Biophysical mechanism of T-cell receptor triggering in a reconstituted system

Abstract

A T-cell-mediated immune response is initiated by the T-cell receptor (TCR) interacting with peptide-bound major histocompatibility complex (pMHC) on an infected cell. The mechanism by which this interaction triggers intracellular phosphorylation of the TCR, which lacks a kinase domain, remains poorly understood. Here, we have introduced the TCR and associated signalling molecules into a non-immune cell and reconstituted ligand-specific signalling when these cells are conjugated with antigen-presenting cells. We show that signalling requires the differential segregation of a phosphatase and kinase in the plasma membrane. An artificial, chemically controlled receptor system generates the same effect as TCR–pMHC, demonstrating that the binding energy of an extracellular protein–protein interaction can drive the spatial segregation of membrane proteins without a transmembrane conformational change. This general mechanism may extend to other receptors that rely on extrinsic kinases, including, as we demonstrate, chimaeric antigen receptors being developed for cancer immunotherapy.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Regulatable TCR triggering in an engineered HEK cell line.
Figure 2: The exclusion of CD45 phosphatase is necessary and sufficient for TCR triggering.
Figure 3: TCR–pMHC binding and triggering can be physically and temporally uncoupled.
Figure 4: The TCR–pMHC interaction drives protein exclusion at conjugate regions.
Figure 5: Artificial receptor systems can cause CD45 exclusion and triggering.
Figure 6: A model for steps in TCR-mediated segregation based on membrane bending and energy minimization.

References

  1. 1

    Smith-Garvin, J. E., Koretzky, G. A. & Jordan, M. S. T cell activation. Annu. Rev. Immunol. 27, 591–619 (2009)

    CAS  Article  Google Scholar 

  2. 2

    Lemmon, M. A. & Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 141, 1117–1134 (2010)

    CAS  Article  Google Scholar 

  3. 3

    Palacios, E. H. & Weiss, A. Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation. Oncogene 23, 7990–8000 (2004)

    CAS  Article  Google Scholar 

  4. 4

    Love, P. E. & Hayes, S. M. ITAM-mediated signaling by the T-cell antigen receptor. Cold Spring Harb. Perspect. Biol. 2, a002485 (2010)

    Article  Google Scholar 

  5. 5

    Au-Yeung, B. B. et al. The structure, regulation, and function of ZAP-70. Immunol. Rev. 228, 41–57 (2009)

    CAS  Article  Google Scholar 

  6. 6

    van der Merwe, P. A. & Dushek, O. Mechanisms for T cell receptor triggering. Nature Rev. Immunol. 11, 47–55 (2011)

    CAS  Article  Google Scholar 

  7. 7

    Xu, C. et al. Regulation of T cell receptor activation by dynamic membrane binding of the CD3ε cytoplasmic tyrosine-based motif. Cell 135, 702–713 (2008)

    CAS  Article  Google Scholar 

  8. 8

    Davis, S. J. & van der Merwe, P. A. The kinetic-segregation model: TCR triggering and beyond. Nature Immunol. 7, 803–809 (2006)

    CAS  Article  Google Scholar 

  9. 9

    Lillemeier, B. F. et al. TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation. Nature Immunol. 11, 90–96 (2010)

    CAS  Article  Google Scholar 

  10. 10

    Varma, R., Campi, G., Yokosuka, T., Saito, T. & Dustin, M. L. T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster. Immunity 25, 117–127 (2006)

    CAS  Article  Google Scholar 

  11. 11

    Szymczak, A. L. et al. Correction of multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A peptide-based retroviral vector. Nature Biotechnol. 22, 589–594 (2004)

    CAS  Article  Google Scholar 

  12. 12

    Aleksic, M. et al. Dependence of T cell antigen recognition on T cell receptor–peptide MHC confinement time. Immunity 32, 163–174 (2010)

    CAS  Article  Google Scholar 

  13. 13

    Holst, J. et al. Scalable signaling mediated by T cell antigen receptor-CD3 ITAMs ensures effective negative selection and prevents autoimmunity. Nature Immunol. 9, 658–666 (2008)

    CAS  Article  Google Scholar 

  14. 14

    Irving, B. A. & Weiss, A. The cytoplasmic domain of the T cell receptor ζ chain is sufficient to couple to receptor-associated signal transduction pathways. Cell 64, 891–901 (1991)

    CAS  Article  Google Scholar 

  15. 15

    Deindl, S. et al. Structural basis for the inhibition of tyrosine kinase activity of ZAP-70. Cell 129, 735–746 (2007)

    CAS  Article  Google Scholar 

  16. 16

    Bergman, M. et al. The human p50csk tyrosine kinase phosphorylates p56lck at Tyr-505 and down regulates its catalytic activity. EMBO J. 11, 2919–2924 (1992)

    CAS  Article  Google Scholar 

  17. 17

    Hermiston, M. L., Xu, Z. & Weiss, A. CD45: a critical regulator of signaling thresholds in immune cells. Annu. Rev. Immunol. 21, 107–137 (2003)

    CAS  Article  Google Scholar 

  18. 18

    Saunders, A. E. & Johnson, P. Modulation of immune cell signalling by the leukocyte common tyrosine phosphatase, CD45. Cell. Signal. 22, 339–348 (2010)

    CAS  Article  Google Scholar 

  19. 19

    Chen, J. L. et al. Structural and kinetic basis for heightened immunogenicity of T cell vaccines. J. Exp. Med. 201, 1243–1255 (2005)

    CAS  Article  Google Scholar 

  20. 20

    Monks, C. R., Freiberg, B. A., Kupfer, H., Sciaky, N. & Kupfer, A. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395, 82–86 (1998)

    ADS  CAS  Article  Google Scholar 

  21. 21

    Altan-Bonnet, G. & Germain, R. N. Modeling T cell antigen discrimination based on feedback control of digital ERK responses. PLoS Biol. 3, e356 (2005)

    Article  Google Scholar 

  22. 22

    Manz, B. N., Jackson, B. L., Petit, R. S., Dustin, M. L. & Groves, J. T-cell triggering thresholds are modulated by the number of antigen within individual T-cell receptor clusters. Proc. Natl Acad. Sci. USA 108, 9089–9094 (2011)

    ADS  CAS  Article  Google Scholar 

  23. 23

    Valitutti, S., Dessing, M., Aktories, K., Gallati, H. & Lanzavecchia, A. Sustained signaling leading to T cell activation results from prolonged T cell receptor occupancy. Role of T cell actin cytoskeleton. J. Exp. Med. 181, 577–584 (1995)

    CAS  Article  Google Scholar 

  24. 24

    Johnson, K. G., Bromley, S. K., Dustin, M. L. & Thomas, M. L. A supramolecular basis for CD45 tyrosine phosphatase regulation in sustained T cell activation. Proc. Natl Acad. Sci. USA 97, 10138–10143 (2000)

    ADS  CAS  Article  Google Scholar 

  25. 25

    Leupin, O., Zaru, R., Laroche, T., Muller, S. & Valitutti, S. Exclusion of CD45 from the T-cell receptor signaling area in antigen-stimulated T lymphocytes. Curr. Biol. 10, 277–280 (2000)

    CAS  Article  Google Scholar 

  26. 26

    Irles, C. et al. CD45 ectodomain controls interaction with GEMs and Lck activity for optimal TCR signaling. Nature Immunol. 4, 189–197 (2003)

    CAS  Article  Google Scholar 

  27. 27

    He, X., Woodford-Thomas, T. A., Johnson, K. G., Shah, D. D. & Thomas, M. L. Targeting of CD45 protein tyrosine phosphatase activity to lipid microdomains on the T cell surface inhibits TCR signaling. Eur. J. Immunol. 32, 2578–2587 (2002)

    CAS  Article  Google Scholar 

  28. 28

    Choudhuri, K., Wiseman, D., Brown, M. H., Gould, K. & van der Merwe, P. A. T-cell receptor triggering is critically dependent on the dimensions of its peptide-MHC ligand. Nature 436, 578–582 (2005)

    ADS  CAS  Article  Google Scholar 

  29. 29

    Porter, D. L., Levine, B. L., Kalos, M., Bagg, A. & June, C. H. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N. Engl. J. Med. 365, 725–733 (2011)

    CAS  Article  Google Scholar 

  30. 30

    Shaw, A. S. & Dustin, M. L. Making the T cell receptor go the distance: a topological view of T cell activation. Immunity 6, 361–369 (1997)

    CAS  Article  Google Scholar 

  31. 31

    Qi, S. Y., Groves, J. T. & Chakraborty, A. K. Synaptic pattern formation during cellular recognition. Proc. Natl Acad. Sci. USA 98, 6548–6553 (2001)

    ADS  CAS  Article  Google Scholar 

  32. 32

    Weikl, T. R. & Lipowsky, R. Pattern formation during T-cell adhesion. Biophys. J. 87, 3665–3678 (2004)

    CAS  Article  Google Scholar 

  33. 33

    Coombs, D., Dembo, M., Wofsy, C. & Goldstein, B. Equilibrium thermodynamics of cell–cell adhesion mediated by multiple ligand–receptor pairs. Biophys. J. 86, 1408–1423 (2004)

    ADS  CAS  Article  Google Scholar 

  34. 34

    Alakoskela, J. M. et al. Mechanisms for size-dependent protein segregation at immune synapses assessed with molecular rulers. Biophys. J. 100, 2865–2874 (2011)

    ADS  CAS  Article  Google Scholar 

  35. 35

    Burroughs, N. J. & Wulfing, C. Differential segregation in a cell–cell contact interface: the dynamics of the immunological synapse. Biophys. J. 83, 1784–1796 (2002)

    ADS  CAS  Article  Google Scholar 

  36. 36

    Yokosuka, T. et al. Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76. Nature Immunol. 6, 1253–1262 (2005)

    CAS  Article  Google Scholar 

  37. 37

    Campi, G., Varma, R. & Dustin, M. L. Actin and agonist MHC–peptide complex-dependent T cell receptor microclusters as scaffolds for signaling. J. Exp. Med. 202, 1031–1036 (2005)

    CAS  Article  Google Scholar 

  38. 38

    Bunnell, S. C. et al. T cell receptor ligation induces the formation of dynamically regulated signaling assemblies. J. Cell Biol. 158, 1263–1275 (2002)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank A. van der Merwe and V. Cerundolo for the 1G4 TCR sequence, A. Weiss for cell lines and advice, C. June for the CD19 CAR construct, N. Stuurman and K. Thorn for microscopy help and members of the Vale laboratory for discussions. R.D.V. is a Howard Hughes Medical Institute investigator and J.R.J. is a fellow of the Jane Coffin Childs Memorial Fund.

Author information

Affiliations

Authors

Contributions

J.R.J. conceived the study, collected the data and conducted the analyses. J.R.J. and R.D.V. designed the experiments and wrote the manuscript.

Corresponding author

Correspondence to Ronald D. Vale.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-8 and the legends to Supplementary Movies 1-5. (PDF 2624 kb)

Supplementary Movie 1

The movie shows that the pervanadate treatment of reconstituted cells causes ZAP70 membrane recruitment - see Supplementary Information file for full legend. (AVI 110 kb)

Supplementary Movie 2

This movies contains a 3D reconstruction of the HEK-1G4:APC conjugate - see Supplementary Information file for full legend. (AVI 1710 kb)

Supplementary Movie 3

In this movie we see that ZAP70 rapidly translocates to the cell-cell interface on TCR/pMHC binding - see Supplementary Information file for full legend. (AVI 2432 kb)

Supplementary Movie 4

In this movie shows monitoring ZAP70 recruitment to signalling-competent regions with time - see Supplementary Information file for full legend. (AVI 988 kb)

Supplementary Movie 5

This movie shows that chimaeric-antigen receptor triggering can lead to extensive convolution of the HEK plasma membrane- see Supplementary Information file for full legend. (AVI 1321 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

James, J., Vale, R. Biophysical mechanism of T-cell receptor triggering in a reconstituted system. Nature 487, 64–69 (2012). https://doi.org/10.1038/nature11220

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing