Limits...
Identification of phenotypically and functionally heterogeneous mouse mucosal-associated invariant T cells using MR1 tetramers.

Rahimpour A, Koay HF, Enders A, Clanchy R, Eckle SB, Meehan B, Chen Z, Whittle B, Liu L, Fairlie DP, Goodnow CC, McCluskey J, Rossjohn J, Uldrich AP, Pellicci DG, Godfrey DI - J. Exp. Med. (2015)

Bottom Line: These cells include CD4(-)CD8(-), CD4(-)CD8(+), and CD4(+)CD8(-) subsets, and their frequency varies in a tissue- and strain-specific manner.Mouse MAIT cells have a CD44(hi)CD62L(lo) memory phenotype and produce high levels of IL-17A, whereas other cytokines, including IFN-γ, IL-4, IL-10, IL-13, and GM-CSF, are produced at low to moderate levels.These observations contrast with previous reports that MAIT cells from Vα19 TCR transgenic mice are PLZF(-) and express a naive CD44(lo) phenotype.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity and Australian Research Council Centre of Excellence in Advanced Molecular Imaging, University of Melbourne, Parkville, Victoria 3010, Australia Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity and Australian Research Council Centre of Excellence in Advanced Molecular Imaging, University of Melbourne, Parkville, Victoria 3010, Australia.

Show MeSH

Related in: MedlinePlus

Cytokine production by MAIT cells. (A) CD24-depleted mouse thymocytes were stimulated for 4 h in PMA/ionomycin, after which time the cells were labeled with MR1-Ag tetramer and then fixed and permeabilized before staining with anti–IFN-γ or IL-17A. Data are representative of three experiments with a combined total of six mice per group. (B) MAIT cells, NKT cells (CD1d–α-GalCer tetramer+ T cells), and conventional MR1 tetramer− TCR-β+ T cells were sorted from WT spleen and thymus and then added at 10,000 cells/well and stimulated by plate-bound CD3 and CD28. Supernatants were harvested at 24 h, and cytokines were analyzed by CBA. Graphs depict the mean concentration of cytokines ± SEM from three independent experiments for thymus and spleen, each using MAIT cells pooled from 10 mice. Dashed lines are the cutoff at 10 pg/ml for clearly detectable cytokine production.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4493408&req=5

fig5: Cytokine production by MAIT cells. (A) CD24-depleted mouse thymocytes were stimulated for 4 h in PMA/ionomycin, after which time the cells were labeled with MR1-Ag tetramer and then fixed and permeabilized before staining with anti–IFN-γ or IL-17A. Data are representative of three experiments with a combined total of six mice per group. (B) MAIT cells, NKT cells (CD1d–α-GalCer tetramer+ T cells), and conventional MR1 tetramer− TCR-β+ T cells were sorted from WT spleen and thymus and then added at 10,000 cells/well and stimulated by plate-bound CD3 and CD28. Supernatants were harvested at 24 h, and cytokines were analyzed by CBA. Graphs depict the mean concentration of cytokines ± SEM from three independent experiments for thymus and spleen, each using MAIT cells pooled from 10 mice. Dashed lines are the cutoff at 10 pg/ml for clearly detectable cytokine production.

Mentions: Previous studies of Vα19 TCR transgenic mice have suggested that, similar to NKT cells, mouse MAIT cells can produce high levels of a range of cytokines including IL-4, IL-5, IL-10, IFN-γ, and TNF (Kawachi et al., 2006). Here, we stimulated MAIT cells from thymus, lung, and spleen of WT mice using PMA and ionomycin for 4 h and measured acute cytokine production by intracellular cytokine staining (Fig. 5 A and not depicted). These data suggested that very few MAIT cells were able to produce IFN-γ (Fig. 5 A) and IL-4 (not depicted). In contrast, the majority of NKT cells in the same experiments were clearly capable of producing both IFN-γ (Fig. 5 A) and IL-4 (not depicted). However, the majority of MAIT cells from the thymus, spleen, and lung were able to produce very high levels of the proinflammatory cytokine IL-17A, whereas expression of this cytokine was limited to a small subset of NKT cells from the same organs (Fig. 5 A). We also examined production of these cytokines by MAIT cell subsets defined by CD4/CD8 expression but did not observe any clear differences (not depicted). To investigate cytokine production in response to TCR ligation, we next isolated MAIT cells from thymus, spleen, and lung and stimulated them in vitro with plate-bound CD3 and CD28 antibodies, measured cytokine secretion into the culture supernatant after 24 h (Fig. 5 B and not depicted), and compared them with NKT cells and conventional T cells. This confirmed that thymic MAIT cells are capable of producing a broad range of cytokines including IL-4, IL-10, IL-13, GM-CSF, IFN-γ, and TNF, although generally at low levels (<100 pg/ml). In line with the intracellular cytokine staining data, IL-17A was produced at extremely high levels by thymic MAIT cells, at least 10-fold higher than that of NKT cells and conventional T cells in the same experiments (Fig. 5 B). Generally lower levels of cytokines were detected from spleen (Fig. 5 B) and lung MAIT cells (not depicted) although IL-17A was the main cytokine produced by these cells in these assays.


Identification of phenotypically and functionally heterogeneous mouse mucosal-associated invariant T cells using MR1 tetramers.

Rahimpour A, Koay HF, Enders A, Clanchy R, Eckle SB, Meehan B, Chen Z, Whittle B, Liu L, Fairlie DP, Goodnow CC, McCluskey J, Rossjohn J, Uldrich AP, Pellicci DG, Godfrey DI - J. Exp. Med. (2015)

Cytokine production by MAIT cells. (A) CD24-depleted mouse thymocytes were stimulated for 4 h in PMA/ionomycin, after which time the cells were labeled with MR1-Ag tetramer and then fixed and permeabilized before staining with anti–IFN-γ or IL-17A. Data are representative of three experiments with a combined total of six mice per group. (B) MAIT cells, NKT cells (CD1d–α-GalCer tetramer+ T cells), and conventional MR1 tetramer− TCR-β+ T cells were sorted from WT spleen and thymus and then added at 10,000 cells/well and stimulated by plate-bound CD3 and CD28. Supernatants were harvested at 24 h, and cytokines were analyzed by CBA. Graphs depict the mean concentration of cytokines ± SEM from three independent experiments for thymus and spleen, each using MAIT cells pooled from 10 mice. Dashed lines are the cutoff at 10 pg/ml for clearly detectable cytokine production.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4493408&req=5

fig5: Cytokine production by MAIT cells. (A) CD24-depleted mouse thymocytes were stimulated for 4 h in PMA/ionomycin, after which time the cells were labeled with MR1-Ag tetramer and then fixed and permeabilized before staining with anti–IFN-γ or IL-17A. Data are representative of three experiments with a combined total of six mice per group. (B) MAIT cells, NKT cells (CD1d–α-GalCer tetramer+ T cells), and conventional MR1 tetramer− TCR-β+ T cells were sorted from WT spleen and thymus and then added at 10,000 cells/well and stimulated by plate-bound CD3 and CD28. Supernatants were harvested at 24 h, and cytokines were analyzed by CBA. Graphs depict the mean concentration of cytokines ± SEM from three independent experiments for thymus and spleen, each using MAIT cells pooled from 10 mice. Dashed lines are the cutoff at 10 pg/ml for clearly detectable cytokine production.
Mentions: Previous studies of Vα19 TCR transgenic mice have suggested that, similar to NKT cells, mouse MAIT cells can produce high levels of a range of cytokines including IL-4, IL-5, IL-10, IFN-γ, and TNF (Kawachi et al., 2006). Here, we stimulated MAIT cells from thymus, lung, and spleen of WT mice using PMA and ionomycin for 4 h and measured acute cytokine production by intracellular cytokine staining (Fig. 5 A and not depicted). These data suggested that very few MAIT cells were able to produce IFN-γ (Fig. 5 A) and IL-4 (not depicted). In contrast, the majority of NKT cells in the same experiments were clearly capable of producing both IFN-γ (Fig. 5 A) and IL-4 (not depicted). However, the majority of MAIT cells from the thymus, spleen, and lung were able to produce very high levels of the proinflammatory cytokine IL-17A, whereas expression of this cytokine was limited to a small subset of NKT cells from the same organs (Fig. 5 A). We also examined production of these cytokines by MAIT cell subsets defined by CD4/CD8 expression but did not observe any clear differences (not depicted). To investigate cytokine production in response to TCR ligation, we next isolated MAIT cells from thymus, spleen, and lung and stimulated them in vitro with plate-bound CD3 and CD28 antibodies, measured cytokine secretion into the culture supernatant after 24 h (Fig. 5 B and not depicted), and compared them with NKT cells and conventional T cells. This confirmed that thymic MAIT cells are capable of producing a broad range of cytokines including IL-4, IL-10, IL-13, GM-CSF, IFN-γ, and TNF, although generally at low levels (<100 pg/ml). In line with the intracellular cytokine staining data, IL-17A was produced at extremely high levels by thymic MAIT cells, at least 10-fold higher than that of NKT cells and conventional T cells in the same experiments (Fig. 5 B). Generally lower levels of cytokines were detected from spleen (Fig. 5 B) and lung MAIT cells (not depicted) although IL-17A was the main cytokine produced by these cells in these assays.

Bottom Line: These cells include CD4(-)CD8(-), CD4(-)CD8(+), and CD4(+)CD8(-) subsets, and their frequency varies in a tissue- and strain-specific manner.Mouse MAIT cells have a CD44(hi)CD62L(lo) memory phenotype and produce high levels of IL-17A, whereas other cytokines, including IFN-γ, IL-4, IL-10, IL-13, and GM-CSF, are produced at low to moderate levels.These observations contrast with previous reports that MAIT cells from Vα19 TCR transgenic mice are PLZF(-) and express a naive CD44(lo) phenotype.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity and Australian Research Council Centre of Excellence in Advanced Molecular Imaging, University of Melbourne, Parkville, Victoria 3010, Australia Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity and Australian Research Council Centre of Excellence in Advanced Molecular Imaging, University of Melbourne, Parkville, Victoria 3010, Australia.

Show MeSH
Related in: MedlinePlus