A three-stage intrathymic development pathway for the mucosal-associated invariant t cell lineage

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

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Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS DIFFERENTIAL CONTROLS OF MAIT CELL EFFECTOR POLARIZATION BY MTORC1/MTORC2 VIA INTEGRATING CYTOKINE AND COSTIMULATORY SIGNALS

Article Open access 01 April 2021 DIFFERENTIAL LOCATION OF NKT AND MAIT CELLS WITHIN LYMPHOID TISSUE Article Open access 08 March 2022 MR1 ANTIGEN PRESENTATION TO MAIT CELLS AND OTHER

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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.).

AUTHOR INFORMATION Author notes * Dale I Godfrey and Daniel G Pellicci: These authors jointly directed this work. AUTHORS AND AFFILIATIONS * Department of Microbiology and Immunology, Peter

Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Australia Hui-Fern Koay, Nicholas A Gherardin, Liyen Loh, Laura K Mackay, Catarina F Almeida, Brendan E

Russ, Sammy Bedoui, Zhenjun Chen, Alexandra J Corbett, Sidonia B G Eckle, Bronwyn Meehan, Katherine Kedzierska, Stuart P Berzins, James McCluskey, Adam P Uldrich, Dale I Godfrey & Daniel

G Pellicci * Cancer Immunology Research Program, Peter MacCallum Cancer Centre, East Melbourne, Australia Nicholas A Gherardin * Department of Immunology, John Curtin School of Medical

Research, Canberra, Australia Anselm Enders & Chris C Goodnow * Australian Research Council Centre of Excellence for Advanced Molecular Imaging, University of Melbourne, Melbourne,

Australia Laura K Mackay, Adam P Uldrich, Dale I Godfrey & Daniel G Pellicci * Department of Paediatrics, Monash University, Clayton, Australia Claudia A Nold-Petry & Marcel F Nold *

The Ritchie Centre, Hudson Institute of Medical Research, Clayton, Australia Claudia A Nold-Petry & Marcel F Nold * Royal Children's Hospital, Flemington Road, Parkville, Australia

Yves d'Udekem & Igor E Konstantinov * Department of Obstetrics and Gynaecology, Obstetrics, Nutrition and Endocrinology Group, University of Melbourne, Heidelberg, Australia Martha

Lappas * Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Australia Martha Lappas * Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia

Ligong Liu & David P Fairlie * Australian Research Council Centre of Excellence for Advanced Molecular Imaging, University of Queensland, Brisbane, Australia Ligong Liu & David P

Fairlie * Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Australia Jamie Rossjohn * Institute of Infection and Immunity, Cardiff

University, School of Medicine, Heath Park, Cardiff, UK Jamie Rossjohn * Australian Research Council Centre of Excellence for Advanced Molecular Imaging, Monash University, Clayton,

Australia Jamie Rossjohn * St Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia, Mark M Chong * Department of Medicine (St Vincent's), University of Melbourne,

Fitzroy, Australia., Mark M Chong * Collaborative Research Network, Federation University, Ballarat, Australia Stuart P Berzins * Fiona Elsey Cancer Research Institute, Ballarat, Australia

Stuart P Berzins * The Division of Molecular Immunology, Walter and Eliza Hall Institute of Medical Research, Parkville, Australia Gabrielle T Belz * Department of Medical Biology,

University of Melbourne, Parkville, Australia Gabrielle T Belz Authors * Hui-Fern Koay View author publications You can also search for this author inPubMed Google Scholar * Nicholas A

Gherardin View author publications You can also search for this author inPubMed Google Scholar * Anselm Enders View author publications You can also search for this author inPubMed Google

Scholar * Liyen Loh View author publications You can also search for this author inPubMed Google Scholar * Laura K Mackay View author publications You can also search for this author

inPubMed Google Scholar * Catarina F Almeida View author publications You can also search for this author inPubMed Google Scholar * Brendan E Russ View author publications You can also

search for this author inPubMed Google Scholar * Claudia A Nold-Petry View author publications You can also search for this author inPubMed Google Scholar * Marcel F Nold View author

publications You can also search for this author inPubMed Google Scholar * Sammy Bedoui View author publications You can also search for this author inPubMed Google Scholar * Zhenjun Chen

View author publications You can also search for this author inPubMed Google Scholar * Alexandra J Corbett View author publications You can also search for this author inPubMed Google

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inPubMed Google Scholar * Yves d'Udekem View author publications You can also search for this author inPubMed Google Scholar * Igor E Konstantinov View author publications You can also

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can also search for this author inPubMed Google Scholar * Chris C Goodnow View author publications You can also search for this author inPubMed Google Scholar * David P Fairlie View author

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author inPubMed Google Scholar * James McCluskey View author publications You can also search for this author inPubMed Google Scholar * Adam P Uldrich View author publications You can also

search for this author inPubMed Google Scholar * Dale I Godfrey View author publications You can also search for this author inPubMed Google Scholar * Daniel G Pellicci View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS 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.

CORRESPONDING AUTHORS Correspondence to Dale I Godfrey or Daniel G Pellicci. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. INTEGRATED

SUPPLEMENTARY INFORMATION 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 _Drosha__fl/+__ CD4-Cre_ heterozygous control mice and

_Drosha__fl/fl__ CD4-Cre_ mice. Absolute numbers and percentage NKT cells of TCRβ+ cells in thymus, spleen and lymph nodes of _Drosha__fl/+__ CD4-Cre_ heterozygous control mice and

_Drosha__fl/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. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES Supplementary Figures 1–6 and Supplementary Tables 1 and 2 (PDF 1562 kb) RIGHTS AND PERMISSIONS

Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 27 May 2016 * Accepted: 22 August 2016 * Published: 26 September 2016 *

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