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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.

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MAIT cells proliferate upon Ag stimulation in vitro. Splenocytes were labeled with CTV and cultured in the presence of 5-OP-RU, 6-FP, or no Ag. After 72 h, MAIT cell numbers and CTV dilution were measured by flow cytometry. (A) Left column shows representative flow cytometry profiles showing percentage of MAIT cells (MR1–5-OP-RU-tetramer+) of total T cells. Plots depict lymphocytes with B220+ B cells excluded by electronic gating. Right column shows MAIT cell and T cell proliferation based on decreased CTV dye dilution. Black histograms, MAIT cells; gray histograms, MR1 tetramer− T cells. Data are representative of a minimum of two separate experiments with a combined total of four mice. (B) Bar graph depicts percentage of expanded MAIT cells, gated as shown in A (mean ± SEM) from two independent experiments and a combined total of four mice. (C) Representative flow cytometry profiles showing CTV dilution of MAIT cells at each Ag dose. Data are representative of a minimum of two separate experiments with a combined total of four mice. Black histograms, MAIT cells; gray histograms, MR1 tetramer− T cells.
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fig7: MAIT cells proliferate upon Ag stimulation in vitro. Splenocytes were labeled with CTV and cultured in the presence of 5-OP-RU, 6-FP, or no Ag. After 72 h, MAIT cell numbers and CTV dilution were measured by flow cytometry. (A) Left column shows representative flow cytometry profiles showing percentage of MAIT cells (MR1–5-OP-RU-tetramer+) of total T cells. Plots depict lymphocytes with B220+ B cells excluded by electronic gating. Right column shows MAIT cell and T cell proliferation based on decreased CTV dye dilution. Black histograms, MAIT cells; gray histograms, MR1 tetramer− T cells. Data are representative of a minimum of two separate experiments with a combined total of four mice. (B) Bar graph depicts percentage of expanded MAIT cells, gated as shown in A (mean ± SEM) from two independent experiments and a combined total of four mice. (C) Representative flow cytometry profiles showing CTV dilution of MAIT cells at each Ag dose. Data are representative of a minimum of two separate experiments with a combined total of four mice. Black histograms, MAIT cells; gray histograms, MR1 tetramer− T cells.

Mentions: To determine whether WT mouse MAIT cells can respond directly to challenge with the 5-OP-RU Ag, B6 mouse splenocytes were cultured in the presence of graded doses of this Ag for 3 d, and MAIT cell proliferation was measured by dilution of the cell trace violet (CTV) fluorescent dye (Fig. 7, A and B). These data show that mouse MAIT cells responded in a dose-dependent manner (Fig. 7, B and C) to the 5-OP-RU Ag, whereas they did not respond to the control MR1-binding nonagonist 6-FP (Fig. 7, A and B). Cytokines, including IFN-γ, were also detected in these cultures (not depicted), although the amounts were lower and more variable than in cultures of purified MAIT cells (Fig. 5), likely because of the low frequency of MAIT cells in the bulk culture. When MAIT cells were purified by flow cytometric sorting of MR1–5-OP-RU tetramer+TCR-β+ cells and stimulated with 5-OP-RU Ag in the presence of MR1+ Ag-presenting cells, we detected clear production of IL-17A in response to Ag, albeit at lower levels than that seen after CD3 and CD28 stimulation (not depicted). Accordingly, these data demonstrate that WT mouse MAIT cells can recognize and respond specifically to the agonist ligand, 5-OP-RU in a dose-dependent manner.


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)

MAIT cells proliferate upon Ag stimulation in vitro. Splenocytes were labeled with CTV and cultured in the presence of 5-OP-RU, 6-FP, or no Ag. After 72 h, MAIT cell numbers and CTV dilution were measured by flow cytometry. (A) Left column shows representative flow cytometry profiles showing percentage of MAIT cells (MR1–5-OP-RU-tetramer+) of total T cells. Plots depict lymphocytes with B220+ B cells excluded by electronic gating. Right column shows MAIT cell and T cell proliferation based on decreased CTV dye dilution. Black histograms, MAIT cells; gray histograms, MR1 tetramer− T cells. Data are representative of a minimum of two separate experiments with a combined total of four mice. (B) Bar graph depicts percentage of expanded MAIT cells, gated as shown in A (mean ± SEM) from two independent experiments and a combined total of four mice. (C) Representative flow cytometry profiles showing CTV dilution of MAIT cells at each Ag dose. Data are representative of a minimum of two separate experiments with a combined total of four mice. Black histograms, MAIT cells; gray histograms, MR1 tetramer− T cells.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig7: MAIT cells proliferate upon Ag stimulation in vitro. Splenocytes were labeled with CTV and cultured in the presence of 5-OP-RU, 6-FP, or no Ag. After 72 h, MAIT cell numbers and CTV dilution were measured by flow cytometry. (A) Left column shows representative flow cytometry profiles showing percentage of MAIT cells (MR1–5-OP-RU-tetramer+) of total T cells. Plots depict lymphocytes with B220+ B cells excluded by electronic gating. Right column shows MAIT cell and T cell proliferation based on decreased CTV dye dilution. Black histograms, MAIT cells; gray histograms, MR1 tetramer− T cells. Data are representative of a minimum of two separate experiments with a combined total of four mice. (B) Bar graph depicts percentage of expanded MAIT cells, gated as shown in A (mean ± SEM) from two independent experiments and a combined total of four mice. (C) Representative flow cytometry profiles showing CTV dilution of MAIT cells at each Ag dose. Data are representative of a minimum of two separate experiments with a combined total of four mice. Black histograms, MAIT cells; gray histograms, MR1 tetramer− T cells.
Mentions: To determine whether WT mouse MAIT cells can respond directly to challenge with the 5-OP-RU Ag, B6 mouse splenocytes were cultured in the presence of graded doses of this Ag for 3 d, and MAIT cell proliferation was measured by dilution of the cell trace violet (CTV) fluorescent dye (Fig. 7, A and B). These data show that mouse MAIT cells responded in a dose-dependent manner (Fig. 7, B and C) to the 5-OP-RU Ag, whereas they did not respond to the control MR1-binding nonagonist 6-FP (Fig. 7, A and B). Cytokines, including IFN-γ, were also detected in these cultures (not depicted), although the amounts were lower and more variable than in cultures of purified MAIT cells (Fig. 5), likely because of the low frequency of MAIT cells in the bulk culture. When MAIT cells were purified by flow cytometric sorting of MR1–5-OP-RU tetramer+TCR-β+ cells and stimulated with 5-OP-RU Ag in the presence of MR1+ Ag-presenting cells, we detected clear production of IL-17A in response to Ag, albeit at lower levels than that seen after CD3 and CD28 stimulation (not depicted). Accordingly, these data demonstrate that WT mouse MAIT cells can recognize and respond specifically to the agonist ligand, 5-OP-RU in a dose-dependent manner.

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.

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Related in: MedlinePlus