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Département de Biologie Cellulaire, Institut National de la Santé et de la Recherche Médicale, Unité 567, Center National de la Recherche Scientifique, Unité Mixte de Recherche 8104, Université René Descartes, Institut Cochin, Paris, France; and Laboratoire Labelisé par la Ligue Nationale contre le Cancer, Paris, France
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Introduction |
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-1, CD3
, and linker for activation of T cells (1). Consistent with these findings, CD5 is one of the molecules that contributes to set the threshold of positive and negative selection in the thymus (4, 5, 6 ; for review, see Ref.7). CD5 expression on mature, single-positive thymocytes also directly parallels the avidity of the positively selecting TCR-MHC-ligand interaction (8). In peripheral T cells too, CD5 inhibition of TCR signaling has been observed. CD5-negative T cells show enhanced proliferation to TCR triggering (1, 9). Moreover, when lymph node T cells are cultured in vitro, their CD5 level decreases rapidly in the absence of MHC-TCR interactions, and they show in parallel an increased response to TCR engagement (10). Thus, in mature T cells, as in thymocytes, the expression level and the modulatory effect of CD5 are finely tuned by the intensity of the TCR-dependent signal. How can CD5 exert its inhibitory effect? The nature of the physiological ligand of CD5 is ignored. It has been proposed that CD72 could be this ligand (11), but this is questionable (2). At present, even the fact that such a ligand exists is unclear. The analysis of the signaling events controlled by CD5 has been based on experiments in which CD5 was clustered with Ag receptors in B or T cells. This type of approach has allowed dissection of the tyrosine phosphorylation of the cytoplasmic tail of CD5 after stimulation of Ag receptors (12, 13, 14) and, at least in B cells, demonstration of the role played by the Src-like autophosphorylation motif of this tail in CD5 inhibition (15). We did not explore whether CD5-Ag receptor aggregation constituted a biased approach in these experiments. However, artificial clustering is not always indispensable for CD5 inhibition, since it can be observed after stimulation of the TCR alone with Abs (5, 9) or after Ag presentation (9, 10). These findings might be related to the constitutive association of a fraction of CD5 with Ag receptors (16, 17).
In T cells Ag recognition takes place in a structure called the immunological synapse (IS).
3This structure results from the assembly of a supramolecular complex including a number of transmembrane proteins (CD3, CD2, CD28, LFA-1), excluding others (CD43), and recruiting an array of intracellular molecules involved in signal transduction (Fyn, Lck, ZAP-70, Vav, SH2 domain-containing leukocyte protein of 76 kDa, phospholipase C-, protein kinase C 
) (for reviews, see Refs.7 ,18 , and 19). An accumulation of CD5 at the IS has been recently reported in Jurkat cells (20). However, it has not been established whether CD5 was recruited at the IS under conditions that would be compatible with an inhibitory effect exerted there. It is not known in particular if CD5 at the IS exerts an inhibitory effect by interfering with synapse formation and steadiness and/or with TCR signaling. Moreover, is this inhibition all-or-none or finely tuned? We show here that CD5 can be recruited to various human and murine IS. In one particular IS, we have analyzed in detail the kinetics of its recruitment and its Ag dependence. Simultaneous video recordings of CD5 recruitment together with either CD3 recruitment or Ca2+ responses were performed. We conclude that a role of CD5 in IS formation or stability is unlikely, and that CD5 acts mostly or entirely by inhibiting signaling at the synapse. As in B cells (12), our results also suggest a role for the pseudo-immunoreceptor tyrosine-based activation motif (ITAM) of CD5 in this process.
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Materials and Methods |
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The following mouse mAbs were used: anti-human CD3
(UCHT1, ascite), anti-human CD5 (O490D, a gift from Dr. L. Boumsell, Institut National de la Santé et de la Recherche Médicale, Unité 448, Creteil, France), and anti-phosphotyrosine (4G10; Upstate Biotechnology, Lake Placid, NY). PE- or biotin-conjugated hamster anti-murine CD3 Abs (145-2C11) were purchased from BD PharMingen (San Diego, CA). Rhodamine Red-X-conjugated and PE-conjugated goat F(ab')2 anti-mouse IgG secondary Abs were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA) and Immunotech (Marseille, France), respectively. Streptavidin Alexa Fluor 488 conjugate was purchased from Molecular Probes (Eugene, OR).
Cells
The murine T cell hybridoma T8.1, provided by Dr. O. Acuto (Institut Pasteur, Paris, France), expresses a human CD4 and a chimeric human (V
,V
)-mouse (C,C) TCR specific for a tetanus toxin peptide (tt830–843; Neosystem, Strasbourg, France; referred to as TTP) restricted to HLA-DRB1*1102 (21). The murine cell transfectants used as APC (L625.7) express HLA-DR*1102 and endogenous B7.1, ICAM-2, and, at a low level, ICAM-1 (data not shown). T8.1 was maintained in DMEM with 10% FCS (Life Technologies, Gaithersburg, MD), 2 mM L-glutamine, and antibiotics (complete medium) supplemented with 400 nM methotrexate, 1 mg/ml G418, and 50 µM 2- ME. T8.1 stable transfectants were maintained in the same medium supplemented with 1 µg/ml puromycin (Sigma-Aldrich, Saint-Quentin Fallavier, France). L625.7 cells were cultured in complete medium in the presence of 250 µg/ml G418. Jurkat J77cl20 (J77) T cells and Raji B cells were cultured in RPMI 1640 medium with 10% FCS.
Plasmid constructs
CD5 was amplified by PCR using the primers 5'-TACCCGGCCAGACACCCTCACCTG-3' and 5'-GCAGCCTCTGAGCCCCATGCAG-3' and the full-length cDNA of human CD5 into the pRC vector as a template. The PCR product was introduced in the TOPO TA cloning vector (Invitrogen, Leek, The Netherlands) according to the manufacturer’s instructions, then subcloned after EcoRI digestion into the pEGFP-N1 or pECFP-N1 vector (Clontech Laboratories, Palo Alto, CA) in-frame with the N-terminal sequence of the green or cyan fluorescent protein (GFP or CFP). Constructs (CD5-GFP, CD5-CFP) were verified by sequencing. The CD3-yellow fluorescent protein (YFP) retroviral construct was a gift from Dr. M. Malissen (Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique, Marseilles-Luminy, France). The CD5 retroviral vector was constructed by inserting the full-length cDNA of human CD5 into a murine stem cell virus-retroviral vector (CD5-MSCV).
To make the desired deletion of the pseudo-ITAM motif (YSQPPRNSRLSAYPAL) in the CD5cytoplasmic domain, the following mutagenic oligonucleotides were used: 5'-GCCTCCCACGTGGATAACGAAGAAGGGGTTCTGCATCGCTCC-3' and 5'-GGAGCGATGCAGAACCCCTTCTTCGTTATCCACGTGGGAGGC-3', according to the manufacturer’s protocol of the Quick Change site-directed mutagenesis kit (Stratagene, La Jolla, CA), and using CD5-murine stem cell virus-retroviral vector as a template. The construct was verified by DNA sequencing.
Cell transfection
Stable T8.1 transfectants were obtained as follows: cells (107 in DMEM supplemented with 10% FCS) were electroporated in a Gene Pulse cuvette (Bio-Rad, Hercules, CA) with 5 µg of CD5-GFP or CD5-CFP plasmid at 960 µF at 250 V. Cells were cotransfected with 20 µg of Sr plasmid to confer puromycin resistance. Forty-eight hours after transfection, cells were cloned in 96-well tissue culture plates in DMEM containing 400 nM methotrexate, 1 mg/ml G418, and 1 µg/ml puromycin. Puromycin-resistant transfectants were then selected for CD5-GFP expression by flow cytometry (EPICSXL; Beckman Coulter, Miami, FL) and for equivalent expression of CD3, CD28, LFA-1, and CD43 markers with wild-type cells. T8.1 cells expressing both CD5-CFP and CD3-YFP were obtained by infecting cells stably expressing CD5-CFP with a retrovirus (pMFG-B2) containing CD3-YFP. Retroviral production was obtained by transfecting the Plat-E packaging cell line (22) using Fugene 6 (Roche, Meylan, France). After 24 h at 37°C, fresh medium was added, and cells were further cultured for 24 h at 32°C. Supernatant was then added to T8.1 CD5-CFP cells for 24 h at 32°C, and YFP-positive cells were sorted with a fluorescence cell sorter (Elite; Beckman Coulter). Cells expressing different levels of CD5 were obtained by infecting T8.1 cells with the retrovirus construct containing CD5, followed by limited dilution cloning and analysis for CD5 expression by flow cytometry. J77 cells were transfected with 10 µg of CD5-GFP at 960 µF and 320 V and were used 24 h after transfection.
Cell preparation
C57BL/6 splenic dendritic cells (DCs) were purified as previously described (23), and macrophages were purified by magnetic depletion of splenic T cells, B cells, and DCs with a mixture of appropriate rat anti-mouse receptor Abs, followed by incubation with Dynabeads (Dynal Biotech, Lake Success, NY) coupled to anti-rat IgG. Splenic T cells from P14 TCR-transgenic mice were purified by negative depletion with a mixture of anti-CD45RB (Cedarlane, Hornby, Canada), anti-MAC1 (BD PharMingen), and anti-CD11c Abs (23).
Conjugate formation and fluorescence analysis
Twelve to 16 h before the experiment, L625.7 fibroblasts (3 x 105/ml) were plated in their culture medium with or without peptide Ag and incubated on glass coverslips at 37°C. Raji cells were incubated with 1 µg/ml staphylococcal enterotoxin E (SEE) superantigen for 10 min at 37°C and plated for 10 min on poly-L-lysine-coated glass coverslips. Splenic DCs and macrophages were incubated with 10-6 M LCMV gp33 peptide (Neosystem, Strasbourg, France) for 90 min at 37°C, then plated for 10 min on poly-L-lysine-coated glass coverslips. T cells (5 x 105), washed once in mammalian saline (140 mM NaCl, 5 mM KCl, 10 mM HEPES (pH 7.3), 1 mM CaCl2, 1 mM MgCl2, and 1 mg/ml glucose), were added to APCs, and the cells were incubated at 37°C for various periods of time. Cells were then fixed with 3% paraformaldehyde in PBS for 10 min and, after several washes, incubated for 20 min with 0.1 M glycine in PBS. Cells were permeabilized, or not, in a PBS-0.1% Triton X-100 solution for 10 min and labeled in PBS-0.2% BSA with the different Abs. After washing, coverslips were mounted in Mowiol (Sigma-Aldrich). Immunofluorescence and transmission light images were acquired on an Eclipse TE300 inverted microscope (Nikon, Badhoevedorp, The Netherlands) equipped with a cooled CCD camera (CoolSNAPFx; Roper Scientific, Evry, France). Image capture and quantitative analysis were realized with Metafluor and Metamorph software (Universal Imaging, West Chester, PA).
Single-cell Ca2+ and fluorescence video imaging
L625.7 or Raji cells were prepared and pulsed with Ag as described above and plated on glass coverslips mounted on 25-mm petri dishes. T8.1 or J77 cells (5 x 105) were incubated for 20 min at 37°C with 1 µM Fura-2/AM (Molecular Probes) and added to the APC in mammalian saline at 37°C. After T8.1 or J77 addition, GFP, CFP, YFP, and Fura-2 fluorescence were followed with a TE300 inverted microscope and the Metafluor software. Averaged Ca2+ responses were calculated in cells showing a Ca2+ increase >150 nM. To avoid a filter effect on the averaged Ca2+ trace due to the asynchrony of the individual responses, the peak Ca2+ responses of all cells were synchronized before averaging. Transmission light images were taken every 12 s in turn with fluorescence images.
Adhesion assay
Fibroblasts were laid on glass coverslips for 16–20 h in the presence of 1 µg/ml TTP. Cell density was adjusted so as to achieve a uniform monolayer. To quantify their adhesion, T8.1 cells labeled with 10 µM CFSE (Molecular Probes) and suspended in mammalian saline were introduced into a parallel plate flow chamber (Immunetics, Cambridge, MA) that was affixed to the coverslip bearing the fibroblast monolayer. In this system a flow rate of 2 ml/h resulted in a shear stress of 0.09 dynes/cm2. Image acquisition was started as soon as the first CFSE-labeled T cells appeared in the chamber and was stopped after 2 min. The initial adhesion phase was then quantified by ratioing the number of cells remaining adherent and the total number of cells that had passed through the microscopic field. Thereafter, cells were left adhering for 8 min more, and then increasing shear stresses were applied, ranging from 0.09–4.5 dynes/cm2. Remaining cells were quantified for each condition, and results were expressed as the percentage of cells remaining adherent.
Quantitative analysis of CD3 or CD5 recruitment at the synapse
Dynamic synaptic recruitment was assessed with the Metafluor software as the ratio between the fluorescent intensity in a region centered on the IS and the intensity over the whole cell. Results are expressed as the fold increase compared with ratio observed in the same regions before contact formation. For averaging purposes, time zero for each response was that of the initial detectable cell-cell contact. For synaptic phosphotyrosine signal and whole CD5 or CD5 
ITAM synaptic recruitment, we measured the intensity ratio between a region in the synapse and a region of same area outside the synapse.
Statistics
Data are expressed as the mean ± SD, and the significance of differences between two series of results was assessed using Student’s t test.
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Results |
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The distribution of CD5 molecule at the surface of unstimulated T cells is usually even, with no sign of spontaneous polarization or clustering. However, when examined after 15 min of APC-T cell interaction, CD5 was found clustered at the contact zone. Such a clustering was observed at the synapse formed between Jurkat T cells (J77 cells transiently transfected with a construction coding for a CD5-GFP fusion protein) and Raji B cells presenting the superantigen SEE (Fig. 1A). It was also observed in conjugates formed between P14 T cells, which express a transgenic TCR specific for lymphocytic choriomeningitis virus glycoprotein peptide gp33 and H-2Db (24), and either dendritic cells (Fig. 1, B and C) or macrophages (Fig. 1D), both pulsed with gp33. A double labeling of CD5 and CD3 revealed a tight colocalization of the two molecules in the different cell systems. In most conjugates formed between Jurkat T cells and SEE-loaded Raji B cells, CD5 was clustered with CD3 in a small area in the center of the immune synapse (Fig. 1A). In P14-dendritic conjugate, either a densely packed localization (Fig. 1B) or a distribution over most of the contact zone (Fig. 1C) was observed. However, in both situations, CD3 and CD5 remained precisely colocalized.
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The recruitment dynamics of CD5 were further analyzed at the IS formed between T8.1 cells (a CD5- murine hybridoma T cells) stably transfected with the full-length CD5 molecule fused to GFP and MHC class II-expressing fibroblasts as APC (25, 26). Fig. 2A, left panel, shows the expression of CD5-GFP in T8.1-transfected cells compared with T8.1 wild-type cells. Both GFP fluorescence and CD5 levels analyzed with a CD5-specific Ab were measured. By fluorescence microscopy, CD5-GFP appeared homogeneously distributed at the plasma membrane of single T8.1 cells (right panel).
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) = 1.8 ± 0.3 min). This recruitment was often unstable, a phenomenon that was manifest in single-cell videos (see movie 1),
4but not in the average shown in Fig. 2C. APCs pulsed with 0.1 µg/ml induced a recruitment that affected a larger amount of CD5 molecules and took more time to reach a plateau ( = 5.3 ± 0.5 min). With APCs pulsed with a larger dose of Ag (1 µg/ml), an even larger proportion of CD5 accumulated at the synapse with similar kinetics ( = 4.8 min ± 0.2). Under these conditions we calculated that 60 ± 10.6% of total CD5 (n = 40) accumulated in the IS. The Ag-dependent recruitment of CD5 was quite stable as shown in single-cell videos (see movie 2). Very similar kinetics was observed with Jurkat cells interacting with SEE-pulsed Raji cells (not shown). CD5 and CD3 recruitment at the IS
As CD5 recruitment is influenced by Ag recognition, we next examined the relationship between CD3 and CD5 movements, monitored simultaneously. To this end we used T8.1 cells expressing both CD5-CFP and CD3-YFP, interacting with Ag-pulsed fibroblasts. Fig. 3A shows in a representative cell that the recruitment of CD3 and CD5 occurred with similar time courses. A quantitative analysis of the kinetics of CD3 and CD5 clustering, measured as the relative increase in CD5-CFP and CD3-YFP fluorescence at the IS, also demonstrated the parallel recruitment of CD5 and CD3 (Fig. 3B, left panel). The duration required for these increases to reach half-maximum values was then measured in 26 cells (Fig. 3B, right panel). In the majority of cells these durations were quite similar. In a few cases CD5 recruitment lasted slightly longer than CD3 recruitment. The average increase half-times were 3.7 ± 2.5 and 4.4 ± 3 min for CD3 and CD5, respectively; these values were not significantly different. In addition, the mean maximum fold increases at the IS were similar for CD5 and CD3 (2.4 ± 1.1 and 2.6 ± 0.9, respectively).
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-specific Ab. Conjugates formed between Ag-pulsed APCs and T8.1 cells were analyzed after 5, 20, or 60 min of interaction (30 conjugates for each condition). The fraction of recruited CD3 was not significantly different in T8.1 cells expressing, or not, CD5 (Fig. 3C). Moreover, at each time point, the number of conjugates showing CD3 clustering at the contact zone was unaffected by the presence of CD5 (data not shown). A similar conclusion was reached when following CD3 in cells expressing CD3-YFP (data not shown). CD5 expression does not alter initial and late T cell adhesion to APCs
Besides the CD3/TCR complex, various molecules, including adhesion molecules, are involved to initiate and stabilize the interaction of a T cell with an APC. We therefore analyzed the influence of CD5 expression on initial and late adhesion steps between T8.1 cells and L625.7 cells interacting in a parallel plate flow chamber. We first assessed the ability of CD5- and CD5+ T cells to stop rolling on a monolayer of Ag-pulsed fibroblasts under a low shear stress of 0.09 dynes/cm2. The fraction of T cells arrested in a 2-min assay of rolling on APCs was not significantly different for the two cells types, i.e., 13.2 ± 2.9% of CD5- T cells and 17.4 ± 4% of CD5+ T cells (Fig. 4A). Next, cells that had been adhering for 10 min on Ag-pulsed APCs were submitted to an increasing shear stress (by steps, up to 4.5 dynes/cm2) to assess the strength of their adherence. Again, the percentage of cells remaining on the monolayer under a fast flow was not significantly different regardless of whether T cells expressed CD5 (Fig. 4B).
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Having established that CD5 is recruited at the IS, but does not alter its formation, we then analyzed CD5-mediated inhibition of T cell signaling induced by APC at the single-cell level. The level of tyrosine phosphorylation (
-Tyr) and Ca2+ responses were monitored in wild-type CD5- T8.1 cells and in T8.1 cells selected for intermediate or high levels of the molecule (Fig. 5A). -Tyr was assessed with the anti-phosphotyrosine mAb 4G10 in conjugates formed between the different T8.1 cells and Ag-loaded APCs. Fig. 5B shows typical conjugates obtained after 5 min of cell-cell contact. The amount of -Tyr was significantly lower in cells expressing CD5. To further analyze this inhibition, kinetic experiments were performed. They showed that both the percentage of cells with increased -Tyr (Fig. 5C) as well as the level of -Tyr at the IS (Fig. 5D) were reduced by CD5. This inhibition was of greater magnitude in cells with the highest expression of CD5. Thus, CD5 impairs Ag-induced tyrosine phosphorylation in T cells, correlating with the expression level of the molecule.
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50% of the cases, CD5 was already concentrated at the synapse before the Ca2+ started to rise (Fig. 6A, left), whereas in the other half, high levels of CD5 recruitment coincided with the Ca2+ response (Fig. 6A, right). In addition, Ag-free L625.7 fibroblasts never triggered Ca2+ responses in T8.1 cells, whereas some extent of CD5 recruitment was observed under those conditions, as mentioned above. These findings demonstrate that the initial CD5 recruitment is not triggered by TCR-dependent signaling events (such as a Ca2+ rise). By comparing CD5- and CD5+ T8.1 cells, we examined whether CD5 exerted an inhibitory effect on these APC-induced Ca2+ responses by affecting the probability of observing a response or by altering its amplitude or its time course. The percentage of responding T8.1 cells was 74 ± 7% for CD5+ and 70 ± 2% for CD5- cells (n = 3 experiments), i.e., the probability of observing a Ca2+ response was unaffected by CD5 expression. However, as shown in Fig. 6B, CD5 expression resulted in a reduced amplitude of the Ca2+ responses in the numerous individual cells analyzed (upper panel) and in the mean responses without affecting their time course, as shown by the overlap of the two scaled responses (lower panel). Knowing that the pseudo-ITAM of CD5 is required to impair B cell receptor-induced Ca2 in B cells (12), we finally tested its contribution to the inhibition of the Ca2+ response triggered by Ag presentation in T cells. The amplitude of the response was quite similar in CD5- cells and in cells expressing the CD5 molecule lacking the pseudo-ITAM, well above the Ca2+ levels observed in cells expressing the wild-type receptor (Fig. 6C).
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Discussion |
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The kinetics of CD5 recruitment at the IS comprise a fast Ag-independent and a slower Ag-dependent component. Everything happens as if bringing the T cell and the APC in close contact, thanks to ill-defined adhesion molecules, was sufficient to rapidly induce (with a time constant of 1.8 min) the recruitment of a limited amount of CD5 at the forming IS. It has been reported that T cell engagement and activation initiate before IS is fully formed (27). Therefore, CD5 probably controls TCR signaling from the moment the T cell and the APC come into contact. However, some signals associated with Ag recognition are necessary to stabilize and further increase CD5 at the IS. This process requires a few minutes (time constant 5 min for the IS under study). Interestingly, the amount of Ag on the APC seems to affect mainly the level of CD5 being recruited at the IS and to poorly affect its rapid initial increase after the contact. This is exactly what one would expect from a fine-tuner of TCR signaling.
By monitoring CD5 recruitment simultaneously with CD3 clustering, one can see that the two events occur in a very narrow window time. The time constants of CD3 and CD5 recruitment are most often similar; in only one-quarter of the cases is CD3 recruitment slightly faster than that of CD5. Besides, CD3 clustering is clearly unaffected by the presence of CD5. Thus, CD5 does not act by slowing down CD3 recruitment to the synapse. These data are consistent with an independent recruitment of CD3 and CD5. This is not to contradict the above conclusion that efficient Ag presentation and TCR signaling contribute to the stabilization/enrichment of CD5 at the IS.
One could have expected that CD5 could act at the IS as one more adhesion molecule if its elusive ligand was present on L625.7 cells. This ligand is unlikely to be CD72, as initially proposed (11), but could be a broadly expressed cell surface protein yet to be identified (28). The functional importance of this ligand is unclear, since it has been recently reported that inhibition of TCR signaling by CD5 during intrathymic selection did not require the CD5 extracellular domain (29). A distinct, and opposite role of CD5 at the IS could have been that, by inhibiting T cell signaling, CD5 would exert an inhibitory effect on cell-cell adhesion. Neither of these hypotheses was validated by experiments measuring both adhesion events associated with synapse formation and the strength of the bonds formed. On the whole, therefore, CD5 seems neutral for synapse formation and stability.
In contrast, single-cell functional assays demonstrated that CD5 expression resulted in an inhibition of the Ca2+ response. Two tyrosine residues of CD5 are significantly phosphorylated in T cells, Y429 and Y463 (13, 14), the former being in the pseudo-ITAM motif of CD5, whose deletion abolished the inhibitory effect of the molecule in B cells (12). Our present results suggest that this motif is also essential for CD5 to inhibit the Ca2+ response induced by Ag presentation in T cells. Although we do not know what is the mechanism(s) underlying this pseudo-ITAM-dependent inhibition, this finding was confirmatory of previous results indicating that CD5 expression was inversely correlated with Ca2+ elevation triggered through the CD3/TCR complex. In particular, it has recently been shown that peripheral T cells expressing transgenic TCR were able in vitro to alter their CD5 level depending on the presence or the absence of MHC-peptide complexes stimulating their TCR; for a given TCR, increasing CD5 expression resulted in reduced, but not suppressed, APC-induced Ca2+ responses in T cell populations (10). Our data also showed that CD5 expression was not accompanied by a failure to trigger Ca2+ responses, but by a reduction of its amplitude. Similarly, we found a reduction of the increase in tyrosine phosphorylation at the IS after Ag presentation in the presence of CD5. CD5 strongly affected the amplitude of the response at the earliest time point studied, in good agreement with the calcium studies. Moreover, tyrosine phosphorylations were clearly titrated down by the molecule. Thus, CD5, rapidly recruited to the IS, does not influence its formation, contrary to other inhibitory receptors such as CD43 (30), but is an early regulator of the signaling events occurring at the IS to control T cell activation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr Georges Bismuth, Département de Biologie Cellulaire, Institut Cochin, 22 rue Méchain, 75014 Paris, France. E-mail address: bismuth{at}cochin.inserm.fr
3 Abbreviations used in this paper: IS, immunological synapse; CFP, cyan fluorescent protein; DC, dendritic cell; GFP, green fluorescent protein; 
-Tyr, phosphotyrosine; SEE, staphylococcal enterotoxin E; TTP, tetanus toxin peptide; -Tyr, level of tyrosine phosphorylation; YFP, yellow fluorescent protein; ITAM, immunoreceptor tyrosine-based activation motif.
4 The on-line version of this article contains supplemental material.
Received for publication July 31, 2002. Accepted for publication February 24, 2003.
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