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International Immunology Advance Access originally published online on August 7, 2006
International Immunology 2006 18(10):1433-1441; doi:10.1093/intimm/dxl076
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

The TCR Vß signature of bacterial superantigens spreads with stimulus strength

Martin Llewelyn1,2, Shiranee Sriskandan1, Nadia Terrazzini2, Jonathan Cohen2 and Daniel M. Altmann1

1 Department of Infectious Diseases, Faculty of Medicine, Imperial College London W12 0NN, UK
2 Division of Medicine, Brighton and Sussex Medical School, Medical Research Building, University of Sussex, Falmer BN1 9PS, UK

Correspondence to: M. Llewelyn; E-mail: m.j.llewelyn{at}bsms.ac.uk


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Superantigens (Sags) induce large-scale stimulation of T lymphocytes by a mechanism distinct from conventional antigen presentation, involving direct MHC binding and stimulation of TCR families based on Vß gene usage. The specific Vß targets of a given Sag have, since the earliest studies in murine models, been considered a hallmark of that toxin. Bacterial Sags are implicated in the aetiology of a wide range of human diseases, although their role has been most clearly defined in toxic shock syndrome. While Sags have been defined by the Vß-specific changes in T cell repertoire they induce, human studies of in vitro stimulation or analysis of cells from infected patients have produced inconsistent findings. Here we have evaluated the contribution of HLA allelic polymorphisms and strength of stimulus to this response. We show that there are differences in binding and presentation of the staphylococcal Sag, staphylococcal enterotoxin A (SEA), by different HLA-DR alleles. We also show that the TCR Vß response, previously thought to be a fixed property defining a given Sag, varies with stimulus strength such that a broader repertoire of response is seen at higher concentrations or following presentation by high-binding class II types. Responses of human Vß8 and Vß1 to SEA, Vß5 to SEB and of Vß12 and Vß13 to streptococcal pyrogenic exotoxin A are absolutely dependent on stimulus strength. These findings have important implications for heterogeneity in the response to Sags and the consequent differences in susceptibility to severe toxic shock.

Keywords: bacterial, human, superantigens, T cells, TCRs


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Superantigens (Sags) are powerful T cell stimulatory proteins that cross-link the variable regions of particular TCR ß chains (TCR Vß) with MHC class II molecules on the surface of antigen-presenting cells (APCs) (1, 2). T cell stimulation by Sag is characterized by Vß-specific changes in T cell repertoire (3). For example, in mice SEB stimulates T cells bearing Vß3, 7, 8.1, 8.2, 8.3 and 17, while in man Vß3, 12, 14 and 17 are targeted and in contrast toxic shock syndrome toxin (TSST)-1 stimulates Vß3, 10, 15 and 17 in mice and Vß2 in man (4–6).

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 (7). While the magnitude of T cell response may be important in toxic shock syndrome, qualitative differences in the TCR Vß-specific T cell response to Sag may also be important in the aetiology in diseases with a putative Sag aetiology, such as Kawasaki's disease, atopic eczema and autoimmunity (8–10). It is unclear why such heterogeneity in the response to Sag exposure should exist. Both genetic factors and factors related to the nature of Sag exposure are likely to be important. It was thought that MHC class II polymorphisms were unimportant with respect to responses to Sag compared with the presentation of conventional peptides bound in the groove of the heterodimer (11). However, Kotb et al. (12) recently showed an influence of HLA genotype on disease susceptibility to septic shock following infection with group A streptococci. A mechanism for these observations was provided by our demonstration that HLA class II polymorphisms can determine the magnitude of the T cell response to Sags (13). In the course of these experiments, we noted that HLA class II polymorphisms associated with greater T cell proliferation to a recombinant streptococcal Sag, streptococcal pyrogenic exotoxin A (SPEA), were associated with greater involvement of certain Vß types in the T cell response (13). We set out to study this effect in more detail specifically looking at how Sag stimulus strength impacts on the Vß-specific response. We looked at the influence of HLA class II in view of the HLA associations which exist for autoimmunity, Kawasaki's disease and toxic shock (8, 12, 14). We looked at the influence of concentration since during the course of streptococcal or staphylococcal infection, different compartments of the immune system may encounter widely ranging concentrations of Sag depending on the site of infection (15).

To widen the base of our earlier observations, we began by determining the influence of HLA-DR ß chain polymorphisms on the binding and presentation of the Sag staphylococcal enterotoxin A (SEA). Whereas SPEA is prototypic of a family of Sags which interact with the class II molecule through the {alpha} chain, SEA is prototypic of a family of Sags which interact with the class II molecule through both a high-affinity interaction with the class II ß chain and a lower affinity interaction with the {alpha} chain which allows cross-linking (16). Whereas SPEA is predominantly presented by HLA-DQ, SEA is predominantly presented by HLA-DR. First, we show that SEA binding is greater to HLA-DR4 and -DR15 than to DR11 and that T cell proliferation is greater in response to SEA presented by DR4 than DR11. For SEA, SEB and SPEA, a relationship exists between Sag concentration and Vß repertoire of T cell response such that the response is narrow at the lowest concentrations used and progressively broadens as concentration rises. Importantly though, the Vß specificity of response is preserved with only a small minority of Vß types responding even at the highest concentrations studied. For SEA and SPEA, which bind polymorphic regions of the class II molecule, we show that class II types modulate the effect of Sag concentration such that class II types associated with greater binding also support a broader range of Vß T cell response at any given Sag concentration. Thus, in the setting of human exposure to bacterial Sags, there is likely to be diversity in the TCR expansions, depending on HLA type and the exact nature of the exposure. This may explain heterogeneity in the immunological sequelae of Sag exposure and may explain the difficulty in finding consistent Vß-specific changes in T cell repertoire following clinical exposure to even individual Sags. Our findings also give additional insights into the purpose Sags serve in S. aureus and S. pyogenes.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Toxins
Recombinant SPEA was expressed in transformed Escherichia coli BL21 as described previously (15). Unconjugated and biotinylated purified SEA and SEB and biotinylated recombinant SPEA were purchased from Toxin Technology (Sarasota, FL, USA).

B cell lines
A panel of HLA class II homozygous B-lymphoblastoid cell lines (B-LCLs) was used for Sag-binding assays and for Sag presentation to T cells in proliferation assays (Table 1). An EBV-transformed bare lymphocyte syndrome cell line was used as a class II negative control (17).


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  		Table 1 B-LCL genotypes and serotypes

 
Preparation of human PBMCs and T cells
PBMCs were obtained by Ficoll gradient separation. Class II negative T cells were purified by negative selection using a method modified from Lavoie et al. (18). A total of 5 x 106 cells were incubated with L243 (anti-HLA-DR), WR18 (anti-pan-class II), Leu14 (anti-CD14) and Leu19 (anti-CD19), followed by two rounds of depletion using anti-mIg DynalTM 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 co-incubated with APCs.

Flow cytometric binding assays
A total of 5 x 105 cultured B cells were incubated with biotinylated SEA and washed and after binding were visualized using extravidin–PE (Sigma, Poole, UK), by FACS (Facscalibur, Cellquest software, Becton-Dickinson, Oxford, UK). A total of 20 000 cells falling within a healthy lymphocyte gate were analysed. SEA binding was measured as mean fluorescence intensity (MFI) of cells incubated with biotinylated SEA and extravidin–PE divided by MFI of cells incubated with unbiotinylated SEA and extravidin–PE. Level of HLA-DR expression was measured using the mouse mAb L243 which recognizes an epitope on the monomorphic DR {alpha} chain, and FITC-labelled anti-mouse Ig second layer. DR expression for each cell line was then expressed as a percentage of the highest DR-expressing cell line. To correct the amount of SEA binding by a cell line for its level of DR expression, the measured SPEA binding was divided by the percentage of highest DR expression and multiplied by 1000.

Purified T cell stimulation assays
To circumvent the possibility of Sag interactions with donor HLA class II expressed on activated T cells, the role of HLA class II in Sag presentation was studied as follows. B-lymphoblastoid cells were incubated in the presence of different concentrations of Sag, washed once and then fixed using 1% PFA. Purified T cells were then incubated with B-lymphoblastoid cells. Negative controls were fixed B-lymphoblastoid cells without Sag pre-incubation. At 48 h, wells were pulsed with 1 µCi of 3H-thymidine. At 72 h, cells were harvested using a betaplate harvester and radioactive incorporation measured by scintillation counting (Wallac, Milton Keynes, UK).

TCR repertoire usage assays
PBMCs from HLA typed donors were stimulated using Sag or PHA, with recombinant human IL-2 (Sigma, Poole, UK) 20 iu ml–1 added at 72 h. After 7 days, cells were harvested, stained with anti-CD4–FITC and anti-Vß–PE and analysed by FACS (Facscalibur, Cellquest software, Becton-Dickinson). For each Vß, percentages of CD4-positive lymphocytes falling within a resting lymphocyte gate for unstimulated cells or a blasting-lymphocyte gate for stimulated cells were recorded.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Influence of HLA-DR type on SEA binding
Binding of SEA to APCs is at the class II ß chain predominantly of HLA-DR but also HLA-DQ. Although other cell surface factors, particularly co-stimulatory pathways, are involved in T cell activation by Sags, binding at the cell surface is primarily mediated by HLA class II (1). Since DR expression is ~10-fold higher than DQ (19), observed binding to B-LCLs reflects predominantly binding to HLA-DR. Binding of SEA to HLA-DR was assessed using a panel of HLA class II homozygous B-LCLs (Fig. 1). Despite showing comparable and, in some instances, lower levels of DR expression, cell lines homozygous for HLA-DR4 and HLA-DR15 showed significantly greater SEA binding than cell lines expressing HLA-DR11 (Fig. 1A). When expressed as SEA bound corrected for level of DR expression (Fig. 1B), this difference was statistically significant.


Figure 1
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  		Fig. 1 Binding of biotinylated SEA to B cell lines expressing HLA-DR. Bindings of SEA 5 µg ml–1 to cell lines expressing HLA-DR4 (WT51, BOLETH, PRIESS) ({blacksquare}), HLA-DR11 (IDF, TISI, SWEIG) ({circ}), HLA-DR15 (PGF, TOK, SCHU) ({diamondsuit}) and a class II negative cell line (bare lymphocyte syndrome) (•) are shown (A). SEA binding corrected for level of DR expression is also shown (B) to allow statistical analysis. P values comparing binding to HLA-DR4 or HLA-DR15 with HLA-DR11 by t-test, HLA-DR0401, P = 0.019; HLA-DR15, P = 0.002.

 
Influence of HLA-DR type on SEA presentation
The ability of high- and low-binding HLA-DR types to present SEA to purified HLA class II negative donor T cells was assessed. As was previously noted for SPEA, class II-Sag-binding differences were associated with differences in the magnitude of the T cell response such that high-binding HLA-DR types were associated with greater T cell proliferation than the low-binding HLA-DR11. Figure 2 shows a representative experiment in which T cells from a single donor are stimulated with SEA presented by HLA-DR4 or HLA-DR11 homozygous B-LCLs. Maximum levels of proliferation were approximately twice as high for SEA presented by HLA-DR4 as for presentation by HLA-DR11. SEA was around three log more potent a stimulator of proliferation in the presence of HLA-DR4 than in the presence of HLA-DR11. Levels of proliferation to SEA presented by DR15 were similar to DR4 and again greater than DR11 (data not shown). No differences in T cell proliferation were observed in either the absence of stimulus or the presence of PHA.


Figure 2
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  		Fig. 2 Influence of HLA-DR on proliferation of T cells in response to SEA. Mean responses of purified single donor T cells to SEA presented by HLA-DR4 homozygous APCs (WT51, BOLETH, PRIESS) (•) and homozygous APCs HLA-DR11 (TISI, IDF, SWEIG) ({circ}) are shown (±1SD).

 
Influence of Sag concentration on the Vß repertoire of the T cell response
Our observation that the magnitude of the T cell response to Sag is determined by its affinity for the HLA class II involved in presentation caused us to question whether Sag stimulus strength might determine the Vß repertoire of response. We approached this question first by determining whether Sag concentration influences the Vß response. We studied a range of Sags exemplifying different modes of interaction with HLA class II, SPEA (which binds to the polymorphic DQ {alpha} chain), SEB (which binds the non-polymorphic DR {alpha} chain) and SEA (which binds to the DR {alpha} and ß chains).

Looking first at SEB and SPEA responses, in view of our observation that HLA class II influences responses to SPEA (13), HLA-DQ3 homozygous donors were used. For SEB, the same donors were used but since SEB interacts with HLA class II through the non-polymorphic HLA-DR {alpha} chain, an influence of HLA class II haplotype would not in any case be expected.

For each Sag, three or more separate analyses were performed using three or more donors. In every case, it was observed that the TCR Vß repertoire of response depended on Sag concentration. Data from representative studies of T cell Vß repertoire response to SEB and SPEA are shown in Fig. 3. PHA stimulation produced no changes in T cell repertoire compared with unstimulated cells (data not shown). At the lowest Sag concentrations, a narrow Vß repertoire of response was seen. At higher concentrations, the repertoire was expanded to include additional TCR Vß types. This was not a generalization of the Sag response to include all Vßs since the Vß repertoire of responding T cells remained markedly skewed towards a few Vß types. Since proportions of the total T cell repertoire are measured, the proportions of those Vßs, which respond at the lowest concentrations, fall as concentration rises and other Vß types are drawn into the response. The Vß3 and Vß17 responses to SEB in Fig. 3(A) show this. Because the response to Sags is Vß specific, non-responsive Vß types are essentially absent from blasting lymphocytes analysed by FACS. Vß types which are absent from the response at low concentrations but appear in the response at high concentrations are therefore Sag-responsive Vß types even though they may at a particular concentration be only present at frequencies comparable with PHA-stimulated cells. The Vß13 response to SPEA in Fig. 3(B) would be an example. Such Vßs are nevertheless clearly Sag responsive since their contribution to the total population of T cell blasts has increased with Sag concentration. Some of the Vß responses noted have not been described before, for example, the response of Vß5 to SEB and Vß3 to SPEA. Furthermore, responses of some established TCR Vß targets were sometimes lost at the lowest concentrations used. For example, at the lowest concentrations of SPEA used, expansion of only Vß14 and not Vß12 was observed.


Figure 3
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  		Fig. 3 Vß-specific changes in CD4 T cell repertoire following Sag stimulation. The response to SEB for eight Vß types is shown (A). Characteristic skewing of the Vß repertoire towards Vß3, 17, 12 and 14 is seen at the lowest concentrations used. As concentration rises, the contribution of these Vß types to the total CD4 response falls as other Vßs, notably Vß5, become drawn into the response. Vßs 8 and 13.1 remain absent at even the highest concentrations used here. The response to SPEA for seven Vß types is shown (B). At the lowest concentration used, only a Vß14 response is apparent. As concentration rises, Vß12, 13.1 and 3 are sequentially drawn into the response and the contribution of Vß14 T cells to the total repertoire diminishes. Other Vß types remain absent from the response at the highest concentrations used. Control responses to PHA stimulation are also shown in each case. Bars show means (n = 4) ± 1SD.

 
Looking next at SEA responses, the Vß-specific response to SEA involves multiple Vß types; Vß1, 5, 8, 9, 16 and 22 have all been suggested as targets (20). Since HLA-DR homozygous donors are uncommon, it is very difficult to find multiple donors of comparable HLA-DR type to study the influence of SEA concentration on Vß-specific response in the way used for studying SPEA and SEB responses in Fig. 3. For these reasons, the relationship between SEA concentration and Vß repertoire of the response to SEA is shown for each studied donor separately. A representative experiment is shown in Fig. 4. Exactly the same relationship between Vß response and Sag concentration was found as for SEB and SPEA. At the lowest concentration at which a Vß-specific effect is apparent (1 pg ml–1), a response of only Vß22 is observed. At higher concentrations, Vß9, 8, 1 and 5.1 are drawn into the response.


Figure 4
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  		Fig. 4 Influence of SEA dose on Vß-specific changes in T cell repertoire. Data from 10 Vß types are shown for two individuals in panels (A) and (B). At the lowest concentration for which a Vß-specific change is apparent, a response of only Vß22 is observed. As concentration rises, other Vßs are drawn into the response sequentially, Vß9, 8, 1 and 5.1. Other Vß types are absent from the response at the irrespective on SEA concentration.

 
Influence of HLA class II on the Vß T cell response
We previously reported that Vß13.1 responses to SPEA could be detected in HLA-DQA1*01 homozygous donors but not in HLA-DQA1*03/05 homozygous donors. Here we have shown that involvement of Vß13.1 in response to SPEA is, in part, related to concentration or stimulus strength (Fig. 3). To explore this relationship in more detail, we observed the Vß-specific T cell response of purified T cells to SPEA or SEA presented by HLA homozygous B-LCLs. In each case, presentation by HLA types associated with high binding (DQA1*01 in the case of SPEA and DR4 in the case of SEA) was associated with Vß responses which were not seen when Sag presentation was by low-binding HLA types (DQA1*05 in the case of SPEA and DR11 in the case of SEA). Specifically, in the case of SPEA, Vß13.1 and Vß3 responses are seen at 500 ng ml–1 SPEA in the presence of HLA-DQA1*01 but not DQA1*05. In the case of SEA, while Vß5 is absent from the T cell response at both 1 and 10 pg ml–1 SEA, the proportion of Vß9 T cells is increased above base line when presentation is by DR4 but not DR11 (Fig. 5).


Figure 5
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  		Fig. 5 Influence of HLA class II on Vß-specific T cell response to SPEA or SEA. Selected Vßs are shown. For SPEA, presentation is by either HLA-DQA1*01 homozygous APCs (PGF, TOK, WT46) (•) or HLA-DQA1*05 homozygous APCs (IDF, TISI) ({circ}). For SEA, presentation is by either HLA-DR4 homozygous APCs (WT51, BOLETH, PREISS) ({blacksquare}) or HLA-DR11 homozygous APCs (IDF, TISI, SWEIG) ({square}). Error bars show ±1SD.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Since the immunological behaviour of the bacterial Sags was first characterized by the laboratories of Kappler, Marrack and others several years ago, there has been a growing appreciation of the role played by Sag-induced T cell activation in a number of human disease states including toxic shock, Kawasaki's disease and autoimmunity (2, 9, 21, 22). Our current findings help considerably in terms of explaining why disease processes associated with Sag exposure are so unpredictable and heterogeneous in comparison to the results of experimental studies.

Our observations of differential SEA binding and presentation by HLA-DR types extend our earlier observations regarding SPEA presentation by HLA-DQ. Furthermore, they have allowed us to undertake a detailed analysis of variations in Vß repertoire following exposure to three different Sags (SEA, SEB and SPEA) which exemplify different modes of interaction with HLA class II. Sags interact with the class II molecule in a variety of configurations but utilize what are essentially only two binding sites (20). One, a high-affinity interaction site, is focused on a highly conserved histadine residue at position 81 of the class II ß chain but also includes sites which are polymorphic, for example, position 70 (23). The other, lower affinity site on the class II {alpha} chain is polymorphic in HLA-DQ but not in HLA-DR (24). SEA interacts at both these sites but the ß chain site is most important (25). SPEA interacts with the DQ {alpha} chain site and SEB interacts with the DR {alpha} chain site (26). Our observations suggest that most, if not all, interactions between Sags and HLA class II are likely to be subject to the influence of HLA class II polymorphism. Analysis of the DR ß chain sequence suggests that polymorphism at ß70 may explain differential SEA binding. The DR15 and DR4 (DRB1*0401) ß chains have glutamine at this position, while DR11 has aspartate at position 70 (27). An alternative explanation for our observations relating to SEA, but not SPEA or SEB binding, is that differences in antigenic peptide are responsible. An influence of peptide on binding of SEA and TSST-1 has been clearly demonstrated (18, 28, 29). The crystal structures of these Sags in complex with class II demonstrate interactions with peptide (30, 31). However, an influence of peptide on SEB, and by inference SPEA, has been sought and not demonstrated (32). This is in keeping with the crystal structure of the SEB–HLA-DR complex which shows no interaction with peptide (26). In any event, since antigenic peptide presentation is HLA class II restricted, the potential role of antigenic peptide does not detract from the importance of the observation that HLA class II polymorphisms influence Sag presentation.

Our observation, here and previously (13), that HLA class II polymorphisms associated with greater Sag binding are also associated with greater magnitude of T cell response leaves open the question, what is the mechanism behind differences in the magnitude of response? The observations presented here provide a plausible mechanism. We have shown that a wider range of T cell Vß types are amenable to stimulation by a Sag in the context of HLA class II polymorphisms associated with higher binding. Thus, the proportion of the total T cell repertoire amenable to stimulation will evidently be greater. Our findings are in keeping with a previously reported relationship between the affinity of Sag-class II interactions and the Vß-specific T cell response. In that study, Kotb et al. used mutations of SEA targeted at the class II {alpha}- and ß-chain-binding sites. Altered T cell mitogenicity and Vß-specific changes in T cell repertoire were observed (33).

Whether or not a T cell responds to Sag stimulation is likely to be determined by the overall characteristics of the trimolecular TCR-Sag-class II interaction (34). This hypothesis is supported by our observation here that both higher Sag concentration and higher Sag-class II-binding affinity are associated with widening repertoire of Vß response. Although contamination of purified Sags with minute concentrations of other Sags could plausibly explain the observed widening of Vß response at high concentrations, this is not the case for our observations concerning SPEA which was made with recombinant toxin purified from E. coli. Furthermore, this would not explain the marked narrowing of Vß repertoire at the lowest concentrations used. Rather it seems to us that the T cell response is determined by stimulus strength which will be determined by characteristics of the Vß region and the class II involved and the concentration at which the Sag is acting. The Vß type targeted by SPEA and SEB in the mouse is mVß8.2 and the sites at which SPEA and SEB interact with this Vß region have been precisely defined (35, 36). The sites overlap extensively and this undoubtedly explains the overlapping Vß specificities of these Sags. The SPEA interaction involves more hydrogen bonds with more side-chain atoms than does the SEB interaction. Thus, the SPEA interaction with TCR is likely to be more dependent on TCR Vß amino acid sequence and this probably explains the narrower repertoire of the Vßs response to SPEA (36). Comparison of the TCR Vß amino acid sequence at sites of SPEA interaction identified by Mariuzza et al. is very much in keeping with our observed hierarchy of Vß responsiveness; Vß14, 12, 13, 3 (35–37). For example, E94 of SPEA forms a hydrogen bond with the side chain of N28 in the mVß8.2 TCR Vß region. The majority of human TCRs has a glycine at this position and hydrogen bond with SPEA would not form (38). Among human Vßs, Vß14, 12 and 13 have N28 while Vß3 uniquely has D28 which should allow hydrogen bond formation with E94 of SPEA (38).

We are aware of six previous reports of the human Vß targets of SPEA (14, 39–43). Many of these studies were conducted before recombinant SPEA was available and when only limited panels of mAbs to different Vß types were available. Vß12 and Vß14 are consistently reported as targets of SPEA in these papers. Vß2 and Vß8 were identified in some early papers as targets of SPEA probably because of contamination of purified SPEA by other Sags. These Vß types share none of the Vß amino acid sequence features of mVß8.2 which are involved in SPEA binding. Of these six reports, only two looked at Vß13 and Vß3. Fleischer et al. (42) used T cell hybridomas-expressing human Vßs of these types and found no responses to SPEA at up to 1 µg ml–1 using RAJI cells (DQA1*0501) for antigen presentation. Their failure to observe response of these Vß types is therefore in keeping with our observation of Vß3 and Vß13 responses only above this concentration and in the presence of HLA-DQA1*01-expressing APCs. Yoshioka et al. (14) reported Vß repertoire changes in PBMCs stimulated by SPEA at 1 µg ml–1 and found Vß13 responses in six out of nine donors of unknown HLA type; Vß3 was not studied.

A Vß-specific change in T cell repertoire is a sine qua non of superantigenicity. Such changes are often regarded as evidence that a disease has a Sag aetiology; Kawasaki's disease is a prime example of this. The observation of variable changes in Vß repertoire in Kawasaki's disease has been interpreted as meaning that the disease may be the common end-point of exposure to one of several different Sags (9). Attempts to establish HLA associations with Kawasaki's have largely yielded negative results (44, 45). However, the recently reported HLA association in patients with Kawasaki's disease characterized by Vß2 and Vß6 changes and associated with streptococcal pyrogenic exotoxin C-producing strains of S. pyogenes is in keeping with the idea of HLA-associated Sag responses (14). In toxic shock syndrome, attempts to define the Vß-specific changes seen in clinical samples have produced conflicting results. Choi et al. found expansion of Vß2 T cells, while Mitchie et al. found reduction of Vß2 T cell numbers in patients with toxic shock. Differences in these studies could have arisen from differences in the timing of sampling or from different Sags being present (46, 47). An alternative explanation for these observations is that Vß-specific changes are not a fixed property of an individual Sag but determined by factors including concentration at the site of T cell activation and the HLA context in which the Sag is acting.

Infection by Sag toxin-producing strains of S. pyogenes and S. aureus is followed by toxic shock syndrome in only a small proportion of cases. Furthermore, staphylococcal toxic shock characteristically follows superficial infections while streptococcal toxic shock only rarely follows superficial infections and Kawaskai's disease appears to follow exposure to these same toxins in the nasopharynx (22). The data presented here explain how both genetic factors and the nature of exposure contribute to different outcomes following Sag exposure. Additionally, these data show how we must be mindful of HLA class II and the nature of exposure in searching for Vß-specific changes in T cell repertoire of patients with putative Sag-mediated diseases.


   Acknowledgements
 
This work was funded through a Medical Research Council (UK) Fellowship to Martin Llewelyn.


   Abbreviations
 
APC, antigen-presenting cells
B-LCL, B-lymphoblastoid cell line
MFI, mean fluorescence intensity
Sag, superantigen
SEA, staphylococcal enterotoxin A
SPEA, streptococcal pyrogenic exotoxin A
TSST, toxic shock syndrome toxin

   Notes
 
Transmitting editor: M. Feldman

Received 17 August 2005, accepted 11 July 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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