotoxic activity, but high levels of expression of GrA and GrK.Finally, we show that MAIT cells are highly proliferative in response to antigenic and cytokine stimulation, maintaining high expression of GrB, perforin, and GrA, but reduced expression of GrK following antigenic proliferation.

View Article: PubMed Central - PubMed

Affiliation: Peter Medawar Building for Pathogen Research, Nuffield Department of Medicine, University of Oxford, Oxford, UK.

ABSTRACT
Mucosal-associated invariant T (MAIT) cells are an innate-like T-cell population restricted by the non-polymorphic, major histocompatibility complex class I-related protein 1, MR1. MAIT cells are activated by a broad range of bacteria through detection of riboflavin metabolites bound by MR1, but their direct cytolytic capacity upon recognition of cognate target cells remains unclear. We show that resting human MAIT cells are uniquely characterized by a lack of granzyme (Gr) B and low perforin expression, key granule proteins required for efficient cytotoxic activity, but high levels of expression of GrA and GrK. Bacterial activation of MAIT cells rapidly induced GrB and perforin, licensing these cells to kill their cognate target cells. Using a novel flow cytometry-based killing assay, we show that licensed MAIT cells, but not ex vivo MAIT cells from the same donors, can efficiently kill Escherichia coli-exposed B-cell lines in an MR1- and degranulation-dependent manner. Finally, we show that MAIT cells are highly proliferative in response to antigenic and cytokine stimulation, maintaining high expression of GrB, perforin, and GrA, but reduced expression of GrK following antigenic proliferation. The tightly regulated cytolytic capacity of MAIT cells may have an important role in the control of intracellular bacterial infections, such as Mycobacterium tuberculosis.

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E.coli-stimulated mucosal-associated invariant T (MAIT) cells can efficiently kill target cells in an MR1- and degranulation-dependent manner. B-cell lines (BCLs) were either incubated with paraformaldehyde (PFA)-fixed E. coli or sterility control and stained with carboxyfluorescein succinimidyl ester (CFSE) and CellTrace Violet (CTV) dyes, respectively, and cocultured with enriched CD8+ T cells. (a) Percentage of specific killing of target BCLs by ex vivo MAIT cells at various E:T ratios. Mean±s.e.m. of duplicate results of three independent experiments shown (n=10). (b) Percentage of specific killing of target cells by ex vivo MAIT cells with and without an anti-MR1-blocking antibody at E:T=50:1. (n=9). (c) Example plots showing the frequency of live E. coli-exposed BCLs and negative control BCLs without effector cells (left), with ex vivo MAIT cells (middle), or with MAIT cells stimulated with E. coli for 6 days (right), added at E:T=10:1. (d) Percentage of specific killing of target cells by ex vivo and E. coli-stimulated MAIT cells from the same donors at E:T=10:1. Mean±s.e.m. of duplicate results of three independent experiments are shown. (n=8). (e) Inhibition of killing at E:T=10:1 by E. coli-stimulated MAIT cells in presence of anti-MR1 antibody, ethylene glycol tetra-acetic acid (EGTA), or anti-FasL antibody, compared with killing with no inhibitor. Repeated-measures one-way analysis of variance (ANOVA), with Bonferroni's multiple comparisons test. (n=5) (f) Cumulative data showing increase in %CD107α+ cells in indicated CD8+ T-cell populations compared with wells with T cells only. Analyzed by two-way ANOVA, comparing increase in CD107α+ expressing cells when target cells are added, within each CD8+ T-cell population, with Bonferroni's multiple comparisons test. CFSE+E. coli-exposed BCL=Target cells, CTV+ negative control BCL=Neg cells. (n=8). (g) Representative plots of ex vivo and E. coli-stimulated CD8 T cells from the killing assays. Histograms showing expression of CD107α, and CFSE and CTV acquired through trogocytosis in CD161++CD8+ T cells. (h) Trogocytosis of CFSE from E. coli-exposed BCL membranes onto degranulating CD161++CD8+ T cells (n=8). Analyzed by two-way ANOVA, comparing wells with ex vivo and E. coli-stimulated cells, with Bonferroni's multiple comparisons test. NS, nonsignificant. *P<0.05, ***P<0.001, ****P<0.0001.
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fig3: E.coli-stimulated mucosal-associated invariant T (MAIT) cells can efficiently kill target cells in an MR1- and degranulation-dependent manner. B-cell lines (BCLs) were either incubated with paraformaldehyde (PFA)-fixed E. coli or sterility control and stained with carboxyfluorescein succinimidyl ester (CFSE) and CellTrace Violet (CTV) dyes, respectively, and cocultured with enriched CD8+ T cells. (a) Percentage of specific killing of target BCLs by ex vivo MAIT cells at various E:T ratios. Mean±s.e.m. of duplicate results of three independent experiments shown (n=10). (b) Percentage of specific killing of target cells by ex vivo MAIT cells with and without an anti-MR1-blocking antibody at E:T=50:1. (n=9). (c) Example plots showing the frequency of live E. coli-exposed BCLs and negative control BCLs without effector cells (left), with ex vivo MAIT cells (middle), or with MAIT cells stimulated with E. coli for 6 days (right), added at E:T=10:1. (d) Percentage of specific killing of target cells by ex vivo and E. coli-stimulated MAIT cells from the same donors at E:T=10:1. Mean±s.e.m. of duplicate results of three independent experiments are shown. (n=8). (e) Inhibition of killing at E:T=10:1 by E. coli-stimulated MAIT cells in presence of anti-MR1 antibody, ethylene glycol tetra-acetic acid (EGTA), or anti-FasL antibody, compared with killing with no inhibitor. Repeated-measures one-way analysis of variance (ANOVA), with Bonferroni's multiple comparisons test. (n=5) (f) Cumulative data showing increase in %CD107α+ cells in indicated CD8+ T-cell populations compared with wells with T cells only. Analyzed by two-way ANOVA, comparing increase in CD107α+ expressing cells when target cells are added, within each CD8+ T-cell population, with Bonferroni's multiple comparisons test. CFSE+E. coli-exposed BCL=Target cells, CTV+ negative control BCL=Neg cells. (n=8). (g) Representative plots of ex vivo and E. coli-stimulated CD8 T cells from the killing assays. Histograms showing expression of CD107α, and CFSE and CTV acquired through trogocytosis in CD161++CD8+ T cells. (h) Trogocytosis of CFSE from E. coli-exposed BCL membranes onto degranulating CD161++CD8+ T cells (n=8). Analyzed by two-way ANOVA, comparing wells with ex vivo and E. coli-stimulated cells, with Bonferroni's multiple comparisons test. NS, nonsignificant. *P<0.05, ***P<0.001, ****P<0.0001.

Mentions: To test the capacity of ex vivo MAIT cells to kill target cells, a flow cytometry-based killing assay was developed, based on the published FATAL assay.28 Briefly, Epstein–Barr virus-transformed B-cell lines (BCLs) were either incubated with PFA-fixed E. coli or sterility control overnight and stained with carboxyfluorescein succinimidyl ester (CFSE) and CellTrace Violet (CTV) dyes, respectively. These were mixed at a 1:1 ratio and cocultured with enriched CD8+ T cells at various E:T ratios. Specific killing of CFSE+ target cells, but not CTV+ control cells, was then calculated based on the ratio of CFSE+ and CTV+ cells in wells without effector cells. In addition, taking advantage of the capacity of modern flow cytometers to measure a greater number of parameters, CD107α externalization by the CD161++CD8+ T cells was measured. Therefore, by combining the FATAL assay with the LAMP-1 assay29 and phenotyping the effector cells, our assay allows the identification of the cell population responsible for cytolysis; thus, removing the necessity to sort enrich specific or rare effector populations. The gating strategy is shown in Supplementary Figure S4A. Using this modified FATAL assay, we found that resting MAIT cells only killed 30% of E. coli-exposed BCLs even the highest E:T ratios, in an MR1-dependent manner (Figure 3a and b).


MAIT cells are licensed through granzyme exchange to kill bacterially sensitized targets.

Kurioka A, Ussher JE, Cosgrove C, Clough C, Fergusson JR, Smith K, Kang YH, Walker LJ, Hansen TH, Willberg CB, Klenerman P - Mucosal Immunol (2014)

E.coli-stimulated mucosal-associated invariant T (MAIT) cells can efficiently kill target cells in an MR1- and degranulation-dependent manner. B-cell lines (BCLs) were either incubated with paraformaldehyde (PFA)-fixed E. coli or sterility control and stained with carboxyfluorescein succinimidyl ester (CFSE) and CellTrace Violet (CTV) dyes, respectively, and cocultured with enriched CD8+ T cells. (a) Percentage of specific killing of target BCLs by ex vivo MAIT cells at various E:T ratios. Mean±s.e.m. of duplicate results of three independent experiments shown (n=10). (b) Percentage of specific killing of target cells by ex vivo MAIT cells with and without an anti-MR1-blocking antibody at E:T=50:1. (n=9). (c) Example plots showing the frequency of live E. coli-exposed BCLs and negative control BCLs without effector cells (left), with ex vivo MAIT cells (middle), or with MAIT cells stimulated with E. coli for 6 days (right), added at E:T=10:1. (d) Percentage of specific killing of target cells by ex vivo and E. coli-stimulated MAIT cells from the same donors at E:T=10:1. Mean±s.e.m. of duplicate results of three independent experiments are shown. (n=8). (e) Inhibition of killing at E:T=10:1 by E. coli-stimulated MAIT cells in presence of anti-MR1 antibody, ethylene glycol tetra-acetic acid (EGTA), or anti-FasL antibody, compared with killing with no inhibitor. Repeated-measures one-way analysis of variance (ANOVA), with Bonferroni's multiple comparisons test. (n=5) (f) Cumulative data showing increase in %CD107α+ cells in indicated CD8+ T-cell populations compared with wells with T cells only. Analyzed by two-way ANOVA, comparing increase in CD107α+ expressing cells when target cells are added, within each CD8+ T-cell population, with Bonferroni's multiple comparisons test. CFSE+E. coli-exposed BCL=Target cells, CTV+ negative control BCL=Neg cells. (n=8). (g) Representative plots of ex vivo and E. coli-stimulated CD8 T cells from the killing assays. Histograms showing expression of CD107α, and CFSE and CTV acquired through trogocytosis in CD161++CD8+ T cells. (h) Trogocytosis of CFSE from E. coli-exposed BCL membranes onto degranulating CD161++CD8+ T cells (n=8). Analyzed by two-way ANOVA, comparing wells with ex vivo and E. coli-stimulated cells, with Bonferroni's multiple comparisons test. NS, nonsignificant. *P<0.05, ***P<0.001, ****P<0.0001.
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Related In: Results  -  Collection

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fig3: E.coli-stimulated mucosal-associated invariant T (MAIT) cells can efficiently kill target cells in an MR1- and degranulation-dependent manner. B-cell lines (BCLs) were either incubated with paraformaldehyde (PFA)-fixed E. coli or sterility control and stained with carboxyfluorescein succinimidyl ester (CFSE) and CellTrace Violet (CTV) dyes, respectively, and cocultured with enriched CD8+ T cells. (a) Percentage of specific killing of target BCLs by ex vivo MAIT cells at various E:T ratios. Mean±s.e.m. of duplicate results of three independent experiments shown (n=10). (b) Percentage of specific killing of target cells by ex vivo MAIT cells with and without an anti-MR1-blocking antibody at E:T=50:1. (n=9). (c) Example plots showing the frequency of live E. coli-exposed BCLs and negative control BCLs without effector cells (left), with ex vivo MAIT cells (middle), or with MAIT cells stimulated with E. coli for 6 days (right), added at E:T=10:1. (d) Percentage of specific killing of target cells by ex vivo and E. coli-stimulated MAIT cells from the same donors at E:T=10:1. Mean±s.e.m. of duplicate results of three independent experiments are shown. (n=8). (e) Inhibition of killing at E:T=10:1 by E. coli-stimulated MAIT cells in presence of anti-MR1 antibody, ethylene glycol tetra-acetic acid (EGTA), or anti-FasL antibody, compared with killing with no inhibitor. Repeated-measures one-way analysis of variance (ANOVA), with Bonferroni's multiple comparisons test. (n=5) (f) Cumulative data showing increase in %CD107α+ cells in indicated CD8+ T-cell populations compared with wells with T cells only. Analyzed by two-way ANOVA, comparing increase in CD107α+ expressing cells when target cells are added, within each CD8+ T-cell population, with Bonferroni's multiple comparisons test. CFSE+E. coli-exposed BCL=Target cells, CTV+ negative control BCL=Neg cells. (n=8). (g) Representative plots of ex vivo and E. coli-stimulated CD8 T cells from the killing assays. Histograms showing expression of CD107α, and CFSE and CTV acquired through trogocytosis in CD161++CD8+ T cells. (h) Trogocytosis of CFSE from E. coli-exposed BCL membranes onto degranulating CD161++CD8+ T cells (n=8). Analyzed by two-way ANOVA, comparing wells with ex vivo and E. coli-stimulated cells, with Bonferroni's multiple comparisons test. NS, nonsignificant. *P<0.05, ***P<0.001, ****P<0.0001.
Mentions: To test the capacity of ex vivo MAIT cells to kill target cells, a flow cytometry-based killing assay was developed, based on the published FATAL assay.28 Briefly, Epstein–Barr virus-transformed B-cell lines (BCLs) were either incubated with PFA-fixed E. coli or sterility control overnight and stained with carboxyfluorescein succinimidyl ester (CFSE) and CellTrace Violet (CTV) dyes, respectively. These were mixed at a 1:1 ratio and cocultured with enriched CD8+ T cells at various E:T ratios. Specific killing of CFSE+ target cells, but not CTV+ control cells, was then calculated based on the ratio of CFSE+ and CTV+ cells in wells without effector cells. In addition, taking advantage of the capacity of modern flow cytometers to measure a greater number of parameters, CD107α externalization by the CD161++CD8+ T cells was measured. Therefore, by combining the FATAL assay with the LAMP-1 assay29 and phenotyping the effector cells, our assay allows the identification of the cell population responsible for cytolysis; thus, removing the necessity to sort enrich specific or rare effector populations. The gating strategy is shown in Supplementary Figure S4A. Using this modified FATAL assay, we found that resting MAIT cells only killed 30% of E. coli-exposed BCLs even the highest E:T ratios, in an MR1-dependent manner (Figure 3a and b).

Bottom Line: Mucosal-associated invariant T (MAIT) cells are an innate-like T-cell population restricted by the non-polymorphic, major histocompatibility complex class I-related protein 1, MR1.We show that resting human MAIT cells are uniquely characterized by a lack of granzyme (Gr) B and low perforin expression, key granule proteins required for efficient cytotoxic activity, but high levels of expression of GrA and GrK.Finally, we show that MAIT cells are highly proliferative in response to antigenic and cytokine stimulation, maintaining high expression of GrB, perforin, and GrA, but reduced expression of GrK following antigenic proliferation.

View Article: PubMed Central - PubMed

Affiliation: Peter Medawar Building for Pathogen Research, Nuffield Department of Medicine, University of Oxford, Oxford, UK.

ABSTRACT
Mucosal-associated invariant T (MAIT) cells are an innate-like T-cell population restricted by the non-polymorphic, major histocompatibility complex class I-related protein 1, MR1. MAIT cells are activated by a broad range of bacteria through detection of riboflavin metabolites bound by MR1, but their direct cytolytic capacity upon recognition of cognate target cells remains unclear. We show that resting human MAIT cells are uniquely characterized by a lack of granzyme (Gr) B and low perforin expression, key granule proteins required for efficient cytotoxic activity, but high levels of expression of GrA and GrK. Bacterial activation of MAIT cells rapidly induced GrB and perforin, licensing these cells to kill their cognate target cells. Using a novel flow cytometry-based killing assay, we show that licensed MAIT cells, but not ex vivo MAIT cells from the same donors, can efficiently kill Escherichia coli-exposed B-cell lines in an MR1- and degranulation-dependent manner. Finally, we show that MAIT cells are highly proliferative in response to antigenic and cytokine stimulation, maintaining high expression of GrB, perforin, and GrA, but reduced expression of GrK following antigenic proliferation. The tightly regulated cytolytic capacity of MAIT cells may have an important role in the control of intracellular bacterial infections, such as Mycobacterium tuberculosis.

Show MeSH
Related in: MedlinePlus