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The Journal of Immunology, 2004, 173: 384-393.
Copyright © 2004 by The American Association of Immunologists

Constitutive and Ligand-Induced TCR Degradation 1

Marina von Essen*, Charlotte Menné Bonefeld*, Volkert Siersma{dagger}, Anette Bødker Rasmussen*, Jens Peter H. Lauritsen*, Bodil L. Nielsen* and Carsten Geisler2,*

* Institute of Medical Microbiology and Immunology and {dagger} Institute of Public Health, Department of Biostatistics, The Panum Institute, University of Copenhagen, Copenhagen, Denmark


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Modulation of TCR expression levels is a central event during T cell development and activation, and it probably plays an important role in adjusting T cell responsiveness. Conflicting data have been published on down-regulation and degradation rates of the individual TCR subunits, and several divergent models for TCR down-regulation and degradation have been suggested. The aims of this study were to determine the rate constants for constitutive and ligand-induced TCR degradation and to determine whether the TCR subunits segregate or are processed as an intact unit during TCR down-regulation and degradation. We found that the TCR subunits in nonstimulated Jurkat cells were degraded with rate constants of ~0.0011 min–1, resulting in a half-life of ~10.5 h. Triggering of the TCR by anti-TCR Abs resulted in a 3-fold increase in the degradation rate constants to ~0.0033 min–1, resulting in a half-life of ~3.5 h. The subunits of the TCR complex were down-regulated from the cell surface and degraded with identical kinetics, and most likely remained associated during the passage throughout the endocytic pathway from the cell surface to the lysosomes. Similar results were obtained in studies of primary human V{beta}8+ T cells stimulated with superantigen. Based on these results, the simplest model for TCR internalization, sorting, and degradation is proposed.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T cell activation is initiated by TCR recognition of antigenic peptides bound to MHC-encoded molecules. The TCR is a multisubunit complex composed of at least eight subunits organized in four dimers: Ti{alpha}{beta}, CD3{gamma}{epsilon}, CD3{delta}{epsilon}, and {zeta}{zeta} dimers (1, 2, 3, 4). The {alpha}- and {beta}-chains of the Ti{alpha}{beta} dimer are covalently linked by a disulfide bridge as are the {zeta}-chains of the {zeta}{zeta} homodimer, whereas the chains of the CD3 dimers are noncovalently linked. Assembly of the TCR begins in the endoplasmic reticulum with the formation of hexameric {alpha}{beta}{gamma}{epsilon}{delta}{epsilon} complexes and is completed in the Golgi apparatus when an {alpha}{beta}{gamma}{epsilon}{delta}{epsilon} complex associates with a {zeta}{zeta} homodimer to form the complete octameric TCR (5, 6, 7, 8). Failure of the {alpha}{beta}{gamma}{epsilon}{delta}{epsilon} complex to associate with the {zeta}{zeta} homodimer in the Golgi apparatus results in rapid sorting and lysosomal degradation of the {alpha}{beta}{gamma}{epsilon}{delta}{epsilon} complex, probably mediated by the unmasked di-leucine motif of CD3{gamma} (9, 10, 11). Thus, only completely assembled octameric TCR are stably expressed at the T cell surface.

The TCR expression level at the T cell surface is the result of a dynamic equilibrium maintained by the membrane expression of newly synthesized TCR, internalization, recycling to the cell surface, and degradation (12). Early studies demonstrated that the TCR is a spontaneously cycling receptor (13, 14). Thus, at steady state a certain amount of TCR is endocytosed, while at the same time an equal amount of TCR is exocytosed. Several studies seem to agree that the constitutive endocytic rate constant for the TCR in resting T cells is ~0.01 min–1, meaning that 1% of the cell surface-expressed TCR is internalized each minute (11, 15, 16, 17). However, whether the TCR is endocytosed and further processed as an intact receptor or as individual subunits with different endocytic, exocytic, and degradation rate constants remains unclear. Some studies indicated that the TCR disassembles during endocytosis, and the individual subunits are subsequently processed differently (18, 19, 20); other studies indicated that the TCR is endocytosed and further processed as an intact receptor (21, 22, 23, 24).

TCR triggering induces down-regulation (16, 17, 25, 26, 27, 28, 29, 30, 31) and degradation (21, 22, 32) of the TCR in a dose-dependent manner. In theory, TCR down-regulation can be accomplished by an increase in the endocytic rate constant, a decrease in the exocytic rate constant, or a combination of both. Most studies found that TCR down-regulation is caused by an increase in the endocytic rate constant after TCR triggering (13, 15, 17, 28, 33); however, some studies indicated that TCR ligation induces TCR down-regulation by a reduction in the exocytic rate constant rather than by an increase in the endocytic rate constant (16). Divergent models for TCR degradation also exist. Thus, some studies indicated that the degradation rate constant increases after TCR triggering and that all TCR subunits are degraded in parallel (21), whereas others found that TCR triggering transiently diverts the Ti{alpha}{beta} from degradation and that the TCR subunits segregate and are degraded with different degradation rate constants (19, 20).

A precise understanding of the mechanisms and factors that regulate TCR expression levels is important because they determine the ability of the T cell to recognize ligand and to be activated. In this study we determine the rate constants for constitutive and ligand-induced TCR degradation and whether the TCR subunits segregate or are processed as an intact unit during TCR down-regulation and degradation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and Abs

The human J76.25.20 Jurkat T cell line, the CD3{gamma}-negative variant E3 (34), and the B cell line Raji (American Type Culture Collection, Manassas, VA) were cultured in complete medium (RPMI 1640 medium supplemented with 0.5 IU/L penicillin, 500 mg/L streptomycin, and 10% FCS) at 37°C in 5% CO2. Primary human V{beta}8+ T cells were generated from freshly isolated blood mononuclear cells (BMNC). 3 BMNC were isolated by density gradient centrifugation using Lymphoprep (Nycomed Pharma, Oslo, Norway) and were washed three times in PBS/0.1% BSA. V{beta}8+ T cells were subsequently isolated by positive selection using the MX6 mAb (anti-V{beta}8, a gift from A. W. Boylston, University of Leeds, Leeds, U.K.) and M-450 goat anti-mouse IgG-conjugated Dynabeads (Dynal Biotech, Oslo, Norway) according to the manufacturer. The isolated cell population was afterward expanded by incubation of 5 x 105 cells/ml complete medium supplemented with 1 x 103 U/ml recombinant human IL-2 (Proleukin; Chiron, Emeryville, CA) and 1 x 105 CD3-CD28 expander beads/ml (Dynal Biotech). The expander beads were removed from the culture 1 day before the experiments. Abs against the TCR complex used included F101.01 (35), UCHT1 and PE-UCHT1 (anti-CD3{epsilon}; DAKO, Glostrup, Denmark), rabbit polyclonal A452 (anti-CD3{epsilon}; DAKO), MX6 (anti-V{beta}8), PE-BV8 (anti-V{beta}8; BD, San Diego, CA), and 6B10.2 (anti-{zeta}; Santa Cruz Biotechnology, Santa Cruz, CA). Other Abs used included PE-goat anti-mouse Ig and Cy5-donkey anti-mouse Ig (Jackson ImmunoResearch Laboratories, West Grove, PA) and HRP-swine anti-rabbit Ig and HRP-rabbit anti-mouse Ig (DAKO). Streptavidin-FITC was obtained from BD PharMingen (San Diego, CA).

Cell stimulation and TCR down-regulation

For stimulation of J76.25.20 by TCR cross-linking, flat-bottom Nunclon microwell plates (Life Technologies, Paisley, U.K.) were coated with the indicated concentrations of anti-Ti{beta} mAb MX6 or anti-CD3 mAb F101.01 for 16 h at 4°C and were subsequently washed three times with PBS. Cells were adjusted to 5 x 105 cells/ml complete medium and incubated in the mAb-precoated wells for the time indicated. For stimulation of J76.25.20 and primary human V{beta}8+ T cells with the Staphylococcus aureus enterotoxin E (SEE) superantigen (Toxin Technology, Sarasota, FL), Raji cells were pulsed for 50 min at 37°C with the indicated concentrations of SEE, stained with 2',7'-bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein acetoxymethyl ester (BCECF/AM; Molecular Probes, Eugene, OR) for 10 min at 37°C, and washed four times. Finally, nonpulsed or SEE-pulsed Raji cells were cocultured with either Jurkat or primary human V{beta}8+ cells at a Raji to T cell ratio of 2:1. For TCR expression analysis, cells were transferred to 4°C and incubated with saturating amounts of mAb for 30 min at 4°C. When the stimulating Ab and the staining Ab were the same, indirect staining methods were used. For direct staining, cells were incubated with PE-UCHT1 (anti-CD3{epsilon}) or PE-BV8 (anti-V{beta}8). When analyzing for TCR expression on SEE-stimulated cells, cells with green fluorescence (BCECF/AM) were gated out. The mean fluorescence intensity (MFI) was determined by flow cytometry and used to calculate the percentage of anti-TCR binding: (MFItreated cells/MFIuntreated cells) x 100%.

Surface biotinylation and deglycosylation

For surface biotinylation, cells were washed twice in PBS and resuspended in a freshly prepared solution of 0.5 mg/ml sulfo-NHS-biotin/PBS (Pierce, Rockford, IL). One milliliter of sulfo-NHS-biotin solution was used per 16 x 106 cells. The cells were incubated on ice for 30 min and gently mixed each fifth minute. Subsequently, the cells were washed twice in PBS and resuspended in complete medium to a concentration of 1 x 106 cells/ml for degradation/dissociation studies. For the deglycosylation experiments, 16 x 106 J76.25.20 were biotinylated, pelleted, lysed in lysis buffer (50 mM Tris-base (pH 7.5), 150 mM NaCl, and 1 mM MgCl2) and 1% digitonin, precleared with protein A-agarose beads (Kem-En-Tec, Østerbro, Denmark), and immunoprecipitated with anti-CD3{epsilon} mAb (UCHT1) and protein A-agarose beads. The beads were washed five times in digitonin washing buffer (50 mM Tris-base (pH 7.5), 150 mM NaCl, and 1 mM MgCl2) and 0.5% digitonin and resuspended in incubation buffer (20 mM Na3PO4 (pH 7.5), 0.02% NaN3, 0.1% SDS, and 50 mM 2-ME) and boiled for 5 min. The supernatant was divided into two aliquots and transferred to new tubes. Nonidet P-40 was added to a final concentration of 0.75%, and one of the samples was treated with N-glycanase F (Genzyme, Boston, MA). The tubes were incubated overnight at 37°C, boiled for 5 min in sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 0.01% bromophenol, and 2% 2-ME), and run on a 10% polyacrylamide gel. The proteins were transferred to nitrocellulose sheets, and Western blotting was performed using HRP-conjugated streptavidin (Pierce) after visualization with ECL (Amersham Pharmacia Biotech, Little Chalfont, U.K.). For intracellular staining experiments, J76.25.20 cells were biotinylated as described; fixed with PBS/1% formaldehyde for 10 min at room temperature; washed twice in PBS; permeabilized for 20 min at room temperature using PBS, 10 mM HEPES, and 0.1% saponin; and subsequently incubated for 20 min at room temperature with anti-CD3{epsilon} (UCHT1) diluted in PBS, 10 mM HEPES, 0.1% saponin, and 2% BSA. The cells were washed twice in PBS, 10 mM HEPES, and 0.1% saponin; incubated with secondary Cy5-donkey anti-mouse Ig and streptavidin-FITC in PBS, 10 mM HEPES, 0.1% saponin, and 2% BSA; and finally washed twice in PBS. Cells were examined by confocal microscopy (Carl Zeiss, Jena, Germany) as previously described (8).

Immunoprecipitation and Western blot analysis

Biotinylated cells (4 x 106 cells/sample) were stimulated for the times indicated and lysed in lysis buffer supplemented with 1 mg/ml Pefabloc SC, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 5 mM EDTA, 10 mM NaF, 1 mM Na3VO4, and 1% Triton X-100 for degradation studies or 1% digitonin for dissociation experiments. Cell lysates were precleared with protein A-agarose beads, immunoprecipitated with MX6 (anti-V{beta}8) plus protein A-agarose beads or with streptavidin-agarose beads (Pierce), subjected to SDS-PAGE, and analyzed by Western blotting using either HRP-conjugated streptavidin or anti-CD3{epsilon} (A452)/anti-{zeta} (6B10.2), respectively. For the dissociation experiments immunoprecipitations were performed with MX6 (anti-V{beta}8) plus protein A-agarose beads and Western blotting with HRP-conjugated streptavidin.

To determine the efficiency of the streptavidin-agarose beads to precipitate biotinylated proteins, 8 x 106 J76.25.20 cells were biotinylated, lysed, divided into two aliquots, and precleared with protein A-agarose beads. The biotinylated proteins of the first aliquot were precipitated with streptavidin-agarose beads, and the remaining Ti{beta}-chains were subsequently precipitated with MX6 (anti-V{beta}8) plus protein A-agarose beads. The TCR {beta}-chains of the second aliquot were directly precipitated with MX6 plus protein A-agarose beads. The samples were subjected to Western blotting using HRP-conjugated streptavidin. The ECL-exposed films were scanned into a computer using a transillumination scanner (AcusII; Agfa, Cologne, Germany), and the intensity of the bands was quantified by the program Quantity One (Bio-Rad, Hercules, CA). Multiple exposure times were applied to each membrane, because Quantity One only accepts films that have not been overexposed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ti{alpha}{beta} and CD3{gamma}{epsilon}/{delta}{epsilon} subunits are down-regulated with equivalent kinetics after TCR triggering with superantigen or anti-TCR/CD3 mAb in Jurkat cells

Some studies have indicated that the TCR subunits are down-regulated from the cell surface with different kinetics after TCR triggering (20). To analyze the down-regulation kinetics of the TCR subunits during TCR triggering, Jurkat cells were stimulated with anti-CD3 mAb, anti-Ti{beta} mAb, or Raji cells pulsed with SEE. The cell surface expression levels of the Ti{alpha}{beta} and CD3{gamma}{epsilon}/{delta}{epsilon} subunits were subsequently determined by flow cytometry. The expression level of the disulfide-linked Ti{alpha}{beta} dimers was determined by using anti-Ti{beta} mAb, and the levels of the CD3{gamma}{epsilon} and CD3{delta}{epsilon} dimers were determined using anti-CD3{epsilon} mAb. As Abs against the small extracellular part of the {zeta}-chain were not available, the kinetics of {zeta}{zeta} dimer down-regulation could not be directly determined by these analyses. Treatment of the cells with mAb against the TCR induced equivalent down-regulation with identical kinetics of the Ti{alpha}{beta} and CD3{gamma}{epsilon}/{delta}{epsilon} subunits (Fig. 1, A–C). Likewise, when the cells were stimulated with superantigen, the Ti{alpha}{beta} and CD3{gamma}{epsilon}/{delta}{epsilon} subunits were down-regulated with identical kinetics (Fig. 1D). Decreasing the strength of the stimuli led to a reduced degree of TCR down-regulation in agreement with previous studies (12, 21, 30, 36). Importantly, the individual TCR subunits followed equivalent kinetics of down-regulation for each concentration of stimuli tested (data not shown). These data indicated that the Ti{alpha}{beta} and CD3{gamma}{epsilon}/{delta}{epsilon} subunits are down-regulated with identical kinetics after TCR triggering. This is in agreement with some previous studies (21, 22, 23, 24) and indicates that the TCR is endocytosed as an intact octameric complex.



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  		FIGURE 1. The Ti{alpha}{beta} and CD3{gamma}{epsilon}/{delta}{epsilon} subunits are down-regulated with equivalent kinetics after TCR triggering. A, Flow cytometry profiles showing down-regulation of cell surface-expressed CD3{epsilon} after stimulation of cells with the anti-CD3 mAb F101.01 for the indicated time. The abscissa gives the fluorescence intensity in a logarithmic scale, and the ordinate shows the cell number in an arbitrary scale. B–D, J76.25.20 cells were stimulated for the time indicated with mAb against CD3{epsilon} (B; F101.01, 1 µg/ml), mAb against Ti{beta} (C; MX6, 1 µg/ml), or Raji cells pulsed with SEE (D; 2 ng/ml); transferred to ice; and subjected to flow cytometric analysis. The MFI was determined and used to calculate the percentage of anti-Ti{beta} or anti-CD3{epsilon} binding of stimulated cells compared with nonstimulated cells as (MFItreated cells/MFIuntreated cells) x 100%. The abscissa gives the time in minutes, and the ordinate shows the relative Ti{beta}/CD3{epsilon} expression. The data are given as the mean ± SD of three independent experiments.

 
Cell surface-expressed Ti{beta}-, CD3{epsilon}-, and {zeta}-chains can be efficiently biotinylated

Several studies seem to agree that the constitutive endocytic rate constant for the TCR in resting T cells is ~0.01 min–1 (11, 15, 16, 17) and that the TCR is a recycling receptor (13, 14, 23). However, a fraction of the endocytosed TCR is degraded, and the size of this fraction vs the recycling fraction is unknown. To increase our knowledge on TCR regulation and trafficking, we determined the rate constant for constitutive TCR degradation. Selective and efficient labeling of cell surface-expressed molecules is required to follow the fate of endocytosed TCR. Each chain of the human TCR complex contains lysine residues in its extracellular part and therefore, in theory, should be biotinylated by use of sulfo-NHS-biotin. However, some lysine residues might be inaccessible for the sulfo-NHS-biotin, thereby preventing efficient labeling. To investigate the efficiency of sulfo-NHS-biotin in biotinylation of the individual TCR chains, a pool of cells was incubated with sulfo-NHS-biotin on ice for 30 min. These cells were subsequently washed, lysed in digitonin lysis buffer, and precipitated with anti-CD3 mAb UCHT1. The precipitate was divided into two aliquots, one of which was treated with N-glycanase F, and the other was left untreated. The deglycosylated and control aliquots were subjected to SDS-PAGE under reducing conditions, transferred to nitrocellulose sheets, and analyzed by Western blot using HRP-conjugated streptavidin. All the TCR chains became biotinylated, particularly the Ti{beta}-chain (Fig. 2A). The strong labeling of the Ti{beta}-chain compared with the other TCR chains may be due to multiple exposed lysine residues of the Ti{beta}-chain being accessible to the sulfo-NHS-biotin. We selected the Ti{beta}-, the CD3{epsilon}-, and the {zeta}-chains as representatives for the Ti{alpha}{beta}, CD3{gamma}{epsilon}/CD3{delta}{epsilon}, and {zeta}{zeta} subunits, respectively, because they became satisfactorily biotinylated, and mAb against these TCR chains were available.



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  		FIGURE 2. Cell surface biotinylation and precipitation of the TCR chains by streptavidin-agarose beads. A, J76.25.20 cells were biotinylated on ice, lysed in 1% digitonin buffer, and immunoprecipitated using anti-CD3 mAb. The precipitated proteins were divided into two aliquots, one of which was left untreated (–) and the other of which was treated with N-glycanase F (+). Proteins were subjected to SDS-PAGE under reducing conditions (untreated proteins in lane 1, deglycosylated proteins in lane 2), followed by Western blotting using HRP-conjugated streptavidin. The positions of the TCR chains are indicated (dg, deglycosylated). B, Efficiency of streptavidin-agarose beads to precipitate biotinylated proteins. J76.25.20 cells were biotinylated and lysed. The cell lysate was divided into two aliquots and precleared with protein A-agarose beads. The biotinylated proteins of the first aliquot were precipitated with streptavidin-agarose beads (lane 1), and the remaining Ti{beta}-chains in the supernatant were subsequently precipitated with MX6 (anti-V{beta}8) plus protein A-agarose beads (lane 2). The Ti{beta}-chains of the second aliquot were directly precipitated with MX6 plus protein A-agarose beads (lane 3). The precipitated proteins were subjected to SDS-PAGE under reducing conditions, followed by Western blotting using HRP-conjugated streptavidin. The position of the Ti{beta}-chain is indicated. C, To analyze whether TCR components located inside the cells became biotinylated, the TCR cell surface-positive J76.25.20 (lanes 2, 4, and 6) and the TCR cell surface-negative E3 (lanes 1, 3, and 5) cells were biotinylated. Biotinylated proteins were immunoprecipitated from the cell lysates with streptavidin-agarose beads and subjected to Western blotting using anti-{zeta} mAb (lanes 1 and 2) or anti-CD3{epsilon} mAb (lanes 3 and 4). As a loading control, the membrane was reprobed with HRP-conjugated streptavidin (lanes 5 and 6). D, Confocal microscopic analysis. J76.25.20 cells were biotinylated, fixed, permeabilized, and labeled with anti-CD3{epsilon} mAb, followed by Cy5-donkey anti-mouse Ig (picture 1, CD3{epsilon} staining in red) and streptavidin-FITC (picture 2, biotin staining in green), and analyzed by confocal microscopy. An overlay of pictures 1 and 2 is shown in picture 3 (colocalization of biotin and CD3{epsilon} in yellow).

 
To determine the efficiency of the streptavidin-agarose beads to precipitate biotinylated proteins, Jurkat cells were biotinylated and lysed. The cell lysate was divided into two aliquots and precleared with protein A-agarose beads. The biotinylated proteins of the first aliquot were precipitated with streptavidin-agarose beads (Fig. 2B, lane 1), and the remaining Ti{beta}-chains in the supernatant were subsequently precipitated with MX6 (anti-V{beta}8) plus protein A-agarose beads (Fig. 2B, lane 2). The Ti{beta}-chains of the second aliquot were directly precipitated with MX6 plus protein A-agarose beads (Fig. 2B, lane 3). The samples were subjected to Western blotting using HRP-conjugated streptavidin. The amount of Ti{beta} precipitated in lane 2 was compared with the total amount of Ti{beta} precipitated in lane 3. This demonstrated that >95% of the biotinylated Ti{beta}-chains were precipitated by the streptavidin-agarose beads.

Furthermore, when studying the fate of cell surface-expressed TCR, it is mandatory that the biotinylation procedure does not lead to biotinylation of intracellular TCR components. Our biotinylation procedure was tested for intracellular labeling by parallel biotinylation of the J76.25.20 cell line that expresses the TCR complex at the cell surface and the J76.25.20 variant E3 that does not express any TCR chains at the cell surface due to lack of CD3{gamma} (34). CD3{epsilon} is retained in the endoplasmic reticulum in CD3{gamma}-deficient Jurkat cells either as part of partial TCR complexes or as isolated chains (34), whereas the {zeta}-chains are found in both the endoplasmic reticulum and the Golgi apparatus (8). Biotinylated proteins from J76.25.20 and E3 lysates were precipitated with streptavidin-agarose beads and subjected to Western blotting using anti-{zeta} mAb and anti-CD3{epsilon} mAb. Although E3 and J76.25.20 cells contain similar amounts of intracellular {zeta} and CD3{epsilon} (34), only the {zeta}-chain (Fig. 2C, lanes 1 and 2) and CD3{epsilon}-chain (Fig. 2C, lanes 3 and 4) from J76.25.20 cells were biotinylated. To further analyze for localization of biotinylated proteins, J76.25.20 cells were biotinylated, fixed, permeabilized, stained with streptavidin-FITC and anti-CD3{epsilon} mAb, followed by Cy5-donkey anti-mouse Ig, and finally analyzed by confocal microscopy (Fig. 2D). Taken together, these experiments demonstrate that the labeling procedure selectively caused biotinylation of cell surface-expressed molecules and that it did not cause biotinylation of intracellular proteins in general or of intracellular TCR subunits in particular.

A close correlation exists between ligand-induced TCR down-regulation and {zeta} degradation

Conflicting data concerning the correlation between TCR down-regulation and {zeta} degradation have been published (18, 20, 21). To analyze whether a correlation between ligand-induced TCR down-regulation and {zeta} degradation existed, cells were biotinylated and incubated for 6 h with different concentrations of immobilized anti-CD3 mAb F101.01. The cells were lysed in lysis buffer containing 1% Triton X-100, and the biotinylated molecules were subsequently precipitated using streptavidin-agarose beads. The amount of biotinylated {zeta}-chain present after 6 h of incubation at each mAb concentration was determined by quantification of the intensity of the bands obtained by Western blotting using the anti-{zeta} mAb 6B10.2. In parallel, the TCR surface level of cells treated with each of the mAb concentrations was determined by flow cytometry. Cell surface expression of the TCR complex decreased in a dose-dependent manner as previously reported (12, 21, 30, 36) (Fig. 3A). Likewise, a close correlation between the concentration of stimulating mAb and the degree of {zeta} degradation was observed (Fig. 3). Thus, low concentrations of mAb resulted in low {zeta} degradation, which increased with increasing concentrations of the mAb. Taken together, these experiments demonstrated that a close correlation exists among the strength of the stimulus, TCR down-regulation, and {zeta} degradation. For the following experiments, we chose the close-to-maximal concentration of the anti-CD3 mAb of 1000 ng/ml.



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  		FIGURE 3. Correlation between ligand-induced TCR down-regulation and {zeta} degradation. A, J76.25.20 cells were biotinylated and incubated for 6 h at 37°C in the presence of various amounts of stimulating anti-CD3 mAb F101.01 (0–3000 ng/ml). The cells were lysed, and biotinylated proteins were precipitated with streptavidin-agarose beads. The amount of biotinylated {zeta}-chain present at each concentration of stimulating mAb was estimated from {zeta} blots, as shown in B. The {zeta} degradation data shown in A represent the mean ± SD of three independent experiments. The TCR cell surface level was assayed in parallel. The TCR down-regulation data are from one representative experiment.

 
Ti{alpha}{beta}, CD3{gamma}{epsilon}/CD3{delta}{epsilon}, and {zeta}{zeta} subunits have similar constitutive degradation rate constants in Jurkat cells

The constitutive degradation rate constants for the subunits of the completely assembled cell surface-expressed TCR have only been analyzed in a few studies. Furthermore, none of these studies examined all the TCR subunits simultaneously; rather, conflicting results have been obtained (15, 18, 19, 20, 21, 22). To determine the rate constants for constitutive degradation of the individual TCR subunits, Jurkat cells were biotinylated and subsequently incubated at 37°C for various times. The cells were then lysed, and the biotinylated proteins were precipitated using streptavidin-agarose beads. By this approach, all biotinylated proteins were precipitated independently of their association with other proteins. The precipitated proteins were subsequently analyzed by Western blotting using mAb directed against the individual subunits. Because no Abs were available against the denatured form of Ti{beta} in Western blots, another approach was used to determine the amount of biotinylated Ti{beta}. In this case, the lysates were immunoprecipitated with anti-V{beta}8 mAb, and Western blotting was performed using HRP-conjugated streptavidin. A gradual decrease in the amount of precipitated biotinylated TCR chains was observed during the time frame of the assay (Fig. 4). To ensure that the observed decrease in the biotinylated bands actually was caused by constitutive degradation of the TCR in living cells rather than by simple cell death, vital staining of the individual cell samples was performed in parallel. This demonstrated that the cells were vital and dividing, and that only a minor fraction of ~5% of the cells was dead throughout the experiments (Table I). The experiments were repeated three times for Ti{beta} and CD3{epsilon} and five times for analyses of {zeta} degradation. The intensity of the biotinylated bands was quantified, and the percent intensity at each time points was calculated, setting the intensity of the bands at time zero to 100%. The calculated values were subsequently plotted against time (Fig. 5A, dots). The values decreased exponentially, as seen for reactions that follow first-order kinetics. In general, it is reasonable to assume first-order kinetics for protein degradation when conditions are kept constant (37, 38). Thus, the degradation process of the biotinylated TCR chains could be described very simply as:

(1)
where [TCR]b(t) denotes the amount of biotinylated TCR at time t, and k is the degradation rate constant. The solution to equation 1 is:

(2)
where [TCR]b(t = 0) denotes the amount of biotinylated TCR at the start of the experiment at time zero.



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  		FIGURE 4. Constitutive and ligand-induced degradation of the TCR. A, J76.25.20 cells were biotinylated and incubated with or without stimulating anti-CD3 mAb for the times indicated. The lysates were precipitated with anti-Ti{beta} mAb and subjected to Western blotting using HRP-conjugated streptavidin. B, J76.25.20 cells were biotinylated and incubated with or without stimulating anti-CD3 mAb for the times indicated. The lysates were precipitated with streptavidin-agarose beads and subjected to Western blotting using anti-{zeta} mAb (middle panel), anti-CD3 mAb (lower panel), or HRP-conjugated streptavidin (upper panel).

 

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  		Table I. Cell numbers and percentages of dead cells during the degradation experiments

 


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  		FIGURE 5. Correlation between experimental and theoretical TCR degradation. A, Constitutive degradation of Ti{beta}, CD3{epsilon}, and {zeta} obtained from the experimental data is shown as dots from one representative experiment. The theoretical values of [TCR]b(t) at the various time points were found by inserting the estimated values of k and [TCR]b(t = 0) into equation 2 and are given as lines. B, Ligand-induced degradation of Ti{beta}, CD3{epsilon}, and {zeta} obtained from the experimental data is shown as dots from one representative experiment. The theoretical values of [TCR]b(t) at the various time points were found by inserting the estimated values of k and [TCR]b(t = 0) into equation 2 and are given as lines. The abscissas give the time in hours, and the ordinates show the percentage of biotinylated protein relative to time zero.

 
The degradation rate constant k and [TCR]b(t = 0) were estimated by nonlinear regression from equation 2 using the values of [TCR]b(t) at t = 0, 1, 2, 3, 4, 6, 10, 20, and 24 h obtained from the independent experiments. The corresponding half-lives for the individual TCR chains were subsequently calculated as t1/2 = ln2/k. The estimated k values obtained for the Ti{beta}-, CD3{epsilon}-, and {zeta}-chains were within reasonable limits, and the data showed no evidence of them being different (by Wald test, p = 0.12). The calculated mean degradation rate constant of the TCR was 0.0011 min–1, meaning that ~0.1% of the cell surface-expressed TCR complex was degraded each minute (Table II). The half-life for the TCR in nonstimulated Jurkat cells was then calculated to be 630 min = 10.5 h.


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  		Table II. Degradation rate constants (k) and half-lives (t1/2) of the TCR subunits in Jurkat cells

 
To further evaluate whether the assumption of first-order kinetics and thereby the estimated degradation rate constants were reasonable, theoretical curves for degradation of the TCR subunits were made. The curves were made by inserting the estimated values of k and [TCR]b(t = 0) into equation 2 (Fig. 5A, lines). The theoretical curves for degradation of the TCR subunits closely followed the curves obtained from experimental observations, strongly indicating that the estimations closely reflected the real values.

TCR triggering increases the degradation rate constants of the Ti{alpha}{beta}, CD3{gamma}{epsilon}/CD3{delta}{epsilon}, and {zeta}{zeta} subunits to the same extent in Jurkat cells

Next, we wanted to determine how TCR triggering affected the degradation rate constants of the TCR subunits. Accordingly, Jurkat cells were treated exactly as described above, except the cells were incubated in wells coated with 1000 ng/ml anti-CD3 mAb F101.01. A more pronounced decrease in the amount of precipitated biotinylated TCR chains was observed during the time frame of the assay compared with nonstimulated cells (Fig. 4). Some T cell lines, including some variants of the Jurkat cell line, undergo activation-induced cell death after TCR triggering (39, 40). To ensure that the decrease in the amount of precipitated biotinylated TCR chains was not simply a result of cell death, vital staining of the individual cell samples was performed in parallel with the precipitation experiments. This demonstrated that stimulation of the cells did not induce cell death, in agreement with previous studies, which found that regular Jurkat cells do not undergo TCR-induced cell death (40). The cells were vital and dividing throughout the experiment, and only a minor constant fraction of ~5% of the cells was dead (Table I). The experiments were repeated three times for Ti{beta} and CD3{epsilon} and five times for analyses of {zeta} degradation. The intensity of the biotinylated bands was quantified, and the percent intensity at each time point was calculated, setting the intensity of the bands at time zero to 100%. The calculated values were subsequently plotted against time (Fig. 5B, dots). The degradation rate constant k and [TCR]b(t = 0) were estimated by nonlinear regression from equation 2 using the values of [TCR]b(t) at t = 0, 1, 2, 3, 4, 6, 10, 20, and 24 h obtained from the independent experiments. The estimated k values obtained for the Ti{beta}-, CD3{epsilon}-, and the {zeta}-chains were within reasonable limits; however, there was some evidence suggesting a small variation of the k values for the different TCR chains (by Wald test, p = 0.03). The calculated mean degradation rate constant for the TCR was 0.0033 min–1, meaning that ~0.3% of the cell surface-expressed TCR complex was degraded each minute after TCR triggering (Table II). The half-life for TCR in stimulated T cells was then calculated to be 210 min = 3.5 h. Theoretical curves were made by inserting the estimated values of k and [TCR]b(t = 0) into equation 2 (Fig. 5B, lines). The theoretical values of [TCR]b(t) were in good agreement with the experimental values, and from this it could be concluded that the estimated degradation rate constants were reliable and closely reflected the true degradation rate constants.

Taken together, these results demonstrated that the TCR subunits in Jurkat cells are degraded with similar kinetics. In nonstimulated cells, the TCR complex is relatively stable, with a half-life of ~10.5 h. TCR triggering increases the degradation rate constant of the TCR dependent on the signal strength. Close-to-maximal stimulation, as in the present experiments, thus reduced the TCR half-life to ~3.5 h.

TCR does not dissociate during endocytosis and subsequent intracellular sorting

To this point the present study had demonstrated that the Ti{alpha}{beta} and CD3 subunits were down-regulated with equivalent kinetics after TCR triggering and that the Ti{alpha}{beta}, CD3, and {zeta}{zeta} subunits were degraded with equivalent kinetics during constitutive degradation. However, Wald tests of the calculated curves indicated that the TCR subunits were degraded with slightly different degradation rate constants after TCR triggering. If the TCR subunits were degraded with different kinetics after TCR stimulation, a segregation of the TCR complex would be required during endocytosis and further intracellular processing. To further investigate this question, cells were biotinylated and subsequently incubated at 37°C for various times either with or without stimulation. The cells were then lysed in 1% digitonin lysis buffer, which opposed to the 1% Triton X-100 lysis buffer used in the previous experiments does not break up the subunit interactions of the TCR (35). The TCR was subsequently immunoprecipitated with the anti-V{beta}8 mAb MX6. By this procedure, all TCR subunits associated with Ti{beta} were coprecipitated with Ti{beta}. The precipitated, biotinylated TCR chains were detected by Western blotting using HRP-conjugated streptavidin. As shown in the previous experiments, a gradual decrease in the intensity of the bands was observed during the experiments (Fig. 6A). To evaluate whether the decrease was equivalent for the different subunits, the intensities of the bands were quantified and plotted as the percent intensity relative to the intensity at time zero. This demonstrated a similar decrease in band intensity for each of the three representatives of the Ti{alpha}{beta} (Ti{beta}), CD3{gamma}{epsilon}/{delta}{epsilon} (CD3{epsilon}), and {zeta}{zeta} ({zeta}) subunits during constitutive TCR degradation (Fig. 6B, left panel). TCR triggering resulted in an equivalent increase in the degradation rate for all TCR subunits (Fig. 6B, right panel). These results supported previous results of the present study demonstrating that the TCR subunits are down-regulated and degraded with equal kinetics, indicating that the subunits of the TCR stay together and are processed as an intact complex during endocytosis, recycling, and transportation to degradation.



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  		FIGURE 6. The TCR does not dissociate during internalization and degradation. A, J76.25.20 cells were biotinylated and incubated with or without stimulating anti-CD3 mAb for the times indicated. The cells were lysed in 1% digitonin lysis buffer, precipitated with anti-Ti{beta} mAb, and subjected to Western blotting using HRP-conjugated streptavidin. B, The bands were quantified, and the percentages of biotinylated protein relative to time zero (ordinate) were plotted against time (abscissa).

 
TCR down-regulation and degradation follow the same pattern in primary human T cells as in Jurkat cells

To investigate whether TCR down-regulation and degradation followed the same pattern in primary T cells as in Jurkat cells, BMNC were purified, and V{beta}8+ T cells were isolated using MX6 (anti-V{beta}8) and M-450 goat anti-mouse IgG-conjugated Dynabeads. The isolated cell population was subsequently expanded using CD3-CD28 expander beads to obtain the required number of V{beta}8+ T cells. The cell population obtained contained 93% V{beta}8+ T cell (Fig. 7A). TCR down-regulation kinetics were studied by incubation of the primary human V{beta}8+ T cells with SEE-pulsed Raji cells for various times. The cell surface expression levels of the Ti{alpha}{beta} and CD3{gamma}{epsilon}/{delta}{epsilon} subunits were subsequently determined by flow cytometry. The expression level of the Ti{alpha}{beta} dimers was determined using anti-V{beta}8 mAb, and the levels of CD3{gamma}{epsilon} and CD3{delta}{epsilon} expression were determined using anti-CD3{epsilon} mAb. As observed for Jurkat cells, the Ti{alpha}{beta} and CD3{gamma}{epsilon}/{delta}{epsilon} subunits of primary human V{beta}8+ T cells were down-regulated with identical kinetics after stimulation with SEE-pulsed Raji cells (Fig. 7B, compare with Fig. 1D).



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  		FIGURE 7. TCR down-regulation and degradation follow the same pattern in primary human T cells as in Jurkat cells. A, Forward-side scatter dot plots of freshly isolated BMNC and the expanded cell population are given in the upper panel, and the corresponding V{beta}8 histograms are shown in the lower panel. Freshly isolated BMNC contained ~4% V{beta}8+ cells, whereas the expanded cell population contained ~93% V{beta}8+ cells. B, Primary human V{beta}8+ T cells were stimulated for the time indicated with Raji cells pulsed with SEE (20 ng/ml), transferred to ice, and subjected to flow cytometric analysis. The MFI was determined and used to calculate the percentage of anti-Ti{beta} or anti-CD3{epsilon} binding of stimulated cells compared with nonstimulated cells as (MFItreated cells/MFIuntreated cells) x 100%. The abscissa gives the time in minutes, and the ordinate shows the relative Ti{beta}/CD3{epsilon} expression. C, Primary human V{beta}8+ T cells were biotinylated and incubated with either nonpulsed (constitutive) or SEE-pulsed (ligand-induced) Raji cells for the times indicated. The lysates were precipitated with streptavidin-agarose beads and subjected to Western blotting using anti-{zeta} mAb (middle panel), anti-CD3 mAb (lower panel), or HRP-conjugated streptavidin (upper panel). D, The intensity of the biotinylated bands in C was quantified, and the percent intensity at each time point was calculated, setting the intensity of the bands at time zero to 100%.

 
Next, constitutive and ligand-induced degradations of the TCR subunits in primary human V{beta}8+ T cells were examined. For this, biotinylated T cells were incubated for the times indicated with either nonpulsed or SEE-pulsed Raji cells. The cells were lysed and precipitated with streptavidin-agarose beads, and the precipitates were subjected to SDS-PAGE, followed by Western blotting using either anti-{zeta} or anti-CD3{epsilon} mAbs (Fig. 7C, middle and lower panels, respectively). The blots were subsequently stripped and incubated with HRP-conjugated streptavidin to evaluate the loading amount of the samples (Fig. 7C, upper panel). As observed for Jurkat cells, constitutive degradation of {zeta}- and CD3{epsilon}-chains was observed. After stimulation with SEE-pulsed Raji cells, degradation of {zeta}- and CD3{epsilon}-chains was increased. The intensity of the biotinylated bands was quantified, and the percent intensity at each time point was calculated, setting the intensity of the bands at time zero to 100%. The calculated values were subsequently plotted against time (Fig. 7D). These plots indicated that the CD3{epsilon}- and {zeta}-chains were degraded with equivalent kinetics during constitutive TCR degradation. As also seen for Jurkat cells, ligand triggering of the TCR resulted in a similar increase in CD3{epsilon} and {zeta} degradation in primary T cells (Fig. 7D).

Taken together, these experiments indicated that TCR down-regulation and degradation follow the same pattern in primary human T cells as in Jurkat cells. Interestingly, the constitutive degradation rate appeared somewhat lower in primary T cells than in Jurkat cells (Fig. 7D vs Fig. 5A), and furthermore, higher concentrations of SEE were required to obtain similar degrees of down-regulation and degradation in primary T cells and Jurkat cells. This indicated that primary T cells might be less sensitive to TCR triggering than Jurkat cells. One explanation could be the lack of phosphatase and tensin homolog deleted on chromosome 10 in Jurkat cells, which renders this cell line hyper-responsive to TCR stimulation (41).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we found that the TCR subunits are down-regulated and degraded with equivalent kinetics, indicating that completely assembled octameric TCR stick together as an intact complex during endocytosis and intracellular trafficking. The TCR in nonstimulated Jurkat cells were fairly stable, with a degradation rate constant of 0.001 min–1, resulting in a TCR half-life of 10.5 h. TCR triggering resulted in an increase in the degradation rate constant to 0.0033 min–1, resulting in a reduced TCR half-life of 3.5 h.

Our results are in disagreement with some previous studies, which indicated that the TCR chains dissociated at the cell surface and during TCR endocytosis, and that the individual chains subsequently were processed differently inside the cell (18, 19, 20, 42). Ono et al. (18) analyzed constitutive degradation of the TCR subunits in freshly isolated spleen T cells. They found that the {zeta}{zeta} subunit was rapidly degraded within 2 h, whereas the rest of the TCR subunits were highly stable. The discrepancy between the studies by Ono et al. and ours could be explained by the fact that different T cells were examined in the two studies. However, we do not think that this is the only explanation. In the study by Ono et al. (18), biotinylation of molecules expressed at the cell surface was a central technique. However, no controls were included, demonstrating that only cell surface-expressed molecules were biotinylated, and, in fact, some of the figures indicate that intracellular biotinylation took place. This could have disturbed the results, because incompletely assembled TCR subunits are quickly degraded (5). Furthermore, no formal proof that the presumed {zeta} bands in the figures actually represented the {zeta}-chain was included. Finally, it has been reported that the {zeta}-chain is sensitive to proteases from T cells and macrophages, and such proteases could have destroyed the epitope recognized by the anti-{zeta} mAb applied and thereby disturbed interpretation of the observations (43, 44, 45).

Kishimoto et al. (20) investigated TCR down-regulation in freshly isolated spleen T cells after stimulation with anti-CD3{epsilon} and Ti{beta} mAb. They found that incubation of the cells with anti-CD3{epsilon} mAb for a period as short as 15 min induced heavy down-regulation of Ti{alpha}{beta}, but left the CD3{gamma}{epsilon} and CD3{delta}{epsilon} subunits unaffected. In our hands, the degree of mAb-induced TCR down-regulation in freshly isolated spleen T cells observed by Kishimoto et al. (20) requires several hours of stimulation, and their results could simply be explained by the fact that anti-Ti{beta} mAb binding is blocked by most anti-CD3{epsilon} mAb.

In agreement with our results, José and Alarcón (19) found that the TCR subunits were down-regulated from the cell surface with equivalent kinetics after stimulation with anti-TCR mAb and superantigen. However, by using complicated techniques they found that the TCR subunits segregated after endocytosis in both resting and stimulated T cells. These results were difficult to interpret, and some rather complicated divergent models for TCR trafficking in nonstimulated vs stimulated T cells were suggested (19, 42). Other studies of TCR down-regulation are in good agreement with our results (15, 21, 24, 32, 46, 47), and we found that the most plausible conclusion is that the TCR subunits stick together when expressed at the T cell surface and during endocytosis and further processing. It has been demonstrated that TCR assembly is dependent on interactions among nine ionizable transmembrane residues of the TCR chains (48, 49, 50, 51). The strength of such interactions in the membrane environment may be responsible for the observation that the entire TCR remains intact at the cell surface and during trafficking, even though it is composed of eight chains. Furthermore, the transmembrane interactions are also shielded from the lumenal proteases in the late endosomal/lysosomal pathway.

Only a few studies have focused on constitutive degradation of completely assembled TCR. By analyses of the degradation of biosynthetic labeled TCR chains, one group found that the fraction of chains that assembled into complete TCR complexes had a half-life of ~10 h (3, 5). Another study found the half-life of the TCR to be >10 h by treating T cells with cycloheximide to block protein synthesis and then follow the decay of TCR cell surface expression (15). Finally, a third study found the half-life of the TCR to be >10 h by following the decay of surface-iodinated TCR subunits (22). Our results are in good agreement with these studies. We found that the constitutive degradation rate constants for the individual TCR subunits were within reasonable limits to conclude that the TCR is degraded as a unit with a rate constant of ~0.0011 min–1, resulting in a TCR half-life of 10.5 h (ln2/k) and a mean lifetime of 15 h (1/k) in Jurkat cells. In primary T cells, the constitutive degradation rate constant was somewhat lower than that in Jurkat cells. It has been shown that the TCR is a recycling receptor and that the mean time for the TCR to traverse one cycle of endocytosis and exocytosis is ~100 min (13, 14, 17). From this and the present results, it can be deduced that a TCR traverses ~9–10 cycles of endocytosis and exocytosis, on the average, before it is sorted to degradation. The constitutive recycling could be part of a quality control of the TCR. The long cytoplasmic tails of the {zeta}-chains might be attacked by intracellular proteases (43, 44, 45), and this could lead to defective TCR signaling. However, at the same time proteolysis of the cytoplasmic tail of {zeta} probably unmasks receptor-sorting signals in the CD3 chains, resulting in sorting of the defective TCR from the recycling pathway to the lysosomal pathway (10, 11). Another possibility is that constitutive TCR degradation is caused by a basic protein tyrosine kinase activity. Thus, several studies have demonstrated that Lck tyrosine kinase activity results in down-regulation and lysosomal degradation of the TCR (52, 53, 54).

Previous studies have indirectly indicated that TCR triggering induces TCR degradation (55, 56, 57), and this has later been directly confirmed (21, 22, 32). In agreement with this, we found that TCR triggering resulted in a dose-dependent increase in TCR degradation with a close-to-maximal degradation rate constant of ~0.0033 min–1, resulting in a decrease in the TCR half-life to 3.5 h in both Jurkat cells and primary T cells. As for constitutive TCR degradation, we found that the individual TCR subunits are degraded with similar rate constants during ligand-induced degradation, indicating that the TCR is endocytosed and sorted for degradation as an intact complex. This was further supported by the coprecipitation studies showing identical degradation curves for the TCR subunits coprecipitated with Ti{beta}. If the TCR dissociated at the cell surface or during endocytosis and further trafficking to the lysosomes, identical degradation curves could not have been obtained in the coprecipitation studies. The exact mechanisms responsible for ligand-induced TCR degradation are still not precisely known, but increased Lck tyrosine kinase activation and ubiquitination of the CD3 and {zeta}-chains probably play important roles (12, 54, 58, 59, 60, 61). Ligand-induced TCR degradation causes a prolonged reduction in the level of TCR expression. Thus, >72 h were required for normalization of the TCR expression level after ligand-induced TCR down-regulation (24). This might protect the stimulated T cell against activation-induced apoptosis and allow it to proliferate and differentiate to effector T cells.

Although densitometry of ECL-exposed films is not linear, we believe that the relative band intensities determined in the present study precisely reflected true values, as supported by the good correlation between experimental and theoretical obtained values (Fig. 5).

In conclusion, the present study indicates that after complete assembly, the TCR chains remain associated during subsequent trafficking, including internalization, sorting, and degradation, in both resting and stimulated T cells.


   Footnotes
 
1 This work was supported by the Danish Cancer Society, the Danish Medical Research Council, the Carlsberg Foundation, the Foundation of Vilhelm Pedersen and Wife by recommendation of the Novo Nordisk Foundation, the A. P. Møller Foundation for the Advancement of Medical Sciences, and the Astrid Thaysen Foundation for Basic Medical Sciences. M.v.E., C.M.B., and J.P.H.L. were recipients of Ph.D. scholarships from University of Copenhagen. Back

2 Address correspondence and reprint requests to Dr. Carsten Geisler, Institute of Medical Microbiology and Immunology, The Panum Institute, Building 22.5, University of Copenhagen, Blegdamsvej 3C, DK-2200 Copenhagen, Denmark. E-mail address: c.geisler{at}immi.ku.dk Back

3 Abbreviations used in this paper: BMNC, blood mononuclear cell; BCECF/AM, 2',7'-bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein acetoxymethyl ester; MFI, mean fluorescence intensity; SEE, Staphylococcus aureus enterotoxin. Back

Received for publication November 10, 2003. Accepted for publication April 19, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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