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Differential Roles of Lck and Itk in T Cell Response to Antigen Recognition Revealed by Calcium Imaging and Electron Microscopy¹

Emmanuel Donnadieu^*, Valérie Lang^2,*, Georges Bismuth^*, Wilfried Ellmeier, Oreste Acuto, Frédérique Michel^3, and Alain Trautmann^3,4,*

^* Laboratoire d’Immuno-Pharmacologie, Centre National de la Recherche Scientifique, Paris, France; Institute of Immunology at Vienna International Research Cooperation Center, University of Vienna, Vienna, Austria; and Molecular Immunology Unit, Department of Immunology, Institut Pasteur, Paris, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ag recognition triggered at the interface between a T cell and an APC is conditioned by cell-cell adhesion and cytoskeletal remodeling. The role played in these phenomena by Lck and Itk, two protein tyrosine kinases essential for T cell signaling, was examined. Early T cell responses (membrane ruffling, Ca²⁺ response, APC-T cell adhesion) were monitored in T cells overexpressing kinase-defective (KD) Lck and Itk mutants by combining fluorescence imaging and electron microscopy. Neither Lck nor Itk appears to be involved in the Ag-independent formation of a small and labile contact interface between T cells and APCs. By contrast, the Ag-induced Ca²⁺ response in a cell population is similarly blunted in both KD transfectants. However, the underlying mechanisms are strikingly different for the two kinases. The major effect of Lck-KD is to reduce the probability of giving rise to quasi-normal Ca²⁺ responses, whereas overexpression of Itk-KD results in a tuning down of all single-cell Ca²⁺ responses. In addition, Lck, but not Itk, is required for the formation of a stable T/APC conjugate and for T cell polarization after Ag stimulation. Overall, our results lead to a clear distinction between Lck and Itk. Lck plays an ignition role, controlling all the downstream events tested here, whereas Itk amplifies the Ca²⁺ response, but is dispensable for APC-induced adhesive and morphological responses.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
One of the earliest events occurring during TCR signaling is the activation of different protein tyrosine kinases (PTKs).⁵ Thus, it has been shown that Src kinases (Fyn and Lck) are involved in the TCR-induced phosphorylation of the different CD3 chains on their immunoreceptor-based activation motif. This allows the SH2-dependent recruitment and the activation of ZAP-70, which leads, in turn, to the phosphorylation of key downstream signaling molecules, such as linker for activation of T cells (LAT) and SH2 domain-containing leukocyte protein of 76 kDa (SLP)-76 (1, 2, 3). The Tec family kinases also participate in signal transduction pathways downstream of the TCR (for review, see Refs. 4 and 5). In T cells, at least three members of this family, Itk, Rlk/Txk, and Tec, are expressed. Genetic studies have underlined the importance of Itk in T cell function, as T cells from Itk-deficient mice show impaired proliferation and Th2 differentiation (6, 7). Analysis of signaling events reveal that Itk^-/- and Itk^-/- Rlk^-/- T cells have markedly decreased phospholipase C-1 (PLC-1) tyrosine phosphorylation, inositol trisphosphate production and Ca²⁺ mobilization, while the overall pattern of immediate tyrosine phosphorylation appears to be normal (8, 9). Thus, Itk in T cells is reminiscent of Btk, another Tec kinase, which controls the Ag-dependent Ca²⁺ response in B cells (10). Recent data suggest that Itk, through its interaction with the scaffolding membrane protein LAT and the adaptor protein SLP-76, is involved in an amplification step that is necessary for a full activation of PLC- and, therefore, of the Ca²⁺ response (11, 12, 13). Most of these conclusions have been drawn from analysis of the tyrosine phosphorylations induced by anti-CD3 Abs, which have been very informative, but which fail to take into account cell-cell interactions and, in particular, the spatial constraints imposed by Ag recognition.

The importance of these spatial constraints has been underlined in a number of recent reports. Thus, we and others have shown the importance of the cytoskeleton alterations that can be detected together with and even before the Ca²⁺ response induced in T cells by APC (Refs. 14, 15, 16 ; for a review, see Ref. 3). Disruption of the active cytoskeleton leads to inhibition of the Ca²⁺ response and abrogation of T cell activation (16, 17).

In addition, major redistribution of cell surface molecules is observed when T lymphocytes recognize an Ag on APCs. Analysis of fixed T cell/APC conjugates reveals that cell surface molecules segregate into distinct regions or clusters, referred to as supramolecular activation clusters, with the TCR/peptide-MHC accumulating in a central region surrounded by a peripheral ring of adhesion molecules (18). Recently, through the use of live T cells and lipid bilayer in which fluorescently labeled molecules have been incorporated, the dynamic nature of these redistributions has been underlined (19). These reorganizations appear to be required for full T cell activation. Hence, immunological synapse formation correlates with full T cell activation, and any processes known to prevent T cell activation such as antagonist peptides, fail to trigger the redistribution of surface molecules (19).

The intracellular signaling events required for these earliest responses are to a large extent undetermined. In a previous work we have begun to characterize the signaling pathway linking Ag recognition to T cell membrane ruffling and Ca²⁺ increase in murine naive T lymphocytes during their interaction with B cells (16). Using a pharmacologic approach, our study highlighted the major role played by PTKs because Ag-induced cytoskeletal responses were entirely blocked by PTKs inhibitors.

Here we have analyzed more precisely the role played by two PTKs, Lck and Itk, in the earliest steps of Ag recognition. To this end, we have used a dominant-negative approach based on overexpression of catalytically inactive mutants of these PTKs in a murine T cell hybridoma. The effects of Lck and Itk kinase-defective (KD) mutants in early Ag-induced T cell responses (conjugate formation, membrane ruffling, Ca²⁺ response, T-APC adhesion) were then monitored by combining video imaging, electron microscopy, and adhesion measurements. Our results show that none of these PTKs is involved in Ag-independent adhesion, and that both contribute by distinct mechanisms to a full-blown Ca²⁺ response. In addition, Lck-KD and Itk-KD differ in that only Lck, not Itk, is strictly required for the control of Ag-driven morphological and adhesive responses.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells

T8.1 is a murine T cell hybridoma expressing a chimeric human-mouse TCR specific for a tetanus toxin peptide (tt_830–843) restricted by HLA-DRB1*1102. The DR-expressing murine fibroblasts L625.7 used as APC also express endogenous B7-1 and ICAM-2 (data not shown). This T/APC system has been previously characterized and used to study biochemical events triggered by Ag recognition (20, 21). T8.1 was maintained in DMEM with 10% FCS, 2 mM L-glutamine, and antibiotics (complete medium) supplemented with 400 nM methotrexate, 1 mg/ml G418, and 50 µM 2-ME. L625.7 cells were cultured in complete DMEM with 250 µg/ml G418. Stable transfectants expressing PTKs-KD were grown in the presence of 1 µg/ml puromycin. Stable transfectants overexpressing Lck-KD have been previously used and characterized (21).

Plasmid construct

The murine Itk cDNA in pBS was mutated at the ATP binding site (R390F) and subcloned in the eukaryotic expression vector, pSR-puro, a derivative of pcDL-Sr296 in which a puromycin resistance cassette had been inserted (22). The mutant was referred to as Itk-KD.

Cell transfections

Stable transfectants expressing different amounts of Itk-KD were obtained as previously described (21). T8.1 cells (1 x 10⁷ in DMEM and 20% FCS) were electroporated in a Gene Pulse cuvette (Bio-Rad, Hercules, CA) with 30 µg of plasmid at 960 µF and 250 V. After 48 h in normal medium, cells were placed in 96-well tissue culture plates in DMEM containing methotrexate (400 nM), G418 (1 mg/ml), and puromycin (1 µg/ml). Puromycin-resistant transfectants were selected for expression of the T cell markers CD3, CD4, CD28, and CD45 similar to that of parental T8.1 cells.

Detection of PTK expression

PTK expression was measured by flow cytometry, fluorescence microscopy, and Western blot. For intracellular detection by flow cytometry, T8.1 cells and KD transfectants were washed and resuspended in 0.5% PBS-BSA (10⁶ cells in 100 µl). Cells were fixed at room temperature by adding 500 µl of a 4% paraformaldehyde PBS-BSA solution for 20 min, washed with PBS-BSA, and resuspended for 30 min in PBS containing 0.1% saponin and specific Abs or control isotypes. Monoclonal anti-Lck and anti-Itk were obtained from Transduction Laboratory (Lexington, KY) and Upstate Biotechnology (Lake Placid, NY), respectively. Cells were washed three times in PBS-BSA-saponin and stained with a FITC-conjugated secondary Ab. After two washes in PBS-saponin and three in PBS-BSA, cells were subjected to flow cytometry (FACScan; Becton Dickinson, San Jose, CA).

For intracellular detection of Lck by fluorescence microscopy, cells plated on microgrid coverslips were washed twice in PBS and fixed at room temperature with 500 µl of a 3% paraformaldehyde PBS solution for 10 min. The fixed cells were washed in PBS and incubated for 20 min in PBS containing 0.1 M glycine, then permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature and blocked with PBS containing 0.2% BSA for 20 min. The anti-Lck mAb (clone 3A5; Santa Cruz Biotechnology, Santa Cruz, CA) was used at 1 µg/ml in blocking solution for 30 min at room temperature. Then cells were washed three times and incubated with a 100-fold dilution of FITC-labeled goat anti-mouse Ab. After washing, the coverslip was mounted on a slide using ProLong antifade reagent (Molecular Probes, Eugene, OR). Immunofluorescence and transmission light images were acquired with Image Pro Plus (Media Cybernetics, Carlsbad, CA) on a Leica microscope equipped with a Sensys 400 cooled CCD camera (Photometrics, Huntington Beach, CA). Quantitative analysis of Lck fluorescence staining was performed with NIH Image on regions of interest defined around individual cells.

PTK expression was measured by Western blot as previously described (23). In brief, cells were rapidly pelleted and lysed for 20 min on ice in 1% Nonidet P-40 lysis buffer containing 20 mM Tris-HCl (pH 7.5), 140 mM NaCl, and 1 mM EDTA in the presence of proteases inhibitors. Proteins in postnuclear supernatant were then separated on a 8% SDS-polyacrylamide gels and blotted onto nitrocellulose. Itk was detected with the Abs used for intracellular detection. For Lck, immunoblotting was performed with Abs from Upstate Biotechnology. Enhanced chemiluminescence was used to reveal specific PTKs.

Single cell Ca²⁺ video imaging

Twelve to 20 h before the experiment L625.7 cells (0.4 x 10⁶) were plated on glass coverslip-mounted petri dishes. Fibroblasts were pulsed with 5 µg/ml of tt_830–843 peptide unless otherwise stated. For successive measurements of Ca²⁺ and Lck levels in the same cells, fibroblasts were plated on 175-µm CELLocate microgrid coverslips (Eppendorf, Hamburg, Germany).

T cells (5 x 10⁵) were incubated for 20 min at 37°C with 1 µM fura-2/AM (Molecular Probes). Cells were then washed and added to fibroblasts. Measurements of the intracellular calcium concentration were performed at 37°C in mammalian saline buffer (140 mM NaCl, 5 mM KCl, 10 mM HEPES (pH 7.3), 1 mM CaCl₂, and 1 mM MgCl₂) with a Diaphot 300 microscope (Nikon, Melville, NY) and an IMSTAR imaging system as described previously (14). Averaged Ca²⁺ responses were calculated either for all cells or for responding cells, i.e., cells showing an amplitude of Ca²⁺ increase of at least 80 nM. When several Ca²⁺ traces were averaged, the rising phases of the traces were first synchronized so that the time course of the average was not "filtered" by the asynchrony of the different responses. Transmission light images acquired to visualize the APCs interacting with the T cells were taken every 10 s in turn with fluorescence images.

Adhesion assay

Ag-independent and -dependent adhesion between murine T cells hybridoma and L625.7 cells was quantified as follows. Fibroblasts were cultured overnight on petri dishes mounted with glass coverslips, in the presence of 0, 0.5, or 5 µg/ml of peptide. Fura-2-loaded T8.1 cells were left to adhere to fibroblasts at 37°C in 2 ml of medium. After 30 min of interaction, fluorescent images of several microscopic fields with at least 50 T cells were acquired. Standardized washes were then applied to cells. To this end, the medium was rapidly removed, and 2 ml of prewarmed solution was added either at the center of the dish for strong washes or at the periphery of the dish for mild washes. Two rounds of washes were preformed for each condition. The number of adherent T cells was counted from processed fluorescent images before and after washes. The percentage of remaining cells was calculated from two or three independent experiments.

Scanning electron microscopy

Twelve to 20 h before the experiment L625.7 cells (0.4 x 10⁶) were plated on glass coverslip-mounted petri dishes. Fibroblasts were pulsed with 5 µg/ml of tt_830–843 peptide unless otherwise stated. T8.1 cells and transfectants were allowed to adhere to L625.7 for 5 and 20 min at 37°C in mammalian saline buffer. Cells were fixed at room temperature with 2.5% prewarmed glutaraldehyde for 30 min. After washing, the samples were dehydrated in five successive and graded ethanol baths (from 25 to 100%), dried by the critical point method using liquid CO₂, coated with gold by sputtering, and observed with a scanning electron microscope (JSM.840.A, JEOL, Peabody, MA).

Quantitation of shape changes

The percentage of polarized T cells able to form a lamellipodium during their interaction with peptide-pulsed L625.7 cells was scored from transmission light images. At least 30 cells were analyzed from each separate experiment. T/APC contact length was quantitated using NIH Image software. The largest visible diameter of the T cell protrusion at the contact interface between the two cells was measured from scanning electron microscope images.

Statistics

Data are expressed as mean ± SD, and the significance of differences between two series of results was assessed using Student’s t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characteristics of stable transfectants overexpressing KD forms of Lck and Itk

To delineate the functional role of Lck and Itk in controlling Ca²⁺, morphological, and adhesive responses induced in T cells by APCs, we used a dominant-negative mutant approach in the murine T cell hybridoma T8.1 (20). Stable transfectants expressing different amounts of KD mutants of Lck (21) and Itk were used.

The level of expression of each kinase in two different clones for each type of transfectants was compared with the level of expression of the wild-type endogenous counterpart detected in T8.1 cells (Fig. 1). Overexpression of PTKs-KD was evaluated by Western blot and flow cytometry after intracellular staining. Analysis of the intracellular staining allows measurement not only of the average expression of the mutant PTK but also of the variability of their expression in the cell population. This distribution appeared homogeneous and unimodal in all cases. As previously reported (21) the level of Lck detected by Western blot in T8.1 cells was very low. T8.1 cells also express a barely detectable level of endogenous Itk. Surface expressions of CD3, CD4, CD28, and CD45 were similar in transfectants and T8.1 cells (data not shown). IL-2 production in response to Ag stimulation was inhibited in T8.1 cells expressing PTKs-KD; the inhibition was positively correlated to the expression of PTKs-KD (data not shown).

View larger version (33K): [in this window] [in a new window]   FIGURE 1. PTK expression measured in KD transfectants and T8.1 cells. PTK expression was measured in two ways: intracellular staining (A) and Western blots (B). Two different clones for each condition are presented.

The normal alterations in the morphology and the intracellular Ca²⁺ concentration of T8.1 cells induced by Ag stimulation were first visualized by scanning electron microscopy and fluorescence imaging of fura-2-loaded T cells. T cells interactions were monitored using as APC, class II-transfected murine fibroblast, L625.7 cells (20). At the beginning of a typical 20-min experiment, T8.1 cells were allowed to settle on a monolayer of L625.7 cells that had been previously pulsed, or not, with 5 µg/ml of tetanus toxin (tt_830–843) peptide for 12 h.

Fig. 2A and movie 1⁶ show a sequence of images captured every 18 s in one T8.1 cell interacting with peptide-pulsed APCs. The first detectable event was the formation of a stable T/APC conjugate in which the T cell contacted an APC through fine cellular processes (marked by arrows). The T cell origin of these processes is clearly visible on movie 1. After an average delay of 207 ± 87 s (n = 14 cells), this contact led to a large and sustained Ca²⁺ increase. Three individual Ca²⁺ responses elicited in T8.1 cells by L625.7 are shown in Fig. 2B. The percentage of Ca²⁺-responding cells was then analyzed. Cells were scored as responsive when the minimal Ca²⁺ increase was >80 nM. Due to the high frequency of T8.1 cells presenting a Ca²⁺ response (71 ± 15% of the cells in seven separate experiments in which >30 cells were analyzed/experiment), the average response of all cells (dotted line) is close to the average response of responding cells (continuous line).

View larger version (67K): [in this window] [in a new window]   FIGURE 2. Ag-induced cytoskeletal and Ca2+ responses in wild-type T8.1 cells. A, Example of one T8.1-L625.7 cell interaction. Image interval, 18 s. The arrows indicate the small contact interface observed before the Ca2+ increase. B, Three single-cell Ca2+ responses and the average Ca2+ response measured only in responding cells (—) or in all cells (----). C, Scanning electron microscope analysis of conjugates between T8.1 cells and L625.7 cells. Bars = 1 µm.

The detailed structure of the contact zone cannot be visualized in transmitted light images, but can be further analyzed by scanning electron microscope (Fig. 2C). In the absence of Ag, T8.1 interacted with L625.7 cells by a bundle of filopodia forming a structure with an average diameter at the contact interface of 2.03 ± 0.63 µm (n = 7 cells; left panel of Fig. 2C). In the presence of Ag, this initial phase was followed by marked morphological changes, first characterized by the appearance of extended lamellipodia projected from the T cell body toward the APC. This cell polarization, which "blossoms" in a few seconds (as judged from video recordings), is illustrated in an electron micrograph shown in the middle panel of Fig. 2C where a T cell had been in contact with the APC for 5 min. At this stage, two distinct compartments can be distinguished in the T cell: a small and shrunken cell body and a spectacular lamellipodium. The average diameter of the lamellipodium measured at the cell-cell contact interface was 7.18 ± 2.61 µm (n = 8 cells). The cell body appears rounded and dented, whereas the lamellipodium is smooth, with a prominent leading edge. Note that the surfaces of both structures display very few microvilli, which contrasts with the spherical unstimulated T cells, which are covered with short microvilli. After a few minutes, T cells revert to a more spherical shape, but an active membrane ruffling persists, essentially restricted to the contact interface between the two cells. One example of a T8.1 cell in contact with L625.7 cells for 20 min is illustrated in the right panel of Fig. 2C. A typical sequence of events occurring in a T8.1 cell in interaction with an Ag-pulsed APC, captured with the imaging setup, is shown in movie 2.

Effect of Lck-KD on Ag-induced Ca²⁺ responses and morphological alterations

The specific Ca²⁺ and morphological responses elicited in Lck-KD transfectants were examined next. Fig. 3A and movie 3 show an example of three individual Lck-KD 3.4 cells that have been added to peptide-pulsed APCs. In two of the three T cells shown, no Ca²⁺ increase or shape changes were detected despite the existence of a contact with the APC. However, one T cell (marked by an arrow in Fig. 3A) displayed marked cytoskeletal and Ca²⁺ responses.

View larger version (70K): [in this window] [in a new window]   FIGURE 3. Different effects of Lck-KD on Ag-induced T cells responses. A, Examples of three Lck-KD 3.4 transfectants added to L625.7 cells. Image interval, 23 s. The arrow indicates the cell that displays near-normal cytoskeletal and Ca2+ responses. B, Three single-cell Ca2+ responses and the average Ca2+ response measured only in responding cells (—) or in all cells (----). C, Scanning electron microscope analysis of conjugates between Lck-KD 3.4 transfectants and L625.7 cells. Bars = 1 µm.

Remarkably, in the cells expressing Lck-KD that did show a Ca²⁺ response, the amplitude of this Ca²⁺ increase was only slightly smaller than the control response detected in T8.1 cells (in the two Lck-KD transfectants, 8% smaller, on the average, than the response measured in T8.1 cells). Examples of individual Ca²⁺ responses elicited in Lck-KD 3.4 transfectants by L625.7 cells are shown in Fig. 3B. Note that the rate of rise of the Ca²⁺ increase in Lck-KD cells was significantly slower than that in wild-type cells and that two distinct phases could be distinguished, as if the Ca²⁺ response hesitated to start. Thus, the global Ca²⁺ response elicited in a cell population was smaller in Lck-KD transfectants essentially because the percentage of responding cells was reduced. As a result, this global Ca²⁺ response differed from the average response of responding cells (Fig. 3B), contrary to what had been observed in T8.1 cells.

We next assessed the influence of Lck on the morphological modifications induced in T8.1 cells by APCs. Scanning electron microscope experiments showed that the small protrusion formed by the Lck-KD 3.4 (Fig. 3C, left panel) transfectant toward unpulsed APCs was very similar to that observed in control T8.1 cells. The diameter of the small protrusion measured in Lck-KD 3.4 cells (1.52 ± 0.94 µm; n = 6 cells) was comparable to that measured in T8.1 cells (2.03 ± 0.63 µm; n = 7 cells). By contrast, the percentage of Lck-KD 3.4 transfectant able to form a conspicuous lamellipodium in the presence of Ag (34 ± 12%; n = 5 experiments in which >30 cells were scored/experiment) was very much reduced compared with that in parental T8.1 cells (73 ± 16%; n = 9; p < 0.0003). Similarly, the diameter of the T/APC contact zone for Lck-KD 3.4 cells (4.24 ± 2.86; n = 13) was significantly smaller than that of parental T8.1 cells (7.18 ± 2.61 µm; n = 8; p = 0.029). The middle and right panels of Fig. 3C illustrate the morphology of Lck-KD 3.4 transfectants after 5 min of interaction with peptide-pulsed APC. A majority of cells did not change their shape (middle panel), whereas a few cells displayed large, well-shaped lamellipodia (right panel).

Next we examined why the Ca²⁺ response in Lck-KD transfectants seems to be almost all or none. One hypothesis is that the amount of dead kinase expressed in a given transfectant cell controls the ignition of the response, and that in the lowest Lck-KD expressors, partial inhibition of the endogenous Lck is not sufficient to inhibit the triggering of the signal. This hypothesis was tested by examining whether there is a correlation between the expression of Lck-KD measured at the single-cell level and the existence of an APC-induced Ca²⁺ response in the very same cells. Abs against Lck recognize both endogenous and Lck-KD. However, the staining of endogenous Lck in wild-type cells was so faint (data not shown) that all significant staining in transfectants can be attributed to Lck-KD.

Lck-KD 3.4 transfectants were allowed to settle on a monolayer of peptide (5 µg/ml)-pulsed L625.7 cells. The Ca²⁺ level in individual cells was monitored during the first 10 min of T/APC interaction. Cells were then fixed, permeabilized, and stained with an anti-Lck mAb followed by a FITC-labeled goat anti-mouse Ab. Fig. 4A shows the Ca²⁺ level and staining of Lck in three individuals Lck-KD 3.4 cells interacting with peptide-pulsed APC. It is clear in this example that Ca²⁺ responses are observed in the two cells showing the lowest level of Lck (i.e., essentially Lck-KD).

View larger version (38K): [in this window] [in a new window]   FIGURE 4. Correlation between the amount of Lck-KD and the inhibition of the Ca2+ response. A, Measurement of the Ca2+ level and the amount of Lck in the same cells. Upper panel, Ca2+ level in three Lck-KD 3.4 cells in interaction with L625.7 cells for 5 min (left) and the corresponding transmission light image (right). Lower panel, Amount of Lck in the same three cells revealed by immunofluorescence (left) and the corresponding transmission light image (right). After 10 min of interaction, cells were fixed, permeabilized, and stained with an anti-Lck mAb followed by FITC-conjugated goat anti-mouse IgG Ab. The fluorescence level was coded with an 8-bit gray scale under nonsaturating conditions. B, Distribution of Lck-KD level in Ca2+ responding and nonresponding cells. The amount of Lck was determined by measuring the fluorescent intensity of every Ca2+-responding and nonresponding individual cells. Each dot represents one cell. The mean for each group is indicated by a white dot. Values were pooled from three independent experiments. The mean ± SD for each group were calculated and compared using unpaired t test.

Effect of Itk-KD on Ag-induced Ca²⁺ responses and morphological alterations

Fig. 5A and movie 4 illustrate typical Ca²⁺ and morphological responses of two Itk-KD 2.5 transfectants interacting with Ag-pulsed APCs. In one cell marked by the arrow a weak Ca²⁺ increase was observed despite extensive cell shape changes (open arrow). The other cell remained unresponsive during the course of the experiment.

View larger version (72K): [in this window] [in a new window]   FIGURE 5. Overexpression of Itk-KD mutant inhibits the Ag-induced Ca2+ response, but not cytoskeletal alterations. A, Examples of two Itk-KD 2.5 transfectants added to L625.7 cells. Image interval, 40 s. The black arrow points to the cell displaying a weak Ca2+ increase despite extensive morphological changes. The white arrows point to the lamellipodium, which was too thin to give rise to a detectable fluorescent signal. B, Three single-cell Ca2+ responses and the average Ca2+ response measured only in responding cells (—) or in all cells (---). B, Scanning electron microscope analysis of conjugates between Itk-KD 2.5 transfectants and L625.7 cells. Bars = 1 µm.

If it did not markedly affect the percentage of responding cells, KD-Itk had profound effects on the characteristics of the Ag-induced Ca²⁺ response. These responses were not only smaller, but also briefer, than their control counterparts. Examples of such Ca²⁺ responses, measured in single Itk-KD 2.5 cells, are shown in Fig. 5B.

In contrast to these strongly impaired Ca²⁺ responses, the changes in T cell shape induced by Ag-loaded APCs were not markedly altered. When L625.7 cells were pulsed with 5 µg/ml of Ag, a majority (53 ± 15%; n = 5 experiments in which >30 cells were scored/experiment) of Itk-KD 2.5 cells formed a conspicuous lamellipodium, i.e., slightly less than in T8.1 cells (73 ± 16%; p < 0.03). The contact length measured in Itk-KD 2.5 interacting with APCs (6.05 ± 2.37; n = 11) was not statistically different from that measured in T8.1 cells interacting with APCs (7.18 ± 2.61 µm; n = 8). Examples of shape changes at 5 and 20 min after the initial contact are illustrated in Fig. 5C (middle and right panels).

Comparison between Ag-induced Ca²⁺ responses in Lck-KD and Itk-KD transfectants

As mentioned above and shown in more detail in Fig. 6A, the percentage of cells showing a Ca²⁺ response in the presence of Ag-pulsed APCs was more profoundly affected by the inhibition of Lck than by that of Itk. The difference between the two KD PTKs was most obvious at a nonsaturating Ag concentration (0.5 µg/ml).

View larger version (29K): [in this window] [in a new window]   FIGURE 6. Effects of Lck and Itk-KD mutants on the characteristics of the Ag-induced Ca2+ response. A, Percentage of Ca2+-responding T8.1 cells and transfectants as a function of the Ag concentration. Only cells presenting an increase in Ca2+ >80 nM and a clear contact with L625.7 were taken into account. B, Average Ca2+ response of T8.1, Lck-KD, and Itk-KD cells measured either in all cells or only in responding cells.

Effects of Lck-KD and Itk-KD on Ag-induced T/APC adhesion

Because full T cell activation requires a long-lasting APC-T cell interaction (24), it is important to evaluate not only the initiation of TCR signaling, but how it can be sustained by a prolonged T-APC interaction. The impact of Lck and Itk-KD was thus evaluated on the adhesion of T cells to L625.7 cells pulsed, or not, with the peptide.

T8.1 cells were left to adhere to L625.7 cells for 30 min, a duration allowing maximum adhesion (25). At this time cells were counted before and after standardized washes. By using washes of two different intensities, either mild or strong, we were able to distinguish among three levels of adhesion: nonspecific, light, and strong.

A monolayer of HeLa cells was used as a negative control for the evaluation of nonspecific binding. Thus, 16 ± 2.8% of T8.1 cells were still in contact with HeLa cells after mild washes (and zero after strong washes). In contrast, when T cells were left to adhere to unpulsed L625.7 cells, 64 ± 19% of the T cells were still in contact with L cells after mild washes. Thereafter, strong washes removed all these T cells. Thus, significant light adhesion was measured between T8.1 cells and APC-expressing MHC class II, B7-1, and ICAM-2 in the absence of Ag (Fig. 7A). The Ag-independent adhesion to L625.7 cells, measured after mild washes was not statistically different between T8.1 cells and Lck and Itk-KD transfectants (Fig. 7A).

View larger version (28K): [in this window] [in a new window]   FIGURE 7. Ag-independent and -dependent adhesion measured in Lck-KD, Itk-KD, and T8.1 cells. A, In the absence of Ag, the percentage of remaining cells was scored after mild washes. The control represents nonspecific binding of T cells to HeLa cells. B, In the presence of Ag, the percentage of remaining cells was scored after strong washes.

Taken together these data show that none of the KD mutants affected the basal light Ag-independent adhesion. In addition, KD forms of Lck had severe effects on the stability of the conjugates, while Itk-KD did not affect this stability.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present work by combining Ca²⁺ imaging, electron microscopy, and cell-cell adhesion measurements, we have analyzed the functional importance of Lck and Itk in Ag recognition by T cells. To compare the roles of the two PTKs in the same T/APC system, KD forms of these PTKs were stably overexpressed in a murine T cell hybridoma. With this type of approach, one cannot exclude that overexpression of one PTK-KD may result in the inhibition of a group of PTKs (for instance, overexpression of Itk-KD may inhibit not only Itk but also other members of the same family, such as Rlk or Tec). However, this should not affect our main conclusion concerning the differential regulation exerted by kinases typified by Lck and Itk, during T-APC interaction.

Lck and Itk have been shown to play an important role in T cell development and TCR signaling (for a review, see Ref. 26). However, many questions remain unanswered, in particular the participation of Lck and Itk in the signaling pathways leading to alterations of the T cell cytoskeleton. This last point is of importance, because actin remodeling is thought to be essential for T cell activation (3, 16, 17). In a previous study using anti-CD3-coated beads and variants of Jurkat T cells defective in Lck and ZAP-70, Lowin-Kropf et al. pointed to the participation of these two kinases in actin remodeling and reorientation of the microtubule-organizing center (27). However, anti-CD3 stimulation does not necessarily mimic all aspects of APC stimulation. By using a more physiological setting, we have examined in more detail the APC-induced changes in T cell shape. They begin with the rapid formation of a large lamellipodium protruding from the T cell body toward the APC. This allows the T cell to translocate most of its cytoplasmic and plasma membrane material to the contact interface. Indeed, a redistribution of both cytoplasmic vesicles and cell surface molecules toward the contact zone has been described (for a review, see Ref. 28). In addition, the large lamellipodia protruded toward the APC increases the contact zone size, which is thought to stabilize the contact between T cells and APCs. We have shown that these events are dependent on Lck, but not on Itk. Recent data have provided insight into the molecular chain linking the TCR to actin. A multimolecular complex including SLP-76, SLP-76-associated protein (Fyb/SLAP), Nck, Vav, and Wiskott-Aldrich syndrome protein (WASP) is suggested to play an important role in these processes (29, 30, 31, 32, 33). Based on our results, one could hypothesize that Lck, but not Itk, should be involved in the formation of this complex.

Another important question that has been addressed here is that of the APC-T cell adhesion. We and others have previously shown that T-APC adhesion can take place in the absence of Ag and presumably constitutes a first checkpoint in Ag recognition (34, 35). Even when Ag is present at the APC surface, it is likely that the initial T-APC interaction occurs in an Ag-independent way, because the avidity of a number of costimulatory molecules, such as CD2 and CD28, for their respective ligands is likely to be much larger than that of TCR for cognate MHC-peptide complexes. The main difference does not reside so much in the affinities but, rather, in the relative abundance of the ligands for CD2 and CD28 compared with the very small number of specific MHC-peptide complexes sufficient to trigger a response (34, 36, 37). We have shown here that this Ag-independent adhesion is weak, but measurable, and that it is not affected by the inhibition of activity of Lck and Itk. Additional experiments indicate that KD forms of Fyn and ZAP-70 are unable to inhibit this Ag-independent adhesion (data not shown). In transmitted light images, one can simply ascertain that an Ag-independent contact exists, as judged namely by the sudden immobilization of the T cell, which emits poorly resolved cellular processes. The characteristics of this tiny contact zone were revealed by scanning electron microscopy: a bundle of filopodia (which might have been former microvilli) forming a small protrusion <1 µm in diameter. It is tempting to draw a parallel between the initial T/APC interaction and the contact between a leukocyte and an endothelial cell even though adhesion molecules are probably different in both cases. Initial contact between a leukocyte and an endothelial cell is made by adhesion molecules (L-selectin, ₄ integrin) preferentially localized at the tip of leukocyte microvilli (38, 39). Indeed, microvilli are particularly well suited to promote the initial T/APC interaction. Beside the presence of adhesion receptors, microvilli are enriched in actin bundles and cytoskeletal proteins such as ezrin, which link adhesion molecules to actin (40, 41). The functional importance of microvilli in the context of T/APC Ag-independent interaction deserves further investigation.

Interestingly, Ag-independent interaction with L625.7 cells never triggered Ca²⁺ responses in T8.1 cells, contrary to what has been reported when T cells interact with dendritic cells (34, 42). It is conceivable that the dendritic cell-induced signal requires a molecule that is uniquely expressed on dendritic cells, such as DC-SIGN (43). Alternatively, the specific ability of dendritic cells to induce an Ag-independent signal may result from their high expression level of costimulatory molecules (35).

Ag-induced Ca²⁺ responses are obviously conditioned by the adhesion events just discussed. However, some additional, specific features of Ag-induced Ca²⁺ responses deserve further examination. The inhibition of Lck and Itk leads to an apparently similar reduction of the Ag-induced Ca²⁺ responses averaged over a number of T cells. However, the single-cell analysis performed in the present work allowed us to make a clear distinction between the two modes of inhibition. The inhibition of Lck resulted essentially in a reduced probability of triggering a Ca²⁺ response, but in the few cells that did respond, the amplitude of the Ca²⁺ response was 90% that of the control (even though the kinetics of the response were slowed). This trend toward an all-or-none inhibition of the Ca²⁺ response is suggestive of a threshold effect; when a sufficiently small fraction of Lck is inactivated, the signaling process remains unaltered, and when enough Lck is inactivated, the signaling cascade is completely blocked. Although in the two clones used the expression of the dead kinases was unimodal, as judged by its measurement by flow cytometry, we have shown that the difference between the low and high expressors contributes to allow the lowest dead kinase expressors to escape from the inhibition.

The situation was different when Itk was inhibited. In this case, a Ca²⁺ response could still be triggered in a large fraction of the T cells (70% of the control), but the amplitude of these responses was strongly reduced (by >50%). In this case, there was no all-or-none effect but, rather, a global tuning of the whole response.

These results suggest that in Ag-induced Ca²⁺ signaling Lck plays an ignition role, while Itk plays an amplification role. This conclusion is reinforced by the observation that, for Lck, the triggering of a Ca²⁺ response is clearly dependent upon the amount of KD-PTK expressed in the clone. By contrast, once the response is initiated, it becomes almost insensitive to the amount of KD-PTK, as the amplitude of the Ca²⁺ signal is nearly as high as that in T8.1 control cells. The situation is different with Itk-KD transfectants. This is consistent with recent reports indicating that in anti-CD3-stimulated T cells, Itk is activated downstream of Src and Syk kinases (for review, see Ref. 4). In fact, in T cells from Itk-deficient T mice, the overall pattern of immediate tyrosine phosphorylation triggered by anti-CD3 stimulation is unaffected, whereas PLC- phosphorylation is greatly reduced (8, 9). Similar results have been observed in Itk-KD transfectants after anti-CD3 triggering (F. Michel and O. Acuto, unpublished observations).

Our results are also informative on the relation between Ca²⁺ responses and adhesion. In particular, Itk-KD transfectants adhere to L625.7 cells as well as T8.1 cells despite a markedly reduced Ca²⁺ response. This result is surprising knowing the relation between the initial Ca²⁺ increase and the subsequent adhesion (14, 15, 44). It is possible that the small and transient Ca²⁺ response detected in Itk-KD cells is large enough to promote a T-APC stable conjugate.

In conclusion, from this and other studies, our present view of the sequence of the events occurring during the T/APC interaction is the following. 1) An initial Ag-independent contact through molecules present at the tip of a small bundle of filopodia leads to a light adhesion that occurs even if either Fyn, Lck, ZAP-70, or Itk is inhibited. 2) In the presence of Ag this first step is followed by engagement of the few TCRs present in the initial structure. Once the resulting initial signal reaches a certain threshold (the level of which is set by Lck, but not by Itk), signals are further amplified, and cellular responses become explosive. This leads to an intense membrane ruffling, which may allow additional membrane receptors, including TCRs, to be engaged and a stronger stabilization of the T-APC conjugate. Concomitant to cytoskeletal reorganizations, a strong Ca²⁺ increase is triggered dependent on Lck and Itk. 3) During the sustained phase of the response, a more compact contact zone is formed, which allows firm adhesion compatible with a mobility visible at both the cellular and molecular levels (18, 19, 45).

	Acknowledgments

 
We thank Clotilde Randriamampita, Jérôme Delon, and Patrick Revy for their comments on the manuscript.

	Footnotes

 
¹ This work was supported by grants from Centre National de la Recherche Scientifique and by Ligue Nationale Centre le Cancer.

² Current address: Division of Cellular Immunology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, U.K. NW7 1AA.

³ F.M. and A.T. contributed equally to this work.

⁴ Address correspondence and reprint requests to Dr. Alain Trautmann, Laboratoire d’Immuno-Pharmacologie, Centre National de la Recherche Scientifique UPR 415, Institut Cochin de Génetique Moléculaire, 22 rue Méchain, 75014 Paris, France.

⁵ Abbreviations used in this paper: PTK, protein tyrosine kinase; PLC-1, phospholipase C-1; KD, kinase-defective. LAT, linker for activation of T cells; SLP-76, SH2 domain-containing leukocyte protein of 76 kDa.

⁶ The on-line version of this article contains supplemental material.

Received for publication August 8, 2000. Accepted for publication January 17, 2001.

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