T-cell development made simple


The thymus is the primary site of T-cell lymphopoiesis. However, the precise molecular interactions that enable the thymus to carry out this function are only recently being elucidated. Although several important molecular players have been identified, including soluble factors, extracellular matrix components, and integral membrane receptors and their ligands, the precise role of these molecules in thymocyte differentiation has yet to be fully characterized. In this regard, the advent of a simple and efficient culture system for the generation of T cells from stem cells, as discussed here, should greatly facilitate the study of T-cell development.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Intrathymic T-cell development.
Figure 2: A simple schematic overview of Notch signalling.
Figure 3: T-cell development made simple: a schematic overview of stem cell–OP9-DL1 cell co-cultures, and potential applications/experimental approaches of this model system.


  1. 1

    Miller, J. F. The discovery of thymus function and of thymus-derived lymphocytes. Immunol. Rev. 185, 7–14 (2002).

  2. 2

    Anderson, G. & Jenkinson, E. J. Lymphostromal interactions in thymic development and function. Nature Rev. Immunol. 1, 31–40 (2001).

  3. 3

    Petrie, H. T. Role of thymic organ structure and stromal composition in steady-state postnatal T-cell production. Immunol. Rev. 189, 8–19 (2002).

  4. 4

    Miller, J. F. A. P. Immunological function of the thymus. Lancet 2, 748 (1961).

  5. 5

    Kondo, M., Weissman, I. L. & Akashi, K. Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 91, 661–672 (1997).

  6. 6

    Allman, D. et al. Thymopoiesis independent of common lymphoid progenitors. Nature Immunol. 4, 168–174 (2003).

  7. 7

    Igarashi, H., Gregory, S. C., Yokota, T., Sakaguchi, N. & Kincade, P. W. Transcription from the RAG1 locus marks the earliest lymphocyte progenitors in bone marrow. Immunity 17, 117–130 (2002).

  8. 8

    Hirose, J. et al. A developing picture of lymphopoiesis in bone marrow. Immunol. Rev. 189, 28–40 (2002).

  9. 9

    Wang, H. & Spangrude, G. J. Aspects of early lymphoid commitment. Curr. Opin. Hematol. 10, 203–207 (2003).

  10. 10

    Henderson, A. J. & Dorshkind, K. In vitro models of B lymphocyte development. Semin. Immunol. 2, 181–187 (1990).

  11. 11

    Anderson, G., Moore, N. C., Owen, J. J. & Jenkinson, E. J. Cellular interactions in thymocyte development. Annu. Rev. Immunol. 14, 73–99 (1996).

  12. 12

    Godfrey, D. I., Kennedy, J., Suda, T. & Zlotnik, A. A developmental pathway involving four phenotypically and functionally distinct subsets of CD3CD4CD8 triple negative adult mouse thymocytes defined by CD44 and CD25 expression. J. Immunol. 150, 4244–4252 (1993).

  13. 13

    Ceredig, R. & Rolink, T. A positive look at double-negative thymocytes. Nature Rev. Immunol. 2, 888–897 (2002).

  14. 14

    Lind, E. F., Prockop, S. E., Porritt, H. E. & Petrie, H. T. Mapping precursor movement through the postnatal thymus reveals specific microenvironments supporting defined stages of early lymphoid development. J. Exp. Med. 194, 127–134 (2001).

  15. 15

    Prockop, S. E. et al. Stromal cells provide the matrix for migration of early lymphoid progenitors through the thymic cortex. J. Immunol. 169, 4354–4361 (2002).

  16. 16

    Porritt, H. E., Gordon, K. & Petrie, H. T. Kinetics of steady-state differentiation and mapping of intrathymic-signaling environments by stem cell transplantation in nonirradiated mice. J. Exp. Med. 198, 957–962 (2003).

  17. 17

    Norment, A. M. & Bevan, M. J. Role of chemokines in thymocyte development. Semin. Immunol. 12, 445–455 (2000).

  18. 18

    Anderson, G., Jenkinson, E. J., Moore, N. C. & Owen, J. J. T. MHC class II-positive epithelium and mesenchyme cells are both required for T-cell development in the thymus. Nature 362, 70–73 (1993).

  19. 19

    Kamarck, M. E. & Gottlieb, P. D. Expression of thymocyte surface alloantigens in the fetal mouse thymus in vivo and in organ culture. J. Immunol. 119, 407–415 (1977).

  20. 20

    DeLuca, D., Mandel, T. E., Luckenbach, G. A. & Kennedy, M. M. Tolerance induction by fusion of fetal thymus lobes in organ culture. J. Immunol. 124, 1821–1829 (1980).

  21. 21

    Asamoto, H. & Mandel, T. E. Thymus in mice bearing the Steel mutation. Morphologic studies on fetal, neonatal, organ-cultured, and grafted fetal thymus. Lab. Invest. 45, 418–426 (1981).

  22. 22

    Ceredig, R., Jenkinson, E. J., MacDonald, H. R. & Owen, J. J. Development of cytolytic T lymphocyte precursors in organ-cultured mouse embryonic thymus rudiments. J. Exp. Med. 155, 617–622 (1982).

  23. 23

    Sekaly, R. P., Ceredig, R. & MacDonald, H. R. Generation of thymocyte subpopulations in organ culture: correlated analysis of Lyt-2 phenotype and cell cycle status by flow microfluorometry. J. Immunol. 131, 1085–1089 (1983).

  24. 24

    Jenkinson, E. J., Franchi, L., Kingston, R. & Owen, J. J. T. Effects of deoxyguanosine on lymphopoiesis in the developing thymus rudiment in vitro: application in the production of chimeric thymus rudiments. Eur. J. Immunol. 12, 583–587 (1982).

  25. 25

    Jenkinson, E. J. & Owen, J. J. T. T cell differentiation in thymus organ culture. Semin. Immunol. 2, 51–58 (1990).

  26. 26

    Takahama, Y. Differentiation of mouse thymocytes in fetal thymus organ culture. Methods Mol. Biol. 134, 37–46 (2000).

  27. 27

    Jenkinson, E. J. & Anderson, G. Fetal thymic organ cultures. Curr. Opin. Immunol. 6, 293–297 (1994).

  28. 28

    Lu, L., Xiao, M., Shen, R. N., Grigsby, S. & Broxmeyer, H. E. Enrichment, characterization, and responsiveness of single primitive CD34 human umbilical cord blood hematopoietic progenitors with high proliferative and replating potential. Blood 81, 41–48 (1993).

  29. 29

    Dexter, T. M., Allen, T. D. & Lajtha, L. G. Conditions controlling the proliferation of haemopoietic stem cells in vitro. J. Cell. Physiol. 91, 335–344 (1977).

  30. 30

    Johnson, G. R. Colony formation in agar by adult bone marrow multipotential hemopoietic cells. J. Cell. Physiol. 103, 371–383 (1980).

  31. 31

    Kubota, K. & Preisler, H. D. Comparison of agar and methylcellulose culture methods for human erythroid colony formation. Exp. Hematol. 10, 292–299 (1982).

  32. 32

    Landreth, K. S. & Dorshkind, K. Pre-B cell generation potentiated by soluble factors from a bone marrow stromal cell line. J. Immunol. 140, 845–852 (1988).

  33. 33

    Collins, L. S. & Dorshkind, K. A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis. J. Immunol. 138, 1082–1087 (1987).

  34. 34

    Cumano, A., Dorshkind, K., Gillis, S. & Paige, C. J. The influence of S17 stromal cells and interleukin 7 on B cell development. Eur. J. Immunol. 20, 2183–2189 (1990).

  35. 35

    Kodama, H., Nose, M., Niida, S. & Nishikawa, S. Involvement of the c-kit receptor in the adhesion of hematopoietic stem cells to stromal cells. Exp. Hematol. 22, 979–984 (1994).

  36. 36

    Ueno, H. et al. A stromal cell-derived membrane protein that supports hematopoietic stem cells. Nature Immunol. 4, 457–463 (2003).

  37. 37

    Nakano, T., Kodama, H. & Honjo, T. Generation of lympho–hematopoietic cells from embryonic stem cells in culture. Science 265, 1098–1101 (1994).

  38. 38

    Nakano, T., Kodama, H. & Honjo, T. In vitro development of primitive and definitive erythrocytes from different precursors. Science 272, 722–724 (1996).

  39. 39

    Nakano, T. Lymphohematopoietic development from embryonic stem cells in vitro. Semin. Immunol. 7, 197–203 (1995).

  40. 40

    Cho, S. K., Bourdeau, A., Letarte, M. & Zúñiga-Pflücker, J. C. Expression and function of CD105 during the onset of hematopoiesis from Flk1+ precursors. Blood 98, 3635–3642 (2001).

  41. 41

    Cho, S. K. et al. Functional characterization of B lymphocytes generated in vitro from embryonic stem cells. Proc. Natl Acad. Sci. USA 96, 9797–9802 (1999).

  42. 42

    Nakayama, N., Fang, I. & Elliott, G. Natural killer and B-lymphoid potential in CD34+ cells derived from embryonic stem cells differentiated in the presence of vascular endothelial growth factor. Blood 91, 2283–2295 (1998).

  43. 43

    Carlyle, J. R. et al. Identification of a novel developmental stage marking lineage commitment of progenitor thymocytes. J. Exp. Med. 186, 173–182 (1997).

  44. 44

    Williams, N. S. et al. Differentiation of NK1. 1+, Ly49+ NK cells from flt3+ multipotent marrow progenitor cells. J. Immunol. 163, 2648–2656 (1999).

  45. 45

    Pear, W. S. & Radtke, F. Notch signaling in lymphopoiesis. Semin. Immunol. 15, 69–79 (2003).

  46. 46

    Radtke, F. et al. Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity 10, 547–558 (1999).

  47. 47

    Pui, J. C. et al. Notch1 expression in early lymphopoiesis influences B versus T lineage determination. Immunity 11, 299–308 (1999).

  48. 48

    Washburn, T. et al. Notch activity influences the αβ versus γδ T cell lineage decision. Cell 88, 833–843 (1997).

  49. 49

    Wolfer, A., Wilson, A., Nemir, M., MacDonald, H. R. & Radtke, F. Inactivation of Notch1 impairs VDJβ rearrangement and allows pre-TCR-independent survival of early αβ lineage thymocytes. Immunity 16, 869–879 (2002).

  50. 50

    Fowlkes, B. J. & Robey, E. A. A reassessment of the effect of activated Notch1 on CD4 and CD8 T cell development. J. Immunol. 169, 1817–1821 (2002).

  51. 51

    Izon, D. J. et al. Notch1 regulates maturation of CD4+ and CD8+ thymocytes by modulating TCR signal strength. Immunity 14, 253–264 (2001).

  52. 52

    Robey, E. et al. An activated form of Notch influences the choice between CD4 and CD8 T cell lineages. Cell 87, 483–492 (1996).

  53. 53

    Deftos, M. L. & Bevan, M. J. Notch signaling in T cell development. Curr. Opin. Immunol. 12, 166–172 (2000).

  54. 54

    De Smedt, M. et al. Active form of Notch imposes T cell fate in human progenitor cells. J. Immunol. 169, 3021–3029 (2002).

  55. 55

    Han, H. et al. Inducible gene knockout of transcription factor recombination signal binding protein-J reveals its essential role in T versus B lineage decision. Int. Immunol. 14, 637–645 (2002).

  56. 56

    Hozumi, K., Abe, N., Chiba, S., Hirai, H. & Habu, S. Active form of notch members can enforce T lymphopoiesis on lymphoid progenitors in the monolayer culture specific for B cell development. J. Immunol. 170, 4973–4979 (2003).

  57. 57

    Jaleco, A. C. et al. Differential effects of Notch ligands Delta-1 and Jagged-1 in human lymphoid differentiation. J. Exp. Med. 194, 991–1002 (2001).

  58. 58

    Wilson, A., MacDonald, H. R. & Radtke, F. Notch 1-deficient common lymphoid precursors adopt a B cell fate in the thymus. J. Exp. Med. 194, 1003–1012 (2001).

  59. 59

    Anderson, G., Pongracz, J., Parnell, S. & Jenkinson, E. J. Notch ligand-bearing thymic epithelial cells initiate and sustain Notch signaling in thymocytes independently of T cell receptor signaling. Eur. J. Immunol. 31, 3349–54 (2001).

  60. 60

    Harman, B. C., Jenkinson, E. J. & Anderson, G. Entry into the thymic microenvironment triggers Notch activation in the earliest migrant T cell progenitors. J. Immunol. 170, 1299–1303 (2003).

  61. 61

    Harman, B. C., Jenkinson, E. J. & Anderson, G. Microenvironmental regulation of Notch signalling in T cell development. Semin. Immunol. 15, 91–97 (2003).

  62. 62

    Felli, M. P. et al. Expression pattern of Notch1, 2 and 3 and Jagged1 and 2 in lymphoid and stromal thymus components: distinct ligand-receptor interactions in intrathymic T cell development. Int. Immunol. 11, 1017–1025 (1999).

  63. 63

    Kaneta, M. et al. A role for pref-1 and HES-1 in thymocyte development. J. Immunol. 164, 256–264 (2000).

  64. 64

    Schmitt, T. M. & Zúñiga-Pflücker, J. C. Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity 17, 749–756 (2002).

  65. 65

    Lehar, S. M. & Bevan, M. J. T cell development in culture. Immunity 17, 689–692 (2002).

  66. 66

    Koch, U., Yuan, J. S., Harper, J. A. & Guidos, C. J. Fine-tuning Notch1 activation by endocytosis and glycosylation. Semin. Immunol. 15, 99–106 (2003).

  67. 67

    Shutter, J. R. et al. Dll4, a novel Notch ligand expressed in arterial endothelium. Genes Dev. 14, 1313–1318 (2000).

  68. 68

    Dorsch, M. et al. Ectopic expression of Delta4 impairs hematopoietic development and leads to lymphoproliferative disease. Blood 100, 2046–2055 (2002).

  69. 69

    Poussier, P. & Julius, M. Speculation on the lineage relationships among CD4 CD8+ gut-derived T cells and their role(s). Semin. Immunol. 11, 293–303 (1999).

  70. 70

    Lancrin, C. et al. Major T cell progenitor activity in bone marrow-derived spleen colonies. J. Exp. Med. 195, 919–929 (2002).

  71. 71

    Garcia-Ojeda, M. E., Dejbakhsh-Jones, S., Weissman, I. L. & Strober, S. An alternate pathway for T cell development supported by the bone marrow microenvironment: recapitulation of thymic maturation. J. Exp. Med. 187, 1813–1823 (1998).

  72. 72

    Wilson, A., Ferrero, I., MacDonald, H. R. & Radtke, F. Cutting edge: an essential role for Notch-1 in the development of both thymus-independent and -dependent T cells in the gut. J. Immunol. 165, 5397–5400 (2000).

  73. 73

    Williams, G. T., Kingston, R., Owen, M. J., Jenkinson, E. J. & Owen, J. J. T. A single micromanipulated stem cell gives rise to multiple T-cell receptor gene rearrangements in the thymus in vitro. Nature 324, 63–64 (1986).

  74. 74

    Michie, A. M. et al. Clonal characterization of a bipotent T cell and NK cell progenitor in the mouse fetal thymus. J. Immunol. 164, 1730–1733 (2000).

  75. 75

    Ikawa, T., Kawamoto, H., Fujimoto, S. & Katsura, Y. Commitment of common T/natural killer (NK) progenitors to unipotent T and NK progenitors in the murine fetal thymus revealed by a single progenitor assay. J. Exp. Med. 190, 1617–1626 (1999).

  76. 76

    McManus, M. T. & Sharp, P. A. Gene silencing in mammals by small interfering RNAs. Nature Rev. Genet. 3, 737–747 (2002).

  77. 77

    Anderson, M. S. et al. Projection of an immunological self shadow within the thymus by the aire protein. Science 298, 1395–1401 (2002).

  78. 78

    Huang, E. Y., Gallegos, A. M., Richards, S. M., Lehar, S. M. & Bevan, M. J. Surface expression of Notch1 on thymocytes: correlation with the double-negative to double-positive transition. J. Immunol. 171, 2296–2304 (2003).

  79. 79

    Artavanis-Tsakonas, S., Rand, M. D. & Lake, R. J. Notch signaling: cell fate control and signal integration in development. Science 284, 770–776 (1999).

  80. 80

    Taniguchi, Y. et al. Notch receptor cleavage depends on but is not directly executed by presenilins. Proc. Natl Acad. Sci. USA 99, 4014–4019 (2002).

  81. 81

    Wu, L. et al. MAML1, a human homologue of Drosophila mastermind, is a transcriptional co-activator for NOTCH receptors. Nature Genet. 26, 484–489 (2000).

  82. 82

    Kao, H. Y. et al. A histone deacetylase co-repressor complex regulates the Notch signal transduction pathway. Genes Dev. 12, 2269–2277 (1998).

Download references


I thank the Canadian Institutes for Health Research and the National Cancer Institute of Canada for their support. Apologies to all colleagues whose work was not cited owing to space constraints.

Author information

Ethics declarations

Competing interests

The author declares no competing financial interests.

Related links

Related links









delta-like 1

delta-like 4


Notch 1

Further information

Juan Carlos Zúñiga-Pflücker's homepage



(AIRE). A transcription factor that promotes the ectopic expression of peripheral tissue-restricted antigens by medullary epithelial cells of the thymus.


The deletion of self-reactive thymocytes in the thymus. Thymocytes that express T-cell receptors that strongly recognize self-peptide bound to self-MHC molecules undergo apoptosis in response to the signalling generated by high-affinity binding.


These mice are deficient in macrophage colony- stimulating factor (MCSF) owing to a naturally occurring recessive mutation, osteopetrosis (op), in the coding region of the MCSF gene.


The maturation of immature CD4+CD8+ precursor thymocytes induced by T-cell receptor (TCR) signals that result from binding to self-peptide–MHC ligands on thymic epithelial cells. This process selects thymocytes that express TCRs that can interact with self-MHC moelcules.


(siRNA). RNA interference (RNAi) is a phenomenon by which the expression of a specific gene is inhibited when a double-stranded complementary RNA (siRNA) is introduced into the organism.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Zúñiga-Pflücker, J. T-cell development made simple. Nat Rev Immunol 4, 67–72 (2004). https://doi.org/10.1038/nri1257

Download citation

Further reading