Abstract
Mucosal-associated invariant T cells (MAIT cells) detect microbial vitamin B2 derivatives presented by the antigen-presenting molecule MR1. Here we defined three developmental stages and checkpoints for the MAIT cell lineage in humans and mice. Stage 1 and stage 2 MAIT cells predominated in thymus, while stage 3 cells progressively increased in abundance extrathymically. Transition through each checkpoint was regulated by MR1, whereas the final checkpoint that generated mature functional MAIT cells was controlled by multiple factors, including the transcription factor PLZF and microbial colonization. Furthermore, stage 3 MAIT cell populations were expanded in mice deficient in the antigen-presenting molecule CD1d, suggestive of a niche shared by MAIT cells and natural killer T cells (NKT cells). Accordingly, this study maps the developmental pathway and checkpoints that control the generation of functional MAIT cells.
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Acknowledgements
We thank the staff of the University of Melbourne DMI and MBC flow cytometry facilities and M. Camilleri, D. Taylor and animal house staff for animal husbandry and assistance with genotyping; J.C. Zúñiga-Pflücker (University of Toronto) for OP9 and OP9-DL1 cells; T. Hansen (Washington University School of Medicine) for the MR1-blocking antibody 8F2.F9; and the clinical research midwives G. Christophers, G. Pell and R. Murdoch and the Obstetrics and Midwifery staffs of the Mercy Hospital for Women for assistance with collection of the cord blood samples. Supported by the National Health and Medical Research Council of Australia (1083942, 1013667 and 1016629; CDF 1035858 to A.E.; ECF 1054431 to D.G.P.; Senior Principal Research Fellowships 1020770 and 1027369 to D.I.G. and D.P.F.; Australia Fellowship AF50 to J.R.; CDF2 Fellowship 1047025 to M.C.; CDF2 Fellowship 1023294 to K.K.; and CDF1 Fellowship 1106004 to L.K.M.), the Australian Research Council (CE140100011 and LE110100106; Future Fellowship FT140100278 to A.P.U.), the Leukaemia Foundation of Australia (Postgraduate Scholarship for N.A.G.), the National Heart Foundation of Australia (Future Leader Fellowship for C.A.N.-P.), the Hudson Institute (Star Recruitment Fellowship for M.F.N.) and the Ritchie Centre (Victor Yu Fellowship for M.F.N.).
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H.-F.K., N.A.G., C.F.A. and D.G.P. performed experiments; H.-F.K. prepared figures; A.E., L.Lo., L.K.M., B.E.R., C.A.N.-P., M.F.N., S.B., Z.C., A.J.C., S.B.G.E., B.M., Y.d.U., I.E.K., M.L., L.Li., C.C.G., D.P.F., J.R., M.M.C., K.K., S.P.B., G.T.B. and J.M. facilitated experiments and/or provided reagents and tissue samples; H.-F.K., A.P.U., D.I.G. and D.G.P. planned experiments, interpreted data and prepared the manuscript; and D.I.G. and D.G.P. led the investigation.
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Supplementary Figure 1 RORγt expression on MAIT cells in RORγt-GFP reporter mice.
Flow cytometric analysis of MAIT cells from MR1-5-OP-RU tetramer enriched RORγt-GFP reporter mouse thymi for CD24, CD44, and GFP expression. Data are representative of a total of 9 mice with 3-pooled mouse thymi from 2 independent experiments.
Supplementary Figure 2 Thymic MAIT cell subsets in mice with transgenic expression of the TRAV1-TRAJ33 TCR.
Flow cytometric analysis of MR1-5-OP-RU tetramer reactive MAIT cells in TRAV1-TRAJ33 Cα-/- TCR transgenic mouse thymus for expression of CD4, CD8, CD24 and CD44. Data are representative of 3 independent experiments.
Supplementary Figure 3 NKT cells in Drosha-deficient mice and germ-free mice.
(a) Flow cytometric analysis of CD1d-PBS44 tetramer+ TCRβ+ NKT cells from thymus, spleen, lymph nodes from Droshafl/+ CD4-Cre heterozygous control mice and Droshafl/fl CD4-Cre mice. Absolute numbers and percentage NKT cells of TCRβ+ cells in thymus, spleen and lymph nodes of Droshafl/+ CD4-Cre heterozygous control mice and Droshafl/fl CD4-Cre mice. (b) Flow cytometric analysis of NKT cells from thymus, spleen, lymph nodes from control (SPF) mice and germ-free (GF) mice. Absolute numbers and percentage NKT cells of TCRβ+ cells in thymus and spleen of SPF mice and GF mice. *P<0.1 **P<0.01 ***P<0.001 NS = not significant (Mann-Whitney rank sum U test (a, b)). Data are representative of 3 independent experiments with a total of 8 mice per group (a; mean ± SEM) or of 2 independent experiments with a combined total of 11-15 mice per group (b; mean ± SEM).
Supplementary Figure 4 MAIT cells in IL-18-deficient mice and IL-18Rα-deficient mice.
(a) Flow cytometric analysis of MAIT cells from MR1-5-OP-RU tetramer enriched thymus, spleen and lymph nodes from WT and IL-18-deficient mice for CD24 and CD44 expression. (b) Absolute numbers and percentage MAIT cells of TCRβ+ cells in individual thymus, spleen and lymph nodes of WT and IL-18-deficient mice. (c) Flow cytometric analysis of MAIT cells from MR1-5-OP-RU tetramer enriched thymus, spleen and lymph nodes from WT and IL-18Rα-deficient mice for CD24 and CD44 expression. (d) Absolute numbers and percentage MAIT cells of TCRβ+ cells in individual thymus, spleen and lymph nodes of WT and IL-18Rα-deficient mice. *P<0.1 **P<0.01 ***P<0.001 NS = not significant (Mann-Whitney rank sum U test (b, d)). Data are representative of 3 independent experiments with a total of 12 mice per group (a, b; mean ± SEM) or 2 independent experiments with a total of 10 mice per group (c, d; mean ± SEM).
Supplementary Figure 5 MAIT cells in C57BL/6 CD1d-deficient mice.
Flow cytometric analysis of MAIT cells from thymus, MR1-5-OP-RU enriched thymus and spleen from C57BL/6 WT and C57BL/6 CD1d-deficient mice for CD24, CD44 and CD4/CD8 co-receptor expression. Data are representative of 3 independent experiments with a total of 6 mice per group.
Supplementary Figure 6 Schematic of the three development stages of MAIT cells.
Mouse and human MAIT cell development in the thymus can be defined by three separate stages. Mouse thymic MAIT cells can be defined by a three-stage sequential pathway from CD24+CD44− (stage 1), via CD24−CD44− (stage 2), to CD24−CD44+ (stage 3), while in humans thymic MAIT cells can be defined by three distinct stages from CD161−CD27− (stage 1), via CD161−CD27+ (stage 2), to CD161+CD27+/lo (stage 3). These stages are regulated by several factors including MR1, PLZF, Drosha and commensal bacteria.
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Koay, HF., Gherardin, N., Enders, A. et al. A three-stage intrathymic development pathway for the mucosal-associated invariant T cell lineage. Nat Immunol 17, 1300–1311 (2016). https://doi.org/10.1038/ni.3565
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DOI: https://doi.org/10.1038/ni.3565
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