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Distinct TCR signaling pathways drive proliferation and cytokine production in T cells

Nature Immunology volume 14, pages 262–270 (2013)Cite this article

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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.

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Figure 1: Ligation of complexes with low CD3 ITAM multiplicity fails to induce c-Myc expression.
Figure 2: Components of the Notch pathway are necessary for T cell proliferative responses but not cytokine responses.
Figure 3: Initiation of canonical TCR signaling events after ligation of TCR-CD3 complexes with low ITAM multiplicity.
Figure 4: Maturation of the immunological synapse requires high TCR–CD3 ITAM multiplicity.
Figure 5: Components of the Notch1-activation pathway coincide with formation of the immunological synapse and show diminished intermolecular interactions with lower ITAM multiplicity.
Figure 6: Low ITAM multiplicity results in less recruitment of Vav1 to the CD3 complex.
Figure 7: Stimulation with weak agonists results in defective induction of c-Myc expression.

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References

  1. Wucherpfennig, K.W., Gagnon, E., Call, M.J., Huseby, E.S. & Call, M.E. 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).

    PubMed  PubMed Central  Google Scholar 

  2. Chan, A.C., Iwashima, M., Turck, C.W. & Weiss, A. ZAP-70: a 70 kd protein-tyrosine kinase that associates with the TCR zeta chain. Cell 71, 649–662 (1992).

    CAS  PubMed  Google Scholar 

  3. Finco, T.S., Yablonski, D., Lin, J. & Weiss, A. The adapter proteins LAT and SLP-76 are required for T-cell activation. Cold Spring Harb. Symp. Quant. Biol. 64, 265–274 (1999).

    CAS  PubMed  Google Scholar 

  4. Guy, C.S. & Vignali, D.A. Organization of proximal signal initiation at the TCR:CD3 complex. Immunol. Rev. 232, 7–21 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  6. 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).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 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  PubMed  PubMed Central  Google Scholar 

  9. 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  PubMed  PubMed Central  Google Scholar 

  10. Holst, J., Vignali, K.M., Burton, A.R. & Vignali, D.A. Rapid analysis of T-cell selection in vivo using T cell-receptor retrogenic mice. Nat. Methods 3, 191–197 (2006).

    CAS  PubMed  Google Scholar 

  11. Holst, J. 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).

    CAS  PubMed  Google Scholar 

  12. Szymczak, A.L. 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).

    CAS  PubMed  Google Scholar 

  13. Douglas, N.C., Jacobs, H., Bothwell, A.L. & Hayday, A.C. Defining the specific physiological requirements for c-Myc in T cell development. Nat. Immunol. 2, 307–315 (2001).

    CAS  PubMed  Google Scholar 

  14. Lindsten, T., June, C.H. & Thompson, C.B. Multiple mechanisms regulate c-myc gene expression during normal T cell activation. EMBO J. 7, 2787–2794 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  16. Ong, C.T., Sedy, J.R., Murphy, K.M. & Kopan, R. Notch and presenilin regulate cellular expansion and cytokine secretion but cannot instruct Th1/Th2 fate acquisition. PLoS ONE 3, e2823 (2008).

    PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  19. Minter, L.M. 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).

    CAS  PubMed  Google Scholar 

  20. Rebay, I. 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).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  22. Luty, W.H., Rodeberg, D., Parness, J. & Vyas, Y.M. Antiparallel segregation of notch components in the immunological synapse directs reciprocal signaling in allogeneic Th:DC conjugates. J. Immunol. 179, 819–829 (2007).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  24. Delwig, A. & Rand, M.D. Kuz and TACE can activate Notch independent of ligand. Cell. Mol. Life Sci. 65, 2232–2243 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Bates, M., Huang, B., Dempsey, G.T. & Zhuang, X. Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317, 1749–1753 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Fischer, K.D., Tedford, K. & Penninger, J.M. Vav links antigen-receptor signaling to the actin cytoskeleton. Semin. Immunol. 10, 317–327 (1998).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  29. James, J.R. & Vale, R.D. Biophysical mechanism of T-cell receptor triggering in a reconstituted system. Nature 487, 64–69 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    PubMed  PubMed Central  Google Scholar 

  31. Schmitter, T. 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).

    CAS  PubMed  Google Scholar 

  32. Gil, D., Schamel, W.W., Montoya, M., Sanchez-Madrid, F. & Alarcon, B. 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).

    CAS  PubMed  Google Scholar 

  33. Lettau, M. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Rogers, P.R., Grey, H.M. & Croft, M. 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).

    CAS  PubMed  Google Scholar 

  35. Radtke, F., Fasnacht, N. & Macdonald, H.R. Notch signaling in the immune system. Immunity 32, 14–27 (2010).

    CAS  PubMed  Google Scholar 

  36. Yuan, J.S., Kousis, P.C., Suliman, S., Visan, I. & Guidos, C.J. Functions of notch signaling in the immune system: consensus and controversies. Annu. Rev. Immunol. 28, 343–365 (2010).

    PubMed  Google Scholar 

  37. Joshi, I. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  40. Kopan, R. & Ilagan, M.X. The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137, 216–233 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  42. Lillemeier, B.F. 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).

    CAS  PubMed  Google Scholar 

  43. Buday, L., Wunderlich, L. & Tamas, P. The Nck family of adapter proteins: regulators of actin cytoskeleton. Cell. Signal. 14, 723–731 (2002).

    CAS  PubMed  Google Scholar 

  44. Lubman, O.Y., Korolev, S.V. & Kopan, R. Anchoring notch genetics and biochemistry; structural analysis of the ankyrin domain sheds light on existing data. Mol. Cell 13, 619–626 (2004).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  47. Szymczak, A.L. 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).

    CAS  PubMed  Google Scholar 

  48. Galla, M., Will, E., Kraunus, J., Chen, L. & Baum, C. Retroviral pseudotransduction for targeted cell manipulation. Mol. Cell 16, 309–315 (2004).

    CAS  PubMed  Google Scholar 

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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.).

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Authors and 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. Department of Pathology and NYU Cancer Institute, Howard Hughes Medical Institute, New York University School of Medicine, New York, New York, USA

    Camille Lobry & Iannis Aifantis

  7. Department of Microbiology and Immunology, Howard Hughes Medical Institute, Stanford School of Medicine, Palo Alto, California, USA

    Jianming Xie & Mark M Davis

  8. Department of Cancer Biology, Mayo Clinic Cancer Center, Jacksonville, Florida, USA

    Howard C Crawford

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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.

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Correspondence to Dario A A Vignali.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 2258 kb)

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. (AVI 15161 kb)

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. (AVI 26129 kb)

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. (AVI 13606 kb)

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Guy, C., Vignali, K., Temirov, J. et al. Distinct TCR signaling pathways drive proliferation and cytokine production in T cells. Nat Immunol 14, 262–270 (2013). https://doi.org/10.1038/ni.2538

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