TCR dynamics on the surface of living T cells

Benoit Favier, Nigel J. Burroughs1, Lucy Wedderburn2 and Salvatore Valitutti

INSERM U395, Institut Claude de Préval, CHU Purpan, 31059 Toulouse Cedex 3, France
1 Department of Mathematics, University of Warwick, Coventry CV4 7AL, UK
2 Institute of Child Health, University College London, London WC1N 1EH, UK

Correspondence to: S. Valitutti


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
T lymphocyte activation by specific antigen requires prolonged TCR occupancy and sustained signaling. This is accomplished by the formation of a specialized signaling domain, the immunological synapse, at the T cell–antigen-presenting cell contact site. Surface receptors and signaling components are progressively recruited into this domain where they are organized in defined three-dimensional structures. To better understand how TCR are supplied to the signaling domain during the activation process, we measured (using confocal microscopy and photo-bleaching recovery techniques) lateral mobility of GFP-tagged TCR on living Jurkat cell surface. We show that: (i) surface-expressed TCR exhibit an intrinsic, actin cytoskeleton-independent, lateral mobility which allows them to passively diffuse over the entire T cell surface within ~60 min and (ii) non-stimulated TCR rapidly enter the signaling domain. Our results indicate that TCR lateral mobility per se is sufficient to ensure TCR supply to the immunological synapse in the course of sustained T cell activation.

Keywords: GFP, fusion proteins, lateral mobility, signal transduction


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
T lymphocytes are activated by engagement of their antigen receptors (TCR) with peptide–MHC complexes displayed on the surface of antigen-presenting cells (APC) (1). It is well established that activation of the T cell biological response is the result of sustained TCR engagement and signaling (2). However, it is also known that T cells are exquisitely sensitive to antigenic stimulation since they can proliferate and produce cytokines in response to APC displaying as few as one to a few hundreds specific peptide–MHC complexes (3–7). An important contribution to the understanding of how a very small number of antigenic ligands can induce sustained signaling in T cells has come from several studies based on time-lapse video recording of T cell–APC interactions (8–11). These studies have contributed to the definition of a new concept in cellular immunology: that T cells actively `collect' stimuli on the surface of APC rather than passively `receive' them from APC. A functional actin cytoskeleton is required for this process as it allows T cells to sample the APC surface and to form areas of tight adhesion with the opposing cell membrane (12).

We have previously observed that the number of triggered TCR far exceeds the number of antigenic ligands offered during sustained and dynamic interaction between T cells and APC (5). We therefore proposed that many TCR are serially engaged and triggered by a few peptide–MHC complexes displayed on the APC surface, resulting in a sustained and amplified signal (5).

Recent studies have provided evidence for the existence of specialized signaling domains at the contact areas between T cells and APC, where TCR engagement and sustained signaling take place (13–16). These observations support the concept that the process of T cell antigen recognition is the result of dynamic and reversible re-localization of surface molecules and signaling components (17).

No information is presently available on the lateral mobility of TCR expressed on the T cell surface. The measurement of this parameter is critical to the understanding of whether an increasing number of TCR may be triggered at the T cell–APC immunological synapse.

In the present work we transfected a TCR-deficient Jurkat T cell line with a GFP-tagged TCRß chain and calculated TCR lateral mobility by measuring TCR photo-bleaching recovery. We show that surface-expressed TCR exhibit lateral mobility that does not depend on a functional actin cytoskeleton. We also show that TCR rapidly diffuse into the contact site between T cells and polystyrene beads coated with anti-TCR–CD3. We discuss our results in the context of the proposed models of T cell activation.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell transfection
The HA1.7 TCRß cDNA was amplified from HAß-pJ6{omega} using oligonucleotides (5'-ATGGGAATCAGGCTCCTCTGT-3') and (5'-AAAGGATCCCGAAATCCTTTCTCTTGAC-3'). The PCR product was cloned into PCR-0-Blunt and re-subcloned in-frame with GFP as an EcoRI–BamHI fragment into the pEGFP-N2 vector (Clontech, Palo Alto, CA).

TCR{alpha} cDNA from HA{alpha}-PJ6{omega} was digested with EcoRI and cloned into the PCR3 expression vector (Invitrogen, Carlsbad, CA).

The ß chain-deficient Jurkat cells (1x107, clone 31.13, kindly provided by Dr Oreste Acuto, Institut Pasteur, Paris, France) (18) were transiently transfected with 8 µg TCRß-EGFP, TCR{alpha}-PCR3 and pEF TAg at 280 V and 900 µF in a BioRad (Hercules, CA) Gene Pulser; 36–48 h after transfection cells were either sorted for GFP and used immediately for experiments or used without previous enrichment.

The correct mol. wt of GFP attached to the TCRß chain was checked by Western blot analysis on cell lysates using a rabbit antibody against GFP (kindly provided by Dr M. Peter, ISREC, Epalinges, Switzerland).

Measurement of TCR down-regulation
Sorted GFP+ 31.13 cells (1x105) were conjugated for 3 h at 37°C with 2x105 polystyrene beads either untreated or coated with either anti-CD3 (OKT3; ATCC, Rockville, MD) or anti-Vß3 (Jovi-3) (19) mAb. Polystyrene latex micro-spheres (diameter 6 µm) were purchased from Polysciences (Warrington, PA). Antibodies were absorbed onto the beads as previously reported (20). TCR expression levels were measured by FACS analysis as previously described (5). Briefly, surface TCR–CD3 complexes were stained either with OKT3 or with Jovi-3 followed by a goat anti-mouse IgG phycoerythrin-labeled antibody (Southern Biotechnology Associates, Birmingham, AL). The TCR–CD3 (FL2) and the GFP (FL1) fluorescence was analyzed on a FACScan (Becton Dickinson, Mountain View, CA) equipped with CellQuest software.

[Ca2+]i measurement
[Ca2+]i measurement was performed as previously described (10).

Photo-bleaching experiments
Fluorescence recovery after photo-bleaching (FRAP) was performed at room temperature on a confocal microscope (LSM 510; Carl Zeiss, Jena, Germany) using a x63 objective and the 488 nm line of a 400 mW Kr/Ar laser. A rectangular region defined by the boxed area was illuminated with the laser at 50% power and 100% transmission. The entire field of view was sequentially imaged (50% laser power and 2% transmission) both before and after photo-bleaching. The time lapse between images was 2.5 s. Regions were drawn at the T cell surface in the bleached area and on the remaining cell surface, and the GFP fluorescence values were plotted against time and used to measure the TCR diffusion constant. In some experiments cells were either treated overnight with 10 µg/ml cycloheximide (CHX) or with 10 µM cytochalasin D (added 10 min before the beginning of the assay). The drugs were present throughout the assay. Overnight treatment with CHX did not affect cell viability as assessed by Trypan blue exclusion test (data not shown).

Diffusion constant (D) determination
Analysis of fluorescence recovery was based upon a one-dimensional diffusion model. Recovery is closely approximated by the formula (for t > 0 and the end of the bleaching interval occurring at t = 0):

where c is a constant with value close to 1, w is the length of the beached region along the T cell surface, U(t) is the fluorescence in the bleached region at time t after bleaching and Uinfinity is the final florescence in the bleached region. We estimated Uinfinity, D and c concurrently with a standard non-linear minimization of the sum of squares statistic for each experiment. Use of the equation considerably simplified the fitting relative to the exact solution. The initial pre-bleach fluorescence intensity of the region U0 was estimated by five consecutive measurements prior to bleaching. The recovery ratio Uinfinity/U0 defines the mobility fraction of the TCR (21). Fluorescence measurement error was found to be relative (based on pre-bleach measurements), i.e. the SD of a measurement U was 0.02 * U.

Statistical analysis
To assess whether experimental conditions or cell batches made any difference to the diffusion constant and mobility, we compared different conditions by ANOVA, paired t-tests and the non-parametric Mann–Whitney rank test. Weak trends were observed in each batch (photo-bleaching performed on the same day on cells from the same culture), but there was variation between batches. To minimize the effect of this variation on comparisons we used the sum of the paired t-statistics for each day, which is approximately normally distributed. P values for this test are quoted.

Fluorescence loss in photo-bleaching (FLIP)
Experiments were performed at room temperature using the above-described technical set-up. A rectangular region was repeatedly illuminated with the laser at 50% power and 100% transmission. Between each intense illumination the entire field of view was imaged (2% transmission) to assess the extent of loss of fluorescence outside the rectangle as a consequence of photo-bleaching within the rectangle. The time lapse between images was 2.5 s. Every 20 images the selected rectangle was newly bleached (50% power and 100% transmission).

Regions were drawn at the T cell surface in the bleached area and on the remaining cell surface and the GFP fluorescence values were plotted against time. No significant photo-bleaching was observed due to imaging at low transmission in the recovering cell since control cells in the field did not lose significant fluorescence intensity during the time followed.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Expression of GFP-tagged TCR on the T cell surface
A well-established method to evaluate lateral mobility of cell surface molecules is based on the expression of GFP-tagged proteins and measurement of fluorescence recovery after photo-bleaching (21–24). Therefore, we generated TCR–CD3 complexes tagged with GFP. To this end we produced a TCRß chain–GFP chimera (Vß3.1 from the HA1.7 TCR) (25) and tested its expression by Western blot analysis with anti-GFP antibodies after transient transfection. This analysis resulted in the detection of a band with the expected molecular weight of the Vß3–GFP chimera (data not shown).

Transient transfection of the Vß3–GFP chimera in a ß chain-deficient Jurkat T cell line (31.13 cells) (18) resulted only in a partial and heterogeneous surface expression of GFP-tagged TCR–CD3 complexes in the cell population (data not shown). This is most likely due to the fact that endogenous {alpha} chain expression was also repressed in 31.13 cells because of chronic ß chain absence (26). Co-transfection of Vß3–GFP with the {alpha} chain derived from the same HA1.7 TCR (V{alpha}1.2) (25) resulted in very high and homogeneous expression of TCR–CD3 complexes, as detected by staining with anti-CD3 and anti-Vß3 antibodies (Fig. 1A and BGo). Surface-expressed receptors were internalized following stimulation with anti-CD3 or anti-Vß3-coated polystyrene beads (Fig. 1C and DGo). Similar results were obtained after stimulation with plastic-bound antibodies (data not shown).



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Fig. 1. Expression of a functional TCR–GFP in ß chain-deficient cells. The ß chain-deficient Jurkat cells (clone 31.13) were transiently transfected with TCR Vß3–GFP together with a TCR V{alpha}1.2 chain. After 48 h cells were conjugated with polystyrene beads either not coated (A and B) or coated with anti-CD3 (C) or anti-TCR antibodies (D). After 3 h cells were stained with anti-CD3 (A and C) or anti-TCR Vß3 antibodies (B and D) to measure TCR–CD3 expression. The FL2 intensity of the dot-plots is indicated. No staining with either anti-CD3 or anti-TCR Vß3 antibodies was detected in 31.13 cells either non-transfected or transfected with GFP only, or with TCR{alpha} only (not shown). In parallel experiments, cells were loaded with Indo-1 and [Ca2+]i was measured either in non-stimulated cells (E) or in cells stimulated with soluble anti-Vß3 (F) or anti-CD3 (G), cross-linked with a goat anti-mouse Ig. Data are from one representative experiment out of four (A–D) or out of two (E –G).

 
Finally, treatment of doubly transfected cells with 1 µM phorbol myristate acetate resulted in a down-regulation of 80.7 ± 5.5% of total surface TCR in three independent experiments. This value is comparable to phorbol myristate acetate-induced TCR internalization in non-transformed human T cells (27), indicating that efficient GFP-tagged TCR internalization is induced not only by anti-TCR antibodies, but also by pharmacological activation of protein kinase C (28). To test whether the GFP-tagged receptors were coupled to the signal transduction machinery we measured calcium mobilization in transfected cells following stimulation with either anti-CD3 or anti-Vß3 antibodies. Both these treatments induced a clear [Ca2+]i rise (Fig. 1EG).

Taken together, the above observations indicate that transfection of V{alpha}1.2 + Vß3–GFP into TCR- cells results in the expression of complete and functional GFP-tagged TCR–CD3 complexes.

Lateral mobility of surface-expressed TCR
To measure TCR–GFP lateral mobility we used the FRAP technique which is based on time-lapse measurement of fluorescence recovery into a bleached region of the cell (24). Since V{alpha}1.2/Vß3–GFP double transfection resulted in a high and homogeneous TCR–CD3 expression in TCR- cells (Fig. 1Go), we used doubly transfected cells for FRAP experiments. Cells were irradiated by a single photo-bleach applied into a large rectangular region across the T cell surface. Fluorescence recovery was measured in narrow regions drawn around the plasma membrane both in the bleached area and at the remaining T cell surface (Fig. 2Go).



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Fig. 2. Rapid TCR–GFP photo-bleaching recovery. (A) A typical photo-bleaching experiment is depicted. Cells were irradiated with a strong laser beam within a large rectangular region. Two T cells either non-stimulated (upper panels) or conjugated with anti-TCR Vß3-coated beads (lower panels) are shown either before (left) or immediately after photo-bleaching (right). Two regions were drawn at the T cell surface—one in the bleached area and one on the remaining cell surface for measurement of GFP fluorescence values. (B) A typical fluorescence recovery plot for a non-stimulated cell and for a cell conjugated with an antibody-coated bead is shown. Data are from one representative experiment out of four.

 
As show in Fig. 2Go, where a typical FRAP experiment is depicted, TCR fluorescence recovery reached a plateau within ~50 s after irradiation.

To estimate the lateral mobility of TCR, experimental data were fitted to a mathematical model for calculation of TCR extent of recovery (R) and diffusion constants (D) (see Methods). As show in Table 1Go and in Fig. 3Go this analysis revealed that TCR exhibit lateral mobility with a mean diffusion constant of ~0.12 µm2/s in non-stimulated T cells. This value is similar to diffusion constant values measured for different transmembrane surface molecules (29).


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Table 1. Values of TCR diffusion constants (D) and mobile fractions (R) (% of the total) in different conditions
 


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Fig. 3. TCR lateral mobility in T cells following inhibition of protein synthesis, blockade of actin cytoskeleton function and conjugation with anti-TCR-coated beads. Photo-bleaching was performed as described in Fig. 2Go. Cells were either left untreated, pre-treated with 10 µg/ml CHX, pre-treated with 10 µM cytochalasin D or conjugated with beads coated with anti-TCR Vß3 antibodies. Symbols represent individual FRAP recordings from at least three independent sessions for each experimental condition.

 
To exclude the possibility that a small fraction of cytosolic TCR/GFP (not completely excluded from the narrow gate drawn at the T cell surface) could contribute to fluorescence recovery, we measured FRAP in T cells in which protein synthesis was blocked several hours before bleaching experiments. These T cells provide a `bona fide' appropriate model to discriminate between surface TCR lateral mobility and intracellular TCRß–GFP diffusion. Indeed, it is likely that a few hours after blocking protein synthesis all the GFP-tagged ß chain, not assembled in TCR complexes, have been degraded (30).

As shown in Table 1Go and in Fig. 3Go, overnight treatment with 10 µg/ml CHX to deprive cells of newly synthesized proteins did not affect the extent and rapidity of fluorescence recovery. This result indicates that fluorescence recovery values reflect TCR lateral mobility and are not due to intracellular ß chain–GFP diffusion.

Surprisingly, also treatment with cytochalasin D at a concentration largely sufficient to poison the T cell actin cyto-skeleton (10 µM) did not affect FRAP of TCR–GFP chimeras (Table 1Go and Fig. 3Go).

Taken together the above results indicate that, in resting conditions, surface-expressed TCR diffuse freely on the T cell surface, via an actin cytoskeleton-independent mechanism.

TCR rapidly enter the signaling domain
We next asked whether GFP-tagged TCR would be efficiently supplied to the signaling area. The ideal experiment to address this question would be to measure FRAP at the contact site between T cells and peptide pulsed APC. However, in our cell system the transfected TCRß–GFP can randomly associate with either the transfected or the endogenous TCR{alpha} chain, making unambiguous interpretation of antigen stimulation experiments difficult. Conversely, using anti-CD3- and anti-Vß3-coated polystyrene beads, a massive and homogeneous down-regulation of GFP-tagged TCR was induced (Fig. 1Go). We therefore studied FRAP at the contact site between T cells and beads coated with anti-Vß3 antibodies (Fig. 2Go). Strikingly, TCR lateral diffusion constants measured at the contact site between T cells and anti-TCR-coated beads were even higher than those measured in resting cells (Table 1Go and Fig. 3Go). This indicates that TCR diffuse rapidly into the signaling zone where they can be potentially exchanged with previously engaged receptors.

Interestingly, the values of R at the signaling area were moderately (but significantly) lower than those obtained in non-stimulated cells (Table 1Go). This indicates that even though non-engaged TCR can rapidly enter the signaling domain, a portion of engaged TCR is trapped by immobilized antibodies and is released at a much slower rate. This is consistent with previously reported data showing that high-affinity ligands, such as immobilized antibodies, do not serially engage TCR and are therefore relatively inefficient in activating T cells (31–33).

To better illustrate TCR lateral diffusion we used a non-quantitative photo-bleaching-based technique named FLIP. This method allows estimation of the fraction of mobile molecules among a total fluorescent population by assessing whether repeated photo-bleaching of a cell area affects or not fluorescence of the remaining cell surface (24). In non-stimulated T cells, repeated bleaches of a defined cell region induced a rapid decrease of fluorescence on the remaining cell surface. Total GFP fluorescence was reduced to ~40% in only 400s (Fig. 4AGo). Interestingly, when the repeated bleaches were applied to the T cell–bead contact site, a comparable FLIP of the remaining T cell surface was observed (Fig. 4BGo).



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Fig. 4. A large fraction of total TCR is bleached by sequential photo-bleaching of a defined T cell region. Cells were sequentially irradiated with a strong laser beam in the indicated region. Two T cells either non-stimulated (A) or conjugated with anti-TCR Vß3-coated beads (B) are shown either before (left) or immediately after the first photo-bleach (center) or after the last photo-bleaching (right). Regions were drawn at the T cell surface in the bleached area and on the remaining cell surface (as shown in Fig. 2Go), and the GFP fluorescence values were plotted against time for non-stimulated (C) and for antibody-stimulated (D) T cells. Data are from one representative experiment out of three.

 
These results parallel those obtained using the FRAP technique and indicate that a large fraction of surface-expressed TCR is mobile.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the present work we provide the first direct measurement of lateral mobility of TCR on the surface of living T lymphocytes. As GFP-tagged TCR–CD3 complexes employed in this study are well expressed on the T cell surface and are coupled to signal transduction machinery, we assume they are likely to give accurate data for wild-type TCR–CD3 complexes.

We show that TCR–CD3 complexes exhibit a relatively fast lateral mobility that resembles that previously measured for other surface molecules (29). As such, these results resolve the issue of whether TCR freely move on the T cell surface and diffuse into the signaling domain (12). Two recent reports have suggested that TCR might exhibit reduced lateral diffusion (13,24). These observations raised questions about how TCR could be efficiently supplied to the signaling area, in order to be engaged by peptide–MHC complexes in a large scale (12).

In the first report, Sloan-Lancaster et al. showed that GFP-tagged TAC/{zeta} chain chimeras expressed on the surface of HeLa cells exhibit a reduced grade of mobility as detected by FRAP and FLIP techniques (24). This observation does not conflict with our results as we measured lateral mobility of fully assembled TCR–CD3 complexes. In addition, these two studies may suggest that complete TCR–CD3 complexes are more mobile than TAC/{zeta} chain chimeras. The molecular mechanisms of such differences in lateral mobility are presently elusive. However, this notion is consistent with previous reports showing that {zeta} chain chimeras may not always completely mimic TCR–CD3{zeta} biological function (34).

In the second report, Grakoui et al. showed the absence of a rapid FRAP of fluorescein-labeled peptide–MHC complexes accumulated in the mature immunological synapse formed between T cells and peptide–MHC complexes embedded in planar lipid bilayers (13). This indicates that TCR-engaged MHC–peptide complexes within the mature immunological synapse are isolated from the free peptide–MHC complexes in the bilayer and may imply that peptide–MHC complexes are kept immobile by engagement with TCR. However, lateral mobility of fully assembled TCR–CD3 complexes was not directly measured in this report. Conversely, our results indicate that, at least in the present experimental system, TCR do not have any particular obstacles to freely diffuse on T cell surface.

It should be noted that our experiments did not address the issue of whether TCR already bound to peptide–MHC complexes present in the immunological synapse may be exchanged. We show here that TCR rapidly diffuse on the T cell surface both in resting conditions and following stimulation, regardless of whether a portion of engaged TCR is trapped in the signaling area (Table 1Go). Hence, our results do not conflict with, but are complementary to the data reported by Grakoui et al. (13).

Strikingly, even the destruction of the T cell actin cytoskeleton did not substantially affect TCR lateral mobility. This observation is important to a better understanding of the role of the actin cytoskeleton in T cell activation. It is well established that T cell antigen recogniton is strictly dependent on a functional actin cytoskeleton (8,10). However, it was not clear whether the actin cytoskeleton might also control TCR lateral mobility, in addition to its role in favoring T cell–APC dynamic interaction (8,10) and in supporting the organization of the immunological synapse (14,35). Here we show that TCR diffuse on the T cell surface independently of a functional actin cytoskeleton. Thus, the actin cytoskeleton may be required to trap and re-organize surface molecules at the immunological synapse, whereas constitutive mobility ensures receptor supply to the synapse. This is in agreement with previously reported data showing that: (i) actin cytoskeleton disruption only moderately affects the dynamics of TCR not yet organized in a mature immunological synapse (14) and (ii) T cell responses to soluble ligands such as anti-TCR–CD3 antibodies are unaffected by cytoskeleton disruption (10,36).

Our results are compatible with the described kinetics of TCR engagement and down-regulation at the T cell–APC contact site (37). Antigen-induced TCR down-regulation has a very fast initial rate since it can be measured as early as 30–60 s after conjugate formation (S. Müller and S. Valitutti, unpublished observations) and may involve up to ~40% of the total TCR within 15 min (37).

Our estimates of TCR diffusion constants (~0.12 µm2/s) are compatible with such a rapid mechanism of TCR engagement and down-regulation (Fig. 3Go). Indeed, assuming that an activated T lymphocyte could be compared to a sphere with a diameter of 10–12 µm, we estimate that individual TCR would be able to move over the entire T cell surface (314–452 µm2) within ~60 min.

In addition, TCR lateral mobility appears to be in excess compared to the rate of TCR down-regulation, in particular at later time points after T cell–APC conjugation when the TCR down-regulation rate slows down (37). This indicates that only a fraction of potentially available receptors is productively engaged at the T cell–APC contact site.

As an explanation to this phenomenon, we have recently proposed that the massive degradation of ZAP-70, which occurs in parallel with TCR engagement and down-regulation, may limit the rate of TCR productive engagement and may therefore act as a `pace-maker' of TCR serial engagement (27).

Finally, our results also provide an explanation for an unresolved issue which concerns the total extent of antigen-induced TCR down-regulation. Indeed down-regulation of ~90% of total TCR has been observed at high antigen concentrations (5), implying that most of the surface-expressed TCR should be motile. Our results, obtained with the FLIP technique, are consistent with this figure since we show that in ~7 min a large fraction of total TCR has been already photo-bleached (Fig. 4Go).

In conclusion, in the present work we provide new insights to the understanding of the mechanisms of signaling cascade assembly at the T cell–APC contact site. It is well established that the process of T cell antigen recognition is the result of dynamic and reversible re-localization of surface molecules and signaling components. To be efficient such a mechanism of activation requires rapid dynamics of TCR and accessory molecules on the T lymphocyte surface. Here we show that TCR possess the intrinsic lateral mobility that allows them to fulfil this task.


    Acknowledgments
 
We thank Thierry Laroche for help in confocal microscopy, and Denis Hudrisier, Sabina Mueller, Andrew Quest and Anne Wilson for discussion and critical reading of the manuscript. This work was supported by grants from the Ligue Contre le Cancer, La Association pour la Recherche sur le Cancer, La Fondation pour la Recherche Medicale, La Fondation Gabriella Giorgi Cavaglieri. L. W. was supported by a grant from the Wellcome Trust.


    Abbreviations
 
APC antigen-presenting cell
[Ca2+]i intracellular Ca2+ concentration
CHX cycloheximide
FLIP fluorescence loss in photo-bleaching
FRAP fluorescence recovery after photo-bleaching

    Notes
 
Transmitting editor: D. R. Littman

Received 9 April 2001, accepted 30 August 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
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