| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
CUTTING EDGE |
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
ß TCR initiates activation of T cells by recognition of peptide
fragments of Ag bound in the groove of MHC molecules. Bacterial
superantigens
(b-SAgs),3 as
exemplified by the Staphylococcus aureus enterotoxins, such
as SEA and SEB, are powerful T cell mitogens produced by certain
bacteria that activate T cells by binding to the Vß region of the TCR
(1, 2). SAgs do not require Ag processing, but efficient
presentation requires binding of the SAg to an MHC class II molecule at
the surface of an Ag presenting cell (3, 4, 5). b-SAgs bind
to class II at sites distinct from the conventional peptide binding
groove on the MHC class II protein. The precise binding sites between
b-SAgs and MHC class II vary for different b-SAgs. SEB binds to a site
on the -chain of class II (6). SEA binds to two sites
on the class II molecule: a high affinity site on the ß-chain that
depends on a Zn2+ ion coordinated between
His81 on the ß-chain and a
Zn2+-binding site on SEA, and a low affinity site
on the -chain similar to the SEB-binding site (7, 8). The interaction between TCR and SAg is mediated by the Vß region of the TCR (9, 10, 11, 12). The binding sites on the b-SAgs for the TCR Vß, and on Vß for the b-SAg, are well defined from mutagenesis and structural studies (10, 11, 12, 13, 14, 15, 16). Residues on one face of the b-SAg contact predominantly main-chain atoms of the Vß, rather than side chain residues, resulting in the ability of certain b-SAgs to interact with several different Vß elements (11, 12).
The affinity between a MHC:peptide complex and its TCR ligand is comparatively weak, with dissociation constants in the micromolar range (17, 18). Due to the strong activation of the T cell by b-SAg recognition, it had been assumed that the biochemical interactions between these proteins would show different, and likely stronger, binding kinetics and affinities than for conventional MHC:peptide ligands (1). Data for the binding of b-SAgs to class II show surprisingly low affinity binding, in the micromolar range for SEB, and very fast on and off rates (19, 20). Binding of TCR to b-SAg also shows weak and fast binding kinetics (14, 20, 21, 22). A synergistically increased signal was obtained when SEB was premixed with soluble class II protein and allowed to bind to immobilized TCR, although the kinetics of this trimolecular interaction were not determined (20).
In this work we analyze the interaction between soluble, heterodimeric TCR, a soluble b-SAg (SEA), and the mouse MHC class II molecule I-Ek. Kinetic measurements of the interactions between the three components are presented, including binding of the TCR to the MHC class II-b-SAg complex. These measurements demonstrate synergy in the binding of TCR to b-SAg in the presence of class II that result in affinities close to those found for TCR interacting with MHC:peptide complexes.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
The 2B4 TCR recognizes I-Ek with peptide
from pigeon cytochrome c residues 81–104. Its TCR is
AV11S2J56, BV3S1A1D2J2S5 (23, 24). The -chain gene was
cloned from 2B4 cDNA with the V11 primer 5'-CAT GCC TCC GAG AAA AGA
GAG GCT GAA GCT AAT TCA AAA GCT AAT CAG-3', and the C primer was
5'-CGT GGT ACC TGC GGC CGC ATC ACA GGG AAC GTC TGA-3'. The ß-chain
gene was cloned with the Vß3 primer 5'-CAT CGC TCC GAG AAA AGA
GAG GCT GAA GCT GAT CAG GTG GAG CAG AGT CCT-3' and Cß primer
5'-GTA TCG CGG CCG CAC AGT CTG CTC GGC CCC-3'. TCR was expressed using
Pichia pastoris expression vectors pPIC9 and pPIC9K
(25) (Invitrogen, San Diego, CA), as described
(17). The TCR -chain was cloned into a modified pPIC9K,
with a deletion in the HIS4 gene (612 bp). The TCR ß-chain
was cloned first into pPIC9, then into pPIC9K.
Expression in P. pastoris
Transfection (by electroporation) of P. pastoris
strain GS115 and selection for recombinant clones was accomplished
using the Invitrogen protocols. Briefly, sequential selection for
growth on RDB plates (without histidine), followed by selection for
growth on G418 yeast extract/peptone/dextrose (YPD) plates, selected
for colonies with both - and ß-chain genes integrated. These were
screened for their
Mut+/Muts phenotype
by comparative growth on minimal dextrose (MD) and minimal methanol
(MM) plates. Replicate MM plates were blotted onto nitrocellulose, and
- and ß-chain secreting clones were detected as described using
anti-C and Cß mAbs (26). For induction of
methanol metabolism, cells were grown to mid-log phase in buffered
glycerol-complex medium (BMGY) then resuspended in buffered
methanol-complex medium (BMMY) (BMGY with 0.5% methanol, according to
Invitrogen protocols), and grown for up to 72 h. In some
experiments, P. pastoris was grown in a Biostat E
fermentation unit (Braun Melsungen, Allentown, PA), in volumes up to
10 L.
Purification and characterization of TCR protein
After concentration of the culture supernatant, TCR was purified
by ion-exchange chromatography on DE52 anion exchange resin (Whatman,
Hills-boro, OR) and eluted with a salt gradient (10–500 mM NaCl).
Aggregates and monomeric TCR proteins were then removed by secondary
purification on Superdex 200 HR and Superdex 75 columns
(17). Fractions from the gel filtration column were
assayed by surface plasmon resonance using a BIAcore 2000 Biosensor
(BIAcore; Pharmacia, Uppsala, Sweden). Anti-Cß Ab H57-597 was
immobilized on a BIAcore chip, and Ab-captured TCR was tested for
heterodimer content (TCR ß) by flowing an anti-C Ab,
H28-710 over the surface. Integrity and purity of the protein was
assessed by nonreducing SDS-PAGE followed by Coomassie blue staining.
The presence of the heterodimeric TCR chains were detected by reducing
SDS-PAGE followed by immunoblotting. Purified protein was detected with
either anti-C or anti-Cß Abs, followed by amplification
with biotinylated goat anti-hamster IgG and streptavidin-HRP
(Vector Laboratories, Burlingame, CA) and visualization using an ECL
detection kit (DuPont, Boston, MA).
Soluble MHC class II proteins
I-Ek was purified from Triton X-100 lysates of CH1 (H-2k) B lymphoma cells by affinity chromatography on a 14-4-4S mAb column as previously described (27). I-Ek preparations were tested for serologic integrity by ELISA and for purity by SDS-PAGE, with protein visualized by silver staining. Proteins were stored at -20°C at 50–200 µg/ml in 0.25% n-octylglucoside, 10 mM Tris (pH 8.0), and 150 mM NaCl.
Surface plasmon resonance analysis
This was performed largely as described (17).
Before analysis of purified proteins by surface plasmon resonance, both
ligand for immobilization and analyte proteins were passed over a
Superdex HR200 column to remove aggregates. A flow of PBS (150 mM NaCl
and 0.005% surfactant P20 (pH 7.4)) was maintained throughout the
immobilization procedure. About 1200–2800 response units (RU) of
protein were immobilized. Both TCR proteins and mAbs were injected at a
flow rate of 10 µl/min at 25°C. All analyses were done using the
nonlinear fit method of O’Shannessy (28) and
BIAevaluation 2.0 software (BIAcore). The bimolecular interactions were
described by the simple binding model (Langmuir equation [A
+ B = AB]). A noncompetitive analyte
binding model was used to derive the rate constants for the
TCR-SEA-class II interactions (see Fig. 2
). This model describes the
binding of one analyte molecule (A1) to the
immobilized ligand (B), which is followed by a second step
in which another analyte molecule (A2)
can bind to the existing ligand-analyte complex
 |
 |
 |
 |
 |
|
|
Results and Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
In this study, we have used surface plasmon resonance measurements
to determine the kinetics of the binding among soluble 2B4 TCR, the
b-SAg SEA, MHC class II (I-Ek), and the kinetics
of formation of the complex of these molecules. First, we measured the
binding of SEA to immobilized I-Ek (Fig. 1A). SEA bound to
I-Ek with a relatively fast association rate,
kon = 3.2 x
104
M-1s-1, and a fairly slow
dissociation rate, koff = 3.5 x
10-3 s-1 (Table I). This gives a
Kd of 1.3 x
10-7 M for the SEA-class II interaction which is
similar to the Kd determined at
steady-state from a Scatchard plot
(Keq = 3.2 x
10-7 M) (Fig. 1A). The affinity of
the binding of SEA to class II molecules on, or purified from a cell
surface, has previously been found to range between 3 x
10-7 and 3 x 10-8 M
(5, 7, 10, 29), but the affinity is dependent on the
nature of the peptide bound to class II, which can lower the affinity
for a particular molecule to 1 x 10-6 M
(8). The higher affinities probably represent the
biologically important interactions as far as T cell activation is
concerned, and the on rate that we measure is close to the fastest of
those measured in the peptide-specific SEA-binding experiments
(8). The off rate measured here is intermediate compared
with binding to specific class II:peptide complexes, which range
between 2 x 10-2 and 2 x
10-4 s-1
(8). It is likely that the peptides that lead to weak SEA
binding act by interfering with the interaction of histidine 81 of the
class II ß-chain with the Zn2+-dependent
binding of SEA, because His81ß is involved in
the interaction with peptide (6). SEB-class II binding
appears to have a much lower affinity of 
10-6
M (19, 20). This difference is likely to be due to the
lack of the high affinity binding site for MHC class II that is present
in SEA.
|
|
B).
The Vß3 element of the 2B4 ß-chain recognizes SEA well (1, 2, 9, 10). The 2B4 TCR bound to I-Ek with
extremely low affinity, demonstrating very fast binding kinetics for
both the association and dissociation phases. This result is not
surprising given that the specific cytochrome c peptide is
not present. A very small increase in binding signal over that observed
on a control surface was observed (Fig. 1B), and as such the
association and dissociation constants of this weak interaction could
not be measured. We estimate that the
Kd is >2 x
10-4 M. In contrast, the affinity of the TCR for
the immobilized b-SAg, SEA, although relatively weak, was significantly
higher than those for TCR-class II interactions (Figs. 1, B
and C). Although the on rate was too fast to be measured,
the TCR-SEA binding kinetics revealed a detectable off-rate
(koff = 0.065
s-1). Keq
measurement by Scatchard plot analysis gave an affinity of 6.9 x
10-5 M. Scatchard analysis of 2B4 TCR binding to
another b-SAg for which the Vß3 is reactive, SED, gave a
Kd of 1.01 x
10-4 M (data not shown). These data indicate
that the TCR binds to b-SAg with higher affinity than for a class II
molecule without the relevant peptide, whereas binding affinity of SEA
to I-Ek was more than 200-fold higher than for
binding to TCR. As the intrinsic affinity of the TCR for both its
ligands is still rather weak, this suggests that binding may be
enhanced during the formation of the ternary complex with SEA and class
II, to enable T cell activation by b-SAgs to occur at such low
concentrations. We believe this to be the first measurement of the
binding between TCR and SEA.
Previous analysis of the TCR-SEB interaction showed a relatively fast
on-rate of 104
M-1s-1 (20),
in the range of the faster on-rates for TCR binding to MHC:peptide
complexes (18). The off-rate for the human TCR binding to
SEB (10-2 s-1)
(20) was similar that determined here for TCR-SEA
(6.5 x 10-2 s-1),
and in the range for off-rates for MHC:peptide (18). The
affinity of the TCR for SEB (8 x 10-7 M)
was thus stronger than the interaction of TCR with class II:peptide but
at the top of the range for class I restricted receptors binding
MHC:peptide (17, 18). In contrast, it has been reported
that murine Vß8.2 TCR can bind to SEB with low affinity; 1.4 x
10-4 M (21), and SEC with higher
affinity; 1 or 5 x 10-6 M for SEC2
(21, 22) and 4.5–8.5 x 10-6
M for SEC3 (14, 21). Thus the binding of the human TCR to
SEB may be particularly high for a TCR-b-SAg interaction
(2).
Trimolecular interactions
Until now, there has not been a quantitative assessment of the
trimolecular complex between TCR, SEA, and MHC class II, although a
qualitative experiment using human TCR, MHC class II and the b-SAg SEB
demonstrated that this was more than the sum of its parts
(20). We decided to determine the affinity and the
kinetics of the binding of TCR to the complex of SAg and class II
proteins. As shown in Fig. 1A, SEA binding to
I-Ek was easily detectable, and while the on-rate
was fast, SEA dissociated from I-Ek in a
relatively short time (half-life = 2.9 min). This was not long
enough to provide a stable surface for TCR to bind. Thus, sequential
injection of SEA followed by TCR created a decaying surface for the TCR
to bind to the SEA-class II complex that was unsuitable to measure
formation of a stable trimolecular complex (Fig. 2A). However, compared with
the injection of TCR over a class II surface (Fig. 1B), the
sequential injection of TCR after SEA over the class II surface
revealed different kinetics, apparently indicating the formation of a
transient trimolecular complex (Fig. 2A). A mixed injection
of SEA and TCR produced better results: a higher binding response and a
more stable complex of TCR-SEA-class II, with different binding
kinetics from those of SEA-class II, was observed (Fig. 2B).
Therefore, binding of SEA and TCR to I-Ek was
measured by coinjecting SEA and TCR together over immobilized
I-Ek (Fig. 2, B and C).
With I-Ek immobilized on the sensor surface,
coinjected TCR and SEA bound stably to the MHC class II protein
surface. Due to the biphasic nature of the binding curves, the simple
Langmuir model of binding (A + B =
AB) failed to provide a good fit, and Scatchard analysis
showed a nonlinear relationship (Fig. 2C). The analysis of
two-step interactions is complex, and thus the equilibrium dissociation
and association constants were measured for the trimolecular complex
using a model that describes "noncompetitive binding of two analytes
to the same ligand" (see Materials and Methods). We found
that the curves gave a better fit to this model
(
2 = 0.037). The first step of the
interactions apparently describes the binding of SEA to class II,
whereas the second step describes the binding of TCR to this complex.
As shown in Table I, the kon and
koff of the first step are virtually
identical with those calculated for SEA binding to class II. The
formation of the ternary complex, described in the second step, gave a
kon of 4.83 x
103
M-1s-1 and a
koff of 0.011
s-1. The half-life of the TCR bound to the
SEA-class II complex is therefore 36.5 s, compared with 10.7
s for binding to the SEA alone. Thus, during formation of the
trimolecular complex of TCR-SEA-class II, two different complexes were
being formed sequentially. It is likely that the first complex formed
is that of SEA and class II because of their higher affinity compared
with TCR-SEA and TCR-class II, and because of the similarity of the
kinetics of the first stage of the reaction to the bimolecular
interaction between these molecules (Table I). The SEA-class II complex
then serves as a ligand to which the TCR can bind, resulting in the
formation of the ternary complex TCR-SEA-class II. In addition, the
difference between TCR-SEA-class II binding and TCR-SEA binding
probably represents the stability which is added by the interaction
between TCR and class II. This may be due to the influence from the TCR
-chain interaction with class II (12). Crystal
structures of TCR-ß complexed to b-SAgs, of b-SAgs complexed to class
II, and of TCR-ß, have been used to assemble models for the
TCR-ß interacting with the b-Sag-class II complex (11, 12). These findings show that the Vß domain has no contact
with class II, but indicate that the V domain interacts with the
class II ß-chain, providing an explanation for the influence of V
on certain SAg responses (12). Our results suggest that
the V interaction with class II provides extra stability in the
binding of TCR to b-SAg-class II over binding to b-SAg alone.
The dissociation constant (Kd) of the trimolecular complex of TCR bound to SEA-class II is calculated as 2.27 x 10-6 M. It is both surprising and interesting that this affinity is close to what has been reported for the higher range of TCR-peptide:MHC interactions with agonist ligands (17, 18). It has been reported that a small number of MHC:peptide complexes are able to cause activation of a large number of TCRs through serial triggering (30). MHC:peptide Ags and b-SAgs are both very effective at T cell activation. In contrast, monovalent anti-CD3 Abs are much less efficient at triggering TCR, perhaps because the high affinity of binding prevents them from dissociating and therefore from serially triggering TCRs. This finding implies that there is a middle ground for TCR affinity, which is optimal for T cell activation (30). Several b-SAgs, including SEA, have more than one binding site for MHC class II, and this may contribute to cross-linking of the class II on APC, with increased potency for SAg stimulation of T cells. Interestingly, there has been a recent report (31) of a potent, novel b-SAg from Streptococcus pyogenes, named SPE-G. The binding affinity of SPE-G for MHC class II is the highest so far determined for any toxin (15 nM). However, SPE-G is a weaker stimulator of human T cells than SEA and other SAgs tested, indicating that affinity for MHC class II does not equate with potency toward T cells. This result concurs with data presented in our own work. Our data show that the binding of a TCR to a b-SAg-class II complex is comparable in affinity to binding of a strong agonist MHC:peptide ligand to TCR. Thus the b-SAg, despite its low affinity for both TCR and class II, is able to mimic the activation of T cells by MHC:peptide.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. N. R. J. Gascoigne, Department of Immunology, IMM1, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address:
3 Abbreviations used in this paper: SAg; superantigen, b-SAg; bacterial SAg; SEA, B etc., Staphylococcus aureus enterotoxin A, B, etc.; RU, response units.
Received for publication March 19, 1999. Accepted for publication April 30, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
ß T cell receptors. Annu. Rev. Immunol. 16:523.[Medline]
ß T-cell receptor and its superantigen and class II-MHC/peptide ligands. Mol. Immunol. 6:493.
gene segments. Nature 317:430.[Medline]
This article has been cited by other articles:
|
|
 |
|
  O. Dauwalder, D. Thomas, T. Ferry, A.-L. Debard, C. Badiou, F. Vandenesch, J. Etienne, G. Lina, and G. Monneret Comparative inflammatory properties of staphylococcal superantigenic enterotoxins SEA and SEG: implications for septic shock J. Leukoc. Biol., October 1, 2006; 80(4): 753 - 758. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Llewelyn, S. Sriskandan, N. Terrazzini, J. Cohen, and D. M. Altmann The TCR V{beta} signature of bacterial superantigens spreads with stimulus strength Int. Immunol., October 1, 2006; 18(10): 1433 - 1441. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. D. Pless, G. Ruthel, E. K. Reinke, R. G. Ulrich, and S. Bavari Persistence of Zinc-Binding Bacterial Superantigens at the Surface of Antigen-Presenting Cells Contributes to the Extreme Potency of These Superantigens as T-Cell Activators Infect. Immun., September 1, 2005; 73(9): 5358 - 5366. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. M. Lavoie, A. R. Dumont, H. McGrath, A.-E. Kernaleguen, and R.-P. Sekaly Delayed expansion of a restricted T cell repertoire by low-density TCR ligands Int. Immunol., July 1, 2005; 17(7): 931 - 941. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Zavala-Ruiz, I. Strug, B. D. Walker, P. J. Norris, and L. J. Stern A hairpin turn in a class II MHC-bound peptide orients residues outside the binding groove for T cell recognition PNAS, September 7, 2004; 101(36): 13279 - 13284. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Blanchard, M. Decraene, K. Yang, F. Miro-Mur, S. Amigorena, and C. Hivroz Strong and Durable TCR Clustering at the T/Dendritic Cell Immune Synapse Is Not Required for NFAT Activation and IFN-{gamma} Production in Human CD4+ T Cells J. Immunol., September 1, 2004; 173(5): 3062 - 3072. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Zavala-Ruiz, E. J. Sundberg, J. D. Stone, D. B. DeOliveira, I. C. Chan, J. Svendsen, R. A. Mariuzza, and L. J. Stern Exploration of the P6/P7 Region of the Peptide-binding Site of the Human Class II Major Histocompatability Complex Protein HLA-DR1 J. Biol. Chem., November 7, 2003; 278(45): 44904 - 44912. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. K. McCormick, T. J. Tripp, A. S. Llera, E. J. Sundberg, M. M. Dinges, R. A. Mariuzza, and P. M. Schlievert Functional Analysis of the TCR Binding Domain of Toxic Shock Syndrome Toxin-1 Predicts Further Diversity in MHC Class II/Superantigen/TCR Ternary Complexes J. Immunol., August 1, 2003; 171(3): 1385 - 1392. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Petersson, H. Pettersson, N. J. Skartved, B. Walse, and G. Forsberg Staphylococcal Enterotoxin H Induces V{alpha}-Specific Expansion of T Cells J. Immunol., April 15, 2003; 170(8): 4148 - 4154. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Borovsky, G. Mishan-Eisenberg, E. Yaniv, and J. Rachmilewitz Serial Triggering of T Cell Receptors Results in Incremental Accumulation of Signaling Intermediates J. Biol. Chem., June 7, 2002; 277(24): 21529 - 21536. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. G. Burrows, Y. K. Chou, C. Wang, J. W. Chang, T. P. Finn, N. E. Culbertson, J. Kim, D. N. Bourdette, D. A. Lewinsohn, D. M. Lewinsohn, et al. Rudimentary TCR Signaling Triggers Default IL-10 Secretion by Human Th1 Cells J. Immunol., October 15, 2001; 167(8): 4386 - 4395. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. M. Lavoie, H. McGrath, N. H. Shoukry, P.-A. Cazenave, R.-P. Sekaly, and J. Thibodeau Quantitative Relationship Between MHC Class II-Superantigen Complexes and the Balance of T Cell Activation Versus Death J. Immunol., June 15, 2001; 166(12): 7229 - 7237. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Hogan, J. VanBeek, D. R. Broussard, S. L. Surman, and D. L. Woodland Identification of MHC Class II-Associated Peptides That Promote the Presentation of Toxic Shock Syndrome Toxin-1 to T Cells J. Immunol., June 1, 2001; 166(11): 6514 - 6522. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. S. Andersen, C. Geisler, S. Buus, R. A. Mariuzza, and K. Karjalainen Role of the T Cell Receptor Ligand Affinity in T Cell Activation by Bacterial Superantigens J. Biol. Chem., August 31, 2001; 276(36): 33452 - 33457. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. S. Andersen, C. Menne, R. A. Mariuzza, C. Geisler, and K. Karjalainen A Response Calculus for Immobilized T Cell Receptor Ligands J. Biol. Chem., December 21, 2001; 276(52): 49125 - 49132. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
