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
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Abstract |
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Keywords: GFP, fusion proteins, lateral mobility, signal transduction
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Introduction |
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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 peptideMHC 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 (1316). 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 cellAPC 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-TCRCD3. We discuss our results in the context of the proposed models of T cell activation.
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Methods |
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TCR cDNA from HA
-PJ6
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-PCR3 and pEF TAg at 280 V and 900 µF in a BioRad (Hercules, CA) Gene Pulser; 3648 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 TCRCD3 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 TCRCD3 (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):
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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 MannWhitney 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.
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Results |
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Transient transfection of the Vß3GFP 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 TCRCD3 complexes in the cell population (data not shown). This is most likely due to the fact that endogenous chain expression was also repressed in 31.13 cells because of chronic ß chain absence (26). Co-transfection of Vß3GFP with the
chain derived from the same HA1.7 TCR (V
1.2) (25) resulted in very high and homogeneous expression of TCRCD3 complexes, as detected by staining with anti-CD3 and anti-Vß3 antibodies (Fig. 1A and B
). Surface-expressed receptors were internalized following stimulation with anti-CD3 or anti-Vß3-coated polystyrene beads (Fig. 1C and D
). Similar results were obtained after stimulation with plastic-bound antibodies (data not shown).
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Taken together, the above observations indicate that transfection of V1.2 + Vß3GFP into TCR- cells results in the expression of complete and functional GFP-tagged TCRCD3 complexes.
Lateral mobility of surface-expressed TCR
To measure TCRGFP 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 V1.2/Vß3GFP double transfection resulted in a high and homogeneous TCRCD3 expression in TCR- cells (Fig. 1
), 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. 2
).
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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 1 and in Fig. 3
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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As shown in Table 1 and in Fig. 3
, 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 chainGFP 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 TCRGFP chimeras (Table 1 and Fig. 3
).
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 TCRßGFP can randomly associate with either the transfected or the endogenous TCR 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. 1
). We therefore studied FRAP at the contact site between T cells and beads coated with anti-Vß3 antibodies (Fig. 2
). 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 1
and Fig. 3
). 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 1). 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 (3133).
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. 4A). Interestingly, when the repeated bleaches were applied to the T cellbead contact site, a comparable FLIP of the remaining T cell surface was observed (Fig. 4B
).
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Discussion |
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We show that TCRCD3 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 peptideMHC complexes in a large scale (12).
In the first report, Sloan-Lancaster et al. showed that GFP-tagged TAC/ 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 TCRCD3 complexes. In addition, these two studies may suggest that complete TCRCD3 complexes are more mobile than TAC/
chain chimeras. The molecular mechanisms of such differences in lateral mobility are presently elusive. However, this notion is consistent with previous reports showing that
chain chimeras may not always completely mimic TCRCD3
biological function (34).
In the second report, Grakoui et al. showed the absence of a rapid FRAP of fluorescein-labeled peptideMHC complexes accumulated in the mature immunological synapse formed between T cells and peptideMHC complexes embedded in planar lipid bilayers (13). This indicates that TCR-engaged MHCpeptide complexes within the mature immunological synapse are isolated from the free peptideMHC complexes in the bilayer and may imply that peptideMHC complexes are kept immobile by engagement with TCR. However, lateral mobility of fully assembled TCRCD3 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 peptideMHC 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 1). 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 cellAPC 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-TCRCD3 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 cellAPC contact site (37). Antigen-induced TCR down-regulation has a very fast initial rate since it can be measured as early as 3060 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. 3). Indeed, assuming that an activated T lymphocyte could be compared to a sphere with a diameter of 1012 µm, we estimate that individual TCR would be able to move over the entire T cell surface (314452 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 cellAPC 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 cellAPC 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. 4).
In conclusion, in the present work we provide new insights to the understanding of the mechanisms of signaling cascade assembly at the T cellAPC 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.
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Acknowledgments |
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Abbreviations |
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APC antigen-presenting cell |
[Ca2+]i intracellular Ca2+ concentration |
CHX cycloheximide |
FLIP fluorescence loss in photo-bleaching |
FRAP fluorescence recovery after photo-bleaching |
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Notes |
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Received 9 April 2001, accepted 30 August 2001.
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References |
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