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The Journal of Immunology, 2001, 166: 6514-6522.
Copyright © 2001 by The American Association of Immunologists

Identification of MHC Class II-Associated Peptides That Promote the Presentation of Toxic Shock Syndrome Toxin-1 to T Cells1

Robert J. Hogan2,*, Josine VanBeek2,{dagger}, Dana R. Broussard{dagger}, Sherri L. Surman{dagger} and David L. Woodland3,*

* Trudeau Institute, Saranac Lake, NY 12983; and {dagger} Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies have shown that the DM-deficient cell line, T2-I-Ab, is very inefficient at presenting toxic shock syndrome toxin 1 (TSST-1) to T cells, suggesting that I-Ab-associated peptides play an essential role in the presentation of this superantigen. Consistent with this, the loading of an I-Ab-binding peptide, staphylococcal enterotoxin B 121–136, onto T2-I-Ab cells enhanced TSST-1 presentation >1000-fold. However, despite extensive screening, no other peptides have been identified that significantly promote TSST-1 presentation. In addition, the peptide effect on TSST-1 presentation has been demonstrated only in the context of the tumor cell line T2-I-Ab. Here we show that peptides that do not promote TSST-1 presentation can be converted into "promoting" peptides by the progressive truncation of C-terminal residues. These studies result in the identification of two peptides derived from IgGV heavy chain and I-E{alpha} proteins that are extremely strong promoters of TSST-1 presentation (47,500- and 12,000-fold, respectively). We have also developed a system to examine the role of MHC class II-associated peptides in superantigen presentation using splenic APC taken directly ex vivo. The data confirmed that the length of the MHC class II-bound peptide plays a critical role in the presentation of TSST-1 by splenic APC and showed that different subpopulations of APC are equally peptide dependent in TSST-1 presentation. Finally, we demonstrated that the presentation of staphylococcal enterotoxin A, like TSST-1, is peptide dependent, whereas staphylococcal enterotoxin B presentation is peptide independent.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bacterial superantigens (SAg)4 are toxins that hyperstimulate the immune system through the direct activation of T cells and APCs (1). SAg exposure often induces a severe, sometimes lethal, toxic shock-like syndrome, which is accompanied by fever and immune dysfunction (2). The activity of SAgs is mediated through the cross-linking of MHC class II molecules on the APC with the TCR on the T cell (3, 4). Recent studies from our laboratory and others have shown that MHC class II-associated peptides have a profound impact on this interaction (5, 6, 7, 8). Kozono et al. (7) have used soluble DR1 molecules engineered to contain a single, covalently attached peptide to show that the N-terminal end of the peptide directly affected staphylococcal enterotoxin A (SEA) binding to class II. This is consistent with the fact that a high affinity binding site for SEA lies at the N-terminal end of the class II MHC peptide-binding groove. Similarly, using the DM-deficient cell line, T2-I-Ab, which can be efficiently loaded with a single peptide, we identified a peptide (SEB121–136) that controlled the binding of toxic shock syndrome toxin 1 (TSST-1) to class II (5, 6). In the case of TSST-1 presentation, the SEB121–136 peptide, or an alanine-substituted version, SEB121–136 (I132->A), enhanced TSST-1 presentation by T2-I-Ab cells by ~3,000- and 10,000-fold, respectively (6). Interestingly, an overlapping peptide, SEB127–142, did not enhance TSST-1 presentation to T cells. Further mutagenic and N- and C-terminal truncation analysis of these two peptides showed that residues at the C terminus of the class II-associated peptide profoundly influenced TSST-1 presentation (6). These data suggested that C-terminal extensions of the peptide sterically blocked TSST-1 binding to class II, consistent with crystallographic studies showing that the {beta}4{beta}5 loop of TSST-1 extends over the class II-associated peptide (9). Although the data strongly support a role for MHC class II-associated peptides in TSST-1 presentation, only 1 peptide of 15 tested (SEB121–136) has been shown to promote TSST-1 presentation to T cells (6). Moreover, because this peptide is itself derived from a SAg, it was possible that its impact on TSST-1 presentation resulted from an unexpected interaction of the peptide with I-Ab outside the peptide-binding groove (10, 11). Thus, it is not clear whether enhancement of TSST-1 presentation is unique to the SEB121–136 peptide or whether the appropriate types of peptides have simply not yet been identified. In addition, all of the data have been obtained exclusively with the T2-I-Ab tumor cell line leaving the possibility that these observations were not generally applicable to other APCs, especially those derived ex vivo. In the current report, we address these issues by identifying two additional peptides that promote TSST-1 presentation and showing that TSST-1 presentation is peptide dependent in APCs obtained directly ex vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6J mice and the H-2 Ma-deficient mice (B6;129S-H2-DMatm1Luc; (referred to as H-2 Ma mice) (12, 13) were obtained from The Jackson Laboratory (Bar Harbor, ME).

SAgs and peptides

TSST-1, SEA, and SEB was purchased from Toxin Technology (Sarasota, FL). The peptides SEB121–136, SEB121–136 (I132->A), SEB127–142, I-E{alpha}52–64, I-E{alpha}52–68, IgG V heavy chain (IgGVH)60–70, IgGVH60–71, HN421–436, and HN418–433 have been described previously, and their sequences and references are presented in Table IGo. The peptides were synthesized at St. Jude Children’s Research Hospital Center for Biotechnology on an Applied Biosystems model 433A peptide synthesizer (Applied Biosystems, Berkeley, CA). Peptide purity was evaluated using reverse phase HPLC analysis. The truncated peptides described in Fig. 1Go were made using the Multipin Synthesis System (Chiron Mimotopes, Clayton, Australia). Peptide synthesis was monitored by including a standard peptide sequence that was subjected to HPLC analysis. Cleaved peptides were resuspended in PBS (pH 7.4) with 3.75% DMSO and used according to the average yield determined by the manufacturer.


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  		Table I. Peptide sequences and references

 


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  		FIGURE 1. C-terminal truncation of I-Ab-binding peptides strongly influences the TSST-1 response of hybridoma 2484. SAg-specific response was determined by assessing IL-2 secretion in response to titered amounts of SAg presented by T2-I-Ab cells that had been preincubated in the presence of the indicated peptides, or left unpulsed. For each peptide, a full TSST-1 dose-response curve was generated, and the concentration of TSST-1 required for half-maximal stimulation was determined. Data are presented as responses relative to unpulsed T2-I-Ab cells (no peptide), defined as 1.0 (see Hybridoma stimulation assays). Results were obtained by analyzing a single TSST-1 reactive hybridoma. However, all peptides were screened with multiple TSST-1-specific hybridomas and similar results were obtained (data not shown). Data are representative of two independent screening experiments.

 
Cell lines

I-Ab-transfected L cells (DCEK Hi7) were a generous gift from Dr. R. Germain (National Institutes of Health, Bethesda, MD) (14). T2-I-Ab and the parental T1-I-Ab cells were generous gifts from Dr. Ned Braunstein, (Columbia University, New York, NY) (15). Hybridomas 2484 and 2508 were derived from the fusion of TSST-1-activated C57BL/6 splenocytes with BW5147, as previously described (6). Both hybridomas express V{beta}15+TCR. Staphylococcal enterotoxin B (SEB)-specific hybridomas 603 and 610 have been described previously (16, 17, 18). The fusion partner BWZ.36 was a gift from Dr. Nilabh Shastri (University of California, Berkeley, CA) (19). The SEB-specific lacZ-inducible hybridoma 5326 was generated in the laboratory in an earlier fusion (our unpublished data).

T cell proliferation assay

Spleen cells were cultured in duplicate at 4 x 105 cells/well for 4 days in the presence of titered concentrations of TSST-1 and SEB121–136 (I132->A) peptide. Proliferation was measured in the final 18 h of incubation by incorporation of 0.5 µCi [3H]thymidine (Amersham, Arlington Heights, IL) per well. Cells were harvested using a 96-well automated harvester and [3H]thymidine was measured using a {beta}-scintillation counter.

Generation of lacZ-inducible T cell hybridomas

T cell fusions were performed as described previously (16). Briefly, spleen cells were harvested from C57BL/6 mice and stimulated with 50 µg/ml of either SEA or TSST-1 for 72 h in vitro. Activated spleen cells (1 x 107) were then fused with 1 x 107 BWZ.36 cells and subsequently cultured in flat-bottom 96-well plates under limiting dilution conditions with hypoxanthine-aminopterin-thymidine selection. The efficiency of the TSST-1 fusion was 13.9%, and a total of 88 clones were obtained. An efficiency of 33.6% was observed in the SEA fusion, and 47 clones were obtained.

Hybridoma stimulation assays

SAg presentation to conventional T cell hybridomas was determined in a standard IL-2 assay as previously described (Figs. 1Go and 2Go) (16, 20). Briefly, 3 x 105 T2-I-Ab or T1-I-Ab cells or 1 x 105 I-Ab L cells were seeded into 96-well plates in complete culture medium (21). Where indicated, synthetic peptides were added and incubated for 2–4 h at 37°C. Next, titered amounts of TSST-1 or SEB were added to the cultures (in a volume of 25 µl) followed by 1 x 105 SAg-reactive hybridoma cells in 75 µl. The cultures were incubated for an additional 24 h at 37°C, and the culture supernatants were harvested to assess IL-2 secretion in a standard bioassay (16, 20). One unit of recombinant human IL-2 (R&D Systems, Minneapolis, MN) is equivalent to 160 U in our assay. SAg presentation to lacZ T cell hybridomas ( Figs. 3–7GoGoGoGoGo) was determined in a {beta}-galactosidase assay as described previously (22). Cultures were set up as for the standard IL-2 assay except that 1 x 106 spleen cells, or purified splenic subpopulations, were used as APC (dendritic cell APC were used at 5 x 104/well). After 24 h in culture, cells were fixed by the addition of 100 µl PBS containing 2% formaldehyde and 0.2% glutaraldehyde for 5 min at 4°C. The plates were washed with PBS and then overlaid with 50 µl of a solution containing 1 mg/ml 5-bromo-4-chloro-3-indolyl-{beta}-D-galactoside, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 2 mM MgCl2 in PBS. Cultures were examined microscopically and the number of blue cells counted after 6–8 h incubation at 37°C or overnight at 4°C. In all experiments, a full dose-response curve was generated for each hybridoma/Ag combination and the concentration of SAg required to stimulate the half-maximal response was calculated. Data are presented as normalized values in which the SAg-specific response to the individual SAgs presented by T2-I-Ab is defined as 1.0. Thus, the data represent the relative shifts in a dose-response curve compared with the relevant control.



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  		FIGURE 2. The influence of C-terminally truncated I-Ab-binding peptides on the response of hybridomas 2484 (A) and 2508 (B) to TSST-1 presented by T2-I-Ab. The data are derived from full dose-response curves as described in the legend to Fig. 1Go. Data are presented as the concentration of TSST-1 required for half-maximal stimulation of the hybridomas and the SD from three independent experiments. Two strongly SEB-reactive hybridomas, 603 and 610, were also tested in this assay. These hybridomas responded only weakly to SEB presented by T2-I-Ab, and this response was not influenced by the presence of absence of the peptides described above (data not shown).

 


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  		FIGURE 3. Proliferation of spleen cells from C57BL/6 (A) or H-2 Ma (B) mice to various doses of TSST-1 in the presencedifferent concentrations of SEB121–136 (I132->A) peptide. Data are representative of three independent experiments.

 


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  		FIGURE 4. Optimal presentation of TSST-1 to three TSST-1 lacZ hybridomas by H-2 Ma spleen cells is dependent on MHC class II-associated peptides. The TSST-1 dose-response curves of three lacZ hybridomas, H55137 (A and B), H5470 (C and D), and H55177 (E and F), were determined using spleen cells as APC from either H-2 Ma (A, C, and E) or C57BL/6 (B, D, and F) mice. In each case, the indicated peptides were added to the assay at a concentration of 50 µM as described in Materials and Methods. The hybridoma response was determined using a colorimetric {beta}-galactosidase assay. For each peptide, a full TSST-1 dose-response curve was generated, and the concentration of TSST-1 required for half-maximal stimulation was determined. Data are presented as the concentration of TSST-1 required for half-maximal stimulation of the hybridomas and are representative of four independent experiments.

 


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  		FIGURE 5. Optimal presentation of TSST-1 to a lacZ hybridoma by different APC populations is dependent on MHC class II-associated peptides. The TSST-1 dose-response curves of H55137 was determined using unsorted spleen cells (A and B), B cells (C and D), macrophages (E and F), or dendritic cells (G and H), as APC from either H-2 Ma (A, C, E, and G) or C57BL/6 (B, D, F, and H) mice. In each case, the indicated peptides were added to the assay at a concentration of 50 µM as described in Materials and Methods. Data are presented as the concentration of TSST-1 required for half-maximal stimulation of the hybridomas (as described in the legend to Fig. 4Go) and are representative of three independent experiments.

 


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  		FIGURE 6. Presentation of TSST-1 to a TSST-1 lacZ hybridoma by dendritic cells is dependent on MHC class II-associated peptides. The TSST-1 dose-response curves of H55137 and H5470 were determined using bone marrow-derived dendritic cells derived from either H-2 Ma (A) or C57BL/6 (B) mice. In each case, the indicated peptides were added to the assay at a concentration of 50 µM as described in Materials and Methods. The hybridoma response was determined using a colorimetric 5-bromo-4-chloro-3-indolyl-{beta}-D-galactoside assay. Data are presented as the concentration of TSST-1 required for half-maximal stimulation of the hybridomas (as described in the legend to Fig. 4Go) and are representative of two independent experiments.

 


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  		FIGURE 7. Presentation of SEA (A and B) or SEB (C and D) to lacZ hybridomas. The SAg dose-response curves of an anti-SEA hybridoma H54157 (A and B) or an anti-SEB hybridoma H5326 (C and D) was determined using spleen cells from either H-2 Ma (A and C) or C57BL/6 (B and D) mice. In each case, the indicated peptides were added to the assay at a concentration of 50 µM as described in Materials and Methods. Data are presented as the concentration of TSST-1 required for half-maximal stimulation of the hybridomas (as described in the legend to Fig. 4Go) and are representative of two independent experiments.

 
Dendritic cell cultures

Bone marrow obtained from the femurs and tibias of C57BL/6 and H-2 Ma mice were cultured at a concentration of 1 x 107/ml in complete tumor medium supplemented with 10 ng/ml IL-4 and 1000 U/ml GM-CSF in Primaria T75 flasks (Becton Dickinson Labware, Franklin Lakes, NJ; Refs.23 and 24). Every other day the medium was replaced. At day 8, the cells were stained to confirm the presence of CD11b, CD11c, CD80, and I-Ab and the absence of CD19 and TCR, and used in the assay. Dendritic cells were used in the assays at a concentration of 5 x 104/ml.

Flow cytometry and cell sorting

All Abs were purchased from PharMingen (San Diego, CA). For flow cytometry, 2 x 105 cells were stained with the indicated Abs in 96-well round-bottom plates. Briefly, nonspecific Fc binding was blocked using anti-CD16/CD32 (Fc{gamma}III/II). The cells were then incubated with directly conjugated Abs for 20 min on ice. A minimum of 5000 gated events was collected using a FACScan flow cytometer, and the data were analyzed using CellQuest software (Becton Dickinson Immunocytometry Systems, San Jose, CA). For sorting, spleen cells were separated into four populations: B cells; macrophages; dendritic cells; and a mock sorted population. After FcR blocking, the spleen cells were stained with anti-CD45R-Cy5, anti-CD11b-PE, and anti-CD11c-FITC. The appropriate populations were then sorted using either a FACStar Plus (Becton Dickinson Immunocytometry Systems) or MoFlo cell sorter (Cytomation, Fort Collins, CO) based on the presence of their specific marker and the absence of the two other markers. Sorted cell populations were generally >90% pure. After sorting, the cells were transferred into 96-well flat-bottom plates. The mock sorted cells and the B cells were plated at a concentration of 1 x 105/ml, whereas macrophages and dendritic cells were plated at a concentration of 5 x 104/ml.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
C-terminal truncation of I-Ab-binding peptides dramatically enhances their ability to promote TSST-1 presentation to T cells

Previous data demonstrated that TSST-1 presentation by Ag-processing defective T2-I-Ab cells (which expresses I-Ab that is either empty or associated with invariant chain-derived peptides) can be dramatically enhanced by SEB121–136 but not by the overlapping peptide SEB127–142 (6). These and other data suggested that C-terminal residues of MHC class II-associated peptides directly control the functional presentation of TSST-1 to T cells (5, 9). Moreover, the data suggested that this effect was not mediated by a cognate interaction between the peptide and TSST-1 molecule but rather was due to simple steric hindrance, or blockade, of the TSST-1-binding site on MHC class II molecules (6). However, despite extensive screening, we have previously been unable to identify other synthetic peptides that strongly promote TSST-1 presentation. On the basis of the observations with SEB121–136 and SEB127–142, we hypothesized that one approach for identifying peptides that promote TSST-1 presentation would be to successively truncate the C-terminal residues of other I-Ab-binding peptides (25). To test this hypothesis, we focused on two previously described I-Ab-binding peptides, I-E{alpha}52–68 and IgGVH60–74, that do not promote TSST-1 presentation in our standard T2-I-Ab assay (data not shown and Refs. 26, 27, 28). Successive truncations of each peptide were synthesized as a pepset on multipins and tested for the ability to promote TSST-1 presentation to a TSST-1-reactive hybridoma (hybridoma 2484). In each case, a full dose-response curve was generated, and the data are presented as shifts in the half-maximal response relative to TSST-1 presented by T2-I-Ab in the absence of peptide. As shown in Fig. 1Go, the full-length peptides had only a minimal impact on TSST-1 presentation to T cells. However, two of the truncated peptides greatly enhanced TSST-1 presentation, I-E{alpha}52–64 (>680-fold) and IgGVH60–70 (>5300-fold). The SEB-derived control peptides showed the same pattern of enhanced TSST-1 presentation as previously described. Thus, SEB121–136 was a strong promoter of TSST-1 presentation, whereas SEB127–142 did not promote TSST-1 presentation (Fig. 1Go; Ref. 6). In addition, an altered SEB121–136(I132->A) peptide, in which residue 132 is replace by an alanine is even more potent than SEB at promoting TSST-1 presentation, consistent with an increased affinity of this peptide for I-Ab (6).

Because these data were generated with relatively impure pepset peptides, we resynthesized and purified the two peptides that showed the most dramatic effect on TSST-1 presentation (I-E{alpha}52–64 and IgGVH60–70), along with two control peptides (I-E{alpha}52–68 and IgGVH60–71). As shown in Fig. 2GoA, the resynthesized I-E{alpha}52–64 and IgGVH60–70 peptides greatly enhanced TSST-1 presentation to hybridoma 2484 when loaded onto T2-I-Ab cells (~1,000- and 12,000-fold, respectively). In contrast, the IgGVH60–71, with one additional amino acid at the C terminus, only modestly enhanced TSST-1 presentation to the same hybridoma (Fig. 2GoA), confirming the data generated from the pepsets. Similar results were obtained with the I-E{alpha}52–68 peptide, which was >100-fold less efficient at promoting TSST-1 presentation than the I-E{alpha}52–64 peptide. TCR-specific effects were ruled out by using a second TSST-1-reactive hybridoma, 2508. As shown in Fig. 2GoB, an even more dramatic enhancement was observed with the shorter I-E{alpha}52–64 and IgGVH60–70, peptides (5,300- and 47,500-fold, respectively), compared with little enhancement with the longer I-E{alpha}52–65, and IgGVH60–71 peptides (2- and 11-fold, respectively). Thus, these data identify two additional peptides that promote TSST-1 presentation and demonstrate that the original observation with SEB121–136 was not unique to that particular peptide. In addition, the data provide very strong support that the length of the C terminus of the peptide plays a key role in the capacity of the peptide to present TSST-1 to T cells.

We also examined the capacity of the peptides to promote the binding of TSST-1 to T2-I-Ab cells using a flow cytometric assay (6). Neither the I-E{alpha}52–64 nor the IgGVH60–70 peptides enhanced TSST-1 binding to T2-I-Ab over the background. However, this result is difficult to interpret given that peptide-loaded T2-I-Ab cells are significantly less efficient than I-Ab L cells at TSST-1 presentation to T cell hybridomas. This suggests that the peptides described here are relatively weak at promoting TSST-1 binding to I-Ab compared with some peptides present on wild-type cells (Fig. 2Go). Thus, the binding assay may not be sensitive enough to detect weak TSST-1 binding over the background. In this regard, it is likely that each peptide-Ab complex on the surface of a normal APC binds TSST-1 with distinct affinity resulting in a range of affinities on the population level. We may have identified peptides that are at the lower end of this affinity range.

Deficient TSST-1 presentation in DM knockout mice can be reversed by the addition of appropriate I-Ab-binding peptides

Previous studies have reported that H-2 Ma mice (which lack DM expression) are deficient at TSST-1 presentation (29). This deficiency is not due to poor class II expression, because these mice express high levels of I-Ab (30). Given our observation that MHC class II-associated peptides control TSST-1 presentation on the T2-I-Ab tumor cell line, we hypothesized that the deficiency in TSST-1 presentation by H-2 Ma mice was due to the absence of appropriate peptides and could be reversed by the addition of exogenous peptide. To test this hypothesis, we analyzed the proliferation of H-2 Ma and C57BL/6 spleen cells to TSST-1 in the presence or absence of the SEB121–136 (I132->A) peptide. As shown in Fig. 3Go, H-2 Ma spleen cells proliferated only poorly to TSST-1 in the absence of peptide compared with C57BL/6 spleen cells, in terms of both the dose of TSST-1 required and the maximal level of proliferation. However, the proliferation of H-2 Ma spleen cells was substantially enhanced by inclusion of the SEB121–136 (I132->A) peptide in the cultures. The addition of high concentrations of SEB121–136 (I132->A) (100 µg/ml) restored the sensitivity to TSST-1 to that of C57BL/6 spleen cells (i.e., half-maximal proliferation was observed at ~1 ng/ml TSST-1). However, the overall level of proliferation was still substantially less than that seen with the C57BL/6 spleen cells (50,000 vs 100,000 cpm, respectively). Because equal numbers of T cells were used in the C57BL/6 and H-2 Ma cultures, these data suggest that not all of the T cells were able to respond in the H-2 Ma cultures. Consistent with this, other studies have shown that CD4+ T cell responses are defective in H-2 Ma mice due to abnormal positive selection in the thymus (29, 30, 31, 32).

The potential T cell deficiency in H-2 Ma mice makes it difficult to investigate the effect of I-Ab-associated peptides on SAg presentation using the T cell proliferation approach. Therefore, as an alternative, we took advantage of T cell hybridomas to directly compare superantigen presentation by H-2 Ma and C57BL/6 spleen cells. It was not possible to use conventional IL-2-producing hybridomas for these studies due to the potential interference by IL-2 produced by contaminating T cells in the stimulator populations. To overcome this obstacle, we generated TSST-1- and SEA-reactive T cell hybridomas by fusion with the BWZ.36 fusion partner (19). BWZ.36 is transfected with the lacZ gene under the control of the IL-2 enhancer element, and hybridomas generated with this cell line make {beta}-galactosidase when stimulated through the TCR (19, 33, 34). Hybridoma responses can be readily determined by a simple colorimetric assay, which is not affected by concurrent responses by the APC population. Altogether, 135 hybridomas were tested for their reactivity to either SEA or TSST-1, and autoreactive hybridomas were excluded by testing the clones in the absence of any SAg. In total, 5 TSST-1-reactive and 18 SEA-reactive hybridomas were obtained. Three TSST-1-reactive hybridomas (55137, 5470, and 55177), a SEA-reactive hybridoma (54157), and a previously described SEB-reactive hybridoma (5326) were selected for further study. The three TSST-1-specific hybridomas were all V{beta}15+, the SEA-specific hybridoma was V{beta}11+, and the SEB-specific hybridoma was V{beta}8+ (as determined by flow cytometry and RT-PCR, data not shown). This pattern of V{beta} expression is consistent with published data on the murine V{beta} specificity of these SAgs (35).

We next tested the ability of the hybridomas to recognize titered amounts of their respective superantigens on H-2 Ma and C57BL/6 spleen cells. As shown in Fig. 4Go, H-2 Ma spleen cells were not able to present TSST-1 to three distinct TSST-1-reactive T cell hybridomas, whereas the wild-type C57BL/6 spleen cells were strong presenters. However, the deficit in TSST-1 presentation by H-2 Ma spleen cells was restored for all three hybridomas by the addition of the SEB121–136 (I132->A), I-E{alpha}52–64, or IgGVH60–70 peptides. In contrast, as had been seen with T2-I-Ab, the SEB127–141, I-E{alpha}52–68, and IgGVH60–71 peptides did not promote TSST-1 presentation to any of the hybridomas. As expected, none of the peptides enhanced the already strong capacity of C57BL/6 spleen cells to present TSST-1 to T cells. These data demonstrate that the effects of MHC class II-associated peptides described with the T2-I-Ab tumor cells line can also be replicated with spleen cells isolated directly ex vivo. In addition, the data confirm the identification of I-E{alpha}52–64 and IgGVH60–70 as peptides capable of promoting TSST-1 presentation.

Different subpopulations of APCs depend on peptides for TSST-1 presentation

The previous data had shown that spleen cells were dependent on MHC class II-associating peptides for TSST-1 presentation to T cell hybridomas. This system allowed us to ask whether distinct APC populations differed in their peptide dependency for TSST-1 presentation. Thus, spleen cells from C57BL/6 and H-2 Ma mice were sorted into the three populations, B cells, macrophages, and dendritic cells. Also a mock sort was included in which the cells were run through the sorter but were not divided in the different populations. Each population was loaded with the same panel of peptides as in the previous experiment. As shown in Fig. 5Go, all three populations isolated from C57BL/6 spleen were strong presenters of TSST-1, and the addition of the different promoting and nonpromoting peptides did not affect this efficiency. In contrast, none of the H-2 Ma populations presented SAg in the absence of exogenously added peptide. When the appropriate peptides were added, there were clear differences between the sorted populations in presentation capacity, with dendritic cells being the most efficient and B cells the least efficient. However, the same basic pattern of presentation was observed for each population with the SEB121–136 (I132->A) peptide being the best and the I-E{alpha} peptide the least efficient at promoting TSST-1 presentation. Similar data were observed using bone marrow-derived dendritic cells that were cultured in vitro with IL-4 and GM-CSF (Fig. 6Go). Taken together, these data indicate that TSST-1 presentation by each of the cell populations was dependent on the class II-associated peptide.

SEA and SEB presentation by H-2 Ma spleen cells

Having shown a clear peptide effect for TSST-1, we next investigated the role of MHC class II-associated peptides in the presentation of SEA and SEB. Previous studies have suggested that the amino terminus of the peptide might affect SEA presentation (5, 7) and TSST-1 (5, 6, 9), whereas no clear peptide effects have been shown for SEB presentation (4, 8, 36, 37). One of the problems in analyzing the presentation of SAgs with the T2-I-Ab tumor line system is that there is no clear positive control (the parental DM-positive T1-I-Ab parental line expresses human class II molecules), unless a peptide is defined that clearly promotes superantigen presentation. As an alternative approach to address this issue, we analyzed SEA and SEB presentation by H-2 Ma spleen cells using C57BL/6 spleen cells as a control. As shown in Fig. 7Go, C and D, spleen cells from H-2 Ma mice were highly efficient at presenting SEB to SEB-reactive hybridomas. In terms of the dose-response curves, H-2 Ma spleen cells were ~5-fold more efficient APC than the control C57BL/6 spleen cells. These data provide the first clear evidence that either empty or invariant chain peptide intermediate (CLIP)-associated I-Ab is sufficient for optimal presentation of this SAg. In contrast, the ability of H-2 Ma spleen cells to present SEA to a T cell hybridoma was substantially lower (<100-fold) than that of C57BL/6 spleen cells (Fig. 7Go, A and B). This deficiency in SEA presentation was not due to an inherent defect in the capacity of splenic H-2 Ma cells to present Ag, given that they were highly efficient at presenting SEB (Fig. 7GoB) and TSST-1 (with the appropriate peptide; Fig. 4Go). Thus, these data suggest that MHC class II-associated peptides play a critical role in SEA presentation to T cells. However, the addition of any of the peptides described earlier (I-E{alpha}52–64, I-E{alpha}52–65, IgGVH60–70, or IgGVH60–71), or the HN421–436 and HN418–433 peptides reported previously, did not enhance SEA presentation, indicating that the appropriate peptides remain to be identified.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have shown that the binding and presentation of TSST-1 to T cells by the I-Ab MHC class II molecule is dependent on the structure of I-Ab-associated peptides (5). However, only one peptide (SEB121–136) had been shown to mediate this effect. Because SEB121–136 peptide is derived from SEB, it was unclear whether additional peptides not derived from bacterial SAgs might also promote the TSST-1 presentation. In the current study, we hypothesized that the appropriate peptides had been missed because they expressed C-terminal extensions that sterically inhibited TSST-1 binding. On this basis, we reasoned that sequential truncation of any I-Ab binding peptide would generate a peptide that promotes TSST-1 presentation, provided that the truncated peptide could still bind to I-Ab. The data in the current report strongly support this idea by showing that truncation of two I-Ab-binding peptides enhanced their capacity to promote TSST-1 presentation by as much as 1000-fold. In addition, these data demonstrate that the capacity to promote TSST-1 presentation is not unique to the SEB121–136 peptide. Furthermore, we have also shown that peptide-dependent TSST-1 presentation is not restricted to the T2-I-Ab cell line but can also be demonstrated in spleen cells and within different subpopulations of APC.

The underlying mechanism by which peptides influence TSST-1 presentation is currently unclear. One possibility is that promoting peptides induce some conformational change in the MHC molecule, including SDS stability, which allows the presentation of TSST-1. Alternatively, there may be a positive interaction between the SAg and the peptide. This would imply that some other structural feature of the peptide could be important, and this could explain why certain peptides were more effective in enhancing TSST-1 presentation than others. However, based on published data and the data presented here, it is likely that residues extending out of the C-terminal end of the peptide-binding groove sterically block TSST-1 binding. This is consistent with the crystal structure of TSST-1 bound to DR1, which suggests a direct contact between TSST-1 and the C-terminal region of the peptide (9). Interestingly, it has been reported that most of the MHC class II molecules on the H-2 Ma APCs are CLIP associated or empty and can be loaded with exogenous peptide (12, 13, 30, 38). This suggests that TSST-1 cannot bind to either empty class II molecules and that the normal set of CLIP peptides in the peptide-binding groove have C-terminal extensions that block TSST-1 binding. Thus replacement of CLIP with permissive peptides, such as those described here, may be the mechanism which facilitates TSST-1 binding to I-Ab. Interestingly, a similar mechanism may be operating to control the binding of the Mycoplasma arthritidis-derived mitogen to human MHC class II molecules (39).

Our data are consistent with findings that TSST-1 binds to only a subset of available DR1 molecules because naturally processed peptides are known to vary in length (37, 40). For example, sequence analysis of peptides isolated from both mouse and human class II molecules have shown that many class II binding peptides are nested sets, varying at the N- and C-terminal ends (26, 41, 42, 43). In addition, only a minority of processed peptides on the surface of APC allow TSST-1 presentation (6). It is currently unclear whether the binding of TSST-1 to a peptide-defined subset of class II molecules plays an important role in the biology of this SAg. However, we have speculated that peptide modulation of TSST-1 presentation may be a mechanism to optimize T cell activation by inducing low density, but high affinity binding of SAg to MHC class II molecules (25). Thus, the SAg mimics the presentation of conventional peptide Ags (which are present at low density on available class II molecules), yet is able to stimulate a high frequency of T cells by degenerate interaction with the TCR (3, 44, 45, 46). In this regard, it is interesting that a peptide that promotes TSST-1 presentation is itself of staphylococcal origin. Thus, it is possible that the pathogen may modulate the SAg response by altering the peptide display on the APC. However, this seems unlikely because only a fraction of class II molecules on the APC surface are likely to contain peptides of pathogen origin. In addition, the current data demonstrate that peptides of host origin are also able to promote TSST-1 presentation.

A concern with using T2-I-Ab cells to study TSST-1 presentation is that it is a tumor cell line and may not reflect the properties of normal APC. To circumvent this problem, we have developed a system in which spleen cells from DM-deficient H-2 Ma mice are used. These studies clearly demonstrate that TSST-1 presentation by several different subsets of APC is generally dependent on class II-associated peptides. It has been proposed that this peptide dependency enables SAgs to distinguish between different APCs (25) because different APCs probably present a distinct array of peptides on their MHC class II molecules (47). Indeed, several studies have documented differences in SAg presentation by identical MHC class II molecules expressed on different APCs (37, 48, 49, 50, 51). This is an interesting possibility that warrants further investigation.

The development of the H-2 Ma system also allowed us to reevaluate the role of peptide in SEA and SEB presentation, because we could now directly compare SAg presentation on DM-deficient and wild-type spleen cells (52). The data clearly showed that SEB presentation by DM-deficient APC was not dependent on exogenously added peptide. Thus, CLIP-associated and/or empty class II molecules are efficient at presenting SEB. In contrast, SEA presentation was significantly impaired on DM-deficient APC, providing strong support for the idea that SEA presentation is peptide dependent. Consistent with this, previous studies indicated a role for the N terminus of the peptide (6, 7). However, we did not identify any peptides promoting SEA presentation in this study using splenic H-2 Ma cells as presenters.

Taken together, the data presented here extend previous studies by identifying two additional peptides that promote TSST-1 presentation to T cells. This rules out the possibility that the previously described SEB121–136 peptide was somehow unique in this regard and indicates that this peptide dependence is a general feature of TSST-1 presentation. Importantly, the length of the C-terminal region of the peptide seems to play a critical role in determining the efficacy of a given peptide in promoting TSST-1 presentation. In addition, the data indicate that MHC class II-associated peptides also affect the presentation of SEA to T cells, although the relevant peptides have not been defined.


   Acknowledgments
 
We thank Drs. Marcia Blackman and Charles Hardy for critical reading of the manuscript and Michael Coppola and Edward Usherwood for helpful discussions.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants CA-56570 (D.L.W.) and AI-09585 (D.R.B), National Institutes of Health Cancer Center Support CORE Grant P30 CA-21765, the Trudeau Institute, and the American Lebanese Syrian Associated Charities. Back

2 R.J.H. and J.V. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. David L. Woodland, Trudeau Institute, 100 Algonquin Avenue, Saranac Lake, NY 12983. E-mail address: dwoodland{at}trudeauinstitute.org Back

4 Abbreviations used in this paper: SAg, superantigen; CLIP, invariant chain peptide intermediates; SEA, staphylococcal enterotoxin A; SEB, staphylococcal enterotoxin B; TSST-1, toxic shock syndrome toxin-1; IgGVH, IgG V heavy chain. Back

Received for publication December 8, 2000. Accepted for publication March 20, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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