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The Journal of Immunology, 2007, 178: 3076-3083.
Copyright © 2007 by The American Association of Immunologists, Inc.

HLA Transgenic Mice Provide Evidence for a Direct and Dominant Role of HLA Class II Variation in Modulating the Severity of Streptococcal Sepsis1

Mohammed M. Nooh*,{dagger},{ddagger},§, Nagala El-Gengehi{dagger},{ddagger},§, Rita Kansal{dagger},{ddagger},§, Chella S. David and Malak Kotb2,*,{dagger},{ddagger},§

* Department of Molecular Sciences, {dagger} Department of Surgery, and {ddagger} Mid-South Center for Biodefense and Security, University of Tennessee Health Science Center, Memphis, TN 38163; § Research Center, Veterans Affairs Medical Center, Memphis, TN 38104; and Department of Immunology, Mayo Clinic, Rochester, MN 55905


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Our epidemiologic studies on invasive Group A Streptococci (GAS) infections identified specific HLA class II haplotypes/alleles conferring high-risk or protection from streptococcal toxic shock syndrome with a strong protection conferred by the DRB1*15/DQB1*06 haplotype. We used HLA-transgenic mice to provide an in vitro and in vivo validation for the direct role of HLA class II allelic variation in streptococcal toxic shock syndrome. When splenocytes from mice expressing the protective HLA-DQB1*06 (DQ6) allele were stimulated with a mixture of streptococcal superantigens (SAgs), secreted by the prevalent M1T1 strain, both proliferative and cytokine responses were significantly lower than those of splenocytes from mice expressing the neutral DRB1*0402/DQB1*0302 (DR4/DQ8) alleles (p < 0.001). In crisscross experiments, the presentation of SAgs to pure T cells from either the DQ6 or the DR4/DQ8 mice resulted in significantly different levels of response depending on the HLA type expressed on the APCs. Presentation by HLA-DQ6 APCs elicited significantly lower responses than the presentation by HLA-DR4/DQ8 APCs. Our in vitro data were supported by in vivo findings, as the DQ6 mice showed significantly longer survival post-i.v. infection with live M1T1 GAS (p < 0.001) and lower inflammatory cytokine responses as compared with the DR4/DQ8 mice (p < 0.01). The data presented here provide evidence for a direct role of HLA class II molecules in modulating responses to GAS SAgs and underscore the dominant role of HLA class II allelic variation in potentiating the severity of GAS systemic infections.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Group A streptococci (GAS)3 are strictly human pathogens that cause a wide spectrum of diseases ranging from mild skin infections and uncomplicated pharyngitis to very severe life-threatening infections, e.g., streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF) (1, 2). In genetically susceptible individuals, GAS infections can sometimes trigger autoimmune disorders such as rheumatic fever, rheumatic heart disease, glomerulonephritis, and possibly a number of neurological disorders (3, 4, 5, 6). Since the early 1980s, there has been a sharp resurgence of severe invasive GAS infections such as STSS and NF (2). GAS produce many virulence factors that contribute to their pathogenesis, but the streptococcal pyrogenic exotoxins (Spes), which belong to the family of microbial superantigens (SAgs), play a crucial role in STSS and NF (3, 7, 8, 9, 10). As SAgs, the Spes bind to both MHC class II and TCR molecules triggering very potent inflammatory cytokine responses, which in certain individuals leads to severe systemic diseases (SSDs) manifested by STSS and multiple organ failure (3, 10). Our previous studies have provided conclusive evidence that GAS SAgs play a pivotal role in the pathogenesis of severe invasive illnesses (7, 9, 10, 11); however, we noted that only a few individuals infected with SAg-producing GAS develop SSDs (10, 11, 12). One possible explanation for the differences in the severity of invasive disease is the fact that some GAS strains are more virulent than others. Another mutually nonexclusive possibility is that the disease outcome is modulated by host immunogenetic variations.

Although there are over 100 known serotypes of GAS, the resurgence of severe invasive infection coincided with the emergence or re-emergence of virulent strains belonging to ancient serotypes that have apparently acquired genetic material via horizontal transfer (13, 14). In particular, a specific subclone of the M1 strain that has disseminated globally, persisting as the most frequently isolated clone from clinical cases over the last 25 years seemed to have acquired two unique prophages, each encoding virulence factors that are not found in the ancestral M1 strain (14). However, we showed that the same M1T1 clonal strain causes starkly different manifestations in different individuals and was in fact isolated from cases with SSDs, STSS, NF, nonsevere invasive disease, as well as from individuals with uncomplicated pharyngitis (12). Similar findings are observed with other prevalent serotypes, e.g., the M3 serotype (15). These findings provided a compelling argument that, despite the resurgence of virulent strains, host factors must be contributing significantly to modulating the outcome of invasive GAS infections.

We and others (10, 16, 17) have shown that the magnitude of inflammatory responses triggered by the same SAg can vary considerably in vitro as well as in vivo in different responders. A logical explanation for these findings is that variations in certain host immunogenetic factors modulate responses to the SAgs and, accordingly, the severity of the systemic disease. Indeed, our epidemiological studies demonstrated a significant association between specific alleles of the HLA class II and the severity of invasive GAS infection (10). While the haplotype DRB1*15/DQB1*06 (DR15/DQ6) conferred strong protection from SSDs, the haplotype DRB1*14/DQB1*05 (DR14/DQ5) was associated with predisposition to SSDs and other haplotypes, e.g., DRB1*04/DQB1*0302 (DR4/DQ8) were neutral with respect to the disease outcome. We also showed that inflammatory responses to the M1T1 SAgs, in the presence of the protective haplotype, were much attenuated as compared with the high-risk and neutral haplotypes (10).

To provide an in vivo validation to these findings, we needed an appropriate animal model for SAg-mediated GAS pathogenesis. Animal models have contributed significantly to the knowledge of the mechanisms involved in Gram-positive bacterial toxic shock; however, regular mice are much less sensitive to SAg-mediated effects than humans (18, 19, 20). This difference relates, in part, to the lower binding affinity of SAgs to murine MHC class II as compared with human HLA class II (21). The advent of the HLA class II transgenic (HLA-tg) mice provided an opportunity to study the pathogenesis of GAS in a readily manipulated animal model with a sensitivity to SAgs similar to that of humans (22, 23, 24, 25, 26). Indeed, several studies have shown that HLA-tg mice mount potent responses to GAS SAgs (20, 23, 25, 27, 28).

In this study, we examined the in vitro and in vivo responses to GAS SAgs in HLA-tg mice expressing HLA-DQ6 (SSD protective allele) or HLA-DR4/DQ8 (SSD neutral alleles). The work presented here provides evidence that HLA class II allelic variation contributes directly to streptococcal invasive disease outcome and confirms our previous epidemiological findings of the protective effect of the HLA-DQ6 allele.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Transgenic mice

HLA-tg, MHC class II-deficient (H-2 Ab°) mice expressing human DR4/DQ8 and DQ6 alleles were generated in the laboratory of C. S. David (Mayo Clinic, Rochester, MN) (29, 30, 31). Mice were bred and maintained in accordance with the guidelines established by our institutional animal care committee. We confirmed the presence of the appropriate HLA class II transgenes by PCR-based genotyping using the following allele specific oligonucleotide primers: DQ6 sense 5'-AGG ATT TCG TGC TCC AGT TTA AGG CCA TG-3' and DQ6 antisense 5'-TCT GCA AGA TCC CGC GGA ACG CC-3'; DQ8 sense 5'-AGG ATT TGG TGT ACC AGT TTA AGG GCA T-3' and DQ8 antisense 5'-TGC AAG GTC GTG CGG AGC TCC AA-3'; and DR4 sense 5'-GTT TCT TGG AGC AGG TTA AAC A-3' and DR4 antisense 5'-CTG CAC TGT GAA GCT CTC AC-3'. We also confirmed expression of HLA class II molecules on mice splenocytes by flow cytometry using mAbs specific to HLA-DQ (FITC, Leu-10; catalog. no. 347453, BD Bioscience) or to HLA-DR (L227) followed after washing by FITC-goat F(ab')2 specific to mouse IgG (Accurate Chemical & Science). Analysis was conducted on a FACSCalibur (BD Biosciences).

Preparation of partially purified streptococcal SAgs from GAS culture supernatants

We used two representative invasive isolates of the clonal M1T1 strain to prepare a partially purified mixture of the native secreted streptococcal SAgs. Isolate 5622 (isolate A) was from a severe invasive case and isolate 6021 (isolate B) from a nonsevere invasive case. Previous studies determined that these two isolates are genetically indistinguishable, belonging to the global M1T1 strain (12). The bacteria were streaked on blood agar plates, subcultured in Todd-Hewitt broth supplemented with 1.5% yeast extract, and grown overnight at 37°C under static conditions. The recovered culture supernatants were mixed with ice-cold absolute ethanol (1/3 v/v) and incubated at –20°C overnight for complete precipitation of the secreted proteins, which include the SAgs. The precipitate was dissolved in distilled water containing 0.5 mM of the protease inhibitor PMSF and subjected to extensive dialysis against deionized water at 4°C. The dialysate was filter-sterilized with a 0.2-µm syringe filter (Sarstedt) and treated with polymyxin B agarose to remove any traces of endotoxin, as described previously (32). Aliquots of the dialyzed, LPS-depleted, and sterilized native mixture of M1T1 SAgs were stored at –20°C until used.

Generation of recombinant GAS SAgs

Recombinant SpeA2 (rSpeA2), rSmeZ1, rSpeG, and rSpeJ were expressed as histidine-tagged fusion proteins according to the manufacturers’ recommendations (Promega and Qiagen). Primer pairs used for the cloning were as follows: SpeA2 (672 bp-5'-G AGG CCT CAA CAA GAC CCC GAT C-3' forward and 5'-G AAG CTT ACT TGG TTG TTA GGT AGA CT-3' reverse), Smez1 (645 bp-5'G AGG CCT TTA GAA GTA GAT AAT AAT TC3' forward and 5'CA AAG CTT AGG AGT CAA TTT C3' reverse), SpeG (630 bp-5' T AGG CCT GAT GAA AAT TTA AAAG3' forward and 5'-C AAG CTT CTA GTG CGT TTT TAA-3' reverse), and SpeJ (630 bp-5'-G AGG CCT ATG AAA AGA ATA ATA AAA ACA A-3' forward and 5'-G AAG CTT ATT TAG TCC AAA GGT AA-3' reverse). The forward and reverse primers contained StuI and HindIII restriction sites, respectively. The amplified PCR products were purified, subcloned in pGEM-T Easy vector (Promega) and sequenced (University of Tennessee Health Science Center Molecular Resource Center). The correct cloned inserts were digested with StuI and HindIII, moved to the pQE30Xa vector (Qiagen), propagated in DH5{alpha} Escherichia coli cells, and subsequently used to transform M15 E. coli cells. rSAg protein expression was induced with 1 mM isopropyl-beta-D-thiogalactopyranoside for 3 h. Proteins in the bacterial cell lysate were resolved by SDS-PAGE, and the specific expression of each rSAg protein was determined by immunoblotting with the RGS-His HRP Conjugate kit (Qiagen) and specific rabbit polyclonal anti-Spe Ab. rSAgs were purified as His-fusion proteins using Ni-NTA Superflow according to manufacturer’s recommendations (Qiagen), and their purity was confirmed by resolving the eluted fractions on SDS-PAGE and silver staining to detect any contaminating proteins. The purified rSAgs were treated with polymyxin B agarose (Boehringer Mannheim) to remove any contaminating endotoxin. The activity and Vbeta specificity of our rSmeZ1 was found to be comparable to rSmeZ1 that was provided by Dr. T. Proft (University of Auckland, Auckland, New Zealand).

T cell proliferation assays

Spleens were aseptically removed from the HLA-tg mice and dispersed into single-cell suspensions. The cells were treated with NH4Cl RBC lysis buffer, washed three times in HBSS, and resuspended to 2 x 106cells/ml in RPMI 1640 medium with 1% (v/v) heat-inactivated FBS, 4 mM L-glutamine, 25 mM HEPES, 50 U/ml penicillin/50 µg/ml streptomycin, and 50 µM 2-ME (RPMI 1640 complete medium). The cells were seeded at 2 x 105/well into U-bottom 96-well microtiter plates (Costar) and stimulated with optimal dilutions/doses of 1) mixture of native secreted M1T1 SAgs, 2) individual rSAgs produced by the clonal M1T1 strain (0.1–100 ng/ml rSpeA2, rSpeG, rSpeJ, or rSmeZ1), or 3) Con A (1 µg/ml). After incubation for 72 h at 37°C in a 5% CO2 and 95% humidity, the cultures were pulsed for the final 6 h with 1 µCi/well [3H]thymidine (specific activity = 6.7 Ci/mmol; Dupont), harvested onto glass fiber filters, washed, and the [3H]thymidine uptake was measured by counting on a Matrix 97 direct ionization beta counter (Packard Instrument). All samples measurements were performed in triplicates, and the data were presented as mean cpm [3H]thymidine uptake ± SD. Each experiment was repeated at least three times.

Crisscross SAg presentation assays

Mouse APC-depleted T cells were purified from splenocytes by negative selection using the mouse pan T cell magnetic isolation kit and AutoMax according to the manufacturer’s instructions (Miltenyi Biotec). Pure APC-depleted T cells were cultured in RPMI 1640 complete medium, and their purity was confirmed physically by flow cytometry and functionally by the lack of response to stimulation by M1T1 SAgs. Experiments in which pure T (cultured alone without any APCs) cells showed a response to Con A or SAgs were discarded. We prepared APCs from each HLA-tg mouse strain by treating whole splenocytes with mitomycin C (Sigma-Aldrich) 50 µg/5 x 107 cell/ml for 20 min at 37°C, washing them five times with HBSS, and suspending them in RPMI 1640 complete medium. The efficacy of mitomycin C treatment was established by the nonresponsiveness of APCs to stimulation with Con A or M1T1 SAgs. Pure, APC-depleted T cells (1 x 105/well) from a specific HLA-tg mouse strain were cocultured in U-bottom 96-well microtiter plates, with either autologous APCs (5 x 104/well) or with the same number of APCs from the other HLA-tg mouse. Proliferation assays were conducted as described above.

In similar assays, pure human T cells were used as responders instead of the pure murine T cells. To prepare human T cells, we used Ficoll hypaque gradient centrifugation to obtain the PBMC from healthy donors’ blood. We purified the T cells from PBMC by one cycle of erythrocyte rosetting, followed by negative selection using the human pan T cell magnetic isolation kit and AutoMax (Miltenyi Biotec). The purity of the APC-depleted T cells was confirmed by flow cytometry and by the lack of response to PHA or the M1T1 SAgs as detailed above.

Cytokine measurement

Splenocytes were cultured as described under the proliferation assays (see above). Culture supernatants were collected from the individual wells after 24, 48, and 72 h of incubation, cleared of cells by centrifugation, and stored at –80°C for subsequent analysis. The cytokines IL-2, IFN-{gamma}, and TNF-{alpha} were simultaneously quantified using the mouse cytometric bead array kit (BD Pharmingen) according to the manufacturer’s instructions. A FACSCalibur (BD Biosciences) was used to determine the fluorescence intensity and the assay results were analyzed using the BD cytometric bead array software (BD Biosciences). The amounts of cytokines produced in picograms per milliliter were assessed by comparison to standards run in parallel with the test samples. Where human T cells were the responding population, we used the human cytokine Lincoplex kit (Linco Research).

Analysis of the TCR Vbeta repertoire by flow cytometry

Relative quantitative analysis of TCR Vbeta repertoire was conducted by flow cytometry using the mouse Vbeta TCR screening panel kit (BD Pharmingen). CD3-PE-Cy5 conjugate (BD Pharmingen) was used as an additional marker to enable proper gating on T cells only. Approximately 5 x 105 splenocyte/tube in 100 µl of PBS containing 1% BSA were stained with mAbs for different Vbeta according to the manufacturer’s instructions. Using a FACSCalibur flow cytometer (BD Biosciences), we gated on the CD3-PE-Cy5 cells and analyzed individual TCR Vbeta per tube. The data were analyzed using FlowJo software (Tree Star). A minimum of 30,000 cell events were acquired for each analysis.

In vivo infection studies

In preparation for infections, a representative isolate of the clonal M1T1 strain was grown for 17 h at 37°C without shaking in Todd-Hewitt broth supplemented with 1.5% yeast extract. The bacteria were washed twice in sterile PBS and diluted to the appropriate CFU/ml in PBS. DQ6 and DR4/DQ8 mice (n = 10) were infected with 5 x 106 CFU/mouse in 250 µl of bacterial suspension via the tail vein. Mouse survival was monitored twice daily over 7 days. We also assessed the systemic inflammatory responses elicited as a result of the GAS infection by collecting heparinized blood (~150 µl) at 24 h postinfection from the retro-orbital plexus. The levels of inflammatory cytokines TNF-{alpha} and IFN-{gamma} were determined as described above. Bacteremia was determined by plating serial dilutions on blood agar plates. For assessing bacterial load in the organs, infected mice were killed by CO2 asphyxiation, and bacteria were evaluated by preparing organs homogenates in PBS and plating 10-fold serial dilutions on blood agar plates. beta-Hemolytic colonies were counted after incubation at 37°C for 24 h.

Statistical analysis

Data are expressed as mean of triplicates or quadruplicates ± SD. Statistical differences were calculated on original values using the nonparametric Mann-Whitney U test. Kaplan-Meier survival curves were generated to compare mice survival following infection and the significant differences in survival curves were calculated by using the log-rank test. A value of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Differential in vitro response of HLA-tg mice splenocytes to M1T1 SAgs

To determine the effect of HLA class II variation on the response to GAS SAgs, spleen cells from mice expressing different HLA class II alleles were stimulated in vitro with partially purified mixture of native SAgs secreted by two different M1T1 isolates: isolate A from severe invasive STSS case and isolate B from nonsevere invasive case of mild bacteremia. Mice expressing the high-risk haplotype/alleles DR14/DQ5 are not yet available, and thus, we compared mice expressing the protective DQ6 allele to mice expressing the DR4/DQ8 alleles that are neither associated with protection nor with high risk for SSDs, i.e., neutral. As expected, the response to M1T1 SAgs from both isolates was comparable, reflecting the clonality of this strain. However, the proliferative response of splenocytes with DQ6 or DR15 alleles, both of which are present on the protective HLA class II haplotype in our epidemiological studies, was significantly lower (p < 0.001) than that of splenocytes expressing the neutral DR4 and/or DQ8 alleles (Fig. 1A). These differences were specific to the SAgs response inasmuch as the Con A responses were similar regardless the type of the HLA class II alleles expressed on mice splenocytes (Fig. 1A). Splenocytes from the non-tg background mice strains failed to respond to the M1T1 SAgs (data not shown).


Figure 1
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  		FIGURE 1. HLA allelic variation affects in vitro response to M1T1 SAgs. In vitro stimulation of spleen cells from various HLA-tg mice with M1T1 SAgs. A, Proliferative responses of the indicated HLA-tg mice to the native mixture of M1T1 SAgs. Spleen cells (2 x 105/well) were stimulated in triplicates by an optimal dilution (1/500) of the mixture of partially purified native SAgs secreted by M1T1 streptococcal isolate A from a severe invasive case, isolate B from a nonsevere invasive case, or by Con A (1 µg/ml). B, Cytokine responses of splenocytes from the indicated of HLA tg mice to native M1T1 SAgs mixture. DQ6 or DR4/DQ8 spleen cells were incubated with the SAgs mixture in triplicates, and at the indicated time points the culture supernatant was collected and analyzed for the production of IL2, TNF-{alpha}, and IFN-{gamma}. C, Proliferative response of splenocytes from DQ6 or DR4/DQ8 mice to individual or a mixture of rSAgs produced by the clonal M1T1 strain. Spleen cells (2 x 105/well) from DQ6 or DR4/DQ8 mice were stimulated with the indicated concentrations of individual rSAg (0.1–100 ng/ml) as well as a rSAg mixture. Individual rSAgs were mixed in equal amounts and 0.1–100 ng/ml of this mixture was used to stimulate mouse spleen cells. Spleen cells from the non-tg ancestral background mice were similarly stimulated with the same rSAgs mixture. Each point represents the mean of triplicate ± SD; shown is a representative of one from at least four independent experiments. **, p < 0.01, and ***, p < 0.001, based on all experiments using the Mann-Whitney U test.

 
In support of the proliferation data, splenocytes from the DQ6 mice produced significantly lower levels of TNF-{alpha}, IFN-{gamma}, and IL-2 than those from the DR4/DQ8 mice in response to the same M1T1 SAgs (Fig. 1B). Cytokine responses peaked at the 72 h; those of the DQ6 mice were 6-, 30-, and 60-fold lower than those of the DR4/DQ8 mice for TNF-{alpha} (p < 0.001), IFN-{gamma} (p < 0.01), and IL-2 (p < 0.001), respectively.

To further confirm that the response is specific to the M1T1 SAgs, we also compared the proliferative response of the splenocytes from the DR4/DQ8 and DQ6 mice to rSpeA2, rSpeG, rSpeJ, and rSmeZ1, individually as well as in a mixture. The pattern of the response to rSAgs mixture reflected that obtained with the native partially purified mixture of SAgs where the response of the DQ6 splenocytes was consistently and significantly less than that of DR4/DQ8 splenocytes and both responses were higher than the response of the non-tg background mice splenocytes (Fig. 1C). This was not related to differences in HLA allele expression inasmuch as flow cytometric analysis showed comparable expression of DQ6 and DQ8 (Table I). DR4 expression was lower than that of DQ6 and DQ8 because cDNA was used for the generation of the DR4-mice, and genomic DNA was used to create the DQ6 and DQ8 tg-mice (29, 30, 33).


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  		Table I. Percentages of splenocytes expressing respective HLA class II transgenes and the mean fluorescent intensity of the expressiona

 
The enhanced responsiveness of the DR4/DQ8 mice compared with DQ6 and DR15 mice was unrelated to expression of double vs single alleles because, as demonstrated in Fig. 1A, mice expressing DQ8 only were also significantly higher responders to the M1T1 SAgs as compared with DR15 and DQ6 mice.

Dominance of the effect of HLA allelic variation in potentiating M1T1 SAgs responses

The possibility that TCR Vbeta repertoire variability may be a major factor in the stark differences in responses between the various mice was addressed. We found the Vbeta repertoire in DQ6 and DR4/DQ8 to be quite comparable with few exceptions (Fig. 2). TCR Vbeta 5 was higher in the DQ6 mice while 10 and 14 were higher in the DR4/DQ8 mice but these elements are not specific to any of the SAgs produced by the M1T1 strain (34, 35). The expression of TCR Vbeta11 and 12, which are specific for SmeZ1, was higher in the DQ6 mice, yet these mice were low responders. Thus, we conclude that variation in the TCR Vbeta repertoire does not account for the enhanced responsiveness of DR4/DQ8 mice to M1T1 SAgs.


Figure 2
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  		FIGURE 2. Comparison of the TCR Vbeta repertoire of the DQ6 and DR4/DQ8 mice. Splenocytes from age-matched DR4/DQ8 and DQ6 tg mice (n = 3 mice/strain) were stained for CD3 and TCR Vbeta-specific fluorochrome-conjugated Abs and analyzed by flow cytometry. Each bar represents the mean ± SD of the percentages of cells expressing a specific TCR Vbeta subfamily within the CD3+ population.

 
To further confirm this, we performed crisscross SAg presentation assays, where pure T cells from the DQ6 or the DR4/DQ8 mice were stimulated with the same M1T1 SAgs, presented either by their autologous HLA class II-expressing APCs or by APCs from the other HLA-tg mouse. The presentation of the M1T1 SAgs by DQ6-expressing APC resulted in a 2-fold lower proliferative response than the presentation by the DR4/DQ8-expressing APCs (p < 0.01), regardless the source of the responding T cells. The dominant effects of the HLA class II allelic variation on the response was also reflected in TNF-{alpha}, IFN-{gamma}, and IL-2 responses, where in the presence of the DQ6 APCs there was, respectively, 2-fold (p < 0.01), 13-fold (p < 0.01), and 3-fold (p < 0.001) lower cytokine level compared with cultures containing the DR4/DQ8 APCs (Fig. 3B). These data demonstrate that T cells from the DQ6 mice are perfectly capable of mounting potent responses to the streptococcal SAgs, if presented by the DR4/DQ8 APCs, but that the magnitude of the response is governed mainly by the HLA class II allele type expressed on the presenting APCs.


Figure 3
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  		FIGURE 3. Cross-presentation of the M1T1 SAgs by APCs from DQ6 and DR4/DQ8 mice to each other’s T cells reveals that variation in HLA class II is the main controller of the magnitude of SAg. A, Proliferative responses. DQ6 (protective) and DR4/DQ8 (neutral) T cells were separated from their autologous APCs. The native mixture of M1T1 SAgs (at 1/500 dilution) was presented to the pure T cells (1 x 105/well) from DQ6 or DR4/DQ8 mice by mitomycin C-treated APCs (0.5 x 105/well) from either DQ6 or DR4/DQ8 mice. B, Cytokine response of pure T cells from DQ6 mice to native M1T1 SAgs presented by APCs from either DQ6 or DR4/DQ8 mice. The culture supernatants were collected at 24, 48, and 72 h culture and analyzed for of IL-2, TNF-{alpha}, and IFN-{gamma} production. Differences in cytokine levels are in support of the observed proliferative responses. The results shown represent one out of four separate experiments. *, p < 0.05, **, p < 0.01, and ***, p < 0.001, based on all experimental results using Mann-Whitney U test.

 
To further confirm the dominant role of class II allelic variation in modulating the SAg response, we investigated the effect of different human HLA class II alleles expressed by murine APCs on the SAg response of pure human T cells. Similar to the murine T cells, human T cells proliferative (Fig. 4A) and cytokine (Fig. 4B) responses to the same M1T1 SAgs were significantly (p < 0.01) lower in the presence of the DQ6 APCs as compared with the DR4/DQ8 APCs.


Figure 4
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  		FIGURE 4. Differential proliferative and cytokine responses of APC-depleted human T cells to native M1T1 SAgs presented by APCs from DQ6 or DR4/DQ8 mice. A, Pure human T cells (1 x 105/well) were stimulated with 1/500 dilution of M1T1 SAgs in the context of mitomycin C-treated APCs (0.5 x 105/well) from DQ6 or DR4/DQ8. B, Human T cells were stimulated in the same way as above and culture supernatants were collected at 24, 48, and 72 h culture, and analyzed for IL-2, TNF-{alpha}, and IFN-{gamma} expression. Each data set represents mean ± SD of triplicates. Shown are the results representing one of three independent experiments. *, p < 0.05, **, p < 0.01, and ***, p < 0.001, based on results from all experiments using Mann-Whitney U test.

 
HLA class II allelic variation directly modulates the severity of systemic disease and mouse survival following infection with M1T1 GAS

We have shown that individuals carrying the protective DR15/DQ6 alleles are significantly more protected against SSDs in invasive infection, compared with others infected with the same GAS strain, but who are not carrying those alleles (10). Therefore, it was important to compare in vivo responses of DQ6 and DR4/DQ8 mice and see if they are comparable to what we found in humans and whether the significant differences observed in our in vitro studies would translate to considerable biologic variation in vivo. Accordingly, we infected the HLA-tg mice i.v., with 5 x 106 CFU of the M1T1 bacteria and recorded their mortality over a period of 7 days. DR4/DQ8 mice started to succumb to the infection within 48 h with a mortality rate of 80% by day 4 after inoculation. By contrast, none of the DQ6 mice died during the observation period (Fig. 5A).


Figure 5
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  		FIGURE 5. A, In vivo differential susceptibility of HLA-tg mice to M1T1 GAS sepsis. (5 x 106 CFU) live M1T1 bacteria in 250 µl PBS were injected i.v. into DQ6 ({blacktriangleup}) or DR4/DQ8 ({blacksquare}) mice. Mice survival was monitored twice daily and death was recorded over 7 days. B, IFN-{gamma} and TNF-{alpha} levels in plasma of HLA-tg mice, 24h post infection. C, Bacterial load in the blood, liver, and spleen of DQ6 ({blacktriangleup}) or DR4/DQ8 ({blacksquare}) mice i.v. infected with 5 x 106 CFU bacteria. *, p < 0.05, and **, p < 0.01.

 
The differential susceptibility of the HLA-tg mice to infection with M1T1 bacteria was also reflected in the systemic levels of the inflammatory cytokines TNF-{alpha} and IFN-{gamma}. Plasma levels of TNF-{alpha} and IFN-{gamma} at 24 h after i.v., infection with M1T1 bacteria were significantly lower in the DQ6 mice than in the DR4/DQ8 mice (p < 0.01 and p < 0.05, respectively). In contrast to the significant difference in the survival and systemic inflammatory cytokines results, both mice strains had comparable bacteremia at 24 h postinfection as well as comparable bacterial load in the liver at 24 and 48 h postinfection (Fig. 5C). However, DR4/DQ8 spleens showed significantly higher bacterial load than those of DQ6 only at 24 h postinfection (p = 0.03), but there was no significant difference at the 48 h in both strains. These observations underscore the role of HLA class II as a major controller of the inflammatory cytokine response and hence the severity of invasive GAS infection.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The resurgence of invasive GAS infection pandemic in the 1980s, with aggressive manifestations such as STSS and NF, has been receiving much attention for the past 25 years (1, 2, 36, 37). Throughout this pandemic, the clonal M1T1 strain was the most frequently isolated strain from severe and nonsevere invasive cases (38, 39, 40). Among the many virulence factors produced by the bacteria, the streptococcal SAgs are mainly responsible for triggering potent inflammatory responses, which mediate the systemic effects seen in patients with STSS (3, 41, 42). Our previous work provided evidence that certain individuals are prone to be high responders to GAS SAgs, whereas others mount moderate or low inflammatory responses (10, 17). We also found that among patients who recovered from invasive GAS infections, those who are prone to be high responders to GAS SAgs in vitro were the ones who had developed severe invasive systemic disease and STSS and mounted very high inflammatory cytokine response during the acute infection, whereas those who developed mild invasive disease were consistently low responders. Differences between the high and low responders reflected host genetic variations that were found to be associated with the HLA class II locus. Specifically, the strongest association was with DRB1*15/DQB1*06 haplotype, which seemed to confer strong protection from the severe manifestations of the invasive disease (10). However, there was still a question of whether the observed association is directly related to HLA class II allelic variation or to other genes that are in linkage disequilibrium with these HLA haplotypes. In this study, we provide evidence for direct role of HLA class II allelic variation in modulating the severity of GAS sepsis and demonstrate the dominant effect of the protective alleles in attenuating responses to SAgs.

Although well-designed epidemiological association studies have an advantage of being conducted in patients with clear clinical outcomes, these studies are limited by the inability to design certain controlled experimentation to delineate disease pathogenesis. Despite limitations, animal models for diseases remain indispensable to address such drawbacks. Regular mice are generally not a model of choice for STSS because they respond poorly to SAgs, particularly to the streptococcal SAgs (18, 19, 20). To elicit appropriate responses in these mice, investigators have resorted to using doses of SAgs that are much higher than physiologic (43, 44), have augmented SAg effects by coadministering LPS (19, 45), or treated the mice with D-galactosamine to prolong the biologic half-life of cytokines by delaying their hepatic clearance (18). Whereas these models provided valuable information, these manipulations may not represent actual events of the human disease.

By contrast, the humanized HLA class II-tg mice have shown high sensitivity to bacterial SAgs; T cells from these mice respond vigorously to bacterial SAgs in a way similar to human T cells without any manipulation, suggesting a SAg presentation pattern comparable to that in humans (24, 25). Several studies have used HLA-tg mice to investigate immune responses to SAgs and SAg-producing bacteria (20, 24, 25, 26). We were particularly interested in HLA-tg mice carrying the HLA class II alleles associated with protection or high-risk for SSDs, as identified in our epidemiological study (10). However, as mentioned earlier, mice carrying the high-risk alleles are not yet available, so we compared responses to M1T1 SAgs of mice carrying the neutral alleles DR4 and/or DQ8 alleles to mice carrying the protective DQ6 allele.

Our previous studies showed that PBMCs isolated from healthy individuals or recovered from patients expressing HLA alleles associated with protection from STSS mounted significantly lower proliferative and cytokine responses to SAgs than PBMCs from individuals carrying neutral or high-risk alleles (10). In a nicely reproducible manner, the in vitro responses of HLA-tg mice splenocytes to M1T1 SAgs were markedly lower for the DQ6 mice than for the DR4/DQ8 mice. Also, in human invasive GAS infections, we showed that patients with SSDs who lack the DR15/DQ6 protective alleles mount significantly higher levels of inflammatory cytokine responses, in both acute and convalescent phase of the disease, than those with mild nonsevere disease (10, 17). We found the same pattern in the HLA-tg mice where splenocytes from the DQ6 mice mounted significantly lower levels of the inflammatory cytokines TNF-{alpha} and IFN-{gamma} in vitro and in vivo than those of the DR4/DQ8 mice. In addition, the DQ6 mice were highly resistant to GAS sepsis, whereas the DR4/DQ8 mice were susceptible and quite overwhelmed by the infection despite having comparable bacterial load in liver and spleen. These findings emphasize the overriding role of HLA class II in governing the outcome of invasive GAS infection by modulating the systemic inflammatory cytokine responses to SAgs. Additionally, the SAgs-crisscross presentation assays where APCs from one HLA-tg mouse were used to present the SAgs to T cells from the other mouse or to human T cells showed reproducibly and significantly lower proliferative and cytokine responses in the context of DQ6 APCs as compared with the DR4/DQ8 APCs. Thus, results from the HLA-tg mice paralleled our findings in humans and confirmed a direct and dominant role of HLA class II allelic variation in controlling the magnitude of response to streptococcal SAgs, with little or no impact of variability in host T cells on disease severity. The dominance of the protective effect of the DQ6 allele seen in humans was also confirmed in these HLA-tg mice.

Differences in proliferative and inflammatory cytokine responses to the M1T1 SAgs when presented by DR4/DQ8 as compared with DQ6 or DR15 alleles may be attributed to difference in binding affinities or to the ensuing cellular signals in the APCs and T cells. Our previous studies (46) indicated no correlation between binding affinity and response level. However, ongoing studies in our lab are addressing this possibility in greater depth as well as examining if differences in the stability of the trimolecular complex (composed of class II-SAg-TCR) taken as a whole may establish the overall strength of the response. Although our present study points to the HLA class II molecule as the principal controller of the SAg-induced response, we still cannot rule out the likelihood that associated polymorphism in other host genes could contribute to susceptibility to STSS.

These findings should facilitate our ongoing studies of the temporal events that take place in the pathogenesis of invasive GAS infection and will help us identify specific molecular and cellular mechanisms involved in regulating SAg responses in the presence of distinct HLA class II alleles in vivo.


   Acknowledgments
 
We thank Sarah Row, Dennis Carrigan, and Yin Su for excellent mouse breeding, husbandry, and typing and Michele Smart for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant AI40198-06 (to M.K.), U.S. Army Medical Research Acquisition Activity Grant W81XWH-05-1-0227 (to M.K.), and Research and Development Office, Medical Research Service, Department of Veterans Affairs (Merit Award) (to M.K.). Back

2 Address correspondence and reprint requests to Dr. Malak Kotb, University of Tennessee Health Science Center, 956 Court Avenue, Suite A-202, Memphis, TN 38163. E-mail address: mkotb{at}utmem.edu Back

3 Abbreviations used in this paper: GAS, Group A streptococci; NF, necrotizing fasciitis; SAg, superantigen; Spe, streptococcal pyrogenic exotoxin; SSD, severe systemic disease; STSS, streptococcal toxic shock syndrome; tg, transgenic. Back

Received for publication August 2, 2006. Accepted for publication December 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Stevens, D. L.. 1992. Invasive group A streptococcus infections. Clin. Infect. Dis. 14: 2-11. [Medline]
  2. Low, D. E., B. Schwartz, A. Mcgeer. 1998. The Reemergence of Severe Group A Streptoccal Disease: An Evolutionary Perspective ASM Press, Washington DC.
  3. Kotb, M.. 1995. Bacterial pyrogenic exotoxins as superantigens. Clin. Microbiol. Rev. 8: 411-426. [Abstract]
  4. Kotb, M.. 1995. Infection and autoimmunity: a story of the host, the pathogen, and the copathogen. Clin. Immunol. Immunopathol. 74: 10-22. [Medline]
  5. Guedez, Y., A. Kotby, M. El-Demellawy, A. Galal, G. Thomson, S. Zaher, S. Kassem, M. Kotb. 1999. HLA class II associations with rheumatic heart disease are more evident and consistent among clinically homogeneous patients. Circulation 99: 2784-2790. [Abstract/Free Full Text]
  6. Cunningham, M. W.. 2000. Pathogenesis of group A streptococcal infections. Clin. Microbiol. Rev. 13: 470-511. [Abstract/Free Full Text]
  7. Watanabe-Ohnishi, R., D. E. Low, A. McGeer, D. L. Stevens, P. M. Schlievert, D. Newton, B. Schwartz, B. Kreiswirth, M. Kotb. 1995. Selective depletion of Vbeta-bearing T cells in patients with severe invasive group A streptococcal infections and streptococcal toxic shock syndrome: Ontario Streptococcal Study Project. J. Infect. Dis. 171: 74-84. [Medline]
  8. Norrby-Teglund, A., R. Kaul, D. E. Low, A. McGeer, D. W. Newton, J. Andersson, U. Andersson, M. Kotb. 1996. Plasma from patients with severe invasive group A streptococcal infections treated with normal polyspecific IgG inhibits streptococcal superantigen-induced T cell proliferation and cytokine production. J. Immunol. 156: 3057-3064. [Abstract]
  9. Norrby-Teglund, A., P. Thulin, B. S. Gan, M. Kotb, A. McGeer, J. Andersson, D. E. Low. 2001. Evidence for superantigen involvement in severe group a streptococcal tissue infections. J. Infect. Dis. 184: 853-860. [Medline]
  10. Kotb, M., A. Norrby-Teglund, A. McGeer, H. El-Sherbini, M. T. Dorak, A. Khurshid, K. Green, J. Peeples, J. Wade, G. Thomson, B. Schwartz, D. E. Low. 2002. An immunogenetic and molecular basis for differences in outcomes of invasive group A streptococcal infections. Nat. Med. 8: 1398-1404. [Medline]
  11. Kaul, R., A. McGeer, A. Norrby-Teglund, M. Kotb, B. Schwartz, K. O’Rourke, J. Talbot, D. E. Low. 1999. Intravenous immunoglobulin therapy for streptococcal toxic shock syndrome–a comparative observational study. The Canadian Streptococcal Study Group. Clin. Infect. Dis. 28: 800-807. [Medline]
  12. Chatellier, S., N. Ihendyane, R. G. Kansal, F. Khambaty, H. Basma, A. Norrby-Teglund, D. E. Low, A. McGeer, M. Kotb. 2000. Genetic relatedness and superantigen expression in group A streptococcus serotype M1 isolates from patients with severe and nonsevere invasive diseases. Infect. Immun. 68: 3523-3534. [Abstract/Free Full Text]
  13. Cleary, P. P., D. LaPenta, R. Vessela, H. Lam, D. Cue. 1998. A globally disseminated M1 subclone of group A streptococci differs from other subclones by 70 kilobases of prophage DNA and capacity for high-frequency intracellular invasion. Infect. Immun. 66: 5592-5597. [Abstract/Free Full Text]
  14. Aziz, R. K., R. A. Edwards, W. W. Taylor, D. E. Low, A. McGeer, M. Kotb. 2005. Mosaic prophages with horizontally acquired genes account for the emergence and diversification of the globally disseminated M1T1 clone of Streptococcus pyogenes. J. Bacteriol. 187: 3311-3318. [Abstract/Free Full Text]
  15. Johnson, D. R., J. T. Wotton, A. Shet, E. L. Kaplan. 2002. A comparison of group A streptococci from invasive and uncomplicated infections: are virulent clones responsible for serious streptococcal infections?. J. Infect. Dis. 185: 1586-1595. [Medline]
  16. Herman, A., G. Croteau, R. P. Sekaly, J. Kappler, P. Marrack. 1990. HLA-DR alleles differ in their ability to present staphylococcal enterotoxins to T cells. J. Exp. Med. 172: 709-717. [Abstract/Free Full Text]
  17. Norrby-Teglund, A., S. Chatellier, D. E. Low, A. McGeer, K. Green, M. Kotb. 2000. Host variation in cytokine responses to superantigens determine the severity of invasive group A streptococcal infection. Eur. J. Immunol. 30: 3247-3255. [Medline]
  18. Miethke, T., C. Wahl, K. Heeg, B. Echtenacher, P. H. Krammer, H. Wagner. 1992. T cell-mediated lethal shock triggered in mice by the superantigen staphylococcal enterotoxin B: critical role of tumor necrosis factor. J. Exp. Med. 175: 91-98. [Abstract/Free Full Text]
  19. Blank, C., A. Luz, S. Bendigs, A. Erdmann, H. Wagner, K. Heeg. 1997. Superantigen and endotoxin synergize in the induction of lethal shock. Eur. J. Immunol. 27: 825-833. [Medline]
  20. Sriskandan, S., M. Unnikrishnan, T. Krausz, H. Dewchand, S. Van Noorden, J. Cohen, D. M. Altmann. 2001. Enhanced susceptibility to superantigen-associated streptococcal sepsis in human leukocyte antigen-DQ transgenic mice. J. Infect. Dis. 184: 166-173. [Medline]
  21. Fremont, D. H., W. A. Hendrickson, P. Marrack, J. Kappler. 1996. Structures of an MHC class II molecule with covalently bound single peptides. Science 272: 1001-1004. [Abstract]
  22. Taneja, V., C. S. David. 1999. HLA class II transgenic mice as models of human diseases. Immunol. Rev. 169: 67-79. [Medline]
  23. Unnikrishnan, M., D. M. Altmann, T. Proft, F. Wahid, J. Cohen, J. D. Fraser, S. Sriskandan. 2002. The bacterial superantigen streptococcal mitogenic exotoxin Z is the major immunoactive agent of Streptococcus pyogenes. J. Immunol. 169: 2561-2569. [Abstract/Free Full Text]
  24. DaSilva, L., B. C. Welcher, R. G. Ulrich, M. J. Aman, C. S. David, S. Bavari. 2002. Humanlike immune response of human leukocyte antigen-DR3 transgenic mice to staphylococcal enterotoxins: a novel model for superantigen vaccines. J. Infect. Dis. 185: 1754-1760. [Medline]
  25. Welcher, B. C., J. H. Carra, L. DaSilva, J. Hanson, C. S. David, M. J. Aman, S. Bavari. 2002. Lethal shock induced by streptococcal pyrogenic exotoxin A in mice transgenic for human leukocyte antigen-DQ8 and human CD4 receptors: implications for development of vaccines and therapeutics. J. Infect. Dis. 186: 501-510. [Medline]
  26. Roy, C. J., K. L. Warfield, B. C. Welcher, R. F. Gonzales, T. Larsen, J. Hanson, C. S. David, T. Krakauer, S. Bavari. 2005. Human leukocyte antigen-DQ8 transgenic mice: a model to examine the toxicity of aerosolized staphylococcal enterotoxin B. Infect. Immun. 73: 2452-2460. [Abstract/Free Full Text]
  27. Rajagopalan, G., K. Iijima, M. Singh, H. Kita, R. Patel, C. S. David. 2006. Intranasal exposure to bacterial superantigens induces airway inflammation in HLA class II transgenic mice. Infect. Immun. 74: 1284-1296. [Abstract/Free Full Text]
  28. Sriskandan, S., M. Ferguson, V. Elliot, L. Faulkner, J. Cohen. 2006. Human intravenous immunoglobulin for experimental streptococcal toxic shock: bacterial clearance and modulation of inflammation. J. Antimicrob. Chemother. 58: 117-124. [Abstract/Free Full Text]
  29. Bradley, D. S., G. H. Nabozny, S. Cheng, P. Zhou, M. M. Griffiths, H. S. Luthra, C. S. David. 1997. HLA-DQB1 polymorphism determines incidence, onset, and severity of collagen-induced arthritis in transgenic mice. Implications in human rheumatoid arthritis. J. Clin. Invest. 100: 2227-2234. [Medline]
  30. Taneja, V., N. Taneja, M. Behrens, S. Pan, T. Trejo, M. Griffiths, H. Luthra, C. S. David. 2003. HLA-DRB1*0402 (DW10) transgene protects collagen-induced arthritis-susceptible H2Aq and DRB1*0401 (DW4) transgenic mice from arthritis. J. Immunol. 171: 4431-4438. [Abstract/Free Full Text]
  31. Cheng, S., J. Baisch, C. Krco, S. Savarirayan, J. Hanson, K. Hodgson, M. Smart, C. David. 1996. Expression and function of HLA-DQ8 (DQA1*0301/DQB1*0302) genes in transgenic mice. Eur. J. Immunogenet. 23: 15-20. [Medline]
  32. Norrby-Teglund, A., R. Lustig, M. Kotb. 1997. Differential induction of Th1 versus Th2 cytokines by group A streptococcal toxic shock syndrome isolates. Infect. Immun. 65: 5209-5215. [Abstract]
  33. Szanto, S., T. Bardos, Z. Szabo, C. S. David, E. I. Buzas, K. Mikecz, T. T. Glant. 2004. Induction of arthritis in HLA-DR4-humanized and HLA-DQ8-humanized mice by human cartilage proteoglycan aggrecan but only in the presence of an appropriate (non-MHC) genetic background. Arthritis Rheum. 50: 1984-1995. [Medline]
  34. Wilson, R. K., E. Lai, P. Concannon, R. K. Barth, L. E. Hood. 1988. Structure, organization and polymorphism of murine and human T cell receptor {alpha} and beta chain gene families. Immunol. Rev. 101: 149-172. [Medline]
  35. Proft, T., J. D. Fraser. 2003. Bacterial superantigens. Clin. Exp. Immunol. 133: 299-306. [Medline]
  36. Stevens, D. L.. 1996. Invasive group A streptococcal disease. Infect Agents Dis. 5: 157-166. [Medline]
  37. Bryant, A. E., C. R. Bayer, J. D. Huntington, D. L. Stevens. 2006. Group A streptococcal myonecrosis: increased vimentin expression after skeletal-muscle injury mediates the binding of Streptococcus pyogenes. J. Infect. Dis. 193: 1685-1692. [Medline]
  38. Cleary, P. P., E. L. Kaplan, J. P. Handley, A. Wlazlo, M. H. Kim, A. R. Hauser, P. M. Schlievert. 1992. Clonal basis for resurgence of serious Streptococcus pyogenes disease in the 1980s. Lancet 339: 518-521. [Medline]
  39. Cockerill, F. R., III, K. L. MacDonald, R. L. Thompson, F. Roberson, P. C. Kohner, J. Besser-Wiek, J. M. Manahan, J. M. Musser, P. M. Schlievert, J. Talbot, et al 1997. An outbreak of invasive group A streptococcal disease associated with high carriage rates of the invasive clone among school-aged children. J. Am. Med. Assoc. 277: 38-43. [Abstract/Free Full Text]
  40. Muotiala, A., H. Seppala, P. Huovinen, J. Vuopio-Varkila. 1997. Molecular comparison of group A streptococci of T1M1 serotype from invasive and noninvasive infections in Finland. J. Infect. Dis. 175: 392-399. [Medline]
  41. Schlievert, P. M.. 1993. Role of superantigens in human disease. J. Infect. Dis. 167: 997-1002. [Medline]
  42. Bernal, A., T. Proft, J. D. Fraser, D. N. Posnett. 1999. Superantigens in human disease. J. Clin. Immunol. 19: 149-157. [Medline]
  43. Marrack, P., M. Blackman, E. Kushnir, J. Kappler. 1990. The toxicity of staphylococcal enterotoxin B in mice is mediated by T cells. J. Exp. Med. 171: 455-464. [Abstract/Free Full Text]
  44. Proft, T., S. L. Moffatt, C. J. Berkahn, J. D. Fraser. 1999. Identification and characterization of novel superantigens from Streptococcus pyogenes. J. Exp. Med. 189: 89-102. [Abstract/Free Full Text]
  45. Stiles, B. G., S. Bavari, T. Krakauer, R. G. Ulrich. 1993. Toxicity of staphylococcal enterotoxins potentiated by lipopolysaccharide: major histocompatibility complex class II molecule dependency and cytokine release. Infect. Immun. 61: 5333-5338. [Abstract/Free Full Text]
  46. Norrby-Teglund, A., G. T. Nepom, M. Kotb. 2002. Differential presentation of group A streptococcal superantigens by HLA class II DQ and DR alleles. Eur. J. Immunol. 32: 2570-2577. [Medline]



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