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.
Similar content being viewed by others
Accession codes
References
Bhandoola, A., von Boehmer, H., Petrie, H.T. & Zuniga-Pflucker, J.C. Commitment and developmental potential of extrathymic and intrathymic T cell precursors: plenty to choose from. Immunity 26, 678–689 (2007).
Kondo, M., Weissman, I.L. & Akashi, K. Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 91, 661–672 (1997).
Martin, C.H. et al. Efficient thymic immigration of B220+ lymphoid-restricted bone marrow cells with T precursor potential. Nat. Immunol. 4, 866–873 (2003).
Gounari, F. et al. Tracing lymphopoiesis with the aid of a pTα-controlled reporter gene. Nat. Immunol. 3, 489–496 (2002).
Krueger, A. & von Boehmer, H. Identification of a T lineage-committed progenitor in adult blood. Immunity 26, 105–116 (2007).
Allman, D. et al. Thymopoiesis independent of common lymphoid progenitors. Nat. Immunol. 4, 168–174 (2003).
Bell, J.J. & Bhandoola, A. The earliest thymic progenitors for T cells possess myeloid lineage potential. Nature 452, 764–767 (2008).
Wada, H. et al. Adult T-cell progenitors retain myeloid potential. Nature 452, 768–772 (2008).
Schlenner, S.M. et al. Fate mapping reveals separate origins of T cells and myeloid lineages in the thymus. Immunity 32, 426–436 (2010).
Masuda, K. et al. Thymic anlage is colonized by progenitors restricted to T, NK, and dendritic cell lineages. J. Immunol. 174, 2525–2532 (2005).
Harman, B.C. et al. T/B lineage choice occurs prior to intrathymic Notch signaling. Blood 106, 886–892 (2005).
Desanti, G.E. et al. Clonal analysis reveals uniformity in the molecular profile and lineage potential of CCR9+ and CCR9− thymus-settling progenitors. J. Immunol. 186, 5227–5235 (2011).
Rodewald, H.R., Kretzschmar, K., Takeda, S., Hohl, C. & Dessing, M. Identification of pro-thymocytes in murine fetal blood: T lineage commitment can precede thymus colonization. EMBO J. 13, 4229–4240 (1994).
Carlyle, J.R. & Zuniga-Pflucker, J.C. Requirement for the thymus in αβ T lymphocyte lineage commitment. Immunity 9, 187–197 (1998).
Douagi, I., Colucci, F., Di Santo, J.P. & Cumano, A. Identification of the earliest prethymic bipotent T/NK progenitor in murine fetal liver. Blood 99, 463–471 (2002).
Yoshida, H. et al. Expression of α4β7 integrin defines a distinct pathway of lymphoid progenitors committed to T cells, fetal intestinal lymphotoxin producer, NK, and dendritic cells. J. Immunol. 167, 2511–2521 (2001).
Possot, C. et al. Notch signaling is necessary for adult, but not fetal, development of RORγt+ innate lymphoid cells. Nat. Immunol. 12, 949–958 (2011).
Douagi, I., Vieira, P. & Cumano, A. Lymphocyte commitment during embryonic development, in the mouse. Semin. Immunol. 14, 361–369 (2002).
Luc, S. et al. The earliest thymic T cell progenitors sustain B cell and myeloid lineage potential. Nat. Immunol. 13, 412–419 (2012).
Rodewald, H.R., Kretzschmar, K., Swat, W. & Takeda, S. Intrathymically expressed c-kit ligand (stem cell factor) is a major factor driving expansion of very immature thymocytes in vivo. Immunity 3, 313–319 (1995).
Porritt, H.E. et al. Heterogeneity among DN1 prothymocytes reveals multiple progenitors with different capacities to generate T cell and non-T cell lineages. Immunity 20, 735–745 (2004).
Luche, H. et al. In vivo fate mapping identifies pre-TCRalpha expression as an intra- and extrathymic, but not prethymic, marker of T lymphopoiesis. J. Exp. Med. 210, 699–714 (2013).
Scollay, R., Smith, J. & Stauffer, V. Dynamics of early T cells: prothymocyte migration and proliferation in the adult mouse thymus. Immunol. Rev. 91, 129–157 (1986).
Donskoy, E. & Goldschneider, I. Thymocytopoiesis is maintained by blood-borne precursors throughout postnatal life. A study in parabiotic mice. J. Immunol. 148, 1604–1612 (1992).
Ceredig, R., Bosco, N. & Rolink, A.G. The B lineage potential of thymus settling progenitors is critically dependent on mouse age. Eur. J. Immunol. 37, 830–837 (2007).
Jotereau, F.V. & Le Douarin, N.M. Demonstration of a cyclic renewal of the lymphocyte precursor cells in the quail thymus during embryonic and perinatal life. J. Immunol. 129, 1869–1877 (1982).
Coltey, M., Jotereau, F.V. & Le Douarin, N.M. Evidence for a cyclic renewal of lymphocyte precursor cells in the embryonic chick thymus. Cell Differ. 22, 71–82 (1987).
Sambandam, A. et al. Notch signaling controls the generation and differentiation of early T lineage progenitors. Nat. Immunol. 6, 663–670 (2005).
Wu, L., Li, C.L. & Shortman, K. Thymic dendritic cell precursors: relationship to the T lymphocyte lineage and phenotype of the dendritic cell progeny. J. Exp. Med. 184, 903–911 (1996).
Cherrier, M., Sawa, S. & Eberl, G. Notch, Id2, and RORγt sequentially orchestrate the fetal development of lymphoid tissue inducer cells. J. Exp. Med. 209, 729–740 (2012).
Itoi, M., Kawamoto, H., Katsura, Y. & Amagai, T. Two distinct steps of immigration of hematopoietic progenitors into the early thymus anlage. Int. Immunol. 13, 1203–1211 (2001).
Rodewald, H.R. Thymus organogenesis. Annu. Rev. Immunol. 26, 355–388 (2008).
Uehara, S., Grinberg, A., Farber, J.M. & Love, P.E. A role for CCR9 in T lymphocyte development and migration. J. Immunol. 168, 2811–2819 (2002).
Radtke, F., Fasnacht, N. & Macdonald, H.R. Notch signaling in the immune system. Immunity 32, 14–27 (2010).
Kieusseian, A., Brunet de la Grange, P., Burlen-Defranoux, O., Godin, I. & Cumano, A. Immature hematopoietic stem cells undergo maturation in the fetal liver. Development 139, 3521–3530 (2012).
Havran, W.L. & Allison, J.P. Origin of Thy-1+ dendritic epidermal cells of adult mice from fetal thymic precursors. Nature 344, 68–70 (1990).
Ikuta, K. et al. A developmental switch in thymic lymphocyte maturation potential occurs at the level of hematopoietic stem cells. Cell 62, 863–874 (1990).
Jotereau, F., Heuze, F., Salomon-Vie, V. & Gascan, H. Cell kinetics in the fetal mouse thymus: precursor cell input, proliferation, and emigration. J. Immunol. 138, 1026–1030 (1987).
Belyaev, N.N., Biro, J., Athanasakis, D., Fernandez-Reyes, D. & Potocnik, A.J. Global transcriptional analysis of primitive thymocytes reveals accelerated dynamics of T cell specification in fetal stages. Immunogenetics 64, 591–604 (2012).
Balciunaite, G., Ceredig, R. & Rolink, A.G. The earliest subpopulation of mouse thymocytes contains potent T, significant macrophage, and natural killer cell but no B-lymphocyte potential. Blood 105, 1930–1936 (2005).
Lu, M. et al. The earliest thymic progenitors in adults are restricted to T, NK, and dendritic cell lineage and have a potential to form more diverse TCRβ chains than fetal progenitors. J. Immunol. 175, 5848–5856 (2005).
Liu, C. et al. Coordination between CCR7- and CCR9-mediated chemokine signals in prevascular fetal thymus colonization. Blood 108, 2531–2539 (2006).
Wurbel, M.A., Malissen, B. & Campbell, J.J. Complex regulation of CCR9 at multiple discrete stages of T cell development. Eur. J. Immunol. 36, 73–81 (2006).
Schwarz, B.A. et al. Selective thymus settling regulated by cytokine and chemokine receptors. J. Immunol. 178, 2008–2017 (2007).
Scimone, M.L., Aifantis, I., Apostolou, I., von Boehmer, H. & von Andrian, U.H. A multistep adhesion cascade for lymphoid progenitor cell homing to the thymus. Proc. Natl. Acad. Sci. USA 103, 7006–7011 (2006).
Kawamoto, H., Ohmura, K. & Katsura, Y. Presence of progenitors restricted to T, B, or myeloid lineage, but absence of multipotent stem cells, in the murine fetal thymus. J. Immunol. 161, 3799–3802 (1998).
Ikawa, T. et al. Identification of the earliest prethymic T-cell progenitors in murine fetal blood. Blood 103, 530–537 (2004).
Godin, I., Garcia-Porrero, J.A., Dieterlen-Lievre, F. & Cumano, A. Stem cell emergence and hemopoietic activity are incompatible in mouse intraembryonic sites. J. Exp. Med. 190, 43–52 (1999).
Jin, Y.H. et al. Beta-catenin modulates the level and transcriptional activity of Notch1/NICD through its direct interaction. Biochim. Biophys. Acta 1793, 290–299 (2009).
Sengupta, A. et al. Deregulation and cross talk among Sonic hedgehog, Wnt, Hox and Notch signaling in chronic myeloid leukemia progression. Leukemia 21, 949–955 (2007).
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.).
Author information
Authors and Affiliations
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.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
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.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–6 and Supplementary Table 1 (PDF 678 kb)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ni.2782
- Springer Nature America, Inc.
This article is cited by
-
A double-negative thymocyte-specific enhancer augments Notch1 signaling to direct early T cell progenitor expansion, lineage restriction and β-selection
Nature Immunology (2022)
-
Exploring the stage-specific roles of Tcf-1 in T cell development and malignancy at single-cell resolution
Cellular & Molecular Immunology (2021)
-
Leaving no one behind: tracing every human thymocyte by single-cell RNA-sequencing
Seminars in Immunopathology (2021)
-
Neonatal mucosal immunology
Mucosal Immunology (2017)
-
Asynchronous lineage priming determines commitment to T cell and B cell lineages in fetal liver
Nature Immunology (2017)