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.

Continuous T cell receptor signaling required for synapse maintenance and full effector potential

Abstract

Although signals through the T cell receptor (TCR) are essential for the initiation of T helper cell activation, it is unclear what function such signals have during the prolonged T cell–antigen-presenting cell contact. Here we simultaneously tracked TCR-CD3 complex and phosphoinositide 3-kinase activity in single T cells using three-dimensional video microscopy. Despite rapid internalization of most of the TCR-CD3, TCR-dependant signaling was still evident up to 10 h after conjugate formation. Blocking this interaction caused dissolution of the synapse and proportional reductions in interleukin 2 production and cellular proliferation. Thus TCR signaling persists for hours, has a cumulative effect and is necessary for the maintenance of the immunological synapse.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Retroviral expression of CD3ζ-CFP and PH(AKT)-YFP did not affect the degree of T cell proliferation in response to antigen.
Figure 2: Antigen-induced PI3K activity colocalized with TCR-CD3 complexes within the nascent immunological synapse and remained mainly synapse associated at later stages despite substantial TCR internalization.
Figure 3: PI3K activity remained synapse-associated for more than 10 h in an antigen-dependent way.
Figure 4: Continued calcium mobilization and the integrity of the immunological synapse depend on TCR signals.
Figure 5: Sustained TCR signaling exerted a cumulative effect on T cell activation.

Similar content being viewed by others

References

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

    Article  CAS  Google Scholar 

  2. Dustin, M.L., Bromley, S.K., Davis, M.M. & Zhu, C. Identification of self through two-dimensional chemistry and synapses. Annu. Rev. Cell. Dev. Biol. 17, 133–157 (2001).

    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  CAS  Google Scholar 

  4. Lee, K.H. et al. T cell receptor signaling precedes immunological synapse formation. Science 295, 1539–1342 (2002).

    Article  CAS  Google Scholar 

  5. Kupfer, H., Monks, C.R. & Kupfer, A. Small splenic B cells that bind to antigen-specific T helper (Th) cells and face the site of cytokine production in the Th cells selectively proliferate: immunofluorescence microscopic studies of Th-B antigen- presenting cell interactions. J. Exp. Med. 179, 1507–1515 (1994).

    Article  CAS  Google Scholar 

  6. Delon, J., Stoll, S. & Germain, R.N. Imaging of T-cell interactions with antigen presenting cells in culture and in intact lymphoid tissue. Immunol. Rev. 189, 51–63 (2002).

    Article  CAS  Google Scholar 

  7. Richie, L.I. et al. Imaging synapse formation during thymocyte selection: inability of CD3z to form a stable central accumulation during negative selection. Immunity 16, 595–606 (2002).

    Article  CAS  Google Scholar 

  8. Stinchcombe, J.C., Bossi, G., Booth, S. & Griffiths, G.M. The immunological synapse of CTL contains a secretory domain and membrane bridges. Immunity 15, 751–761 (2001).

    Article  CAS  Google Scholar 

  9. Kane, L.P., Lin, J. & Weiss, A. Signal transduction by the TCR for antigen. Curr. Opin. Immunol. 12, 242–249 (2000).

    Article  CAS  Google Scholar 

  10. Valitutti, S., Muller, S., Cella, M., Padovan, E. & Lanzavecchia, A. Serial triggering of many T-cell receptors by a few peptide-MHC complexes. Nature 375, 148–151 (1995).

    Article  CAS  Google Scholar 

  11. Liu, H., Rhodes, M., Wiest, D.L. & Vignali, D.A. On the dynamics of TCR:CD3 complex cell surface expression and downmodulation. Immunity 13, 665–675 (2000).

    Article  CAS  Google Scholar 

  12. Iezzi, G., Karjalainen, K. & Lanzavecchia, A. The duration of antigenic stimulation determines the fate of naive and effector T cells. Immunity 8, 89–95 (1998).

    Article  CAS  Google Scholar 

  13. Zal, T., Zal, M.A. & Gascoigne, N.R. Inhibition of T cell receptor-coreceptor interactions by antagonist ligands visualized by live FRET imaging of the T-hybridoma immunological synapse. Immunity 16, 521–534 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Krummel, M.F., Sjaastad, M.D., Wulfing, C. & Davis, M.M. Differential clustering of CD4 and CD3zeta during T cell recognition. Science 289, 1349–1352 (2000).

    Article  CAS  Google Scholar 

  16. Botelho, R.J. et al. Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis. J. Cell. Biol. 151, 1353–1368 (2000).

    Article  CAS  Google Scholar 

  17. Costello, P.S., Gallagher, M. & Cantrell, D.A. Sustained and dynamic inositol lipid metabolism inside and outside the immunological synapse. Nat. Immunol. 3, 1082–1089 (2002).

    Article  CAS  Google Scholar 

  18. Harriague, J. & Bismuth, G. Imaging antigen-induced PI3K activation in T cells. Nat. Immunol. 3, 1090–1096 (2002).

    Article  CAS  Google Scholar 

  19. Marshall, J.G. et al. Restricted accumulation of phosphatidylinositol 3-kinase products in a plasmalemmal subdomain during Fc g receptor-mediated phagocytosis. J. Cell Biol. 153, 1369–1380 (2001).

    Article  CAS  Google Scholar 

  20. Valitutti, S., Muller, S., Salio, M. & Lanzavecchia, A. Degradation of T cell receptor (TCR)-CD3-z complexes after antigenic stimulation. J. Exp. Med 185, 1859–1864 (1997).

    Article  CAS  Google Scholar 

  21. Ward, S.G. & Cantrell, D.A. Phosphoinositide 3-kinases in T lymphocyte activation. Curr. Opin. Immunol. 13, 332–338 (2001).

    Article  CAS  Google Scholar 

  22. Feske, S., Giltnane, J., Dolmetsch, R., Staudt, L.M. & Rao, A. Gene regulation mediated by calcium signals in T lymphocytes. Nat. Immunol. 2, 316–324 (2001).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  24. Lollo, B.A., Chan, K.W., Hanson, E.M., Moy, V.T. & Brian, A.A. Direct evidence for two affinity states for lymphocyte function- associated antigen 1 on activated T cells. J. Biol. Chem. 268, 21693–21700 (1993).

    CAS  PubMed  Google Scholar 

  25. Labadia, M.E., Jeanfavre, D.D., Caviness, G.O. & Morelock, M.M. Molecular regulation of the interaction between leukocyte function- associated antigen-1 and soluble ICAM-1 by divalent metal cations. J. Immunol. 161, 836–842 (1998).

    CAS  PubMed  Google Scholar 

  26. Lyons, A.B. & Parish, C.R. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171, 131–137 (1994).

    Article  CAS  Google Scholar 

  27. Banchereau, J. & Steinman, R.M. Dendritic cells and the control of immunity. Nature 392, 245–252 (1998).

    Article  CAS  Google Scholar 

  28. Mellman, I., Turley, S.J. & Steinman, R.M. Antigen processing for amateurs and professionals. Trends Cell Biol. 8, 231–237 (1998).

    Article  CAS  Google Scholar 

  29. Gunzer, M. et al. Antigen presentation in extracellular matrix: interactions of T cells with dendritic cells are dynamic, short lived, and sequential. Immunity 13, 323–332 (2000).

    Article  CAS  Google Scholar 

  30. Miller, M.J., Wei, S.H., Parker, I. & Cahalan, M.D. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296, 1869–1873 (2002).

    Article  CAS  Google Scholar 

  31. Stoll, S., Delon, J., Brotz, T.M. & Germain, R.N. Dynamic imaging of T cell-dendritic cell interactions in lymph nodes. Science 296, 1873–1876 (2002).

    Article  Google Scholar 

  32. Schrum, A.G. & Turka, L.A. The proliferative capacity of individual naive CD4+ T cells is amplified by prolonged T cell antigen receptor triggering. J. Exp. Med. 196, 793–803 (2002).

    Article  CAS  Google Scholar 

  33. Bird, J.J. et al. Helper T cell differentiation is controlled by the cell cycle. Immunity 9, 229–237 (1998).

    Article  CAS  Google Scholar 

  34. Iezzi, G., Scotet, E., Scheidegger, D. & Lanzavecchia, A. The interplay between the duration of TCR and cytokine signaling determines T cell polarization. Eur. J. Immunol. 29, 4092–4101 (1999).

    Article  CAS  Google Scholar 

  35. Lee, K.M. et al. Molecular basis of T cell inactivation by CTLA-4. Science 282, 2263–2266 (1998).

    Article  CAS  Google Scholar 

  36. Egen, J.G., Kuhns, M.S. & Allison, J.P. CTLA-4: new insights into its biological function and use in tumor immunotherapy. Nat. Immunol. 3, 611–618 (2002).

    Article  CAS  Google Scholar 

  37. Egen, J.G. & Allison, J.P. Cytotoxic T lymphocyte antigen-4 accumulation in the immunological synapse is regulated by TCR signal strength. Immunity 16, 23–35 (2002).

    Article  CAS  Google Scholar 

  38. Harty, J.T., Tvinnereim, A.R. & White, D.W. CD8+ T cell effector mechanisms in resistance to infection. Annu. Rev. Immunol. 18, 275–308 (2000).

    Article  CAS  Google Scholar 

  39. Kaech, S.M. & Ahmed, R. Memory CD8+ T cell differentiation: initial antigen encounter triggers a developmental program in naive cells. Nat. Immunol. 2, 415–222 (2001).

    Article  CAS  Google Scholar 

  40. van Stipdonk, M.J., Lemmens, E.E. & Schoenberger, S.P. Naive CTLs require a single brief period of antigenic stimulation for clonal expansion and differentiation. Nat. Immunol. 2, 423–429 (2001).

    Article  CAS  Google Scholar 

  41. Carbone, F.R., Kurts, C., Bennett, S.R., Miller, J.F. & Heath, W.R. Cross-presentation: a general mechanism for CTL immunity and tolerance. Immunol. Today 19, 368–373 (1998).

    Article  CAS  Google Scholar 

  42. Schoenberger, S.P., Toes, R.E., van der Voort, E.I., Offringa, R. & Melief, C.J. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393, 480–483 (1998).

    Article  CAS  Google Scholar 

  43. Bennett, S.R. et al. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393, 478–480 (1998).

    Article  CAS  Google Scholar 

  44. Ozato, K., Mayer, N. & Sachs, D.H. Hybridoma cell lines secreting monoclonal antibodies to mouse H-2 and Ia antigens. J. Immunol. 124, 533–540 (1980).

    CAS  PubMed  Google Scholar 

  45. Reay, P.A. et al. Determination of the relationship between T cell responsiveness and the number of MHC-peptide complexes using specific monoclonal antibodies. J. Immunol. 164, 5626–5634 (2000).

    Article  CAS  Google Scholar 

  46. Fredrickson, G.G. & Basch, R.S. Early thymic regeneration after irradiation. Dev. Comp. Immunol. 18, 251–263 (1994).

    Article  CAS  Google Scholar 

  47. Pawelec, G., Ziegler, A. & Wernet, P. Dissection of human allostimulatory determinants with cloned T cells: stimulation inhibition by monoclonal antibodies TU22, 34, 35, 36, 37, 39, 43, and 58 against distinct human MHC class II molecules. Hum. Immunol. 12, 165–176 (1985).

    Article  CAS  Google Scholar 

  48. Unkeless, J.C. Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors. J. Exp. Med. 150, 580–596 (1979).

    Article  CAS  Google Scholar 

  49. 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  CAS  Google Scholar 

Download references

Acknowledgements

We thank M.F. Kuhns, M.F. Krummel, R. Sciammas and L.C. Wu and for advice and discussions. We thank P.J. Ebert, C. Gerke, M. Krogsgaard, B.F. Lillemeier, Q.-J. Li and M. A. Purbhoo for comments on this manuscript. We thank N. Prado for technical assistance and S.M. Wheaton for final preparation of the manuscript. J.B.H. was a fellow of the Cancer Research Institute and M.G. is a predoctoral fellow of the Howard Hughes Medical Institute. This work was supported by grants from the Howard Hughes Medical Institute and the National Institutes of Health.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Mark M Davis.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1.

Antigen specific downregulation of TCR surface expression amounts. (a) Flow cytometry shows downregulation of TCR surface expression amounts. Day 9 5c.c7 T cell blasts were left untreated or pooled with CH27 B cells that had been left untreated or preloaded with MCC peptide (0.4 µM). 30 min after cell pooling, cells were treated with antibody to FcgIII/II, stained with PE-conjugated monoclonal antibody to TCR Vb3 (KJ25) and FITC-conjugated monoclonal antibody to CD4 (RM4-5). TCR expression is shown for CD4 + cells. (b) Kinetics of surface TCR downregulation. Day 9 5c.c7 T cell blasts were pooled as in (a) with CH27 B cells in the absence (negative control) and presence of 0.4µM MCC peptide and incubated for the times indicated. TCR surface expression was analyzed by flow cytometry as in (a). Values refer to mean fluorescence intensities. (PDF 27 kb)

Supplementary Fig. 2.

Selective interference with the 5c.c7 TCR-IEk-MCC interaction as opposed to blocking potential non-antigen mediated interactions involving MHC class II molecules leads to decline in synaptic signaling activity and disruption of the immunological synapse. (a) Sustained PI3K activity depends on the presence of stimulatory pMHC ligands but not irrelevant pMHC complexes. 5c.c7 TCR transgenic T cell blasts expressing PH(AKT)YFP were added to CH27 B cells that had been preloaded with the MCC peptide. About 90% of the T cells were found in conjugates 15 minutes after cell pooling. Three hours after cell pooling monoclonal antibodies (20 µg/ml of the anti-antibodies IAk 10-3.6 and 11-5.2 and 20µg/ml of the antibody to IEk/MCC antibody D4) were added to the conjugates and T cells were inspected for localization of PH(AKT)-YFP 15 minutes later. The ratio of synapse-associated fluorescence intensity to cytoplasmic fluorescence intensity served as a relative measure for PI3K activity. Error bars represent the standard deviation derived from 25 T cells inspected in each group. (b) Sustained calcium signal depends on the presence of stimulatory pMHC ligands but not irrelevant pMHC complexes. 5c.c7 TCR transgenic T cell blasts were added to CH27 B cells that had been preloaded with the MCC peptide (0.4µM). About 90% of the T cells were found in conjugates 15 minutes after cell pooling. Three hours after cell pooling monoclonal antibodies (20 µg/ml of the anti-antibodies IAk 10-3.6 and 11-5.2 and 20µg/ml of the antibody to IEk/MCC antibody D4) and the calcium-sensitive dye fura-2 were added to the conjugates and T cells were inspected for calcium signaling status as described in figure 3 (N/data group=25). (c) The integrity of the immunological synapse depends on the presence of stimulatory pMHC ligands but not irrelevant pMHC complexes. 5c.c7 TCR transgenic T cell blasts were added to CH27 B cells expressing ICAM-1-GFP that had been preloaded with the MCC peptide (0.4µM). About 90% of the T cells were found in conjugates 15 minutes after cell pooling. Three hours after cell pooling monoclonal antibodies (20 µg/ml of the anti-antibodies IAk 10-3.6 and 11-5.2 and 20µg/ml of the antibody to IEk/MCC antibody D4). 15 minutes after antibody addition conjugates were inspected for ICAM-1-GFP surface redistribution as described in figure 3. n=25 (PDF 29 kb)

Supplementary video.

Three-dimensional representations of image stacks (derived from Fig. 2) accentuate gradual internalization of the TCR from the IS. (MOV 559 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Huppa, J., Gleimer, M., Sumen, C. et al. Continuous T cell receptor signaling required for synapse maintenance and full effector potential. Nat Immunol 4, 749–755 (2003). https://doi.org/10.1038/ni951

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni951

This article is cited by

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