Critical and Multiple Roles for the CD3ε Intracytoplasmic Tail in Double Negative to Double Positive Thymocyte Differentiation

Thymic selection shapes the T cell repertoire to ensure maximal antigenic coverage against pathogens while preventing autoimmunity. Recognition of self-peptides in the context of peptide-MHC complexes by the TCR is central to this process, which remains partially understood at the molecular level. In this study we provide genetic evidence that the Nck adapter proteins are essential for thymic selection. In vivo Nck deletion resulted in a reduction of the thymic cellularity, defective positive selection of low-avidity T cells, and impaired deletion of thymocytes engaged by low-potency stimuli. Nck-deficient thymocytes were characterized by reduced ERK activation, particularly pronounced in mature single positive thymocytes. Taken together, our findings identify a crucial role for the Nck adapters in enhancing TCR signal strength, thereby fine-tuning the threshold of thymocyte selection and shaping the preimmune T cell repertoire.

Section: Discussionmentioning

confidence: 99%

“…The Nck adapters have been implicated in the regulation of the surface levels of the TCR (11,22,23). The levels of TCR expression are finely regulated at the DP stage (24), as both positive and negative selection depend on the strength of TCR signaling (25).…”

Section: Nck Deletion Results In Tcr Overexpression On Dp Thymocytesmentioning

confidence: 99%

Fine Tuning of the Threshold of T Cell Selection by the Nck Adapters

Roy

Togbé

Holdorf

et al. 2010

“…Bone marrow reconstitution with CD3ε PRS mutants in CD3ε-deficient mice, Nck overexpression in primary T cells, Nck knockout mice, and PRS knockin (KI) mice have all been used to study the role of the PRS and Nck in T cell development and mature T cell activation (14)(15)(16)(17)(18)(19)(20). Some experiments have suggested that the PRS is important for thymic maturation, but not for mature T cell activation in vitro (15), although experiments with bone marrow chimeras indicate that the PRS is important to activate mature T cells by weak, but not by strong, agonists in vitro (19).…”

mentioning

confidence: 99%

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

Borroto

Arellano

Blanco

et al. 2014

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.

“…Bone marrow reconstitution with CD3ε PRS mutants in CD3ε-deficient mice, Nck overexpression in primary T cells, Nck knockout mice, and PRS knockin mice have all been used to study the role of PRS and Nck in T cell development (9)(10)(11)(12)(13)(14)(15). Some experiments have suggested that the PRS is important for thymic maturation, but not for mature T cell activation in vitro (10), although experiments with bone marrow chimeras indicate that the PRS is important to activate mature T cells by weak but not by strong agonists in vitro (14).…”

mentioning

confidence: 99%

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

Borroto

Arellano

Dopfer

et al. 2013

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.