Testing the Efficiency and Kinetics of Negative Selection Using Thymic Slices

  • Tyng-An Zhou
  • Chia-Lin Hsu
  • Ivan Lilyanov DzhagalovEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2111)


Central tolerance is an efficient barrier to autoimmunity and negative selection of self-reactive thymocytes is one of its major manifestations. Because of its importance, negative selection has been studied extensively through numerous in vitro and in vivo approaches that have tremendously increased our understanding of the process. Recently, in situ experimental systems using thymus slices have been developed that combine some of the advantages of in vitro assays such as ease of manipulation and high throughput with the existence of three dimensional mature thymus microenvironment. These approaches offer unprecedented opportunity to study negative selection. Here, we describe how thymic slices can be used to measure the kinetics and magnitude of negative selection. Taking the OT-1/Ova system as an example, we provide detailed guidance on cutting thymic slices, labeling and overlaying thymocytes on them and reading out the extent of negative selection by flow cytometry. The system can easily be adapted to evaluate the effects of various mutations or treatments on negative selection or to study the behavior of different cells in the thymus through time-lapse imaging.

Key words

Thymus Thymocytes Negative selection Central tolerance Flow cytometry Thymic slices Vibratome OT-1 Ova 



We would like to thank Hsing-Kai Feng and Yun-Tzu Chen for help with taking the photos and carefully reviewing the manuscript. This work was supported by grants from MOST, 106-2320-B-010-026-MY3 (ILD), 107-2320-B-010-016-MY3 (ILD), 104-2628-B-010-002-MY4 (CLH), and 107-2320-B-010-020 (CLH), and a grant from the Yen Tjing Ling Medical Foundation, CI-108-5 (ILD).


  1. 1.
    Kappler JW, Roehm N, Marrack P (1987) T cell tolerance by clonal elimination in the thymus. Cell 49:273–280CrossRefGoogle Scholar
  2. 2.
    Anderson MS, Venanzi ES, Chen Z et al (2005) The cellular mechanism of Aire control of T cell tolerance. Immunity 23:227–239. Scholar
  3. 3.
    Le Borgne M, Ladi E, Dzhagalov IL et al (2009) The impact of negative selection on thymocyte migration in the medulla. Nat Immunol 10:823–830. Scholar
  4. 4.
    Hu Z, Lancaster JN, Sasiponganan C, Ehrlich LIR (2015) CCR4 promotes medullary entry and thymocyte-dendritic cell interactions required for central tolerance. J Exp Med 212:1947–1965. Scholar
  5. 5.
    Ferrero I, Anjuère F, MacDonald HR, Ardavín C (1997) In vitro negative selection of viral superantigen-reactive thymocytes by thymic dendritic cells. Blood 90:1943–1951CrossRefGoogle Scholar
  6. 6.
    Mcgargill MA, Hogquist KA (1999) Antigen-induced coreceptor down-regulation on thymocytes is not a result of apoptosis. J Immunol 162:1237–1245PubMedGoogle Scholar
  7. 7.
    Guerri L, Peguillet I, Geraldo Y et al (2013) Analysis of APC types involved in CD4 tolerance and regulatory T cell generation using reaggregated thymic organ cultures. J Immunol 190:2102–2110. Scholar
  8. 8.
    Bhakta NR, Oh DY, Lewis RS (2005) Calcium oscillations regulate thymocyte motility during positive selection in the three-dimensional thymic environment. Nat Immunol 6:143–151. Scholar
  9. 9.
    Melichar HJ, Ross JO, Herzmark P et al (2013) Distinct temporal patterns of T cell receptor signaling during positive versus negative selection in situ. Sci Signal 6:ra92–ra92. Scholar
  10. 10.
    Ross JO, Melichar HJ, Au-Yeung BB et al (2014) Distinct phases in the positive selection of CD8+ T cells distinguished by intrathymic migration and T-cell receptor signaling patterns. Proc Natl Acad Sci 111:E2550–E2558. Scholar
  11. 11.
    Dzhagalov IL, Chen KG, Herzmark P, Robey EA (2013) Elimination of self-reactive T cells in the thymus: a timeline for negative selection. PLoS Biol 11:e1001566. Scholar
  12. 12.
    Melichar HJ, Ross JO, Taylor KT, Robey EA (2015) Stable interactions and sustained TCR signaling characterize thymocyte-thymocyte interactions that support negative selection. J Immunol 194:1057–1061. Scholar
  13. 13.
    Ki S, Thyagarajan HM, Hu Z et al (2017) EBI2 contributes to the induction of thymic central tolerance in mice by promoting rapid motility of medullary thymocytes. Eur J Immunol 14:377–312. Scholar
  14. 14.
    Au-Yeung BB, Melichar HJ, Ross JO et al (2014) Quantitative and temporal requirements revealed for Zap70 catalytic activity during T cell development. Nat Immunol 15:687–694. Scholar
  15. 15.
    Weist BM, Kurd N, Boussier J et al (2015) Thymic regulatory T cell niche size is dictated by limiting IL-2 from antigen-bearing dendritic cells and feedback competition. Nat Immunol 16:635–641. Scholar
  16. 16.
    Ueda Y, Katagiri K, Tomiyama T et al (2012) Mst1 regulates integrin-dependent thymocyte trafficking and antigen recognition in the thymus. Nat Commun 3:1098–1013. Scholar
  17. 17.
    Ehrlich LIR, Oh DY, Weissman IL, Lewis RS (2009) Differential contribution of chemotaxis and substrate restriction to segregation of immature and mature thymocytes. Immunity 31:986–998. Scholar
  18. 18.
    Halkias J, Melichar HJ, Taylor KT et al (2013) Opposing chemokine gradients control human thymocyte migration in situ. J Clin Invest 123:2131–2142. Scholar
  19. 19.
    Dzhagalov IL, Melichar HJ, Ross JO, et al (2012) Two-photon imaging of the immune system. Curr Protoc Cytom Chapter 12: Unit12.26.
  20. 20.
    Ross JO, Melichar HJ, Halkias J, Robey EA (2016) Studying T cell development in thymic slices. Methods Mol Biol 1323:131–140. Scholar
  21. 21.
    Lancaster JN, Ehrlich LIR (2017) Analysis of thymocyte migration, cellular interactions, and activation by multiphoton fluorescence microscopy of live thymic slices. In: Wells CM, Parsons M (eds) Cell migration. Springer New York, New York, NY, pp 9–25Google Scholar
  22. 22.
    Hogquist KA, Jameson SC, Heath WR et al (1994) T cell receptor antagonist peptides induce positive selection. Cell 76:17–27. Scholar
  23. 23.
    Sood A, Dong M, Melichar HJ (2016) Preparation and applications of organotypic thymic slice cultures. J Vis Exp 114:e54355. Scholar

Copyright information

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

Authors and Affiliations

  • Tyng-An Zhou
    • 1
  • Chia-Lin Hsu
    • 1
  • Ivan Lilyanov Dzhagalov
    • 1
    Email author
  1. 1.Institute of Microbiology and ImmunologyNational Yang-Ming UniversityTaipeiTaiwan

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