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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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Granzyme (Gr) B and perforin upregulation in E. coli-stimulated mucosal-associated invariant T (MAIT) cells is rapid and associated with Blimp1 and T-bet expression. (a–d) Peripheral blood mononuclear cells (PBMCs) were stimulated with (a, b) paraformaldehyde (PFA)-fixed E. coli, or (c, d) anti-CD3/CD28/CD2 coated beads, and stained for GrB and perforin at the indicated time points (n=3). Asterisks (*) indicate significant increases in effector molecules in MAIT cells compared with the unstimulated controls, by a two-way analysis of variance (ANOVA), with Bonferroni's multiple comparisons test. (e–i) PBMCs were stimulated with PFA-fixed E. coli for 24 h with and without blocking antibodies to MR1 and interleukin (IL)-12, and stained for GrB, T-bet, and Blimp1 (n=7). (e) Cumulative data for frequency of GrB expressing MAIT cells and (f) representative example of GrB expression in MAIT cells after E. coli stimulation, ±blocking antibodies to MR1 or IL-12. One-way ANOVA, Bonferroni's multiple comparisons test. (g) Representative T-bet and Blimp1 expression in MAIT cells unstimulated or stimulated with PFA-fixed E. coli±blocking antibodies to MR1 or IL-12. For figure legend, see panel f. (h) Frequency of GrB-expressing cells in T-bet+ or T-bet− MAIT cells. (i) Frequency of GrB-expressing cells in Blimp1+ or Blimp1− MAIT cells. Results analyzed by two-way ANOVA with Bonferroni's multiple comparisons test. Bars indicate mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
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fig4: Granzyme (Gr) B and perforin upregulation in E. coli-stimulated mucosal-associated invariant T (MAIT) cells is rapid and associated with Blimp1 and T-bet expression. (a–d) Peripheral blood mononuclear cells (PBMCs) were stimulated with (a, b) paraformaldehyde (PFA)-fixed E. coli, or (c, d) anti-CD3/CD28/CD2 coated beads, and stained for GrB and perforin at the indicated time points (n=3). Asterisks (*) indicate significant increases in effector molecules in MAIT cells compared with the unstimulated controls, by a two-way analysis of variance (ANOVA), with Bonferroni's multiple comparisons test. (e–i) PBMCs were stimulated with PFA-fixed E. coli for 24 h with and without blocking antibodies to MR1 and interleukin (IL)-12, and stained for GrB, T-bet, and Blimp1 (n=7). (e) Cumulative data for frequency of GrB expressing MAIT cells and (f) representative example of GrB expression in MAIT cells after E. coli stimulation, ±blocking antibodies to MR1 or IL-12. One-way ANOVA, Bonferroni's multiple comparisons test. (g) Representative T-bet and Blimp1 expression in MAIT cells unstimulated or stimulated with PFA-fixed E. coli±blocking antibodies to MR1 or IL-12. For figure legend, see panel f. (h) Frequency of GrB-expressing cells in T-bet+ or T-bet− MAIT cells. (i) Frequency of GrB-expressing cells in Blimp1+ or Blimp1− MAIT cells. Results analyzed by two-way ANOVA with Bonferroni's multiple comparisons test. Bars indicate mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Mentions: Next, we probed the kinetics of this MAIT cell licensing by examining GrB and perforin upregulation in response to E. coli. MAIT cells showed no change in the expression of GrB and perforin within the first 6 h of stimulation (Figure 4a and b), but markedly upregulated these effector molecules by 18 h, with most cells expressing GrB and perforin by 30 h. This upregulation was specific to the MAIT cell population, the kinetics of which was similar to anti-CD3/CD28/CD2 stimulation (Figure 4c and d).


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)

Granzyme (Gr) B and perforin upregulation in E. coli-stimulated mucosal-associated invariant T (MAIT) cells is rapid and associated with Blimp1 and T-bet expression. (a–d) Peripheral blood mononuclear cells (PBMCs) were stimulated with (a, b) paraformaldehyde (PFA)-fixed E. coli, or (c, d) anti-CD3/CD28/CD2 coated beads, and stained for GrB and perforin at the indicated time points (n=3). Asterisks (*) indicate significant increases in effector molecules in MAIT cells compared with the unstimulated controls, by a two-way analysis of variance (ANOVA), with Bonferroni's multiple comparisons test. (e–i) PBMCs were stimulated with PFA-fixed E. coli for 24 h with and without blocking antibodies to MR1 and interleukin (IL)-12, and stained for GrB, T-bet, and Blimp1 (n=7). (e) Cumulative data for frequency of GrB expressing MAIT cells and (f) representative example of GrB expression in MAIT cells after E. coli stimulation, ±blocking antibodies to MR1 or IL-12. One-way ANOVA, Bonferroni's multiple comparisons test. (g) Representative T-bet and Blimp1 expression in MAIT cells unstimulated or stimulated with PFA-fixed E. coli±blocking antibodies to MR1 or IL-12. For figure legend, see panel f. (h) Frequency of GrB-expressing cells in T-bet+ or T-bet− MAIT cells. (i) Frequency of GrB-expressing cells in Blimp1+ or Blimp1− MAIT cells. Results analyzed by two-way ANOVA with Bonferroni's multiple comparisons test. Bars indicate mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
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fig4: Granzyme (Gr) B and perforin upregulation in E. coli-stimulated mucosal-associated invariant T (MAIT) cells is rapid and associated with Blimp1 and T-bet expression. (a–d) Peripheral blood mononuclear cells (PBMCs) were stimulated with (a, b) paraformaldehyde (PFA)-fixed E. coli, or (c, d) anti-CD3/CD28/CD2 coated beads, and stained for GrB and perforin at the indicated time points (n=3). Asterisks (*) indicate significant increases in effector molecules in MAIT cells compared with the unstimulated controls, by a two-way analysis of variance (ANOVA), with Bonferroni's multiple comparisons test. (e–i) PBMCs were stimulated with PFA-fixed E. coli for 24 h with and without blocking antibodies to MR1 and interleukin (IL)-12, and stained for GrB, T-bet, and Blimp1 (n=7). (e) Cumulative data for frequency of GrB expressing MAIT cells and (f) representative example of GrB expression in MAIT cells after E. coli stimulation, ±blocking antibodies to MR1 or IL-12. One-way ANOVA, Bonferroni's multiple comparisons test. (g) Representative T-bet and Blimp1 expression in MAIT cells unstimulated or stimulated with PFA-fixed E. coli±blocking antibodies to MR1 or IL-12. For figure legend, see panel f. (h) Frequency of GrB-expressing cells in T-bet+ or T-bet− MAIT cells. (i) Frequency of GrB-expressing cells in Blimp1+ or Blimp1− MAIT cells. Results analyzed by two-way ANOVA with Bonferroni's multiple comparisons test. Bars indicate mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Mentions: Next, we probed the kinetics of this MAIT cell licensing by examining GrB and perforin upregulation in response to E. coli. MAIT cells showed no change in the expression of GrB and perforin within the first 6 h of stimulation (Figure 4a and b), but markedly upregulated these effector molecules by 18 h, with most cells expressing GrB and perforin by 30 h. This upregulation was specific to the MAIT cell population, the kinetics of which was similar to anti-CD3/CD28/CD2 stimulation (Figure 4c and d).

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