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The Journal of Immunology, 2005, 175: 3140-3149.
Copyright © 2005 by The American Association of Immunologists

Selective Defect in Antigen-Induced TCR Internalization at the Immune Synapse of CD8 T Cells Bearing the ZAP-70(Y292F) Mutation1

Suzel Davanture2,*, Julie Leignadier2,*, Pascale Milani*, Philippe Soubeyran{dagger}, Bernard Malissen*, Marie Malissen*, Anne-Marie Schmitt-Verhulst* and Claude Boyer3,*

* Centre d’Immunologie de Marseille-Luminy, Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/Université de la Méditerranée, and {dagger} Institut National de la Santé et de la Recherche Médicale, Unité 624, Stress Cellulaire, Parc Scientifique de Luminy, Marseille, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 CD3{zeta} 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The Ag-specific TCR, TCR{alpha}{beta} is expressed at the cell surface when associated with the CD3 complex composed of the CD3{gamma}/CD3{epsilon}, CD3{delta}/CD3{epsilon}, and CD3{zeta}-{zeta} dimers. Its level of surface expression is finely tuned by mechanisms controlling its assembly, membrane dynamics, and degradation, which vary along T cell development and as a consequence of T cell activation. In particular, T cell stimulation by APC presenting the cognate peptide/MHC complex leads to a rapid decrease in the level of surface-expressed TCR. This phenomenon, known as TCR/CD3 down-modulation, occurs mainly by internalization of the whole receptor complex through endosomal pathways (1, 2), although mechanisms invoking exocytosis of vesicles containing the receptor have also been described (3).

Ag-induced TCR down-modulation is intimately linked to TCR signaling and, in particular, to protein tyrosine kinase (PTK)4 activation, including Src family PTKs p56lck and p59fyn. These kinases phosphorylate tyrosines in the ITAM motifs of CD3 cytoplasmic domains, allowing for binding of the ZAP-70 PTK via its SH2 domains. Subsequent phosphorylation of adaptor molecules and protein-protein interactions further transduce positive and negative signals for functional activation of the T cells (4). In this context, TCR/CD3 down-modulation has been suggested to be one of the mechanisms that contribute to the arrest of signaling initiated by Ag. Alternatively, it could be reasoned that the failure to disengage peptide/MHC complexes by receptor clearance would prevent the serial engagement of new receptors that is required for optimal T cell stimulation by the limiting amount of peptide/MHC complexes available on the APCs (4, 5).

Approaches at unraveling the complexities of TCR dynamics and signaling have included the analysis of T cells expressing TCR/CD3 complexes modified at potential internalization motifs, or in components of the receptor endocytosis/degradation pathway. In particular, each CD3 cytoplasmic domain contains tyrosine-based motifs in their ITAMs that are able to bind to the µ2 chain of the clathrin adaptor AP2 (6). Furthermore, the CD3{gamma} and CD3{delta} chains each present a di-leucine motif (7) capable of binding the {beta}1 subunit of the AP2 complex (8). The CD3{gamma} chain di-leucine motif appears necessary for TCR/CD3 constitutive cycling (9), whereas the proximal serine-126 on CD3{gamma} is additionally required for phorbol ester-induced TCR/CD3 internalization (10), a protein kinase C (PKC)-dependent event that involves receptor sequestration without subsequent degradation (11). For mAb-induced TCR/CD3 down-regulation, a process dependent upon tyrosine kinase activity (12, 13, 14), but not PKC, absence of both CD3{gamma} and CD3{delta} cytoplasmic domains led to deficient receptor down-modulation, suggesting that upon T cell activation, unveiling of the di-leucine and tyrosine-based motifs present in each of these chains may be involved (1). However, Ag-induced TCR down-modulation was not affected by the combined truncations of CD3{gamma} and CD3{delta} cytoplasmic domains (15), suggesting that a distinct mechanism involving ZAP-70 activity (14), was rate limiting in this process.

The particularity of Ag-induced TCR down-modulation is that it occurs upon engagement of the TCR by peptide/MHC complexes within the immune synapse (IS) formed at the T cell/APC contact zone. This specialized structure, associated with a redistribution of membrane receptors, develops over a 5–30 min period. It subdivides the T cell/APC interface into supramolecular activation clusters (SMACs) that are spatially organized into a central (c-SMAC) region where TCR-MHC/peptide and CD28-CD80 interactions take place, and a peripheral (p-SMAC) area enriched in cytoskeletal components and adhesion molecules such as LFA-1 (16, 17), and CD2 (18). However, the rapid induction of PTK activation upon Ag-induced T cell stimulation precedes the redistribution of the TCR/CD3 complex in the c-SMAC (19, 20, 21). The observation that surface TCR disappeared from the c-SMAC may signify its localized internalization (21).

Tyrosine-292 in ZAP-70, when phosphorylated upon TCR activation, is a binding site for c-cbl and cbl-b via the cbl tyrosine kinase binding domain (22, 23, 24). Contrasting results were reported for thymocytes of mice expressing an altered c-cbl mutated in its tyrosine kinase binding domain, because this led to constitutive activation of rac without affecting ZAP-70 phosphorylation (25). However, the effect of this mutation could not be studied in peripheral T cells, where mostly cbl-b is expressed.

Adaptor proteins of the Cbl-interacting protein of 85 kDa (CIN85) family have recently been shown to bind both cbl and endophilins, and thereby to regulate the endocytosis of activated receptor tyrosine kinases (RTK) (26, 27, 28, 29). Cbl is also involved in RTK monoubiquitination allowing for receptor binding to the clathrin adaptor Eps15 (28, 30, 31). In T cells, the CIN85 family member CD2-associated protein (CD2AP) has recently been implicated in the pathway of TCR/CD3 down-modulation (32, 33). Furthermore, we have recently described that T cells from mice expressing ZAP-Y292F presented hyper-tyrosine-phosphorylation in response to TCR/CD3 engagement but were partially inhibited in Ag-induced TCR down-modulation (34).

In the present work, we analyzed the mechanisms by which TCR down-modulation is impaired in CD8 T cells expressing the ZAP-Y292F mutation. Our results showed that the defect in TCR down-modulation was specific for Ag-induced TCR internalization and was associated with a delayed recruitment of TCR and cbl at the T/APC synapse zone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Knockin mice mutated at ZAP-Y292F and crossed for expression of the transgenic P14 TCR specific for the LCMV gp33/H-2Db on the C57BL/6 mice background (TCR-P14 or P14) have been described (34). Control mice were genetically similar except for the lack of the mutation and were designated as ZAP-70WT.

Ag-presenting cells

Tap-2-deficient RMA-S H-2b cells (35) were generally used as APCs, and similar results were obtained when LPS blasts from Tap-1°/° mice were used (results not shown). Before loading with peptide (LCMV gp33 (36) or control peptide HY (37)), Tap-negative cells were incubated overnight at 31°C in RPMI 1640 with 5% dialyzed FCS.

T cell preparation

The CD8 T cell population was purified (to >95%) from lymph node (LN) cells by negative selection as previously described (38).

Abs and reagents for immunofluorescence

mAbs B20.1 (anti-V{alpha}2-TCR; Ref.39), H-57.101.2 (anti-TCR{beta} (H57; Ref.40)), H146.968 (anti-CD3{zeta} chain; Ref.41), as well as anti-CD3{epsilon} mAbs 145.2C11 (42) and KT3 (43) were used for TCR/CD3 analysis. Anti-LFA-1 mAb was H35-89-9 (44). Ganglioside GM1 for raft localization was visualized with the cholera toxin {beta}-chain (CTX{beta}) coupled to FITC (Sigma-Aldrich), or Alexa488 (Molecular Probes). F-actin was visualized using phalloidin Alexa488 (Molecular Probes). Rabbit anti-cbl Ring domain was prepared as described (26), and revealed with anti-rabbit Ig Zenon A546 (Molecular Probes). The specificity of the Ab for cbl was further established in T cell lysates, because only the cbl band (at 120 kDa) was detected by immunoblot (results not shown).

Fluorescence analysis

Measurement of surface fluorescence and FACS analysis were as described using mean fluorescence intensity (MFI) values (15). TCR down-modulation was calculated as (1 – MFI experimental/MFI medium) x 100. For intracellular CD3{zeta} staining, cells were washed (4 min; 4°C) in PBS before 5-min fixation with 2% paraformaldehyde (PAF) at room temperature (RT), washed again with PBS, and permeabilized for 10 min at RT with 0.5% saponin in PBS, 2% FCS, and 0.02% NaN3 containing 1 mM vanadate (saponin buffer). Biotinylated mAb H146.968 was diluted in the same saponin buffer and added for 30 min at 4°C. After permeabilization, cells were washed and centrifuged at 2000 rpm for 5 min at 4°C. For calibration, we set up optimal mAb dilutions to obtain a linear decrease in labeling in the presence of increasing amounts of the CD3 peptide recognized by the mAb. CD3{zeta} degradation was calculated as follows: (1 – MFI experimental/MFI medium) x 100.

Measurement of internalized TCR

Our approach involved first biotinylation of cell surface proteins with SS-biotin 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). We then chose to prelabel T cells with anti-TCR mAb at 4°C, before Ag stimulation at 37°C, followed by acid treatment to release mAb remaining at the cell surface (as described in Ref.2). The anti-TCRV{alpha}2 B20.1 mAb was unsuitable for this approach because it rapidly dissociates from the TCR. Therefore anti-TCR{beta} (H57), which stably binds the TCR, was used for this study, as follows. T cells were incubated at 4°C with anti-TCR{beta} (H57) mAb A488 (Molecular Probes kit) before Ag stimulation at 37°C. At different times, samples were divided, and one-half was directly used to measure total fluorescence, the other half was treated with an acid solution (0.15 M acetic acid, 0.15 M NaCl) for 5 min on ice before two washes in medium and measurement of remaining fluorescence corresponding to internalized mAb (2). Prelabeling of T cells with anti-TCR{beta} (H57) did not per se activate the P14 T cells and did not prevent subsequent Ag-induced TCR down-modulation and signaling for CD69 expression (see Fig. 2).



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  		FIGURE 2. The ZAP-Y292F mutation affects the rate of Ag-induced TCR internalization. Purified CD8 LN T cells from P14 ZAP-WT or ZAP-Y292F mice were preincubated with anti-TCR{beta} (H57-A488) mAb at 4°C, washed, and cultured in different conditions. At the indicated times, anti-TCR{beta} MFI was measured either directly on half the sample (NT), or after acid treatment (AT) on the other half. A, NT 90-min medium 37°C (thin solid line), AT 90-min medium 0°C (dashed line), AT 90-min medium 37°C (dotted line), and AT 90-min APC/gp33 10–7 M 37°C (bold solid line). Numbers represent anti-TCR{beta}-A488 MFI, italic for cells maintained at 0°C for 90 min. B, Percentage of internalized TCR (AT/NT x 100) for ZAP-WT ({diamondsuit}, {blacksquare}, •, {blacktriangleup}; solid line) and ZAP-Y292F ({diamond}, {square}, {circ}, {triangleup}; dashed line) CD8 T cells. The mean values for three independent experiments are reported with SD, shown only for Ag-stimulated samples. For all other samples, SDs were <7%, and values were similar for ZAP-WT and ZAP-Y292F T cells. Of note: TCR down-modulation induced by 10–6 M gp33 was, respectively, 64.9 ± 3.3 and 59.3 ± 2.7% for ZAP-WT T cells preincubated or not with mAb H57, and 46.0 ± 7.0 and 32.5 ± 6% for ZAP-Y292F T cells preincubated or not with mAb H57, and was <20% for both ZAP-WT and ZAP-Y292F T cells preincubated with mAb H57 in the presence of irrelevant HY peptide. Prelabeling with mAb H57 also did not induce the activation marker CD69 (MFI, <10) in T cells incubated with irrelevant peptide, and did not prevent induction of the activation marker in the presence of gp33 (MFI = 70 at 90 min).

 
Confocal analysis

Glass plates were treated with 1% poly-lysine (Sigma-Aldrich). For some experiments, APC were labeled the day before the experiment with Cy-5 Mono NHS Ester (Cy5; Amersham Biosciences) as described (21). Briefly, PBS-washed cells (107 cell/ml) were labeled for 15 min on ice with 10 µg/ml Cy5 before blocking the reaction with FCS and two washes with RPMI 1640/FCS, and overnight incubation at 31°C in medium with dialyzed FCS. This treatment does not interfere with a subsequent Ag stimulation (not shown), as described in the preceding section. APC were loaded with peptide just before the experiment. For some experiments, purified CD8 T cells were prelabeled at 4°C with anti-TCR{beta} (H57) coupled to Alexa A546 (Molecular Probes). T/APC conjugates were prepared in tubes (RPMI 1640 containing 0.5% FCS; 30-s centrifugation at 1200 rpm), and then gently poured onto a glass plate coated with poly-lysine as previously described (12). Alternatively, RPMI 1640-washed APC were first laid on the glass plate to which T cells in culture medium were subsequently added. Incubations were at 37°C for the indicated times. Cells were fixed at RT with 2% PAF in PBS, and permeabilized or not in saponin buffer (PBS containing 0.5% saponin, 2% FCS, 0.02% NaN3, 1 mM Na orthovanadate). A relevant control Ab was used for each type of labeling. For surface TCR/CD3 labeling, rat mAb KT3 was used and revealed by goat anti-rat-IgG-biotin (Chemicon International) and streptavidin A-546 (Molecular Probes). The same procedure was used to label LFA-1. Next, CTX labeling was performed on fixed cells, and F-actin on fixed and permeabilized cells, before washes and fixation in Moviol as described (1). Confocal analysis was performed on a Zeiss Axiovert 200 microscope, using Zeiss LSM 510 software. Three-dimensional deconvolution used images spaced by 0.3 µm. The three-dimensional representation used the Imaris software (Bitplane) and isosurface method.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Ag-induced TCR down-modulation is defective in ZAP-Y292F compared with ZAP-wild-type (WT) CD8 T cells

Our previous results indicated that TCR down-modulation in response to antigenic stimulation was partially inhibited in CD8 T cells from mice expressing ZAP-Y292F molecules (34). We first confirmed this finding in a kinetic experiment shown in Fig. 1. As seen in Fig. 1A, exposure of ZAP-WT CD8 T cells to APC loaded with the gp33 LCMV peptide at 10–7 M decreased the level of surface TCR from 830 to 400 MFI units after 60 min, and to MFI 113 after 270 min. A similar treatment of the ZAP-Y292F CD8 T cells decreased the level of surface TCR from 870 to 590 after 60 min, and to 330 after 270 min. The rate of TCR down-modulation in response to gp33 10–7 M/APC by the ZAP-Y292F CD8 T cells was about half (20%/h) that observed for the ZAP-WT CD8 T cells (38%/h) (Fig. 1B). The large SDs of the 60-min time points correspond to the variability of TCR down-modulation observed from one experiment to the other at that early time, but the respective relative values for mutant to WT T cells were very reproducible in the different experiments (decreased rate of TCR down-modulation 52 ± 11%). The loading of the RMA-S used as APC was performed with gp33 concentrations between 10–7 and 10–9 M, and we observed a linear dose effect on TCR down-modulation and activation, with a differential rate of TCR down-modulation between ZAP-WT and ZAP-Y292F T cells maintained at all concentrations tested (results not shown).



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  		FIGURE 1. The ZAP-Y292F mutation affects the rate of Ag-induced, but not anti-CD3-induced TCR down-modulation. LN T cells from P14 ZAP-WT or ZAP-Y292F mice were activated for different times with APC (RMA-S loaded with 10–7 M gp33, or HY peptide), or with soluble anti-CD3 at 7 µg/ml. Gated CD8 T cells were analyzed for surface TCR level with anti-V{alpha}2 mAb. A, FACS analysis of surface TCR during stimulation with APC/HY at 270 min (thin solid line), APC/gp33 at 60 min (dashed line), and at 270 min (bold solid line). Numbers in A represent the MFI of anti-TCR-V{alpha}2 mAb. B, Kinetics of APC/gp33- or anti-CD3- induced TCR down-modulation for ZAP-WT ({diamondsuit}, •, {blacktriangleup}) or ZAP-Y292F ({diamond}, {circ}, {triangleup}) CD8 T cells. The mean value of anti-V{alpha}2 MFI and SD from four independent experiments are shown. No difference was observed for TCR levels of cells incubated in medium or with APC/HY (not shown). C, TCR down-modulation measured after 150 min as a function of the concentration of soluble anti-CD3 mAb 145.2C.11. Data represent the mean of two complete dose response experiments and were representative of more than four experiments using 1 and 7 µg/ml mAb (not shown).

 
We next analyzed the effect of this mutation on other means of inducing TCR down-modulation, such as anti-CD3 mAb or PMA stimulations (15, 47). The rate of TCR down-modulation induced by anti-CD3 mAb was the same for the ZAP-WT and ZAP-Y292F T cells, i.e., ~55%/h (Fig. 1B). Receptor down-modulation was also similar for the two types of T cells at any dose of anti-CD3 mAb used, whether soluble (Fig. 1C) or plastic-coated (results not shown). When cells were stimulated by PMA, we also observed a similar rate of TCR down-modulation for both strains (results not shown) (see Fig. 2B).

All experiments were controlled for T cell activation by measuring the induction of CD69, and we confirmed that there was no difference between the two strains in that assay (34).

In conclusion, the ZAP-Y292F mutation caused a reduced rate (40–50% decrease) of TCR down-modulation in response to antigenic stimulation compared with that observed in ZAP-WT T cells within a range of antigenic peptide from 10–7 to 10–9 M. Furthermore, this study showed that the ZAP-Y292F mutation selectively affects Ag-induced, and not anti-CD3 mAb- or PMA-induced receptor down-modulation.

Use of brefeldin A (BFA) revealed that the ZAP-Y292F mutation does not affect spontaneous TCR cycling and shows no recycling of anti-CD3 mAb- or Ag-induced TCR down-modulation in both types of CD8 T cells

Constitutive TCR endocytosis and cycling can be demonstrated by the effect on receptor surface expression of BFA, which inhibits export from the endoplasmic reticulum/Golgi and endosome recycling (45). We have examined the effect of BFA on constitutive TCR down-modulation, as well as on Ag- or anti-CD3-induced TCR down-modulation of ZAP-WT and ZAP-Y292F CD8 T cells. Results shown in Table I are expressed as the ratio of TCR down-modulation between untreated and BFA-treated T cells. As previously described for naive T cells (48), P14 CD8 T cells do not thrive when cultured unstimulated at 37°C. They showed a gradual decrease of TCR expression (~5%/h), which was slower than the rate previously described for naive CD4 T cells (45). The presence of BFA increased receptor down-modulation (15–20%/h), so that, after 2 h, the ratio of TCR down-modulation between untreated and BFA-treated T cells was of 0.34 for ZAP-WT and 0.38 for ZAP-Y292F T cells, respectively (Table I), indicating that both types of T cells have a similar level of constitutive TCR/CD3 cycling.


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  		Table I. BFA similarly affects TCR cycling in ZAP-Y292F and ZAP-WT CD8 T cells and has no effect on TCR down-modulation induced by Ag or anti-CD3 mAb

 
Ag- or anti-CD3-induced TCR down-modulation was the same, whether BFA was present or not, for both ZAP-WT and ZAP-Y292F T cells (Table I). As a control for the activity of the BFA treatment, we observed that no CD69 expression was induced when T cells were stimulated with Ag (results not shown), as predicted by the inhibitory effect of BFA on protein secretion (49). Furthermore, when cycloheximide was added to prevent de novo receptor synthesis during the incubation with or without BFA, no difference appeared for the two types of T cells either.

Together, these results suggest that Ag- and anti-CD3-induced TCR down-modulation are not affected by prevention of TCR recycling by BFA.

Ag-induced TCR internalization is affected in ZAP-Y292F compared with ZAP-WT CD8 T cells

The level of surface TCR expression is the result of an equilibrium between de novo synthesis, recycling, internalization, and degradation of its constituents. To establish which step was affected in the Ag-induced TCR/CD3 down-modulation observed for ZAP-Y292F T cells, we next determined the rate of receptor internalization in mutant and WT T cells. For this, surface receptor has to be labeled before induction of internalization. As described in Materials and Methods, we prelabeled the surface TCR with anti-TCR{beta} (H57) mAb (A488) and visualized TCR internalization either after acid treatment by FACS analysis or directly by confocal microscopy (see Fig. 4B). One can note that anti-TCR{beta} (H57) mAb binding was stable at 37°C (Fig. 2A). Furthermore, this treatment did not per se induce TCR down-modulation or T cell activation (measured by CD69 surface expression), and it did not affect Ag-induced TCR down-modulation (Fig. 2).



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  		FIGURE 4. Confocal analysis of TCR redistribution and internalization in T/APC conjugates. A, Purified CD8 T cells from ZAP-WT (left side) or ZAP-Y292F (right side) were incubated with Cy5 labeled RMA-S (blue) loaded with 10–6 M gp33 or HY (irrelevant peptide) for 15 min before CD3 labeling (red), followed by F-actin (green). xyz three-dimensional deconvolution was analyzed with the cells described on the figure. B, Purified CD8 LN T cells from P14 ZAP-WT (left side), or ZAP-Y292F (right side) mice were prelabeled at 4°C for 60 min with anti-TCR{beta} (H57)-A546 (red), and mixed with APC loaded with peptide gp33 (10–7 M) (ratio T/APC, 1:1) for conjugate formation, and incubated for 15 or 45 min, before fixation and labeling with CTX-A488 (green). Nonactivated T cells alone in medium for 45 min are shown as controls. Note that RMA-S APC were poorly labeled with CTX. Statistical analyses are represented in Table II.

 
The ratio between the MFI on acid-treated (AT; measuring internalized Ab) and untreated cells (not treated (NT); measuring total mAb at the cell surface and inside) from the same sample corresponds to the fraction of internalized TCR. After acidic treatment, the total fluorescence dropped to 10 and 9 when, respectively, ZAP-WT and ZAP-Y292F CD8 T cells were maintained at 0°C for 90 min, and to 22 and 16 when they were incubated for the same time at 37°C. This corresponds for both mouse strains to ~30% internalized TCR in medium at 37°C, compared with ~14% at 0°C (Fig. 2B). When cells were incubated with 10–7 M gp33 loaded APCs, acid-resistant fluorescence increased to MFI of 47 for the ZAP-WT and to only 33 for the ZAP-Y292F cells (Fig. 2A), corresponding, respectively, to 70 and 45% internalized TCR after 60-min stimulation (Fig. 2B). By contrast, when cells were stimulated with PMA, we observed a similar rate of TCR internalization (70% internalized TCR after 60-min stimulation; Fig. 2B) for both strains.

Because the assay for TCR internalization follows the fate of mAb H57 and not the TCR per se, we cannot exclude the possibility that, in late endosomes, the acidic pH induces the release of the mAb from the TCR. However, in previous studies, using an anti-clonotypic TCR mAb on a CTL clone, we could readily observe the presence of both the mAb and the complete TCR/CD3 components in the late endosomal compartment (1, 12). With the restriction discussed above, our results suggest that, in naive CD8 T cells, different stimuli (Ag or PMA, and also anti-CD3 (results not shown)) induce TCR internalization at different rates. Moreover, the ZAP-Y292F mutation affects the rate of Ag-induced TCR internalization in P14 CD8 T cells by ~40% (for Ag peptide concentrations up to 10–7 M), but does not alter the kinetics of PMA-induced TCR internalization. Furthermore, in contrast to its effect on Ag-induced TCR down-modulation and internalization, the ZAP-Y292F mutation does not affect receptor endocytosis and recycling (Table I). These results are compatible with previous data indicating an increased rate of TCR internalization in response to Ag (46), compared with spontaneous receptor cycling, which contrast with other data (45).

Analysis of the fate of CD3{zeta} during TCR internalization

Using anti-CD3{zeta} mAb H146.968, we developed a sensitive intracellular FACS analysis to measure CD3{zeta} levels (see Materials and Methods). We previously compared this intracellular fluorescence measurement with immunoblots on cell lysates using the same mAb to CD3{zeta} and obtained comparable results (M. Buferne and C. Boyer, unpublished results), indicating that a decrease in anti-CD3{zeta} MFI corresponds to the degradation of intracellular CD3{zeta}. All cultures were performed at 37°C in a medium containing cycloheximide to prevent protein synthesis.

In Fig. 3A, we observe similar levels of CD3{zeta} for P14 ZAP-WT or ZAP-Y292F CD8 T cells when cultured in medium (MFI, 433 and 384, respectively). In the ZAP-WT T cells, a pronounced decrease in CD3{zeta} was observed in response to APC/gp33 (10–5 M) (MFI, 217), whereas its level remained high in control cultures with APC/HY (MFI 362). This decrease corresponds to 50% CD3{zeta} degradation in ~60% of the cells (Fig. 3, A and B). For P14 ZAP-Y292F CD8 T cells, by contrast, we observed a moderate decrease in CD3{zeta} in response to APC/gp33 (10–5 M) (MFI, 260) compared with its level in control cultures with APC/HY (MFI, 332). This corresponds to 38% CD3{zeta} degradation in only 25% of the cells (Fig. 3, A and B). Note that CD3{zeta} degradation and TCR down-modulation both increase in proportion to Ag dose (Fig. 3, compare B and C), in agreement with recent work (46).



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  		FIGURE 3. Degradation of CD3{zeta} is proportional to TCR down-modulation. LN T cells from P14 ZAP-WT or ZAP-Y292F mice were stimulated for 270 min in medium containing 10 µg/ml cycloheximide to prevent protein synthesis (this does not affect TCR down-modulation for those cells; results not shown) in the presence of APC loaded with 10–5, 10–7, 10–9 M gp33, or 10–5 M HY peptide, 7 µg/ml anti-CD3, or 10 ng/ml PMA. In A and B, total CD3{zeta} was measured, and in B, results are shown as CD3{zeta} degradation. In C, TCR down-modulation is shown as in Fig. 1. A, Medium (dashed line), APC/HY (thin solid line), APC/gp33 (10–5 M) (bold solid line). B and C, ZAP-WT ({blacksquare}), ZAP-Y292F ({square}).

 
Contrasting results were obtained for T cells responding to anti-CD3 mAb or PMA. Stimulation with anti-CD3 led to very efficient TCR down-modulation (~80% after 4 h) and extensive CD3{zeta} degradation (60–70%) in CD8 T cells from both strains, whereas the PMA-induced TCR down-modulation (50%), led to only minor CD3{zeta} degradation (15%). The latter pattern was also similar for ZAP-WT and ZAP-Y292F CD8 T cells (Fig. 3, B and C). This observation is in agreement with published work (11), and with the notion that PMA induces TCR sequestration, without driving the receptor to the degradation pathway.

Thus, ligand-induced receptor degradation is dependent upon its internalization, but all internalization, such as that induced by PMA, does not necessarily lead to degradation.

Together, these results suggest that ZAP-Y292F CD8 T cells are selectively affected at a step of Ag-induced receptor internalization and that, once internalized, the CD3{zeta} component is similarly degraded. However, these results do not exclude the possibility that the ZAP-Y292F mutation may additionally affect the degradation of the CD3 complex.

To further address the stage at which the fate of the TCR/CD3 complex is affected in the ZAP-Y292F CD8 T, we examined the dynamics of the receptor with respect to T cell/APC interaction and formation of the IS.

Ag-induced surface TCR redistribution at the T/APC contact is defective and delayed for ZAP-Y292F CD8 T cells

To examine whether the decreased rate of Ag-induced TCR internalization was associated with differences in TCR redistribution during the T/APC interaction, we used two different approaches using confocal microscopy. In the first one (Fig. 4A), we analyzed the TCR/CD3 complex at the T cell surface during T/APC conjugate formation by labeling the conjugates with an anti-CD3 mAb after cell fixation with PAF (see Materials and Methods). In the second approach, we analyzed the fate of anti-TCR{beta} mAb H57 used to prelabel the T cells before conjugate formation (Fig. 4B).

Fig. 4A shows conjugates formed after 15 min of P14 CD8 T cell interaction with antigenic gp33/APC or nonantigenic HY/APC. The surface of the APC is shown in blue, CD3 at the T cell surface is in red, and phalloidin staining of F-actin in green. The choice of T/APC conjugates was on the basis of the relocalization of F-actin at the T/APC contact zone. Analysis of the ZAP-WT T cells (Fig. 4A, left) showed ~70% of T/APC conjugates with a patched concentration of CD3 mainly in the central contact zone and/or in a region where F-actin was concentrated (Table II). Three-dimensional deconvolution analysis (xyz) showed a concentrated patched CD3 in the T/APC contact zone (Fig. 4A, third row, left). A similar distribution was also observed after 45 min conjugate formation (Table II). For the T/gp33 APC conjugates formed by ZAP-Y292F CD8 T cells, the striking difference was that CD3 was poorly patched at the T/APC contact zone (Fig. 4A, right). A rather homogenous distribution of CD3 was observed on most of the conjugated ZAP-Y292F T cells (~85% of the analyzed conjugates; Table II), which is also shown in three-dimensional deconvolution (Fig. 4A, third row, right). By contrast, F-actin was concentrated at the T/APC contact zone of ZAP-Y292F T cells as for ZAP-WT T cells. The poor CD3 redistribution in ZAP-Y292F T cell conjugates was partly compensated after 45 min, where we observed that 40% of the T cells had a patched CD3 distribution (Table II). For both types of CD8 T cells, the TCR/CD3 was not redistributed if the irrelevant peptide HY was presented by the APC (Fig. 4A, first row). One can notice that, in these conjugates, TCR/CD3 and F-actin are homogenously distributed around the cell, and that F-actin is poorly labeled in unactivated T cells.


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  		Table II. Statistical analysis of TCR redistribution in T-gp33/APC conjugatesa

 
In the second set of experiments (Fig. 4B), CD8 T cells were prelabeled with anti-TCR{beta} (H57 coupled to Alexa 546), before incubation with APC. After 15 or 45 min, T/APC conjugates were fixed and treated with CTX coupled to Alexa 488 for GM1 gangliosides and raft labeling (50) and analyzed by confocal microscopy. As a control, T cells were also incubated alone, and we observed a homogenous cell surface labeling for both TCR (red) and CTX (green), yielding a yellow-to-orange colocalization labeling (Fig. 4B, first row, no APC, 45 min). For the P14 ZAP-WT CD8 T cells, after 15-min T/gp33 APC conjugate formation, we observed a redistribution of the anti-TCR, indicating a localization of the TCR either at the plasma membrane in the T/APC interaction zone, or inside the cell where it is no longer colocalized with the green CTX membrane staining (Fig. 4B, left panel, second row). Furthermore, some (but not all) of the internalized TCR appeared in organelles budding from the T/APC synapse zone, as already suggested by others (21). After 45 min of conjugate formation, most of the TCR was in organelles inside the cell (Fig. 4B, third row) and did not colocalize with the surface-labeled CTX. This pattern was observed for 90% of T/APC conjugates analyzed, both after 15 and 45 min of T/APC contact. One can notice that CTX is rather poorly relocalized in the T/APC contact zone, a pattern previously described for human CD8 T cells (51), in contrast to the CTX concentration in the T/APC zone described for mouse CD4 T cell lines (52). Additionally, the RMA-S cells used as APC were very poorly labeled with CTX, consequently CTX could be used to mark CD8 T cell membranes.

TCR redistribution was different for P14 ZAP-Y292F CD8 T cells, as shown in Fig. 4B (right panels). After 15-min T/gp33 APC conjugate formation, we observed very few TCR patches and no TCR redistribution to the T/APC contact zone. Both TCR and surface CTX were still homogeneously distributed at the T cell surface. However, after 45-min conjugation, we observed some TCR patches inside the T cell (see also Table II), as well as remaining TCR cell surface expression, suggesting that for the ZAP-Y292F T cells, TCR redistribution and internalization was delayed and at a lower rate, in agreement with the previous results (Figs. 2 and 4A). Together, these results indicate that TCR redistribution and internalization, in the T/APC zone are delayed and partially inhibited by the ZAP-Y292F mutation. By contrast, anti-CD3 mAb-induced TCR patching and internalization was not affected by the ZAP-Y292F mutation when analyzed by confocal microscopy (results not shown).

Recruitment of cbl at the T/APC contact zone is delayed and decreased in ZAP-Y292F compared with ZAP-WT T cells

Because the TCR clustering at the T/APC conjugate contact zone was delayed in ZAP-Y292F T cells, we were interested in analyzing the localization of cbl, to establish whether the loss of the cbl-binding site on ZAP-Y292F would affect its redistribution. Indeed, cbl was previously observed to be recruited to the T/APC contact zone (53), and to be colocalized with the signaling phosphotyrosine zone (54). Therefore, we compared the localization of cbl and F-actin in T/APC conjugates formed for different periods of time (15–45 min). This study was performed by confocal microscopy on fixed and permeabilized T/APC conjugates further labeled for cbl (rabbit anti-Ring cbl/anti-rabbit-A546, red), and for F-actin (phalloidin-A488, green), as described in Materials and Methods (Fig. 5).



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  		FIGURE 5. Confocal analysis of cbl and LFA-1 redistribution. Purified CD8 LN T cells from P14 ZAP-WT (left panels), or ZAP-Y292F (right panels) mice were incubated with APC (prelabeled with Cy5) presenting peptide HY or gp33 (10–6 M) for conjugate formation, and incubated for 15 or 45 min, before fixation with 2% PAF and permeabilization with saponin buffer. A, Phalloidin A488 (green) was used to label F-actin (green), rabbit anti-cbl and Zenon A546 (red)/anti-rabbit Ig was used to label cbl. No staining was observed with normal rabbit serum and the second reagent (not shown). Three independent experiments were analyzed. B, LFA-1 labeling was performed with mAb H35.89.9 followed by anti-rat Ig-biotin and streptavidin A-546 (red) on PAF-fixed cells before permeabilization and F-actin labeling with phalloidin (green). C, A quantitative analysis of the fluorescence distribution is shown as a histogram through a section of the conjugate indicated with the white-stippled arrow in A and B. The colors are similar to those used in A and B and allow distinction of the boundary of the APC as a blue histogram. Corresponding numbers indicate which conjugates from A and B are analyzed. Statistical analyses of the conjugates are represented in Table II.

 
Because cbl and F-actin are highly expressed by the RMA-S cells used as APCs, we labeled the RMA-S cell with Cy5 (blue) to visualize its surface. In nonactivated CD8 T cells, F-actin was localized more or less uniformly around the cell membrane, and cbl was rather dispersed inside the cell (Fig. 5A, first row, HY peptide). As for the TCR/CD3 analysis, T/APC conjugates were chosen on the basis of increased and redistributed F-actin staining. At 15 min, for both types of T cells, cbl was partially recruited to the T/APC synapse zone, mostly under F-actin (Fig. 5A, second row, ZAP-WT and ZAP-Y292F). However, most of the ZAP-WT T/APC conjugates (65%; Table II) presented an additional localization in the middle of the contact zone (Fig. 5A, second row, ZAP-WT; see bold arrow). Such a localization was observed in only 30% of the ZAP-Y292F T/APC conjugates (Table II). A histogram analysis of the fluorescence distribution (Fig. 5C, Cbl 1) showed that the cbl labeling was concentrated at the T/APC contact zone, bulging out of the F-actin zone within the T cell, before the blue (Cy5) boundary of the APC membrane. The frequency of this pattern increased for both strains at 45 min, reaching 80% for the ZAP-WT and 50% for the ZAP-Y292F T/APC conjugates (Table II). Despite variability between experiments, the difference between the two strains was always observed (three independent experiments).

In Fig. 5B, we analyzed the distribution of LFA-1 in T/APC conjugates. A high redistribution of LFA-1 at the T/gp33 APC contact zone was observed for both ZAP-WT and ZAP-Y292F CD8 T cells (Fig. 5B, second row), which was not observed in the T/HY APC conjugates (first row). On the histograms representing the fluorescence distribution within T/APC conjugates, one can observe that LFA-1 labeling is concentrated at the T/APC contact zone on the T cell side, proximal to the blue boundary of the APC membrane. The LFA-1 redistribution remained rather homogenous at the T/APC contact zone even at 45 min, as described for other markers (PKC{theta} and Talin) in naive CD8 T cell/APC conjugates (55). This contrasts with the description of a p-SMAC distribution after 30-min conjugate formation in other T cell lines including naive CD4 T cells (21, 56).

These results showed that the redistribution of LFA-1 and F-actin at the T/APC conjugate contact zone was not affected by the ZAP-Y292F mutation. In contrast, the recruitment of cbl to the central region, which was readily observed in the ZAP-WT T/APC conjugates at an early time, was defective in the ZAP-Y292F T cells. Whether this recruitment corresponds to an endosomal localization of cbl associated with the activated TCR complex needs further investigation. It is consistent with the fact that cbl and associated CMS/CIN85/CD2AP can be involved in endosome organization via rab4 (57), and with the described effect of the ZAP-Y292F mutation as preventing the ZAP-cbl association (58). However, this effect was partially compensated with time, which suggests that other cbl associations may secondarily develop in the IS. These may involve vav linked to ZAP Y315 (59), CD28 via PI3K (60), the src PTK (61), CD2AP binding to CD2 (62), or connection of CD2AP/CIN85 proteins to the actin cytoskeleton (63, 64).


   Discussion
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In the present work, we report that naive CD8 P14 T lymphocytes expressing the mutated ZAP-Y292F were affected in Ag-induced TCR down-modulation, and presented a defect and delay in TCR and cbl recruitment at the T/APC conjugate interaction zone compared with ZAP-WT T cells. However, LFA-1 and F-actin were clearly redistributed in the ZAP-Y292F T/APC contact zone as in ZAP-WT T/APC conjugates.

The TCR down-modulation defect of the ZAP-Y292F T cells appeared to be a consequence of a reduced rate of receptor internalization, which was paralleled by CD3{zeta} degradation in response to APC. The experimental approach included the use of mAb H57 to trace the TCR (Figs. 2 and 4B). As previously shown, this mAb did not per se induce TCR internalization (15), and did not affect Ag-induced TCR down-modulation (Fig. 2). Furthermore, the observation that the reduced rate of TCR/CD3 down-modulation in response to Ag by ZAP-Y292F T cells was also observed when TCR recycling was inhibited by BFA (Table I) further suggests that TCR internalization is affected by the ZAP-Y292F mutation, using an assay independent of a mAb ligand. However, it has not been ruled out that the ZAP-Y292F mutation may additionally affect the degradation of some of the TCR/CD3 components. Constitutive endocytosis, which depends mainly on a CD3{gamma} di-leucine motif (9), or on several motifs from all CD3 components (45), was not altered by the ZAP-Y292F mutation. Anti-CD3- and PMA-induced TCR internalization, which depend on CD3{gamma} or CD3{gamma}{delta} cytoplasmic domains (1, 65), were not affected either. Therefore, the observed defects, although partial, would appear to implicate the ZAP-Y292/cbl interaction (58) at a step that selectively affects APC-induced TCR internalization.

Binding of the cbl adaptor to phosphorylated ZAP-292Y (22) has a negative effect on the activity of ZAP-70, which was mainly attributed to the E3-Ubiquitin ligase activity of cbl and its Ring domain (66). Consistent with this, cbl was found to be involved in ubiquitination of ZAP-70 (67) and of CD3{zeta} (68). Cbl has also recently been implicated at various steps of RTK surface receptor internalization and degradation. First, cbl may participate in the formation of clathrin-coated vesicles by adapting endophilin via a protein of the CIN85 family; additionally, the ubiquitin-ligase activity of cbl allows the ubiquitinated receptor to be recognized by ubiquitin interacting motif domain bearing proteins like Eps15, which is a clathrin adaptor (26, 27, 28, 29, 31).

In T cells, the double genetic deletion of c-cbl/cbl-b was associated with a partial deficiency in anti-CD3-induced TCR internalization, whereas the effect on Ag stimulation was not reported in that work (23). CD2AP, a member of the CIN85 family (62) capable of binding cbl (69), was recently reported to play a major role in T cell signaling and TCR down-modulation (32). Indeed, T cells from CD2AP knockout mice failed to down-modulate their TCR in response to Ag stimulation, and showed defects in the fine structure of the c-SMAC and p-SMAC (32, 33). However, in contrast to the c-cbl/cbl-b double knockout, CD2AP knockout T cells were not affected in mAb-induced TCR down-modulation, at least at early steps (32).

The fact that the defect in ZAP/cbl association selectively affects APC-induced, and not constitutive or anti-CD3-induced TCR internalization, together with the observation of the delay in TCR and cbl recruitment to the T-APC interaction zone in ZAP-Y292F T cells strongly suggests that the defect in TCR internalization may be the consequence of its delayed recruitment to the IS. The alternative, that the delay in TCR recruitment and c-SMAC formation would be the consequence of a defect in TCR internalization, appears difficult to entertain. Indeed, in contrast to receptor engagement by mAb, stimulation of TCRs by peptide/MHC complexes on the APC is most efficient when receptors are concentrated within the IS. Consistent with this, internalized TCR is mainly detected at the site of the IS (Ref.21 ; Fig. 4B), whereas mAb-induced internalization is observed all around the plasma membrane (results not shown). The molecular basis for the defect in c-SMAC formation in the absence of CD2AP is not fully elucidated (32). Similarly, it is not clear in the ZAP-Y292F T cells 1) whether the delay in recruiting the TCR/CD3 at the IS is due to the absence of CD2AP/cbl/ZAP binding, and 2) which molecular interaction of a putative CD2AP/cbl/ZAP complex is required to form the IS. Although such CD2AP/cbl/epidermal growth factor receptor complexes have been described to connect to the actin cytoskeleton via the actin-related protein 2/3 complex (64), it is not clear whether such connections function in the TCR pathway. They have been suggested for CD2 signaling (63).

Together, these results are compatible with a dual role for cbl-b in the control of TCR-dependent signaling upon its recruitment to activated ZAP-70 (Refs.24 and 32 ; for review, see Ref.70). Through 1) the ubiquitination pathway, cbl may dampen strong PTK signals, independently from the IS, and through 2) its association with CD2AP, it may contribute to the concentration of TCR/CD3/ZAP complexes within a c-SMAC, a step required for sustained signaling in the absence of apoptosis (32). The latter contribution of the ZAP-70/cbl-b interaction to promote T cell activation may be balanced by the increased rate of TCR/CD3 complex internalization within the c-SMAC.

The unique properties of TCR/CD3 redistribution within the T/APC interface may explain why distinct motifs within the receptor complex appear to control its constitutive or mAb-induced internalization (45) that have implicated di-leucine motifs (9), and APC-induced internalization for which phospho-CD3-associated ZAP-Y292 appears to be a rate-limiting motif. However, the fact that the defect in the ZAP-Y292F T was partial, suggests that other mechanisms may lead to APC-induced TCR/CD3 internalization. These may involve other cbl/CD2AP binding sites within the receptor complex, cbl being a scaffold protein that may bind other proximal ligands (25). It is also possible that another ubiquitin ligase is able to bind to the activated TCR complex, akin to the Nedd4 ubiquitinin ligase that was also localized in the raft fraction of IgE in activated RBL cells (71).

Concerning the relative control of TCR/CD3 complex internalization and degradation, we found that for Ag-, as for anti-CD3-induced receptor down-modulation, CD3{zeta} degradation was proportional to receptor internalization and down-modulation (Fig. 3). This result is in agreement with other work showing that during constitutive, and mAb-induced TCR internalization, all TCR/CD3 components are degraded with a rate that is proportional to the rate of TCR down-modulation (46) through a mechanism that is dependent on lysosomal activity (72). However, an exception exists for PMA-induced TCR internalization that leads to poor CD3{zeta} degradation (Fig. 3).

Together with data from the literature, the present results are compatible with a role for cbl in the Ag-induced TCR/CD3 internalization/degradation following schemes also proposed for the epidermal growth factor receptor (29, 30, 33). Clearly, questions remain about the mechanisms by which the TCR/CD3 complex is driven into the endocytic pathway at the IS, and in particular whether, as for the BCR, internalization via clathrin-coated vesicles is dependent on integral raft components (73, 74).


   Acknowledgments
 
We thank the members of the Centre d’Immunologie de Marseille-Luminy (CIML) Imaging Service, Marc Barad, Nicole Brun, and Mathieu Fallet for their help, Corinne Béziers-Guigue for artwork, Stéphane Méresse and Nicolas Lapaque for reagents, and the CIML personnel involved in animal care. We thank L. Leserman, N. Auphan, A. Guimezanes, H.-T. He, and G. Verdeil for critical reading of the manuscript.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by institutional grants from Institut National de la Santé et de la Recherche Médicale and Centre National de la Recherche Scientifique and by a grant from Association pour la Recherche sur le cancer. Back

2 S.D. and J.L. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Claude Boyer, Centre d’Immunologie de Marseille-Luminy, Parc Scientifique de Luminy, Case 906, 13288 Marseille, Cedex 9, France. Back

4 Abbreviations used in this paper: PTK, protein tyrosine kinase; PKC, protein kinase C; IS, immune synapse; SMAC, supramolecular activation cluster; c-SMAC, central SMAC; p-SMAC, peripheral SMAC; CIN85, Cbl-interacting protein of 85 kDa; RTK, receptor tyrosine kinase; CD2AP, CD2-associated protein; LN, lymph node; CTX, cholera toxin; MFI, mean fluorescence intensity; PAF, paraformaldehyde; RT, room temperature; BFA, brefeldin A; WT, wild type; AT, acid treated; NT, not treated. Back

Received for publication October 19, 2004. Accepted for publication June 13, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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