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

Nature Immunology volume 7, pages 995–1003 (2006)Cite this article

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 CD4−CD8− 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.

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Figure 1: Characterization of Tcrd-H2BEGFP mice.
Figure 2: H2BEGFP expression in DN thymocytes.
Figure 3: Mapping the Tcrd locus opening.
Figure 4: A single checkpoint controls γδ cell development.
Figure 5: Cell proliferation is induced by γδ-selection.
Figure 6: Phenotype of EGFPhi DN thymocytes.

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References

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

    Article  CAS  Google Scholar 

  2. Hoffman, E.S. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Livak, F., Petrie, H.T., Crispe, I.N. & Schatz, D.G. In-frame TCR δ gene rearrangements play a critical role in the αβ/γδ T cell lineage decision. Immunity 2, 617–627 (1995).

    Article  CAS  Google Scholar 

  6. Wilson, A., Capone, M. & MacDonald, H.R. Unexpectedly late expression of intracellular CD3ε and TCR γδ proteins during adult thymus development. Int. Immunol. 11, 1641–1650 (1999).

    Article  CAS  Google Scholar 

  7. Krotkova, A., Smith, E., Nerz, G., Falk, I. & Eichmann, K. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Kanda, T., Sullivan, K.F. & Wahl, G.M. Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr. Biol. 8, 377–385 (1998).

    Article  CAS  Google Scholar 

  11. Kontgen, F., Suss, G., Stewart, C., Steinmetz, M. & Bluethmann, H. Targeted disruption of the MHC class II Aa gene in C57BL/6 mice. Int. Immunol. 5, 957–964 (1993).

    Article  CAS  Google Scholar 

  12. Boucontet, L., Sepulveda, N., Carneiro, J. & Pereira, P. 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).

    Article  CAS  Google Scholar 

  13. Alt, F.W., Blackwell, T.K., DePinho, R.A., Reth, M.G. & Yancopoulos, G.D. Regulation of genome rearrangement events during lymphocyte differentiation. Immunol. Rev. 89, 5–30 (1986).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. 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).

    Article  CAS  Google Scholar 

  17. Laurent, J., Bosco, N., Marche, P.N. & Ceredig, R. New insights into the proliferation and differentiation of early mouse thymocytes. Int. Immunol. 16, 1069–1080 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. Tan, J.B., Visan, I., Yuan, J.S. & Guidos, C.J. Requirement for Notch1 signals at sequential early stages of intrathymic T cell development. Nat. Immunol. 6, 671–679 (2005).

    Article  CAS  Google Scholar 

  20. Aifantis, I., Mandal, M., Sawai, K., Ferrando, A. & Vilimas, T. Regulation of T-cell progenitor survival and cell-cycle entry by the pre-T-cell receptor. Immunol. Rev. 209, 159–169 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  25. Aifantis, I., Feinberg, J., Fehling, H.J., Di Santo, J.P. & von Boehmer, H. Early T cell receptor β gene expression is regulated by the pre-T cell receptor-CD3 complex. J. Exp. Med. 190, 141–144 (1999).

    Article  CAS  Google Scholar 

  26. Malissen, M., Pereira, P., Gerber, D.J., Malissen, B. & DiSanto, J.P. The common cytokine receptor γ chain controls survival of γ/δ T cells. J. Exp. Med. 186, 1277–1285 (1997).

    Article  CAS  Google Scholar 

  27. Kang, J., Coles, M. & Raulet, D.H. 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).

    Article  CAS  Google Scholar 

  28. Ye, S.K. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  31. Vicari, A.P., Mocci, S., Openshaw, P., O'Garra, A. & Zlotnik, A. 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).

    Article  CAS  Google Scholar 

  32. Lees, R.K., Ferrero, I. & MacDonald, H.R. 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).

    Article  CAS  Google Scholar 

  33. Azuara, V., Grigoriadou, K., Lembezat, M.P., Nagler-Anderson, C. & Pereira, P. Strain-specific TCR repertoire selection of IL-4-producing Thy-1 dull γδ thymocytes. Eur. J. Immunol. 31, 205–214 (2001).

    Article  CAS  Google Scholar 

  34. Taghon, T., Yui, M.A., Pant, R., Diamond, R.A. & Rothenberg, E.V. Developmental and molecular characterization of emerging β- and γδ-selected pre-T cells in the adult mouse thymus. Immunity 24, 53–64 (2006).

    Article  CAS  Google Scholar 

  35. Silva-Santos, B., Pennington, D.J. & Hayday, A.C. Lymphotoxin-mediated regulation of γδ cell differentiation by αβ T cell progenitors. Science 307, 925–928 (2005).

    Article  CAS  Google Scholar 

  36. Pennington, D.J., Silva-Santos, B. & Hayday, A.C. γδ T cell development-having the strength to get there. Curr. Opin. Immunol. 17, 108–115 (2005).

    Article  CAS  Google Scholar 

  37. Radtke, F., Wilson, A., Mancini, S.J. & MacDonald, H.R. Notch regulation of lymphocyte development and function. Nat. Immunol. 5, 247–253 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  39. Capone, M., Hockett, R.D., Jr . & Zlotnik, A. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  41. Shortman, K., Egerton, M., Spangrude, G.J. & Scollay, R. The generation and fate of thymocytes. Semin. Immunol. 2, 3–12 (1990).

    CAS  PubMed  Google Scholar 

  42. Vasseur, F., Le Campion, A. & Penit, C. Scheduled kinetics of cell proliferation and phenotypic changes during immature thymocyte generation. Eur. J. Immunol. 31, 3038–3047 (2001).

    Article  CAS  Google Scholar 

  43. Crompton, T., Moore, M., MacDonald, H.R. & Malissen, B. Double-negative thymocyte subsets in CD3ζ chain-deficient mice: absence of HSA+CD44−CD25− cells. Eur. J. Immunol. 24, 1903–1907 (1994).

    Article  CAS  Google Scholar 

  44. Hayes, S.M., Li, L. & Love, P.E. TCR signal strength influences αβ/γδ lineage fate. Immunity 22, 583–593 (2005).

    Article  CAS  Google Scholar 

  45. Ardouin, L., Ismaili, J., Malissen, B. & Malissen, M. 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).

    Article  CAS  Google Scholar 

  46. Malissen, B., Ardouin, L., Lin, S.Y., Gillet, A. & Malissen, M. Function of the CD3 subunits of the pre-TCR and TCR complexes during T cell development. Adv. Immunol. 72, 103–148 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  48. Ferrero, I., Wilson, A., Beermann, F., Held, W. & MacDonald, H.R. T cell receptor specificity is critical for the development of epidermal γδ T cells. J. Exp. Med. 194, 1473–1483 (2001).

    Article  CAS  Google Scholar 

  49. Xiong, N., Kang, C. & Raulet, D.H. Positive selection of dendritic epidermal γδ T cell precursors in the fetal thymus determines expression of skin-homing receptors. Immunity 21, 121–131 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  52. Prinz, I., Klemm, U., Kaufmann, S.H. & Steinhoff, U. Exacerbated colitis associated with elevated levels of activated CD4+ T cells in TCRα chain transgenic mice. Gastroenterology 126, 170–181 (2004).

    Article  CAS  Google Scholar 

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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.).

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  1. 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

Authors and 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, Marseille, 13288, France

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

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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.

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Correspondence to Bernard Malissen.

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Supplementary information

Supplementary Fig. 1

Generation and identification of Tcrd-H2BEGFP mice. (PDF 2726 kb)

Supplementary Fig. 2

Schematic of rearrangement, transcription and translation of the targeted Tcrd gene. (PDF 669 kb)

Supplementary Fig. 3

Normal percentages and numbers of thymocyte subsets in lymphoid organs of Tcrd-H2BEGFP mice. (PDF 55 kb)

Supplementary Fig. 4

Comparable numbers of T cells, NK cells and NKT cells in lymphoid organs of WT and Tcrd-H2BEGFP mice. (PDF 17 kb)

Supplementary Fig. 5

Lineage-negative bone marrow cells from Tcrd-H2BEGFP mice efficiently generate γδ T cells during competitive reconstitution. (PDF 17 kb)

Supplementary Fig. 6

Tcrd transcription occurs in the absence of Jak3. (PDF 89 kb)

Supplementary Fig. 7

Turnover of EGFPhigh DN3 and EGFPhigh DN4 cells from Tcrd-H2BEGFP mice. (PDF 11 kb)

Supplementary Fig. 8

Earliest steps of αβ and γδ cell development in adult thymus. (PDF 21 kb)

Supplementary Methods (PDF 164 kb)

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Prinz, I., Sansoni, A., Kissenpfennig, A. et al. Visualization of the earliest steps of γδ T cell development in the adult thymus. Nat Immunol 7, 995–1003 (2006). https://doi.org/10.1038/ni1371

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