Constitutive and Ligand-Induced TCR Degradation

Essen, Marina Rode von; Bonefeld, Charlotte M.; Siersma, Volkert; Rasmussen, Anette Bødker; Lauritsen, Jens Peter H.; Nielsen, Bodil; Geisler, Carsten

doi:10.4049/jimmunol.173.1.384

Cited by 51 publications

(55 citation statements)

References 61 publications

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“…Note that CD3 degradation and TCR downmodulation both increase in proportion to Ag dose (Fig. 3, com-pare B and C), in agreement with recent work (46).…”

Section: Analysis Of the Fate Of Cd3 During Tcr Internalizationsupporting

confidence: 91%

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Selective Defect in Antigen-Induced TCR Internalization at the Immune Synapse of CD8 T Cells Bearing the ZAP-70(Y292F) Mutation

Davanture

Leignadier

Milani

et al. 2005

The Journal of Immunology

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Cbl proteins have been implicated in ligand-induced TCR/CD3 down-modulation, but underlying mechanisms are unclear. We analyzed the effect of mutation of a cbl-binding site on ZAP-70 (ZAP-Y292F) on dynamics, internalization, and degradation of the TCR/CD3 complex in response to distinct stimuli. Naive CD8 T cells expressing the P14 transgenic TCR from ZAP-Y292F mice were selectively affected in TCR/CD3 down-modulation in response to antigenic stimulation, whereas neither anti-CD3 Ab-, and PMA-induced TCR down-modulation, nor constitutive receptor endocytosis/cycling were impaired. We further established that the defect in TCR/CD3 down-modulation in response to Ag was paralleled by an impaired TCR/CD3 internalization and CD3ζ degradation. Analysis of T/APC conjugates revealed that delayed redistribution of TCR at the T/APC contact zone was paralleled by a delay in TCR internalization in the synaptic zone in ZAP-Y292F compared with ZAP-wild-type T cells. Cbl recruitment to the synapse was also retarded in ZAP-Y292F T cells, although F-actin and LFA-1 redistribution was similar for both cell types. This study identifies a step involving ZAP-70/cbl interaction that is critical for rapid internalization of the TCR/CD3 complex at the CD8 T cell/APC synapse.

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“…Note that CD3 degradation and TCR downmodulation both increase in proportion to Ag dose (Fig. 3, com-pare B and C), in agreement with recent work (46).…”

Section: Analysis Of the Fate Of Cd3 During Tcr Internalizationsupporting

confidence: 91%

“…Our approach involved first biotinylation of cell surface proteins with SSbiotin to discriminate between surface and internalized TCR (45,46). However, this treatment prevented subsequent Ag-induced P14-TCR down-modulation and T cell activation as measured by CD69 expression (results not shown).…”

Section: Measurement Of Internalized Tcrmentioning

confidence: 99%

Selective Defect in Antigen-Induced TCR Internalization at the Immune Synapse of CD8 T Cells Bearing the ZAP-70(Y292F) Mutation

Davanture

Leignadier

Milani

et al. 2005

The Journal of Immunology

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“…Because TCR recycling was not considered in this model, the rate constants for TCR internalization, B and T , are also the rate constants for TCR degradation. Thus, the B and T values used here were based on published rate constants for TCR internalization (34)(35)(36)(37). Labeled TCRs that reach the edge of the computational region (corresponding to the entire immunological synapse) revert to their original, unlabeled state.…”

Section: Methodsmentioning

confidence: 99%

T cell receptor binding kinetics required for T cell activation depend on the density of cognate ligand on the antigen-presenting cell

González

Carreño

Coombs

et al. 2005

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

148

123

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CD8 ؉ T cells recognize peptides of eight to nine amino acid residues long in the context of MHC class I molecules on the surface of antigen-presenting cells (APCs). This recognition event is highly sensitive, as evidenced by the fact that T cells can be activated by cognate peptide͞MHC complex (pMHC) at extremely low densities (1-50 molecules). High sensitivity is particularly valuable for detection of antigens at low density, such as those derived from tumor cells and intracellular pathogens, which can down-modulate cognate pMHCs from the surface of APCs to evade recognition by the adaptive immune system. T cell activation is only triggered in response to interactions between the T cell receptor (TCR) and the pMHC ligand that reach a specific half-life threshold. However, interactions with excessively long half-lives result in impaired T cell activation. Thus, efficient T cell activation by pMHC on the surface of APCs requires an optimal dwell time of TCR-pMHC interaction. Here, we show that, although this is a requirement at low cognate pMHC density on the APC surface, at high epitope density there is no impairment of T cell activation by extended TCR-pMHC dwell times. This observation was predicted by mathematical simulations for T cell activation by pMHC at different densities and supported by experiments performed on APCs selected for varied expression of cognate pMHC. According to these results, effective T cell activation depends on a complex interplay between inherent TCR-pMHC binding kinetics and the epitope density on the APC.

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“…Prior studies (2,3,18,19) indicated that internalized TcR/ CD3 in stimulated cells is targeted for degradation; therefore, we examined colocalization of the reporter or CD3ζ-eGFP with lysosomes using immunofluorescent staining with the lysosomal markers LAMP1 or LysoTracker Red. In unstimulated cells, only a fraction of internalized CD3ζ colocalized with fluorescent lysosomes (Fig.…”

Section: Intracellular Organization Of Cd3ζmentioning

confidence: 99%

“…The dually phosphorylated ITAMs serve as a binding platform for molecules involved in TcR/CD3-proximal signaling, most importantly the tyrosine kinase ZAP-70, which phosphorylates downstream targets (1). Activation-dependent tyrosine phosphorylation of the ITAM-containing chains of the CD3 complex leads to TcR/CD3 internalization and, eventually, degradation in lysosomes (2,3).…”

mentioning

confidence: 99%

Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3ζ in the endosomal compartment

Yudushkin

Vale²

2010

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

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Phosphorylation of the T-cell receptor complex (TcR/CD3) mediates the survival and antigen-induced activation of T cells. TcR/CD3 phosphorylation is usually monitored using phospho-specific antibodies, which precludes dynamic measurements. Here, we have developed genetically encoded, live-cell reporters that enable simultaneous monitoring of the phosphorylation state and intracellular trafficking of CD3ζ, the major signal-transducing subunit of the TcR/CD3. We show that these reporters provide accurate readouts of TcR/CD3 phosphorylation and are sensitive to the local balance of kinase and phosphatase activities acting upon TcR/CD3. Using these reporters, we demonstrate that, in addition to the expected activation-dependent phosphorylation at the plasma membrane, tyrosine-phosphorylated CD3ζ accumulates on endosomal vesicles distinct from lysosomes. These results suggest that an intracellular pool of phosphorylated CD3ζ may help to sustain TcR/ CD3 signaling after the receptor internalization. Recent studies using high-resolution imaging have demonstrated that signaling via TcR/CD3 displays complex spatial organization. TcR/CD3 molecules are preclustered at the plasma membrane and, upon ligation, nucleate downstream signaling components seconds after T-cell activation (4, 5). TcR/CD3 is constitutively internalized and recycled to the plasma membrane in naïve and activated T cells (reviewed in ref. 6), but the function of this turnover as well as the phosphorylation state of internalized receptor remain unknown.Currently, phosphorylation of TcR/CD3 in cells and in vitro is monitored using phospho-specific antibodies, and there are no methods that enable dynamic monitoring of the spatial organization of CD3 phosphorylation in live cells. Here, we have constructed genetically encoded fluorescent reporters compatible with imaging of live and fixed cells and demonstrate that they accurately monitor the dynamics and intracellular organization of CD3ζ phosphorylation in Jurkat T cells. Using the reporters, we observed that in addition to the expected activationdependent phosphorylation at the plasma membrane, tyrosinephosphorylated CD3ζ accumulated in the perinuclear endosomal vesicles. Our results demonstrate that endosomal CD3ζ remains signaling-competent and suggest the possibility that internalized CD3ζ pool may help to sustain long-term signaling in T cells. Results Design and Characterization of the CD3ζ Phosphorylation Reporters.To generate a Förster's resonant energy transfer (FRET)-based monomolecular reporter for phosphorylation of the key CD3 signal-transducing subunit ζ, we fused the C terminus of CD3ζ to a pair of green and red fluorescent proteins, eGFP and mCherry, linked by a flexible spacer and followed by the tandem SH2 domains of human ZAP-70 (residues 1-259; tSH2 ). We reasoned that intramolecular binding of the SH2 domains to tyrosine-phosphorylated ITAMs of CD3ζ (7) would result in conformational rearrangement of the adjacent fluorescent proteins and change in the FRET efficiency between the dono...

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Constitutive and Ligand-Induced TCR Degradation

Cited by 51 publications

References 61 publications

Selective Defect in Antigen-Induced TCR Internalization at the Immune Synapse of CD8 T Cells Bearing the ZAP-70(Y292F) Mutation

Selective Defect in Antigen-Induced TCR Internalization at the Immune Synapse of CD8 T Cells Bearing the ZAP-70(Y292F) Mutation

T cell receptor binding kinetics required for T cell activation depend on the density of cognate ligand on the antigen-presenting cell

Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3ζ in the endosomal compartment

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