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The Journal of Immunology, 2001, 166: 7229-7237.
Copyright © 2001 by The American Association of Immunologists

Quantitative Relationship Between MHC Class II-Superantigen Complexes and the Balance of T Cell Activation Versus Death1

Pascal M. Lavoie*,{dagger}, Helen McGrath{ddagger}, Naglaa H. Shoukry*,§, Pierre-André Cazenave, Rafick-Pierre Sékaly*,{dagger},{ddagger},§ and Jacques Thibodeau2,||

* Laboratoire d’Immunologie, Département de Microbiologie et Immunologie, Université de Montréal, Montréal, Canada; {dagger} Department of Experimental Medicine, McGill School of Medicine, Montréal, Canada; {ddagger} Laboratoire d’Immunologie, Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montréal, Canada; § Department of Microbiology and Immunology, McGill University, Montréal, Canada; Institut Pasteur, Unité d’Immunochimie Analytique, Paris, France; and || Laboratoire d’Immunologie Moléculaire, Université de Montréal, Montréal, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The binding of bacterial superantigens (SAgs) is profoundly affected by the nature of the MHC class II-associated antigenic peptide. It was proposed that this limitation in the density of SAgs displayed at the surface of APCs is important for efficient TCR serial triggering as well as for preventing apoptosis of the responding T lymphocytes. Here, we have addressed quantitatively the size of this SAg-receptive pool of HLA-DR molecules that are available to bind and present staphylococcal enterotoxin A (SEA) at the surface of B lymphocytes. Our binding curves, depletion experiments, and quantitative immunoprecipitations show that about half the HLA-DR class II molecules on B cells are refractory to SEA binding. Yet, as compared with typical nominal Ags, an unusually high amount of class II-SAg complexes can be presented to T cells. This characteristic appears to be necessary for SAg-induced T cell apoptosis. When <0.3% of the total cell surface MHC class II molecules are occupied by SEA, T cells undergo a normal sequence of early activation events. However, presentation of a ligand density beyond this threshold results in T cell activation that is readily aborted by apoptosis but only after a few cell divisions. Thus, we confirm the existence of MHC class II subsets that are structurally unable to present SEA and provide a quantitative framework to account for the ability of bacterial SAgs to induce peripheral activation vs tolerance in the host.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Superantigens (SAgs)3 are bacterial and viral products capable of inducing T cell deletion and anergy through their interaction with both the MHC class II molecule and the TCR. They are produced by a large spectrum of microorganisms, including potential human pathogens (1), and may be insidiously involved in the development of autoimmune diseases by triggering autoreactive T cells (2, 3). The ability of SAgs to stimulate a high proportion of the T cell repertoire can be explained by their distinct interaction with the complementarity-determining regions of the TCR {beta}-chain variable domain (4). T cell stimulation by SAgs leads to suppression of IL-2 production, anergy, and apoptosis of SAg-reactive T cells (5, 6). The determinants responsible for the induction of T cell activation and/or deletion by SAgs remain enigmatic and thus far cannot be explained by their kinetics and/or topology of interaction with the TCR (7). Indeed, it has been demonstrated by surface plasmon resonance that the affinity of SAg-class II complexes for soluble TCRs is well within the range defined for agonist peptide ligands (8, 9). A better understanding of the molecular parameters allowing SAgs to induce a tolerance state in the host should allow a more rational use of these toxins in the development of immunomodulatory therapies.

Bacterial SAgs are presented in their native form and bind conserved regions of MHC class II molecules outside the peptide-binding groove (10). As such, they should bind to the totality of cell surface MHC class II molecules and thus engage the TCR with a higher avidity than nominal Ags. However, we and others have shown that SAg binding and presentation is markedly affected by the structural microheterogeneity of the MHC class II molecules, probably imparted by the groove-occupying peptide (11, 12, 13, 14, 15, 16). Whether that phenomenon modulates the activity of SAgs on T cells has not been addressed in previous studies. In vivo, the proportion of MHC molecules bound by a given antigenic peptide appears to be naturally limited by inherent peptide processing or loading mechanisms. This possibly prevents induction of high avidity T cell tolerance against nonself Ags (17). Similarly, the restriction imposed by the groove peptide on the binding of SAgs to class II molecules might limit functional inactivation of the responding T cells at low doses (16, 18). Moreover, given that the TCR machinery is sensitive to very low amounts of antigenic peptide ligands (19, 20) or SAg (21), others have proposed that a limited number of SAg-MHC class II complexes is necessary to insure proper serial triggering of the TCR (16).

In the present study, we investigated quantitatively how the structural microheterogeneity in MHC class II molecules affects the binding of staphylococcal enterotoxin A (SEA) to B lymphocytes. We have determined 1) the proportion of MHC class II-peptide complexes on B cells that allows binding of SEA, 2) the minimal number of complexes required for T cell activation, and 3) the number of SAg-MHC complexes required to induce T cell death. Our results show that the amount of SEA receptors on APCs largely exceeds the proportion of class II-ligand complexes required for T cell activation or deletion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines, reagents, and Abs

The human EBV-transformed B cell line LG2 (DR1/DR1) was provided by L. J. Stern (Massachusetts Institute of Technology, Cambridge, MA) and grown in RPMI 1640 (Life Technologies, Rockville, MD) supplemented with 5–10% FCS and 2-ME. The pan anti-DR Ab L243 (IgG2a) (HB55; American Type Culture Collection, Manassas, VA) recognizes a conformational epitope situated in the {alpha}1 domain (22). The anti-DR{beta} XD5.117 (23) and anti-DR{alpha} DA6.147 (24) mAbs have been previously described. In all the biochemical experiments, SEA from three different sources was used and yielded similar results. Purified SEA was obtained from Toxin Technology (Sarasota, FL), and purified recombinant SEA was a kind gift of P. Antonsson (Active Biotech Research, Lund, Sweden) and W. Mourad (Université Laval, Québec, Canada). The L243 hybridoma was grown in RPMI 1640 medium supplemented with synthetic 2% HY (Life Technologies) and purified on a column of protein G-Sepharose 4 Fast Flow (Amersham Pharmacia Biotech, Quebec, Canada). The Fab portion (L243-Fab) was prepared using a commercially available kit (Pierce, Rockford, IL) and yielded a single band (50 kDa) on SDS-PAGE under nonreducing conditions. The concentration was determined by absorbance at 280 nm on a standard spectrophotometer. The SEA or L243-Fab (referred to as SEA-bio or L243-bio) was biotinylated using the sulfo-NHS-LC-biotin as described by the manufacturer (Pierce). The biotinylated proteins were dialyzed in PBS and stored at -20°C in 50% glycerol. Sepharose-SEA or -L243 was prepared by coupling proteins to cyanogen bromide-activated Sepharose 4B beads (Pharmacia Biotech, Uppsala, Sweden) according to the supplier’s recommendations. In brief, SEA or L243 (600 µg) in 0.1 M NaHCO3 and 0.5 M NaCl was incubated to cyanogen bromide-activated Sepharose gel (0.75 g) for 2 h at 4°C with constant agitation. The beads were then blocked with ethanolamine, washed, and kept at 4°C.

Purification of DR1 molecules

Soluble HLA-DR1 was produced in a baculovirus-based system (from the pACDR1 virus obtained from L. J. Stern) and purified according to protocols described (25). Purified membrane HLA-DR1 was obtained as follows: Cells (2 x 108) were lysed in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 µM ZnSO4, 1 mM PefaBloc SC (Roche, Montréal, Canada), 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1% Triton X-100, and the insoluble materiel was spun at 14,000 rpm for 20 min. The supernatant was passed over a L243 column and eluted (see above). The concentration of the DR1 preparation was determined immediately by the Bradford method, and Triton was added to a final concentration of 1%. The HLA-DR1 heterodimer migrated as a homogeneous compact band (55 kDa) in SDS-PAGE under nonboiled conditions.

Surface binding experiments and Scatchard analyses

A dozen completely independent experiments were performed over a several-month period. The concentration of each batch of purified SEA or L243-Fab was carefully determined on Coomassie-stained SDS-PAGE and by Bradford protein dosage (data not shown). The radiolabeling and fractionation of SEA or L243 was performed as described (12). SDS-PAGE separation of the labeled fraction confirmed that the ligand of interest was responsible for >95% of the total specific activity. The binding assay was performed by incubating 125I-radiolabeled SEA or L243-Fab (25–100 ng, depending on the experiments) with 200,000–106 LG2 cells (varied between the different experiments) for 4 h in DMEM (2% FCS-0.05% NaN3) in the presence or absence of a 0.1- to 100-fold excess of unlabeled competitor. The bound fraction was separated from free ligand by centrifugation of cells over a silicon oil cushion (16% mineral oil and 84% silicon oil; Sigma, St. Louis, MO) and counted.

The free fraction of ligand (F), as well as the total (nonspecific and specific) amount of ligand bound (Btot) at a given concentration, was calculated using the following equations:

The amount of ligand bound specifically (Bsp) was determined by subtracting the nonspecific binding (Bnsp) from the total binding (Btot) at each ligand concentration ([F]) according to the following equation:

where when [F] >> Kd (i.e., when the specific binding is saturated); L signifies the amount of cold ligand added; L* signifies the amount of 125I-labeled ligand added; [F] signifies the concentration of free ligand in solution; Kd signifies equilibrium dissociation constant of the interaction; and Bmax signifies the number of receptor molecules.

Human PBL purification, cytokine production, and [3H]thymidine incorporation assays

Peripheral blood obtained from a DR1 donor was diluted (2/1) in PBS and underlayered with Ficoll-Hypaque (Pharmacia Biotech) at room temperature. After centrifugation, the interface was collected and washed three to four times in PBS-2% FCS. MHC class II-negative PBLs were obtained using a modified protocol of the goat anti-mouse-conjugated IsoCell enrichment columns (Pierce). Cells were incubated for 45 min with anti-CD14 (Leu-14), -CD16 (Leu-16), -CD19 (Leu-19), -CD56 (Leu-56) (BD Biosciences, San Jose CA), and L243 Abs at 4°C and passed through the enrichment column. Cell fractions were collected and washed in RPMI 1640–10% FCS. The quality of the purification was confirmed by staining for HLA-DR (using the 50D6 mAb) anti-TCR (Beckman Coulter, Miami FL), and CD4 and CD8 (Simultest; BD Biosciences). For [3H]thymidine incorporation assays, cells were cultured at 37°C in complete RPMI 1640 medium supplemented with 5% FCS. Total PBMCs alone or PBLs with LG2 cells were incubated in the presence of SEA in round-bottom 96-well plates. Following incubation, 1 µCi [3H]thymidine was added for 16 h at 37°C. Cells were harvested, and [3H]thymidine incorporation was measured using a beta plate counter (Pharmacia LKB, Gaithersburg, MD). For [3H]thymidine incorporation assays using purified T cells, LG2 cells were washed three to four times in PBS, treated for 1.5 h with 100 µg/ml mitomycin C (Sigma) at a density of 107 cells/ml, and washed again three to four times in PBS 2%-FCS to block DNA synthesis. IL-2 production was assayed by incubating SEA with total PBMCs (105) for 24 or 36 h. IL-2 in the supernatant was quantified using a CTLL-based assay as previously described (11). Intracellular IL-2 and IFN-{gamma} were measured after 8 h of stimulation using procedures previously described (26). In brief, PBMCs (2 x 106/ml) were incubated with no stimulus, PMA (25 ng/ml) and ionomycin (1 µg/ml), or SEA for 8 h. Brefeldin A (10 µg/ml) was added for the final 6 h of stimulation. After stimulation, cells were permeabilized and stained with either anti-IL-2 or anti-IFN-{gamma}, anti-CD69, and either anti-CD4 or anti-TCR. Abs, isotype controls, and lysing and permeabilizing solutions were purchased from BD Biosciences. Control Ab for permeabilization was purchased from Medicorp (Montreal, Quebec, Canada). Flow cytometry analyses were performed on a FACScan using CellQuest software (BD Biosciences). In lymphocyte stimulation experiments, recombinant SEA was used.

Quantitative immunoprecipitations and Western blotting

Cell surface HLA-DR1 molecules were immunoprecipitated using SEA-bio or L243-bio. Unless specified otherwise, LG2 cells (3 x 105) were incubated for 4 h in presence of either SEA-bio (80 µg/ml; 3 µM), or L243-bio (60 µg/ml; 0.6 µM) at 37 or 4°C in culture medium stabilized with 0.01% sodium azide. After incubation, the cells were washed eight times in 1 ml of PBS to eliminate background, as controlled using an irrelevant biotinylated mAb. Cells were then lysed in 200 µl lysis buffer and kept on ice for 15 min. The insoluble material was spun down, and the supernatant was transferred to a fresh Eppendorf tube containing 100 µl of streptavidin-coated nonporous beads (Dynal M-280; Dynal Biotech, Oslo, Norway). After 1 h at 4°C, 1 ml of PBS was added, and the beads were centrifuged for 10 s. The beads were again washed 10 times (lysis buffer with 0.5% Triton X-100) and resuspended in 50 µl of 2x Laemmli protein-loading buffer. The samples were boiled for 5 min, loaded on 10% SDS-PAGE, and analyzed by Western blotting using the XD5.117 and/or DA6.147 Abs as previously described (12). A peroxydase-coupled goat anti-mouse secondary Ab reactive to the Fc portion of mouse Igs was used for chemiluminescence detection (Pierce).

Depletion experiments

LG2 cells (2 x 105) were incubated as indicated above in 50 µg/ml SEA-bio for 4 h at 37°C before washings and immunoprecipitation with streptavidin-coupled beads. The lysate was transferred to an Eppendorf tube containing fresh SEA-bio-streptavidin beads. After a 4-h agitation at 4°C, the beads were centrifuged, washed eight times with lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 µM ZnSO4, 1 mM PefaBloc SC, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1% Triton X-100), and resuspended in 50 µl of 2x Laemlli buffer. The lysate was then reincubated for three more rounds of depletion using saturating amounts of SEA-coupled Sepharose beads (400 µg/ml SEA). Alternatively, similar results were obtained (data not shown) by extraction of the lysate for 4 h at 4°C in the presence of SEA-bio and immunoprecipitation by transferring the lysate to a fresh Eppendorf tube containing a pellet of 3 x 108 streptavidin beads. The lysate was then transferred to a fresh tube containing 75 µl of L243-coupled Sepharose beads and incubated at 4°C for an additional 4 h before washings. The samples were loaded on SDS-PAGE and analyzed by Western blotting.

CFSE, annexin V, and 7-amino actinomycin D (7-AAD) experiments

The CFSE dye was obtained from Molecular Probes (Eugene, OR). CFSE (0.5–1.25 µM, depending on the preparation) was added to 2 x 107 PBMC and incubated with gentle mixing at room temperature for 10 min. The reaction was quenched by addition of an equal volume of FCS, and cells were washed three times with PBS containing 5% FCS. The cells were cultured at 37°C at 5% CO2 at 1.5 x 106/ml in RPMI 1640 containing 3% FCS overnight to obtain a stable fluorescence intensity between 103 and 104 logs. Labeling of cells with 7-AAD and annexin was performed as previously described (27). For the experiment in Fig. 7Go, cells stained with CFSE according to the above-mentioned method were stimulated with SEA or PHA for 3 days and then labeled with 7-AAD before analysis by flow cytometry.



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  		FIGURE 7. High-dose SEA-activated T cells need to proliferate before undergoing apoptosis. CFSE-stained PBMCs (5 x 105) were stimulated with optimal (10-10 M) and supraoptimal (10-5 M) doses of SEA or PHA as a control, and stained with 7-AAD. The percentages of 7-AAD+ or 7-AAD- cells in the dividing population (CFSElow) are indicated. Results were obtained by gating on CD4+ T cells using a PE-conjugated CD4-specific Ab.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SEA binds a substantial but restricted fraction of cell surface HLA-DR1 molecules

Scatchard analyses. We have previously shown that B cell lines or primary lymphoid cells expressing the class II-associated invariant chain and HLA-DM can bind and present SAgs most efficiently (12). To determine the fraction of MHC class II molecules that are permissive to SEA binding, we performed Scatchard analyses using radiolabeled SEA. For this purpose, we used the representative DR1-expressing B cell line LG2 because, in those cells, the great majority of MHC class II molecules form SDS-stable complexes containing a mixture of oligopeptides (25). Although B lymphocytes express other class II isotypes, HLA-DR is the major receptor for this SAg (28). The reproducibility of the number of complexes obtained by Scatchard plots is shown by SDs of <2% obtained between each of the independent experiments (see Table IGo). A representative set of data is shown in Fig. 1GoA. Of note, in each of these experiments, the binding of either SEA (n = 7) or the L243 anti-DR Fab (n = 4) appeared to follow a first-order binding ligand-receptor interaction as previously described by others (29, 30). Binding of SEA occurs mostly to the high affinity {beta}-chain site, as occupancy of the low affinity of the {alpha}-chain site (Kd >= 10-5 M) is insignificant in the range of concentrations used (29, 30, 31, 32). Using these data, we calculated that LG2 cells express a total of (3.2 ± 0.5) x 106 SEA receptors/cell. An average Kd of (4 ± 2) x 10-8 M was derived, which is in accordance with affinities reported on a number of cell types (12, 33). Similarly, we found a total number of (5.2 ± 2.6) x 106 DR1 receptors/cell as measured using an anti-DR Fab portion (Table IGo). These experiments show that the proportion of SAg-binding sites averages 60% of the total number of surface MHC class II molecules, suggesting that some class II molecules on the surface of APCs are unable to interact with SEA.


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  		Table I. Results from independent experiments of the quantification of the number of SEA receptors on LG2 cells (obtained from Scatchard data)

 


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  		FIGURE 1. Quantification of the number of SEA or L243 receptors on LG2 B cells. A, A representative set of binding data using 125I-L243 ({blacksquare}) or -SEA ({circ}) is shown in two graphical representations. In the two bottom panels, the curves represent the results of nonlinear regressions (y = [Bmax x x]/[x + Kd]) performed on the whole set of binding data compiled from multiple independent experiments using the same LG2 cells. B, The amount of DR1 at the surface of LG2 cells was confirmed by immunoprecipitation using saturating concentration (60 µg/ml) of a L243 Fab portion and compared with soluble DR1 standards purified by immunoaffinity from baculovirus-infected insect cells (25 ) (right lanes) and from detergent-solubilized LG2 cells (left lanes). The data of a densitometric scanning (bottom inset) of the autoradiogram confirm a linear relationship between the amount of protein and the chemiluminescence signal obtained from the Western blot.

 
Immunoprecipitation and depletion analysis. Given the extent of the experimental variations in the binding of the anti-DR Ab obtained with Scatchard analyses (Table IGo), we used independent approaches to confirm the number of DR1 molecules expressed at the surface of LG2 cells. As shown in Fig. 1GoB, the amount of cell surface DR1 molecules immunoprecipitated using saturating concentrations of the L243 Fab portion was compared with DR1 standards of known concentrations. In at least five independent experiments, the total number of HLA-DR1 receptors found ((5.3 ± 0.6) x 106 molecules/cell) was highly consistent with results obtained on Scatchard plots.

The heterogeneity of antigenic peptides bound to MHC class II molecules at the cell surface may give rise to interactions with SEA of different affinities (13), thus skewing our estimate of the total number of binding sites derived from Scatchard data. In principle, such lower-affinity sites can be discerned from nonbinding sites by using saturating amounts of the ligand. We have confirmed the binding of SEA to a fraction of class II molecules by quantitatively comparing the total surface HLA-DR molecules immunoprecipitated using microgram amounts of L243 vs SEA. The amount of DR1 was determined by Western blotting using the XDS.117 Ab, which recognizes a linear epitope on the DR {beta}-chain (34). Fig. 2GoA shows that about half (49 ± 8.3%) of MHC class II molecules were immunoprecipitated with SEA as compared with L243. Similar results were obtained in five different experiments independently of the temperature used for incubation, and these are in accordance with Scatchard results (see Table IGo). Importantly, the nonconformer anti-DRb Ab XD5.117 precipitated an amount of DR1 comparable to that obtained with L243, indicating that the latter does not bind to a limited subset of MHC class II in itself (data not shown).



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  		FIGURE 2. SEA binds a fraction of HLA-DR1 molecules on LG2. A, Quantitative immunoprecipitation of DR1 molecules showing that saturating concentrations of SEA immunoprecipitates only 49 ± 8.3% of HLA-DR1 molecules as determined by densitometric scanning of the autoradiograms. The signals were scanned, and linearity was assured in independent experiments by using different amounts of material and multiple film exposures of the same Western blot (data not shown). Under the conditions used for these experiments, no class II molecules could be detected on Western blots when using an irrelevant Ab (data not shown). B, Rounds (1–5) of HLA-DR1 immunodepletion from LG2 cell lysates using biotinylated SEA (SEA-bio), followed by immunoprecipitation using an Fab portion of the anti-HLA-DR Ab L243. The complexes were run on SDS-PAGE and analyzed by Western blotting.

 
We reasoned that if a subset of MHC class II molecules is unable to bind SEA, it should not be possible to deplete HLA-DR from a cell lysate using this SAg. Thus, we performed serial rounds of immunodepletion using saturating doses of SEA (a saturating dose was determined from the results of Fig. 1GoA), followed by a final immunoprecipitation using the anti-DR Ab L243. We specifically chose to use this conformation-dependent mAb, which recognizes only fully folded post-Golgi {alpha}{beta} heterodimers of importance here (35). Fig. 2GoB is a representative experiment in which we completely depleted SEA-binding HLA-DR receptors from the LG2 lysate after three to four rounds of immunoprecipitation. However, it was not possible to deplete all of the HLA-DR molecules from the cell extract, as demonstrated by the presence of a strong residual signal for DR molecules immunoprecipitated using L243. Altogether, the results obtained from Scatchard plots, immunoprecipitation, and depletion experiments provide a rigorous quantification of the number of MHC class II-peptide complexes that are structurally competent to bind SEA. These receptors represent ~45–60% of the total surface HLA-DR molecules, thus suggesting that some class II-bound oligopeptides might restrict the binding of SAgs on live cells.

The number of cell surface MHC class II-SAg complexes required for T cell stimulation

LG2 cells were used in proliferation assays to characterize the relationship between the number of class II-SAg complexes and the extent of the T cell response. Fig. 3GoA compares a typical proliferation dose-response curve to the proportion of HLA-DR1 molecules that are occupied by SEA at different concentrations. Independently of the T cell:LG2 ratio, proliferation was detected starting at concentrations as low as 10-14 M SEA (Fig. 3GoA), as previously described (36). Similarly, blastic transformation was evident by flow cytometry at 10-15 M SEA (Fig. 3GoB). These concentrations correspond to an average SEA occupancy of ~0.1 (0.000003125%) to 1 (0.00003125%) DR1 molecules/cell (Fig. 3GoC). Most likely, at these concentrations, only some APCs are occupied by a few SEA ligands per cell and are therefore able to stimulate a small number of T cells, possibly those of highest affinity for the SAg-class II complex. The potency of SAgs such as SEA is remarkable because an extremely low number of SEA-MHC class II complexes are sufficient to trigger T cell activation in the conditions established in the present assay. Many more potential SAg-MHC class II complexes can be formed than the minimal number required for T cell activation (see Fig. 3GoA). Maximal T cell proliferation was achieved at SEA concentrations corresponding to ~10,000–20,000 occupied MHC class II molecules (0.2 nM SEA).



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  		FIGURE 3. Minimal number of SEA-MHC complexes required to stimulate human peripheral blood T cells. A, SEA dose-response of purified T cells. A total of 1 x 104 ({blacktriangleup}), 3 x 104 ({circ}) or no (x) LG2 APCs were incubated with T cells (105) in the presence of increasing concentrations of SEA. [3H]thymidine incorporation was determined 3 days after stimulation. The results are compared with the proportion of DR1 molecules occupied by SEA expressed as a percentage (100% = 3.2 x 106 molecules) ({blacksquare}). B, Representative data of the SEA-induced blastic transformation of T cells as assessed by flow cytometry (change in side/forward scatter) 48 h after stimulation. C, The data in B is compared ({circ}) to the incorporation of [3H]thymidine ({blacksquare}) as a function of the dose of SEA. For a given concentration, the calculated average proportion of SEA-MHC complexes per LG2 APC is indicated (0.1, 1, 10, 100) with an estimation of the error range. D, Purified T cells were incubated with SEA-pulsed LG2 cells. After the specified amount of time, the T cells were quenched on ice, and cells were analyzed by flow cytometry (gated on live lymphocytes) using an anti-V{beta}22 Ab. The TCR internalization was strictly dependent on the presence of the LG2 cells and on the formation of T cell-APC conjugates (data not shown). The expression of V{beta}22 is represented as a percentage. Incubation with SEA concentrations >10-7 M reproducibly yielded undetectable TCR levels (NS, nonstimulated).

 
We consistently measured decreasing levels of proliferation at SEA concentrations higher than this optimal number of complexes (10,000–20,000 SEA-MHC/cell) (see Fig. 3GoA), suggesting that a higher density of SAg ligands leads to an impaired T cell response. This decrease in proliferation could not be explained by a lower efficiency of TCR engagement, as evidenced by the strong internalization of the TCR in SEA-reactive V{beta}22+ T cells (which account for a large part of the SEA-responsive T cells; data not shown) above 0.2 nM SEA. As shown in Fig. 3GoD, TCR internalization was rapid and occurred within the first hour of stimulation. The level of TCR rapidly decreased by ~10% within an hour under conditions in which ~10 SAg-MHC class II complexes were formed. At saturating doses of SEA (10-7 M or ~2 x 106 SEA-MHC complexes/cell), >90% of TCRs were internalized from the cell surface. As a control, TCR internalization was not observed in the non-SEA-responsive TCR V{beta}2 cells (data not shown).

Presentation of supraoptimal number of SEA-MHC class II complexes leads to impaired cytokine production by T cells

The next series of experiments were designed to study the functional response of T cells to supraoptimal doses of SEA as defined above. The decreased T cell proliferation observed after 3 days at high SEA concentrations (Fig. 3GoA) was not exclusive to presentation by LG2 cells, as the same results were obtained using class II-autologous PBLs as a source of APCs (Fig. 4GoA). At supraoptimal SEA concentrations, T cells underwent a normal dose-dependent kinetics of induction of early (3–18 h) CD69 and late (24–48 h) CD25 activation markers (see Fig. 4Go, B and C). However, above 10-10 M, a selective dose-dependent inhibition of IL-2 production was observed (Fig. 5GoA). For example, at 10-7 M SEA (Fig. 5GoB), we measured a 2-fold reduction in the proportion of cytokine (IL-2 and IFN-{gamma})-producing cells among activated (CD69+) lymphocytes (Fig. 5GoC) as early as 8 h after stimulation.



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  		FIGURE 4. Dose-dependent stimulation of human PBMCs with high doses of SEA. A, T cell proliferation measured by [3H]thymidine incorporation at 3 days after stimulation with SEA. Dose-dependent kinetics of CD69 (B) or CD25 (C) expression. Human PBMCs (106) were cultured with different concentrations of SEA. The expression of CD69 or CD25 was determined by flow cytometry in live CD4+ lymphocytes using a PE conjugated anti-CD4 Ab.

 


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  		FIGURE 5. Supraoptimal dose of SEA induces a selective inhibition of IL-2 production. A, IL-2 production was measured in the supernatant of SEA-stimulated PBMCs (106) using a CTLL-based assay described previously (11 ). B, IL-2, but also IFN-{gamma}, production was specifically measured in activated (CD69+) T cells early (8 h) after stimulation using a intracellular cytokine assay (see Materials and Methods). The proportion of CD69+ T cells is shown in C. Similar results were consistently obtained in different experiments from at least four different blood donors.

 
T cell apoptosis at high SEA concentrations occurs as a late event after cell division

Despite the normal induction of CD69 and CD25, the proliferative response of T cells at high doses of SEA dropped after 3 days (see Figs. 3Go and 4Go, A). At day 5, the number of cells recovered at 10-7 M SEA was barely increased over the unstimulated control (Fig. 6GoA). In contrast, at the optimal dose of 10-11 M SEA, considering that SEA triggers only ~10–15% of total population of T cells, a 7-fold expansion was observed. Importantly, the reduction in expansion at high SEA doses was not due to nutrient depletion following a massive expansion, because stimulation with PHA, which roughly triggers proliferation of 10-fold more T cells, yielded a significant expansion (see Fig. 6GoA).



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  		FIGURE 6. Reduced expansion of T cells at high doses of SEA by specific induction of apoptosis. A, The expansion of T cells is compared with nonstimulated (NS) cells by counting the total number of cells after 5 days in response to different doses of SEA (or PHA as a control). A representative experiment is shown. B, In similar conditions, the amount of T cell proliferation ([3H]thymidine incorporation, curve) and the proportion of cell death (7-AAD+ cells, histogram) were measured as a function of the dose of SEA to demonstrate a significantly higher amount of cell death with high concentrations of SEA, whereas an optimal SEA concentration (10-10 M) yielded a high proliferation with miminal cell death. In C, the induction of apoptosis at a high dose of SEA was evidenced by staining live cells (7-AAD- cells) with the early apoptotic marker annexin V. The engagement of cells from proliferating blasts (annexin Vmed/forward light scatter (FSC)high) to apoptotic cells (annexin Vhigh/FSClow) is represented. As a control, nonapoptotic proliferating blasts (annexin Vmed/FSChigh) are represented in conditions of minimal apoptosis (10-14 M SEA).

 
A likely explanation for the reduced T cell expansion observed at high concentrations is that a high density of SEA ligands results in an abortive activation through programmed cell death, reflecting the physiological response to SAgs in vivo (5, 6). As shown in Fig. 6GoB, the proportion of nonviable (7-AAD+) cells after 48 h was significantly increased to >50% as cells were stimulated by supraoptimal (10-5 M) doses of SEA. The commitment of activated T cells to apoptosis was further confirmed by the presence of annexin V+7-AAD- cells (a phenotype associated with early apoptosis (27)) in the cells stimulated by SEA.

In additional experiments, we used the CFSE dye that specifically tracks dividing cells to confirm the relationship between the proliferation of SEA-responding cells and their commitment to apoptosis. As described previously, CFSE is a marker of cell division because the fluorescent dye is halved in each daughter cell (37). A representative experiment is shown in Fig. 7Go. Activated T cells expanded under all three conditions tested, consistent with our observation that the induction of activation markers is unaffected by supraoptimal levels of SEA in the first 48 h after stimulation (Fig. 4Go, A and B). Interestingly, we observed a late occurrence of cell death (as measured by staining with 7-AAD) specifically after three to four rounds of division in cells stimulated with SEA. This result is consistent with the absolute requirement for T cell proliferation in SAg-induced apoptosis in vivo (38). Although we detected some degree of cell death in all conditions tested, apoptosis increased in a dose-dependent manner at supraoptimal vs optimal SEA doses (53 vs 13%) (see Fig. 7Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The presentation of SAgs such as SEA and toxic shock syndrome toxin-1 is modulated by MHC class II-bound ligands, most likely including Ags present in the MHC class II peptides groove (11, 12, 13, 14, 15, 16, 39). For example, using MHC class II molecules loaded with single peptides, Kappler and collaborators have measured by surface-plasmon resonance relatively wide variations in the affinity of these complexes for SEA (13). To better define the extent to which these phenomenon are relevant to the presentation of SAgs in vivo, we have used a number of independent biochemical approaches to define unambiguously the proportion of MHC class II molecules acting as SEA receptors on B cells (12). Our Scatchard analysis suggests that, among the 5 x 106 DR1 molecules expressed on LG2 cells, a substantially high proportion (35–60%) is unable to bind and present SEA. We have confirmed that only a fraction of DR molecules bind SEA by performing 1) quantitative immunoprecipitations on cell surface molecules using either the L243 Fab or SEA and 2) depletion experiments on total cell lysates. In these experiments, the maximal concentrations of SEA used were high enough to have picked up any remaining pool of MHC class II complexes with physiologically relevant affinity for the SAg. Therefore, we conclude that there are some class II-bound ligands that are simply nonpermissive for the binding of SEA. Earlier quantification of the proportion of SAg-binding MHC class II molecules, using I-E molecules encapsulated in planar membranes, suggested that most molecules (76%) can bind SEA. These results were interpreted as evidence for the fact that SEA binds MHC class II molecules in an unrestricted fashion (40), although a negative effect of some MHC class II-bound peptides on SAgs binding was not purposely addressed.

The inability of SAgs to bind all the cell surface HLA-DR molecules is a likely a consequence of their indispensable need to interact in close proximity to the polymorphic and structurally diverse peptide groove to reach the TCR (41). Although our results demonstrate that a majority of peptides are permissive to SEA binding, we cannot formally exclude that variations in the affinity of different MHC-peptide complexes may be important for T cell activation. Indeed, among the 35–60% of surface DR1s that bind SEA, ligand-induced structural variations may give rise to a spectrum of affinities for the SAg (13). If the intrinsic affinity of MHC molecules is high (i.e., >4 x 10-8 M), then the existence of progressively lower affinity sites should keep the amount of bound SAg within a range optimal for T cell proliferation. This would be consistent with the idea that a mechanism to limit SAg density is important for inducing a proliferative T cell response as opposed to deletion (16). However, the affinity of SEA as measured herein (4 x 10-8 M) is identical with the highest affinities reported using homogenous soluble MHC class II molecules covalently attached to single peptides. This suggests that, for those MHC complexes that are permissive to SEA binding, the mixture of peptides does not significantly modulate the affinity for the SAg.

The structural difference between SEA-binding vs non-SEA-binding subsets of MHC class II molecules remains to be determined. In addition to MHC class II-bound oligopeptides, other glycoproteins associated in complex with HLA-DR molecules may directly interfere with the binding of SAgs (12, 42). For example, HLA-DR was found to be associated in B cells with molecules such as CD40 (43), HLA-DM (44), and members of the tetraspan protein family (45).

A low amount of SEA, corresponding to 15,000 ligands/APC (0.3% of total cell surface HLA-DR molecules), triggers a strong proliferative response with very little apoptosis. Interestingly, in vivo studies looking at the induction of peripheral deletion by SAgs have used relatively (~1–50 µg/mouse) high doses (6, 46, 47), whereas studies using low doses of SAgs showed that they could stimulate T cell expansion just like antigenic peptides (48, 49). Above a given threshold of SEA-MHC complexes, this balance is tipped toward induction of apoptosis. These observations suggest that the potential for a large pool of MHC-bound SEA is essential to the induction of peripheral tolerance in vivo. In the physiological setting, T cells typically respond to Ags presented by ~102–104 molecules of the restricting allele occupied by a given peptide (50). Interestingly, these estimates compare well to our calculation of the number of SAg ligands needed to achieve a sustained T cell expansion. This interpretation is consistent with recent experiments showing that the affinity of MHC class II-bound SAgs for the TCR is within the range of purely agonist antigenic ligands (8, 9).

In vivo treatment with high doses of SEA leads to a selective suppression of IL-2 production that occurs before and independently of T cell deletion (6, 46). In addition to IL-2, we observed a selective block in the production of IFN-{gamma} that seemingly occurred before the induction of apoptosis in these cells. Indeed, at the earliest time points of TCR engagement (between 6 and 8 h) and in the absence of any detectable cells undergoing apoptosis, we observed a drop in the IL-2 produced, despite a normal induction of the CD69 activation marker (Figs. 2Go and 4Go). Interestingly, a comparable dose-dependent inhibition of the number of cytokine-producing cells has been reported in specific CD8+ T cells responding to supraoptimal lymphocytic choriomeningitis viral loads (51). It is tempting to speculate that prolonged, sustained engagement of the TCR by SAgs limits the cytokine response of T cells perhaps by virtue of the excess number of TCR down-regulated. In our system, addition of exogenous rIL-2 (50, 100, or up to 500 U/ml) at the beginning or daily in the culture did not rescue these cells from programmed death (data not shown). Finally, apoptosis occurred as a relatively late event after initiation of cell division. Based on the level of CFSE in the apoptotic cells, we estimate that these cells die only after having gone through about three to four cell divisions. Interestingly, a similar observation has recently been reported using staphylococcal enterotoxin B (52).

Primed or naive T cells may likely differ in their sensitivity to ligand-induced apoptosis (53, 54). Here, a minimal fraction of the PBLs was composed of primed T cells before stimulation with SEA, as evidenced by the expression of either CD69 or CD25 in <1% of these cells (data not shown). Our data mostly reflect the combined response of naive T cells differing with respect to their affinity for SEA. However, it may be interesting to investigate the functional difference existing between memory and naive T cells or cells otherwise differing in their TCR V{beta} expression.

In conclusion, our results show that despite the existence of different pools of class II molecules relative to their ability to bind SEA, the potential number of SAg-MHC class II complexes largely exceeds the prerequisite for inducing peripheral tolerance in the host. This data should help define the optimal dosage for SAgs in anti-tumor therapies (55)


   Acknowledgments
 
We thank J. P. Corre for expert technical assistance.


   Footnotes
 
1 This work was funded by grants from the National Cancer Institute (to R.P.S.), the Medical Research Council of Canada (awarded to R.P.S and J.T.), and the Cancer Research Society (awarded to J.T.). P.M.L. is supported by a studentship from the Medical Research Council of Canada, R.P.S. holds a Medical Research Council Scientist Award, and J.T. holds a Medical Research Council fellowship. Back

2 Address correspondence and reprint requests to Dr. Jacques Thibodeau, Laboratoire d’Immunologie Moléculaire, Département de Microbiologie et Immunologie, Faculté de Médecine, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, Canada H3C 3J7. E-mail address: jacques.thibodeau{at}UMontreal.CA Back

3 Abbreviations used in this paper: SAg, superantigen; SEA, staphylococcal enterotoxin A; 7-AAD, 7-amino actinomycin D; FSC, forward light scatter. Back

Received for publication January 30, 2001. Accepted for publication April 12, 2001.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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