Antigen-induced TCR–CD3 down-modulation does not require CD3{delta} or CD3{gamma} cytoplasmic domains, necessary in response to anti-CD3 antibody

Valérie Legendre, Annick Guimezanes, Michel Buferne, Marc Barad, Anne-Marie Schmitt-Verhulst and Claude Boyer

Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, Case 906, 13288 Marseilles, Cedex 9, France

Correspondence to: C. Boyer


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
We studied cytotoxic T lymphocyte (CTL) clones expressing cytoplasmic domain-deleted CD3{delta} and CD3{gamma} chains. These cells retained efficient antigen-specific cytolysis. Because the cytoplasmic domains of native CD3{delta} and CD3{gamma} chains contain a dileucine-based and a tyrosine-based motif thought to be important for receptor endocytosis, we compared TCR–CD3 down-modulation on the CTL clones expressing or not these domains. We found that antigen-induced TCR–CD3 down-modulation was not dependent on either the CD3{delta} or CD3{gamma} cytoplasmic domains. This contrasts with phorbol ester- and anti-CD3 mAb (soluble or plastic-coated)-induced TCR–CD3 down-modulation, that are respectively dependent on CD3{gamma} and on either CD3{delta} or CD3{gamma} cytoplasmic domains, suggesting that differences may exist between the mechanisms of TCR–CD3 down-modulation in response to the three stimuli. TCR–CD3 down-modulation in response to antigen was demonstrated by confocal microscopy to be associated with TCRß chain internalization, whether CD3{delta} and CD3{gamma} were native or truncated. Inhibition by the protein tyrosine kinase inhibitor PP1 of TCR–CD3 down-modulation in response to antigen was also similar whether CD3{delta} and CD3{gamma} cytoplasmic domains were present or not. These properties of receptor down-modulation are discussed with respect to the requirements for TCR engagement on antigen-presenting cells.

Keywords: cell–cell interactions, cytotoxic T lymphocyte, protein kinases, rodent, TCR


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
The TCR–CD3 complex is composed of the TCR{alpha}ß disulfide-linked heterodimer recognizing peptide antigen presented by MHC molecules and the CD3 transduction complex. The latter is composed of CD3{gamma}{varepsilon} and CD3{delta}{varepsilon} non-covalent dimers as well as CD3{zeta} family disulfide-linked homo- or heterodimers (1). Surface TCR–CD3 expression is regulated at the level of the assembly of the multimeric complex (1), as well as by its down-modulation from the cell surface following antigen stimulation (2,3). A TCR–CD3 down-modulated phenotype has been correlated with a status of peripheral tolerance in some TCR transgenic models (4) as well as in T cells exposed to superantigens in vivo (5). In T cell immunization, once a T cell precursor has been optimally triggered, its chance to re-engage a limited set of relevant peptide–MHC complexes should be lowered to favor engagement of naive T cells. Down-modulation of antigen-engaged TCR complexes is a means to achieve this (3,6).

Down-modulation of receptors by internalization involves specific motifs in their cytoplasmic domains which bind to adaptor proteins (AP) associated with the endocytic pathway (reviewed in 7). Thus, AP-2 functions to associate plasma membrane receptors to clathrin-coated vesicles (7). The receptor motifs either are constitutively accessible within the cytoplasmic domain, as for nutrient receptors that constantly cycle, or need to be unmasked following receptor triggering by a ligand, as for receptors with intrinsic protein tyrosine kinase (PTK) activity (7). Two major types of motifs have been characterized, one containing a tyrosine (Y) residue (7) and the other a dileucine (LL) sequence (8,9), which are both found in the cytoplasmic domains of the CD3 chains: (i) each of the CD3 chains contains one or several Y-based motifs which are part of the imunoreceptor tyrosine-based activation motif (ITAM) sequence involved in PTK signaling (10); and (ii) CD3{gamma} and CD3{delta} each have a dileucine-based motif (8). Low-level TCR–CD3 cycling has been correlated with CD3{gamma} phosphorylation (11) and TCR–CD3 down-modulation can be further induced in different ways. (i) Phorbol esters that directly activate protein kinase C (PKC) induce a transient down-modulation (12,13) for which phosphorylation of Ser-126 near the dileucine motif of the CD3{gamma} chain was clearly demonstrated to be responsible (9,14). (ii) CD3-specific and some TCR-specific mAb (13,15) induce a long-term TCR–CD3 down-modulation as a result of its internalization via clathrin-coated vesicles (13). Internalization is preceded by a genistein-sensitive step of TCR–CD3 redistribution (16) and proceeds through early and late endosomes, where the TCR–CD3 was found associated with protein kinase activity (17) and where the ligand is degraded (13). (iii) Antigen-presenting cell (APC)- (3,18) and superantigen (19,20)-induced TCR–CD3 down-modulation were associated with the degradation of some CD3 components (21). It is not clear whether TCR–CD3 down-modulation in response to these different stimuli shares common mechanisms.

We reasoned that the development of a TCR–CD3-internalization-deficient variant would allow us to address questions concerning the influence of this mechanism on the physiology of T cell–target cell interaction in vitro and if similar receptor complexes were established in mice, the supposed role for this mechanism in in vivo tolerance induction could be established. Therefore, we constructed a variant cytotoxic T lymphocyte (CTL) clone expressing surface TCR–CD3 complexes deficient in both CD3{delta} and CD3{gamma} cytoplasmic domains (22) and showed that these receptor complexes resisted both phorbol myristate acetate (PMA)-induced and anti-CD3 mAb-induced TCR–CD3 internalization, but that only PMA- and not CD3 ligand-induced internalization was dependent on PKC activation (22). In the present study, we showed that APC-induced TCR–CD3 down-modulation and internalization, however, still occurred in CTL expressing a TCR–CD3 complex deleted of dileucine motifs, suggesting that distinct motifs control PMA-, anti-CD3- and APC-induced TCR–CD3 internalization. Together with our previous results (22), this showed that in the antigen response of CTL effectors no specific signaling function could be attributed to either the CD3{delta} or CD3{gamma} cytoplasmic domains, favoring the notion of redundancy within the cytoplasmic domains of CD3 components. We also showed that the PTK inhibitor PP1 affects APC-induced receptor down-modulation whether CD3{gamma}/{delta} cytoplasmic domains are present or not within the receptor complex.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Cells
BM3.3, a murine clone specific for the H-2Kb alloantigen, was maintained in culture as described (22). Clones D6{delta}t{gamma} and F1{delta}t{gamma}t originated from a BM3.3 variant which had spontaneously lost expression of both CD3{gamma} and CD3{delta} chain mRNA, and was transfected to re-express native ({gamma}) or cytoplasmic domain deleted ({gamma}t) human CD3{gamma}, and cytoplasmic domain-deleted mouse CD3{delta} chain ({delta}t), as described (22). RMA-S, a TAP-2-negative variant of RMA (H-2b), was used for presentation of antigenic peptides in association with H-2Kb as described (23). BM3.3 recognizes H-2Kb plus a specific peptide (Guimezanes et al., in preparation), whereas the ovalbumin (OVA) peptide was used as a control peptide, as it binds to H-2Kb but was not recognized by the BM3.3 TCR (24).

Antibodies
Ti98, referred to as anti-TCR mAb, is specific for the BM3.3 TCR (23). mAb 145.2.C11 (25) and H57.597 (H57) (26) are specific respectively for CD3{varepsilon} and TCRß.

Reagents
PP1 from Calbiochem (La Jolla, CA) and cytochalasin D (cyto D) from Sigma (St Louis, MO) were used from stock solutions in DMSO. Phycoerythrin (PE)-coupled streptavidin was from Immunotech (Marseilles, France).

Activation-induced modulation of the TCR–CD3 complex and fluorescence analysis
Cells (105) were incubated in round-bottom plates with mAb or with APC (2x105) as indicated, washed and analyzed for surface TCR or CD3{varepsilon} expression by staining with biotinylated mAb and PE–streptavidin. In a preliminary study it was shown that the RMA-S cells expressed high level surface CD44, whereas the CTL clones used here were always negative for this marker. Therefore RMA-S APC were `gated out' using FITC-labeled anti-CD44 mAb (Caltag, San Francisco, CA). Data from flow cytometric analysis performed on a FACScan (Becton Dickinson, Mountain View, CA) were analyzed using CellQuest software (Becton Dickinson).

Confocal microscopy
Cells (106) were incubated with FITC-conjugated anti-TCRß (10 µg/ml) for 1 h at 4°C in RPMI/FCS, washed once, incubated as indicated and prepared for confocal microscopy as described (16).

Cytolysis assay
RMA-S were labeled with 51Cr (Na chromate; NEN, Boston, MA) overnight at 26°C, washed and incubated at 2x106 cells/ml for 1 h at 37°C with different concentrations of peptide, and 104 cells were incubated in the presence of various numbers of CTL clones at 37°C for 4 h as described (24).


    Results and discussion
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Cytolytic function and TCR–CD3 down-modulation in response to peptide-presenting target cells is retained in CTL clones lacking CD3{delta} and CD3{gamma} cytoplasmic domains
The alloreactive CTL clone BM3.3 is specific for an endogenous peptide associated with H-2Kb (24) and efficiently kills mouse target cells expressing H-2Kb in conditions that did not lead to detectable receptor down-modulation (22 and results not shown). Here we used the TAP-2-negative H-2b RMA-S target cells, that require exogenous addition of specific peptide, to evaluate the dose of peptide needed for CTL activity and for TCR–CD3 down-modulation by the BM3.3 clone and its variants F1{delta}t{gamma}t (cytoplasmic-deleted CD3{delta} and cytoplasmic-deleted CD3{gamma}) and D6{delta}t{gamma} (cytoplasmic-deleted CD3{delta}, but native CD3{gamma}) (22). Results show (Fig. 1BGo) that 50% maximum CTL activity is obtained at ~10–11 M peptide for BM3.3, and ~10–10 and 10–9 M respectively for clones F1{delta}t{gamma}t and D6{delta}t{gamma}. None of the clones kill target cells expressing Kb with the OVA peptide. Thus, deletion of the cytoplasmic domain of both CD3{delta} and CD3{gamma} does not prevent CTL activity, and the requirement for higher doses of peptide for the variant clones is probably related to the generally lower activity of transfected clones, even when re-expressing native CD3{delta} and CD3{gamma} (22,23). TCR–CD3 down-modulation was also observed in a peptide dose-dependent fashion for the three clones (shown after 1 h in Fig. 1AGo, with kinetics shown in Fig. 2CGo), half maximal down-modulation being obtained with ~3x10–9 M peptide for clone BM3.3, and requiring ~10-fold more peptide for clones F1{delta}t{gamma}t and D6{delta}t{gamma} (Fig. 1AGo). Thus for CTL clones expressing either native or cytoplasmic-deleted CD3{delta} and CD3{gamma}, half-maximal TCR–CD3 down-modulation was obtained at concentrations of peptide ~300-fold higher than those inducing half-maximal CTL activity. This is in agreement with data obtained with human CTL clones (27) and consistent with our failure to detect receptor down-modulation in response to H-2b cells with endogenous peptide.



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Fig. 1. CTL activity and TCR–CD3 down-modulation as a function of dose of peptide. BM3.3 ({blacktriangleup}), F1{delta}t{gamma}t ({blacksquare}) or D6{delta}t{gamma} (•) were incubated with RMA-S cells preloaded with different concentrations of specific peptide or control OVA peptide as described in Methods to measure TCR–CD3 down-modulation after 2 h (A) or cytolytic activity (B) after 4 h. In (A), samples were stained with biotin-conjugated anti-TCR mAb plus streptavidin–PE and for each point the mean fluorescence intensity (MFI) was normalized to that of cells incubated in the presence of RMA-S cells loaded with the OVA peptide; this ratio (anti-TCR fluorescence) is represented on the ordinate. OVA peptide did not induce a significant change in expression of the TCR as ratios of TCR expression on clones incubated in the presence of OVA/RMA-S as compared to medium were respectively 1.05 (BM3.3), 0.92 (F1{delta}t{gamma}t) and 1.14 (D6{delta}t{gamma}). In (B), percent 51Cr released from 104 RMA-S target cells loaded with the specific peptide is shown at an effector:target ratio of 9:1. Percent 51Cr released from RMA-S target cells loaded with the OVA peptide, shown for the BM3.3 effector ({triangleup}), was <3% for the other effector cells.

 


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Fig. 2. TCR down-modulation in response to APC or anti-CD3 mAb on clones BM3.3, D6{delta}t{gamma} and F1{delta}t{gamma}t. (A and B) Clone BM3.3 (upper panel), F1{delta}t{gamma}t (middle panel) or D6{delta}t{gamma} (lower panel) were incubated in the presence of: (A) RMA-S cells loaded with 10–6 M specific (solid line) or control (dashed line) peptide for 1 h at 37°C, or (B) medium (dashed line) or anti-CD3{varepsilon} mAb (10 µg/ml) (solid line) for 1 h at 37°C. Staining was as in Fig. 1Go(A). (C and D) BM3.3 ({blacktriangleup}), F1{delta}t{gamma}t ({blacksquare}) or D6{delta}t{gamma} (•) were treated as described in (A) and (B) for the indicated time and stained as in (A) and (B). For each time point, the MFI was normalized to that of cells incubated in the presence of RMA-S cells loaded with control OVA peptide (C) or of medium (D). This ratio (anti-TCR fluorescence) is represented on the ordinate as mean ± SD of three (C) or five (D) independent experiments. This representation is valid since OVA peptide did not induce a significant change in expression of the TCR as ratios of TCR expression on clones incubated in the presence of OVA/RMA-S as compared to medium were respectively 1.0 (BM3.3), 1.0 (F1{delta}t{gamma}t) and 1.1 (D6{delta}t{gamma}) after 1 h, and 0.9 (BM3.3), 1.0 (F1{delta}t{gamma}t) and 1.3 (D6{delta}t{gamma}) after 4 h. No significant variations of the expression of the TCR for clones incubated in medium alone was observed either.

 
Cytoplasmic domains of either CD3{delta} or CD3{gamma} are necessary for anti-CD3 mAb-induced, but both are dispensable for antigen-induced TCR–CD3 down-modulation.
We next analyzed the kinetics of TCR down-modulation following antigen (Fig. 2A and CGo) or anti-CD3 mAb (Fig. 2B and DGo) stimulation of clone BM3.3 and its variants F1{delta}t{gamma}t and D6{delta}t{gamma}. In Fig. 2Go(A), TCR surface expression is shown 1 h after stimulation with specific as compared to control peptide. Clone BM3.3, as well as variants F1{delta}t{gamma}t and D6{delta}t{gamma} efficiently down-modulated TCR surface expression (in MFI units: from 700 to 80 for both BM3.3 and F1{delta}t{gamma}t, and from 700 to 200 for D6{delta}t{gamma}). No consistent difference was observed between the three CTL clones in terms of kinetics of TCR–CD3 down-modulation in response to antigen as indicated in Fig. 2CGo (mean of three experiments). The level of TCR–CD3 expression was already decreased after 10 min, reached 30% after 1 h for each of the three clones (Fig. 2CGo) and remained low at 48 h (not shown). Antigen-induced CD3 down-modulation (measured with mAb 145.2C11) was similar to that observed for the TCR (results not shown), in contrast to a previous report (28). As a comparison, we also show TCR down-modulation in response to T cell treatment with anti-CD3 mAb at 10 µg/ml, previously found to be optimal (Fig. 2B and DGo). This confirms our previous data (22) indicating that receptor down-modulation was observed for clones BM3.3 and D6{delta}t{gamma} (TCR level down to 60 and 30% at 2 and 20 h respectively; Fig. 2DGo), but not for F1{delta}t{gamma}t (TCR level of 120 and 90% at 2 and 20 h respectively; Fig. 2DGo). To test whether the observed differences could be related to the degree of receptor cross-linking by soluble anti-CD3 as compared to APC-associated MHC + peptide, we analyzed TCR–CD3 down-modulation in response to plastic-coated anti-CD3 mAb, in conditions previously shown to activate the CTL (29). For the BM3.3 and D6{delta}t{gamma} clones, this was less efficient, however, than soluble anti-CD3 at inducing TCR–CD3 down-modulation (TCR level down to 70% of the initial level after 2 h) and for clone F1{delta}t{gamma}t, there was no TCR–CD3 down-modulation induced (results not shown). It should be noted that the BM3.3 TCR is of high affinity for the specific peptide–H-2Kb complex (Guimezanes et al., in preparation), as already suggested by its characteristic CD8-independent interaction with H-2Kb (29).

Thus in the model presented here, results clearly showed (i) a major difference between antigen- and anti-CD3 ligand-induced TCR–CD3 down-modulation, since only the former did not require either CD3{delta} or CD3{gamma} cytoplasmic domains; and (ii) that antigen on APC can be more efficient at inducing TCR–CD3 down-modulation than anti-CD3 mAb, whether soluble or plastic-coated, suggesting that the efficiency of the APC cannot solely be attributed to their capacity to induce receptor oligomerization. The latter point is in agreement with previous data (30).

Antigen-induced TCR–CD3 down-modulation is associated with receptor internalization for CTL clones expressing either native or cytoplasmic domain truncated CD3{delta} and CD3{gamma}
The fate of the modulated TCR–CD3 complexes was analyzed by confocal microscopy. To follow the TCR, the cells were first stained with FITC-conjugated anti-TCRß mAb at 4°C. This mAb was used because it was previously shown to be unable to induce TCR–CD3 down-modulation (16,31) and because it was not inhibitory for antigen recognition by the BM3.3 TCR (results not shown). Upon incubation with RMA-S loaded with specific peptide, TCR fluorescence was redistributed in small patches, and was internalized in both clones BM3.3 (Fig. 3AGo) and F1{delta}t{gamma}t (Fig. 3BGo), but not upon incubation with RMA-S loaded with OVA peptide (Fig. 3C and DGo). These results indicate that the down-modulation of the TCR–CD3 from the cell surface was associated with internalization of the TCR in response to the specific antigen whether the CD3{gamma} and CD3{delta} cytoplasmic domains were present or not. It should be noted that the dileucine motifs which are uniquely present in those domains have also been characterized as directing the CD3{gamma} and CD3{delta} isolated chains towards lysosomes (8), and may thus be important for a subsequent degradation step of the internalized receptor (17,21).



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Fig. 3. TCR internalization in response to antigen on clones BM3.3 and F1{delta}t{gamma}t. Clones BM3.3 (A and C) and F1{delta}t{gamma}t (B and D) were stained with FITC-conjugated anti-TCRß mAb (H57) at 4°C for 1 h. Cells were washed and incubated in the presence of RMA-S cells loaded with 10–6 M specific (+) (A and B) or control (–) (C and D) peptide for 20 min at room temperature. Fluorescence was visualized by confocal microscopy.

 
Antigen-induced TCR–CD3 down-modulation is sensitive to the PTK inhibitor PP1 for TCR–CD3 complexes containing or not dileucine motifs
For the insulin receptor, it was recently suggested that both a dileucine motif and tyrosine-based motifs were involved in the control of the association of the receptor with clathrin-coated pits, and that only the latter mechanism was dependent on the receptor kinase activity (32). For the epidermal growth factor receptor, kinase activity was found necessary for efficient ligand-induced receptor down-modulation and intracellular routing (33,34). The role of protein kinase activation for TCR–CD3 receptor down-modulation is not entirely clear as conflicting data exist in the literature (16,20,35,36). We asked whether a difference in sensitivity to protein kinase inhibitors could be detected depending on the presence of the dileucine motifs within the TCR–CD3 complex.

The strong PTK inhibitor PP1, described as a preferential inhibitor of src-family PTK (37), was found to efficiently inhibit cytolytic activity of the CTL (90% at 5 µM; result not shown). It partially inhibited APC-induced TCR–CD3 down-modulation on clone BM3.3 (50% inhibition from 1 to 18 h at 10 µM PP1; Fig. 4AGo). The inhibition of anti-CD3-induced TCR–CD3 down-modulation was partial at early time points (50% up to 2 h) but was consistently not observed at later time points (Fig. 4BGo). The lower sensitivity of anti-CD3-induced TCR–CD3 down-modulation to inhibition by PP1 could be consistent with the importance of the dileucine-containing cytoplasmic domains in the internalization induced by the CD3 ligand. Therefore, we tested whether the sensitivity to inhibition by PP1 was increased for the APC-induced down-modulation of a receptor complex depleted of dileucine motifs. The results showed that at 2 h 10 µM PP1 inhibited ~35% of the APC-induced receptor down-modulation on both clone F1{delta}t{gamma}t and clone D6{delta}t{gamma} (Table 1Go). There was thus no evidence that in the absence of dileucine-containing motifs in the receptor complex (clone F1{delta}t{gamma}t), APC-induced TCR–CD3 down-modulation became more sensitive to a PTK inhibitor. Altogether these results may indicate that inhibition of the src-family PTK (p56lck and p59fyn) required to initiate TCR–CD3 signaling also affects the internalization of the TCR–CD3 complex, as recently suggested (36). This result is also in agreement with the previously described inhibition of anti-TCR-induced TCR redistribution and internalization by genistein (16), and with the role described for p56lck activation in TCR–CD3 internalization (36). Differences between antibody- and APC-induced TCR down-modulation are apparent, however, with respect to inhibition by various PTK inhibitors: genistein does not inhibit APC-induced TCR–CD3 down-modulation/internalization (18,20 and results not shown), whereas PP1 inhibition is more effective on APC- than on antibody-induced TCR down-modulation (Fig. 4A and BGo). It cannot be excluded that PP1 may inhibit, in addition to p56lck and p59fyn, another PTK (38) involved in a step distinct from the actual TCR–CD3 internalization. This could involve the establishment of stable CTL–APC interactions that are required for efficient sequential engagements of the TCR by MHC + peptide complexes on the APC (39).



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Fig. 4. APC-induced TCR–CD3 down-modulation is inhibited by PP1. In (A) and (B), clone BM3.3 was preincubated for 30 min at 37°C without (•) or with ({blacksquare}) PP1 (10 µM) and was further incubated in the same conditions for the indicated time at 37°C after addition of RMA-S loaded with 10–6 M specific or control peptide (A) or 10 µg/ml soluble anti-CD3 mAb or medium (B). For each time point, the MFI was normalized to that of cells incubated in the presence of RMA-S cells loaded with control OVA peptide (A) or of medium (B). This ratio (anti-TCR fluorescence) was represented on the ordinate. For samples incubated for 20 h, ratios of TCR expression on clones incubated in the presence of OVA/RMA-S as compared to medium were respectively 0.74 (BM3.3), 1.1 (F1{delta}t{gamma}t) and 1.2 (D6{delta}t{gamma}). The presence of PP1 did not affect the level of cell surface expression of the TCR.

 

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Table 1. Partial inhibition of antigen-induced TCR down-modulation with src-family PTK inhibitor PP1
 
Concluding remarks
Results presented in this work showed that TCR–CD3 receptor internalization in response to anti-CD3 mAb (whether soluble or plastic-coated) and APC is differentially controlled, since absence of both CD3{delta} and CD3{gamma} cytoplasmic domains totally inhibited anti-CD3 mAb-induced, without affecting APC-induced receptor internalization.

Peptides corresponding to D/ExxxLL CD3{gamma}/{delta} motifs have recently been shown to bind AP-2 in vitro (9). In vivo, chimeric proteins containing this motif in their cytoplasmic tail were shown to selectively compromise the targeting of dileucine-motif-containing proteins (40). Additionally, the YQPL sequence of one of the ITAM motifs present in both CD3{delta} and CD3{gamma} has been shown to confer internalization activity in a chimeric protein system (8,40), whereas the complete CD3{zeta} cytoplasmic domain which contains different ITAM sequences failed to do so in a similar assay (40). The cytoplasmic domains of CD3{delta} and CD3{gamma} therefore appeared to be particularly well equipped to control the internalization and lysosomal targeting (8) of the TCR–CD3 complex. So why are these motifs not functional for TCR–CD3 down-modulation in response to APC? Two major possibilities exist. First, redundancy of motifs among the different CD3 components could lead to internalization of the TCR–CD3 complex via association with AP of the endocytic pathway of motifs such as NPxY (CD3{varepsilon}) or ITAM (all CD3 chains). However, it was previously shown that neither the CD3{zeta} (40) nor the CD3{varepsilon} (8) cytoplasmic domain could confer internalization in a chimeric construct. It remains unclear whether in the context of the complete receptor and as a consequence of T cell activation, changes in the tyrosine phosphorylation of particular ITAM motifs may alter their properties: they may prevent their function as AP binders, akin to the situation for CTLA-4 (41), or may lead to the recruitment of SH2-containing proteins such as Grb2 recently implicated in endocytosis for other receptors (42). Second, although TCR–CD3 down-modulation in response to APC was associated with the internalization of TCRß (this study) and CD3{zeta} (21), and that there is a coordinate disappearance of CD3{varepsilon} and TCR from the cell surface (18,21 and our results not shown), no formal proof exists that it involves classical receptor endocytosis. Alternative mechanisms, such as receptor ubiquitination (43), may be at play.

The unique characteristics of APC-induced TCR–CD3 internalization have also to be considered as the MHC–peptide ligand is cell bound and the receptor dissociates from the ligand before its internalization. T cell engagement by APC leads to an active process of organization of the contact area between the two cells that depends on an intact actin cytoskeleton on the T cell that contributes to the recruitment in this area of molecules such as ICAM-1 on the APC (44). It is thus not clear whether an intact actin cytoskeleton (as indicated by inhibition of APC-induced TCR–CD3 down-modulation by cyto D; our results not shown) and an active PTK (Fig. 4Go) are required for efficient TCR engagement by the APC rather than for the receptor internalization step itself. It is also not clear how this cytoskeleton reorganization may affect the accessibility of the receptor to the endocytic pathway. It can thus not be excluded that within the area of T cell–APC interaction, the local concentration of TCR–CD3 complexes and other recruited molecules as well as the reorganization of the cytoskeleton (45) lead to the dissociation of the TCR–CD3 complex (46), with individual components being independently routed intracellularly. For instance, the proline-rich region of CD3{varepsilon} (22) could associate with cytoskeletal components independently of CD3{zeta}. In such a model, alterations of individual motifs within the cytoplasmic domains of the TCR–CD3 complex would not prevent the dissociation and down-modulation of the complex. Our results suggest that dileucine-based and tyrosine-based internalization motifs that are unique to the CD3{delta} and CD3{gamma} chains are not involved in the rate-limiting step of the mechanism by which the TCR–CD3 complex is internalized as a result of T cell–APC interaction. Further characterization of this process will require the development of tools adapted to the analysis of events occurring at the interaction zone between T cell and APC.


    Acknowledgments
 
We thank Corinne Béziers La Fosse for artwork. 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 grants from Association pour la Recherche sur le Cancer (ARC), the Ligue Nationale Française contre le Cancer (LNFCC) and the LNFCC-Comité des Bouches du Rhône. V. L. was the recipient of a fellowship from ARC.


    Abbreviations
 
AP adaptor protein
APC antigen-presenting cell
CTL cytotoxic T lymphocyte
cyto D cytochalasin D
ITAM immunoreceptor tyrosine-based activation motif
MFI mean fluorescence intensity
OVA ovalbumin
PE phycoerythrin
PKC protein kinase C
PMA phorbol myristate acetate
PTK protein tyrosine kinase

    Notes
 
Transmitting editor: M. M. Davis

Received 18 January 1999, accepted 7 July 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 

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