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Retroviral Gene Transduction into T Cell Progenitors for Analysis of T Cell Development in the Thymus

  • Ryunosuke Muro
  • Hiroshi Takayanagi
  • Takeshi NittaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2111)

Abstract

The thymus is an organ where T cells develop throughout life. Using mice as a model animal, molecular mechanisms of intrathymic T cell development have been studied. Fetal thymus organ culture technique enables ex vivo reconstitution of fetal-specific T cell development, while bone marrow chimera technique allows in vivo reconstitution of T cell development in adult thymus. These techniques can be combined with retroviral gene transduction into the T cell progenitors to evaluate the function of genes of interest in developing T cells. Here, we describe the basic protocols for retrovirus gene transduction into fetal or adult T cell progenitors and reconstitution of thymic T cell development including experimental tips such as using cryopreserved fetal liver or bone marrow cells as sources of T cell progenitors.

Key words

Thymus T cells Retrovirus Fetal thymus organ culture Bone marrow chimera 

Notes

Acknowledgments

We thank S. Nitta and M. Tsutsumi for the technical assistance. This study was supported by Grants-in-Aid for Research from the Japan Society for the Promotion of Science (JSPS) (KAKENHI 15H05703, 16H05202, 16K19102, and 17H05788), the Kanehara-Ichiro Foundation (grants 29–23), and the Kanae Foundation for the promotion of medical science (grant 47th).

References

  1. 1.
    Abramson J, Anderson G (2017) Thymic epithelial cells. Annu Rev Immunol 35:85–118.  https://doi.org/10.1146/annurev-immunol-051116-052320CrossRefPubMedGoogle Scholar
  2. 2.
    Shibata K, Yamada H, Nakamura R et al (2008) Identification of CD25+ gamma delta T cells as fetal thymus-derived naturally occurring IL-17 producers. J Immunol 181(9):5940–5947CrossRefGoogle Scholar
  3. 3.
    Morita S, Kojima T, Kitamura T (2000) Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther 7:1063–1066.  https://doi.org/10.1038/sj.gt.3301206CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Spidale NA, Sylvia K, Narayan K et al (2018) Interleukin-17-producing γδ T cells originate from SOX13. Immunity 49(5):857–872.e5.  https://doi.org/10.1016/j.immuni.2018.09.010CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Muro R, Nitta T, Nakano K et al (2018) γδTCR recruits the Syk/PI3K axis to drive proinflammatory differentiation program. J Clin Invest 128(1):415–426.  https://doi.org/10.1172/JCI95837CrossRefPubMedGoogle Scholar
  6. 6.
    Nitta T, Kochi Y, Muro R et al (2017) Human thymoproteasome variations influence CD8 T cell selection. Sci Immunol 2:eaan5165.  https://doi.org/10.1126/sciimmunol.aan5165CrossRefPubMedGoogle Scholar
  7. 7.
    Hawley RG, Fong AZC, Burns BF et al (1992) Transplantable myeloproliferative disease induced in mice by interleukin 6 retrovirus. J Exp Med 176:1149–1163CrossRefGoogle Scholar
  8. 8.
    Nitta T, Ohigashi I, Takahama Y (2013) The development of T lymphocytes in fetal thymus organ culture. Methods Mol Biol 946:85–102.  https://doi.org/10.1007/978-1-62703-128-8_6CrossRefPubMedGoogle Scholar
  9. 9.
    Jenkinson W, Jenkinson E, Anderson G (2008) Preparation of 2-dGuo-treated thymus organ cultures. J Vis Exp.  https://doi.org/10.3791/906

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Ryunosuke Muro
    • 1
  • Hiroshi Takayanagi
    • 1
  • Takeshi Nitta
    • 1
    Email author
  1. 1.Department of Immunology, Graduate School of Medicine and Faculty of MedicineThe University of TokyoTokyoJapan

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