ITAM-mediated Signaling by the T-Cell Antigen Receptor

Love, Paul E.; Hayes, Sandra M.

doi:10.1101/cshperspect.a002485

Cited by 164 publications

(160 citation statements)

References 75 publications

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“…The TCR has to be able to interpret small differences in the chemical composition of the peptide Ag bound to MHC as quantitatively and qualitatively different signaling outcomes, although the mechanism underlying this process remains poorly understood. The most prevalent, simplistic model proposes two types of cytoplasmic tyrosine kinases as the sole direct effectors of the TCR: Lck and ZAP70 (2). However, direct recruitment of other proteins to the CD3 subunits of the TCR has also been described (2)(3)(4)(5)(6), suggesting that the diversity of signaling outcomes emanating from the TCR may be modulated by the composition of the "TCR signalosome."…”

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confidence: 99%

“…The most prevalent, simplistic model proposes two types of cytoplasmic tyrosine kinases as the sole direct effectors of the TCR: Lck and ZAP70 (2). However, direct recruitment of other proteins to the CD3 subunits of the TCR has also been described (2)(3)(4)(5)(6), suggesting that the diversity of signaling outcomes emanating from the TCR may be modulated by the composition of the "TCR signalosome." Thus, these mechanisms may involve the recruitment and activation of different cytoplasmic and membrane effectors to the TCR.…”

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confidence: 99%

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Relevance of Nck–CD3ε Interaction for T Cell Activation In Vivo

Borroto

Arellano

Blanco

et al. 2014

The Journal of Immunology

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On TCR ligation, the adaptor Nck is recruited through its src homology 3.1 domain to a proline-rich sequence (PRS) in CD3ε. We have studied the relevance of this interaction for T cell activation in vitro and in vivo by targeting the interaction sites in both partners. The first approach consisted of studying a knockin (KI) mouse line (KI-PRS) bearing a conservative mutation in the PRS that makes the TCR incompetent to recruit Nck. This deficiency prevents T cell activation by Ag in vitro and inhibited very early TCR signaling events including the tyrosine phosphorylation of CD3ζ. Most important, KI-PRS mice are partly protected against the development of neurological symptoms in an experimental autoimmune encephalitis model, and show a deficient antitumoral response after vaccination. The second approach consisted of using a high-affinity peptide that specifically binds the src homology 3.1 domain and prevents the interaction of Nck with CD3ε. This peptide inhibits T cell proliferation in vitro and in vivo. These data suggest that Nck recruitment to the TCR is fundamental to mount an efficient T cell response in vivo, and that the Nck–CD3ε interaction may represent a target for pharmacological modulation of the immune response.

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confidence: 99%

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Relevance of Nck–CD3ε Interaction for T Cell Activation In Vivo

Borroto

Arellano

Blanco

et al. 2014

The Journal of Immunology

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“…The TCR has to be able to interpret small differences in the chemical composition of the peptide Ag bound to MHC as quantitatively and qualitatively different signaling outcomes, although the mechanism underlying this process remains poorly understood. The most prevalent, simplistic model proposes two types of cytoplasmic tyrosine kinases as the sole direct effectors of the TCR: Lck and ZAP70 (2). However, direct recruitment of other proteins to the CD3 subunits of the TCR has also been described (2)(3)(4)(5), suggesting that the diversity of signaling outcomes emanating from the TCR may be modulated by the composition of the "TCR signalosome."…”

mentioning

confidence: 99%

Nck Recruitment to the TCR Required for ZAP70 Activation during Thymic Development

Borroto

Arellano

Dopfer

et al. 2013

The Journal of Immunology

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The adaptor protein Nck is inducibly recruited through its SH3.1 domain to a proline-rich sequence (PRS) in CD3ε after TCR engagement. However, experiments with a knockin mutant bearing an 8-aa replacement of the PRS have indicated that Nck binding to the TCR is constitutive, and that it promotes the degradation of the TCR in preselection double-positive (DP) CD4+CD8+ thymocytes. To clarify these discrepancies, we have generated a new knockin mouse line (KI-PRS) bearing a conservative mutation in the PRS resulting from the replacement of the two central prolines. Thymocytes of KI-PRS mice are partly arrested at each step at which pre-TCR or TCR signaling is required. The mutation prevents the trigger-dependent inducible recruitment of endogenous Nck to the TCR but does not impair TCR degradation. However, KI-PRS preselection DP thymocytes show impaired tyrosine phosphorylation of CD3ζ, as well as impaired recruitment of ZAP70 to the TCR and impaired ZAP70 activation. Our results indicate that Nck is recruited to the TCR in an inducible manner in DP thymocytes, and that this recruitment is required for the activation of early TCR-dependent events. Differences in the extent of PRS mutation could explain the phenotypic differences in both knockin mice.

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“…We observed considerable cell-to-cell variation in Ca 2+ dynamics and magnitudes, where responding cells generally assumed peak-plateau-type profiles, elevating within minutes of engagement and returning to baseline at the end of an hour. Similarly, we demonstrated the feasibility of assessing early molecular events such as the phosphorylation states of signaling molecules, whose number and dynamics govern downstream processes (20)(21)(22)(23). As a representative example, we focused on the phosphorylation of extracellular signal-regulated kinase (ppERK), a key player of immunoreceptor signaling pathways mediating a variety of developmental and functional responses (20,22), and measured its level 10 min after tumor cell contact (SI Appendix, Fig.…”

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confidence: 99%

Longitudinal multiparameter assay of lymphocyte interactions from onset by microfluidic cell pairing and culture

Dura

Servos

Barry

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

104

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Resolving how the early signaling events initiated by cell-cell interactions are transduced into diverse functional outcomes necessitates correlated measurements at various stages. Typical approaches that rely on bulk cocultures and population-wide correlations, however, only reveal these relationships broadly at the population level, not within each individual cell. Here, we present a microfluidics-based cell-cell interaction assay that enables longitudinal investigation of lymphocyte interactions at the single-cell level through microfluidic cell pairing, on-chip culture, and multiparameter assays, and allows recovery of desired cell pairs by micromanipulation for off-chip culture and analyses. Well-defined initiation of interactions enables probing cellular responses from the very onset, permitting singlecell correlation analyses between early signaling dynamics and later-stage functional outcomes within same cells. We demonstrate the utility of this microfluidic assay with natural killer cells interacting with tumor cells, and our findings suggest a possible role for the strength of early calcium signaling in selective coordination of subsequent cytotoxicity and IFN-gamma production. Collectively, our experiments demonstrate that this new approach is well-suited for resolving the relationships between complex immune responses within each individual cell.microfluidics | cell pairing | single-cell analysis | cell-cell interactions | multiparameter assay I nitiation and progression of immune responses require direct cell-cell interactions. Molecular interactions at the contact interface mediate cellular cross-talk and trigger a series of wellorchestrated downstream signaling events. The magnitude and dynamics of these signals promote and coordinate immune cell activation, and underlie a broad array of functional outcomes, such as target cell elimination, secretory activity, and proliferation (1, 2). Understanding how these early signaling events are transduced into functional responses demands correlated measurements at different stages as these responses unfold.Typically, these relationships are probed by first activating cells in bulk cocultures, often mixing cell populations and initiating contacts via a brief centrifugal cosedimentation, and then piecing together measurements from independent assays performed at different time points. This approach may determine broad outcomes of intercellular interactions, but it suffers from three major limitations. First, individual assay measurements are averaged over many different combinations of interactions due to the uncontrolled and indefinite nature of cell-cell interactions in bulk cocultures. For example, varying times of initiation and duration of contacts (1, 3), number of interacting partners (4), and differences in antigen presenting cells (5) can all modulate immune cell responses. Unpredictable variation is therefore introduced into measured responses, masking the true intrinsic cellular heterogeneity and blurring the correlations. Second, conventional as...

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ITAM-mediated Signaling by the T-Cell Antigen Receptor

Cited by 164 publications

References 75 publications

Relevance of Nck–CD3ε Interaction for T Cell Activation In Vivo

Relevance of Nck–CD3ε Interaction for T Cell Activation In Vivo

Nck Recruitment to the TCR Required for ZAP70 Activation during Thymic Development

Longitudinal multiparameter assay of lymphocyte interactions from onset by microfluidic cell pairing and culture

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