By
From the Department of Molecular Medicine, University of Auckland, Auckland, New Zealand
The exotoxins produced by Staphylococcus aureus and
Streptococcus pyogenes are prototype molecules for the
larger family of superantigens (SAGs). This family now includes many structurally unrelated molecules of disparate
origins reflecting a wide evolutionary convergence towards
the common goal of subverting T cell antigen recognition.
SAGs bind simultaneously to MHC class II molecules and
TCRs, bringing them together in such a way as to induce
profound T cell activation. How SAGs, and in particular
the staphylococcal enterotoxins do this, has been the subject of intense interest over the last nine years. It's the subject of an elegant paper from Leder et al. at the Center for
Advanced Research at the University of Maryland published in this issue of the Journal of Experimental Medicine
(1). This paper examines the thermodynamics of staphylococcal enterotoxin (SE) B and C3 binding to TCR and ultimately raises questions about how similar peptides and
SAGs are in their engagement and triggering of T cells.
Much is now known about the fine structure of the staphylococcal and streptococcal exotoxins. They are small,
single chain proteins constructed from two globular domains. In all toxins except streptococcal pyrogenic exotoxin C (SPEC), the variable NH2-terminal domain contains a generic binding site for the invariant The currently held model of T cell recognition is that the TCR
discriminates exquisite differences in peptide-MHC (pepMHC) complexes on the basis of small quantitative differences in affinity (3). All pepMHC-TCR interactions measured thus far (barring one [reference 4]) have been found to
exhibit low affinities and short half-lives. This fits well with
a mechanism of sequential engagement of multiple TCR
molecules by a single pepMHC complex elegantly revealed
by Antonio Lanzavecchia and colleagues. They have shown
that at limiting surface concentration, a single pepMHC complex triggers as many as 200 TCR complexes (5). Their
serial triggering model predicts that not only must the interaction between pepMHC and TCR be brief, but also
that increasing binding affinities might inhibit serial triggering and thus reduce efficiency. As an added level of complexity, pepMHC complexes with affinities that fall just below a threshold required for T cell activation are antagonists,
inducing T cell anergy rather than activation. Rabinowitz
et al. (6) have defined this difference in biological outcome
as determined by the ratio of complete (positive) to negative (incomplete) signals via the TCR.
In real terms, affinity differences towards TCR of as little
as 10-fold appear to be sufficient to induce antagonism. For instance, in a well studied Ova/Kb 42.12 TCR model, the
affinity difference between the agonist Ova/Kb complex
(6.5 µM) and two antagonist peptides, E1/Kb and R4/Kb
(22 and 57 µM respectively), are only 3-fold and 9-fold respectively (7). In comparison, the binding affinities for the
SEC3 mutant/mV A further anomaly of the data presented is the finding
that SEB is 10-fold more potent than SEC3 to V So what can we deduce from these results about the response to SEC3 and its similarities to peptide antigens? First,
the affinity and short half-life of the SEC3-mV Where T cells appear to differ markedly in their response
to SEB and SEC3 in comparison to peptides is in their apparent tolerance of significant differences in ligand affinity.
For a pepMHC ligand where even the slightest difference
in affinity results in an altered outcome, a 68-fold decrease
should in theory abrogate the response altogether. This
clearly does not happen even for the weakest binding
SEC3 mutant and points to a fundamental difference between SAGs and peptide ligation of TCR that must reflect
the different ways in which these two antigenic forms are
presented. Unlike peptides, which are for all intents and
purposes bound irreversibly to MHC class II, bacterial
SAGs are in equilibrium between free and bound states.
With affinities towards both MHC class II and TCR in the
micromolar range, the vast majority of SEB or SEC3 molecules will be unbound at the physiological relevant pico-femtomolar range. How then do they trigger T cells at
concentrations many orders of magnitude less than their
dissociation constants? In the case of SEB, any transitional
dimer complex that forms with either TCR or with MHC
class II molecules appears to be stabilized by an additional
TCR-MHC interaction before the TCR is triggered. Serial triggering would require SEB to be bound more securely to MHC class II and only transiently engage TCR.
On the basis of its measured affinities for both molecules,
this does not seem possible.
SEB binds weakly to
MHC class II The second class II binding site of SEA also introduces
another possible way for SAG-mediated T-cell activation,
namely promotion of MHC cross-linking on the surface of
APCs. SEA is a potent activator of APCs promoting cell
adhesion and aggregation (13, 14). It is not yet known
whether MHC class II cross-linking also enhances T cell
signaling directly, but it might be possible that MHC aggregation promotes areas of high local ligand concentration, which in turn increases the avidity for TCR and induces local TCR clustering.
There are at least two more SAGs that are also able to
cross-link class II, both using different mechanisms to SEA.
As shown from its crystal structure, SED has the capability
to form zinc-dependent homodimers by coordinate binding of two Zn2+ ions between the COOH-terminal domain (homologous to the high affinity zinc site of SEA) of
two SED molecules. Binding to MHC class II is proposed
to occur via the SED NH2-terminal domain to the class II
The fact that SAGs have coevolved at least three separate
mechanisms to bind and coalesce MHC class II on the surface of APCs suggests that surface aggregation plays an important function in T cell triggering. However, it doesn't
explain why toxins with only one MHC class II binding
site like SEB, SEC1-3, and TSST are still as potent as those
toxins that have two. We can only assume that every SAG
has been optimized to stimulate efficiently under the limitations of its own modus operandi. The development of
additional modes of MHC class II binding reflects a need
by bacteria to stimulate more T cells by accommodating a
greater range of individual SAG-TCR interactions.
In conclusion, while SAGs and pepMHC complexes
bind to TCRs with similar affinities, the tolerance to a 60-fold decrease in SEC3 affinity for TCR and the apparent
absence of any antagonist responses indicates that T cell activation by SAGs is optimal over a much broader range of
affinities. Perhaps this reflects the importance of avidity
rather than intrinsic affinity in SAG activation. For peptide
activation, the window of affinity for agonist/antagonist responses, set during thymic development, is very narrow indeed and provides for both positive and negative TCR signals. The serial triggering model proposes a mechanism for
peptide-mediated TcR signaling that is unlikely to require TcR dimerization or cross-linking and perhaps relies on its
very absence to discriminate subtle differences in pepMHC
affinities. On the other hand, SAGs have coevolved several
elaborate mechanisms that promote MHC class II coalescence. This could promote TcR signaling through clustering and/or TCR dimerization.
The murky picture we have of SAG-induced T cell activation is not helped by the remarkable variations seen in
SAG binding. A clearer picture is sure to emerge as more
and more SAG-TCR interactions are examined using similar quantitative approaches to those used by Leder et al.
(1). However, the most fundamental question still remains;
why do bacteria produce SAGs in the first place?
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1 domain of
MHC class II. In a subset of toxins including SEA, SED,
SEE, and SPEC, an additional zinc-dependent site is located in the larger more conserved COOH-terminal domain that binds to the highly polymorphic
1 domain of
MHC class II. The TCR binding site is located in a shallow
groove between the two toxin domains (except in toxic
shock syndrome toxin [TSST] and probably also SPEC). In
the paper by Leder et al. (1) the energetic contribution of
individual residues within the TCR binding site of SEC3
and SEB to a soluble form of mV
8.2 TCR have been determined using a combination of sedimentation equilibrium and real-time bio-sensing techniques. A previous
three-dimensional crystal structure of SEC3/V
8.2 is used
as a guide to mutate all those residues that make contact
with V
8.2 (2). The authors generate a thermodynamic map of the TCR site of SEC3 that shows that overall binding energy is shared fairly evenly among all residues but
there are five that are clustered in the center of the binding
site that make significantly more energy contributions than
the others. Two residues, an asparagine at position 23 and a
tyrosine at position 90, are conserved in all toxins, whereas
another, a glutamine at position 210, varies between toxins
and appears to be important in determining V
specificity.
Using these mutants to stimulate V
8.2 transgenic T cells,
the authors reveal a simple almost linear relationship between ligand affinity and biological potency. This might
seem like a rather obvious association, but the relationship
between T cell ligand affinity and the ensuing biological
response is anything but obvious.
8.2 interactions ranged from 3.5 µM to
240 µM, an affinity difference of >60-fold. Moreover,
even the weakest binding SEC3 mutant SEC3 Y90A required a mere 45-fold more toxin to stimulate at wild-type levels. This represents only a small difference in potency
considering that SAG responses are usually measured over
five to six orders of magnitude. No antagonist response was
seen even from the weakest binding SEC3 mutant.
8.2-bearing T cells but showed much weaker binding to soluble
V
8.2. The authors explain this contradiction by introducing the role of MHC class II in the cooperative stabilizing
of SEB/TCR, interactions and indeed there is firm evidence to suggest this does occur. Seth et al. (8) showed that
although SEB has only very weak individual affinities towards either HLA-DR1 and a hV
3 TCR alone, SEB
mixed with HLA-DR1 and TCR together produced a
much more stable trimer with an apparent affinity far
greater than the sum of the two components. It is important to mention that the solution affinities of the SEC3 mutants towards TCR were not calculated in the presence of
MHC class II so we do not know yet whether MHC class
II also stabilized the SEC3-TCR interaction. Therefore it
is possible that the real ligand affinities of SEC3-MHC
complexes to TCR are much higher.
8.2 interaction (Kd = 3.5 µM and 5 s, respectively) in the absence of
class II is very similar to those measured for several pepMHC-
TCR solution complexes. Therefore we cannot attribute
the extreme potency of SEC3 simply to tighter binding to
TCR. In fact, an attempt by the authors to increase the affinity of SEB by engineering its site to look like that of
SEC3 was only partially successful. Although the authors
managed to increase the SEB-TCR affinity 10-fold with a
single V26Y mutation, this achieved only a modest 4-fold
increase in biological response. This result is interesting because it hints that SAGs can't be made to bind more strongly
to TCR than they already do, and is consistent with the
concept of "optimal affinity" proposed for the serial triggering model. We will have to wait and see whether other
SAGs bind to TCRs with similar affinities in order to confirm this.
-chain and relies on continued engagement
between TCR and MHC class II to stabilize an activation complex, whereas SEA has achieved stable binding to the
APC surface in another way. It has developed a second zinc-dependent interaction with the polymorphic MHC class II
chain that is 100-fold stronger than its SEB-like
-chain
interaction (9). This enables one SEA molecule to cooperatively bind and stabilize a second weak
-chain SEA
interaction resulting initially in MHCII-(SEA)2 trimers (12). Indeed, this second high affinity binding is so important to SEA that mutations in the zinc site completely destroy SEA activity even though SEA can still bind to the
chain and presumably ligate TCRs in the same way as SEB.
Stabilizing SEA in this fashion presumably alleviates the reliance on MHCII-TCR compatibility required by SEB
and expands the repertoire of T cells activated by SEA. Interestingly, in humans, SEB really only stimulates V
3 bearing cells en masse while SEA stimulates V
1, 5.3, 6.3, 6.4, 6.9, 7.3, 7.4, 9.1, and 23.1 (11). It also means that SEA
achieves much greater stability on the surface of the APC,
perhaps allowing it to act more like a pepMHCII complex
in sequentially triggering multiple TCRs.
chain in a similar fashion to SEB. This could potentially
result in MHC cross-linking in an MHCII
-SED-SED-MHCII
mode (15). The recent crystal structure of SPEC
indicates that the generic class II
chain binding site in the
NH2-terminal domain has been lost in favor of an SPEC
dimer interface (16). SPEC instead binds only to the
chain by a zinc-mediated mechanism and thereby might
cross-link class II in a MHCII
-SPEC-SPEC-MHCII
mode. This has been supported by the finding that SPEC
readily dimerizes in solution and also cross-links MHC class
II to induce homotypic cell adhesion (16). In contrast to
the cross-linking mechanism of the bivalent SEA molecule,
dimeric SAGs like SED and SPEC might well be able to
promote TCR dimerization, due to the optimal location of
two TCR binding sites in the dimer structure.
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Footnotes |
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Address correspondence to John Fraser, University of Auckland, School of Medicine, Department of Molecular Medicine, Private Bag 92019, Auckland, New Zealand. Phone: 64-9-373-7599; Fax: 64-9-373-7492.
Received for publication 21 January 1998.
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References |
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