Responses to Bacterial Superantigens HLA Class II Polymorphisms Determine
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The Journal of Immunology 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. Altmann2* 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. The Journal of Immunology, 2004, 172: 1719–1726. uperantigens (SAgs)3 are potent immunostimulatory pro- of all T cells. On the TCR, SAgs bind at the -chain variable (V) teins that bind the MHC class II and TCR molecules on the region. Individual SAgs are limited in the V families they stim- S surface of APCs and T lymphocytes (1, 2). The range of ulate (12) and produce a marked skewing of the V repertoire in human diseases with possible SAg etiology includes Kawasaki’s responding T cells known as the V signature. On the MHC class disease (3), psoriasis (4), atopic eczema (5), rheumatic fever (6), II molecule, SAgs adopt four principal binding modes. Some bind Crohn’s disease (7), and sepsis (8). The best-characterized role for the ␣-chain, some the -chain, and some crosslink class II either SAgs in human disease remains that of the SAg exotoxins of by binding both ␣- and -chains or by virtue of two -chain bind- Staphylococcus aureus and Streptococcus pyogenes, which are be- ing sites. SAgs that adopt the same mode of binding do not nec- lieved to trigger the staphylococcal and streptococcal toxic shock essarily compete, suggesting more subtle differences exist within syndromes (9, 10). However, the observation that apparently iden- these binding modes (13). tical toxigenic strains of S. pyogenes or S. aureus cause clinical Mice mount relatively weak in vitro and in vivo responses to syndromes ranging from superficial carriage through pharyngitis to bacterial SAgs compared with humans, a fact that is believed to toxic shock syndrome, suggests that genetic heterogeneity contrib- arise from sequence differences between human and mouse MHC utes to the clinical phenotype following SAg exposure (11). class II, with the mouse class II binding bacterial SAgs poorly Shared structural features allow SAgs to bind, as unprocessed (14). Furthermore, within human HLA class II, differences be- proteins, to the MHC class II molecule and the TCR at sites away tween isotypes in presentation of individual SAgs are also estab- from conventional Ag binding sites, thereby activating up to 20% lished. 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 † *Department of Infectious Diseases, Faculty of Medicine, Imperial College, Depart- streptococcal pyrogenic exotoxin A (SPEA) preferentially use ment of Immunology, Guy’s, Kings and St. Thomas’ School of Medicine, London, United Kingdom; ‡Vaccine Research Center, National Institute of Allergy and Infec- HLA-DQ (15). However, during the early characterization of su- tious Diseases, National Institutes of Health, Bethesda, MD 20892; §Virginia Mason perantigenicity, it became clear that a hallmark of this effect was Research Center, Seattle, WA 98101; and ¶Division of Medicine, Brighton and Sus- sex Medical School, Brighton, United Kingdom the lack of classical MHC restriction. This led historically to a Received for publication June 23, 2003. Accepted for publication November focus on the similarities rather than differences in SAg binding by 10, 2003. different HLA alleles, and with the exception of the observation The costs of publication of this article were defrayed in part by the payment of page that SEA and staphylococcal enterotoxin E bind to DRw53 par- charges. This article must therefore be hereby marked advertisement in accordance ticularly poorly (16), the influence of differences within class II with 18 U.S.C. Section 1734 solely to indicate this fact. isotypes has not been studied in detail. 1 This work was supported by the Medical Research Council (U.K.) through a training fellowship (to M.L.). The worldwide resurgence of streptococcal toxic shock syn- 2 drome since the 1990s has been associated with circulation of Address correspondence and reprint requests to Dr. Daniel M. Altmann, Human ϩ Disease Immunogenetics Group, Department of Infectious Diseases, Hammersmith novel spea strains of S. pyogenes (17). An epidemiological link Hospital, London W12 ONN, U.K. E-mail address: [email protected] between HLA haplotype and susceptibility to the SAg-associated 3 Abbreviations used in this paper: SAg, superantigen; SPEA, streptococcal pyrogenic manifestations of S. pyogenes infection was recently demonstrated exotoxin A; SEA, staphylococcal enterotoxin A; SEB, staphylococcal enterotoxin B; B-LCL, B-lymphoblastoid cell line; MFI, mean fluorescence intensity; BLS; bare by Kotb et al. (18). However, because the patients in the Kotb and lymphocyte syndrome. colleagues study were necessarily infected by different S. pyogenes Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 1720 HLA CLASS II POLYMORPHISM AND BACTERIAL SAgs strains, each carrying multiple different SAg genes (19), it is im- followed by two rounds of depletion using anti-mouse Ig Dynal beads possible to know from such clinical data whether the HLA asso- (Dynal Biotech, Oslo, Norway) according to manufacturer’s instructions. ciation relates to an individual SAg, and if so, to which SAg. This Depletion of all HLA class II expressing cells was confirmed by failure of purified T cells to proliferate in response to SAg stimulation unless coin- makes it difficult to elucidate molecular mechanisms at the level of cubated with APCs. SAg-HLA class II-TCR interactions. In this work we have set out to study in detail the relationship Flow cytometric binding assays between HLA class II polymorphism and the presentation of bac- A total of 5 ϫ 105 cultured B cells were incubated with biotinylated SPEA terial SAg focusing primarily on SPEA as a protype SAg. Because and washed, then binding visualized using Extravidin-PE (Sigma-Aldrich, SPEA binds HLA-DQ specifically and not HLA-DR or HLA-DP Poole, U.K.) by FACS (FACSCalibur using CellQuest software; BD Bio- (15) we have been able to use HLA homozygous B lymphoblastoid sciences). A total of 20,000 cells falling within a healthy lymphocyte gate cell lines (B-LCLs) to screen a range of HLA-DQ molecules for were analyzed. SPEA binding was measured as mean fluorescence inten- differences in SPEA binding. The data presented in this study dem- sity (MFI) of cells incubated with biotinylated SPEA and Extravidin-PE divided by MFI of cells incubated with unbiotinylated SPEA and Extra- onstrate up to 10-fold higher levels of SPEA binding to cells ex- vidin-PE. Level of HLA-DQ expression was measured using Abs recog- pressing HLA-DQ ␣-chains encoded by the gene HLA-DQA1*01 nizing different conserved regions of the DQ molecule Leu10 (DQ1 and than to cells expressing HLA-DQA1*03 or *05 ␣-chains. In ex- DQ3), L2 (pan DQ␣), and SPV-L3 (pan DQ). Binding of each DQ mAb periments using purified HLA class II, we have extended these 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- observations to other staphylococcal SAgs. In addition these data mouse Ig second layer divided by MFI of cells incubated with isotype demonstrate that the magnitude of the T cell response to SPEA is control Ab and second layer. DQ expression assessed by each Ab was then determined by HLA-DQ polymorphisms both in terms of prolif- expressed as a percentage of the highest DQ expressing cell line and an eration and cytokine response. Furthermore the V signature of average taken of the three Abs to generate a single value, for each cell line, SPEA is itself determined by the HLA-DQ involved in its presen- 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 tation to T cells. DQ expression, the measured SPEA binding was divided by the percentage Although previous studies have demonstrated that HLA class II of highest DQ expression and multiplied by 1000. isotypes differ in presentation of individual SAgs, these are the first data to demonstrate that HLA class II polymorphisms determine SAg-soluble HLA class II binding assay both the magnitude and the quality of the T cell response to SAgs. ELISA plates (Merck, Poole, U.K.) were coated with L243 or SPV-L3. The data provide a plausible mechanism for observed interindi- Wells were washed (PBS 0.1% Tween 20) and then blocked (PBS 1% vidual differences in disease phenotype following infection by tox- BSA) for 1 h.