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International Immunology Advance Access originally published online on January 12, 2007
International Immunology 2007 19(3):239-248; doi:10.1093/intimm/dxl141
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

CD2 and TCR synergize for the activation of phospholipase C{gamma}1/calcium pathway at the immunological synapse

Nicolas Espagnolle1, David Depoil3, Rossana Zaru4, Cecile Demeur1, Eric Champagne1, Martine Guiraud1 and Salvatore Valitutti1,2

1 INSERM U563, Lymphocyte Interaction Group, Institut Claude de Préval, CHU Purpan, 31059 Toulouse, France
2 Faculty of Life-Sciences, University Toulouse III, Toulouse, France
3 Present address: Cancer Research UK, London Research Institute, London, WC2A 3PX, UK
4 Present address: School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK

Correspondence to: S. Valitutti; E-mail: svalitu{at}toulouse.inserm.fr


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Upon conjugation with cognate antigen-presenting cells (APCs), T lymphocytes undergo a sustained [Ca2+]i increase resulting from the engagement of TCR and of accessory molecules with ligands expressed on the surface of APCs. We investigated the contribution of the accessory molecule CD2 to the activation of phospholipase C{gamma}1 (PLC{gamma}1)/calcium pathway in antigen-stimulated T cells. We show that CD2 binding with its ligand CD58 expressed on the surface of APCs augments and sustains antigen-induced [Ca2+]i increase in individual T cells interacting with APCs. We also show that in conditions in which CD2–CD58 interaction is impeded, the recruitment of PLC{gamma}1 to the immunological synapse (IS) is reduced. Interestingly, in these conditions PLC{gamma}1 phosphorylation in the regulatory tyrosine 783 is also defective. Our results indicate that TCR- and CD2-derived signals converge for the recruitment and activation of PLC{gamma}1 at the IS and shed new light on the accessory function of CD2 in T cell activation by specific antigen.

Keywords: antigen-presenting cells, CD58, signal transduction, T cell activation


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary data
 References
 
T cells interacting with cognate antigen-presenting cells (APCs) undergo a sustained calcium mobilization, which is required for the activation of cytokine production and proliferation (1, 2). The molecular steps of this signaling pathway are well defined: TCR triggering induces phosphorylation of phospholipase C{gamma}1 (PLC{gamma}1) in regulatory tyrosines (3); this activates the enzymatic activity of PLC{gamma}1 (4) resulting in sustained breakdown of phosphoinositides (5) and sustained [Ca2+]i increase (6).

PLC{gamma}1 has four tyrosine phosphorylation sites, and these are the tyrosine Tyr 771, Tyr 783, Tyr 1254 and Tyr 775. Phosphorylation of the first tyrosine (Tyr 771) has no effect on the activation of PLC{gamma}1 function (7). Conversely, phosphorylation of Tyr 783 is strictly required for the activation of PLC{gamma}1 in NIH 3T3 fibroblasts stimulated by platelet-derived growth factor (7). In these cells, phosphorylation of the third tyrosine (Tyr 1254) allows an optimal activation of PLC{gamma}1 (7). The crucial role of Tyr 783 in the activation of PLC{gamma}1 was also confirmed in Jurkat T cells stimulated with anti-CD3 antibodies (4). A recent study has better characterized PLC{gamma}1 phosphorylation in Jurkat T cells and has further confirmed the role of Tyr 783. In this study, an additional regulatory tyrosine (Tyr 775) has been identified (8). Finally, a recent study has characterized the molecular mechanisms by which Tyr 783 phosphorylation activates PLC{gamma}1 enzymatic activity (9). Together, the above reports highlight the importance of Tyr 783 phosphorylation as being a conserved mechanism in different cellular systems.

While it is well established that the engagement of TCR with peptide–MHC complexes displayed on the surface of APC triggers PLC{gamma}1/calcium pathway, the contribution of accessory molecules to the strength and duration of this signaling pathway in antigen-stimulated T lymphocytes is elusive.

CD2 is an accessory molecule well known to be implicated in several aspects of T cell physiology. CD2 binding with its ligand (CD58 in human and CD48 in mouse) lowers the threshold for T cell activation by specific antigen (10) and is implicated in facilitating T cell adhesion to APC (11, 12). CD2 is also an important component of the immunological synapse (IS) (13). It has been proposed that the binding of CD2 with its ligand allows the formation of areas of tight adhesion between T cells and APCs where TCR engagement with peptide–MHC complexes may be facilitated (13). In addition, CD2 is also known to be involved in T cell signaling. Several transducing enzymes and adapter proteins have been shown to interact with the intracellular portion of CD2 (14). Among these is the CD2 adapter protein (CD2AP), which contains in its sequence multiple protein–protein interaction domains (15).

Experiments performed using anti-CD2 antibodies to stimulate T cells showed that CD2 cross-linking activates PLC{gamma}1 phosphorylation and calcium pathway (16) and that in these conditions PLC{gamma}1 phosphorylation is mediated by the protein tyrosine kinase p59 Fyn (17). Conversely, experiments performed using anti-CD3 antibodies showed that CD3-mediated phosphorylation of PLC{gamma}1 is mediated by other protein tyrosine kinases such as zeta-associated protein-70 (ZAP-70) and IL-2-inducible T cell kinase (Itk) (18–21).

Together, these results suggest that the phosphorylation of PLC{gamma}1 and consequently its activation is downstream of synergistic signaling pathways triggered by TCR and CD2 engagement and rise the question of how each of these two signaling pathways may contribute to [Ca2+]i increase in antigen-stimulated T cells. To address this question, we investigated the impact of CD2/CD58 blocking in the activation of the PLC{gamma}1/calcium pathway in individual T cells interacting with cognate APCs. In a first approach, we measured using time-lapse video recording [Ca2+]i in T cells interacting with APCs in conditions in which the engagement of CD2 with CD58 was either allowed or impeded. Our results show that during the sustained interaction between T cells and APCs, the engagement of CD2 at the IS augments the level of TCR-induced [Ca2+]i increase and sustains signal transduction.

To define in individual T cell–APC conjugates whether TCR/CD3- and CD2-derived signals may converge into the activation of PLC{gamma}1, we studied the impact of CD2/CD58 blocking on the recruitment and phosphorylation of PLC{gamma}1 in the regulatory tyrosine 783 using confocal microscopy. Our results show that while TCR engagement by specific antigen is necessary to induce recruitment and phosphorylation of PLC{gamma}1 in T cell–APC conjugates, it is not sufficient for full activation of this key signaling enzyme. In conclusion, we show that in antigen-stimulated T cells CD2–CD58 interaction is required for full PLC{gamma}1 activation supporting the notion that PLC{gamma}1 is at the intersection between TCR and CD2 triggering at the IS.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary data
 References
 
T cells and APCs
Two DRBI*0101-restricted T cell clones (6396p5.1.2 and 6396p5.1.6) specific for the measles virus fusion protein peptide P5 (F254–268) were used. DR-matched EBV-transformed B cells were used as APCs. T cell clones and EBV-B cell lines were generated and maintained as described previously (22).

[Ca2+]i analysis in single cells
T cells were loaded with 5 µM fura 2-AM (Molecular Probes, Eugene, OR, USA) as described (23). Fura 2-loaded T cells were dropped onto APCs previously pulsed with 100 µM-specific peptide treated with either 10 µg ml–1 anti-CD58 mAb (1C3, BD PharMingen, Mountain View, CA, USA) or anti-MHC class-I (W6/32, American Type Culture Collection, Rockville, MD, USA) attached on a poly-D-lysine-coated slide to form a monolayer in RPMI 5% FCS and 10 mM HEPES. Fluorescence measurements were done on a Zeiss Axiovert 200 M inverted microscope equipped with a CCD camera (either CoolSNAP, Photometrics, Tucson, AZ, USA or i-PentaMAX, Princeton Instruments, Trenton, NJ, USA), an arc xenon lamp and a computer-controlled monochromator (TILL Photonics, Martinsried, Germany) at 37°C, 5% CO2. Cells were consecutively excited with 340 and 380 nm at intervals of 10 s by means of the monochromator and wavelength emission at 510 nm was collected with the CCD camera. The camera output was analyzed using custom calcium-imaging software, MetaFluor, provided by Universal Imaging (West Chester, PA, USA).

Measurement of conjugate formation and of [Ca2+]i by FACS analysis
[Ca2+]i and conjugate formation were measured as previously described (23).

Immunoprecipitation and western blot
G-protein-coupled beads were incubated with 1 µg ml–1 mouse monoclonal anti-PLC{gamma}1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), overnight at 4°C. T cells were conjugated with EBV-B cells as previously described for different times. In some experiments, EBV-B cells were treated for 30 min before conjugation with anti-CD58 mAbs or with anti-MHC class-I mAbs. Activation was stopped with ice-cold PBS 1x plus 2 mM sodium orthovanadate. After centrifugation, cellular pellets were lysed with lysis buffer (NaCl 150 mM, NP-40 1%, dodecyl thiosulphate 0. 5%, SDS 0. 1% and 50 mM Tris pH 8 plus protease inhibitors 1x plus 2 mM sodium orthovanadate). Supernatants were incubated with anti-PLC{gamma}1 beads, overnight at 4°C. Samples were separated by SDS-PAGE and transferred on nitrocellulose membrane. Membranes were blocked with PBS–Tween 0. 05% and 3% BSA plus 1% gelatin (Sigma–Aldrich, France) and incubated with 1 µg ml–1 anti-phosphotyrosine mAbs (Santa Cruz Biotechnology) followed by an incubation with HRP-coupled goat anti-mouse antibodies (Southern Biotechnology Associates, Birmingham, AL, USA). Membranes were revealed with enhanced chemiluminescence kit (Pierce, Rockford, IL, USA). After stripping with glycin buffer, membranes were reprobed with anti-PLC{gamma}1 (Santa Cruz Biotechnology). For quantification of bands, we used Image Quant software.

Intracellular staining
T cells were conjugated with EBV-B cells previously loaded with 0.5 µM Orange-CMTMR (Molecular Probes, Leiden, The Netherlands) either unpulsed or pulsed with the specific peptide as described (24). After washing, T cells were mixed with EBV-B cells in 100 µl RPMI 5% FCS in U-bottomed tubes and centrifuged to allow conjugate formation. In some experiments, EBV-B cells were treated for 30 min before conjugation with 10 µg ml–1 anti-CD58 mAbs at 4°C or with anti-MHC class-I mAbs. After different times at 37°C, the cells were gently re-suspended and laid on poly-L-lysine-coated slides for 3 min at 37°C. The cells were fixed for 10 min at room temperature with 3% PFA, permeabilized for 10 min with HEPES-buffered PBS containing 0.1% saponin and 3% BSA (24) and stained with anti-CD2 (RPA-2.10, BD PharMingen or MEM-65 kindly provided by Vaclav Horejsi, University of Prague), anti-PLC{gamma}1 mAb (Santa Cruz Biotechnology), anti-phosphotyrosine 783 PLC{gamma}1 or anti-phosphotyrosine 493 ZAP-70 rabbit antibodies (Cell Signaling, Beverly, MA, USA), followed by isotype-matched Cy5-labeled goat anti-mouse antibodies (Caltag Laboratories, Burlingame, CA, USA) or FITC-labeled goat anti-mouse antibodies (Southern Biotechnology Associates) or Alexa 488-labeled goat anti-rabbit antibodies (Molecular Probes) as described (24). The samples were mounted and examined using a Carl Zeiss LSM 510 confocal microscope (Carl Zeiss, Jena, Germany) with a 63x Plan-Apochromat objective (1.4 oil). An argon laser at 488 nm was used to detect fluorescein and Alexa 488 fluorochrome. To detect Orange-CMTMR fluorescence, a HeNe laser was filtered at 543 nm. To detect Cy5 fluorescence, a HeNe laser was filtered at 633 nm.

Measurement of intracellular phosphotyrosine 493 ZAP-70 by FACS analysis
The phosphotyrosine 493 ZAP-70 fluorescence was analyzed in fixed and permeabilized T cell–APC conjugates on a FACScan as previously described for total phosphotyrosine staining with minor modification (25).

Image quantification
To evaluate enrichment of PLC{gamma}1, PTyr783 PLC{gamma}1 and CD2 at IS in T cell–APC conjugates, unprocessed images of T cell–APC conjugates were analyzed using the linescan function of the MetaMorph software (Universal Imaging Corporation) as described (26). Briefly, two reference lines were drawn at the center of contact sites between T cells and APCs and outside of synapses on different areas of T cells. The software calculates the mean fluorescence intensity all along the reference lines for 15 pixels of width (7 and 7 laterally to each reference lines) and plots the measurements. The integral of the curves obtained are ratioed and results are reported in Fig. 6.


Figure 6
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  		Fig. 6. Quantitative analysis of PLC{gamma}1 recruitment and phosphorylation in antigen-stimulated T lymphocytes. Measurement of the ratio between the fluorescence intensity at the IS versus the fluorescence intensity in an area outside the IS in T cell–APC conjugates stained with anti-PLC{gamma}1 mAbs (A), anti-CD2 mAbs (B and D) and anti-PTyr783 PLC{gamma}1 antibodies (C); n is the number of T cell–APC conjugates measured. Quantification of fluorescence intensity was performed in the same T cell–APC conjugate for results presented in (A–B) and in (C–D). The Student’s t-test is used for statistical testing between values of two conditions. The horizontal bar represents the mean of all values for each condition. The values between control and anti-CD58-treated cells were significantly different (P values are indicated in the figure).

 

   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary data
 References
 
CD2–CD58 interaction sustains [Ca2+]i increase in antigen-stimulated T lymphocytes
We have previously shown that in T cells interacting with cognate APC, the engagement of CD2 with CD58 at the cell–cell contact site is not required for productive TCR engagement; however, it enhances [Ca2+]i increase and IFN{gamma} production (25). To visualize, at single-cell level, CD2 induced co-stimulation of calcium pathway in antigen-stimulated T lymphocytes, we measured [Ca2+]i in human CD4+ Th interacting with APCs in conditions in which the CD2–CD58 interaction was either allowed or impeded. Fura 2-loaded T cells were seeded onto APCs previously treated either with anti-MHC class-I control antibodies or with anti-CD58 antibodies. The [Ca2+]i levels were measured during 30 min after the initial contact between T cells and APCs using time-lapse video microscopy.

T cells interacting with unpulsed APCs exhibited low-level fluctuations in the [Ca2+]i (Fig. 1A). Figure 1(B and C) depicts typical [Ca2+]i patterns obtained for T cells interacting with peptide-pulsed APCs previously treated with either anti-MHC class-I antibodies or anti-CD58 antibodies. In control conditions, (anti-MHC class-I antibodies) [Ca2+]i exhibited an initial peak followed by a high sustained phase (Fig. 1B). When the CD2/CD58 binding was impeded (with anti-CD58 antibodies), the initial peak of [Ca2+]i was only moderately affected; conversely, the sustained [Ca2+]i increase was significantly inhibited (Fig. 1C). To evaluate the kinetics of calcium mobilization in a statistically valid sample of the T cell population, [Ca2+]i was measured in randomly selected individual T cells and the measurements obtained at defined time points after conjugate formation were compared. As shown in Fig. 1(D), the levels of [Ca2+]i were lower in T cells interacting with APCs treated with anti-CD58 antibodies at all the time points considered. Interestingly, inhibition increased with the increase of time after initial calcium peak. It should be noted that T cell adhesion to APC and arrest following contact with peptide-pulsed APC was not affected by the treatment of the APC with anti-CD58 antibodies. This indicates that CD2–CD58 interaction does not strongly contribute to cell–cell adhesion but rather augments [Ca2+]i signaling (Supplementary movies 1–3 are available at International Immunology Online). To obtain a more quantitative evaluation of the role of CD2 in cell–cell adhesion, we measured by FACS analysis conjugate formation and [Ca2+]i increase in T cells conjugated with APCs (either unpulsed or pulsed with the specific peptide) treated with either anti-CD58-blocking antibodies or control anti-MHC class-I antibodies. As shown in Fig. 2, treatment with anti-CD58 antibodies had only a limited effect on the stability of conjugate formation (Fig. 2A). However and interestingly, this treatment significantly reduced the level of calcium mobilization in conjugated T cells (Fig. 2B) without affecting the number of responding cells in conjugate (Fig. 2C). These results confirm those obtained measuring [Ca2+]i increase in individual T cell–APC conjugates and makes it clear that the decrease of [Ca2+]i response following block of CD2–CD58 interaction does not result from the inhibition of conjugate stability. Taken together, the above results indicate that the binding of CD2 with CD58 plays a key role in sustaining [Ca2+]i increase in antigen-stimulated T cells, thus favoring their activation.


Figure 1
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  		Fig. 1. CD2–CD58 interaction is required for sustained [Ca2+]i increase. Measurement of [Ca2+]i levels in T cells conjugated with unpulsed APCs (A) or peptide-pulsed APCs treated with either anti-MHC class-I mAbs (B) or anti-CD58 mAbs (C). Each color line corresponds to a single-cell measurement. (D) Statistical analysis of calcium data. Symbols represent individual [Ca2+]i measurements from at least three independent sessions for each experimental condition. Bar shows the mean value; n is the number of individual cells analyzed. The values between control (red) and anti-CD58-treated cells (blue) were statistically different (P < 0.001). The percentage of inhibition of calcium mobilization in T cells interacting with APCs treated with anti-CD58 antibodies is indicated (%). Similar results were obtained when the APCs were untreated instead of being treated with anti-class-I blocking antibodies (data not shown).

 

Figure 2
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  		Fig. 2. Anti-CD58-induced inhibition of [Ca2+]i increase is not dependent on CD2/CD58 role in conjugate stability. Indo-1-loaded T cells were conjugated with APCs either unpulsed or pulsed with 100 µM-specific peptide and treated with either anti-MHC class-I (PMHCI) or anti-CD58 (PCD58), at 1:2 (T cell clone:APC) ratio. (A) Percentage of T cells conjugated with APCs at different time points after conjugation. (B) Ratio of 405/530 mean fluorescence intensity emission in T cells at different time points after conjugation. Analysis was performed on conjugated cells only. (C) Percentage of conjugated T cells increasing [Ca2+]i. Data are from one experiment performed in triplicate representative of three. Bars represent standard deviation to the mean. The values between control and anti-CD58-treated cells were significantly different (P values are indicated in the figure).

 
CD2–CD58 interaction augments PLC{gamma}1 phosphorylation in antigen-stimulated T cells
In T lymphocytes, the sustained activation of calcium pathway requires the sustained breakdown of phosphoinositides by PLC{gamma}1 resulting in the accumulation of inositol-3-phosphate (27). It is well established that PLC{gamma}1 is phosphorylated following TCR engagement and that the enzymatic activity of the PLC{gamma}1 is controlled by phosphorylation (3, 7). In order to evaluate the effect of the block of CD2/CD58 binding at the IS on the activation of PLC{gamma}1, we measured PLC{gamma}1 phosphorylation in antigen-stimulated T cells.

Cloned human T cells were conjugated with APCs treated with either control antibodies or anti-CD58 antibodies; PLC{gamma}1 was immunoprecipitated from cellular lysates. As shown in Fig. 3(A), phosphorylation of PLC{gamma}1 was not detectable in T cells alone, in APCs alone or in T cells interacting with unpulsed APCs. Conversely, a strong phosphorylation was observed 3 and 10 min after conjugate formation in antigen-stimulated T cells (Fig. 3A and B). PLC{gamma}1 phosphorylation was inhibited by ~50% in T cell–APC conjugates in which the binding of CD2 with CD58 was blocked, as shown in Fig. 3(C).


Figure 3
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  		Fig. 3. CD2–CD58 interaction is required for PLC{gamma}1 phosphorylation. (A) PLC{gamma}1 phosphorylation detected by western blot analysis with anti-PTyr antibodies after immunoprecipitation of PLC{gamma}1 from lysates of T cell–APC conjugates. (B) Time course of PLC{gamma}1 phosphorylation. (C) Quantification of bands of PLC{gamma}1 phosphorylation. NP = unpulsed APCs, PMHCI = APCs pulsed with specific antigen and treated with anti-MHC class-I antibodies and PCD58 = APCs pulsed with specific antigen and treated with anti-CD58 antibodies. Data are presented as 100% of the PLC{gamma}1 phosphorylation measured in T cells interacting with APCs pulsed with 100 µM peptide and treated with anti-MHC class-I mAbs. Data are from one representative experiment out of two.

 
These results indicate that the phosphorylation of PLC{gamma}1 induced by TCR ligation by specific antigen is defective in the absence of CD2/CD58 binding at the cell–cell contact site.

CD2 and TCR signaling synergize for translocation and activation of PLC{gamma}1 at the IS
It has been shown that binding of PLC{gamma}1 to linker for activation T cell (LAT) and PLC{gamma}1 translocation to the plasma membrane are required for its phosphorylation and activation (28).

In order to evaluate the role of CD2/CD58 binding on the translocation of PLC{gamma}1 to the IS, we measured PLC{gamma}1 intracellular localization and its phosphorylation by confocal microscopy in fixed and permeabilized T cell–APC conjugates in which the CD2–CD58 interaction was either allowed or impeded. The enrichment of different molecules at the IS was evaluated by visual inspection in a blinded study (Table 1) and by measurement of the molecular enrichment at the IS in unprocessed images using the MetaMorph software (Fig. 6).


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  		Table 1. Measurement of the distribution of CD2, PLC{gamma}1 and PTyr783 PLC{gamma}1 at the T cell–APC contact site in T cells conjugated with peptide-pulsed APCs treated with anti-MHC class-I or anti-CD58 mAbs

 
In T cells interacting with unpulsed APCs, PLC{gamma}1 staining was diffused in the cytosol (Fig. 4A–C). PLC{gamma}1 staining was detectable also in the APCs, possibly due to cross-reactivity of antibodies with the PLC{gamma}2 isoform present in B cells or to the expression of PLC{gamma}1 isoform in EBV-transformed human B cells (29, 30). In T cells interacting with peptide-pulsed APCs (treated with anti-MHC class-I control antibodies), PLC{gamma}1 was rapidly translocated to the IS in parallel with CD2 enrichment (Figs 4D–F and 6 and Table 1). The translocation of PLC{gamma}1 was sustained since it was detectable for at least 30 min after conjugate formation (data not shown). When T cells were conjugated with peptide-pulsed APCs previously treated with anti-CD58 antibodies, the enrichment of CD2 was strongly inhibited as previously shown (25). Conversely, PLC{gamma}1 enrichment was partially inhibited (Figs 4G–I and 6 and Table 1). Taken together, these results indicate that when CD2–CD58 interaction is blocked, PLC{gamma}1 translocation is affected.


Figure 4
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  		Fig. 4. CD2–CD58 interaction regulates PLC{gamma}1 translocation to the IS. T cells were conjugated for 5 min at 37°C with APCs (red) which were either unpulsed (A–C) or pulsed with 100 µM-specific peptide (D–I). APCs were either treated with anti-MHC class-I mAbs (D–F) or with anti-CD58 mAbs (G–I). Cells were stained with anti-CD2 mAbs (blue) and anti-PLC{gamma}1 mAbs (green). Data are from one representative experiment out of three.

 
We next investigated whether the interaction between these two accessory molecules was relevant for phosphorylation of PLC{gamma}1 at the IS and therefore for its activation. T cell–APC conjugates were stained with antibodies directed against the phosphorylated form of the regulatory tyrosine 783, which is known to be phosphorylated upon PLC{gamma}1 activation. As shown in Fig. 5(A–C), in T cells interacting with unpulsed APCs no phosphorylation of PLC{gamma}1 was detected. Conversely, phosphorylation of PLC{gamma}1 was observed in T cells conjugated for 5 min with peptide-pulsed APCs (Fig. 5D–F). In T cells interacting with APCs in which the binding of CD2 with CD58 was impeded, the phosphorylation of PLC{gamma}1 was partially inhibited (Fig. 5G–I); however, a minor level of PLC{gamma}1 phosphorylation was detectable in a substantial fraction of T cell–APC conjugates. Thus, a large fraction of conjugates was scored as positive. This resulted in a scoring for PLC{gamma}1 phosphorylation that was not significantly altered by anti-CD58 treatment (Table 1). To obtain an accurate quantification of PLC{gamma}1 recruitment to the IS and of its phosphorylation in the different stimulation condititions, we measured the intensity of PLC{gamma}1 and phospho-PLC{gamma}1 staining at the IS using the MetaMorph software in unprocessed images of T cell–APC conjugates. This analysis revealed that in T cells, in which CD2 engagement was blocked, a 40–50% inhibition of PLC{gamma}1 recruitment and phosphorylation was detectable (Fig. 6).


Figure 5
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  		Fig. 5. CD2–CD58 interaction regulates PLC{gamma}1 phosphorylation on Tyr 783 at the IS. T cells were conjugated for 5 min at 37°C with APCs (red) which were either unpulsed (A–C) or pulsed with 100 µM antigenic peptide (D–I). APCs were treated with either anti-MHC class-I mAbs (D–F) or with anti-CD58 mAbs (G–I). Cells were stained with anti-CD2 mAbs (blue) and anti-PTyr783 PLC{gamma}1 rabbit antibodies (green). Data are from one representative experiment out of three.

 
These results are in agreement with those obtained using biochemical approaches (Fig. 3) and show that the phosphorylation of PLC{gamma}1 is actually occurring in the T cells and not in the APCs. Thus, these results not only confirm but also extend biochemical data. Taken together, these results indicate that the engagement of TCR with peptide–MHC complexes displayed on the APC surface in the absence of CD2 binding is not sufficient for a complete PLC{gamma}1 recruitment and phosphorylation. Thus, CD2 engagement enhances TCR signaling by favoring the assembly of signaling pathways involved in PLC{gamma}1 phosphorylation and activation at the IS.

CD2 and TCR signaling synergize for phosphorylation of ZAP-70 at the IS
To better characterize the role of CD2 in modulating TCR-mediated signal transduction, we studied its role in the activation of ZAP-70, a key signaling molecule upstream of PLC{gamma}1 pathway. To address this question we investigated the possibility of measuring by FACS analysis the intensity of phosphorylation of ZAP-70 in its regulatory tyrosine 493 (31, 32).

To our knowledge, the staining for PTyr 493 ZAP-70 was never previously documented in antigen-stimulated T cells. In preparatory experiments, we initially studied by confocal microscopy whether an enrichment of PTyr 493 ZAP-70 could be detected at the T cell–APC contact site and whether this enrichment could be modulated by CD2–CD58 interaction. As shown in Fig. 7(A), PTyr 493 ZAP-70 is actually enriched at the IS in antigen-stimulated T cells and its enrichment is inhibited by anti-CD58-blocking antibodies. Since the extent of ZAP-70 phosphorylation significantly varied among the different individual T cell–APC conjugates (Fig. 7Ac and Ad), we investigated by FACS analysis the effect that anti-CD58 antibodies may have on the level of ZAP-70 phosphorylation in the whole T cell population. Figure 7(B and C) show that the block of CD2/CD58 binding at the IS reduced the level of phosphorylation of ZAP-70 in the regulatory tyrosine 493. Taken together, these results indicate that CD2 synergizes with TCR for the activation of ZAP-70 at the IS.


Figure 7
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  		Fig. 7. CD2–CD58 interaction enhances ZAP-70 phosphorylation on Tyr 493. (A) T cells were conjugated for 5 min at 37°C with APCs (red) either unpulsed (a) or pulsed with 100 µM antigenic peptide (b–d). APCs were treated with either anti-MHC class-I mAbs (b) or with anti-CD58 mAbs (c and d). Cells were stained with anti-PTyr 493 ZAP-70 rabbit antibodies (green). Data are from one representative experiment out of three. (B and C) FACS analysis measurement of PTyr 493 ZAP-70 staining in T cells conjugated with APCs either unpulsed or pulsed with antigenic peptide, treated with either anti-MHC class-I or anti-CD58 antibodies. (B) A representative PTyr 493 ZAP-70 staining is shown. (C) Measurement of PTyr 493 ZAP-70 at 5 min and at 15 min after conjugate formation in three independent experiments, each one performed in triplicate. Data are expressed as 100% of unpulsed. The mean values of the measurements are shown by the horizontal bar. The values between control and anti-CD58-treated cells were significantly different (P values are indicated in the figure).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary data
 References
 
In the present work, we investigated the role of CD2–CD58 interaction in the activation of the PLC{gamma}1 pathway in antigen-stimulated T lymphocytes. The activation of PLC{gamma}1 pathway and of downstream calcium and protein kinase C pathways are critical events in T cell activation (27). Even though the biochemical steps leading to the activation of PLC{gamma}1 in T cells stimulated by anti-CD3 antibodies are well defined (3), a detailed description of the mechanisms of PLC{gamma}1 activation at the IS is still missing. Here we show that the engagement of TCR and CD2 at the IS synergizes for the activation of PLC{gamma}1 in antigen-stimulated T cells.

The IS is a specialized area of signal transduction formed at the T cell–APC contact site (13, 33, 34). In its original definition, the mature IS was described as a specialized signaling domain formed at the contact site between T cells and APCs, characterized by large-scale molecular segregation of surface receptors and signaling components (13, 33, 34). Current research lead to an expansion of this term, where the IS comprises a multitude of structures all of them having in common that they are mediators of intercellular communication (35). The role of the IS is still elusive, and it may serve several non-mutually exclusive functions. It has been proposed that IS could either augment and sustain activation (35) or extinguish the activation process by accelerating TCR down-regulation (36). Our results support the notion that the IS is indeed a platform for signaling sustenance and amplification (25).

In the present work, we concentrated on the activation of PLC{gamma}1 in antigen-stimulated T cells. We study PLC{gamma}1 activation by detecting its recruitment and phosphorylation at the IS. We show that the activation of this key signaling enzyme has two components. The first component is given by the engagement of TCR with specific peptide–MHC complexes. This event is required for PLC{gamma}1 recruitment and activation. The second component is given by the engagement of CD2 which amplifies the TCR-induced PLC{gamma}1 recruitment and phosphorylation. The molecular mechanisms of such synergy are elusive. Our observation that CD2- and TCR-derived signals synergize for the activation of ZAP-70 at the IS suggest that CD2 may play a central role in enhancing the entire signaling pathway leading to calcium mobilization.

We have previously shown that extracellular signal-regulated kinase (ERK) phosphorylation is unaffected by blocking CD2–CD58 interaction. This observation is not in contrast with our present observation of reduced ZAP-70 phosphorylation. Indeed, work performed in ZAP-70-deficient Jurkat cells or cells derived from patients affected by genetic deficiency of ZAP-70 indicates that the activation of ERK pathway and of PLC{gamma}1/calcium pathway are differently regulated in T cells and that ZAP-70 function is dispensable for the activation of ERK pathway (37–40).

We also previously showed that the block of CD2/CD58 binding does not affect the total level of phosphotyrosine staining in T cells. These data are not in contrast with our present results showing a moderate (~30%) inhibition of ZAP-70 phosphorylation in antigen-stimulated T cells. Experiments performed using P116 cells have shown that ZAP-70 is required for several downstream phosphorylation events following TCR aggregation with anti-CD3 antibodies (39). Even though these results are important and central to our understanding of signaling events in T lymphocytes, they are difficult to apply to our cellular system in which T cells detect antigen on the surface of APCs. In our cell system several surface molecules are engaged in parallel with TCR and modulate signal transduction. This makes it difficult to establish a direct parallelism between observations in Jurkat cells (stimulated by anti-CD3 antibodies) and our results. In addition, it should be noted that while ZAP-70 is reportedly absent in P116, we observe only a moderated inhibition of phosphorylation in one of the ZAP-70 regulatory residues (Fig. 7). Thus, in our system, the ZAP-70 enzymatic activity is not knocked out such as in P116. The remaining ZAP-70 enzymatic activity can be sufficient to activate downstream phosphorylation events. It is likely that in our cellular system (similarly to Jurkat cells), PLC{gamma}1 pathway is more dependent than other pathways to the reduction of ZAP-70 function (37).

In conclusion, how cross-talk among signaling cascades occurs and how it is regulated in antigen-stimulated T cells are presently elusive. It is possible that differences may exist among antigen-stimulated T lymphocytes and Jurkat cells. Addressing this issue will be a challenging task for the TCR signaling community. Our present results provide a first stepping-stone to address this challenging question.

We cannot exclude that additional mechanisms could be implicated in the role of CD2 in enhancing PLC{gamma}1 phosphorylation. It is tempting to speculate that CD2 via the intermediation of associated proteins such as CD2AP may favor the assembly of a signaling scaffold at the IS thus augmenting PLC{gamma}1 recruitment and phosphorylation. It is well established that following TCR engagement, PLC{gamma}1 is associated to tyrosine 132 of the adapter molecule LAT following phosphorylation by ZAP-70 (28, 41). Moreover, it has been proposed that several additional tyrosine kinases such as Itk (21) and/or p59 Fyn (17) are involved in PLC{gamma}1 phosphorylation on regulatory tyrosines following binding to LAT. CD2AP could bind different tyrosine kinases or adapters required for PLC{gamma}1 activation. In agreement with this hypothesis, it has been shown that CD2AP binds phosphoinositide-3-kinase (PI3K), resulting in an increase in PIP3 synthesis (42). PI3K products could participate in the recruitment of PLC{gamma}1 to the IS by binding its pleckstrin homology (PH) domain (43). In addition, this mechanism may favor recruitment of Tec kinases that may bind to PIP3 via their PH domain and therefore be recruited to the TCR signaling area to participate in PLC{gamma}1 phosphorylation. An additional mechanism by which CD2 engagement may favor PLC{gamma}1 activation could result from CD2 localization in the specialized membrane lipid domain named ‘rafts’ (44, 45). The enrichment of CD2 into the IS during antigenic stimulation (Figs 4 and 5) may favor clustering of raft components involved in TCR signaling such as LAT and src kinases.

In conclusion, our results shed new light on the role of CD2 in physiological T lymphocyte activation. It is well known that the cross-linking of CD2 with specific antibodies triggers signal transduction in T cells (16). Conversely, it has been shown that in antigen-stimulated T lymphocytes, CD2 expression is not strictly required for T cell activation, yet its expression lowers the threshold of antigenic stimulation (10). The reason for this apparent discrepancy is not clear. It is tempting to speculate that CD2 cross-linking may induce a massive aggregation of surface molecules and signaling components thus bypassing the requirement for TCR engagement. Our results provide the missing link among these studies. We show that in T lymphocyte–APC conjugates, the interaction between CD2 and CD58 does not autonomously initiate but rather modulate signal transduction.

Interestingly, in the absence of TCR triggering, the engagement of CD2 with its ligand CD58 displayed on the surface of APCs is not sufficient to induce neither CD2 recruitment nor substantial activation of calcium pathway (unpulsed T cell–APC conjugates in Figs 1 and 4–6). When specific TCR ligands are present on the APC surface, CD2 is recruited to the IS and its engagement augments signal transduction even in conditions of optimal antigen stimulation.

Our data are compatible with a model in which CD2 may behave as an amplifier of TCR-induced activation of the PLC{gamma}1/calcium pathway without being intrinsically able to trigger this pathway. In other words, it would behave as the volume control of a radio in which the tuning of the signal is dependent on TCR engagement.


   Supplementary data
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
References
 
Supplementary movies 1–3 are available at International Immunology Online.


   Acknowledgements
 
This work was supported by grants from la Ligue contre le Cancer ‘Equipe labellisee 2005’ and la Fondation BNP Paribas. N.E. is supported by a fellowship from the Fondation pour la Recherche Medicale.


   Abbreviations
 
APC, antigen-presenting cell
CD2AP, CD2 adapter protein
ERK, extracellular signal-regulated kinase
Itk, IL-2-inducible T cell kinase
IS, immunological synapse
LAT, linker for activation T cell
PH, pleckstrin homology
PI3K, phosphoinositide-3-kinase
PLC{gamma}1, phospholipase C{gamma}1
ZAP-70, zeta-associated protein-70

   Notes
 
Transmitting editor: J. Borst

Received 15 June 2006, accepted 13 December 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 

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