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. 2012 Oct 2;137(3):214–225. doi: 10.1111/j.1365-2567.2012.03626.x

Syndecan-2 can promote clearance of T-cell receptor/CD3 from the cell surface

Xavier Rovira-Clavé 1,*, Maria Angulo-Ibáñez 1,*, Oriol Noguer 1,3, Enric Espel 2, Manuel Reina 1
PMCID: PMC3482679  PMID: 22881146

Abstract

T cells express the heparan sulphate proteoglycans syndecan-2 and syndecan-4. Syndecan-4 plays a T-cell inhibitory role; however, the function of syndecan-2 is unknown. In an attempt to examine this function, syndecan-2 was expressed constitutively in Jurkat T cells. Interestingly, the expression of syndecan-2 decreased the surface levels of T-cell receptor (TCR)/CD3 complex, concomitant with intracellular retention of CD3ε and partial degradation of the TCR-ζ chain. Immunofluorescence microscopy revealed that intracellular CD3ε co-located with Rab-4 endosomes. However, the intracellular pool of CD3ε did not recycle to the cell surface. The lower TCR/CD3 surface levels caused by syndecan-2 led to reduced TCR/CD3 responsiveness. We show that the cytosolic PDZ-binding domain of syndecan-2 is not necessary to elicit TCR/CD3 down-regulation. These results identify a previously unrecognized means of controlling surface TCR/CD3 expression by syndecan-2.

Keywords: endocytosis, syndecan, T lymphocytes, T-cell receptor down-regulation, T-cell receptor ζ chain

Introduction

The T-cell receptor (TCR)/CD3 complex is formed by a non-covalent association between the antigen-binding TCR-αβ heterodimer and the CD3δ, ε, γ molecules and TCR-ζ chains, which together are referred to as the CD3 complex and are required for TCR expression, signal transduction and receptor transport.1 The TCR/CD3 complex cycles continuously between the plasma membrane and the intracellular compartment. This balance keeps approximately 75–85% of the cycling TCR/CD3 pool located at the cell surface, with the rest located inside the cell.2–5 Following ligation with antigen or antibody, the TCR/CD3 is internalized and degraded, leading to its decrease on the cell surface and the subsequent attenuation of TCR/CD3 signalling, which contributes to T-cell desensitization.5–8 Indeed, when endocytosis of the TCR/CD3 complex is delayed by interference in the internalization process, this results in increased surface TCR/CD3 density and sustained signalling in both mature T cells9–12 and double-positive thymocytes,10,13,14 which may lead to autoreactivity.8,15 Therefore, regulation of surface TCR/CD3 density is an important mechanism that determines the ability of the T cells to respond to stimuli.

Syndecan (SDC) family proteins are recycling endocytic receptors that can control cell surface dynamics.16–18 Syndecans are also expressed in haematopoietic cells, and SDC2 and SDC4 are up-regulated during T-cell activation.19–21 Recognition of SDC4 by its ligand DC-HIL in antigen-presenting cells impairs T-cell activation, by the action of the tyrosine phosphatase CD148.22 However, the function of SDC2 in T cells is not yet known. This study identifies a new mechanism of regulation of the cell surface levels of the TCR/CD3 complex by SDC2.

Materials and methods

Materials and antibodies

MAR93 monoclonal antibody (mAb) was used to detect human cell surface CD25 (kindly donated by Dr M. Lopez Botet; Universitat Pompeu Fabra). Anti-TCR-ζ clone 6B10.2 (sc-1239) and anti-SDC2 (sc-9492) were obtained from Santa Cruz Biotechnology (Tebu-bio, Barcelona, Spain). Anti-CD3ε clone 33-2A3 is a mouse IgG2a mAb (Inmunostep, Salamanca, Spain). Anti-TCR-αβ clone IP26 was from Biolegend (Grupo Taper, Alcobendas, Madrid, Spain). Anti-transferrin receptor clone RVS10, anti-CD69 clone FN50, FITC-conjugated anti-CD95 clone LT95 and anti-MHC class I clone W6/32 were from Immunotools (Friesoythe, Germany). Allophycocyanin-conjugated anti-CD4 clone RPA-T4 was from Becton Dickinson (San Agustin de Guadalix, Madrid, Spain). Anti-CD28 is a mouse IgM mAb (clone CK248, kindly donated by Dr Pedro Romero; Ludwig Institute, Lausanne, Switzerland) and was used as a hybridoma supernatant for priming of CD4 T cells in combination with phytohaemagglutinin. Rabbit anti-Rab4 was from Abcam (Cambridge, UK) and rabbit anti-Lamp1 conjugated with Cy3 was from Sigma-Aldrich (Tres Cantos, Madrid, Spain). Anti-Erk1/2 (ref. 9102) and anti-phospho-Erk1/2 (ref. 9101) were from Cell Signalling (Izasa, Barcelona, Spain). The anti-syndecan-2 mAb 186C (mouse IgG1, clone sdc2.1.186.CL.C) was described elsewhere.21 This mAb and anti-syndecan-2 mAb 51C (mouse IgG3, clone sdc2.1.51.CL.C), both to human SDC2, were made by immunizing mice with human sdc2-transfected 300.19 cells and showed specific binding by FACS to sdc2-transfected cell lines (300.19, Jurkat and COS) but not to untransfected cells. These anti-syndecan mAb were purified by ammonium sulphate precipitation from concentrated supernatants obtained from hybridoma cultures in Integra-CL-1000 flasks (Integra Biosciences, Cultek, Madrid, Spain).

Western blot

A total cell extract was prepared from 106 cells dissolved in 150 μl SDS-Loading Buffer. The cell extract was incubated at 95° during 30 min before loading on 10% or 12% SDS–polyacrylamide gel and transfer to a nitrocellulose membrane. Western blots were quantified on a Luminescent Image Analyzer LAS-3000 (Fujifilm, Barcelona, Spain) using Gel Evaluation analysis software version 1.35 (FrogDance Software, Dundee, UK) or imagej software (Rasband, W.S., imagej; US National Institutes of Health, Bethesda, MD, http://rsb.info.nih.gov/ij/, 1997–2009).

For TCR-ζ degradation experiments, cells were cultured in Dulbecco's modified Eagle's medium (DMEM) 10% fetal calf serum (FCS) containing 50 μg/ml cycloheximide (Sigma). At each time-point, cells were lysed in SDS loading buffer as described above. Lysates were analysed by immunoblot for anti-actin (AC-40) or TCR-ζ (6B10.2). Immunoblots and quantification were performed as described above.

Plasmids

Human HA-Arf6 and HA-Arf6-T27N were plasmids #10834 and #10831, respectively, from Addgene (Cambridge, MA), deposited by Dr T. Roberts.23 The DNA fragment corresponding to human wild-type sdc2 cDNA was obtained by PCR amplification using the low-error Phusion™ High-Fidelity DNA polymerase (Finnzymes, Thermo Fisher Scientific, Madrid, Spain) and cloned in pcDNA3. Mutation of wild-type sdc2 cDNA was carried out by PCR amplification using primers containing the desired mutations. The 5′ ends of each pair of mutating primers (forward and reverse) overlapped approximately by 15 bp (with a Tm below 55° for the 15-bp overlap). After PCR amplification (15–20 cycles) the amplified DNA was DpnI digested and directly used to transform competent DH5α. The authenticity of the constructs generated was confirmed by sequencing.

Cell culture and transfections

Jurkat E6.1 human cells were obtained from the European Collection of Cell Cultures (ECACC) (Sigma-Aldrich). Jurkat 8B is a CD4-negative Jurkat cell line routinely used in our laboratory.21,24 Both Jurkat cell lines (E6.1 and 8B) were used in all assays described in this study with analogous results. Jurkat cells were routinely cultured in DMEM, supplemented by 10% FCS, 2 mm l-glutamine and penicillin/streptomycin, and maintained at 37° in 5% CO2. The plasmid constructs were transfected into cells by electroporation (Multiporator; Eppendorf, Madrid, Spain). The transfected cells were cultured in complete medium for 24 hr before the selecting antibiotic was added. A total of five Jurkat cell clones over-expressing wild-type SDC2 were obtained and used in this study.

Preparation of human T lymphocytes

The studies with human samples have been reviewed and approved by the University of Barcelona ethics committee. Primary human T cells were obtained from buffy coats from blood donors at the Banc de Sang i Teixits (Blood and Tissue Bank, Barcelona, Spain).

Peripheral blood mononuclear cells were isolated by a Ficoll 1·007 density gradient (Lymphoprep ref. 1114545; Axis-Shield, Reactiva, Barcelona, Spain). CD4 T cells were purified by negative selection, with affinity chromatography (Cedarlane, Tebu-bio, Barcelona, Spain), following the protocol supplied by the manufacturer. Purified CD4 T cells were cultured in DMEM–10% FCS, supplemented by 2 mm glutamine, 200 U/ml penicillin and 200 μg/ml streptomycin.

To prepare primed CD4 T cells, these were activated at 37° using phytohaemagglutinin (10 μg/ml), anti-CD28 mAb (CK248 supernantant) and interleukin-2 (20 U/ml). After 3–4 days, lymphocytes were washed and cultured in normal media (DMEM 10% FCS) for at least 1 day before use in experiments.

Labelling of cycling pool and recycling

The measurement of the cycling pool was performed as described elsewhere.25 To analyse the rate of CD3ε recycling, cells were incubated at 37° for an hour in the presence of anti-CD3ε antibody, then the surface antibody bound to CD3 was eliminated by acid wash by adding 1 volume of 0·5 m NaCl, 0·5 m acetic acid (pH 2·2). In this way 96% of cell surface staining was removed. Cells were immediately layered on top of an FCS cushion and pelleted by centrifugation. Cells were resuspended in cell culture media and incubated at 37° for the times indicated. At each time-point, recycling was stopped by layering cells on ice. Cells were labelled with an Alexa 488-conjugated F(ab')2 rabbit anti-mouse IgG and analysed by flow cytometry. Recycled CD3ε was calculated using the following formula: % Recycled = 100 × (MFI at time point - MFI after acid wash at t=0)/(MFI before acid wash at t=0 - MFI after acid wash at t=0).25

Lymphocyte activation in coated plates

CD4 T cells (2 × 105 cells/well) were seeded in 24-well flat-bottom plates, which had been pre-coated with anti-CD3 or mouse IgG control antibodies. After culturing at 37°, cells were lysed and total RNA was purified.

Flow cytometry

Cells were incubated with saturating amounts of primary antibody in PBS–1% FCS for 30 min at 4°, washed and incubated with Alexa 488-conjugated secondary antibody in PBS–1% FCS for 30 min at 4° in the dark. Non-specific fluorescence was assessed by incubating cells only with the same Alexa 488-conjugated anti-mouse immunoglobulin antibody (grey histograms).

For intracellular labelling, cells were fixed in 1% paraformaldehyde in PBS for 20 min at room temperature, washed and permeabilized with 0·2% saponin in PBS–1% FCS (permeabilization buffer) for 30 min in ice. Cells were centrifuged and resuspended in permeabilization buffer containing primary antibody for 1 hr at 4°. Then, cells were washed and treated with secondary antibody conjugated to Alexa 488 (Invitrogen, Barcelona, Spain) in permeabilization buffer for 45 min at 4°. Cells were washed with permeabilization buffer for 45 min at room temperature, resuspended in PBS–1% FCS and analysed by flow cytometry. Non-specific fluorescence was calculated by incubating cells with a non-specific IgG (clone NCG01 for mouse antibodies and clone DA1E for rabbit antibodies) followed by staining with the same Alexa 488-conjugated antibody. Flow cytometry experiments used a Cytomics FC500 MPL or a Gallios flow cytometer (Beckman Coulter, Inc, Fullerton, CA). The sample was excited by a 488 nm air-cooled argon-ion laser (Cytomics, Beckman Coulter, Izasa, Barcelona, Spain) or 488 and 635 nm lasers (Gallios, Beckman Coulter, Izasa). Fluorescence was collected on the logarithmic scale. Optical alignment was checked with 10-nm fluorescent beads (Flow-Check fluorospheres; Beckman Coulter). Cell population was selected by gating in a forward scatter versus side scatter dot plot, excluding aggregates and cell debris. Fluorescence histograms were represented in single-parameter histograms (1024 channels) and dual-parameter histograms of green versus orange fluorescence, to distinguish auto-fluorescence from Alexa 488 fluorescence.

Immunocytochemical staining and confocal microscopy

Cells were fixed and permeabilized with 0·2% saponin as indicated before. Cells were incubated with 100 μl primary antibody mix (1 μg/100 μl) diluted in permeabilization buffer, for 1 hr at 4°. Rabbit anti-Rab4 and rabbit anti-Lamp1 conjugated with Cy3 were used to detect endosomal or lysosomal intracellular compartments, respectively. Cells were washed and incubated with anti-mouse-Alexa 488 or anti-rabbit-Alexa 546 (both from Invitrogen) antibody diluted in permeabilization buffer. Finally, cells were washed and resuspended in 7 μl Fluoromont mounting medium, which was applied to microscope slides and covered with coverslips. Immunostained samples were viewed with a Leica TCS-SPE confocal microscope with an argon–krypton laser, in a 60 × (NA 0·7, oil) Leitz Plan-apochromatic objective, at room temperature. Pictures were acquired with the Leica Application Suite software (Leica microsistemas, Barcelona, Spain). imagej software was used to quantify the Pearson correlation coefficient for co-localization analysis in individual cells. The results given are the mean of at least five single cells. Final artwork was processed by Adobe Photoshop (Pro CS5) software (Adobe Corp, San Jose, CA).

Real-time quantitative PCR

Cells were lysed and their RNA was isolated. The RNA was reverse-transcribed (Super-Script III, ref. 11752-050; Invitrogen) and real-time PCR was carried out with SybrGreen-based detection (ref. 11761-500; Invitrogen). Oligonucleotides were designed with the Primer 3 program on the www (Steve Rozen and Helen J. Skaletsky, http://frodo.wi.mit.edu). The oligonucleotides used for interleukin-2 were: 5′-AACTCACCAGGATGCTCACA-3′ (sense) and 5′-GCACTTCCTCCAGAGGTTTG-3′ (anti-sense). The oligonucleotides used for TCR-ζ were: 5′-CAGCCTCTTTCTGAGGGAAA-3′ (sense) and 5′-TCTCAGGAACAAGGCAGTGA-3′ (anti-sense). The oligonucleotides to detect 18S rRNA were from TATAA (ref. RRN18S; TATAA Biocenter, Tebu-bio, Barcelona, Spain).

A dilution series (10−1–10−3) of the specific PCR product under study was prepared to determine the standard curve (relative quantification). The samples (in duplicate) were amplified according to the following general protocol: 10 min at 95°, 42 cycles: 15 seconds at 95°, 15 seconds at 60° and 20 seconds at 72°. To control the specificity of the reaction, melting-curve analysis was performed after amplification. Levels of endogenous 18S RNA were used as normalization controls and we calculated relative mRNA levels using the equation: Inline graphic where E is the efficiency of the PCR, Ct is the threshold cycle, s is the stimulated sample and c is the unstimulated control.26

Statistical analysis

All the data are presented as means ± SD from three or more separate experiments. The unpaired t-test was performed for data involving two groups only and data involving more than two groups were analysed by one-way analysis of variance with Tukey's multiple comparison test. The relationship between cell surface expression of SDC2 and that of CD3 was determined by Spearman's rank correlation analysis. The graphpad instat version 3.1a software for Macintosh was used (GraphPad, San Diego, CA). Differences were considered statistically significant when P values were < 0·05.

Results

Over-expression of SDC2 down-regulates TCR/CD3 and promotes degradation of TCR-ζ chain

Human T cells express SDC2 at the cell surface (see Supplementary material, Fig. S1).19,21 To study the role played by SDC2 in T lymphocytes we stably over-expressed SDC2 in Jurkat T cells (Fig. 1a). Interestingly, sdc2-transfected cells (Jurkat-SDC2) showed a marked reduction in surface TCR-αβ and CD3ε, as analysed by flow cytometry (a representative clone of the five analysed is shown in Fig. 1b,c). However, other cell membrane proteins like Fas, CD69 or MHC class I were not down-regulated (see Supplementary material, Fig. S2a). During T cell–antigen-presenting cell interaction, the CD4 co-receptor is down-regulated coordinately with triggered TCR.27 In Jurkat-SDC2 cells, down-regulation of CD4 was also observed, possibly accompanying the reduction of surface TCR/CD3 in these cells (Fig. S2a). Analysis of transferrin receptor (TfR) expression showed substantial heterogeneity between the different clones. The TfR was down-regulated in four clones but up-regulated in another (Fig. S2b).

Figure 1.

Figure 1

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Over-expression of SDC2 down-regulates T-cell receptor (TCR)/CD3 in Jurkat T cells. (a) Expression of SDC2. Cells were incubated with anti-SDC2 monoclonal antibodies (mAb; 186C), followed by staining with F(ab′)2 of Alexa 488-conjugated rabbit anti-mouse Ig (open histograms). For staining of total SDC2 (extracellular and intracellular), cells were fixed and permeabilized before incubation with antibody. Grey histograms, control cells with secondary antibody only (surface labelling) or isotype-matched control primary antibody plus secondary antibody (permeabilized cells). The abscissa gives the fluorescence intensity on a logarithmic scale. (b) Expression of CD3ε. Jurkat-SDC2 and untransfected Jurkat cells were incubated with anti-CD3ε mAb 33-2A3, followed by staining with Alexa 488-conjugated anti-mouse antibody (open histograms). (c) Expression of TCR-αβ and TCR-ζ chain. Cells were incubated with anti-TCR-αβ mAb IP26 or permeabilized and incubated with anti-TCR-ζ chain rabbit antibody followed by staining with Alexa 488-conjugated secondary antibody (open histograms). (d) Quantification of FACS staining shown in (b) and (c). The relative expression ratio is calculated from the mean fluorescence intensity (MFI) values of untransfected Jurkat versus the Jurkat-SDC2 cells (TCR-αβ, n = 3, data from one Jurkat-SDC2 cell clone representative of three; CD3ε, the error bars representing SD among five different cell clones analysed in five independent experiments; TCR-ζ, the error bars represent variations among four different cell clones analysed in four independent experiments). (e) Western blot analysis of TCR-ζ. The blot was reprobed with anti-actin as an internal loading control of the samples and the intensity of the protein bands was quantified using quantitative luminescence. The analysis was repeated with five different Jurkat-SDC2 clones and the ratio TCRζ : actin ± SD is represented (bottom).

To examine whether the decreased levels of surface TCR/CD3 were caused by its intracellular retention, sdc2-transfected cells were fixed, permeabilized and incubated with anti-CD3ε or anti-TCR-ζ chain antibodies to analyse their total content. Interestingly, sdc2-transfected cells contained considerable intracellular amounts of CD3ε (Fig. 1b), but a decreased TCR-ζ chain content (Fig. 1c). Although the surface levels of CD3ε in five sdc2-expressing clones were on average 17-fold lower than on untransfected Jurkat cells, the total cellular levels of CD3ε (surface plus intracellular) were not significantly different (Fig. 1d). As for the total cellular levels of TCR-ζ there was an approximately 50% decrease in Jurkat cells over-expressing SDC2 from the level in untransfected cells (Fig. 1c–e). Therefore, over-expression of SDC2 in Jurkat cells down-regulates TCR/CD3 and reduces TCR-ζ total levels.

To study the effect of SDC2 on TCR-ζ cellular levels in more detail, we analysed whether TCR-ζ was regulated at the transcriptional level. The reduced amount of cellular TCR-ζ in Jurkat-SDC2 was not a consequence of lower transcriptional expression, as the TCR-ζ mRNA steady-state levels in Jurkat and Jurkat-SDC2 cells were comparable (Fig. 2a). This suggests that the intracellular retention of CD3δε and CD3γε dimers in Jurkat-SDC2 cells is accompanied by TCR-ζ degradation. To address this possibility, cells were treated with the protein synthesis inhibitor cycloheximide and total levels of TCR-ζ were evaluated by immunoblotting. Quantitative analysis of the Western blot showed a faster decrease in the TCR-ζ protein in Jurkat-SDC2 cells than in untransfected cells (Fig. 2b,c); its half-life was > 10 hr in untransfected Jurkat cells, but approximately 5 hr when SDC2 was expressed (Fig. 2b,c). These data suggest that SDC2 controls the degradation of TCR-ζ chain.

Figure 2.

Figure 2

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SDC2 induces degradation of T-cell receptor-ζ (TCR-ζ). (a) Total RNA was extracted from aliquots of cells to analyse specifically the levels of TCR-ζ mRNA and 18S RNA by real-time PCR. The TCR-ζ : 18S RNA ratio was calculated and was given an arbitrary value of 1·0 for control Jurkat cells. The analysis was repeated with three different Jurkat-SDC2 clones and the average ± SD is represented. (b) Western blot analysis of TCR-ζ. Jurkat and Jurkat-SDC2 cells were cultured in the presence of cycloheximide for different time intervals, the lysates were immunoblotted with anti-TCR-ζ, and the same membrane was reprobed with anti-actin. (c) The intensity of TCR-ζ and actin bands of the experiment depicted in (b) and additional experiments were quantified and the ratio TCR-ζ : actin at time 0 was given an arbitrary value of 100. Data are the mean ± SD of three experiments with three Jurkat-SDC2 clones (*P ≤ 0·5).

Jurkat-SDC2 cells are poorly activated by CD3ε stimulation

In line with the low surface levels of the TCR/CD3 receptor on sdc2-transfected cells, these cells had a reduced capacity to respond to anti-CD3ε stimulation. Indeed, the analysis of interleukin-2 mRNA and cell surface expression of CD25 and CD69 as read-outs of T-cell activation showed that Jurkat-SDC2 cells were virtually unresponsive to plate-bound anti-CD3ε mAb (Fig. 3). However, the similar responsiveness of untransfected and sdc2-transfected Jurkat cells to activation with phorbol ester indicated that the differences in T-cell activation were not caused by alterations in signalling pathways downstream of the TCR (Fig. 3b–e). Analysis of Erk1/2 phosphorylation upon incubation of cells with phorbol ester confirmed that mitogen-activate protein kinase signalling was not impaired (Fig. 3d,e). Overall, these results indicate that low surface levels of TCR/CD3 receptor on sdc2-transfected cells, make them unresponsive to anti-CD3ε stimulation.

Figure 3.

Figure 3

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Jurkat-SDC2 cells do not respond to CD3 ligation. (a) Cells were starved overnight in DMEM-0·5% FCS and then stimulated with plate-bound anti-CD3ε monoclonal antibody (mAb) or control IgG (4 μg/ml) for 3 hr. The expression of interleukin-2 (IL-2) mRNA was analysed by quantitative PCR and normalized to 18S RNA content. Results are expressed as IL-2 : 18S ratio ± SD, average of three experiments with one Jurkat-SDC2 cell clone, representative of two. (b,c) Relative levels of CD25 and CD69 on the surface of Jurkat-SDC2 cells stimulated with plate-bound anti-CD3ε mAb or PMA were compared with control Jurkat cells by flow cytometry. (b) Representative histograms of CD25 and CD69 labelling show unlabelled control (grey shaded), control IgG-treated cells, and anti-CD3- and PMA-treated cells (open histograms). Although the Jurkat-SDC2 cells show the basal expression of CD69 diminished, this trait was not commonly associated with SDC2 expression in the other clones (see Supplementary material, Fig. S2). (c), The mean fluorescence intensity (MFI) of cells expressing surface CD25 and CD69 is represented as a percentage of the maximum value obtained in cells stimulated with anti-CD3. Data represent the mean + SD of three Jurkat-SDC2 clones analysed in eight experiments. (d) Western blot analysis of phosphorylated erk1/2. Jurkat and Jurkat-SDC2 cells (results from two different clones are shown) were stimulated with PMA for 5 min, the lysates were immunoblotted with anti-phospho-erk1/2, and the same samples were analysed with anti-erk1/2 in a separate immunoblot. (e) The intensities of phospho-erk1/2 and erk1/2 bands of the experiment depicted in (d) were quantified and the ratio (phospho-erk1/2) : (erk1/2) for PMA-stimulated Jurkat-SDC2 samples was given an arbitrary value of 100. Data represent the average of two experiments, each with three cell clones (the error bars represent variations among clones).

CD3ε locates in endosomes in Jurkat-SDC2 cells

The above results showed that the total expression of CD3ε was unaffected by SDC2 over-expression. To determine the fate of CD3ε in Jurkat-SDC2 we analysed the presence of CD3ε in the endosomal and lysosomal compartments using confocal microscopy. CD3ε co-located with endosomal Rab4 in both sdc2-transfected and untransfected Jurkat cells. Moreover, very low co-location between CD3ε and lysosomal Lamp-1 was detected in sdc2-transfected cells (Fig. 4a-c). For comparison, we analysed the distribution of CD3ε in human CD4 T cells that had been stimulated with anti-CD3ε to trigger TCR/CD3 down-regulation and TCR-ζ degradation. The immunoblot analysis of cellular TCR-ζ chain showed its partial degradation in stimulated cells (see Supplementary material, Fig. S3). As for the distribution of CD3ε in CD3-stimulated primary T cells, it was similar to Jurkat-SDC2 cells, showing major co-location of CD3ε with Rab4-endosomes, but low co-location with Lamp1-lysosomes (see Supplementary material, Fig. S4). These data suggest that the degradation of TCR-ζ chain observed in Jurkat-SDC2 cells is accompanied by intracellular storage of CD3γ/ε and CD3δ/ε dimers.

Figure 4.

Figure 4

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Intracellular location of CD3ε in wild-type and sdc2-transfected Jurkat cells, and recycling of CD3ε. Cells were fixed, permeabilized and stained with antibodies against CD3ε (green) and either Lamp-1 (a) or Rab4 (red) (b). Co-location is clearly observed between CD3ε and the early endosomal marker Rab4 (merged image, yellow). There is low co-location with Lamp-1. A magnification of a single cell is shown in the right-hand column. Scale bar: 5 μm. One of three independent experiments is shown. A total of three Jurkat-SDC2 clones were analysed. (c) Quantification of the co-location of internalized CD3 and endosomal Rab4 or lysosomal Lamp1, by measurement of Pearson's correlation coefficient in individual cells (n ≥ 5), as described in the Materials and methods. (d) Down-regulation of CD3ε in Jurkat-SDC2. Jurkat-SDC2 were stimulated for 2 hr with monoclonal antibodies (mAb) against CD3ε (33-2A3, 0·3 μg/ml) or mouse IgG and transferred to ice. The cells were then incubated with saturating amounts of the same anti-CD3ε mAb, followed by staining with Alexa 488-conjugated goat anti-mouse IgG2a antibody and subjected to flow cytometry analysis. Data represent the average of mean fluorescence intensity (MFI) + SD (error bars) of two independent experiments carried out with two separate Jurkat-SDC2 clones. (e) Top panel. Surface CD3ε and total recycling pool of CD3ε in Jurkat-SDC2 and untransfected Jurkat cells. The surface and recycling pools were labelled by incubating cells with anti-CD3ε mAb and an Alexa 488-conjugated rabbit anti-mouse F(ab′)2 antibody on ice to label only surface CD3ε or at 37° for 120 min to label both surface CD3ε and CD3ε that recycled to the cell surface during the 120-min incubation.25 The MFI of the cells were determined by flow cytometry and are represented as a percentage of the values of ice-incubated control cells. Mean + SD of five experiments conducted with the same Jurkat-SDC2 cell clone (*significant increase of fluorescence for cells incubated at 37° as compared with control cells, P < 0·05, paired t-test). Note that the recycling pool is shown in relationship to the surface CD3ε at time 0, which was lower for Jurkat-SDC2 cells. Similar data were obtained with two additional Jurkat-SDC2 clones. Bottom panel. Recycling of previously internalized anti-CD3ε antibody. The percentage of recycling is shown in reference to the cell surface labelling at time 0 (see Materials and methods). Mean + SD of three experiments conducted with the same Jurkat-SDC2 clone (significant increase of recycling for Jurkat-SDC2 cells after incubating for 50 min as compared with time 0, paired t-test). The results are representative of two cell clones. In some Jurkat-SDC2 clones the low level of surface CD3ε precluded the analysis of recycling.

The increased intracellular content of CD3 in sdc2-transfected cells is possibly a direct consequence of the limiting amount of TCR-ζ chain. However, ligation of TCR/CD3 induces TCR down-regulation by increasing the internalization rate2 and preventing recycling.5 Therefore, we analysed the dynamics of TCR/CD3 internalization and recycling, to rule out that changes of these mechanisms contribute to intracellular storage of CD3ε. After the constitutive TCR/CD3 down-regulation of sdc2-expressing cells, we wondered whether these cells could down-regulate the TCR/CD3 further by receptor ligation. Indeed, ligation with anti-CD3ε mAb triggered down-regulation in Jurkat-SDC2 cells (Fig. 4d). This endocytosis response of Jurkat-SDC2 cells suggests that SDC2 does not affect the internalization of TCR/CD3.

To test whether the over-expression of SDC2 regulates recycling of the TCR/CD3 complex we first determined the total CD3ε cycling pool. The sdc2-transfected Jurkat cells were incubated with anti-CD3ε mAb and an Alexa 488-conjugated rabbit anti-mouse F(ab′)2 antibody, either on ice to label only surface CD3ε or at 37° for 120 min to label also the CD3ε that recycled to the cell surface during the incubation.25 An increase of fluorescence because of recycling of TCR/CD3 complexes was observed in both untransfected and sdc2-transfected cells, was proportional to the surface levels of CD3ε (Fig. 4e) and independent of the large intracellular CD3ε reservoir present in Jurkat-SDC2 cells. Therefore, in sdc2-transfected cells the intracellular pool of CD3ε contributes marginally to recycling.

We also measured the rate of CD3ε recycling in the two cell types. The results show that recycling of a previously internalized anti-CD3ε antibody in Jurkat and Jurkat-SDC2 cells proceeds at a similar rate (Fig. 4e). It should be noted that as the Jurkat-SDC2 cells have reduced TCR/CD3 surface levels (Fig. 1), they internalize less receptor than control Jurkat cells (data not shown).

These results suggest that changes in the internalization and recycling of TCR/CD3 do not contribute substantially to the low surface levels of TCR/CD3 in Jurkat-SDC2 cells. This suggests that the SDC2-induced shortage of TCR-ζ chain limits the amount of completed TCR/CD3 complexes reaching the cell surface.

Location of SDC2 and TCR-ζ chain in Jurkat-SDC2 cells

Recycling of syndecans is partially dependent on the small GTPase Arf6.18 To test whether the Arf6-GTP/GDP cycling activity was involved in SDC2-dependent TCR/CD3 internalization, we transfected Jurkat and Jurkat-SDC2 with wild-type Arf6 or the dominant negative mutant Arf6T27N, a form of Arf6 that is unable to exchange GDP for GTP.28 Flow cytometry analysis indicated that expression of wild-type Arf6 or Arf6T27N had no detectable effect on the surface levels of SDC2 and CD3ε (see Supplementary material, Fig. S5). These results suggest that the activity of Arf6 is not required to keep CD3ε down-regulated in Jurkat-SDC2 cells.

Transferrin receptor is a well-characterized recycling receptor that shares early endosomal trafficking with the TCR/CD3 during T-cell stimulation.29–31 Examination of TfR expression on Jurkat-SDC2 cells, showed that four of the five clones had reduced surface TfR, suggesting that the effect of SDC2 expression is not restricted to TCR/CD3, but includes clathrin-dependent endocytosis (Fig. S2b). The fact that CD4, which internalizes by a clathrin-dependent pathway32 was also down-regulated in Jurkat-SDC2, supports this idea (Fig. S2a).

To rule out a close interaction between SDC2 and TCR/CD3 we analysed the subcellular location of TCR-ζ and SDC2 by confocal microscopy. The expression of TCR-ζ chain in sdc2-transfected Jurkat cells was low and therefore, its staining pattern by confocal microscopy was dimmer than in normal Jurkat (Fig. 5a). Despite this limitation, co-location of TCR-ζ chain and SDC2 was observed in a fraction of Jurkat-SDC2 cells (arrowheads in Fig. 5b).

Figure 5.

Figure 5

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Location of SDC2, T-cell receptor-ζ (TCR-ζ) and CD3ε. (a) Staining with anti-TCR-ζ chain antibody of Jurkat and Jurkat-SDC2 cells. Cells were sedimented on poly-lysine-coated coverslips, fixed, permeabilized and stained with antibody. Scale bar: 9 μm. One of the five clones examined is shown. (b) Location of TCR-ζ and SDC2 in sdc2-transfected Jurkat cells. Cells were prepared as in (a) and stained with antibodies against TCR-ζ chain (green) and SDC2 (red) (polyclonal antibody sc-9492). Some co-location of TCR-ζ with SDC2 was observed. Scale bar: 18 μm. One of two independent experiments with four Jurkat-SDC2 clones is shown. (c) Location of CD3ε and SDC2 in sdc2-transfected Jurkat cells. Cells were prepared as in (a) and stained with antibodies against CD3ε (green) and SDC2 (red). Co-location of CD3ε with SDC2 was not observed. Scale bar: 18 μm. One of two independent experiments with two Jurkat-SDC2 clones is shown. (d) Location of CD3ε and SDC2 in CD4 T cells. T lymphocytes were treated with anti-CD3ε 33-2A3 and mouse IgG (top) or with anti-CD3ε and anti-SDC2 186C (bottom) for 10 min at 37°. Then, the cells were fixed and permeabilized. Immunocytochemical staining of CD3ε (green) and SDC2 (red) is shown. Co-location of CD3ε with SDC2 was not observed. Scale bar: 5 μm. One of two independent experiments is shown.

In contrast, the distribution of CD3ε, which accumulates in the cytoplasm of Jurkat-SDC2 cells, was clearly separated from SDC2 without evidence of co-location of the two proteins (Fig. 5c).

A similar analysis of the subcellular location of CD3ε and SDC2 was carried out in primary T cells. Activated T cells were incubated with anti-CD3ε and anti-SDC2 mAb to initiate down-regulation of both receptors,33 and were then stained for analysis by confocal microscopy. CD3ε fluorescence was punctate and distributed throughout cytoplasm, whereas SDC2 fluorescence was more diffuse (Fig. 5d). However, no co-location of CD3ε and SDC2 was observed. These results suggest that the TCR-ζ and CD3ε form different intracellular pools in Jurkat-SDC2 cells and that TCR-ζ and SDC2 may partially interact.

The PDZ binding domain of SDC2 is not necessary to down-regulate TCR/CD3

SDC2 has one recognized interaction motif involved in membrane traffic, the PDZ-binding domain in the C-terminal region, which interacts with syntenin.18 We therefore analysed whether the PDZ-binding domain of SDC2 played a role in TCR/CD3 down-regulation. To do this, we removed the four amino acids (EFYA) essential for binding to PDZ proteins. When the mutant sdc2 (Δefya) was expressed in Jurkat, the down-regulation of CD3ε was observed as in Jurkat-SDC2 cells (Fig. 6). The cell surface levels of CD3ε in transfectants decreased in inverse correlation to the expression of surface SDC2 (ΔEFYA), much like the cells transfected with wild-type sdc2 (Fig. 6). These data indicate that the PDZ-binding domain is not essential to down-regulate the TCR/CD3, and suggest that traffic of SDC2 is not involved in TCR/CD3 down-regulation.18

Figure 6.

Figure 6

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The PDZ-binding domain of SDC2 is not necessary to induce T-cell receptor (TCR)/CD3 down-regulation. (a) A representation of the SDC2 wild-type molecule and the SDC2 with the C-terminal EFYA deletion [SDC2 (ΔEFYA)]: TM, transmembrane region. (b) Correlation between the expression of CD3ε and SDC2 in Jurkat (filled circle), five Jurkat-SDC2 clones (filled square) and three independent Jurkat-SDC2 (ΔEFYA) clones (open squares). The solid line is the curve with the best fit to the data points (Spearman's rank correlation coefficient = −0·88, P = 0·003).

Discussion

This study provided genetic evidence that SDC2 can down-regulate the TCR/CD3. Over-expression of SDC2 in Jurkat cells resulted in partial TCR-ζ chain degradation, reduced surface TCR/CD3 density and reduced cell responsiveness to CD3 stimulation.

Confocal analysis of the subcellular location of CD3 and SDC2 suggests that the two receptors do not interact. Therefore, the down-regulatory effect of SDC2 on TCR/CD3 may be indirect, through control of the early endocytic pathway. The reduced cell surface levels in Jurkat-SDC2 cells of TfR and CD4, which traffic in a similar way to the TCR/CD3 during T-cell stimulation,29–31,34 support this idea. Moreover, when Jurkat cells were transfected with an SDC2 mutant lacking the PDZ-binding domain, the TCR/CD3 was down-regulated too, arguing against a direct involvement of SDC2 trafficking in TCR/CD3 down-regulation.

The results presented here suggest that SDC2 may play a functional role in TCR/CD3 down-regulation. To explore this possibility, we ligated SDC2 with specific antibody in antigen-stimulated or CD3-stimulated T cells in an attempt to increase TCR/CD3 down-regulation. However, no effect of SDC2-specific antibodies on TCR/CD3 down-regulation was observed (data not shown). This suggests that the anti-SDC2 antibodies used may not mimic the natural SDC2 ligand(s), which might increase TCR/CD3 down-regulation in a more physiological setting, this point merits further study.

Considerable attention has been paid to understanding the regulation of TCR/CD3 surface density because of its biological importance. The analysis of TCR/CD3 down-regulation constitutes a measurement of receptor engagement by the ligand.35,36 By increasing the concentration of the TCR/CD3 ligand, a proportional increase in both the down-regulation of TCR/CD3 and the functional activation of the T cell was observed.35,37 Conversely, when the internalization of TCR/CD3 complex is blocked9,10,13 or when the trafficking of internalized TCR/CD3 to the lysosome and degradation are reduced,8,12,25 TCR/CD3 surface density increases and signalling by TCR/CD3 is sustained, which may lead to autoreactivity.8

The TCR/CD3 down-regulation is dependent on its ubiquitination and on recognition of the ubiquitinated receptor by tumour suppressor gene (TSG) 101 at the immunological synapse.8,9 The TCR/CD3 can be differently ubiquitinated, leading to different biological outcomes, such as internalization and degradation8,9,13 or signalling attenuation.38 Recognition of ubiquitinated TCR/CD3 by TSG101 leads to receptor translocation into multivesicular bodies and lysosomes, which causes receptor dephosphorylation and degradation.9 The down-regulation of triggered TCR/CD3 and its subsequent degradation are considered necessary events for limiting sustained TCR signalling, resulting in decreased sensitivity of the T-cell to the antigen.9,36,39–41

In addition to TSG101, other proteins mechanistically associated with the TCR/CD3 down-regulation process have been identified. Mature T cells, when they lack CD2-associated protein41 or intra-flagellar transport proteins,42 show an altered balance between surface and intracellular TCR/CD3. Over-expression of the adaptor proteins HIP-55 (haematopoietic progenitor kinase 1 interacting protein of 55 000 molecular weight) or TRIM (T-cell receptor interacting molecule) in Jurkat modify the TCR/CD3 surface levels.43,44 Similarly, enforced expression of the lysosomal protein LAPTM5 (lysosomal-associated protein transmembrane 5) reduces surface TCR/CD3 levels by promoting TCR-ζ chain degradation without affecting other CD3 proteins.12 More recently, the ligation of CD90 in mouse T cells has been shown to modulate TCR/CD3 down-regulation and T-cell activation.45

Ligation of TCR/CD3 induces TCR down-regulation by increasing the internalization rate2 and preventing recycling.5 Our data show that SDC2 down-regulated TCR/CD3 by increasing TCR-ζ chain degradation, limiting the amount of completed TCR/CD3 complexes reaching the cell surface, and leading to intracellular storage of excess CD3δε and CD3γε dimers. It is tempting to speculate that SDC2 over-expression leads to increased ubiquitination of TCR-ζ chain and hence to increased transport to multivesicular bodies and lysosomes. The incomplete TCR/CD3 complexes lacking TCR-ζ may be retained in a non-recycling pool.

Like SDC2, SDC4 is also up-regulated in activated T cells.19–21 We speculated that it might also down-regulate TCR/CD3, just as SDC2 does. Chung et al.46 over-expressed SDC4 in Jurkat cells but reported no change in the expression of TCR/CD3, which suggests that SDC4 and SDC2 play different roles in T-cell biology.

The results of this study illustrate a unique mechanism for the regulation of surface TCR/CD3 expression by SDC2. This mechanism has the potential to contribute to modulate T-cell responses.

Acknowledgments

We thank the technical staff of the FACS facility for their support. Our thanks also to Manel Bosch for his help with the confocal microscopy analyses and to Anna Pascual for her technical help in plasmid construction. This research was supported by the Spanish Ministry of Education and Science (SAF2004-05481).

Disclosures

The authors declare no competing financial interests.

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Figure S1. Expression of SDC2 in resting and activated primary CD4+ T cells.

imm0137-0214-SD1.docx (23.7KB, docx)

Figure S2. Expression of Fas, CD69, MHC class I, CD4 and transferrin receptor (TfR) in sdc2-transfected and control untransfected Jurkat cells as analysed by flow cytometry.

imm0137-0214-SD2.docx (23.8KB, docx)

Figure S3. CD3-stimulation of CD4 T cells triggers T-cell receptor ζ degradation.

imm0137-0214-SD3.docx (24.8KB, docx)

Figure S4. Intracellular location of internalized CD3ε in CD4 T lymphocytes.

imm0137-0214-SD4.docx (27.1KB, docx)

Figure S5. SDC2-mediated T-cell receptor/CD3 down-regulation is independent of Arf6 activity.

imm0137-0214-SD5.docx (24.7KB, docx)

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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Supplementary Materials

imm0137-0214-SD1.docx (23.7KB, docx)
imm0137-0214-SD2.docx (23.8KB, docx)
imm0137-0214-SD3.docx (24.8KB, docx)
imm0137-0214-SD4.docx (27.1KB, docx)
imm0137-0214-SD5.docx (24.7KB, docx)

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