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


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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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




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.

Corresponding author

Correspondence to Dario A A Vignali.

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The authors declare no competing financial interests.

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).

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