Altered superantigenic ligands demonstrate the quantitative nature of T-cell activation

In a recent study, a superantigen mutated in the TCR binding site (staphylococcal enterotoxin B (SEB)delta61Y) was described, which behaved as a partial agonist for a Vbeta17-expressing T-cell clone. Evidence is now presented to demonstrate that there is distinct heterogeneity in the response of primary T cells to this protein. Some Vbeta17 T cells responded to SEBdelta61Y by modulating surface receptor expression consistent with activation, and by proliferating. Other Vbeta17 T cells did not proliferate, nor did they display a receptor expression phenotype consistent with activation. However, when repeatedly exposed to the altered superantigen, some of these non-responders entered cell cycle. This pattern of responses was not recapitulated by providing additional costimulation via CD28, although such treatment did induce some of the 'unresponsive' Vbeta17 T cells to upregulate the IL-2 receptor, indicative of partial activation. It was also found that the heterogeneous pattern could be replicated using very low doses of native SEB. The data are discussed in the context of models of T-cell activation in which differences in TCR ligand affinity and dose determine qualitatively different response phenotypes.     

Cloning, expression and interaction of human T-cell receptors with the bacterial superantigen SSA.

Superantigens (SAgs) are a class of disease-causing and immunostimulatory proteins of bacterial or viral origin that activate a large number of T-cells through interaction with the Vbeta domain of T-cell receptors (TCRs). In this study, recombinant TCR beta chains were constructed with human variable domains Vbeta5.2, Vbeta1 and Vbeta2.1, expressed as inclusion bodies, refolded and purified. The Streptococcus pyogenes SAg SSA-1 was cloned and expressed as a soluble periplasmic protein. SSA-1 was obtained both as a monomer and a dimer that has an intermolecular disulfide bond. We analyzed the biological activity of the recombinant SAgs by proliferation assays. The results suggest that SSA dimerization occludes the TCR interaction site. Naturally occurring SSA dimerization was also observed in supernatants of S. pyogenes isolates. An SSA mutant [SSA(C26S)] was produced to eliminate the Cys responsible for dimerization. Affinity assays using a resonant biosensor showed that both the mutant and monomeric wild type SSA have affinity for human Vbeta5.2 and Vbeta1 with Kd of 9-11 microm with a fast kass and a moderately fast kdiss. In spite of the reported stimulation of Vbeta2.1 bearing T-cells by SSA, we observed no measurable interaction.     

Superantigen natural affinity maturation revealed by the crystal structure of staphylococcal enterotoxin G and its binding to T-cell receptor Vbeta8.2

The illnesses associated with bacterial superantigens (SAgs) such as food poisoning and toxic shock syndrome, as well as the emerging threat of purpura fulminans and community-associated methicillin-resistant S. aureus producer of SAgs, emphasize the importance of a better characterization of SAg binding to their natural ligands, which would allow the development of drugs or biological reagents able to neutralize their action. SAgs are toxins that bind major histocompatibility complex class II molecules (MHC-II) and T-cell receptors (TCR), in a nonconventional manner, inducing T-cell activation that leads to production of cytokines such as tumor necrosis factor and interleukin-2, which may result in acute toxic shock. Previously, we cloned and expressed a new natural variant of staphylococcal enterotoxin G (SEG) and evaluated its ability to stimulate in vivo murine T-cell subpopulations. We found an early, strong, and widespread stimulation of mouse Vbeta8.2 T-cells when compared with other SAgs member of the SEB subfamily. In search for the reason of the strong mitogenic potency, we determined the SEG crystal structure by X-ray crystallography to 2.2 A resolution and analyzed SEG binding to mVbeta8.2 and MHC-II. Calorimetry and SPR analysis showed that SEG has an affinity for mVbeta8.2 40 to 100-fold higher than that reported for other members of SEB subfamily, and the highest reported for a wild type SAg-TCR couple. We also found that mutations introduced in mVbeta8.2 to produce a high affinity mutant for other members of the SEB subfamily do not greatly affect binding to SEG. Crystallographic analysis and docking into mVbeta8.2 in complex with SEB, SEC3, and SPEA showed that the deletions and substitution of key amino acids remodeled the putative surface of the mVbeta8.2 binding site without affecting the binding to MHC-II. This results in a SAg with improved binding to its natural ligands, which may confer a possible evolutionary advantage for bacterial strains expressing SEG.     

Overexpressed Bcl-x(L) prevents bacterial superantigen-induced apoptosis of thymocytes in vitro

bcl-x, a homologous gene of bcl-2, has an anti-apoptotic function and appears to play a critical role in the development of lymphoid systems. To investigate the effect of overexpressed Bcl-x(L) on the development of T lymphocytes, we established two lines of transgenic mice by using Emu-chicken bcl-x(L) (cbcl-x(L)) transgene, where the cBcl-x(L) protein was expressed mainly in lymphoid cells. Although thymocytes and splenocytes from cbcl-x(L) transgenic mice are resistant to apoptosis in vitro, clonal deletion of thymocytes, recognizing endogenous self-superantigens in the thymus, still normally proceeded and no self-reactive T cells were found in the spleen of the transgenic mice. To dissect clonal deletion, we utilized two in vitro models, thymocytes/antigen presenting cells co-culture system and fetal thymus organ culture system. In both, bacterial superantigen staphylococcus aureus enterotoxin B (SEB) induces apoptosis of T cells with Vbeta8+ T cell receptor (TCR) reacting to SEB, which mimics clonal deletion of self-reactive thymocytes in vivo. SEB-induced depletion of Vbeta8+ T cells from thymocytes when taken from the transgenic mice was effectively inhibited. The data might raise the possibility that cell death process involved in clonal deletion in the thymus is a form of apoptosis inhibited by Bcl-x(L).     

Flow cytometric analysis of the Vbeta repertoire in healthy controls

BACKGROUND: Analysis of the T-cell receptor (TCR)-Vbeta repertoire has been used for studying selective T-cell responses in autoimmune disease, alloreactivity in transplantation, and protective immunity against microbial and tumor antigens. For the interpretation of these studies, we need information about the Vbeta repertoire usage in healthy individuals. METHODS: We analyzed blood T-lymphocyte (sub)populations of 36 healthy controls (age range: from neonates to 86 years) with a carefully selected most complete panel of 22 Vbeta monoclonal antibodies, which together recognized 70-75% of all blood TCRalphabeta(+) T lymphocytes. Subsequently, we developed a six-tube test kit with selected Vbeta antibody combinations for easy and rapid detection of single ("clonal") Vbeta domain usage in large T-cell expansions. RESULTS: The mean values of the Vbeta repertoire usage were stable during aging in blood TCRalphabeta(+) T lymphocytes as well as in the CD4(+) and CD8(+) T-cell subsets, although the standard deviations increased in the elderly. The increased standard deviations were caused by the occurrence of oligoclonal T-cell expansions in the elderly, mainly consisting of CD8(+) T lymphocytes. The 15 detected T-cell expansions did not reach 40% of total TCRalphabeta(+) T lymphocytes and represented less than 0.4 x 10(9) cells per liter in our study. Vbeta usage of the CD4(+) and CD8(+) subsets was comparable for most tested Vbeta domains, but significant differences (P < 0.01) between the two subsets were found for Vbeta2, Vbeta5.1, Vbeta6.7, Vbeta9.1, and Vbeta22 (higher in CD4(+)), as well as for Vbeta1, Vbeta7.1, Vbeta14, and Vbeta23 (higher in CD8(+)). Finally, single Vbeta domain expression in large T-cell expansions can indeed be detected by the six-tube test kit. CONCLUSIONS: The results of our study can now be used as reference values in studies on distortions of the Vbeta repertoire in disease states. The six-tube test kit can be used for detection of single Vbeta domain expression in large T-cell expansions (>2.0 x 10(9)/l), which are clinically suspicious of T-cell leukemia.     

Association between susceptibility to Theiler's virus-induced demyelination and T-cell receptor Jbeta1-Cbeta1 polymorphism rather than Vbeta deletion.

Theiler's murine encephalomyelitis virus (TMEV) induces demyelinating disease in susceptible mouse strains after intracerebral inoculation. The clinical symptoms and histopathology of the central nervous system appear to be similar to those of human multiple sclerosis (MS), and thus, this system provides an excellent infectious animal model for studying MS. The virus-induced demyelination is immune mediated, and the genes involved in the immune response such as those for the T-cell receptor beta-chain and major histocompatibility complex (MHC) haplotypes are known to influence disease susceptibility. To define whether the T-cell receptor Jbeta-Cbeta or Vbeta genes are associated with susceptibility, we have analyzed F2 mice from crosses of susceptible SJL/J (Vbeta(a)-JCbeta(b)) mice and resistant C57L (Vbeta(a)-JCbeta(a)) mice. Our results indicate that susceptibility to TMEV-induced demyelination is associated with restriction fragment length polymorphism reflecting the T-cell receptor Jbeta1-Cbeta1 region rather than the Vbeta polymorphism. This association becomes stronger when the MHC haplotype is considered in the linkage analysis. However, differences in the T-cell receptor alpha-chain haplotype have no significant influence on the pathogenesis of TMEV-induced demyelination.     

High affinity T cell receptors from yeast display libraries block T cell activation by superantigens

The alphabeta T cell receptor (TCR) can be triggered by a class of ligands called superantigens. Enterotoxins secreted by bacteria act as superantigens by simultaneously binding to an MHC class II molecule on an antigen- presenting cell and to a TCR beta-chain, thereby causing activation of the T cell. The cross-reactivity of enterotoxins with different Vbeta regions can lead to stimulation of a large fraction of T cells. To understand the molecular details of TCR-enterotoxin interactions and to generate potential antagonists of these serious hyperimmune reactions, we engineered soluble TCR mutants with improved affinity for staphylococcal enterotoxin C3 (SEC3). A library of randomly mutated, single-chain TCRs (Vbeta-linker-Valpha) were expressed as fusions to the Aga2p protein on the surface of yeast cells. Mutants were selected by flow cytometric cell sorting with a fluorescent-labeled SEC3. Various mutations were identified, primarily in Vbeta residues that are located at the TCR:SEC3 interface. The combined mutations created a remodeled SEC3-binding surface and yielded a Vbeta domain with an affinity that was increased by 1000-fold (K(D)=7 nM). A soluble form of this Vbeta mutant was a potent inhibitor of SEC3-mediated T cell activity, suggesting that these engineered proteins may be useful as antagonists.     

Partial T cell activation with an altered superantigenic ligand

T cells have the capacity to respond to ligands as full, weak, partial or null agonists, or indeed as antagonists. In the present paper, it is reported that staphylococcal enterotoxin B (SEB) mutated in a T cell receptor (TCR) contact site (SEBDelta61Y) behaves as an altered ligand for a T cell clone (AC20) that expresses the Vbeta17 TCR. The T cells were partially activated by SEBDelta61Y, as shown by TCR down-modulation and up-regulation of the IL-2 receptor. However, these cells did not secrete IL-2, IL-3, IL-4 or IFN-gamma, nor did they proliferate. Analysis of intracellular protein tyrosine phosphorylation after cellular activation provided further evidence that SEBDelta61Y could transduce a signal via the Vbeta17 TCR. The events following receptor ligation were clearly different when the T cells were stimulated with SEB or SEBDelta61Y, manifested as both quantitatively and qualitatively different patterns of phosphorylation of intracellular substrates. In contrast, only quantitative differences were apparent when a transfectant expressing the same alpha/beta TCR was stimulated with the different superantigens. Together, these results provide the first demonstration that altered TCR ligands are not restricted to peptides substituted at secondary TCR contact residues. Rather, an altered superantigenic ligand mutated in the TCR binding site can behave as a partial agonist.     

Vbeta14(+) T cells mediate the vaccine-enhanced disease induced by immunization with respiratory syncytial virus (RSV) G glycoprotein but not with formalin-inactivated RSV

Mice immunized with respiratory syncytial virus (RSV) G glycoprotein or with formalin-inactivated RSV (FI-RSV) exhibit severe disease following RSV challenge. This results in type 2 cytokine production and pulmonary eosinophilia, both hallmarks of vaccine-enhanced disease. RSV G-induced T-cell responses were shown to be restricted to CD4(+) T cells expressing Vbeta14 in the T-cell receptor (TCR), and the deletion of these T cells resulted in less severe disease. We therefore examined the role of Vbeta14(+) T cells in FI-RSV-induced disease. BALB/c mice were immunized with vaccinia virus expressing secreted RSV G (vvGs) or with FI-RSV. At the time of challenge with live RSV, mice were injected with antibody to the Vbeta14 component of the TCR. vvGs-immunized mice treated with anti-Vbeta14 had reduced cytokine levels in the lung. Eosinophil recruitment to the lung was also significantly reduced. In contrast, depletion of Vbeta14(+) T cells in FI-RSV-immunized mice had little impact on cytokine production or pulmonary eosinophilia. An analysis of TCR Vbeta chain usage confirmed a bias toward Vbeta14 expression on CD4(+) T cells from vvGs-immunized mice, whereas the CD4(+) T cells in FI-RSV-immunized mice expressed a diverse array of Vbeta chains. These data show that although FI-RSV and vvGs induce responses resulting in similar immunopathology, the T-cell repertoire mediating the response is different for each immunogen and suggest that the immune responses elicited by RSV G are not the basis for FI-RSV vaccine-enhanced disease.     

Potent Neutralization of Staphylococcal Enterotoxin B In Vivo by Antibodies that Block Binding to the T-Cell Receptor

To develop an antibody (Ab) therapeutic against staphylococcal enterotoxin B (SEB), a potential incapacitating bioterrorism agent and a major cause of food poisoning, we developed a “class T" anti-SEB neutralizing Ab (GC132) targeting an epitope on SEB distinct from that of previously developed “class M" Abs. A systematic engineering approach was applied to affinity-mature Ab GC132 to yield an optimized therapeutic candidate (GC132a) with sub-nanomolar binding affinity. Mapping of the binding interface by hydrogen–deuterium exchange coupled to mass spectrometry revealed that the class T epitope on SEB overlapped with the T-cell receptor binding site, whereas other evidence suggested that the class M epitope overlapped with the binding site for the major histocompatibility complex. In the IgG format, GC132a showed ∼ 50-fold more potent toxin-neutralizing efficacy than the best class M Ab in vitro, and fully protected mice from lethal challenge in a toxic shock post-exposure model. We also engineered bispecific Abs (bsAbs) that bound tetravalently by utilizing two class M binding sites and two class T binding sites. The bsAbs displayed enhanced toxin neutralization efficacy compared with the respective monospecific Ab subunits as well as a mixture of the two, indicating that enhanced efficacy was due to heterotypic tetravalent binding to two non-overlapping epitopes on SEB. Together, these results suggest that class T anti-SEB Ab GC132a is an excellent candidate for clinical development and for bsAb engineering.     

Identification of antigenic sites on staphylococcal enterotoxin B and toxoid

The staphylococcal enterotoxins (SEs) are capable of causing both food poisoning and a toxic shock-like illness in man. In addition, SEs are known to act as superantigens, stimulating T-cells according to their T-cell receptor Vβ type. Relatively little is known of their antigenic determinants and how these may relate to the structure and function of the toxins. As a step in the study of these relationships, the entire molecule of SEB was synthesized in duplicate as a series of octapeptides overlapping by seven residues. This series thus represented all the potential linear epitopes of eight residues or less. The reactivity of the octapeptide series with antisera raised to purified SEB and to formaldehyde-inactivated SEB has been used to locate several antigenic sites on native SEB and to identify antigenic differences in the toxoid. Three antigenic peptides identified from the antigenic profile were synthesized and characterized. These represented amino acids 21–32, 93–107 and 202–217 of SEB. None of these peptides affected SEB-induced T-cell proliferation. However, the occurrence or absence of cross-reactivity of these peptides with antibodies to native SEB corresponds to the degree of exposure and/or the rigidity of these regions within SEB.     

Novel Platform for the Detection of Staphylococcus aureus Enterotoxin B in Foods

Staphylococcal contamination of food products and staphylococcal food-borne illnesses continue to be a problem worldwide. Screening of food for the presence of Staphylococcus aureus and/or enterotoxins using traditional methods is laborious. Reliable and rapid multiplex detection methods from a single food extract or culture supernatant would simplify testing. A fluorescence-based cytometric bead array was developed for the detection of staphylococcal enterotoxin B (SEB), using magnetic microspheres coupled with either an engineered, enterotoxin-specific Vβ domain of the T-cell receptor (Vβ-TCR) or polyclonal antibodies. The binding affinity of the Vβ-TCR for SEB has been shown to be in the picomolar range, comparable to the best monoclonal antibodies. The coupled beads were validated with purified enterotoxins and tested in a variety of food matrices spiked with enterotoxins. The Vβ-TCR or antibody was shown to specifically bind SEB in four different food matrices, including milk, mashed potatoes, vanilla pudding, and cooked chicken. The use of traditional polyclonal antibodies and Vβ-TCR provides a redundant system that ensures accurate identification of the enterotoxin, and the use of labeled microspheres permits simultaneous testing of multiple enterotoxins from a single sample.     

Engineered pH-dependent recycling antibodies enhance elimination of Staphylococcal enterotoxin B superantigen in mice

A new modality in antibody engineering has emerged in which the antigen affinity is designed to be pH dependent (PHD). In particular, combining high affinity binding at neutral pH with low affinity binding at acidic pH leads to a novel antibody that can more effectively neutralize the target antigen while avoiding antibody-mediated antigen accumulation. Here, we studied how the in vivo pharmacokinetics of the superantigen, Staphylococcal enterotoxin B (SEB), is affected by an engineered antibody with pH-dependent binding. PHD anti-SEB antibodies were engineered by introducing mutations into a high affinity anti-SEB antibody, 3E2, by rational design and directed evolution. Three antibody mutants engineered in the study have an affinity at pH 6.0 that is up to 68-fold weaker than the control antibody. The pH dependency of each mutant, measured as the pH-dependent affinity ratio (PAR – ratio of affinity at pH 7.4 and pH 6.0), ranged from 6.7–11.5 compared to 1.5 for the control antibody. The antibodies were characterized in mice by measuring their effects on the pharmacodynamics and pharmacokinetics (PK) of SEB after co-administration. All antibodies were effective in neutralizing the toxin and reducing the toxin-induced cytokine production. However, engineered PHD antibodies led to significantly faster elimination of the toxin from the circulation than wild type 3E2. The area under the curve computed from the SEB PK profile correlated well with the PAR value of antibody, indicating the importance of fine tuning the pH dependency of binding. These results suggest that a PHD recycling antibody may be useful to treat intoxication from a bacterial toxin by accelerating its clearance.     

Molecular basis of TCR selectivity, cross-reactivity, and allelic discrimination by a bacterial superantigen: integrative functional and energetic mapping of the SpeC-Vbeta2.1 molecular interface

Superantigens activate large fractions of T cells through unconventional interactions with both TCR beta-chain V domains (Vbetas) and MHC class II molecules. The bacterial superantigen streptococcal pyrogenic exotoxin C (SpeC) primarily stimulates human Vbeta2(+) T cells. Herein, we have analyzed the SpeC-Vbeta2.1 interaction by mutating all SpeC residues that make contact with Vbeta2.1 and have determined the energetic and functional consequences of these mutations. Our comprehensive approach, including mutagenesis, functional readouts from both bulk T cell populations, and an engineered Vbeta2.1(+) Jurkat T cell, as well as surface plasmon resonance binding analysis, has defined the SpeC "functional epitope" for TCR engagement. Although only two SpeC residues (Tyr(15) and Arg(181)) are critical for activation of virtually all human CD3(+) T cells, a larger cluster of four hot spot residues are required for interaction with Vbeta2.1. Three of these residues (Tyr(15), Phe(75), and Arg(181)) concentrate their binding energy on the CDR2 loop residue Ser(52a), a noncanonical residue insertion found only in Vbeta2 and Vbeta4 chains. Plasticity of this loop is important for recognition by SpeC. Although SpeC interacts with the Vbeta2.1 hypervariable CDR3 loop, our data indicate these contacts have little to no influence on the functional interaction with Vbeta2.1. These studies also provide a molecular basis for selectivity and cross-reactivity of SpeC-TCR recognition and reveal a degree of fine specificity in these interactions, whereby certain SpeC mutants are capable of distinguishing between different alleles of the same Vbeta domain subfamily.     

Structure-based design of a bispecific receptor mimic that inhibits T cell responses to a superantigen

Key surface proteins of pathogens and their toxins bind to the host cell receptors in a manner that is quite different from the way the natural ligands bind to the same receptors and direct normal cellular responses. Here we describe a novel strategy for "non-antibody-based" pathogen countermeasure by targeting the very same "alternative mode of host receptor binding" that the pathogen proteins exploit to cause infection and disease. We have chosen the Staphylococcus enterotoxin B (SEB) superantigen as a model pathogen protein to illustrate the principle and application of our strategy. SEB bypasses the normal route of antigen processing by binding as an intact protein to the complex formed by the MHC class II receptor on the antigen-presenting cell and the T cell receptor. This alternative mode of binding causes massive IL-2 release and T cell proliferation. A normally processed antigen requires all the domains of the receptor complex for its binding, whereas SEB requires only the alpha1 subunit (DRalpha) of the MHC class II receptor and the variable beta subunit (TCRVbeta) of the T cell receptor. This prompted us to design a bispecific chimera, DRalpha-linker-TCRVbeta, that acts as a receptor mimic and prevents the interaction of SEB with its host cell receptors. We have adopted (GSTAPPA)(2) as the linker sequence because it supports synergistic binding of DRalpha and TCRVbeta to SEB and thereby makes DRalpha-(GSTAPPA)(2)-TCRVbeta as effective an SEB binder as the native MHC class II-T cell receptor complex. Finally, we show that DRalpha-(GSTAPPA)(2)-TCRVbeta inhibits SEB-induced IL-2 release and T cell proliferation at nanomolar concentrations.     

Mechanisms Mediating Enhanced Neutralization Efficacy of Staphylococcal Enterotoxin B by Combinations of Monoclonal Antibodies

Staphylococcal enterotoxin B (SEB) is a superantigen that cross-links the major histocompatibility complex class II and specific V-β chains of the T-cell receptor, thus forming a ternary complex. Developing neutralizing mAb to disrupt the ternary complex and abrogate the resulting toxicity is a major therapeutic challenge because SEB is effective at very low concentrations. We show that combining two SEB-specific mAbs enhances their efficacy, even though one of the two mAbs by itself has no effect on neutralization. Crystallography was employed for fine-mapping conformational epitopes in binary and ternary complexes between SEB and Fab fragments. NMR spectroscopy was used to validate and identify subtle allosteric changes induced by mAbs binding to SEB. The mapping of epitopes established that a combination of different mAbs can enhance efficacy of mAb-mediated protection from SEB induced lethal shock by two different mechanisms: one mAb mixture promoted clearance of the toxin both in vitro and in vivo by FcR-mediated cross-linking and clearance, whereas the other mAb mixture induced subtle allosteric conformational changes in SEB that perturbed formation of the SEB·T-cell receptor·major histocompatibility complex class II trimer. Finally structural information accurately predicted mAb binding to other superantigens that share conformational epitopes with SEB. Fine mapping of conformational epitopes is a powerful tool to establish the mechanism and optimize the action of synergistic mAb combinations.     

Chimeric anti-staphylococcal enterotoxin B antibodies and lovastatin act synergistically to provide in vivo protection against lethal doses of SEB

Staphylococcal enterotoxin B (SEB) is one of a family of toxins secreted by Staphylococcus aureus that act as superantigens, activating a large fraction of the T-cell population and inducing production of high levels of inflammatory cytokines that can cause toxic shock syndrome (TSS) and death. Extracellular engagement of the TCR of T-cells and class II MHC of antigen presenting cells by SEB triggers the activation of many intracellular signaling processes. We engineered chimeric antibodies to block the extracellular engagement of cellular receptors by SEB and used a statin to inhibit intracellular signaling. Chimeric human-mouse antibodies directed against different neutralizing epitopes of SEB synergistically inhibited its activation of human T-cells in vitro. In the in vivo model of lethal toxic shock syndrome (TSS) in HLA-DR3 transgenic mice, two of these antibodies conferred significant partial protection when administered individually, but offered complete protection in a synergistic manner when given together. Similarly, in vivo, lovastatin alone conferred only partial protection from TSS similar to single anti-SEB antibodies. However, used in combination with one chimeric neutralizing anti-SEB antibody, lovastatin provided complete protection against lethal TSS in HLA-DR3 transgenic mice. These experiments demonstrate that in vivo protection against lethal doses of SEB can be achieved by a statin of proven clinical safety and chimeric human-mouse antibodies, agents now widely used and known to be of low immunogenicity in human hosts.