Article | Published:

Visualization of the earliest steps of γδ T cell development in the adult thymus

Nature Immunology volume 7, pages 9951003 (2006) | Download Citation

Subjects

Abstract

The checkpoint in γδ cell development that controls successful T cell receptor (TCR) gene rearrangements remains poorly characterized. Using mice expressing a reporter gene 'knocked into' the Tcrd constant region gene, we have characterized many of the events that mark the life of early γδ cells in the adult thymus. We identify the developmental stage during which the Tcrd locus 'opens' in early T cell progenitors and show that a single checkpoint controls γδ cell development during the penultimate CD4CD8 stage. Passage through this checkpoint required the assembly of γδ TCR heterodimers on the cell surface and signaling via the Lat adaptor protein. In addition, we show that γδ selection triggered a phase of sustained proliferation similar to that induced by the pre-TCR.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    et al. Thymic selection revisited: how essential is it? Immunol. Rev. 191, 62–78 (2003).

  2. 2.

    et al. Productive T-cell receptor β-chain gene rearrangement: coincident regulation of cell cycle and clonality during development in vivo. Genes Dev. 10, 948–962 (1996).

  3. 3.

    γδ cells: a right time and a right place for a conserved third way of protection. Annu. Rev. Immunol. 18, 975–1026 (2000).

  4. 4.

    et al. Intrathymic δ selection events in γδ cell development. Immunity 7, 83–95 (1997).

  5. 5.

    , , & In-frame TCR δ gene rearrangements play a critical role in the αβ/γδ T cell lineage decision. Immunity 2, 617–627 (1995).

  6. 6.

    , & Unexpectedly late expression of intracellular CD3ε and TCR γδ proteins during adult thymus development. Int. Immunol. 11, 1641–1650 (1999).

  7. 7.

    , , , & Delayed and restricted expression limits putative instructional opportunities of Vγ1.1/Vγ2 γδ TCR in αβ/γδ lineage choice in the thymus. J. Immunol. 173, 25–32 (2004).

  8. 8.

    & Intraepithelial lymphocytes. Anatomical site, not T cell receptor form, dictates phenotype and function. J. Exp. Med. 170, 1569–1581 (1989).

  9. 9.

    et al. Repertoire development and ligand specificity of murine TCR γδ cells. Immunol. Rev. 120, 5–33 (1991).

  10. 10.

    , & Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr. Biol. 8, 377–385 (1998).

  11. 11.

    , , , & Targeted disruption of the MHC class II Aa gene in C57BL/6 mice. Int. Immunol. 5, 957–964 (1993).

  12. 12.

    , , & Mechanisms controlling termination of V-J recombination at the TCRγ locus: implications for allelic and isotypic exclusion of TCRγ chains. J. Immunol. 174, 3912–3919 (2005).

  13. 13.

    , , , & Regulation of genome rearrangement events during lymphocyte differentiation. Immunol. Rev. 89, 5–30 (1986).

  14. 14.

    , & Regulation of the murine Dδ2 promoter by upstream stimulatory factor 1, Runx1, and c-Myb. J. Immunol. 174, 4144–4152 (2005).

  15. 15.

    & A positive look at double-negative thymocytes. Nat. Rev. Immunol. 2, 888–897 (2002).

  16. 16.

    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).

  17. 17.

    , , & New insights into the proliferation and differentiation of early mouse thymocytes. Int. Immunol. 16, 1069–1080 (2004).

  18. 18.

    et al. Notch signaling controls the generation and differentiation of early T lineage progenitors. Nat. Immunol. 6, 663–670 (2005).

  19. 19.

    , , & Requirement for Notch1 signals at sequential early stages of intrathymic T cell development. Nat. Immunol. 6, 671–679 (2005).

  20. 20.

    , , , & Regulation of T-cell progenitor survival and cell-cycle entry by the pre-T-cell receptor. Immunol. Rev. 209, 159–169 (2006).

  21. 21.

    et al. Essential role of LAT in T cell development. Immunity 10, 323–332 (1999).

  22. 22.

    et al. Lat regulates γδ T cell homeostasis and differentiation. Nat. Immunol. 4, 999–1008 (2003).

  23. 23.

    et al. Specific requirement for CD3ε in T cell development. Proc. Natl. Acad. Sci. USA 95, 14909–14914 (1998).

  24. 24.

    et al. Altered T cell development in mice with a targeted mutation of the CD3ε gene. EMBO J. 14, 4641–4653 (1995).

  25. 25.

    , , , & Early T cell receptor β gene expression is regulated by the pre-T cell receptor-CD3 complex. J. Exp. Med. 190, 141–144 (1999).

  26. 26.

    , , , & The common cytokine receptor γ chain controls survival of γ/δ T cells. J. Exp. Med. 186, 1277–1285 (1997).

  27. 27.

    , & Defective development of γ/δ T cells in interleukin 7 receptor-deficient mice is due to impaired expression of T cell receptor γ genes. J. Exp. Med. 190, 973–982 (1999).

  28. 28.

    et al. Induction of germline transcription in the TCRγ locus by Stat5: implications for accessibility control by the IL-7 receptor. Immunity 11, 213–223 (1999).

  29. 29.

    & Development of γδ T cells in the adult murine thymus. Eur. J. Immunol. 23, 1655–1660 (1993).

  30. 30.

    et al. CD5 expression is developmentally regulated by T cell receptor (TCR) signals and TCR avidity. J. Exp. Med. 188, 2301–2311 (1998).

  31. 31.

    , , , & Mouse γδ TCR+NK1.1+ thymocytes specifically produce interleukin-4, are major histocompatibility complex class I independent, and are developmentally related to αβ TCR+NK1.1+ thymocytes. Eur. J. Immunol. 26, 1424–1429 (1996).

  32. 32.

    , & Tissue-specific segregation of TCRγδ+ NKT cells according to phenotype TCR repertoire and activation status: parallels with TCRαβ+ NKT cells. Eur. J. Immunol. 31, 2901–2909 (2001).

  33. 33.

    , , , & Strain-specific TCR repertoire selection of IL-4-producing Thy-1 dull γδ thymocytes. Eur. J. Immunol. 31, 205–214 (2001).

  34. 34.

    , , , & Developmental and molecular characterization of emerging β- and γδ-selected pre-T cells in the adult mouse thymus. Immunity 24, 53–64 (2006).

  35. 35.

    , & Lymphotoxin-mediated regulation of γδ cell differentiation by αβ T cell progenitors. Science 307, 925–928 (2005).

  36. 36.

    , & γδ T cell development-having the strength to get there. Curr. Opin. Immunol. 17, 108–115 (2005).

  37. 37.

    , , & Notch regulation of lymphocyte development and function. Nat. Immunol. 5, 247–253 (2004).

  38. 38.

    et al. The IL-7 receptor controls the accessibility of the TCRγ locus by Stat5 and histone acetylation. Immunity 15, 813–823 (2001).

  39. 39.

    , . & Kinetics of T cell receptor β, γ, and δ rearrangements during adult thymic development: T cell receptor rearrangements are present in CD44+CD25+ pro-T thymocytes. Proc. Natl. Acad. Sci. USA 95, 12522–12527 (1998).

  40. 40.

    Unique features of the pre-T-cell receptor α-chain: not just a surrogate. Nat. Rev. Immunol. 5, 571–577 (2005).

  41. 41.

    , , & The generation and fate of thymocytes. Semin. Immunol. 2, 3–12 (1990).

  42. 42.

    , & Scheduled kinetics of cell proliferation and phenotypic changes during immature thymocyte generation. Eur. J. Immunol. 31, 3038–3047 (2001).

  43. 43.

    , , & Double-negative thymocyte subsets in CD3ζ chain-deficient mice: absence of HSA+CD44CD25 cells. Eur. J. Immunol. 24, 1903–1907 (1994).

  44. 44.

    , & TCR signal strength influences αβ/γδ lineage fate. Immunity 22, 583–593 (2005).

  45. 45.

    , , & The CD3-gammadeltaepsilon and CD3-ζ/ε modules are each essential for allelic exclusion at the T cell receptor β locus but are both dispensable for the initiation of V to (D)J recombination at the T cell receptor-β, -γ, and -δ loci. J. Exp. Med. 187, 105–116 (1998).

  46. 46.

    , , , & Function of the CD3 subunits of the pre-TCR and TCR complexes during T cell development. Adv. Immunol. 72, 103–148 (1999).

  47. 47.

    et al. Mice lacking major histocompatibility complex class I and class II molecules. Proc. Natl. Acad. Sci. USA 90, 3913–3917 (1993).

  48. 48.

    , , , & T cell receptor specificity is critical for the development of epidermal γδ T cells. J. Exp. Med. 194, 1473–1483 (2001).

  49. 49.

    , & Positive selection of dendritic epidermal γδ T cell precursors in the fetal thymus determines expression of skin-homing receptors. Immunity 21, 121–131 (2004).

  50. 50.

    et al. Antigen recognition determinants of γδ T cell receptors. Science 308, 252–255 (2005).

  51. 51.

    et al. Functional immunoglobulin transgenes guide ordered B-cell differentiation in Rag-1-deficient mice. Genes Dev. 8, 1030–1042 (1994).

  52. 52.

    , , & Exacerbated colitis associated with elevated levels of activated CD4+ T cells in TCRα chain transgenic mice. Gastroenterology 126, 170–181 (2004).

Download references

Acknowledgements

We thank C.A. Stewart, D.J. Pennington (University of London, London, UK), W. Held (Ludwig Institute for Cancer Research, Lausanne, Switzerland) and R. Ceredig (Center for Biomedecine, Basel, Switzerland) for discussions; and P. Grenot, M. Barad, F. Danjan, M. Richelme, P. Perrin, C. Grégoire, M. Fallet, S. Sarazin and A. Gillet for advice. Supported by Centre National de la Recherche Scientifique (B.M.), Institut National de la Santé et de la Recherche Médicale (B.M.), Association pour la Recherche contre le Cancer (B.M.), Fondation pour la Recherche Médicale (B.M.), Ministère de l'Education Nationale et de la Recherche (Plate-forme RIO-MNG; B.M.), Agence National de Recherches (B.M.), the European Community (MUGEN Network of Excellence; B.M.) and a Marie Curie Intra-European Fellowship within the 6th European Community Framework Program (I.P.).

Author information

Author notes

    • Adrien Kissenpfennig

    Present address: Infection and Immunity Group, Centre for Cancer Research and Cell Biology, School of Biomedical Sciences, Queen's University, Belfast BT9 7AB, Northern Ireland.

Affiliations

  1. Centre d'Immunologie de Marseille-Luminy, Université de la Méditerranée, Case 906, Institut National de la Santé et de la Recherche Médicale, U631, and Centre National de la Recherche Scientifique, UMR6102, 13288 Marseille, France.

    • Immo Prinz
    • , Amandine Sansoni
    • , Adrien Kissenpfennig
    • , Laurence Ardouin
    • , Marie Malissen
    •  & Bernard Malissen

Authors

  1. Search for Immo Prinz in:

  2. Search for Amandine Sansoni in:

  3. Search for Adrien Kissenpfennig in:

  4. Search for Laurence Ardouin in:

  5. Search for Marie Malissen in:

  6. Search for Bernard Malissen in:

Contributions

All authors contributed to discussions of experimental design and data analysis; I.P. did all experimental studies unless otherwise indicated; A.S. provided technical assistance; A.K. helped design the 'knock-in' strategy; L.A. and M.M. generated the Janus kinase 3–deficient mice; and I.P. and B.M. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Bernard Malissen.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Generation and identification of Tcrd-H2BEGFP mice.

  2. 2.

    Supplementary Fig. 2

    Schematic of rearrangement, transcription and translation of the targeted Tcrd gene.

  3. 3.

    Supplementary Fig. 3

    Normal percentages and numbers of thymocyte subsets in lymphoid organs of Tcrd-H2BEGFP mice.

  4. 4.

    Supplementary Fig. 4

    Comparable numbers of T cells, NK cells and NKT cells in lymphoid organs of WT and Tcrd-H2BEGFP mice.

  5. 5.

    Supplementary Fig. 5

    Lineage-negative bone marrow cells from Tcrd-H2BEGFP mice efficiently generate γδ T cells during competitive reconstitution.

  6. 6.

    Supplementary Fig. 6

    Tcrd transcription occurs in the absence of Jak3.

  7. 7.

    Supplementary Fig. 7

    Turnover of EGFPhigh DN3 and EGFPhigh DN4 cells from Tcrd-H2BEGFP mice.

  8. 8.

    Supplementary Fig. 8

    Earliest steps of αβ and γδ cell development in adult thymus.

  9. 9.

    Supplementary Methods

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/ni1371

Further reading