Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

TCR–peptide–MHC interactions in situ show accelerated kinetics and increased affinity

Nature volume 463, pages 963–967 (2010)Cite this article

Subjects

Abstract

The recognition of foreign antigens by T lymphocytes is essential to most adaptive immune responses. It is driven by specific T-cell antigen receptors (TCRs) binding to antigenic peptide–major histocompatibility complex (pMHC) molecules on other cells1. If productive, these interactions promote the formation of an immunological synapse2,3. Here we show that synaptic TCR–pMHC binding dynamics differ significantly from TCR–pMHC binding in solution. We used single-molecule microscopy and fluorescence resonance energy transfer (FRET) between fluorescently tagged TCRs and their cognate pMHC ligands to measure the kinetics of TCR–pMHC binding in situ. When compared with solution measurements, the dissociation of this complex was increased significantly (4–12-fold). Disruption of actin polymers reversed this effect, indicating that cytoskeletal dynamics destabilize this interaction directly or indirectly. Nevertheless, TCR affinity for pMHC was significantly elevated as the result of a large (about 100-fold) increase in the association rate, a likely consequence of complementary molecular orientation and clustering. In helper T cells, the CD4 molecule has been proposed to bind cooperatively with the TCR to the same pMHC complex. However, CD4 blockade had no effect on the synaptic TCR affinity, nor did it destabilize TCR–pMHC complexes, indicating that the TCR binds pMHC independently of CD4.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: FRET as a sensor for TCR–ligand interactions.
Figure 2: Synaptic off-rates measured through smFRET.
Figure 3: Quantitative image analysis yields effective synaptic 2D  Kd, 2D  Ka and 2D  kon.
Figure 4: Effect of CD4 on TCR signalling and ligand binding.

Similar content being viewed by others

References

  1. Davis, M. M. et al. T cells as a self-referential, sensory organ. Annu. Rev. Immunol. 25, 681–695 (2007)

    Article  CAS  Google Scholar 

  2. Grakoui, A. et al. The immunological synapse: a molecular machine controlling T cell activation. Science 285, 221–227 (1999)

    Article  CAS  Google Scholar 

  3. 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)

    Article  ADS  CAS  Google Scholar 

  4. Corr, M. et al. T cell receptor-MHC class I peptide interactions: affinity, kinetics, and specificity. Science 265, 946–949 (1994)

    Article  ADS  CAS  Google Scholar 

  5. Matsui, K., Boniface, J. J., Steffner, P., Reay, P. A. & Davis, M. M. Kinetics of T-cell receptor binding to peptide/I-Ek complexes: correlation of the dissociation rate with T-cell responsiveness. Proc. Natl Acad. Sci. USA 91, 12862–12866 (1994)

    Article  ADS  CAS  Google Scholar 

  6. Krogsgaard, M. et al. Evidence that structural rearrangements and/or flexibility during TCR binding can contribute to T cell activation. Mol. Cell 12, 1367–1378 (2003)

    Article  CAS  Google Scholar 

  7. Irvine, D. J., Purbhoo, M. A., Krogsgaard, M. & Davis, M. M. Direct observation of ligand recognition by T cells. Nature 419, 845–849 (2002)

    Article  ADS  CAS  Google Scholar 

  8. Sykulev, Y., Joo, M., Vturina, I., Tsomides, T. J. & Eisen, H. N. Evidence that a single peptide-MHC complex on a target cell can elicit a cytolytic T cell response. Immunity 4, 565–571 (1996)

    Article  CAS  Google Scholar 

  9. Bell, G. I. Models for the specific adhesion of cells to cells. Science 200, 618–627 (1978)

    Article  ADS  CAS  Google Scholar 

  10. Dustin, M. L., Ferguson, L. M., Chan, P. Y., Springer, T. A. & Golan, D. E. Visualization of CD2 interaction with LFA-3 and determination of the two-dimensional dissociation constant for adhesion receptors in a contact area. J. Cell Biol. 132, 465–474 (1996)

    Article  CAS  Google Scholar 

  11. Zhu, D. M., Dustin, M. L., Cairo, C. W. & Golan, D. E. Analysis of two-dimensional dissociation constant of laterally mobile cell adhesion molecules. Biophys. J. 92, 1022–1034 (2007)

    Article  ADS  CAS  Google Scholar 

  12. Lillemeier, B. F. et al. TCR and LAT occur in separate membrane domains and concatenate during activation. Nature Immunol. 11, 90–96 (2010)

    Article  CAS  Google Scholar 

  13. Lillemeier, B. F., Pfeiffer, J. R., Surviladze, Z., Wilson, B. S. & Davis, M. M. Plasma membrane-associated proteins are clustered into islands attached to the cytoskeleton. Proc. Natl Acad. Sci. USA 103, 18992–18997 (2006)

    Article  ADS  CAS  Google Scholar 

  14. Schamel, W. W. et al. Coexistence of multivalent and monovalent TCRs explains high sensitivity and wide range of response. J. Exp. Med. 202, 493–503 (2005)

    Article  CAS  Google Scholar 

  15. Garcia, K. C. et al. Structural basis of plasticity in T cell receptor recognition of a self peptide-MHC antigen. Science 279, 1166–1172 (1998)

    Article  ADS  CAS  Google Scholar 

  16. Wang, J. et al. Atomic structure of an αβ T cell receptor (TCR) heterodimer in complex with an anti-TCR Fab fragment derived from a mitogenic antibody. EMBO J. 17, 10–26 (1998)

    Article  Google Scholar 

  17. Kaizuka, Y., Douglass, A. D., Varma, R., Dustin, M. L. & Vale, R. D. Mechanisms for segregating T cell receptor and adhesion molecules during immunological synapse formation in Jurkat T cells. Proc. Natl Acad. Sci. USA 104, 20296–20301 (2007)

    Article  ADS  CAS  Google Scholar 

  18. van der Merwe, P. A., McNamee, P. N., Davies, E. A., Barclay, A. N. & Davis, S. J. Topology of the CD2–CD48 cell-adhesion molecule complex: implications for antigen recognition by T cells. Curr. Biol. 5, 74–84 (1995)

    Article  CAS  Google Scholar 

  19. 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)

    Article  ADS  CAS  Google Scholar 

  20. Xiong, Y., Kern, P., Chang, H. & Reinherz, E. T cell receptor binding to a pMHCII ligand is kinetically distinct from and independent of CD4. J. Biol. Chem. 276, 5659–5667 (2001)

    Article  CAS  Google Scholar 

  21. Li, Q. J. et al. CD4 enhances T cell sensitivity to antigen by coordinating Lck accumulation at the immunological synapse. Nature Immunol. 5, 791–799 (2004)

    Article  CAS  Google Scholar 

  22. Krogsgaard, M. et al. Agonist/endogenous peptide-MHC heterodimers drive T cell activation and sensitivity. Nature 434, 238–243 (2005)

    Article  ADS  CAS  Google Scholar 

  23. Fricke, G. M. & Thomas, J. L. Receptor aggregation by intermembrane interactions: a Monte Carlo study. Biophys. Chem. 119, 205–211 (2006)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  25. Tsumoto, K. et al. Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent–application to a human single-chain Fv fragment. J. Immunol. Methods 219, 119–129 (1998)

    Article  CAS  Google Scholar 

  26. Moertelmaier, M., Brameshuber, M., Linimeier, M., Schütz, G. J. & Stockinger, H. Thinning out clusters while conserving stoichiometry of labeling. Appl. Phys. Lett. 87, 263903 (2005)

    Article  ADS  Google Scholar 

  27. Dai, S. et al. Crossreactive T cells spotlight the germline rules for αβ T cell-receptor interactions with MHC molecules. Immunity 28, 324–334 (2008)

    Article  CAS  Google Scholar 

  28. Tynan, F. E. et al. A T cell receptor flattens a bulged antigenic peptide presented by a major histocompatibility complex class I molecule. Nature Immunol. 8, 268–276 (2007)

    Article  CAS  Google Scholar 

  29. Wu, L. C., Tuot, D. S., Lyons, D. S., Garcia, K. C. & Davis, M. M. Two-step binding mechanism for T-cell receptor recognition of peptide MHC. Nature 418, 552–556 (2002)

    Article  ADS  CAS  Google Scholar 

  30. Huppa, J. B., Gleimer, M., Sumen, C. & Davis, M. M. Continuous T cell receptor signaling required for synapse maintenance and full effector potential. Nature Immunol. 4, 749–755 (2003)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank F. E. Tynan, A. K. Chakraborty and S. R. Quake for helpful discussions, and P. P. J. Ebert for help with the flow cytometry experiments. This work was supported by grants from the National Institutes of Health (RO1 AI52211) and the Howard Hughes Medical Institute to M.M.D. J.B.H. is partly supported by the Immunology Frontier Research Center (iFREC) consortium, M.A.M. received a postdoctoral fellowship from the Wilhelm Macke Stiftung, and E.W.N. from the American Cancer Society’s Steven Stanley and Edward Albert Bielfelt Fund. L.O.K. is supported by a National Science Foundation predoctoral fellowship. M.A., M.B. and G.J.S. received grants from the Austrian Science Fund (project Y250-B10) and the GEN-AU project of the Austrian Ministry for Science and Research.

Author Contributions J.B.H., G.J.S. and M.M.D. conceived the project. Reagents and experimental systems were designed and tested by J.B.H. unless indicated otherwise. M.A., M.A.M. and G.J.S. provided important expertise in single-molecule microscopy. M.A. conducted all single dye tracing experiments and all smFRET experiments performed in Linz. J.B.H. conducted all bulk FRET experiments and all smFRET experiments at Stanford. J.B.H. and M.A.M. laid the experimental foundation in the initial phase of the project. M.A.M. provided important expertise in setting up the microscope system at Stanford. B.F.L. provided the expression system for ICAM-1 and B7-1. E.W.N. conducted all SPR measurements. M.B. conducted all TOCCSL experiments. L.O.K. and B.F.L. contributed important ideas. J.B.H. and M.M.D. wrote the manuscript.

Author information

Author notes

  1. Manuel A. Mörtelmaier & Björn F. Lillemeier

    Present address: Present addresses: Agilent Technologies Austria, Mooslackengasse 17, 1190 Wien, Austria (M.A.M.); Nomis Center for Immunobiology and Microbial Pathogenesis, Waitt Advanced Biophotonics Center, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037, USA (B.F.L.).,

Authors and Affiliations

  1. Department of Microbiology and Immunology, Stanford School of Medicine, and,

    Johannes B. Huppa, Manuel A. Mörtelmaier, Björn F. Lillemeier, Evan W. Newell, Lawrence O. Klein & Mark M. Davis

  2. Howard Hughes Medical Institute, Beckman Center B221, 279 Campus Drive, Stanford, California 94305-5323, USA ,

    Mark M. Davis

  3. Institut für Biophysik, Johannes Kepler-Universität, Altenbergerstrasse 69, A-4040 Linz, Austria ,

    Markus Axmann, Manuel A. Mörtelmaier, Mario Brameshuber & Gerhard J. Schütz

Authors
  1. Johannes B. Huppa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Markus Axmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Manuel A. Mörtelmaier
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Björn F. Lillemeier
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Evan W. Newell
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mario Brameshuber
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lawrence O. Klein
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Gerhard J. Schütz
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Mark M. Davis
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mark M. Davis.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-23 with Legends and Supplementary References. (PDF 2050 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Huppa, J., Axmann, M., Mörtelmaier, M. et al. TCR–peptide–MHC interactions in situ show accelerated kinetics and increased affinity. Nature 463, 963–967 (2010). https://doi.org/10.1038/nature08746

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08746

This article is cited by

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

Advanced 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