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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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Mucosal-associated invariant T (MAIT) cells are readily proliferative in response to E. coli and maintain a highly cytotoxic profile. Peripheral blood mononuclear cells (PBMCs) from healthy donors were CellTrace Violet (CTV)-labeled and cultured with paraformaldehyde (PFA)-fixed E. coli for 6 days. BpC=bacteria per cell ratio. (a) Representative plot showing dilution of CTV against CD161 expression (upper panel) and Vα7.2 expression (lower panel) at 10 BpC or with anti-CD3/CD28/CD2 beads, gated on CD8+ T cells. (b) Cumulative data (left) and representative flow cytometry histograms are shown for each BpC. One-way analysis of variance (ANOVA), with Bonferroni's multiple comparisons test. (c) Representative plot (left) and cumulative data (right) showing Ki67 staining in CD8+ T cells after E. coli-stimulation at 10 BpC (n=4). (d) Proliferation of Vα7.2+CD161++CD8+ T cells in response to E. coli with anti-MR1, interleukin (IL)-12, and IL-18-blocking antibodies at the indicated BpCs. Results pooled from two independent experiments and analyzed by two-way ANOVA, with Bonferroni's multiple comparisons test. (e) GrB and perforin, and (f) GrA and GrK expression in CTV-labeled Vα7.2+CD161++CD8+ T cells following E. coli stimulation for 6 days. Cumulative data (left) and representative staining is shown (right). Gating for granzymes and perforin based on isotype controls. Results analyzed by paired t-test (n=8). Bars indicate mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
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fig5: Mucosal-associated invariant T (MAIT) cells are readily proliferative in response to E. coli and maintain a highly cytotoxic profile. Peripheral blood mononuclear cells (PBMCs) from healthy donors were CellTrace Violet (CTV)-labeled and cultured with paraformaldehyde (PFA)-fixed E. coli for 6 days. BpC=bacteria per cell ratio. (a) Representative plot showing dilution of CTV against CD161 expression (upper panel) and Vα7.2 expression (lower panel) at 10 BpC or with anti-CD3/CD28/CD2 beads, gated on CD8+ T cells. (b) Cumulative data (left) and representative flow cytometry histograms are shown for each BpC. One-way analysis of variance (ANOVA), with Bonferroni's multiple comparisons test. (c) Representative plot (left) and cumulative data (right) showing Ki67 staining in CD8+ T cells after E. coli-stimulation at 10 BpC (n=4). (d) Proliferation of Vα7.2+CD161++CD8+ T cells in response to E. coli with anti-MR1, interleukin (IL)-12, and IL-18-blocking antibodies at the indicated BpCs. Results pooled from two independent experiments and analyzed by two-way ANOVA, with Bonferroni's multiple comparisons test. (e) GrB and perforin, and (f) GrA and GrK expression in CTV-labeled Vα7.2+CD161++CD8+ T cells following E. coli stimulation for 6 days. Cumulative data (left) and representative staining is shown (right). Gating for granzymes and perforin based on isotype controls. Results analyzed by paired t-test (n=8). Bars indicate mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Mentions: Finally, we asked whether the granule content of MAIT cells were sustained during proliferation. When CTV-labeled PBMCs were cultured with PFA-fixed E. coli for 6 days, MAIT cells proliferated in a dose-dependent manner to E. coli (Figure 5a and b). The proliferation of MAIT cells was confirmed by Ki67 staining (Figure 5c), and was inducible when enriched CD8+ T cells were directly stimulated with E. coli-exposed THP1 cells (Supplementary Figure S5A), confirming this is a specific effect and not bystander proliferation. Interestingly, although most cells remained CD161++ (Figure 5a, Supplementary Figure S5A), CD161 was downregulated on MAIT cells with increasing rounds of cell divisions (Supplementary Figure S5B). In addition, although both CD8αα and CD8αβ subsets of MAIT cells proliferated, there was greater proliferation of the CD8αβ subset (Supplementary Figure S5C). To investigate the relative contributions of the TCR signal and innate cytokines for MAIT cell proliferation, blocking antibodies to MR1 or IL-12 and IL-18 were added. Addition of the MR1-blocking antibody, but not IL-12- or IL-18-blocking antibodies, significantly inhibited MAIT cell proliferation (Figure 5d), demonstrating that E. coli-induced MAIT cell expansion is largely TCR dependent; this was further confirmed with enriched CD8+ T cells (Supplementary Figure S5A). Of note, proliferation of MAIT cells at 10BpC could not be completely inhibited by the anti-MR1-blocking antibody, suggesting that other cytokines may be contributing to their proliferation. Indeed, CD161++CD8+ T cells readily proliferated in response to IL-2, IL-15, or IL-12 in the absence of exogenous TCR stimulation (Supplementary Figure S4D–G).


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)

Mucosal-associated invariant T (MAIT) cells are readily proliferative in response to E. coli and maintain a highly cytotoxic profile. Peripheral blood mononuclear cells (PBMCs) from healthy donors were CellTrace Violet (CTV)-labeled and cultured with paraformaldehyde (PFA)-fixed E. coli for 6 days. BpC=bacteria per cell ratio. (a) Representative plot showing dilution of CTV against CD161 expression (upper panel) and Vα7.2 expression (lower panel) at 10 BpC or with anti-CD3/CD28/CD2 beads, gated on CD8+ T cells. (b) Cumulative data (left) and representative flow cytometry histograms are shown for each BpC. One-way analysis of variance (ANOVA), with Bonferroni's multiple comparisons test. (c) Representative plot (left) and cumulative data (right) showing Ki67 staining in CD8+ T cells after E. coli-stimulation at 10 BpC (n=4). (d) Proliferation of Vα7.2+CD161++CD8+ T cells in response to E. coli with anti-MR1, interleukin (IL)-12, and IL-18-blocking antibodies at the indicated BpCs. Results pooled from two independent experiments and analyzed by two-way ANOVA, with Bonferroni's multiple comparisons test. (e) GrB and perforin, and (f) GrA and GrK expression in CTV-labeled Vα7.2+CD161++CD8+ T cells following E. coli stimulation for 6 days. Cumulative data (left) and representative staining is shown (right). Gating for granzymes and perforin based on isotype controls. Results analyzed by paired t-test (n=8). Bars indicate mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
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fig5: Mucosal-associated invariant T (MAIT) cells are readily proliferative in response to E. coli and maintain a highly cytotoxic profile. Peripheral blood mononuclear cells (PBMCs) from healthy donors were CellTrace Violet (CTV)-labeled and cultured with paraformaldehyde (PFA)-fixed E. coli for 6 days. BpC=bacteria per cell ratio. (a) Representative plot showing dilution of CTV against CD161 expression (upper panel) and Vα7.2 expression (lower panel) at 10 BpC or with anti-CD3/CD28/CD2 beads, gated on CD8+ T cells. (b) Cumulative data (left) and representative flow cytometry histograms are shown for each BpC. One-way analysis of variance (ANOVA), with Bonferroni's multiple comparisons test. (c) Representative plot (left) and cumulative data (right) showing Ki67 staining in CD8+ T cells after E. coli-stimulation at 10 BpC (n=4). (d) Proliferation of Vα7.2+CD161++CD8+ T cells in response to E. coli with anti-MR1, interleukin (IL)-12, and IL-18-blocking antibodies at the indicated BpCs. Results pooled from two independent experiments and analyzed by two-way ANOVA, with Bonferroni's multiple comparisons test. (e) GrB and perforin, and (f) GrA and GrK expression in CTV-labeled Vα7.2+CD161++CD8+ T cells following E. coli stimulation for 6 days. Cumulative data (left) and representative staining is shown (right). Gating for granzymes and perforin based on isotype controls. Results analyzed by paired t-test (n=8). Bars indicate mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Mentions: Finally, we asked whether the granule content of MAIT cells were sustained during proliferation. When CTV-labeled PBMCs were cultured with PFA-fixed E. coli for 6 days, MAIT cells proliferated in a dose-dependent manner to E. coli (Figure 5a and b). The proliferation of MAIT cells was confirmed by Ki67 staining (Figure 5c), and was inducible when enriched CD8+ T cells were directly stimulated with E. coli-exposed THP1 cells (Supplementary Figure S5A), confirming this is a specific effect and not bystander proliferation. Interestingly, although most cells remained CD161++ (Figure 5a, Supplementary Figure S5A), CD161 was downregulated on MAIT cells with increasing rounds of cell divisions (Supplementary Figure S5B). In addition, although both CD8αα and CD8αβ subsets of MAIT cells proliferated, there was greater proliferation of the CD8αβ subset (Supplementary Figure S5C). To investigate the relative contributions of the TCR signal and innate cytokines for MAIT cell proliferation, blocking antibodies to MR1 or IL-12 and IL-18 were added. Addition of the MR1-blocking antibody, but not IL-12- or IL-18-blocking antibodies, significantly inhibited MAIT cell proliferation (Figure 5d), demonstrating that E. coli-induced MAIT cell expansion is largely TCR dependent; this was further confirmed with enriched CD8+ T cells (Supplementary Figure S5A). Of note, proliferation of MAIT cells at 10BpC could not be completely inhibited by the anti-MR1-blocking antibody, suggesting that other cytokines may be contributing to their proliferation. Indeed, CD161++CD8+ T cells readily proliferated in response to IL-2, IL-15, or IL-12 in the absence of exogenous TCR stimulation (Supplementary Figure S4D–G).

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