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Two waves of distinct hematopoietic progenitor cells colonize the fetal thymus

Nature Immunology volume 15, pages 27–35 (2014)Cite this article

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Abstract

The generation of T cells depends on the migration of hematopoietic progenitor cells to the thymus throughout life. The identity of the thymus-settling progenitor cells has been a matter of considerable debate. Here we found that thymopoiesis was initiated by a first wave of T cell lineage–restricted progenitor cells with limited capacity for population expansion but accelerated differentiation into mature T cells. They gave rise to αβ and γδ T cells that constituted Vγ3+ dendritic epithelial T cells. Thymopoiesis was subsequently maintained by less-differentiated progenitor cells that retained the potential to develop into B cells and myeloid cells. In that second wave, which started before birth, progenitor cells had high proliferative capacity but delayed differentiation capacity and no longer gave rise to embryonic γδ T cells. Our work reconciles conflicting hypotheses on the nature of thymus-settling progenitor cells.

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Figure 1: DN1 compartment from the thymus at E12 and E13.
Figure 2: DN1a CD135hiCD127+ cells are the most immature progenitors of T cells.
Figure 3: Functional characterization of CRLPs at E13.
Figure 4: Thymic colonization occurs in two successive waves during embryonic development.
Figure 5: TSPs from the two waves have different potential and kinetics of differentiation.
Figure 6: The slow differentiation and high proliferation properties of TSPs at E18 are reflected in their transcriptional profiles.

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Acknowledgements

We thank F. Melchers (Berlin, Germany) for myeloma cell lines transfected with cytokine-encoding cDNA; I. Prinz (Hannover, Germany) and R. Tigelaar (London, UK) for antibody 17D1; A. Bandeira for critical reading of the manuscript and advice; the Freitas laboratory (Pasteur Institute) for Cd3−/− mice; A. Louise, P.-H. Commere and M. Nguyen from the flow cytometry core facility of the Pasteur Institute for technical advice; the staff of the animal facility of the Pasteur Institute for mouse care; V. Villaret and B. Gerstmayer for whole-mouse genome microarrays; and P. de la Grange for biostatistical analysis. Supported by the Pasteur Institute, INSERM, Agence Nationale de Recherche (grant 'Lymphopoiesis' to A.C.), the REVIVE Future Investment Program (A.C.) and La Ligue contre le Cancer (A.C. and C.R.).

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  1. Cyrille Ramond

    Present address: Present address: INSERM U845, Research Center Growth and Signaling, Faculty-Institute Cochin, Paris, France.,

  2. Claire Berthault and Odile Burlen-Defranoux: These authors contributed equally to this work.

Authors and Affiliations

  1. Immunology Department, Unit for Lymphopoiesis, INSERM U668, Paris, France

    Cyrille Ramond, Claire Berthault, Odile Burlen-Defranoux, Ana Pereira de Sousa, Delphine Guy-Grand, Paulo Vieira, Pablo Pereira & Ana Cumano

  2. Université Pierre et Marie Curie, Paris, France

    Cyrille Ramond

  3. Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France

    Claire Berthault

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Contributions

C.R., C.B. and O.B.-D. designed and did experiments and wrote the manuscript; A.P.d.S. contributed to the initial phases of the study; A.C., P.V. and P.P. interpreted data, designed experiments and wrote the manuscript; and D.G.-G. analyzed chimeric mice and contributed to discussions.

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Correspondence to Ana Cumano.

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Integrated supplementary information

Supplementary Figure 1 Gating strategy for CD135 and CD127 expression on DN1a.

(a) Viable Lin- (the lineage cocktail contains CD3, CD4, CD8, NK1.1, CD19, Gr-1, CD25, TER119 and CD11c) CD117+CD44hi cells were separated by CD24 expression into DN1a and DN1b. The upper right panel shows the expression of CD127 and CD135 in DN1a. The CD135 gate was set using DN2 (Lin+CD117+cells) as negative internal control. The bottom histogram shows CD135 expression of DN1aCD135hiCD127+ (in black, open histogram), DN2 (in grey, open histogram), FL CMP (in grey, filled histogram) and FL CLP (in dark grey, filled histogram). (b) CD135 expression in E13 DN2 (in grey, open histogram) compared to newborn (NB) DN2 (in grey, filled histogram), E13 DN1a CD135hi (in black, open histogram) and to NB DN1a CD135hi (dark grey, filled histogram). (c) DN1a CD135hi (black, open histogram), FL CLP (in dark grey, filled histogram), DN2 (in grey open histogram) and FL CMP (in grey, filled histogram). Dot plots are representative of at least twenty-five independent experiments. (d) CD135 expression in NB DN1 subsets (representative of four independent experiments).

Supplementary Figure 2 Phenotype of E13 FL CLPs and E11 CRTPs.

(a) Flow cytometry analysis of E13 FL progenitors (compare with E13 FB in Fig. 2e). (b) E11 FL and FB phenotype (analyzed and stained as for E13 FL and FB) (c) T and B cell potential in purified single E11 FB progenitors were determined as described in Fig. 2f. Results are representative of three independent experiments.

Supplementary Figure 3 CCR9 and CCR7 expression in FB progenitors and ETPs.

(a) relative expression by Q-RT-PCR of CCR9 in CD135+/-CD127- (MPP and MCP), in CD135hiCD127- (LMPP and LMCP), in CD135hiCD127+ CD24+ and CD24lo (CLP and CRLPs). FL (black bars), FB (white bars) and DN1aCD135hiCD127+ (grey bar) (p=0.0003 between FL and FB CD135hi CD127-, p=0.01 between FB CD127+ CD124lo and DN1a) (b) Relative expression of CCR9 in E13 DN1 subsets (DN1aCD135hiCD127+, DN1aCD135lo and DN1b cells) p=0.0001 between CD135hi and CD135lo. AU arbitrary units representing the relative expression to hprt. Error bars represent the mean ± SEM, in one representative experiment. (c) Expression of CCR9 in E17 DN1a subsets (DN1aCD135hi and DN1aCD135lo). (d) Expression of CCR7 in E13 and E17 ETPs (Lin-CD44+CD25-CD117+) compared to DP cells. Results are representative of three independent experiments.

Supplementary Figure 4 Expression of Notch1, Hes1, Ebf1, Pax5 and Rag2 in FL and FB progenitors.

Purified MPP (CD135+/-CD127-), LMPP (CD135hiCD127-), CLP CD24+ (CD127+CD24+) and CD24lo (CD127+CD24lo) from E13 FL (black bars), circulating E13 FB progenitors (white bars) and DN1aCD135hi (grey bar) were analyzed in Q-RT-PCR. AU arbitrary units representing the relative expression to hprt. Results are representative of two to three independent experiments. P=0.02 for Notch1. p=0.0003 for hes1. p=0.002 and p=0.001 for ebf in FL and FB CD127+CD24lo and in FB CD127+CD24lo and DN1a, respectively. p=0.0008 and p=0.01 for pax5 in FL and FB CD127+CD24lo and FB CD127+CD24lo and DN1a, respectively. p=0.002 for rag2 in FL and FB CD127+CD24lo. Error bars represent the mean ± SEM, in one representative experiment.

Supplementary Figure 5 Differentiation potential of E15 and E18 DN1 cells.

The progeny of single E15 DN1a, b, c and e were analyzed for T, B and myeloid potential (a) and single E18 DN1aCD135hi and LMPP for T (in black), NK (in grey) and DC (in white) potential (b) as described in Fig. 1. Results are representative of three independent experiments. ND: not detected (frequency lower than one positive well in 96 wells).

Supplementary Figure 6 Expression of CD127 and Sca-1 in E13 and E18 DN1a thymocytes.

(a) Q-RT-PCR for CD127 transcripts in DN1aCD135hi from E13, E16, E17 and E18. Results are representative of 3 independent experiments. AU arbitrary units representing the relative expression to hprt. P=0.01 in E13 and E16 and p=0.002 in E13 and E17. Error bars represent the mean ± SEM, in one representative experiment. (b) Flow cytometry analysis of CD127 expression in E13 DN1aCD135hiCD127+ (DN1a+ black line), DN2 (light grey line) and FB CLP (dark grey line) (far left panel). CD127 expression in E13 DN1aCD135hi (DN1a+) compared to the same subset from E16 (middle left panel), E17 (middle right panel) and E18 (far right panel) thymi. (c) Flow cytometry analysis of Sca-1 expression on E13 DN1aCD135hiCD127+ in black (DN1a+) compared to E13 FL LMPP (grey line, far left panel) or to CLP CD135hi (CLP+, middle left panel). Sca-1 expression in E18 DN1aCD135hi (DN1a+) compared to E18 FL LMPP (middle right panel) or to CLP CD135hi (CLP+ far right panel). FB cells from E 10.5 (d) and from E 10 (e) were stained with a lineage cocktail, CD127, Sca-1, CD135, CD24 and CD117 antibodies. A progressive gating strategy is shown for E10.5 (>35 somites, upper panels) or for E10 (<30 somites, bottom panels) FB cells. Results are representative of two independent experiments.

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Ramond, C., Berthault, C., Burlen-Defranoux, O. et al. Two waves of distinct hematopoietic progenitor cells colonize the fetal thymus. Nat Immunol 15, 27–35 (2014). https://doi.org/10.1038/ni.2782

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