©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Staphylococcus aureus Enterotoxin B Superantigen Induces Specific T Cell Receptor Down-regulation by Increasing Its Internalization (*)

Florence Niedergang (§) , Agnès Hémar , Colin R. A. Hewitt (1), Michael J. Owen (1), Alice Dautry-Varsat , Andrés Alcover (¶)

From the (1) Unité de Biologie des Interactions Cellulaires, URA CNRS 1960, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Superantigens are able to stimulate T lymphocyte populations expressing T cell antigen receptors (TCR) belonging to particular V families. Moreover, the presence of these superantigens may induce long term unresponsiveness (anergy) of these sensitive cells. Some bacterial toxins are potent superantigens. We have analyzed in vitro the capacity of some Staphylococcus aureus enterotoxin superantigens to modulate T cell antigen receptor expression and the cellular mechanisms involved. Staphylococcus enterotoxin B (SEB) induced rapid down-regulation of surface T cell antigen receptors in V3-expressing T lymphocytes, as assessed by flow cytometry. This phenomenon was a consequence of the direct interaction between the toxin and the TCR since it was observed in the absence of cells expressing major histocompatibility complex class II molecules. The cellular mechanism involved in SEB-induced down-regulation of TCR was further investigated. Immunofluorescence and confocal microscopy experiments showed that toxin B induced intracellular accumulation of TCRCD3 in endocytic vesicles. Moreover, SEB induced an increase in T cell receptor endocytosis as measured using radiolabeled Fab fragments of an anti-CD3 monoclonal antibody. Taken together, our observations indicate that Staphylococcus enterotoxin B superantigen induced changes in the dynamics of surface T cell receptors, which resulted in the fast reduction of membrane receptor numbers.


INTRODUCTION

Superantigens have the capacity to strongly activate oligoclonal populations of T lymphocytes expressing antigen receptors homologous within their chain variable regions (V families). Certain strains of Staphylococcus or Streptococcus produce exotoxins that are potent superantigens. Similar to endogenous murine retroviral superantigens, the presence of bacterial superantigens may shape the T cell repertoire by deletion or inactivation (anergy) of reactive clones. The alteration in immune system homeostasis occurring in infections caused by superantigen-producing bacteria may be the basis for some of the clinical manifestations observed and could play a role in disease pathogenesis (1, 2) .

T cell antigen receptor (TCR)() expressed on the majority of peripheral T lymphocytes is a complex composed of the hypervariable - heterodimer noncovalently linked to the monomorphic CD3,, and - or - chains (TCRCD3). The and chains contain immunoglobulin-like variable regions responsible for antigen and superantigen interactions (3) , whereas the CD3 and the chains play a role in signal transduction (4) . Surface expression of the TCRCD3 complex requires association of all of their polypeptides. Individual chains or partial complexes are retained and degraded before reaching the plasma membrane (5, 6) .

Although both conventional T cell antigens and superantigens drive T cells to proliferate and secrete lymphokines, several characteristics distinguish superantigen from conventional antigen T cell recognition. (i) Superantigen stimulation, although usually requiring the presence of major histocompatibility complex class II (MHC)-presenting cells, is not restricted to one MHC allele. (ii) Processing of superantigens into antigenic peptides is not necessary. (iii) Superantigens are specific for TCR-V domains, with no particular TCR-V domains being required. (iv) Residues involved in superantigen interactions with MHC or TCR are different from those involved in conventional antigen recognition (1, 7, 8, 9, 10) . It has been proposed therefore that superantigens stimulate T cells by bridging MHC molecules on antigen presenting cells with TCR-V domains on T lymphocytes.

Among the known superantigens, S. aureus enterotoxins are the best characterized structurally and functionally. These toxins are structurally related (11) . Mutational analysis revealed one TCR binding site and one or two MHC binding sites located on separate regions of the toxin (1) . S. aureus enterotoxins generally bind to MHC molecules with a much better affinity than to the TCR. Therefore, it was suggested that toxin superantigens might bind to MHC molecules before being able to interact with the TCR (12, 13, 14, 15, 16, 17) . Recently, it has been reported that the formation of the ternary complex, MHC-toxin-TCR, may stabilize the interactions of each of the binary complexes (18) .

Although MHC-presenting cells are usually required for superantigen recognition by T cells, some toxins may be able to interact directly with the TCR in the absence of MHC molecules. Thus, Hewitt et al. (19) reported that the Staphylococcus enterotoxin B (SEB) could stimulate V3 human T cells in the absence of MHC molecules, driving them to a state of anergy (19) . Although the molecular mechanism responsible for anergy remained to be elucidated, it was suggested that T cell receptor down-regulation might contribute to reduce the sensitivity to new stimulations. Consistent with this proposal, treatment of cells with SEB reduced TCR levels in a dose-dependent manner. Moreover, the capacity of the cells to increase cytosolic free Ca concentration in response to a subsequent exposure to optimal concentrations of SEB was proportional to TCR levels, suggesting that down-regulation might be of functional significance (19) . Studies in vivo also support the idea that the level of T cell receptor may be of importance in the mechanism of both T cell activation and anergy (20) . However, other intracellular mechanisms also seem to be involved, since in some experimental models, cell anergy can be induced in vitro (21) or in vivo(22) , without substantial TCR down-regulation.

The cellular mechanisms modulating T cell receptor levels upon interaction with superantigens have not been defined yet. The aim of our study was to elucidate how the interaction with bacterial toxin superantigens may regulate the molecular dynamics of surface T cell receptors. In order to avoid interference from other molecular interactions provided by superantigen-presenting cells, we made use of cells from the human tumor T cell line, Jurkat, which do not express MHC molecules. These cells had been transfected with genes coding for the and chains of the influenza hemagglutinin-specific T cell receptor HA1.7 (V1.2-V3.1) and are able respond to peptide antigen or to SEB by secreting lymphokines. Moreover, incubation of these cells with SEB in the absence of MHC accessory cells had been shown to induce anergy (19) . Here we show that Staphylococcus enterotoxin B superantigen induces a rapid, specific, and dose-dependent down-modulation of surface T cell receptors in these cells. Using confocal microscopy and biochemical approaches, we demonstrated that SEB provokes changes in the intracellular traffic of TCR. Cells treated with SEB show an increased receptor internalization rate and accumulation of TCR in endocytic vesicles.


EXPERIMENTAL PROCEDURES

Materials

Reagents

Staphylococcal enterotoxins SEA, SEB, SED, SEE, and TSST1 were obtained from Toxin Technology, Inc. (Madison, WI) or Sigma. Human transferrin (Sigma) was loaded with iron and coupled to lissamine rhodamine (Eastman Kodak Co.) as described previously (23) . Monoclonal antibodies anti-CD2 (TS2.18, IgG), anti-CD3 (OKT3, IgG), anti-CD4 (OKT4, IgG), and anti-CD45 (GAP8.3, IgG) were obtained from the American Type Culture Collection (Rockville, MD). Anti-CD3 (UCHT1, IgG) was a kind gift of Dr. P. Beverley (Imperial Cancer Research Fund, London), and anti-CD5 (B36.1, IgG) was a kind gift of Dr B. Perussia (Jefferson Cancer Inst., Philadelphia, PA). The anti-V3 mAb (JOVI-3, IgG) has been described previously (24) . Fluorescein-conjugated goat anti-mouse Ig (Becton-Dickinson, San Jose, CA) was used for flow cytometry and fluorescein-conjugated sheep anti-mouse Ig (Amersham Corp.) was used for confocal analysis.

Cell lines

J77Cl20 (TCR V1.2 V8, CD2, CD3, CD4, CD5, CD45, MHC, MHC) is a subclone of the cell line Jurkat. CH7Cl7 (TCR V1.2 V3.1, CD2, CD3, CD4, CD5, CD45, MHC, MHC) is a Jurkat transfectant expressing the and chains of a hemagglutinin-specific TCR HA1.7, described previously (19) . All cells were grown in RPMI 1640 medium supplemented with 10% decomplemented fetal calf serum (FCS), 10 mM HEPES, pH 7.2, 2 mML-glutamine, 1 µg/ml penicillin, and 1 µg/ml streptomycin. CH7Cl7 cells were cultured in the presence of 400 µg/ml hygromycin and 4 µg/ml puromycin.

Methods

Preparation and I-Labeling of Anti-CD3 Fab Fragments

The OKT3 mouse monoclonal antibody was purified from ascites by Protein A affinity chromatography (Bio-Rad). Monovalent Fab fragments were prepared as follows. The OKT3 antibody was digested for 18 h by immobilized papain (Pierce Chemical Co.) at 37 °C following manufacturer's instructions and further purified through a protein A-Sepharose chromatography column. The purity of monovalent Fab fragments was confirmed by SDS-polyacrylamide gel electrophoresis. Fab fragments were radiolabeled with I (Amersham, Corp.) by the chloramine T method to a specific activity of 35 µCi/µg. For labeling, three successive additions of chloramine T were performed within 5 min, at room temperature, to a final concentration of 25 µg/ml. The reaction was stopped after another 5 min, and labeled ligand was separated from free I by passage through an Exocellulose GF-5 column (Pierce Chemical Co.).

Immunofluorescence and Flow Cytometry

Cells at 10 cells/ml were incubated in growth medium containing toxins at the concentrations indicated for various times at 37 °C. Cells were then washed in cold phosphate-buffered saline (PBS), 10 mM phosphate buffer, pH 7.3, 150 mM NaCl, containing 1% FCS (1% FCS-PBS), and cell surface expression of TCR or accessory molecules was assessed by indirect immunofluorescence. Cells were stained for 45 min at 4 °C using saturating concentrations of the appropriate murine monoclonal antibodies, washed twice in cold 1% FCS-PBS, and incubated for an additional 45 min with fluorescein-conjugated goat anti-mouse Ig (1/20, Becton Dickinson, San Jose, CA). Cells were washed twice and resuspended in 1% FCS-PBS. Five thousand viable cells, identified by their ability to exclude propidium iodide, were analyzed immediately after labeling on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). The mean fluorescence intensity was obtained from the recorded data, and the results were expressed as the percentage of fluorescence intensity of cells incubated without toxins.

Immunofluorescence and Confocal Microscopy

Exponentially growing cells were preincubated at 10 cells/ml in serum-free RPMI 1640 medium supplemented with 20 mM HEPES buffer pH 7.2 and 1 mg/ml bovine serum albumin (BSA) for 30 min at 37 °C in order to deplete transferrin from cells. SEB at 10 µg/ml and/or 600 nM rhodamine-transferrin were then added, and cells were incubated for 3 h at 37 °C. Cells were then washed twice in cold PBS containing 1 mg/ml BSA (BSA-PBS) and fixed for 30 min at 4 °C in PBS containing 3.7% paraformaldehyde and 30 mM sucrose. After quenching formaldehyde for 10 min in 50 mM NHCl-PBS, the cells were washed once in BSA-PBS and permeabilized for 15 min at 37 °C in BSA-PBS containing 0.05% saponin. Subsequent steps were performed at room temperature in permeabilizing buffer. Cells were incubated with anti-CD3 antibody (UCHT1) at 1:500 ascites dilution for 45 min. After two washes, the presence of antibodies was revealed by incubating the cells for 45 min with fluorescein isothiocyanate-coupled sheep anti-murine Ig antibody (1/50, Amersham Corp.). After three washes in permeabilizing buffer and one wash in PBS, the cells were mounted on microscope slides in 25 mg/ml Dabco (1,4-diazalbicyclo[2.2.2]octane, Sigma), 100 mg/ml Mowiol (Calbiochem, La Jolla, CA), 25% (v/v) glycerol, 100 mM Tris-HCl, pH 8.5.

The samples were examined under a confocal microscope (Leica) attached to a diaplan microscope (Leitz) equipped with a double laser, argon-krypton. Serial optical sections were recorded at 0.5 µm intervals with a 63 lens. Photographs were taken on Kodak Ektachrome 100 ASA. No immunofluorescence staining was ever observed when second antibodies were used without the first antibody or with an irrelevant first antibody.

Endocytosis of Radiolabeled Anti-CD3 Fab Fragments

Cells, 3 10/point, were preincubated at 37 °C for 30 min in 100 µl of growth medium. The I-labeled Fab fragment of anti-CD3 OKT3 mAb was then added to a final concentration of 40 nM, either alone or together with SEB (final concentration, 10 µg/ml). At the end of the incubation times, cells were chilled in 2 ml of RPMI medium containing 20 mM HEPES buffer pH 7.2 and 1 mg/ml BSA (wash medium). Cells were washed twice at 4 °C to remove unbound ligand. They were then subjected to two successive acid pH treatments. For each treatment, cell pellets were resuspended in 300 µl of acid medium (RPMI 1640 medium, 25 mM sodium acetate, brought to pH 2.8 with HCl) for 2.5 min and then neutralized with 0.8 ml of RPMI 1640 medium brought to pH 9 with NaOH. This treatment removed surface-associated I-Fab fragments with an efficiency of 85-90%. Results are expressed as internalized I-Fab anti-CD3. Data were corrected taking into account the efficiency of the acid wash, and the percentage of internalized receptors was calculated as described previously (25) .


RESULTS

SEB Superantigen Induces a Rapid Down-regulation of TCRCD3 Surface Expression

To study in detail the effect of SEB superantigen on TCRCD3 surface expression, we analyzed the dose-response and the kinetics of down-regulation. To this end, V3-expressing Jurkat cells were treated with various doses of toxin for 18 h, and TCRCD3 surface expression was measured by immunofluorescence and flow cytometry using the anti-CD3 mAb OKT3. As shown in Fig. 1A, SEB induced a dose-dependent down-regulation of the surface TCRCD3 complex. The S. aureus enterotoxins A and D, also able to stimulate V3-expressing T lymphocytes (26) , displayed only minor or undetectable effects. Other S. aureus enterotoxin (SE) superantigens, such as SEE or the toxic shock syndrome toxin 1 (TSST1), which are specific for other V families (11) , did not have any effect on TCRCD3 expression in V3-expressing cells. The kinetics of down-regulation are shown in Fig. 1B. The addition of SEB induced a rapid decrease in TCRCD3 surface expression. Receptor numbers decayed rapidly during the initial 30 min, falling to 50% of the initial levels (Fig. 1B) and then falling more slowly to 30-40%, and they remained stable for the time that the toxin was present (up to 72 h tested) (data not shown). Only a transient minor effect was obtained with SED (Fig. 1B), whereas other enterotoxins tested, such as SEA, SEE, or TSST1, did not induce this effect.


Figure 1: Staphylococcus enterotoxin B induces the rapid down-regulation of TCRCD3 independently of MHC class II molecules. Jurkat transfectants expressing V3 TCR (CH7C17 cells) were incubated for 18 h with various concentrations of Staphylococcus enterotoxin superantigens (panelA) or with various toxins at 10 µg/ml for different times (panelB). Surface TCRCD3 levels were analyzed by immunofluorescence and flow cytometry using saturating concentrations of the anti-CD3 mAb OKT3. Mean fluorescence intensity was measured at each point. Results are given as percentage of control cells (incubated in medium alone). Each point represents the average ± S.D. (n = 5). , SEB; , SED; , SEE; , SEA; , TSST1.



The T cell antigen receptor complex is functionally and physically linked to other surface molecules such as CD2, CD4, CD5, and CD45. These molecules cooperate with the TCR during antigen recognition and participate in signal transduction (27, 28) . In order to analyze whether T cell receptor down-regulation induced by toxin superantigens could affect the expression of any of these ``accessory'' molecules, we treated V3-expressing Jurkat cells with appropriate doses of SEB, and we followed TCRCD3 surface expression together with that of CD2, CD5, or CD45 by flow cytometry. The CD4 molecule was not tested since it is very weakly expressed in these cells. As shown in Fig. 2, only TCRCD3 expression was affected, whereas surface levels of CD2, CD5, or CD45 remained unchanged. No significant changes in CD2, CD5, or CD45 expression were observed upon 16-h treatment at various concentrations of SEB (data not shown). As expected, changes in TCRCD3 expression followed either by an anti-CD3 (OKT3) or an anti-V3 (JOVI-3) mAb led to identical results.


Figure 2: Staphylococcus enterotoxin B induces down-regulation of TCRCD3 but did not change the expression of accessory molecules. Jurkat transfectants expressing V3 TCR (CH7C17 cells) were incubated for various times with SEB at 10 µg/ml. Cells were then washed and stained for immunofluorescence and flow cytometry using mAbs OKT3 anti-CD3 (), JOVI-3 anti-TCRV3 (), TS2.18 anti-CD2 (), B36.1 anti-CD5 (), or GAP8.3 anti-CD45 (). Mean fluorescence intensity was measured at each point. Results are given as percentage of control cells (incubated in medium alone). Each point represents the average ± S.D. (n = 3) for CD3 and TCR, and a representative experiment is shown for the other molecules.



We further investigated whether other toxins able to activate cells expressing T cell receptors belonging to other V families could also induce TCRCD3 down-regulation. Thus, Jurkat cells expressing V8 TCR were tested against a panel of toxins. As shown in Fig. 3, none of the toxins tested, SEA, SEB, SED, SEE, or TSST1, were able to down-regulate TCRCD3 at any of the concentrations used. The lack of effect of these toxins on T cell receptor expression was not due to general unresponsiveness of these cells, since nanomolar concentrations of SED or SEE could induce IL2 secretion, provided that superantigen-presenting cells expressing MHC molecules were present (data not shown). Moreover, SEE, and to a lesser extent SED, induced TCRCD3 down-regulation of V8-expressing Jurkat cells when MHC superantigen presenting cells were present in the assay.() This suggests that SED and SEE, when used in soluble form, have lower affinities for their corresponding T cell receptor than that displayed by SEB for V3.


Figure 3: Staphylococcus enterotoxin superantigens do not induce down-regulation of TCRCD3 in V8-expressing Jurkat cells. J77cl20 cells were incubated for 18 h with various concentrations of toxins. Surface TCRCD3 levels were analyzed by immunofluorescence and flow cytometry using saturating concentrations of the anti-CD3 mAb OKT3. Mean fluorescence intensity was measured at each point. Results are given as percentage of control cells (incubated in medium alone). Each point represents the average ± S.D. (n = 3). , SEB; , SED; , SEE; , SEA; , TSST1.



SEB Superantigen Induces Accumulation of TCRCD3 Complexes in Endocytic Vesicles

Steady state display of surface receptors is the result of a dynamic equilibrium maintained by the membrane expression of newly synthesized molecules, internalization, recycling to the cell surface, and degradation. In order to characterize the mechanism that modulates TCRCD3 expression in the presence of SEB, we looked for changes in any of the components of this equilibrium.

We first estimated the half-life of surface TCRCD3 by measuring the decay of receptor surface expression in the presence of cycloheximide to block protein synthesis. The half-life of surface TCRCD3 in Jurkat cells was estimated to be longer than 10 h (data not shown). The fact that SEB-induced down-regulation was rapid, reaching a plateau at 30 min, indicated that changes in endocytosis or recycling might account for this phenomenon rather than inhibition of the secretory pathway. To address this question, we analyzed the effect of SEB on subcellular localization of TCRCD3 complexes by confocal microscopy. Thus, V3-expressing Jurkat transfectants were treated with SEB for various times and then fixed, permeabilized, and stained using anti-CD3 mAbs and fluorescent second antibodies. The staining of CD3 in untreated cells is shown in Fig. 4A. The immunofluorescence pattern observed within the cytoplasm resembles that of the endoplasmic reticulum (ER). The black area corresponds to the nucleus. Due to the brightness of the ER, membrane staining is not clearly appreciable. Strong ER labeling was expected, since CD3 is synthesized in excess and assembled with other TCRCD3 subunits in the ER. Unassembled single polypeptides or partial complexes are retained in the ER and eventually degraded before reaching the cell surface (5, 6) . Cells treated with SEB showed a different pattern of immunofluorescence, since their TCRCD3 accumulated in intracellular vesicles (Fig. 4B). Vesicular staining was maximal at 2-3 h, disappearing afterwards. No patching or capping of receptors was observed even at shorter times of treatment. After 6 h of continuous incubation with SEB, no clear differences were observed between the immunofluorescence patterns displayed by treated and untreated cells. Accumulated TCRCD3 was not reexpressed on the cell surface, since expression quantitated by flow cytometry remained at 30-40% of control levels for up to 72 h (not shown).


Figure 4: SEB induces accumulation of TCRCD3 in intracellular vesicles. Jurkat transfectants expressing V3 TCR (CH7C17 cells) were incubated for 3 h at 37 °C in medium alone (panelA) or in the presence of 10 µg/ml SEB (panelB). Cells were then fixed, permeabilized, and stained for immunofluorescence using UCHT1 anti-CD3 mAb and fluorescein-coupled second antibodies. A Z series of optical sections was performed at 0.5-µm increments. The image shows a projection of four medial optical cuts of a representative cell. The color scale used ranges from red (weak staining) to yellow (bright staining). Bar, 10 µm.



To analyze whether TCRCD3 complexes observed in intracellular vesicles were endocytosed receptors, we simultaneously labeled the endocytic compartment using transferrin coupled to rhodamine, and we followed both fluorescent labels by dual immunofluorescence and confocal microscopy. The intracellular endocytic pathway of transferrin and its receptor has been extensively studied in numerous cell types. Transferrin accompanies its receptor through the recycling pathway (29, 30) and thus defines early and recycling endocytic organelles. Cells were incubated in the presence or absence of SEB in a medium containing rhodamine-transferrin, and then CD3 was localized on permeabilized cells as described above using a fluorescein-coupled second antibody. The results are shown in Fig. 5 . Untreated cells displayed an immunofluorescence pattern corresponding to CD3, which accumulated mainly in the endoplasmic reticulum (green). In addition, vesicles containing rhodamine transferrin (red) were readily observed in the same cells. As described above, SEB-treated cells displayed CD3 that accumulated in the ER as well as in intracellular vesicles (green). In the computer-generated composite image, the areas of colocalization of both fluorochromes appear as yellow. Most of the intracellular vesicles containing CD3 were stained in yellow. This indicates that, upon SEB interaction, TCRCD3 accumulated in organelles containing transferrin, i.e. in the early endocytic and recycling compartment. This suggests that SEB may provoke changes in TCRCD3 endocytosis and/or recycling.


Figure 5: SEB induces accumulation of TCRCD3 in endocytic vesicles. Jurkat transfectants expressing V3 TCR (CH7C17 cells) were incubated for 3 h in medium containing 600 nM rhodamine transferrin in the absence (A-C) or presence (D-F) of 10 µg/ml SEB. Cells were then fixed, permeabilized, and stained by immunofluorescence using UCHT1 anti-CD3 mAb and fluorescein-coupled second antibodies. A Z series of optical sections was performed at 0.5-µm increments. Measurements of fluorescein and rhodamine emissions were acquired simultaneously. The image shows a medial optical cut of a representative cell. A and D, fluorescein CD3-labeling; B and E, rhodamine-transferrin-labeling; C and F, combined images. Areas of colocalization appear yellow in the computer-generated composite image. Bar, 10 µm.



SEB Treatment Increases the Internalization of TCRCD3 Complex

Interaction of ligands with cell surface receptors may induce receptor internalization through the well described pathway of receptor-mediated endocytosis (31) . The fate of a receptor along its intracellular route can be followed by means of a radiolabeled ligand. Binding of toxin superantigens to TCR is very difficult to measure due to their low affinity (see ``Discussion''). Therefore, we used radiolabeled mAbs to follow the TCRCD3 complex. In order to avoid interference of antibodies with SEB binding to TCR, we used mAbs directed to epitopes on the CD3 chain, rather than antibodies against TCR or chains. Moreover, to prevent the influence of antibody cross-linking on receptor endocytosis (32) , we used purified Fab fragments.

To analyze whether SEB could augment T cell receptor endocytosis, internalization of TCRCD3 was followed using I-Fab fragments of the anti-CD3 mAb OKT3, either alone or in the presence of 10 µg/ml SEB. As shown in Fig. 6, untreated cells rapidly internalized I-Fab fragments, reaching a plateau at 3 min. This shows, in agreement with previously reported data (32, 33) , that TCRCD3 is constitutively endocytosed and recycled. 18% of the total surface receptors were found inside the cells at 7 min. When cells were incubated in the presence of SEB, I-Fab-OKT3 was internalized more efficiently than in control cells (Fig. 6). The receptors internalized at 7 min represent 32% of total surface receptors. These data indicate that TCRCD3 internalization was augmented in the presence of SEB.


Figure 6: SEB increases TCRCD3 internalization. Jurkat transfectants expressing V3 TCR (CH7C17 cells) were incubated in growth medium at 37 °C. Then, I-Fab fragments of the anti-CD3 mAb OKT3 were added alone (opencircles) or together with SEB (closedcircles) to a final concentration of 40 nM for I-Fab and 10 µg/ml for SEB. Aliquots of cells were taken at each time point, and the amount of I-Fab internalized was determined as described under ``Methods.'' The figure shows a representative experiment out of three independent experiments carried out. At 7 min, the amount of I-Fab fragments internalized represented 18% of cell associated ligand in control cells and 32% in SEB-treated cells.




DISCUSSION

The mechanisms involved in the modulation of T cell receptor expression upon antigen or superantigen recognition are not well defined. Different cellular processes that regulate steady state levels of surface molecules may be affected such as synthesis and secretion of new polypeptides, internalization, recycling back to the plasma membrane, and degradation.

Here we show that SEB superantigen induced a rapid down-modulation of TCRCD3 surface expression due, at least in part, to increased receptor internalization. This is indicated by the following observations. First, we observed an accumulation of TCRCD3 complexes in intracellular vesicles that colocalized with internalized transferrin. Since transferrin is endocytosed and recycled together with its receptor (29, 30) , it is a marker widely used for early endocytic and recycling organelles. Accumulation of TCRCD3 in these organelles suggests that SEB induces changes in internalization and/or recycling. Second, SEB treatment augmented the efficiency of internalization of the TCRCD3 complex as measured using radiolabeled Fab fragments of anti-CD3 mAb, thus providing further evidence for a role of endocytosis in T cell receptor down-regulation.

Although an increase in receptor internalization may account for the rapid TCR down-regulation observed, the recycling and/or degradation pathways might also be affected. Some of our observations suggest that accumulated receptors ended up being degraded. Thus, after 6 h of treatment with SEB, accumulation of TCRCD3 complexes in intracellular vesicles was no longer detected by confocal microscopy. In addition, surface levels of the receptor did not recover; on the contrary, they continued diminishing during 18 h and then remained low for the time that the toxin was present (up to 72 h tested). Additional experiments are in progress to formally prove degradation. Furthermore, from our data we cannot rule out the possibility that TCRCD3 that accumulated in intracellular vesicles might be further dispersed in smaller undetectable subcellular structures.

Our experiments suggest that inhibition of the secretory pathway is not responsible for the rapid SEB-induced TCR down-regulation, since the half-life of surface TCRCD3 was estimated to be longer than 10 h, indicating that the TCRCD3 complex is secreted at low rates. It is therefore unlikely that the secretory pathway plays a direct role in the rapid regulation of receptor numbers. However, an effect at the level of gene transcription, protein synthesis, and/or secretion may occur at longer times of incubation with the toxin.

In line with previous results (19) , we observed a biological effect of SEB on V3-transfected Jurkat cells in the complete absence of MHC molecules. This indicates that the toxin can interact directly with the TCR. In agreement with this, Seth et al. (18) recently showed that SEB could form complexes in vitro with a soluble form of the V3 TCR HA1.7. If the interaction between SEB and the TCR were strong enough to form stable complexes, one might expect SEB to be endocytosed with its TCR. To address this point, we labeled SEB with I and carried out ligand internalization experiments. We were unable to detect significant levels of internalized toxin. Moreover, we could not detect SEB inside the cells by means of immunofluorescence using an anti-SEB antiserum. It is worth noting that I-SEB was able to induce down-regulation of surface TCRCD3 with the same efficiency as unlabeled toxin, showing that radiolabeled toxin was biologically active. Our findings indicate that SEB, although capable of interacting with the TCR to induce a biological response, does not bind stably enough to be endocytosed together with the receptor. The rapid dissociation and slow association kinetics reported for the interaction between SEB and V3TCR could explain this fact (18) . Therefore, in this case, TCR internalization does not require stable association with the ligand. It is tempting to speculate that, upon (even unstable) interaction between SEB and TCR, a signal is transduced to the cell that in turn triggers receptor down-regulation. Post-translational modifications (i.e. phosphorylation) of CD3 chains that occur upon T cell activation might be responsible for changes in the intracellular traffic (32, 33, 34) . Moreover, our experiments indicate that low affinity ligands can induce receptor endocytosis without being involved in further intracellular trafficking of the receptor. This may be of physiological relevance for T cells, since the natural ligand for T cell receptors, the peptide antigen/MHC complex, is anchored on the surface of antigen-presenting cells. Interaction between these two structures could provoke TCR internalization, keeping the independence of each receptor on the surface of its own cell.

We further investigated whether other enterotoxin superantigens could down-regulate their specific TCR in a similar fashion to SEB. To this end, Jurkat cells expressing V8 TCR were tested against a panel of toxins. Previous experiments showed that these cells responded to SED and SEE by secreting IL2, provided that accessory cells expressing MHC molecules were present (not shown). Our data showed that none of the toxins tested, including SED and SEE, induced TCR down-regulation in the absence of MHC molecules. This is most likely due to differences in affinity of these toxins for the TCR, since the addition of MHC accessory cells made SEE and SED able to induce TCRCD3 down-regulation on V8 Jurkat cells. Previous binding of toxins to MHC molecules on accessory cells may facilitate binding to TCR and increase the stability of the interaction. This may improve the intracellular signal required to induce down-regulation. Other intercellular interactions provided by other surface molecules may also play an important role in this process.

Down-regulation of signaling receptors upon ligand interaction may alter the biology of a particular receptor system by removing the receptor from the cell surface. This may diminish further responsiveness of the cells simply by the loss of available receptors or by uncoupling them from the signal transduction machinery. Receptor internalization through the receptor-mediated endocytosis pathway is a very efficient cellular mechanism to rapidly reduce the number of surface receptors (31) .

Cellular unresponsiveness and concomitant down-regulation of T cell receptor and/or co-receptors has been observed in some experimental models of tolerance in vitro(19, 26, 35) or in vivo(20, 36) , suggesting that receptor down-modulation may be of functional significance for cellular inactivation. However, anergy is most likely the result of a more complex molecular regulation and not solely the result of modification in the expression of membrane receptors. Thus, in other experimental systems, T cells rendered tolerant to antigen or superantigen, in vitro(21) or in vivo(22) , displayed normal levels of TCR and CD4. To explain the different experimental observations, Arnold et al.(37) proposed a model where multiple levels of tolerance may exist. This may involve down-regulation of TCR and/or CD4 or CD8 coreceptors as well as other intracellular mechanisms and would condition the capacity of T cells to be reactivated. Tolerant T cells would still be susceptible to further tolerogenic signals, driving them to a deeper state of tolerance (37) .

T cell receptor and/or CD4 or CD8 coreceptor down-regulation has been observed upon physiological stimulation of T cell clones with appropriately presented antigen. In this case, down-modulation of T cell receptors may contribute to arrest the activation process and to create the transient period of unresponsiveness that follows cell stimulation (38, 39, 40, 41, 42, 43) .

The experiments that we report here shed further light on the mechanism of T cell receptor down-regulation induced by bacterial toxin superantigens. The understanding of the effect of these bacterial products at the cellular level may help to clarify the strategies that these organisms use to elude the immune response of the host. Moreover, it may provide us with the means to modulate the immune system in novel ways.


FOOTNOTES

*
This work was supported by Grant BIO2-CT92-0164 from European Economic Community and by the Agence Nationale de Recherches sur le Syndrome de l'Immunodeficience Acquise. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Fellowship from Ministère de l'Enseignement Supérieur et de la Recherche.

To whom correspondence should be addressed. Tel.: 33 1 40 61 30 49; Fax: 33 1 40 61 32 38; E-mail: aalcover@pasteur.fr.

The abbreviations used are: TCR, T cell antigen receptor; BSA, bovine serum albumin; FCS, fetal calf serum; MHC, major histocompatibility complex; mAb, monoclonal antibody; PBS, phosphate-buffered saline; SE, Staphylococcus enterotoxin; TSST1, toxic shock syndrome toxin 1; ER, endoplasmic reticulum.

F. Niedergang, A. Dautry-Varsat, and A. Alcover, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Raymond Hellio for help and advice with confocal microscopy and David Ojcius for critical reading of the manuscript.


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