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Superantigens – powerful modifiers of the immune system
Reviews
MOLECULAR MEDICINE TODAY, MARCH 2000 (VOL. 6)
Superantigens – powerful modifiers of the immune system John Fraser, Vickery Arcus, Philip Kong, Edward Baker and Thomas Proft
Superantigens are powerful microbial toxins that activate the immune system by binding to class II major histocompatibility complex and T-cell receptor molecules. They cause a number of diseases characterized by fever and shock and are important virulence factors for two human commensal organisms, Staphylococcus aureus and Streptococcus pyogenes, as well as for some viruses. Their mode of action and variation around the common theme of over-stimulating T cells, provides a rich insight into the constant battle between microbes and the immune system. SUPERANTIGENS (SAGs) are microbial toxins that target the immune system causing massive T-cell activation, cytokine release and systemic shock. They represent some of the most potent bioactive molecules ever discovered. Their mechanism of action has been the subject of intense study over the past decade and has led to speculation that they play a hidden role in promoting autoimmune disease and altering the normal immune responses to viral infections such as HIV1–3. Despite wide variation in their protein structure, SAGs all share a common ability to simultaneously bind the class II major histocompatibility complex (MHC-II) molecules expressed on professional antigen presenting cells (APCs) and the variable region of the T-cell receptor b-chain (TCR Vb). The result is massive stimulation of any T cell that expresses the correct TCR Vb element on its surface (Fig. 1). The complexities of SAG structure and function, and the variety of binding mechanisms that have evolved for different SAGs, indicate that, for some microbes at least, SAGs are an important mechanism for survival. John Fraser PhD* Associate Professor in Molecular Medicine Vickery Arcus PhD Research Fellow Philip Kong MSc Graduate Student Edward Baker PhD Professor of Structural Biology Thomas Proft PhD Research Fellow School of Biological Sciences, Department of Molecular Medicine, University of Auckland, Private Bag, 92019, Auckland, New Zealand. Tel: 164 9 373 7599 ext. 6036 Fax: 164 9 373 7674 *e-mail: [email protected]
Bacterial superantigens The best studied SAGs are the family of staphylococcal enterotoxins (SE) and streptococcal pyrogenic exotoxins (SPEs) secreted by the Gram-positive bacteria Staphylococcus aureus and Streptococcus pyogenes commonly found on the skin, the nose and upper respiratory tract of humans4,5. These toxins are potent virulence factors in diseases caused by these bacteria, but their exact function in the life cycle of either S. aureus or S. pyogenes is still uncertain. The addition of femtogram quantities of purified toxin to a culture of human peripheral blood lymphocytes results in the activation and proliferation of T cells that express a restricted number of TCR Vb domains. Staphylococcal enterotoxin A (SEA), for example, stimulates T cells bearing Vb1, 5.3, 6.3, 6.4, 6.9, 7.4, 9.1 and 23, whereas staphylococcal enterotoxin E (SEE), which differs in amino acid sequence from SEA by only 20%, stimulates a different but overlapping group of T cells bearing Vb1, 5.1, 6.3, 6.4, 6.9, 7.4 and 8.1 (Ref. 6). Approximately 20% of all T cells are activated by SEA. In contrast, normal peptide antigens only stimulate between 0.001% and 0.0001% of T cells because recognition is dependent on both the variable and junctional segments (D and J) of the TCR a and b-chains. This Vb-restricted expansion is the characteristic hallmark of all SAGs. No-one has yet found a SAG that stimulates every T cell or one that binds to the other side of the TCR – the Va domain. For many years, only six staphylococcal (SEA, SEB, SEC, SED, SEE and TSST) and two streptococcal (SPE-A and SPE-C) toxins were known. This number has increased considerably in recent years with the availability of S. aureus and S. pyogenes genome sequencing databases. The number of staphylococcal and streptococcal SAGs now stands at 18 (Fig. 2) and is likely to grow even further as the search for homologous sequences among these databases continues. Moreover, other unrelated bacterial SAGs have also been confirmed from Yersinia pseudotuberculosis and Mycoplasma arthritidis, and viral SAGs include the mouse mammary tumour virus (MMTV) products plus potential SAGs from cytomegalovirus (CMV) and Epstein–Barr virus (EBV) (Table 1). Similarity in amino acid sequences between the staphylococcal and streptococcal SAG family members varies between 20% and 90% and it is clear they have all evolved from a common ancestral gene (Fig. 2). The SEA gene resides on a variable genetic element7 whereas the spe-a gene from S. pyogenes
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T cell
Activation Signal transduction cascade
IL-1β
TCR SEA
TNF-α
IL-2 MHC-II IFN-γ
Macrophage
Molecular Medicine Today
Figure 1. SAGs stimulate high levels of cytokines. The interaction of the MHCII molecule with the TCR regulates the production of an array of cytokines from both the T cell and the antigen presenting cell (APC), in this case a macrophage. Normally, an antigen is processed by the APC into small peptides that are presented on the surface in the distal end of the MHC-II molecule. Only about 1 in 10 000–100 000 T cells possesses a TCR that binds strongly enough to stimulate the T cells. With SAGs, however, as many as 1 in 10 T cells are engaged. This results in a greater production of cytokines, particularly IL2, TNF-a, IFN-g and IL-1 from the APC. This massive release of cytokines causes the acute illness of toxic shock, which is often fatal. Why some SAGs are more efficient at causing toxic shock is unknown but about 70% of all cases appear to arise from the production of the SAG toxic shock syndrome toxin – a Vb2-stimulating toxin from a small site of infection.
is located on streptococcal phage T12 and is found in about 30% of all streptococcal isolates.
Superantigens from other bacteria Unrelated SAGs have also been isolated from Mycoplasma arthritidis and Yersinia pseudotuberculosis, and both have been confirmed by testing the recombinant products in T-cell stimulation assays. M. arthritidis is a rodent pathogen that causes inflammatory infection and chronic joint disease in rats and mice. Although the M. arthritidis mitogen (MAM) has been known for many years, research on this SAG has been hindered by difficulties in its isolation and stability. The MAM gene was finally cloned in 1996 and did not show any significant homology to the staphylococcal or streptococcal SAGs (Ref. 8). MAM is a single 25 kDa protein that preferentially binds to one isotype of the MHC class II protein: murine I-E or its human equivalent HLADR – with DR4, DR7 and DR12 subtypes presenting MAM most efficiently. Little is known about its structure and mechanism of action, but a recent study on the TCRs that respond to MAM indicates that it 126
is likely to contact not just the germ-line encoded TCR b-chain but also the complementarity determining region 3 (CDR3) that is generated through somatic mutation9. This interesting finding reveals for the first time that some SAGs might have a much more restricted TCR repertoire and thus might not leave the obvious Vb ‘footprint’ seen for the prototypic staphylococcal and streptococcal SAGs. This raises the question of how many more semi-restricted SAGs exist in nature that can alter the immune response without producing the visible signs of massive T-cell activation, cytokine release and shock that accompany an acute poisoning by one of the prototype staphylococcal or streptococcal SAGs. The Y. pseudotuberculosis-derived mitogen (YPM) is a small 21 kDa protein that specifically targets human Vb13.1 and -13.2 bearing TCRs (Ref. 10). Like MAM, it shows no significant homology to other SAGs, and its role in the pathogenicity of Y. pseudotuberculosis is unclear.
The structure of superantigen molecules reveals a variety of binding mechanisms Perhaps one of the most intriguing findings has been that individual bacterial SAGs have evolved quite different mechanisms of binding to MHC-II molecules despite a highly conserved structural fold. The crystal structures of SEA (Ref. 11), SEB (Ref. 12), SEC2 (Ref. 13), SED (Ref. 14), TSST (Ref. 15) and SPEC (Ref. 16) reveal a common core-fold based on two globular domains: a C-terminal domain of the b-grasp motif and a smaller N-terminal pseudo b-barrel domain (Fig. 3). The crystal structures of SEB–HLA-DR1 and TSST–DR1 complexes reveal similar but not identical binding mechanisms17,18. Staphylococcal enterotoxin B (SEB) has a single, low-affinity MHCII binding site located in the smaller N-terminal domain. Residues in this region bind to a hydrophobic groove located in the distal region of the invariant a1 domain of HLA-DR (Fig. 3). The region of SEB that binds to the TCR is a shallow groove between the two domains. SEB contacts both the CDR2 and the fourth hypervariable loop (HV4) of the TCR b-chain19. Thus, SEB can be likened most simply to a wedge that fits snugly between the TCR and MHC-II molecule with the TCR and MHC-II bound together, although this analogy is not entirely accurate. A recent comparison of several crystal structures reveals that SEB is likely to tip and rotate the TCR away from its normal position on the MHC-II molecule so that entirely new contacts must be accommodated from those determined during thymic selection20. The crystal structure of TSST–DR1 reveals a different binding mechanism17 (Fig. 3). Although TSST binds to the same region of DRa1 domain as SEB, it covers most of the top of the MHC-II molecule preventing any further contact with the TCR. Unlike SEB, activation by TSST is likely to rely entirely on the affinity of TSST–TCR binding with no contribution from MHC-II–TCR contacts. TSST also makes contact with residues from the bound peptide that limits the number of MHC-II molecules that TSST can bind and also raises the intriguing possibility that different peptides might regulate the potency of SAG-mediated T-cell activation21. A third type of MHC-II binding occurs with a subset of the bacterial SAGs that contain a high affinity zinc-binding site in the C-terminal domain on the opposite side of the molecule to the low affinity MHCII a-chain binding site. Members of this group include SEA, SEE, SED, SSA and SME-Z. The zinc atom forms a stable co-ordination complex with a conserved histidine (His81) in the polymorphic b-chain of the MHC-II molecule so that these SAGs can bind to both sides of the molecule, cross-linking the MHC-II molecule on the surface of the antigen presenting cell (Fig. 4). This leads to TNF-a and IL-1 gene transcription
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MOLECULAR MEDICINE TODAY, MARCH 2000 (VOL. 6)
Table 1. The biochemical and functional properties of human superantigens and their associated diseasesa SAG
MW
Microbe
Crystal structure
Zinc binding
MHC-II binding a/b chain
Human TCR Vb specificity
Disease
SEA
27.1
S. aureus
1
1
1/1
1.1, 5.3, 6.3, 6.4, 6.9, 7.3, 7.4, 9.1, 23.1
Food poisoning
SEB
28.4
S. aureus
1
2
1/2
1.1, 3.2, 6.4, 15.1
Food poisoning
SEC1
27.5
S. aureus
2
2
1/2
3.2, 6.4, 6.9, 15.1
Food poisoning
SEC2
27.6
S. aureus
1
2
1/2
12, 13, 14, 15, 17, 20
Food poisoning
SEC3
27.6
S. aureus
1
2
1/2
5.1
Food poisoning
SED
26.9
S. aureus
1
1
1/?
1.1, 5.3, 6.9, 7.4, 8.1, 12.1
Food poisoning
SEE
26.8
S. aureus
2
1
1/1
5.1, 6.3, 6.4, 6.9, 8.1
Food poisoning
SEG
27.0
S. aureus
2
?
?/?
?
?
SEH
25.6
S. aureus
2
?
?/?
?
Toxic shock syndrome
SEI
24.9
S. aureus
2
?
?/?
?
?
TSST
21.9
S. aureus
1
2
1/2
2.1, 8.1
Toxic shock syndrome
SPE-A
26.0
S. pyogenes
2
2
1/2
2.1, 12.2, 14.1, 15.1
Toxic shock-like syndrome Scarlet fever
SPE-C
24.4
S. pyogenes
1
1
2/1
2.1, 3.2, 12.5, 15.1
Toxic shock-like syndrome
SPE-G
24.6
S. pyogenes
2
1
?/1
2.1, 4.1, 6.9, 9.1, 12.3
?
SPE-H
23.6
S. pyogenes
2
1
?/1
2.1, 7.3, 9.1, 23.1
?
SSA
26.9
S. pyogenes
2
2
?/?
1, 3, 15, 17, 19
Toxic shock-like syndrome
SMEZ
24.3
S. pyogenes
2
1
?/1
2.1, 4.1, 7.3, 8.1
Kawasaki syndrome? Rheumatic fever?
SMEZ-2
24.1
S. pyogenes
1
1
?/1
4.1, 8.1
Kawasaki syndrome? Rheumatic fever?
YPM
21.0
Y. pseudotuberculosis
2
?
3, 9, 13.1, 13.2
Toxic shock-like syndrome? Kawasaki syndrome?
MAM
25.2
M. arthritidis
2
1
?/?
6, 8
Arthritis?
CMV
?
Cytomegalovirus
2
?
?/?
12
?
EBV
?
Epstein–Barr Virus
2
?
?/?
13
?
a The list of superantigens (SAGs) that are known to affect humans continues to grow. There are currently 18 confirmed SAGs produced by S. aureus and S. pyogenes and these are linked to or are directly responsible for a number of diseases. Note that every SAG has a different TCR Vb profile. T cells bearing Vb2 and Vb8 are most commonly targeted by S. pyogenes SAGs. The M. arthritidis mitogen (MAM) and Y. pseudotuberculosis-derived mitogen (YPM) SAGs are not related to the other bacterial SAGs. The cytomegalovirus (CMV) SAG has yet to be isolated and is only known for its ability to enhance HIV replication in human Vb12 T cells. Abbreviations: MHC, major histocompatibility complex; MW, molecular weight; SEA–SEI, staphylococcal enterotoxins A–I; TSST, toxic shock syndrome; SPE, streptococcal pyrogenic exotoxin; SSA, streptococcal superantigen; SMEZ, streptococcal mitogenic exotoxin z; EBV, Epstein–Barr virus.
in the APC6,22,23. It is not clear how APC activation increases the potency of SEA. One current theory is that cross-linking induces localized regions on the APC surface that possess high concentrations of MHC-II, SEA and adhesion molecules such as intracellular adhesion molecule 1 (ICAM-1), and CD80 and CD86, which are surface ligands for T-cell co-stimulation24. These activation clusters might be required for efficient triggering of T cells and recent studies using highresolution confocal microscopy confirm the formation of MHC–TCR supramolecular structures during T-cell activation25. It is likely that this
subgroup of SAGs has evolved a second MHC-II binding site to increase the efficiency at which they concentrate on the APC surface and to cross-link MHC-II molecules into activation clusters. Yet another variation in SAG binding is seen with the streptococcal SAG SPE-C, which has dispensed with MHC-II a-chain binding altogether in favor of a zinc-mediated b-chain binding site. Moreover, the crystal structure of SPE-C reveals a dimer structure formed through an interface where the low-affinity N-terminal binding-site is normally located16,26. Thus the orientation of SPE-C on the MHC-II molecule is 127
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SPE-C SPE-G
A
SMEZ-1 SMEZ-2 SEI SPE-H SEA SEE C SED SEH SEC1 SEC2 SEC3 B
SEB SSA SPE-A SEG TSST
Molecular Medicine Today
Figure 2. The staphylococcal/streptococcal SAG family-tree. The current number of staphylococcal and streptococcal SAGs stands at 18. This tree has been constructed using the program Clustal X (Ref. 50) and is based on a multiple amino-acids-sequence alignment. The bacterial SAG tree can be divided into three distinct groups and these are labelled according to Proft et al.27 Those in groups A and C all require zinc to bind to MHC-II molecules, whereas those in group B all appear to bind in only a single orientation on the MHC-II molecule. SEI and SPE-H share characteristics with both groups A and C, whereas TSST clearly represents a very early deviation from the primordial gene. A few of these SAGs, such as SMEZ, show considerable allelic polymorphism and are expressed at high frequency in the general Group A streptococcal population.
likely to be very different from SEB, requiring the TCR to be in an entirely different orientation in relation to the MHC-II molecule. The recent discovery of three new streptococcal SAGs indicates that SPE-C is not alone in this group. Streptococcal mitogenic exotoxin z 1 (SMEZ1), SMEZ-2, SPE-G and SPE-H all appear to operate in the same fashion as SPE-C with only a single zinc-mediated binding to the MHC-II b-chain27. The essential role of zinc in promoting binding of SEA to the MHC-II molecule is an intriguing departure from normal protein– protein interactions. The zinc atom bridges the two proteins, offering a minimal set of contacts to the MHC-II molecule, thus alleviating the need to include contacts with surrounding polymorphic residues.
How do SAGs bind to TCRs? One of the more intriguing questions about SAGs is how they manage to ligate so many different TCR molecules and whether SAG–TCR binding is any stronger than TCR–MHC-peptide, which would explain the 128
Figure 3. Different modes of binding to MHC-II molecules. At least three separate binding orientations have been found for bacterial SAGs. The extracellular domain of the MHC-II molecule is shown in blue and the MHC-II residues with which SEB interacts are shown as space filling spheres in red. SEB has been moved by a simple translation away from its position on the human HLA-DR1 molecule (from the crystal structure18). TSST forms a complex with the MHC-II molecule using the same face of the toxin (when compared to SEB) but in a different orientation. TSST has also been moved up from its position by a simple translation from the crystal structure17. The residues on the MHC-II molecule that interact with TSST following complex formation include those that interact with SEB (red spheres) and, in addition, those shown as yellow spheres. Note that TSST bridges one end of the peptide-binding groove and interacts with two residues of the b-chain of MHC-II molecule. The orientation of SMEZ-2 (grey) on binding to the MHC-II molecule is not known, although binding is mediated by zinc and His-81 on the MHC-II b-chain (shown as grey spheres), which is believed to form the fourth co-ordination zinc ligand. The three zinc ligands on SMEZ-2 are shown (His162, His202 and Asp204) clustered around a zinc atom.
extreme potency of SAGs. These questions are not easily answered simply because soluble forms of TCR are so hard to produce. Mariuzza and colleagues have succeeded in crystallizing staphylococcal enterotoxin C 3 (SEC3) with a soluble form of the murine Vb8 TCR (Ref. 19). This complex shows that the SAG makes multiple contacts with residues from the CDR2, the third framework region (FR3) and the 4th hypervariable loop (HV4) of the TCR b-chain of murine Vb8. What is most significant to their function is that individual contacts between SAG side-chain residues are predominantly made with atoms in the a-carbon backbone of the TCR, not with atoms of the side-chains, so that sidechain amino acid variation in the TCR is less likely to affect the affinity of binding. The affinity of binding between SEC3 and Vb8 has been calculated at 3.5 mM and mutations of SEC3 residues in the TCR binding site indicate that the contribution to binding energy is spread evenly across 5–6 amino acids that are generally conserved among other SAGs (Ref. 20). Leder et al. have performed a careful mutational analysis of this region in an attempt to correlate binding affinity to T-cell activation. By mutating individual residues in the TCR binding site, then comparing
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using Fas- and FasL-homozygous knockout mice indicate that FasL is predominantly expressed on non-lymphoid tissue such as liver, lung and small intestine rather than lymphoid tissue31. Moreover, the induction of non-lymphoid FasL is not inhibited by cyclosporin, which normally inhibits the induction of FasL on T cells. This suggests a separate pathway for activation of non-lymphoid cells to express FasL. Thus, one function of bacterial SAGs might be to promote the deletion of T cells that help B cells in the immune response against the bacteria.
Viral superantigens Mouse mammary tumor virus
Figure 4. The SAG SEA has two binding sites to cross-link MHC-II molecules. One is identical to the SEB site and the other is a much higher affinity zinc site on the opposite side of the molecule. This picture is looking down from above, with the distal domains of MHC-II a and b-chains shown in blue. On the left of the figure, an SEA molecule (red) is bound via the generic a-chain binding-site in the same fashion as SEB. On the right, another SEA molecule is bound to the same MHCII molecule via a zinc bridge between His81 on the MHC-II b-chain and His187, His225 and Asp227 in SEA. This interaction is about 100 times stronger than the first and so occurs first. There is some cooperation between the two SEA molecules. Once bound, it is clear that cross-linking of another MHC-II molecule can occur via both bound SEA molecules. This image is only inferred from mutational data. The crystal structure of an SEA–MHC-II complex is not yet known.
TCR binding affinity to potency, they were able to show a proportional relationship between binding affinity and potency. Their results were intriguing from the point of view of comparison to normal peptide recognition where even the smallest reduction in affinity results in a complete loss of T-cell stimulation28. In contrast, SEC3 withstands an affinity decrease of over 60-fold without loss of T-cell stimulation. This difference might reflect the different mechanisms of binding and ligation. Whereas peptide–MHC complexes trigger TCRs in a sequential fashion29 and only transiently associate with TCR, SAGs might bind more tightly and induce more stable complexes24.
Superantigens cause peripheral T-cell deletion SAG activation serves as an excellent model to examine the process of peripheral deletion of T cells because of the high proportion of responder cells. In animals injected with purified SEB, there is an immediate Vb8-specific T-cell expansion followed by significant peripheral T-cell deletion of Vb1 T cells30. T cells stimulated by a SAG are refractory to further antigenic stimulation and are rapidly removed from the periphery by a process of Fas–FasL mediated deletion. Recent experiments
The other major group of SAGs are produced by an endogenous mouse retrovirus and are type II membrane proteins with no sequence similarity to the bacterial SAGs. They were first discovered in 1974 by Hillyard Festenstein and were referred to as minor lymphocyte stimulating (Mls) antigens. They were known to generate strong T-cell proliferative responses between strains of mice matched at all MHC loci32. The T-cell response to Mls antigens was identical to the response to bacterial SAGs with expansion of unique Vb subsets. In 1991, several groups showed that the Mls antigens were the products of the endogenous murine retrovirus mouse mammary tumor virus (MMTV). So far, they are the only confirmed SAGs of viral origin. The gene for the MMTV SAG resides in the 39 long terminal repeat of the retroviral genome and is present as an integrated pro-viral form in all laboratory and many wild-type strains of mice2,33. Infectious MMTV is present in the mammary tissue and breast milk of only a few strains of mice. The SAG molecule is an essential component of the life cycle of the virus, providing efficient viral replication in newly infected gut B-cells by recruiting Vb mediated T-cell ‘help’ and promoting B-cell proliferation (Fig. 5). To defend themselves from MMTV infection, mice have continued to express the endogenous SAG gene in thymic stromal cells; thus deleting reactive T cells during development and removing the source of T-cell help from the periphery33. The net effect is large Vb specific deletions occurring in the thymus of newborn mice and the absence of Vb bearing T-cell populations in the peripheral T-cell repertoire of all mice34. The structure of the viral SAGs has not been determined despite many attempts to express it in a number of heterologous systems. The protein sequence is highly conserved among all strains of MMTV except in the last 29–32 C-terminal residues that are highly variable and confer the Vb specificity. The 45 kDa protein is extensively glycosylated and is only weakly expressed in infected B cells. Glycosylation is important for efficient expression, and Grigg et al. have shown that the SAG protein traffics independently of MHC-II protein in B cells35. Evidence also suggests that proteolytic cleavage to release an 18 kDa C-terminal fragment is required for efficient binding to MHC-II molecules once the viral SAG reaches the cell surface36.
Other viral superantigens So far, mice are the only animals that are known for certain to express endogenous viral SAGs. Despite extensive investigations, there is no evidence for Vb specific T-cell deletions or skewing in the peripheral T-cell repertoire in humans. Moreover, the search for other viral SAGs has not revealed any other clear candidates although there have been some tantalizing preliminary studies suggesting skewed Vb responses to some viral infections.
Cytomegalovirus Evidence for a cytomegalovirus (CMV)-encoded SAG has come from the unorthodox observation that HIV replicates preferentially in Vb12 bearing T cells by a factor of up to 100-fold37. This was true both in vivo 129
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(a) Viral superantigens attract help from T cells
Major histocompatibility complex (MHC) – A cluster of genes that code for MHC molecules. Polymorphic class II MHC (MHC-II) molecules, are expressed on antigen presenting cells. They bind small peptides obtained from processing within the cell.
T-cell help
MHC-II TCR B cell
T cell SAG T-cell help is required for B-cell proliferation
MMTV
(b) Endogenous superantigens remove T cells
B cell
T cell
T cells are deleted in the thymus when stimulated by endogenous MMTV SAG
No T-cell help
B cell
Glossary
T cells are missing from the peripheral repertoire Mouse is protected from viral infection Molecular Medicine Today
Figure 5. Endogenous SAGs protect mice from MMTV infection. Viral SAGs in mice are a product of retroviral integration and are used to protect the mouse from infectious wild-type MMTV. MMTV is present at high concentration in breast milk and infects B cells in the gut when the pup suckles from its infected mother. The viral SAG is expressed on the surface of the infected B cell in order to recruit T cells to provide the help necessary for B-cell proliferation and hence viral replication. Mice have incorporated the MMTV genome as a provirus and MMTV protein is expressed in the thymus. The effect is to delete all T cells as they mature in the thymus before they exit to the periphery, thus removing any T cells likely to provide help for virus-infected B cells. Mice that lack a particular Vb subset of T cells are resistant to virus infection.
T-cell receptor (TCR) – Binds MHC–peptide complexes presented by antigen presenting cells. They are highly polymorphic and composed of two polypeptide chains: a- and b-chains. Both chains are coded by multiple germline genes, including variable (V), diversity (D), joining (J) and constant (C) gene segments. TCR diversity is achieved by the random rearrangement of these segments. The V region of the b-chain (Vb) is bound by SAGs. Antigen presenting cell (APC) – A cell that presents foreign peptides to the immune system. APCs include dendritic cells, Langerhans cells, B cells and macrophages. Complementarity determining region (CDR) – Hypervariable loop regions that form the antigen-binding surface of TCRs and immunoglobulins. The TCR has six juxtaposed CDR peptide loop regions, three from each chain. The CDR1 and CDR2 are encoded by the variable gene segment, and the CDR3 region is the most hypervariable region (HV) and represents the junction between V, D and J segments. TCR b-chains have a fourth hypervariable loop (HV4) that does not contact the MHC-peptide complex but does come into contact with SAGs. b-grasp motif – A common protein fold that represents a twisted sheet structure, resembling a grasping hand. Pseudo b-barrel – Protein fold motif formed from two b-sheets. Intracellular adhesion molecule-1 (ICAM-1) – an adhesion molecule on APCs that binds LFA-1 on T cells and provides added adhesive strength to the APC–T-cell interaction. CD80 and CD86 – Two key antigens expressed on APCs that are involved in a second activation signal for T cells. They bind to the TCR molecules CD28 and CTLA-4. Framework region (FR) – The region of the T-cell receptor or immunoglobulin that is not variable but forms the backbone structure on which the variable parts of the molecule are established. Hypervariable (HV) – Those regions of the TCR that vary between different molecules.
(where T cells isolated from normal individuals display a similar pattern of differential permissiveness to HIV) and in vitro (where Vb12bearing T cells might act as a viral reservoir). This selectivity was attributed to a Vb-selecting element that activates these T cells, allowing HIV to replicate in them. Furthermore, this selectivity is MHC-II dependent but not MHC-restricted, and requires the participation of nonT cells. Such an element has the hallmark of a Vb12-specific SAG. The possibility that a ubiquitous virus such as CMV could be responsible for this element was tested. It was shown that the Vb12-selective HIV replication depended on non-T cells that came from CMV-positive individuals. In addition, a monocytic cell-line was able to promote this phenomenon only when the cells themselves were infected with CMV and were in direct contact with the T cells, thus ruling out the possibility of a soluble mediator37. 130
Mycoplasma arthritidis mitogen (MAM) – The superantigen produced by M. arthritidis. Fas–FasL – The Fas–FasL pathway is an important regulator of apoptosis in lymphoid cells. Triggering of FAS on T cells causes induction of cell-death mechanisms. Mouse mammary tumour virus (MMTV) – An endemic retrovirus found in all laboratory strains of mice and most wild-type mice. Minor lymphocyte stimulating (Mls) antigens – Surface-bound molecules encoded by genes in the 39 long terminal repeat region of MMTV.
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The outstanding questions influence do SAGs have on the normal immune system? • What many other types of SAG exist in nature apart from • How the prototype staphylococcal and streptococcal toxins and the MMTV viral SAGs?
Are there any SAGs produced by human viruses? • What effect does SAG activation have on the progression of • an autoimmune response? Why do some bacteria to produce SAGs – what • benefit does it serve to continue the microbe?
Epstein–Barr virus Evidence for an Epstein–Barr virus (EBV) encoded SAG arose from observations that EBV-infected human B-cell lines induced into the lytic cycle with a B-cell mitogen, selectively stimulate Vb13 bearing T cells. This Vb13 specific T-cell activation remains the only tantalizing evidence so far of an EBV-encoded SAG (Ref. 38)
Superantigens in diabetes mellitus
also be caused by streptococcal infection through the release of streptococcal SAGs. However, the site of infection is usually deeper. Both conditions are acute and serious and have a fatality rate of approximately 30%. Treatment usually involves the administration of pooled g globulin, which contains naturally occurring neutralizing anti-toxin antibodies. Patients have no anti-TSST neutralizing responses.
Kawasaki’s disease Kawasaki’s disease (KD) is an acute febrile disease of children aged about five years. Without treatment, it can be fatal. In Japan and the US, it has become one of the most common causes of acquired heart disease in children. Intravenous immunoglobulin therapy is highly effective when given early, suggesting that the agent is a toxin that is neutralized by naturally occurring anti-toxin antibodies. The etiological agent is not known, but several lines of evidence suggest that it is SAG mediated. The symptoms of the disease resemble TSS and several studies have shown that there is a selective expansion of a Vb2 subset of T cells44. In one study of 16 patients, 13 were found to have TSST positive isolates of S. aureus in their pharynx, rectum or groin45. Further investigations have, however, not been able to substantiate the connection with S. aureus TSST strains and there is still debate over whether SAGs really are the culprit in KD. In another article, a link between the Yersinia pseudotuberculosis SAG YPM and KD has been suggested51, although this link has yet to be firmly established.
Recent reports of a SAG expressed from a novel human endogenous retrovirus linked with insulin dependent diabetes mellitus (IDDM) have created considerable excitement. Conrad et al. had previously reported that infiltrating T cells in the pancreas of two acutely diabetic juveniles were biased for the Vb7 domain using immunohistochemistry with Vb specific monoclonal antibodies (mAbs)39. This discovery was followed more recently by a report from the same author on the isolation of a new human retrovirus named IDDMK that is responsible for this activity. Moreover, the putative SAG was encoded within the 39 end of the IDDMK viral envelope region40. IDDMK is highly homologous to members of the HERVK family of human endogenous retroviruses. The HERVK genes are highly abundant (over 50 copies/genome) integrated retroviral elements that are largely silent in humans except in certain malignancies. Evidence for IDDMK viral expression in plasma from 10/10 IDDM patients against 0/10 normal patients was greeted with much excitement. This initial excitement has since been tempered by further scrutiny. Many independent studies have not supported the existence of an active IDDMK virus and instead suggest that the original PCR technique used to amplify the IDDMK’s RNA genes also amplified HERVK sequences41. Experiments in our laboratory using the same methods as Conrad have only uncovered HERVK and we have yet to find any evidence of SAG activity expressed from the region of the HERVK viral genome proposed in the original report.
It has been proposed that SAGs derived from bacteria, viruses or mycoplasmas might contribute to the pathogenesis of autoimmune disease by activating T cells that are specific for self antigens2,3. Early reports observed Vb-specific enrichment of T cells in common diseases such as arthritis. These created much excitement. Paliard et al., for example, analysed the TCR b chain profiles of synovial T cells and found a selective expansion of human Vb14-expressing T cells46. Using an animal model of multiple sclerosis, experimental autoimmune encephelomyelitis (EAE), it has been shown that administration of SEB to mice recovering from EAE triggered a rapid relapse of the disease. This effect was the result of direct stimulation of the Vb3 positive autoreactive MBP peptide specific T cells that initially caused the brain inflammation. This was clear evidence that SAGs could, under the right conditions, break the tolerance or suppression of autoreactive T-cell clones and induce a state of autoimmune disease47–49. Despite these early reports, there has been little clear evidence directly linking a SAG to any autoimmune disease. Nevertheless, sufficient evidence has been provided to suggest that SAGs can and do play a secondary role in autoimmune activation through the relatively indiscriminate stimulation of auto-reactive T cells.
Superantigens and disease
Concluding remarks
Toxic shock syndrome (TSS) is a superantigen-mediated disease. Toxic shock results from the release of TSST-1 into the blood stream by TSST-1 producing strains of S. aureus and is characterized by fever, rash, desquamation, hypotension and major organ involvement. The sites of infection are usually superficial and the most common site is the vagina in menstruating women. An outbreak in the late 1970s was linked to the prolonged use of a particular high absorbency tampon which promoted the rapid growth of S. aureus42. TSST is found in approximately 70% of all toxic-shock inducing strains of S. aureus and studies have shown that TSS patients have elevated levels of Vb2 T cells in the blood during the acute stages of the disease43. TSS can
As a family of molecules, SAGs display a remarkable degree of variation in structure and function around the common goal of bringing the TCR and MHC-II molecules together. They have provided remarkable insight into the mechanisms of T-cell activation and the extraordinary sensitivity of T-cell antigen recognition. Because of the ubiquitous expression and widespread carriage of these toxins by both staphylococci and streptococci, it is clear that our immune systems are under constant challenge from these extremely powerful agents, which are therefore likely to influence our response to other challenges. What benefit these toxins serve the bacteria remains a mystery. SAGs remain a curious enigma – a result of the constant battle between microbes and the immune system.
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References 01 Marrack, P. and Kappler, J. (1990) The Staphylococcal enterotoxins and their relatives. Science 248, 705–711 02 Kotzin, B.L. et al. (1993) Superantigens and their potential role in human disease. Adv. Immunol. 54, 99–166 03 Renno, T. and Acha-Orbea, H. (1996) Superantigens in autoimmune diseases: still more shades of gray. Immunol. Rev. 154, 175–191 04 Wannamaker, L.W. and Schlievert, P.M. (1988) Exotoxins of group A Streptococci. In Bacterial toxins (Vol. 4) (Hardegree, C.M. and Tu, A.T., eds), pp. 267–295, Marcel Dekker 05 Schlievert, P.M. (1993) Role of superantigens in human disease. J. Inf. Dis. 167, 997–1002 06 Hudson, K.R. et al. (1993) Two adjacent residues in staphylococcal enterotoxins A and E determine T cell receptor Vb specificity. J. Exp. Med. 177, 175–184 07 Betley, M.J. and Mekalanos, J.J. (1988) Nucleotide sequence of the type A staphylococcal enterotoxin gene. J Bacteriol. 170, 34–41 08 Cole, B.C. and Atkin, C.L. (1991) The Mycoplasma arthritidis T-cell mitogen, MAM: a model superantigen. Immunol. Today 12, 271–276 09 Hodtsev, A.S. et al. (1998) Mycoplasma superantigen is a CDR3-dependent ligand for the T cell antigen receptor. J. Exp. Med. 187, 319–327 10 Ito,Y. et al. (1995) Sequence analysis of the gene for a novel superantigen produced by Yersinia pseudotuberculosis and expression of the recombinant protein. J. Immunol. 154, 5896–5906 11 Schad, E.M. et al. (1995) Crystal structure of the superantigen staphylococcal enterotoxin type A. Crystal structure of Staphylococcal enterotoxin B, a superantigen. EMBO J. 14, 3292–3301 12 Swaminathan, S. et al. (1992) Crystal structure of Staphylococcal enterotoxin B, a superantigen. Nature 359, 801–806 13 Papageorgiou, A.C. et al. (1995) Crystal structure of the superantigen enterotoxin C2 from Staphylococcus aureus reveals a zinc-binding site. Structure 3, 769–779 14 Sundstrom, M. et al. (1996) The crystal structure of staphylococcal enterotoxin type D reveals Zn21-mediated homodimerization. EMBO Journal 15, 6832–6840 15 Acharya, K.R. et al. (1994) Structural basis of superantigen action inferred from crystal structure of toxic-shock syndrome toxin-1. Nature 367, 94–97 16 Roussel, A. et al. (1997) Crystal structure of the streptococcal superantigen SPE-C: dimerization and zinc binding suggest a novel mode of interaction with MHC class II molecules. Nat. Struct. Biol. 4, 635–643 17 Kim, J., Urban, R., Strominger, J.L. and Wiley, D. (1994) Toxic Shock Syndrome Toxin-1 Complexed with a Major Histocompatibility Molecule HLA-DR1. Science 266, 1870–1874 18 Jardetzky, T.S. et al. (1994) Three-dimensional structure of a human class II histocompatibility molecule complexed with superantigen. Nature 368, 711–718 19 Fields, B.A. et al. (1996) Crystal structure of a T-cell receptor b-chain complexed with a superantigen. Nature 384, 188–192 20 Li, H. et al. (1998) Three-dimensional structure of the complex between a T-cell receptor b-chain and the superantigen staphylococcal enterotoxin B. Immunity 9, 807–816 21 Thibodeau, J. et al. (1994) Subsets of HLA-DR1 molecules defined by SEB and TSST1 binding. Science 266, 1874–1878 22 Mehindate, K. et al. (1995) Cross-linking of major histocompatibility complex class II molecules by staphylococcal enterotoxin A superantigen is a requirement for inflammatory cytokine gene expression. J. Exp. Med. 182, 1573–1577 23 Tiedemann, R.E. and Fraser, J.D. (1996) Cross-linking of MHC class II molecules by staphylococcal enterotoxin A is essential for antigen-presenting cell and T-cell activation. J. Immunol. 157, 3958–3966 24 Proft, T. and Fraser, J.D. (1998) Superantigens: just like peptides only different. J. Exp. Med. 187, 819–821 25 Monks, C.R. et al. (1998) Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395, 82–86
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26 Li, P.L. et al. (1997) The superantigen streptococcal pyrogenic exotoxin C (SPE-C) exhibits a novel mode of action. J. Exp. Med. 186, 375–383 27 Proft, T. et al. (1999) Identification and characterization of novel superantigens from Streptococcus pyogenes. J. Exp. Med. 189, 89–102 28 Leder, L. et al. (1998) A mutational analysis of the binding of staphylococcal enterotoxins B and C3 to the T cell receptor b-chain and major histocompatibility class II. J. Exp. Med. 187, 823–833 29 Valitutti, S. et al. (1995) Serial triggering of many T-cell receptors by a few peptideMHC complexes. Nature 375, 148–151 30 Kawabe, Y. and Ochi, A. (1991) Programmed cell death and extrathymic reduction of Vb81 CD41 T cells in mice tolerant to Staphylococcus aureus enterotoxin B. Nature 349, 245–248 31 Bonfoco, E. et al. (1998) Inducible non-lymphoid expression of Fas ligand is responsible for superantigen-induced peripheral deletion of T cells. Immunity 9, 711–720 32 Festenstein, H. and Kimura, S. (1988) The Mls system: past and present. J. Immunogenet. 15, 183–196 33 Acha-Orbea, H. and MacDonald, H.R. (1995) Superantigens of mouse mammary tumor virus. Ann. Rev. Immunol. 13, 459–486 34 Acha-Orbea, H. et al. (1992) Mls: a link between immunology and retrovirology. Int. Rev. Immunol. 8, 327–336 35 Grigg, M.E. et al. (1998) Mtv-1 superantigen trafficks independently of major histocompatibility complex class II directly to the B-cell surface by the exocytic pathway. J. Virol. 72, 2577–2588 36 Winslow, G.M. et al. (1992) Detection and biochemical characterization of the mouse mammary tumor virus 7 superantigen (Mls-1a). Cell 71, 719–730 37 Dobrescu, D. et al. (1995) Enhanced HIV-1 replication in Vb 12 T cells due to human cytomegalovirus in monocytes: evidence for a putative herpesvirus superantigen. Cell 82, 753–763 38 Sutkowski, N. et al. (1996) An Epstein–Barr virus-associated superantigen. J. Exp. Med. 184, 971–980 39 Conrad, B. et al. (1994) Evidence for superantigen involvement in insulin-dependent diabetes mellitus aetiology. Nature 371, 351–355 40 Conrad, B. et al. (1997) A human endogenous retroviral superantigen as candidate autoimmune gene in type I diabetes. Cell 90, 303–313 41 Lower, R. et al. (1998) Development of insulin-dependent diabetes mellitus does not depend on specific expression of the human endogenous retrovirus HERV-K. Cell 95, 11–14 42 Todd, J. and Tishant, D. (1978) Toxic Shock syndrome associated with phage-group1 Staphylococci. Lancet 2, 1116–1118 43 Choi, Y. et al. (1990) Selective expansion of T cells expressing Vb 2 in toxic shock syndrome. J. Exp. Med. 172, 981–984 44 Abe, J. et al. (1993) Characterization of T cell repertoire changes in acute Kawasaki disease. J. Exp. Med. 177, 791–796 45 Leung, D.Y. et al. (1995) The potential role of bacterial superantigens in the pathogenesis of Kawasaki syndrome. J. Clin. Immunol. 15, 11S–17S 46 Paliard, X. et al. (1991) Evidence for the effects of a superantigen in rheumatoid arthritis. Science 253, 325–329 47 Schiffenbauer, J. et al. (1993) Staphylococcal enterotoxins can reactivate experimental allergic encephalomyelitis. Proc. Natl. Acad. Sci. U. S. A. 90, 8543–8546 48 Racke, M. et al. (1994) Superantigen modulation of experimental allergic encephalomyelitis: activation of anergy determines outcome. J. Immunol. 152, 2051–2059 49 Brocke, S. et al. (1993) Induction of relapsing paralysis in experimental autoimmune encephalomyelitis by bacterial superantigen. Nature 365, 642–644 50 Thompson, J.D. et al. (1997) The ClustalX windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24, 4876–4882 51 Konishi, et al. (1996) A case of Kawasaki’s disease with coronary aneurysms documenting Yersinia pseudotuberculosis infection. Acta Paediatr. 86, 661–664
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Superantigens – powerful modifiers of the immune system John Fraser, Vickery Arcus, Philip Kong, Edward Baker and Thomas Proft
Superantigens are powerful microbial toxins that activate the immune system by binding to class II major histocompatibility complex and T-cell receptor molecules. They cause a number of diseases characterized by fever and shock and are important virulence factors for two human commensal organisms, Staphylococcus aureus and Streptococcus pyogenes, as well as for some viruses. Their mode of action and variation around the common theme of over-stimulating T cells, provides a rich insight into the constant battle between microbes and the immune system. SUPERANTIGENS (SAGs) are microbial toxins that target the immune system causing massive T-cell activation, cytokine release and systemic shock. They represent some of the most potent bioactive molecules ever discovered. Their mechanism of action has been the subject of intense study over the past decade and has led to speculation that they play a hidden role in promoting autoimmune disease and altering the normal immune responses to viral infections such as HIV1–3. Despite wide variation in their protein structure, SAGs all share a common ability to simultaneously bind the class II major histocompatibility complex (MHC-II) molecules expressed on professional antigen presenting cells (APCs) and the variable region of the T-cell receptor b-chain (TCR Vb). The result is massive stimulation of any T cell that expresses the correct TCR Vb element on its surface (Fig. 1). The complexities of SAG structure and function, and the variety of binding mechanisms that have evolved for different SAGs, indicate that, for some microbes at least, SAGs are an important mechanism for survival. John Fraser PhD* Associate Professor in Molecular Medicine Vickery Arcus PhD Research Fellow Philip Kong MSc Graduate Student Edward Baker PhD Professor of Structural Biology Thomas Proft PhD Research Fellow School of Biological Sciences, Department of Molecular Medicine, University of Auckland, Private Bag, 92019, Auckland, New Zealand. Tel: 164 9 373 7599 ext. 6036 Fax: 164 9 373 7674 *e-mail: [email protected]
Bacterial superantigens The best studied SAGs are the family of staphylococcal enterotoxins (SE) and streptococcal pyrogenic exotoxins (SPEs) secreted by the Gram-positive bacteria Staphylococcus aureus and Streptococcus pyogenes commonly found on the skin, the nose and upper respiratory tract of humans4,5. These toxins are potent virulence factors in diseases caused by these bacteria, but their exact function in the life cycle of either S. aureus or S. pyogenes is still uncertain. The addition of femtogram quantities of purified toxin to a culture of human peripheral blood lymphocytes results in the activation and proliferation of T cells that express a restricted number of TCR Vb domains. Staphylococcal enterotoxin A (SEA), for example, stimulates T cells bearing Vb1, 5.3, 6.3, 6.4, 6.9, 7.4, 9.1 and 23, whereas staphylococcal enterotoxin E (SEE), which differs in amino acid sequence from SEA by only 20%, stimulates a different but overlapping group of T cells bearing Vb1, 5.1, 6.3, 6.4, 6.9, 7.4 and 8.1 (Ref. 6). Approximately 20% of all T cells are activated by SEA. In contrast, normal peptide antigens only stimulate between 0.001% and 0.0001% of T cells because recognition is dependent on both the variable and junctional segments (D and J) of the TCR a and b-chains. This Vb-restricted expansion is the characteristic hallmark of all SAGs. No-one has yet found a SAG that stimulates every T cell or one that binds to the other side of the TCR – the Va domain. For many years, only six staphylococcal (SEA, SEB, SEC, SED, SEE and TSST) and two streptococcal (SPE-A and SPE-C) toxins were known. This number has increased considerably in recent years with the availability of S. aureus and S. pyogenes genome sequencing databases. The number of staphylococcal and streptococcal SAGs now stands at 18 (Fig. 2) and is likely to grow even further as the search for homologous sequences among these databases continues. Moreover, other unrelated bacterial SAGs have also been confirmed from Yersinia pseudotuberculosis and Mycoplasma arthritidis, and viral SAGs include the mouse mammary tumour virus (MMTV) products plus potential SAGs from cytomegalovirus (CMV) and Epstein–Barr virus (EBV) (Table 1). Similarity in amino acid sequences between the staphylococcal and streptococcal SAG family members varies between 20% and 90% and it is clear they have all evolved from a common ancestral gene (Fig. 2). The SEA gene resides on a variable genetic element7 whereas the spe-a gene from S. pyogenes
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T cell
Activation Signal transduction cascade
IL-1β
TCR SEA
TNF-α
IL-2 MHC-II IFN-γ
Macrophage
Molecular Medicine Today
Figure 1. SAGs stimulate high levels of cytokines. The interaction of the MHCII molecule with the TCR regulates the production of an array of cytokines from both the T cell and the antigen presenting cell (APC), in this case a macrophage. Normally, an antigen is processed by the APC into small peptides that are presented on the surface in the distal end of the MHC-II molecule. Only about 1 in 10 000–100 000 T cells possesses a TCR that binds strongly enough to stimulate the T cells. With SAGs, however, as many as 1 in 10 T cells are engaged. This results in a greater production of cytokines, particularly IL2, TNF-a, IFN-g and IL-1 from the APC. This massive release of cytokines causes the acute illness of toxic shock, which is often fatal. Why some SAGs are more efficient at causing toxic shock is unknown but about 70% of all cases appear to arise from the production of the SAG toxic shock syndrome toxin – a Vb2-stimulating toxin from a small site of infection.
is located on streptococcal phage T12 and is found in about 30% of all streptococcal isolates.
Superantigens from other bacteria Unrelated SAGs have also been isolated from Mycoplasma arthritidis and Yersinia pseudotuberculosis, and both have been confirmed by testing the recombinant products in T-cell stimulation assays. M. arthritidis is a rodent pathogen that causes inflammatory infection and chronic joint disease in rats and mice. Although the M. arthritidis mitogen (MAM) has been known for many years, research on this SAG has been hindered by difficulties in its isolation and stability. The MAM gene was finally cloned in 1996 and did not show any significant homology to the staphylococcal or streptococcal SAGs (Ref. 8). MAM is a single 25 kDa protein that preferentially binds to one isotype of the MHC class II protein: murine I-E or its human equivalent HLADR – with DR4, DR7 and DR12 subtypes presenting MAM most efficiently. Little is known about its structure and mechanism of action, but a recent study on the TCRs that respond to MAM indicates that it 126
is likely to contact not just the germ-line encoded TCR b-chain but also the complementarity determining region 3 (CDR3) that is generated through somatic mutation9. This interesting finding reveals for the first time that some SAGs might have a much more restricted TCR repertoire and thus might not leave the obvious Vb ‘footprint’ seen for the prototypic staphylococcal and streptococcal SAGs. This raises the question of how many more semi-restricted SAGs exist in nature that can alter the immune response without producing the visible signs of massive T-cell activation, cytokine release and shock that accompany an acute poisoning by one of the prototype staphylococcal or streptococcal SAGs. The Y. pseudotuberculosis-derived mitogen (YPM) is a small 21 kDa protein that specifically targets human Vb13.1 and -13.2 bearing TCRs (Ref. 10). Like MAM, it shows no significant homology to other SAGs, and its role in the pathogenicity of Y. pseudotuberculosis is unclear.
The structure of superantigen molecules reveals a variety of binding mechanisms Perhaps one of the most intriguing findings has been that individual bacterial SAGs have evolved quite different mechanisms of binding to MHC-II molecules despite a highly conserved structural fold. The crystal structures of SEA (Ref. 11), SEB (Ref. 12), SEC2 (Ref. 13), SED (Ref. 14), TSST (Ref. 15) and SPEC (Ref. 16) reveal a common core-fold based on two globular domains: a C-terminal domain of the b-grasp motif and a smaller N-terminal pseudo b-barrel domain (Fig. 3). The crystal structures of SEB–HLA-DR1 and TSST–DR1 complexes reveal similar but not identical binding mechanisms17,18. Staphylococcal enterotoxin B (SEB) has a single, low-affinity MHCII binding site located in the smaller N-terminal domain. Residues in this region bind to a hydrophobic groove located in the distal region of the invariant a1 domain of HLA-DR (Fig. 3). The region of SEB that binds to the TCR is a shallow groove between the two domains. SEB contacts both the CDR2 and the fourth hypervariable loop (HV4) of the TCR b-chain19. Thus, SEB can be likened most simply to a wedge that fits snugly between the TCR and MHC-II molecule with the TCR and MHC-II bound together, although this analogy is not entirely accurate. A recent comparison of several crystal structures reveals that SEB is likely to tip and rotate the TCR away from its normal position on the MHC-II molecule so that entirely new contacts must be accommodated from those determined during thymic selection20. The crystal structure of TSST–DR1 reveals a different binding mechanism17 (Fig. 3). Although TSST binds to the same region of DRa1 domain as SEB, it covers most of the top of the MHC-II molecule preventing any further contact with the TCR. Unlike SEB, activation by TSST is likely to rely entirely on the affinity of TSST–TCR binding with no contribution from MHC-II–TCR contacts. TSST also makes contact with residues from the bound peptide that limits the number of MHC-II molecules that TSST can bind and also raises the intriguing possibility that different peptides might regulate the potency of SAG-mediated T-cell activation21. A third type of MHC-II binding occurs with a subset of the bacterial SAGs that contain a high affinity zinc-binding site in the C-terminal domain on the opposite side of the molecule to the low affinity MHCII a-chain binding site. Members of this group include SEA, SEE, SED, SSA and SME-Z. The zinc atom forms a stable co-ordination complex with a conserved histidine (His81) in the polymorphic b-chain of the MHC-II molecule so that these SAGs can bind to both sides of the molecule, cross-linking the MHC-II molecule on the surface of the antigen presenting cell (Fig. 4). This leads to TNF-a and IL-1 gene transcription
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Table 1. The biochemical and functional properties of human superantigens and their associated diseasesa SAG
MW
Microbe
Crystal structure
Zinc binding
MHC-II binding a/b chain
Human TCR Vb specificity
Disease
SEA
27.1
S. aureus
1
1
1/1
1.1, 5.3, 6.3, 6.4, 6.9, 7.3, 7.4, 9.1, 23.1
Food poisoning
SEB
28.4
S. aureus
1
2
1/2
1.1, 3.2, 6.4, 15.1
Food poisoning
SEC1
27.5
S. aureus
2
2
1/2
3.2, 6.4, 6.9, 15.1
Food poisoning
SEC2
27.6
S. aureus
1
2
1/2
12, 13, 14, 15, 17, 20
Food poisoning
SEC3
27.6
S. aureus
1
2
1/2
5.1
Food poisoning
SED
26.9
S. aureus
1
1
1/?
1.1, 5.3, 6.9, 7.4, 8.1, 12.1
Food poisoning
SEE
26.8
S. aureus
2
1
1/1
5.1, 6.3, 6.4, 6.9, 8.1
Food poisoning
SEG
27.0
S. aureus
2
?
?/?
?
?
SEH
25.6
S. aureus
2
?
?/?
?
Toxic shock syndrome
SEI
24.9
S. aureus
2
?
?/?
?
?
TSST
21.9
S. aureus
1
2
1/2
2.1, 8.1
Toxic shock syndrome
SPE-A
26.0
S. pyogenes
2
2
1/2
2.1, 12.2, 14.1, 15.1
Toxic shock-like syndrome Scarlet fever
SPE-C
24.4
S. pyogenes
1
1
2/1
2.1, 3.2, 12.5, 15.1
Toxic shock-like syndrome
SPE-G
24.6
S. pyogenes
2
1
?/1
2.1, 4.1, 6.9, 9.1, 12.3
?
SPE-H
23.6
S. pyogenes
2
1
?/1
2.1, 7.3, 9.1, 23.1
?
SSA
26.9
S. pyogenes
2
2
?/?
1, 3, 15, 17, 19
Toxic shock-like syndrome
SMEZ
24.3
S. pyogenes
2
1
?/1
2.1, 4.1, 7.3, 8.1
Kawasaki syndrome? Rheumatic fever?
SMEZ-2
24.1
S. pyogenes
1
1
?/1
4.1, 8.1
Kawasaki syndrome? Rheumatic fever?
YPM
21.0
Y. pseudotuberculosis
2
?
3, 9, 13.1, 13.2
Toxic shock-like syndrome? Kawasaki syndrome?
MAM
25.2
M. arthritidis
2
1
?/?
6, 8
Arthritis?
CMV
?
Cytomegalovirus
2
?
?/?
12
?
EBV
?
Epstein–Barr Virus
2
?
?/?
13
?
a The list of superantigens (SAGs) that are known to affect humans continues to grow. There are currently 18 confirmed SAGs produced by S. aureus and S. pyogenes and these are linked to or are directly responsible for a number of diseases. Note that every SAG has a different TCR Vb profile. T cells bearing Vb2 and Vb8 are most commonly targeted by S. pyogenes SAGs. The M. arthritidis mitogen (MAM) and Y. pseudotuberculosis-derived mitogen (YPM) SAGs are not related to the other bacterial SAGs. The cytomegalovirus (CMV) SAG has yet to be isolated and is only known for its ability to enhance HIV replication in human Vb12 T cells. Abbreviations: MHC, major histocompatibility complex; MW, molecular weight; SEA–SEI, staphylococcal enterotoxins A–I; TSST, toxic shock syndrome; SPE, streptococcal pyrogenic exotoxin; SSA, streptococcal superantigen; SMEZ, streptococcal mitogenic exotoxin z; EBV, Epstein–Barr virus.
in the APC6,22,23. It is not clear how APC activation increases the potency of SEA. One current theory is that cross-linking induces localized regions on the APC surface that possess high concentrations of MHC-II, SEA and adhesion molecules such as intracellular adhesion molecule 1 (ICAM-1), and CD80 and CD86, which are surface ligands for T-cell co-stimulation24. These activation clusters might be required for efficient triggering of T cells and recent studies using highresolution confocal microscopy confirm the formation of MHC–TCR supramolecular structures during T-cell activation25. It is likely that this
subgroup of SAGs has evolved a second MHC-II binding site to increase the efficiency at which they concentrate on the APC surface and to cross-link MHC-II molecules into activation clusters. Yet another variation in SAG binding is seen with the streptococcal SAG SPE-C, which has dispensed with MHC-II a-chain binding altogether in favor of a zinc-mediated b-chain binding site. Moreover, the crystal structure of SPE-C reveals a dimer structure formed through an interface where the low-affinity N-terminal binding-site is normally located16,26. Thus the orientation of SPE-C on the MHC-II molecule is 127
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SPE-C SPE-G
A
SMEZ-1 SMEZ-2 SEI SPE-H SEA SEE C SED SEH SEC1 SEC2 SEC3 B
SEB SSA SPE-A SEG TSST
Molecular Medicine Today
Figure 2. The staphylococcal/streptococcal SAG family-tree. The current number of staphylococcal and streptococcal SAGs stands at 18. This tree has been constructed using the program Clustal X (Ref. 50) and is based on a multiple amino-acids-sequence alignment. The bacterial SAG tree can be divided into three distinct groups and these are labelled according to Proft et al.27 Those in groups A and C all require zinc to bind to MHC-II molecules, whereas those in group B all appear to bind in only a single orientation on the MHC-II molecule. SEI and SPE-H share characteristics with both groups A and C, whereas TSST clearly represents a very early deviation from the primordial gene. A few of these SAGs, such as SMEZ, show considerable allelic polymorphism and are expressed at high frequency in the general Group A streptococcal population.
likely to be very different from SEB, requiring the TCR to be in an entirely different orientation in relation to the MHC-II molecule. The recent discovery of three new streptococcal SAGs indicates that SPE-C is not alone in this group. Streptococcal mitogenic exotoxin z 1 (SMEZ1), SMEZ-2, SPE-G and SPE-H all appear to operate in the same fashion as SPE-C with only a single zinc-mediated binding to the MHC-II b-chain27. The essential role of zinc in promoting binding of SEA to the MHC-II molecule is an intriguing departure from normal protein– protein interactions. The zinc atom bridges the two proteins, offering a minimal set of contacts to the MHC-II molecule, thus alleviating the need to include contacts with surrounding polymorphic residues.
How do SAGs bind to TCRs? One of the more intriguing questions about SAGs is how they manage to ligate so many different TCR molecules and whether SAG–TCR binding is any stronger than TCR–MHC-peptide, which would explain the 128
Figure 3. Different modes of binding to MHC-II molecules. At least three separate binding orientations have been found for bacterial SAGs. The extracellular domain of the MHC-II molecule is shown in blue and the MHC-II residues with which SEB interacts are shown as space filling spheres in red. SEB has been moved by a simple translation away from its position on the human HLA-DR1 molecule (from the crystal structure18). TSST forms a complex with the MHC-II molecule using the same face of the toxin (when compared to SEB) but in a different orientation. TSST has also been moved up from its position by a simple translation from the crystal structure17. The residues on the MHC-II molecule that interact with TSST following complex formation include those that interact with SEB (red spheres) and, in addition, those shown as yellow spheres. Note that TSST bridges one end of the peptide-binding groove and interacts with two residues of the b-chain of MHC-II molecule. The orientation of SMEZ-2 (grey) on binding to the MHC-II molecule is not known, although binding is mediated by zinc and His-81 on the MHC-II b-chain (shown as grey spheres), which is believed to form the fourth co-ordination zinc ligand. The three zinc ligands on SMEZ-2 are shown (His162, His202 and Asp204) clustered around a zinc atom.
extreme potency of SAGs. These questions are not easily answered simply because soluble forms of TCR are so hard to produce. Mariuzza and colleagues have succeeded in crystallizing staphylococcal enterotoxin C 3 (SEC3) with a soluble form of the murine Vb8 TCR (Ref. 19). This complex shows that the SAG makes multiple contacts with residues from the CDR2, the third framework region (FR3) and the 4th hypervariable loop (HV4) of the TCR b-chain of murine Vb8. What is most significant to their function is that individual contacts between SAG side-chain residues are predominantly made with atoms in the a-carbon backbone of the TCR, not with atoms of the side-chains, so that sidechain amino acid variation in the TCR is less likely to affect the affinity of binding. The affinity of binding between SEC3 and Vb8 has been calculated at 3.5 mM and mutations of SEC3 residues in the TCR binding site indicate that the contribution to binding energy is spread evenly across 5–6 amino acids that are generally conserved among other SAGs (Ref. 20). Leder et al. have performed a careful mutational analysis of this region in an attempt to correlate binding affinity to T-cell activation. By mutating individual residues in the TCR binding site, then comparing
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using Fas- and FasL-homozygous knockout mice indicate that FasL is predominantly expressed on non-lymphoid tissue such as liver, lung and small intestine rather than lymphoid tissue31. Moreover, the induction of non-lymphoid FasL is not inhibited by cyclosporin, which normally inhibits the induction of FasL on T cells. This suggests a separate pathway for activation of non-lymphoid cells to express FasL. Thus, one function of bacterial SAGs might be to promote the deletion of T cells that help B cells in the immune response against the bacteria.
Viral superantigens Mouse mammary tumor virus
Figure 4. The SAG SEA has two binding sites to cross-link MHC-II molecules. One is identical to the SEB site and the other is a much higher affinity zinc site on the opposite side of the molecule. This picture is looking down from above, with the distal domains of MHC-II a and b-chains shown in blue. On the left of the figure, an SEA molecule (red) is bound via the generic a-chain binding-site in the same fashion as SEB. On the right, another SEA molecule is bound to the same MHCII molecule via a zinc bridge between His81 on the MHC-II b-chain and His187, His225 and Asp227 in SEA. This interaction is about 100 times stronger than the first and so occurs first. There is some cooperation between the two SEA molecules. Once bound, it is clear that cross-linking of another MHC-II molecule can occur via both bound SEA molecules. This image is only inferred from mutational data. The crystal structure of an SEA–MHC-II complex is not yet known.
TCR binding affinity to potency, they were able to show a proportional relationship between binding affinity and potency. Their results were intriguing from the point of view of comparison to normal peptide recognition where even the smallest reduction in affinity results in a complete loss of T-cell stimulation28. In contrast, SEC3 withstands an affinity decrease of over 60-fold without loss of T-cell stimulation. This difference might reflect the different mechanisms of binding and ligation. Whereas peptide–MHC complexes trigger TCRs in a sequential fashion29 and only transiently associate with TCR, SAGs might bind more tightly and induce more stable complexes24.
Superantigens cause peripheral T-cell deletion SAG activation serves as an excellent model to examine the process of peripheral deletion of T cells because of the high proportion of responder cells. In animals injected with purified SEB, there is an immediate Vb8-specific T-cell expansion followed by significant peripheral T-cell deletion of Vb1 T cells30. T cells stimulated by a SAG are refractory to further antigenic stimulation and are rapidly removed from the periphery by a process of Fas–FasL mediated deletion. Recent experiments
The other major group of SAGs are produced by an endogenous mouse retrovirus and are type II membrane proteins with no sequence similarity to the bacterial SAGs. They were first discovered in 1974 by Hillyard Festenstein and were referred to as minor lymphocyte stimulating (Mls) antigens. They were known to generate strong T-cell proliferative responses between strains of mice matched at all MHC loci32. The T-cell response to Mls antigens was identical to the response to bacterial SAGs with expansion of unique Vb subsets. In 1991, several groups showed that the Mls antigens were the products of the endogenous murine retrovirus mouse mammary tumor virus (MMTV). So far, they are the only confirmed SAGs of viral origin. The gene for the MMTV SAG resides in the 39 long terminal repeat of the retroviral genome and is present as an integrated pro-viral form in all laboratory and many wild-type strains of mice2,33. Infectious MMTV is present in the mammary tissue and breast milk of only a few strains of mice. The SAG molecule is an essential component of the life cycle of the virus, providing efficient viral replication in newly infected gut B-cells by recruiting Vb mediated T-cell ‘help’ and promoting B-cell proliferation (Fig. 5). To defend themselves from MMTV infection, mice have continued to express the endogenous SAG gene in thymic stromal cells; thus deleting reactive T cells during development and removing the source of T-cell help from the periphery33. The net effect is large Vb specific deletions occurring in the thymus of newborn mice and the absence of Vb bearing T-cell populations in the peripheral T-cell repertoire of all mice34. The structure of the viral SAGs has not been determined despite many attempts to express it in a number of heterologous systems. The protein sequence is highly conserved among all strains of MMTV except in the last 29–32 C-terminal residues that are highly variable and confer the Vb specificity. The 45 kDa protein is extensively glycosylated and is only weakly expressed in infected B cells. Glycosylation is important for efficient expression, and Grigg et al. have shown that the SAG protein traffics independently of MHC-II protein in B cells35. Evidence also suggests that proteolytic cleavage to release an 18 kDa C-terminal fragment is required for efficient binding to MHC-II molecules once the viral SAG reaches the cell surface36.
Other viral superantigens So far, mice are the only animals that are known for certain to express endogenous viral SAGs. Despite extensive investigations, there is no evidence for Vb specific T-cell deletions or skewing in the peripheral T-cell repertoire in humans. Moreover, the search for other viral SAGs has not revealed any other clear candidates although there have been some tantalizing preliminary studies suggesting skewed Vb responses to some viral infections.
Cytomegalovirus Evidence for a cytomegalovirus (CMV)-encoded SAG has come from the unorthodox observation that HIV replicates preferentially in Vb12 bearing T cells by a factor of up to 100-fold37. This was true both in vivo 129
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(a) Viral superantigens attract help from T cells
Major histocompatibility complex (MHC) – A cluster of genes that code for MHC molecules. Polymorphic class II MHC (MHC-II) molecules, are expressed on antigen presenting cells. They bind small peptides obtained from processing within the cell.
T-cell help
MHC-II TCR B cell
T cell SAG T-cell help is required for B-cell proliferation
MMTV
(b) Endogenous superantigens remove T cells
B cell
T cell
T cells are deleted in the thymus when stimulated by endogenous MMTV SAG
No T-cell help
B cell
Glossary
T cells are missing from the peripheral repertoire Mouse is protected from viral infection Molecular Medicine Today
Figure 5. Endogenous SAGs protect mice from MMTV infection. Viral SAGs in mice are a product of retroviral integration and are used to protect the mouse from infectious wild-type MMTV. MMTV is present at high concentration in breast milk and infects B cells in the gut when the pup suckles from its infected mother. The viral SAG is expressed on the surface of the infected B cell in order to recruit T cells to provide the help necessary for B-cell proliferation and hence viral replication. Mice have incorporated the MMTV genome as a provirus and MMTV protein is expressed in the thymus. The effect is to delete all T cells as they mature in the thymus before they exit to the periphery, thus removing any T cells likely to provide help for virus-infected B cells. Mice that lack a particular Vb subset of T cells are resistant to virus infection.
T-cell receptor (TCR) – Binds MHC–peptide complexes presented by antigen presenting cells. They are highly polymorphic and composed of two polypeptide chains: a- and b-chains. Both chains are coded by multiple germline genes, including variable (V), diversity (D), joining (J) and constant (C) gene segments. TCR diversity is achieved by the random rearrangement of these segments. The V region of the b-chain (Vb) is bound by SAGs. Antigen presenting cell (APC) – A cell that presents foreign peptides to the immune system. APCs include dendritic cells, Langerhans cells, B cells and macrophages. Complementarity determining region (CDR) – Hypervariable loop regions that form the antigen-binding surface of TCRs and immunoglobulins. The TCR has six juxtaposed CDR peptide loop regions, three from each chain. The CDR1 and CDR2 are encoded by the variable gene segment, and the CDR3 region is the most hypervariable region (HV) and represents the junction between V, D and J segments. TCR b-chains have a fourth hypervariable loop (HV4) that does not contact the MHC-peptide complex but does come into contact with SAGs. b-grasp motif – A common protein fold that represents a twisted sheet structure, resembling a grasping hand. Pseudo b-barrel – Protein fold motif formed from two b-sheets. Intracellular adhesion molecule-1 (ICAM-1) – an adhesion molecule on APCs that binds LFA-1 on T cells and provides added adhesive strength to the APC–T-cell interaction. CD80 and CD86 – Two key antigens expressed on APCs that are involved in a second activation signal for T cells. They bind to the TCR molecules CD28 and CTLA-4. Framework region (FR) – The region of the T-cell receptor or immunoglobulin that is not variable but forms the backbone structure on which the variable parts of the molecule are established. Hypervariable (HV) – Those regions of the TCR that vary between different molecules.
(where T cells isolated from normal individuals display a similar pattern of differential permissiveness to HIV) and in vitro (where Vb12bearing T cells might act as a viral reservoir). This selectivity was attributed to a Vb-selecting element that activates these T cells, allowing HIV to replicate in them. Furthermore, this selectivity is MHC-II dependent but not MHC-restricted, and requires the participation of nonT cells. Such an element has the hallmark of a Vb12-specific SAG. The possibility that a ubiquitous virus such as CMV could be responsible for this element was tested. It was shown that the Vb12-selective HIV replication depended on non-T cells that came from CMV-positive individuals. In addition, a monocytic cell-line was able to promote this phenomenon only when the cells themselves were infected with CMV and were in direct contact with the T cells, thus ruling out the possibility of a soluble mediator37. 130
Mycoplasma arthritidis mitogen (MAM) – The superantigen produced by M. arthritidis. Fas–FasL – The Fas–FasL pathway is an important regulator of apoptosis in lymphoid cells. Triggering of FAS on T cells causes induction of cell-death mechanisms. Mouse mammary tumour virus (MMTV) – An endemic retrovirus found in all laboratory strains of mice and most wild-type mice. Minor lymphocyte stimulating (Mls) antigens – Surface-bound molecules encoded by genes in the 39 long terminal repeat region of MMTV.
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The outstanding questions influence do SAGs have on the normal immune system? • What many other types of SAG exist in nature apart from • How the prototype staphylococcal and streptococcal toxins and the MMTV viral SAGs?
Are there any SAGs produced by human viruses? • What effect does SAG activation have on the progression of • an autoimmune response? Why do some bacteria to produce SAGs – what • benefit does it serve to continue the microbe?
Epstein–Barr virus Evidence for an Epstein–Barr virus (EBV) encoded SAG arose from observations that EBV-infected human B-cell lines induced into the lytic cycle with a B-cell mitogen, selectively stimulate Vb13 bearing T cells. This Vb13 specific T-cell activation remains the only tantalizing evidence so far of an EBV-encoded SAG (Ref. 38)
Superantigens in diabetes mellitus
also be caused by streptococcal infection through the release of streptococcal SAGs. However, the site of infection is usually deeper. Both conditions are acute and serious and have a fatality rate of approximately 30%. Treatment usually involves the administration of pooled g globulin, which contains naturally occurring neutralizing anti-toxin antibodies. Patients have no anti-TSST neutralizing responses.
Kawasaki’s disease Kawasaki’s disease (KD) is an acute febrile disease of children aged about five years. Without treatment, it can be fatal. In Japan and the US, it has become one of the most common causes of acquired heart disease in children. Intravenous immunoglobulin therapy is highly effective when given early, suggesting that the agent is a toxin that is neutralized by naturally occurring anti-toxin antibodies. The etiological agent is not known, but several lines of evidence suggest that it is SAG mediated. The symptoms of the disease resemble TSS and several studies have shown that there is a selective expansion of a Vb2 subset of T cells44. In one study of 16 patients, 13 were found to have TSST positive isolates of S. aureus in their pharynx, rectum or groin45. Further investigations have, however, not been able to substantiate the connection with S. aureus TSST strains and there is still debate over whether SAGs really are the culprit in KD. In another article, a link between the Yersinia pseudotuberculosis SAG YPM and KD has been suggested51, although this link has yet to be firmly established.
Recent reports of a SAG expressed from a novel human endogenous retrovirus linked with insulin dependent diabetes mellitus (IDDM) have created considerable excitement. Conrad et al. had previously reported that infiltrating T cells in the pancreas of two acutely diabetic juveniles were biased for the Vb7 domain using immunohistochemistry with Vb specific monoclonal antibodies (mAbs)39. This discovery was followed more recently by a report from the same author on the isolation of a new human retrovirus named IDDMK that is responsible for this activity. Moreover, the putative SAG was encoded within the 39 end of the IDDMK viral envelope region40. IDDMK is highly homologous to members of the HERVK family of human endogenous retroviruses. The HERVK genes are highly abundant (over 50 copies/genome) integrated retroviral elements that are largely silent in humans except in certain malignancies. Evidence for IDDMK viral expression in plasma from 10/10 IDDM patients against 0/10 normal patients was greeted with much excitement. This initial excitement has since been tempered by further scrutiny. Many independent studies have not supported the existence of an active IDDMK virus and instead suggest that the original PCR technique used to amplify the IDDMK’s RNA genes also amplified HERVK sequences41. Experiments in our laboratory using the same methods as Conrad have only uncovered HERVK and we have yet to find any evidence of SAG activity expressed from the region of the HERVK viral genome proposed in the original report.
It has been proposed that SAGs derived from bacteria, viruses or mycoplasmas might contribute to the pathogenesis of autoimmune disease by activating T cells that are specific for self antigens2,3. Early reports observed Vb-specific enrichment of T cells in common diseases such as arthritis. These created much excitement. Paliard et al., for example, analysed the TCR b chain profiles of synovial T cells and found a selective expansion of human Vb14-expressing T cells46. Using an animal model of multiple sclerosis, experimental autoimmune encephelomyelitis (EAE), it has been shown that administration of SEB to mice recovering from EAE triggered a rapid relapse of the disease. This effect was the result of direct stimulation of the Vb3 positive autoreactive MBP peptide specific T cells that initially caused the brain inflammation. This was clear evidence that SAGs could, under the right conditions, break the tolerance or suppression of autoreactive T-cell clones and induce a state of autoimmune disease47–49. Despite these early reports, there has been little clear evidence directly linking a SAG to any autoimmune disease. Nevertheless, sufficient evidence has been provided to suggest that SAGs can and do play a secondary role in autoimmune activation through the relatively indiscriminate stimulation of auto-reactive T cells.
Superantigens and disease
Concluding remarks
Toxic shock syndrome (TSS) is a superantigen-mediated disease. Toxic shock results from the release of TSST-1 into the blood stream by TSST-1 producing strains of S. aureus and is characterized by fever, rash, desquamation, hypotension and major organ involvement. The sites of infection are usually superficial and the most common site is the vagina in menstruating women. An outbreak in the late 1970s was linked to the prolonged use of a particular high absorbency tampon which promoted the rapid growth of S. aureus42. TSST is found in approximately 70% of all toxic-shock inducing strains of S. aureus and studies have shown that TSS patients have elevated levels of Vb2 T cells in the blood during the acute stages of the disease43. TSS can
As a family of molecules, SAGs display a remarkable degree of variation in structure and function around the common goal of bringing the TCR and MHC-II molecules together. They have provided remarkable insight into the mechanisms of T-cell activation and the extraordinary sensitivity of T-cell antigen recognition. Because of the ubiquitous expression and widespread carriage of these toxins by both staphylococci and streptococci, it is clear that our immune systems are under constant challenge from these extremely powerful agents, which are therefore likely to influence our response to other challenges. What benefit these toxins serve the bacteria remains a mystery. SAGs remain a curious enigma – a result of the constant battle between microbes and the immune system.
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