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HLA Class II Polymorphisms Determine Responses to Bacterial Superantigens¹

Martin Llewelyn^*, Shiranee Sriskandan^*, Mark Peakman, David R. Ambrozak, Daniel C. Douek, William W. Kwok, Jonathan Cohen^*,¶ and Daniel M. Altmann^2,*

^* Department of Infectious Diseases, Faculty of Medicine, Imperial College, Department of Immunology, Guy’s, Kings and St. Thomas’ School of Medicine, London, United Kingdom; Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; Virginia Mason Research Center, Seattle, WA 98101; and ^¶ Division of Medicine, Brighton and Sussex Medical School, Brighton, United Kingdom

	Abstract

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The excessive immunological response triggered by microbial superantigens has been implicated in the etiology of a wide range of human diseases but has been most clearly defined for the staphylococcal and streptococcal toxic shock syndromes. Because MHC class II presentation of superantigens to T cells is not MHC-restricted, the possibility that HLA polymorphisms could influence superantigenicity, and thus clinical susceptibility to the toxicity of individual superantigens, has received little attention. In this study, we demonstrate that binding of streptococcal and staphylococcal superantigens to HLA class II is influenced by allelic differences in class II. For the superantigen streptococcal pyrogenic exotoxin A, class II binding is dependent on DQ -chain polymorphisms such that HLA-DQA1*01 -chains show greater binding than DQA1*03/05 -chains. The functional implications of differential binding on T cell activation were investigated in various experimental systems using human T cells and murine V8.2 transgenic cells as responders. These studies showed quantitative and qualitative differences resulting from differential HLA-DQ binding. We observed changes in T cell proliferation and cytokine production, and in the V specific changes in T cell repertoire that have hitherto been regarded as a defining feature of an individual superantigen. Our observations reveal a mechanism for the different outcomes seen following infection by toxigenic bacteria.

	Introduction

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Superantigens (SAgs)³ are potent immunostimulatory proteins that bind the MHC class II and TCR molecules on the surface of APCs and T lymphocytes (1, 2). The range of human diseases with possible SAg etiology includes Kawasaki’s disease (3), psoriasis (4), atopic eczema (5), rheumatic fever (6), Crohn’s disease (7), and sepsis (8). The best-characterized role for SAgs in human disease remains that of the SAg exotoxins of Staphylococcus aureus and Streptococcus pyogenes, which are believed to trigger the staphylococcal and streptococcal toxic shock syndromes (9, 10). However, the observation that apparently identical toxigenic strains of S. pyogenes or S. aureus cause clinical syndromes ranging from superficial carriage through pharyngitis to toxic shock syndrome, suggests that genetic heterogeneity contributes to the clinical phenotype following SAg exposure (11).

Shared structural features allow SAgs to bind, as unprocessed proteins, to the MHC class II molecule and the TCR at sites away from conventional Ag binding sites, thereby activating up to 20% of all T cells. On the TCR, SAgs bind at the -chain variable (V) region. Individual SAgs are limited in the V families they stimulate (12) and produce a marked skewing of the V repertoire in responding T cells known as the V signature. On the MHC class II molecule, SAgs adopt four principal binding modes. Some bind the -chain, some the -chain, and some crosslink class II either by binding both - and -chains or by virtue of two -chain binding sites. SAgs that adopt the same mode of binding do not necessarily compete, suggesting more subtle differences exist within these binding modes (13).

Mice mount relatively weak in vitro and in vivo responses to bacterial SAgs compared with humans, a fact that is believed to arise from sequence differences between human and mouse MHC class II, with the mouse class II binding bacterial SAgs poorly (14). Furthermore, within human HLA class II, differences between isotypes in presentation of individual SAgs are also established. For example, the staphylococcal enterotoxin A (SEA) and enterotoxin B (SEB) use HLA-DR more efficiently than HLA-DQ in T cell activation, whereas some streptococcal SAgs such as streptococcal pyrogenic exotoxin A (SPEA) preferentially use HLA-DQ (15). However, during the early characterization of superantigenicity, it became clear that a hallmark of this effect was the lack of classical MHC restriction. This led historically to a focus on the similarities rather than differences in SAg binding by different HLA alleles, and with the exception of the observation that SEA and staphylococcal enterotoxin E bind to DRw53 particularly poorly (16), the influence of differences within class II isotypes has not been studied in detail.

The worldwide resurgence of streptococcal toxic shock syndrome since the 1990s has been associated with circulation of novel spea⁺ strains of S. pyogenes (17). An epidemiological link between HLA haplotype and susceptibility to the SAg-associated manifestations of S. pyogenes infection was recently demonstrated by Kotb et al. (18). However, because the patients in the Kotb and colleagues study were necessarily infected by different S. pyogenes strains, each carrying multiple different SAg genes (19), it is impossible to know from such clinical data whether the HLA association relates to an individual SAg, and if so, to which SAg. This makes it difficult to elucidate molecular mechanisms at the level of SAg-HLA class II-TCR interactions.

In this work we have set out to study in detail the relationship between HLA class II polymorphism and the presentation of bacterial SAg focusing primarily on SPEA as a protype SAg. Because SPEA binds HLA-DQ specifically and not HLA-DR or HLA-DP (15) we have been able to use HLA homozygous B lymphoblastoid cell lines (B-LCLs) to screen a range of HLA-DQ molecules for differences in SPEA binding. The data presented in this study demonstrate up to 10-fold higher levels of SPEA binding to cells expressing HLA-DQ -chains encoded by the gene HLA-DQA1*01 than to cells expressing HLA-DQA1*03 or *05 -chains. In experiments using purified HLA class II, we have extended these observations to other staphylococcal SAgs. In addition these data demonstrate that the magnitude of the T cell response to SPEA is determined by HLA-DQ polymorphisms both in terms of proliferation and cytokine response. Furthermore the V signature of SPEA is itself determined by the HLA-DQ involved in its presentation to T cells.

Although previous studies have demonstrated that HLA class II isotypes differ in presentation of individual SAgs, these are the first data to demonstrate that HLA class II polymorphisms determine both the magnitude and the quality of the T cell response to SAgs. The data provide a plausible mechanism for observed interindividual differences in disease phenotype following infection by toxigenic strains of bacteria and the recent identification of HLA class II haplotypes associated with susceptibility to severe S. pyogenes infections (18).

	Materials and Methods

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B cell lines

A panel of HLA class II homozygous human B-LCLs was used, including three DQ5 cell lines, HOM2, LWAGS (DQA1*0101/DQB1*0501), and BEC11 (DQA1*0101/DQB1*0503); five DQ6 cell lines, TOK (DQA1*0103/DQB1*0604), PGF, SCHU (DQA1*0102/DQB1*0602), HOR, and WT46 (DQA1*0102/DQB1*0604); two DQ7 cell lines, IDF (DQA1*0501/DQB1*0301) and TISI (DQA1*0505/DQB1*0301); and two DQ8 cell lines WT51 and 600sf (DQA1*0301/DQB1*0302). Bare lymphocyte syndrome (BLS) cell lines retrovirally transfected to express either DQA1*0301/DQB1*0302, DQA1*0102/DQB1*0602, or DQA1*0102/DQB1*0604, as previously described, were also used (20).

Toxins

Recombinant SPEA-1 was expressed as previously described (21) and was biotinylated using EZ-link Sulfo-NHS-LC biotinylation kit (Pierce, Rockford, IL). SPEA, SEA, SEB, and biotin conjugates of each were purchased from Toxin Technology (Sarasota, FL).

HLA class II Abs

L243 (DR), L2 (pan DQ), and SPVL-3 (pan DQ) were purified from mouse hybridoma supernatants. TDR31.1 (Ancell, Bayport, MN), WR18 (pan class II; Serotec, Oxford, U.K.), and Leu10 (DQ; BD Biosciences, Oxford, U.K.) were purchased from manufacturers.

Purification of HLA class II

HLA-DR11 and HLA-DQ3.1 were affinity purified from IDF cells and HLA-DR1501 and HLA-DQ6.2 from PGF cells using L243 and SPV-L3 columns as previously described (22). Purified class II was >98% pure as assessed by SDS-PAGE. The concentration of purified HLA class II was determined by bicinchoninic acid protein assay (Pierce).

Preparation of human PBMC and T cells

PBMCs were obtained by Ficoll gradient separation. The class II negative T cells were purified by negative selection. A total of 5 x 10⁶ cells were incubated with L243, WR18, Leu14 (anti-CD14), and Leu19 (anti-CD19), followed by two rounds of depletion using anti-mouse Ig Dynal beads (Dynal Biotech, Oslo, Norway) according to manufacturer’s instructions. Depletion of all HLA class II expressing cells was confirmed by failure of purified T cells to proliferate in response to SAg stimulation unless coincubated with APCs.

Flow cytometric binding assays

A total of 5 x 10⁵ cultured B cells were incubated with biotinylated SPEA and washed, then binding visualized using Extravidin-PE (Sigma-Aldrich, Poole, U.K.) by FACS (FACSCalibur using CellQuest software; BD Biosciences). A total of 20,000 cells falling within a healthy lymphocyte gate were analyzed. SPEA binding was measured as mean fluorescence intensity (MFI) of cells incubated with biotinylated SPEA and Extravidin-PE divided by MFI of cells incubated with unbiotinylated SPEA and Extravidin-PE. Level of HLA-DQ expression was measured using Abs recognizing different conserved regions of the DQ molecule Leu10 (DQ1 and DQ3), L2 (pan DQ), and SPV-L3 (pan DQ). Binding of each DQ mAb was assessed independently for each cell line in each experiment and was measured as the MFI of cells incubated with Ab and FITC-labeled anti-mouse Ig second layer divided by MFI of cells incubated with isotype control Ab and second layer. DQ expression assessed by each Ab was then expressed as a percentage of the highest DQ expressing cell line and an average taken of the three Abs to generate a single value, for each cell line, of DQ expression compared with the highest expressing cell line in the assay. To correct the amount of SPEA binding by a cell line for its level of DQ expression, the measured SPEA binding was divided by the percentage of highest DQ expression and multiplied by 1000.

SAg-soluble HLA class II binding assay

ELISA plates (Merck, Poole, U.K.) were coated with L243 or SPV-L3. Wells were washed (PBS 0.1% Tween 20) and then blocked (PBS 1% BSA) for 1 h. Reaction tubes containing 30 µg/ml purified HLA class II and biotinylated SAg at a range of concentrations were incubated overnight at 4°C. Negative control tubes contained biotinylated SAg alone. Triplicates were then set up in the ELISA plate for 1 h at room temperature. Detection was with avidin-HRP (Jackson ImmunoResearch Laboratories, West Grove, PA) and tetramethylbenzidine (Sigma-Aldrich), stopped using 1 M H₂SO₄, and read absorbence read at 450 nM.

Binding of SPEA to DQ peptides

Twenty-one amino acid peptides representing the HLA-DQA1*01 -chain 53–73 (KFGGFDPQGALRNMAVAKHNL), a three substitution mutant F/G 61, T/R 64, and I/M 66 (Biosynthesis, Lewisville, TX), and two single substitution mutants F/G 61 and T/R 64 (Dr. R. Edwards, Imperial College, London, U.K.) were synthesized. SPEA binding to the peptides was assessed using peptide, or diluent (DMSO) alone, at concentrations from 1 mg/ml to 1 µg/ml to coat an ELISA plate and capture biotinylated SPEA.

Purified T cell stimulation assays

APCs were incubated with SPEA (1000 ng/ml) or culture medium alone for 40 min at 4°C, washed and fixed using 1% paraformaldehyde. Purified T cells were added at a range of cell concentrations. At 48 h cells were pulsed with 1 µCi [³H]thymidine. At 72 h cells were harvested using a Betaplate harvester (Wallac, Milton Keynes, U.K.)

Mouse splenocyte stimulation assays

APCs were incubated with mitomycin C (100 µg/ml) (Sigma-Aldrich) for 40 min at 37°C and washed. Splenocytes from DO11.10 TCR transgenic mice were incubated with mitomycin-treated APC lines (5 x 10⁴ cells/well) and SPEA (0–1000 ng/ml). Culture supernatants were harvested and [³H]thymidine incorporation assayed.

TCR repertoire usage assays

PBMCs from HLA typed donors were stimulated using SPEA or PHA, with recombinant human IL-2 (Sigma-Aldrich) 20 IU/ml added at 72 h. After 7 days, cells were harvested, stained with anti-CD4-FITC and anti-V-PE, and analyzed by FACS (FACSCalibur using CellQuest software; BD Biosciences). For each V, percentages of CD4-positive lymphocytes falling within a resting lymphocyte gate for unstimulated cells or a blasting-lymphocyte gate for stimulated cells were recorded, and pairwise comparisons made between unstimulated PHA and SPEA stimulated cells by t test. Values for p < 0.05 were considered significant.

	Results

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Binding of SPEA by B cell lines

Binding of SPEA to HLA-DQ expressing cells is almost entirely mediated through the -chain of HLA-DQ and not other class II determinants such as HLA-DR (15). To confirm this, we demonstrated that the Ab L2, which binds the DQ -chain, almost entirely blocked SPEA binding to B-LCLs whereas the DR-specific Ab L243 produced no detectable blocking (data not shown). If all HLA-DQ molecules were equal in their binding of SPEA, SPEA binding to B cell lines would be directly related to level of HLA-DQ expression. However, in six experiments correlating SPEA binding with level of DQ expression on a panel of cell lines expressing common HLA-DQ molecules, we found that HLA-DQ genotype markedly influenced SPEA binding. A representative experiment is shown in Fig. 1, a and b. SPEA binding to cell lines expressing HLA-DQA1*01 -chains was greater than binding to cell lines expressing DQA1*03 or DQA1*05, and SPEA binding to HLA-DQA1*01 expressing cell lines related closely to level of DQ expression, whereas cell lines expressing DQA1*03 or DQA1*05 bound SPEA poorly, despite comparable levels of DQ expression (Fig. 1a). When expressed as SPEA bound corrected for level of DQ expression (Fig. 1b), a statistically highly significant difference between SPEA binding to DQA1*01 and DQA1*03/05 expressing cell lines was found. Because variations in cell cycle and culture conditions can lead to fluctuations in the level of cell surface HLA expression, we analyzed the relationship between HLA-DQ expression and SPEA binding in several independent cultures of two DQA1*01 lines (PGF and WT46) and two DQA1*05 lines (IDF and TISI). Irrespective of minor fluctuations in HLA expression, the hierarchy of SPEA binding was maintained (Fig. 1, c and d), and again there was a statistically highly significant difference between the SPEA binding corrected for level of DQ expression of DQA1*01 expressing cell lines in comparison with the DQA1*05 expressing cell lines.

View larger version (27K): [in this window] [in a new window]   FIGURE 1. Binding of biotinylated SPEA to B cell lines expressing HLA-DQ. a and b, SPEA binding to a panel of B-LCLs expressing different HLA-DQ molecules. HLA-DQA1*01/DQB1*06 cell lines (•), HLA-DQA1*01/DQB1*05 cell lines (), HLA-DQA1*03/DQB1*03, or HLA-DQA1*05/DQB1*03 cell lines (), BLS cell line (). Cell lines used were BLS, 600sf, WT51, IDF, TISI, BEC11, HOM2, LWAGS, HOR, TOK, WT46, PGF, and SCHU, each numbered 1–13, respectively. c, Relationship between level of HLA-DQ expression and SPEA binding for individual cell lines: PGF (•), WT46 (), IDF (), TISI (). b and d, SPEA binding has been corrected for level of DQ expression, cell lines expressing HLA-DQA1*01 -chains (), cell lines expressing HLA-DQA1*03/05 -chains (), p < 0.05 and p < 0.01, respectively, by t test. e and f, SPEA binding corrected for level of DQ expression on BLS cell lines transfected to express different HLA-DQ molecules, cell lines expressing HLA-DQA1*01 -chains (), cell lines expressing HLA-DQA1*03 -chains (), p < 0.05 and p < 0.01, respectively, by t test.

Binding of SAgs to purified HLA class II by ELISA

To establish whether an effect of HLA class II polymorphism on SAg binding could be observed in a cell-free system and extended more broadly to other bacterial SAgs, we designed an ELISA to compare SAg–HLA class II binding using purified HLA class II. The binding of SEA and SEB to HLA-DR4, HLA-DR11, and HLA-DR15 were assessed. SEA binding to HLA-DR is predominantly through the polymorphic DR -chain. Binding to HLA-DR4 and -DR15 was markedly greater than to HLA-DR11 (Fig. 2a), indicating that just as HLA-DQ polymorphisms influence SPEA binding to HLA-DQ, HLA-DR polymorphisms influence SEA binding to HLA-DR. In contrast, SEB binding to HLA-DR is through the nonpolymorphic DR -chain. We found that accordingly, differences in binding of SEB by different HLA-DR molecules were small (Fig. 2b). Using HLA-DQ purified from the same cell lines, we compared SPEA binding to HLA-DQ6.2 (HLA-DQA1*01 -chain) with HLA-DQ3.1 (HLA-DQA1*05 -chain). In keeping with the results from whole cell binding experiments, binding of SPEA by purified HLA-DQ6.2 was markedly superior to binding by purified HLA-DQ3.1 (Fig. 2c). SEA-HLA-DR binding is dependent on the presence of zinc. Performing the binding assay in the presence of 1 mM EDTA, as would be predicted, completely blocked binding of this SAg to purified HLA-DR15 (Fig. 2d). SPEA binding to the HLA-DQ -chain has been predicted by analogy with SEB to be independent of the presence of zinc, although a zinc-binding pocket has been identified in the crystal structure of SPEA (23, 24). SPEA-DQ binding was however not altered in the presence of 1 mM EDTA (Fig. 2e) confirming that like the SEB-DR interaction, SPEA-DQ binding is zinc independent.

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Binding of bacterial SAgs to purified HLA class II by ELISA. SEA (a) and SEB (b) binding by HLA-DR; HLA-DR4 (•), HLA-DR15 (), HLA-DR11 (), no DR control (). c, SPEA binding by HLA-DQ; HLA-DQ6.2 (•), HLA-DQ3.1 (), no DQ control (). d, Influence of zinc chelation on binding of SEA to HLA-DR15 (•) and no DR control (). e, Influence of zinc chelation on binding of SPEA to HLA-DQ6.2 (•) and no DQ control (). Points joined by a dashed line show zinc chelation using 1 mM EDTA. Points show mean OD ± 1 SD.

View this table: [in this window] [in a new window]   Table I. Sequence alignment of the bacterial SAg binding domains of HLA-DQ -chainsa

View larger version (20K): [in this window] [in a new window]   FIGURE 3. Binding of SPEA to DQA1*01 (53–73) peptide (•) and DQA1*01 (53–73)3S mutant () and control (). Points show mean OD ± 1 SD.

The ability of high and low SPEA-binding HLA-DQ molecules to support SPEA activation of T cells was assessed using three approaches. The aim of the first approach was to control maximally for variability attributable to donor T cell differences and the confounding effect of T cell HLA class II expression. This was achieved by using HLA homozygous B-LCLs to present SPEA to murine TCR V8.2 transgenic responder T cells, mV8.2, with this being one of the murine V families targeted in the response to SPEA. APCs expressing DQA1*01 were found to support higher levels of response to SPEA than were APCs expressing DQA1*05, assessed by proliferation or by TNF-, IFN-, or IL-4 (Fig. 4, a–e). Furthermore, the lowest concentration of SPEA associated with detectable T cell activation was two orders of magnitude lower in the presence of DQA1*01 expressing APCs than of DQA1*05 APCs, despite comparable levels of HLA-DQ expression. Elevated TNF- levels are of central importance in the lethality of SAg-mediated shock (26). It is noteworthy therefore that, within the likely in vivo SPEA concentration range (<100 ng/ml), there was an absolute difference in the TNF- response, with DQA1*05 APCs eliciting no TNF- release.

View larger version (29K): [in this window] [in a new window]   FIGURE 4. Influence of HLA-DQ polymorphism on murine TCR V8.2 transgenic T cell proliferation and cytokine response to SPEA. a–d, Presentation by B-LCLs PGF (DQA1*0102/DQB1*0602), IDF (DQA1*0501/DQB1*0301), and BLS (no DQ control). e–h, Presentation by HLA-DQ transfected BLS lines DQA1*0102/DQB1*0602, DQA1*0301/DQB1*0302, no DQ control. a and e, Proliferation in response to presentation of SPEA by DQ6.2 (•), DQ3.1 (), and no DQ control (). b–d and f–h, Cytokine response to presentation by DQ6.2 (), DQ3.1 (), and no DQ control (). Mean ± 1 SD is shown in each case.

View larger version (27K): [in this window] [in a new window]   FIGURE 5. Influence of HLA-DQ on magnitude of proliferation in response to SPEA. Proliferation of purified T cells to SPEA presented by B-LCLs. PGF (DQ6.2), IDF (DQ3.1), BLS (DQ negative). Two levels of stimulation are shown. *, p < 0.01 by t test.

View larger version (15K): [in this window] [in a new window]   FIGURE 6. Proliferation of PBMCs from HLA typed donors. HLA-DQA1*01 homozygous donors (•) HLA-DQA1*03/05 donors (), n = 3 in each group. Means ± 1 SD are shown. Background counts were 150–300 cpm and not statistically different between groups.

Influence of HLA-DQ polymorphism on V repertoire of SPEA-stimulated T cells

SPEA has been previously reported to produce changes in the proportions of V12- and V14-positive lymphocytes (27, 28). To assess impact of HLA-DQ polymorphism on the V repertoire of the T cell response to SPEA, we stimulated PBMCs from donors homozygous for either HLA-DQA1*01 or for HLA-DQA1*03/05 using either PHA or SPEA. The proportion of CD4-positive lymphocytes binding Abs to 21 different V types was assessed before and after stimulation. In three experiments, expansion of V12 and V14 was noted following SPEA stimulation, relative to V percentages of unstimulated or PHA-stimulated PBMCs irrespective of donor HLA type. However in HLA-DQA1*01 homozygous donors, an additional 2-fold expansion of V13.1 CD4 cells was found (p = 0.026 by t test). Changes for these and four representative other Vs are shown in Fig. 7. PHA stimulation produced no changes in the V repertoire compared with unstimulated cells (data not shown) and no differences between donor groups were observed following SPEA stimulation for the other sixteen Vs assessed.

View larger version (20K): [in this window] [in a new window]   FIGURE 7. Influence of donor HLA type on V repertoire of T cells proliferating in response to 100 ng/ml SPEA. HLA-DQA1*01 homozygous (), HLA-DQA1*03/05 homozygous (), prestimulation (hatched bars), and following stimulation (filled bars) are shown. A total n = 3 were in each group, and means ± 1 SD are shown. Expansion of V12 and V14 occurs following SPEA stimulation irrespective of donor HLA-DQA1 genotype whereas only T cells from HLA-DQA1*01 donors show expansion of V13.1. Values for p < 0.05 are by t test.

	Discussion

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Our observation that SPEA binding by cell lines expressing DQA1*01 -chains is a factor of the level of DQ expression whereas SPEA binding to cell lines expressing DQA1*03 or DQA1*05 -chains is virtually undetectable under the conditions used, indicates that DQ -chain polymorphisms influence the binding of this SAg to HLA-DQ. The sites at which SPEA interacts with the HLA-DQ -chain have been predicted from the DR1-SEB crystal structure and the structure of the DQ -chain binding streptococcal SAg (25). Comparison of the amino acid sequences of the DQ -chains encoded by the polymorphic DQA1 gene demonstrates that those -chains coded by DQA1*01 and its variants are virtually nonpolymorphic at sites of SPEA binding, whereas the -chains encoded by DQA1*03 or DQA1*05 share three amino acid substitutions at sites of SPEA binding: F/G 61, T/R 64 and I/M 66 (Table I). Constraints on HLA-DQ heterodimer formation mean that the great majority of HLA-DQ5 and HLA-DQ6 molecules comprise a DQA1*01 -chain paired with a DQB1*05 or DQB1*06 -chain (32), as do all the DQ5 and DQ6 cell lines used in this study. Conversely the DQ3 expressing cell lines used in this study all comprise an -chain encoded by DQA1*03 or DQA1*05 paired with a DQB1*03 -chain. Our observation of poor SPEA binding by HLA-DQA1*03/*05 cell lines compared with HLA-DQA1*01 cell lines, irrespective of level of DQ expression, is likely therefore to be due to DQ -chain polymorphism at sites of SPEA binding.

Using purified HLA class II and peptide fragments of the DQ -chain in ELISAs of SAg–class II binding, we have confirmed that HLA-DQ polymorphisms influence SPEA binding in isolation from other cell surface factors and antigenic peptide. Furthermore we have demonstrated that HLA class II polymorphisms influence the binding of at least one other important bacterial SAg, namely SEA. It is likely, therefore, that HLA class II polymorphisms influence the properties of many, if not all, SAgs.

The differences observed in magnitude of proliferation and cytokine response to SPEA presented by different HLA-DQ alleles were to some extent quantitative, following the trend observed for differences in binding. However, it is noteworthy that qualitative differences were also found. At lower concentrations of SAg such as might be encountered pathologically during sepsis, we observed an absolute difference between strong and weak binding alleles in the presence or absence of a TNF- response, particularly because high TNF- levels are thought to underlie the lethality of toxic shock (26).

Although V-specific T cell expansion is considered to be a hallmark of superantigenicity, definition of the V signature of individual SAgs is complicated by the fact that SAg activation may be variably associated with T cell proliferation or deletion and apparent loss of SAg responsive V types. Previous studies have reported principally V12 and V14 changes associated with SPEA stimulation (27, 28). It is noteworthy that V13 is the closest relative of V12 in humans (33). Our observation of an additional expansion of V13.1, seen only in HLA-DQA1*01 homozygous individuals, is in keeping with previous observations that the nature of the class II SAg interaction determines V specificity. In particular, an effect of mouse MHC class II isotype on the response of V-specific T cell hybridomas has been reported (34) and SAgs with mutations at class II binding sites show both altered T cell mitogenicity and V specificity (35). It is not surprising therefore that V-specific changes following SAg stimulation are determined, at least in part, by intra-isotype HLA class II polymorphisms.

Our findings demonstrate that allelic differences can account for both quantitative and qualitative differences in the SAg response. They therefore offer a molecular explanation for why individuals vary in their susceptibility to toxic shock syndrome during the course of infection by toxigenic organisms. They reinforce and greatly extend the observation by Kotb et al. (18) of an association between HLA haplotype and susceptibility to SAg-mediated manifestations of S. pyogenes infection. Because Kotb and coworkers were using epidemiological data and clinical isolates, they were not able to focus on specific SAgs and elucidate the mechanisms at work. The HLA class II haplotypes they identified contrast with those identified in this study. Kotb found HLA DQA1*0102/DQB1*0602 for example to be protective against the severest manifestations of infection. This is likely to be because the patients in Kotb’s study will necessarily have been infected with different S. pyogenes strains each carrying multiple different SAg genes (19). The association identified may therefore relate to other SAgs such as SMEZ. Kotb et al. (19) demonstrated that HLA class II haplotypes associated with severe disease were also associated with greater proliferation of PBMCs and purified T cells stimulated in the presence of HLA homozygous B-LCLs. However the stimuli used in these experiments were partially purified culture supernatants containing an undetermined range of SAgs. Again therefore, no specific relationship between the SAg and the class II involved in its presentation could be studied. Kotb et al. (19) speculate that the association between class II haplotype and susceptibility may be through modification of the inflammatory response to the SAg. By focusing on individual SAgs and performing detailed binding analyses, the work presented in this study demonstrates that differences in the response to a SAg relate rather to differences in class II binding determined by HLA class II polymorphisms.

In the autoimmunity field, disease associations with HLA-DQ have been both striking and perplexing in light of the relatively poor expression of HLA-DQ by APCs and the fact that HLA-DQ-restricted T cells are not commonly isolated in most systems (36). This pattern of HLA-DQ association with responses to a pathogenic bacterial SAg argues that the list of HLA-DQ associations can now be extended from autoimmunity to bacterial immunity and septic shock. HLA genotype is likely to be a useful predictor of outcome during clinical toxic shock and the molecular interactions analyzed in this study offer clear potential for development of immunotherapeutic interventions.

It is intriguing that S. aureus and S. pyogenes, being distinctly the most pathogenic of the Gram-positive bacteria that infect man, are also distinct in their use of SAgs and have each evolved multiple SAg toxins. The findings we report suggest that host genetic heterogeneity may have played a role in driving the evolution of bacterial SAg diversity.

	Footnotes

 
¹ This work was supported by the Medical Research Council (U.K.) through a training fellowship (to M.L.).

² Address correspondence and reprint requests to Dr. Daniel M. Altmann, Human Disease Immunogenetics Group, Department of Infectious Diseases, Hammersmith Hospital, London W12 ONN, U.K. E-mail address: d.altmann{at}ic.ac.uk

³ Abbreviations used in this paper: SAg, superantigen; SPEA, streptococcal pyrogenic exotoxin A; SEA, staphylococcal enterotoxin A; SEB, staphylococcal enterotoxin B; B-LCL, B-lymphoblastoid cell line; MFI, mean fluorescence intensity; BLS; bare lymphocyte syndrome.

Received for publication June 23, 2003. Accepted for publication November 10, 2003.

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