Abstract

The physiological basis and mechanistic requirements for a large number of functional immunoreceptor tyrosine-based activation motifs (ITAMs; high ITAM multiplicity) in the complex of the T cell antigen receptor (TCR) and the invariant signaling protein CD3 remain obscure. Here we found that whereas a low multiplicity of TCR-CD3 ITAMs was sufficient to engage canonical TCR-induced signaling events that led to cytokine secretion, a high multiplicity of TCR-CD3 ITAMs was required for TCR-driven proliferation. This was dependent on the formation of compact immunological synapses, interaction of the adaptor Vav1 with phosphorylated CD3 ITAMs to mediate the recruitment and activation of the oncogenic transcription factor Notch1 and, ultimately, proliferation induced by the cell-cycle regulator c-Myc. Analogous mechanistic events were also needed to drive proliferation in response to weak peptide agonists. Thus, the TCR-driven pathways that initiate cytokine secretion and proliferation are separable and are coordinated by the multiplicity of phosphorylated ITAMs in TCR-CD3.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , , , & Structural biology of the T-cell receptor: insights into receptor assembly, ligand recognition, and initiation of signaling. Cold Spring Harb. Perspect. Biol. 2, a005140 (2010).

  2. 2.

    , , & ZAP-70: a 70 kd protein-tyrosine kinase that associates with the TCR zeta chain. Cell 71, 649–662 (1992).

  3. 3.

    , , & The adapter proteins LAT and SLP-76 are required for T-cell activation. Cold Spring Harb. Symp. Quant. Biol. 64, 265–274 (1999).

  4. 4.

    & Organization of proximal signal initiation at the TCR:CD3 complex. Immunol. Rev. 232, 7–21 (2009).

  5. 5.

    et al. A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts. Cell 94, 667–677 (1998).

  6. 6.

    , , , & Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395, 82–86 (1998).

  7. 7.

    et al. Functional anatomy of T cell activation and synapse formation. Annu. Rev. Immunol. 28, 79–105 (2010).

  8. 8.

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

  9. 9.

    , , , & T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster. Immunity 25, 117–127 (2006).

  10. 10.

    , , & Rapid analysis of T-cell selection in vivo using T cell-receptor retrogenic mice. Nat. Methods 3, 191–197 (2006).

  11. 11.

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

  12. 12.

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

  13. 13.

    , , & Defining the specific physiological requirements for c-Myc in T cell development. Nat. Immunol. 2, 307–315 (2001).

  14. 14.

    , & Multiple mechanisms regulate c-myc gene expression during normal T cell activation. EMBO J. 7, 2787–2794 (1988).

  15. 15.

    et al. Instruction of distinct CD4 T helper cell fates by different notch ligands on antigen-presenting cells. Cell 117, 515–526 (2004).

  16. 16.

    , , & Notch and presenilin regulate cellular expansion and cytokine secretion but cannot instruct Th1/Th2 fate acquisition. PLoS ONE 3, e2823 (2008).

  17. 17.

    & Notch and Presenilin: regulated intramembrane proteolysis links development and degeneration. Annu. Rev. Neurosci. 26, 565–597 (2003).

  18. 18.

    et al. Notch signaling augments T cell responsiveness by enhancing CD25 expression. J. Immunol. 171, 2896–2903 (2003).

  19. 19.

    et al. Inhibitors of γ-secretase block in vivo and in vitro T helper type 1 polarization by preventing Notch upregulation of Tbx21. Nat. Immunol. 6, 680–688 (2005).

  20. 20.

    et al. Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor. Cell 67, 687–699 (1991).

  21. 21.

    Calcium signalling in lymphocyte activation and disease. Nat. Rev. Immunol. 7, 690–702 (2007).

  22. 22.

    , , & Antiparallel segregation of notch components in the immunological synapse directs reciprocal signaling in allogeneic Th:DC conjugates. J. Immunol. 179, 819–829 (2007).

  23. 23.

    et al. The Notch regulator Numb links the Notch and TCR signaling pathways. J. Immunol. 174, 890–897 (2005).

  24. 24.

    & Kuz and TACE can activate Notch independent of ligand. Cell. Mol. Life Sci. 65, 2232–2243 (2008).

  25. 25.

    & Jagged1 and Notch1 help edit M cell patterning in Peyer's patch follicle epithelium. Dev. Comp. Immunol. 37, 306–312 (2012).

  26. 26.

    , , & Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317, 1749–1753 (2007).

  27. 27.

    , & Vav links antigen-receptor signaling to the actin cytoskeleton. Semin. Immunol. 10, 317–327 (1998).

  28. 28.

    Vav-family proteins in T-cell signalling. Curr. Opin. Immunol. 17, 267–274 (2005).

  29. 29.

    & Biophysical mechanism of T-cell receptor triggering in a reconstituted system. Nature 487, 64–69 (2012).

  30. 30.

    et al. ZAP-70: an essential kinase in T-cell signaling. Cold Spring Harb. Perspect. Biol. 2, a002279 (2010).

  31. 31.

    et al. The granulocyte receptor carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) directly associates with Vav to promote phagocytosis of human pathogens. J. Immunol. 178, 3797–3805 (2007).

  32. 32.

    , , , & Recruitment of Nck by CD3ɛ reveals a ligand-induced conformational change essential for T cell receptor signaling and synapse formation. Cell 109, 901–912 (2002).

  33. 33.

    et al. The adapter protein Nck: role of individual SH3 and SH2 binding modules for protein interactions in T lymphocytes. Protein Sci. 19, 658–669 (2010).

  34. 34.

    , & Modulation of naive CD4 T cell activation with altered peptide ligands: the nature of the peptide and presentation in the context of costimulation are critical for a sustained response. J. Immunol. 160, 3698–3704 (1998).

  35. 35.

    , & Notch signaling in the immune system. Immunity 32, 14–27 (2010).

  36. 36.

    , , , & Functions of notch signaling in the immune system: consensus and controversies. Annu. Rev. Immunol. 28, 343–365 (2010).

  37. 37.

    et al. Notch signaling mediates G1/S cell-cycle progression in T cells via cyclin D3 and its dependent kinases. Blood 113, 1689–1698 (2009).

  38. 38.

    et al. Notch 1 signaling regulates peripheral T cell activation. Immunity 20, 407–415 (2004).

  39. 39.

    Notch signalling: a simple pathway becomes complex. Nat. Rev. Mol. Cell Biol. 7, 678–689 (2006).

  40. 40.

    & The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137, 216–233 (2009).

  41. 41.

    , & The anti-apoptotic effect of Notch-1 requires p56lck-dependent, Akt/PKB-mediated signaling in T cells. J. Biol. Chem. 279, 2937–2944 (2004).

  42. 42.

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

  43. 43.

    , & The Nck family of adapter proteins: regulators of actin cytoskeleton. Cell. Signal. 14, 723–731 (2002).

  44. 44.

    , & Anchoring notch genetics and biochemistry; structural analysis of the ankyrin domain sheds light on existing data. Mol. Cell 13, 619–626 (2004).

  45. 45.

    et al. Tonic ubiquitylation controls T-cell receptor:CD3 complex expression during T cell development. EMBO J. 29, 1285–1298 (2010).

  46. 46.

    , , & Continuous T cell receptor signaling required for synapse maintenance and full effector potential. Nat. Immunol. 4, 749–755 (2003).

  47. 47.

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

  48. 48.

    , , , & Retroviral pseudotransduction for targeted cell manipulation. Mol. Cell 16, 309–315 (2004).

Download references

Acknowledgements

We thank P.J. Dempsey (University of Michigan) for Adam10fl/fl mice; I. Aifantis (New York University School of Medicine) for Notch1fl/fl mice; D. Green (SJCRH) for tamoxifen-inducible c-Myc and for critical review of the manuscript; R. Kubo (CytelCorp) for antibody H146-968 (to CD3ζ); D. Littman (New York University School of Medicine) for constitutively active Lck; J. Gray (SJCRH) for the third-generation lentiviral vector pCML20; K. Forbes and A. McKenna for the maintenance, breeding and genotyping of mouse colonies; members of the Vignali laboratory for assistance with bone marrow collection; and R. Cross, S. Morgan and G. Lennon of the Department of Immunology Flow Lab (SJCRH) for cell sorting; the staff of the Shared Animal Resource Center (SJCRH) for animal husbandry; and the Hartwell Center for Biotechnology and Bioinformatics (SJCRH) for the synthesis of primers and probes for real-time PCR. Images were acquired at the Cell & Tissue Imaging Center (supported by SJCRH and the National Cancer Institute (P30 CA021765)) and the Department of Immunology Imaging Facility (both at SJCRH). Supported by the US National Institutes of Health (R01 AI052199 to D.A.A.V.), the St. Jude National Cancer Institute Comprehensive Cancer Center (CA21765 to D.A.A.V.) and the American Lebanese Syrian Associated Charities (D.A.A.V.).

Author information

Affiliations

  1. Department of Immunology, St. Jude Children's Research Hospital (SJCRH), Memphis, Tennessee, USA.

    • Clifford S Guy
    • , Kate M Vignali
    • , Matthew L Bettini
    • , Abigail E Overacre
    •  & Dario A A Vignali
  2. Cell and Tissue Imaging Shared Resource, SJCRH, Memphis, Tennessee, USA.

    • Jamshid Temirov
  3. Department of Biostatistics, SJCRH, Memphis, Tennessee, USA.

    • Matthew Smeltzer
    •  & Hui Zhang
  4. Center for Pathophysiology, Infectiology and Immunology Institute for Hygiene and Applied Immunology, Immune Recognition Unit, Medical University of Vienna, Austria.

    • Johannes B Huppa
  5. Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan, USA.

    • Yu-Hwai Tsai
    •  & Peter J Dempsey
  6. Howard Hughes Medical Institute, Department of Pathology and NYU Cancer Institute, New York University School of Medicine, New York, New York, USA.

    • Camille Lobry
    •  & Iannis Aifantis
  7. Howard Hughes Medical Institute, Department of Microbiology and Immunology, Stanford School of Medicine, Palo Alto, California, USA.

    • Jianming Xie
    •  & Mark M Davis
  8. Mayo Clinic Cancer Center, Department of Cancer Biology, Jacksonville, Florida, USA.

    • Howard C Crawford

Authors

  1. Search for Clifford S Guy in:

  2. Search for Kate M Vignali in:

  3. Search for Jamshid Temirov in:

  4. Search for Matthew L Bettini in:

  5. Search for Abigail E Overacre in:

  6. Search for Matthew Smeltzer in:

  7. Search for Hui Zhang in:

  8. Search for Johannes B Huppa in:

  9. Search for Yu-Hwai Tsai in:

  10. Search for Camille Lobry in:

  11. Search for Jianming Xie in:

  12. Search for Peter J Dempsey in:

  13. Search for Howard C Crawford in:

  14. Search for Iannis Aifantis in:

  15. Search for Mark M Davis in:

  16. Search for Dario A A Vignali in:

Contributions

C.S.G. designed and did most of the experiments, and wrote the manuscript; K.M.V. generated DNA constructs; J.T. and A.E.O. assisted with microscopic analyses; M.S. and H.Z. did biostatistical analyses; M.L.B., Y.-H.T., C.L. and J.X. generated and/or provided mice; J.B.H., P.J.D., H.C.C., I.A. and M.M.D. provided technical advice and assistance; D.A.A.V. conceived of the project, directed the research and wrote the manuscript; and all authors edited and approved the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Dario A A Vignali.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7

Videos

  1. 1.

    Supplementary Video 1

    Supplementary Movie 1. Live Imaging analysis of CD3 10 ITAM T cells stimulated by planar lipid bilayers containing anti-TCRβ antibodies and ICAM-1.

  2. 2.

    Supplementary Video 2

    Supplementary Movie 2. Live Imaging analysis of CD3 4 ITAM T cells stimulated by planar lipid bilayers containing anti-TCRβ antibodies and ICAM-1.

  3. 3.

    Supplementary Video 3

    Supplementary Movie 3. Live Imaging analysis of CD3 2 ITAM T cells stimulated by planar lipid bilayers containing anti-TCRβ antibodies and ICAM-1.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/ni.2538

Further reading