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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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Identification of MAIT cells using MR1 tetramer in mice. (A) Detection of MAIT cells reactive to MR1–5-OP-RU tetramer in human and mouse blood. Flow cytometry analysis of human and mouse blood showing reactivity to MR1–5-OP-RU tetramer (left) or MR1–Ac-6-FP tetramer (negative control; right). Numbers indicate the percentage of MAIT cells (red gate) of total αβ T cells (black gate). Data are representative of four separate experiments with a combined total of n = 6 human blood samples and 9 mouse blood samples. (B) Scatter plot depicts MAIT cells as a proportion of T lymphocytes in mouse and human blood, gated as shown in A. Bars depict mean ± SEM of n = 6 human blood samples and 9 mouse blood samples derived from four separate experiments. ***, P < 0.001 using a Mann-Whitney rank sum U test. (C) B6, Vα19 Cα−/− transgenic (Tg) MR1+, or B6-MR1−/− spleen cells were stained with MR1–5-OP-RU tetramer (left three plots) or MR1–Ac-6-FP tetramer (far right plot). Numbers indicate the percentage of MAIT cells (red gate) of total αβ T cells (black gate). Data are representative of two independent experiments with a combined total of four mice. (D) Intensity of TCR-β staining on WT and transgenic MAIT cells is depicted as histograms, representative of four independent experiments with a combined total of six mice. Numbers depict mean fluorescence intensity.
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fig1: Identification of MAIT cells using MR1 tetramer in mice. (A) Detection of MAIT cells reactive to MR1–5-OP-RU tetramer in human and mouse blood. Flow cytometry analysis of human and mouse blood showing reactivity to MR1–5-OP-RU tetramer (left) or MR1–Ac-6-FP tetramer (negative control; right). Numbers indicate the percentage of MAIT cells (red gate) of total αβ T cells (black gate). Data are representative of four separate experiments with a combined total of n = 6 human blood samples and 9 mouse blood samples. (B) Scatter plot depicts MAIT cells as a proportion of T lymphocytes in mouse and human blood, gated as shown in A. Bars depict mean ± SEM of n = 6 human blood samples and 9 mouse blood samples derived from four separate experiments. ***, P < 0.001 using a Mann-Whitney rank sum U test. (C) B6, Vα19 Cα−/− transgenic (Tg) MR1+, or B6-MR1−/− spleen cells were stained with MR1–5-OP-RU tetramer (left three plots) or MR1–Ac-6-FP tetramer (far right plot). Numbers indicate the percentage of MAIT cells (red gate) of total αβ T cells (black gate). Data are representative of two independent experiments with a combined total of four mice. (D) Intensity of TCR-β staining on WT and transgenic MAIT cells is depicted as histograms, representative of four independent experiments with a combined total of six mice. Numbers depict mean fluorescence intensity.

Mentions: Using 5-OP-RU–loaded MR1 tetramers, MAIT cells can be readily identified in human peripheral blood and in Vα19 TCR transgenic mice (Reantragoon et al., 2013; Corbett et al., 2014). To directly compare human and mouse MAIT cells, we labeled peripheral blood lymphocytes from both species with either MR1–5-OP-RU (Corbett et al., 2014) or control nonantigenic MR1–acetyl-6-formylpterin (6-FP [Ac-6-FP]) tetramers (Eckle et al., 2014). Human MR1 tetramer was used for the human cells, and mouse MR1 tetramer was used for the mouse cells. These data showed that MAIT cells were much less abundant in B6 mouse blood, equating to ∼0.1% of T cells, compared with human blood where they comprised ∼6% of T cells, which falls within the usual range of human blood MAIT cells (Fig. 1 A; Le Bourhis et al., 2010; Reantragoon et al., 2013). To further investigate the specificity of the mouse MR1–5-OP-RU tetramer, we compared splenocytes from WT B6, Vα19 TCR transgenic.Cα−/− (Kawachi et al., 2006), and MR1−/− mice (Kawachi et al., 2006). In B6 mice, there was a clear population of TCRint tetramer+ cells, of similar frequency to those in peripheral blood, that was not detected with the control MR1–Ac-6-FP tetramer (Fig. 1 C). As expected, the MR1–5-OP-RU tetramer also stained an abundant population of cells in the TCR transgenic mice, yet no population was detected in the MR1−/− (Fig. 1 C), thus confirming the specificity of mouse MR1–5-OP-RU tetramer for MAIT cells. Staining of MR1−/− mouse cells with MR1–Ac-6-FP tetramer also failed to show a population of tetramer+ cells, as expected (not depicted). To formally establish that the population of MR1–5-OP-RU tetramer+ TCRint cells were indeed MAIT cells, single cells were sorted and TCRs sequenced after PCR amplification (Table 1). As expected, of the 21 cells where matched TCR-α and TCR-β chains were successfully identified, all expressed the MAIT cell invariant TRAV1-TRAJ33 (Vα19-Jα33) TCR-α chain, paired with a range of TCR-β chains that were highly enriched for TRBV13-3 and TRBV13-2 (Vβ8.1 or Vβ8.2), consistent with the previously documented MAIT TCR-β bias (Tilloy et al., 1999; Reantragoon et al., 2013; Lepore et al., 2014). In contrast to human MAIT cells, which can also use Jα12 and Jα20 instead of Jα33 (Reantragoon et al., 2013; Gold et al., 2014; Lepore et al., 2014), no alternate Jα gene usage was observed within the 21 productive TCR-α sequences derived from these experiments, suggesting that mouse MAIT cells have a strong dependence on this TCR element. Similar to NKT cells, MAIT cells expressed lower levels of TCR compared with conventional T cells, and furthermore, their TCR levels were also almost twofold lower in normal mice compared with Vα19 TCR transgenic Cα−/− mice (Fig. 1 D), which may reflect transgenic overexpression of the TCR in these mice. Accordingly, MR1–5-OP-RU tetramers can be used to specifically detect MAIT cells in WT mice.


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)

Identification of MAIT cells using MR1 tetramer in mice. (A) Detection of MAIT cells reactive to MR1–5-OP-RU tetramer in human and mouse blood. Flow cytometry analysis of human and mouse blood showing reactivity to MR1–5-OP-RU tetramer (left) or MR1–Ac-6-FP tetramer (negative control; right). Numbers indicate the percentage of MAIT cells (red gate) of total αβ T cells (black gate). Data are representative of four separate experiments with a combined total of n = 6 human blood samples and 9 mouse blood samples. (B) Scatter plot depicts MAIT cells as a proportion of T lymphocytes in mouse and human blood, gated as shown in A. Bars depict mean ± SEM of n = 6 human blood samples and 9 mouse blood samples derived from four separate experiments. ***, P < 0.001 using a Mann-Whitney rank sum U test. (C) B6, Vα19 Cα−/− transgenic (Tg) MR1+, or B6-MR1−/− spleen cells were stained with MR1–5-OP-RU tetramer (left three plots) or MR1–Ac-6-FP tetramer (far right plot). Numbers indicate the percentage of MAIT cells (red gate) of total αβ T cells (black gate). Data are representative of two independent experiments with a combined total of four mice. (D) Intensity of TCR-β staining on WT and transgenic MAIT cells is depicted as histograms, representative of four independent experiments with a combined total of six mice. Numbers depict mean fluorescence intensity.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493408&req=5

fig1: Identification of MAIT cells using MR1 tetramer in mice. (A) Detection of MAIT cells reactive to MR1–5-OP-RU tetramer in human and mouse blood. Flow cytometry analysis of human and mouse blood showing reactivity to MR1–5-OP-RU tetramer (left) or MR1–Ac-6-FP tetramer (negative control; right). Numbers indicate the percentage of MAIT cells (red gate) of total αβ T cells (black gate). Data are representative of four separate experiments with a combined total of n = 6 human blood samples and 9 mouse blood samples. (B) Scatter plot depicts MAIT cells as a proportion of T lymphocytes in mouse and human blood, gated as shown in A. Bars depict mean ± SEM of n = 6 human blood samples and 9 mouse blood samples derived from four separate experiments. ***, P < 0.001 using a Mann-Whitney rank sum U test. (C) B6, Vα19 Cα−/− transgenic (Tg) MR1+, or B6-MR1−/− spleen cells were stained with MR1–5-OP-RU tetramer (left three plots) or MR1–Ac-6-FP tetramer (far right plot). Numbers indicate the percentage of MAIT cells (red gate) of total αβ T cells (black gate). Data are representative of two independent experiments with a combined total of four mice. (D) Intensity of TCR-β staining on WT and transgenic MAIT cells is depicted as histograms, representative of four independent experiments with a combined total of six mice. Numbers depict mean fluorescence intensity.
Mentions: Using 5-OP-RU–loaded MR1 tetramers, MAIT cells can be readily identified in human peripheral blood and in Vα19 TCR transgenic mice (Reantragoon et al., 2013; Corbett et al., 2014). To directly compare human and mouse MAIT cells, we labeled peripheral blood lymphocytes from both species with either MR1–5-OP-RU (Corbett et al., 2014) or control nonantigenic MR1–acetyl-6-formylpterin (6-FP [Ac-6-FP]) tetramers (Eckle et al., 2014). Human MR1 tetramer was used for the human cells, and mouse MR1 tetramer was used for the mouse cells. These data showed that MAIT cells were much less abundant in B6 mouse blood, equating to ∼0.1% of T cells, compared with human blood where they comprised ∼6% of T cells, which falls within the usual range of human blood MAIT cells (Fig. 1 A; Le Bourhis et al., 2010; Reantragoon et al., 2013). To further investigate the specificity of the mouse MR1–5-OP-RU tetramer, we compared splenocytes from WT B6, Vα19 TCR transgenic.Cα−/− (Kawachi et al., 2006), and MR1−/− mice (Kawachi et al., 2006). In B6 mice, there was a clear population of TCRint tetramer+ cells, of similar frequency to those in peripheral blood, that was not detected with the control MR1–Ac-6-FP tetramer (Fig. 1 C). As expected, the MR1–5-OP-RU tetramer also stained an abundant population of cells in the TCR transgenic mice, yet no population was detected in the MR1−/− (Fig. 1 C), thus confirming the specificity of mouse MR1–5-OP-RU tetramer for MAIT cells. Staining of MR1−/− mouse cells with MR1–Ac-6-FP tetramer also failed to show a population of tetramer+ cells, as expected (not depicted). To formally establish that the population of MR1–5-OP-RU tetramer+ TCRint cells were indeed MAIT cells, single cells were sorted and TCRs sequenced after PCR amplification (Table 1). As expected, of the 21 cells where matched TCR-α and TCR-β chains were successfully identified, all expressed the MAIT cell invariant TRAV1-TRAJ33 (Vα19-Jα33) TCR-α chain, paired with a range of TCR-β chains that were highly enriched for TRBV13-3 and TRBV13-2 (Vβ8.1 or Vβ8.2), consistent with the previously documented MAIT TCR-β bias (Tilloy et al., 1999; Reantragoon et al., 2013; Lepore et al., 2014). In contrast to human MAIT cells, which can also use Jα12 and Jα20 instead of Jα33 (Reantragoon et al., 2013; Gold et al., 2014; Lepore et al., 2014), no alternate Jα gene usage was observed within the 21 productive TCR-α sequences derived from these experiments, suggesting that mouse MAIT cells have a strong dependence on this TCR element. Similar to NKT cells, MAIT cells expressed lower levels of TCR compared with conventional T cells, and furthermore, their TCR levels were also almost twofold lower in normal mice compared with Vα19 TCR transgenic Cα−/− mice (Fig. 1 D), which may reflect transgenic overexpression of the TCR in these mice. Accordingly, MR1–5-OP-RU tetramers can be used to specifically detect MAIT cells in WT mice.

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