Superantigen Staphylococcal Enterotoxin Research Articles

The Nitty-Gritty of Protein Interactions

In this issue of Structure , Cho et al. (2005) provide a detailed structural analysis of the molecular mechanism of affinity maturation for the murine T cell receptor Vβ domain. Information from nine X-ray structures of combinations along the maturation pathway and comprehensive thermodynamics reveal the fine details of high-affinity binding.

CT7 (MAGE-C1)-Specific Cellular Immune Responses in the Bone Marrow Microenvironment of Multiple Myeloma Patients.

In previous studies we demonstrated that CT7 (MAGE-C1), a member of the type I MAGE family of Cancer-Testis antigens, was expressed in 82% of primary multiple myeloma specimens. Higher percentages of CT7-expressing cells were correlated with abnormally elevated plasma cell proliferation in these patients, and CT7 was co-expressed in proliferating myeloma cells. Therefore, a tumor vaccine targeting CT7 may preferentially eliminate proliferating myeloma cells, resulting in long-term cures. The type I MAGE were originally identified because they elicited cellular immune responses in melanoma patients whose tumors expressed these antigens. These data suggest the hypothesis that CT7 expression in myeloma cells may similarly elicit T cell immunity in myeloma patients. To test this hypothesis, we examined lymphocytes from myeloma patient bone marrow, the tumor microenvironment. Lymphocytes were isolated from myeloma patient bone marrow aspirates by magnetic bead depletion of CD138 plasma cells followed by plating to remove adherent monocyte. Bone marrow lymphocytes were then expanded by two rounds of stimulation with anti-CD3, anti-CD28 monoclonal antibody (mAb)-conjugated beads and 50 IU/ml recombinant human (rhu) IL-2. Autologous myeloid dendritic cells (mDC) served as antigen-presenting cells for this analysis; they were prepared from peripheral blood monocytes by cultivation with rhu-IL-4 and GM-CSF, then matured with a cytokine cocktail consisting of TNFα , rhu-IL-1β , rhu IL-6, and prostaglandin E2. Myeloid DC were then transduced with in vitro transcribed CT7 mRNA or positive control antigen flu matrix protein (MP) mRNA by electroporation. Negative control mDC were also mock electroporated without mRNA. The dendritic cells were then co-cultured with the expanded bone marrow lymphocytes at 1:1 and 1:20 ratios in IFNγ ELISpot assays. Positive control stimulation with the T cell superantigen Staphylococcal enterotoxin B (SEB) served to assess overall responsiveness of the polyclonal lymphocyte population. Results from four patients are shown in Table 1. Two of four patients, both diagnosed with stage IIIa disease and both expressing CT7 mRNA by RT-PCR, exhibited increases in IFNγ-secreting lymphocytes when co-incubated with CT7-transduced mDC compared to mock electroporated mDC. In contrast, one stage I patient who did not express CT7 mRNA and one stage IIIb, CT7+ patient did not show any increase in IFNγ-secreting cells in response to CT7-transduced mDC stimulation. These data indicated that CT7 expression in myeloma cells can provoke cellular immunity, and suggests that CT7-specific vaccines may boost this immunity. These ongoing studies will also characterize the antigenic T cell epitopes of CT7.Table 1CT7-specific bone marrow lymphocytesPatientStageCT7 mRNA expressionMockCT7Flu MPSEB1IIIa+28.0 ± 7.157.7 ± 5.9nd288 ± 42.12IIIa+6.3 ± 2.327.7 ± 10.117.7 ± 5.390.0 ± 50.33Ia−6.0 ± 1.08.5 ± 0.5nd91.5 ± 54.54IIIb+1.5 ± 0.54.5 ± 0.5nd72.0 ± 43.0IFNγ-secreting cells/50,000 bone marrow lymphocytes

Efficient preservation in a silicon oxide matrix of Escherichia coli, producer of recombinant proteins

The aim of this work was to study the use of silicon oxide matrices for the immobilization and preservation of recombinant-protein-producing bacteria. We immobilized Escherichia coli BL21 transformants containing different expression plasmids. One contained DNA coding for a T-cell receptor beta chain, which was expressed as inclusion bodies in the cytoplasm. The other two encoded bacterial superantigens Staphylococcal Enterotoxin G and Streptococcal Superantigen, which were expressed as soluble proteins in the periplasm. The properties of immobilization and storage stability in inorganic matrices prepared from two precursors, silicon dioxide and tetraethoxysilane, were studied. Immobilized E. coli was stored in sealed tubes at 4 and 20 degrees C and the number of viable cells and level of recombinant protein production were analyzed weekly. Different tests showed that the biochemical characteristics of immobilized E. coli remained intact. At both temperatures selected, we found that the number of bacteria in silicon dioxide-derived matrix was of the same order of magnitude (10(9) cfu ml(-1)) as before immobilization, for 2 months. After 2 weeks, cells immobilized in an alkoxide-derived matrix decreased to 10(4) cfu ml(-1) at 4 degrees C, and no viable cells were detected at 20 degrees C. We found that immobilized bacteria could be used as a starter to produce recombinant proteins with yields comparable to those obtained from glycerol stocks: 15 mg l(-1) for superantigens and 2 mg l(-1) for T-cell receptor beta chain. These results contribute to the development of methods for microbial cell preservation under field conditions.

Endogenous Superantigens Shape Response to Exogenous Superantigens

Endogenous superantigen-mediated thymic negative selection resulted in a paucity of mature T cells bearing T-cell receptor (TCR) Vbeta8 in the periphery. Consequently, the magnitude of immune response to exogenous superantigen staphylococcal enterotoxin B, which activates TCR Vbeta8(+) T cells, was significantly reduced and conferred protection from superantigen-induced mortality.

The Role of SPECs, Small Cdc42-binding Proteins, in F-actin Accumulation at the Immunological Synapse

SPEC1 and SPEC2 are structurally similar Cdc42-binding proteins of 79 and 84 amino acid residues, respectively. We investigated the role of SPEC2 in T cell function due to its high mRNA expression in lymphocytes. Western blot analysis revealed abundant SPEC2 protein in lymphocytes, which in glutathione S-transferase-capture experiments specifically interacted with only GTP-bound Cdc42. Immunofluorescence experiments revealed that the SPEC2 protein was diffusely localized in the cytoplasm and at the cell membrane in unstimulated Jurkat T cells and Raji B cells. Recruitment of SPEC2 within Jurkat T cells to the antigen-presenting cell interface occurred following incubation with staphylococcal enterotoxin E superantigen-loaded B cells and colocalized there with F-actin and Cdc42. T cell receptor (TCR) activation studies using anti-CD3 antibody-coated polystyrene beads showed that SPEC2 was recruited to the site of bead contact, which was not observed with anti-major histocompatibility complex antibody-coated beads. Accumulation of SPEC2 following TCR engagement occurred as early as 5 min, before obvious F-actin accumulation. Biochemical studies with Jurkat T cells demonstrated that N-terminal cysteine residues in SPEC2 were palmitoylated. Overexpression studies of the related SPEC1 showed that it also was recruited to the activated TCR. Mutational analysis revealed that localization of SPEC1 to the TCR required two N-terminal cysteine residues. Furthermore, a SPEC1 Cdc42 Rac-interacting binding mutant, containing an intact N terminus but defective in Cdc42 binding, completely blocked F-actin accumulation at the activated TCR. Taken together these results suggest that SPECs may play important roles in Cdc42-mediated F-actin accumulation at the immunological synapse.

Neuronal, Endocrine, and Anorexic Responses to the T-Cell Superantigen Staphylococcal Enterotoxin A: Dependence on Tumor Necrosis Factor-α

Staphylococcal enterotoxin A (SEA) is a microbial superantigen that activates T-lymphocytes and induces production of various cytokines, including tumor necrosis factor-alpha (TNFalpha). Previously, it was shown that SEA activates the hypothalamic-pituitary-adrenal axis and augments gustatory neophobic behaviors. In the present study, it was hypothesized that these effects involve neuronal activation in forebrain regions mediating fear and/or anxiety and are dependent on the production of TNFalpha. Male C57BL/6J mice were given intraperitoneal injections of 10 microg of SEA and 5 microg of lipopolysaccharide (LPS) or saline and perfused 2 h later for histochemical determination of brain c-Fos immunoreactivity (IR). The results showed increased c-Fos IR in the paraventricular nucleus, arcuate nucleus, central nucleus of the amygdala, bed nucleus of the stria terminalis, and lateral septum. Challenge of TNF-/- mice with SEA did not produce a significant increase in brain c-Fos IR, although c-Fos was increased after exposure to a psychogenic stressor (i.e., open field). In additional experiments, the elevated corticosterone response to SEA was abrogated in TNF-/- mice and was shown to be corticotropin-releasing hormone dependent. Finally, the augmented reduction in novel food intake after SEA challenge was attenuated in TNF-/- mice as well as in wild-type mice administered antibody to TNFalpha. In conclusion, challenge with SEA recruits brain regions mediating stress and anxiety responses, an effect that requires endogenous TNFalpha. Whether this is indicative of all T-cell superantigens remains to be determined, although it stands in contrast to other models of neuroimmunomodulation (e.g., LPS) that involve multiple cytokine influences.

Preclinical evaluation of superantigen (staphylococcal enterotoxin B) in the intravesical immunotherapy of superficial bladder cancer

Superantigens are potent activators of T lymphocytes; therefore, their characteristics can be exploited in diseases where immunomodulation is known to be effective. In this study, we evaluated a new approach for the intravesical therapy of superficial bladder cancer. We investigated in coculture experiments if staphylococcal enterotoxin B (SEB)-activated PBMCs are able to induce apoptosis in human transitional cell carcinoma (TCC) cells. Additionally, we tested the toxicity and efficacy of SEB dissolved in NaCl 0.9% administered intravesically once weekly for 6 weeks in a rat bladder cancer model. To validate the coculture in vitro findings, we evaluated tumor stage, grade, apoptotic cells in the urothelium and stroma of the bladder and infiltration of the bladder wall by lymphocytes, macrophages and mononuclear cells. Coculture experiments revealed that SEB-activated PBMCs are able to kill TCC cells by inducing apoptosis. The intravesical toxicity study with a maximum dose of 100 microg/ml SEB demonstrated no side effects. In the intravesically SEB-treated animals (10 microg/ml), only 3 tumors remained vs. 15 persisting tumors in the control group. The remaining tumors of the therapy group showed a significant amount of apoptosis and granulocytes, mainly in the urothelium, whereas no relevant apoptosis or infiltration of the bladder with lymphocytes or macrophages was found in the control group. These preclinical findings suggest that SEB might be an interesting candidate for further clinical evaluation.

Analysis of T-Cell Receptor Vβ Gene Repertoires after Immune Stimulation and in Malignancy by Use of Padlock Probes and Microarrays

Detection of expanded T-cell clones, identified by their receptor (TCR) repertoires, can assist diagnosis and guide therapy in infectious, inflammatory, and autoimmune conditions as well as in tumor immunotherapy. Analysis of tumor-infiltrating lymphocytes often reveals preferential use of one or a few TCR V beta genes, compared with peripheral blood, indicative of a clonal response against tumor antigens. To simultaneously measure the relative expression of all V beta gene families, we combined highly specific and sensitive oligonucleotide reagents, called padlock probes, with a microarray read-out format. T-Cell cDNA was combined with a pool of V beta subfamily-specific padlock probes. Reacted probes were selectively amplified and the products hybridized to a microarray, from which the V beta subfamily distribution in each sample could be determined relative to a control sample. In lymphocytes stimulated with the superantigen staphylococcal enterotoxin B, we detected expansions at the mRNA level of TCR subfamilies previously shown to respond to staphylococcal enterotoxin B. Expansions of the same V beta families could also be detected by flow cytometry. In samples from two bladder cancer patients, we detected predominant representations of specific V beta subfamilies in both tumor-infiltrating lymphocytes and in the draining lymph nodes, but not in non-tumor-draining lymph nodes or peripheral blood. Several expression profiles from draining lymph nodes in patients with malignant melanoma were divergent from profiles seen in non-tumor-draining lymph nodes. Padlock probe-based parallel analysis of TCR V beta gene distributions provides an efficient method for screening multiple samples for T-cell clonal expansions with reduced labor and time of analysis compared with traditional methods.

Preparation of murine B7.1‐glycosylphosphatidylinositol and transmembrane‐anchored staphylococcal enterotoxin

The authors have previously reported a tumor cell vaccine modified with superantigen staphylococcal enterotoxin A (SEA) and its antitumor effect. The tumor cell vaccines modified with multiple immune activators frequently elicited stronger immune responses against established tumors than single-modified vaccines. The authors explored the effectiveness of a tumor cell vaccine transduced with immune activators, dual-modified using the protein transfer technique. First, a glycosylphosphatidylinositol (GPI)-anchored murine B7.1 (mB7.1-GPI) and a transmembrane-anchored SEA (TM-SEA) were genetically generated. Then, the murine lymphoma EL4 cells were dual modified with the incorporation of mB7.1-GPI and TM-SEA onto the cell surface. Flow cytometry and laser confocal microscopy showed that the incorporation of B7.1 and SEA molecules onto EL4 cells was quite stable. The dual-modified tumor cell vaccine EL4/mB7.1-GPI + TM-SEA elicited significantly stronger antitumor immune responses both in vitro and in vivo when compared with the single-modified tumor cell vaccines EL4/mB7.1-GPI and EL4/TM-SEA. The results of the current study validated the novel approach for preparing tumor cell vaccines modified with dual immune active molecules using the protein transfer technique, and supported the feasibility and effectiveness of the dual-modified tumor cell vaccine.

SEB combined with IL-1ra could prolong the survival of the rat allografts in high-risk corneal transplantation

Purpose To determine whether the superantigen Staphylococcal enterotoxin B (SEB) combined with interleukin-1 receptor antagonist (IL-1ra) prolong allograft survival better than individual agents in high-risk corneal transplantation in a rat model. Methods Fisher 344 donor corneas were transplanted into Lewis recipients. High-risk transplantation meant that the transplants were sutured into the recipient beds with corneal neovascularization induced by placing three interrupted sutures in the host cornea. All of the recipients were divided in blinded fashion into four groups. Group I was injected with saline buffer. Group II was injected intraperitoneally with 0.2 mL SEB (75 μg/kg) at 4-day intervals on three occasions before transplantation. Group III was injected with 0.1 mL IL-1ra (1 mg/mL) subconjunctivally from the first day after transplantation for 2 weeks. Group IV received both SEB and IL-1ra. All transplants were evaluated for signs of rejection for 4 weeks after surgery. Ten days after transplantation, two recipients in each group were sacrificed for histopathological and immunological evaluation. Results The mean survival time of the allografts in the control group was 5.89 ± 0.79 days; in SEB group, 10.70 ± 2.52 days; in IL-1ra group, 8.25 ± 0.71 days; in the SEB and IL-1ra group, 17.36 ± 2.39 days. CD4 + and CD8 + lymphocyte infiltration into the allografts and the percentage of the lymphocytes in the spleen and mandibular lymphatic nodes was significantly decreased among the treated groups with dampened lymphocyte reactivity. The SEB plus IL-1ra combination group showed the strongest inhibition. Conclusion SEB and IL-1ra are most effective in combination to treat high-risk corneal transplants.

CC Chemokine Ligand 18, An Atopic Dermatitis-Associated and Dendritic Cell-Derived Chemokine, Is Regulated by Staphylococcal Products and Allergen Exposure

Atopic dermatitis is a chronic inflammatory skin disease with a steadily increasing prevalence. Exposure to allergens or bacterial superantigens triggers T and dendritic cell (DC) recruitment and induces atopic skin inflammation. In this study, we report that among all known chemokines CCL18/DC-CK1/PARC represents the most highly expressed ligand in atopic dermatitis. Moreover, CCL18 expression is associated with an atopic dermatitis phenotype when compared with other chronic inflammatory skin diseases. DCs either dispersed within the dermis or clustering at sites showing perivascular infiltrates are abundant sources of CCL18. In vitro, microbial products including LPS, peptidoglycan, and mannan, as well as the T cell-derived activation signal CD40L, induced CCL18 in monocytes. In contrast to monocytes, monocyte-derived, interstitial-type, and Langerhans-type DCs showed a constitutive and abundant expression of CCL18. In comparison to Langerhans cells, interstitial-type DCs produced higher constitutive levels of CCL18. In vivo, topical exposure to the relevant allergen or the superantigen staphylococcal enterotoxin B, resulted in a significant induction of CCL18 in atopic dermatitis patients. Furthermore, in nonatopic NiSO4-sensitized individuals, only relevant allergen but not irritant exposure resulted in the induction of CCL18. Taken together, findings of the present study demonstrate that CCL18 is associated with an atopy/allergy skin phenotype, and is expressed at the interface between the environment and the host by cells constantly screening foreign Ags. Its regulation by allergen exposure and microbial products suggests an important role for CCL18 in the initiation and amplification of atopic skin inflammation.

Depletion of activated Vβ8+ T cells disrupts bispecific antibody directed antitumor immunity 1

Activation of Vbeta8+ T cells with superantigen staphylococcal enterotoxin B (SEB) and use of an antitumor, anti-CD3 bispecific antibody (BsAb) leads to tumor protective immunity. We hypothesize that Vbeta8+ T-cell activation in combination with BsAb is crucial for tumor protective immunity in this model. Adolescent C3H/HeN mice were intravenously injected with syngeneic CL62 melanoma to establish pulmonary metastasis. Three days after establishing pulmonary metastasis, predominantly Vbeta8+ T cells are activated with 50 mug of intraperitoneal superantigen SEB. T cells were depleted at different time points in relation to SEB administration to assess the effect on protective immunity against a second tumor challenge. Protective immunity is significantly (P < 0.008) decreased when Vbeta8+ depletion occurs 6 h after SEB injection, as growth of rechallenged CL62 melanoma occurred in 43%. Protective immunity is present at all other time points when mice survive Vbeta8+ T-cell depletion. Survival of animals treated with SEB/BsAb (82%) is significantly better (P < 0.002) than with SEB alone (60%) or nontreated control (0%). Survival when Vbeta8+ T-cell depletion occurred at 6 h and 48 h post-SEB is 72% and 77%, respectfully, and is statistically indistinguishable (P < 0.232 and P < 0.602). If T-cell depletion was conducted before SEB administration, however, the combination of SEB and BsAb did not result in significant protective immunity. T-cell depletion before the use of SEB alone, without BsAb, failed to result in significant protective immunity. Depletion of Vbeta8+ T cells 6 h after activation disrupts the development of protective immunity.

Naive T cells are resistant to anergy induction by anti-CD3 antibodies.

Anti-CD3 mAbs are potent immunosuppressive agents used in clinical transplantation. It has been generally assumed that one of the anti-CD3 mAb-mediated tolerance mechanisms is through the induction of naive T cell unresponsiveness, often referred to as anergy. We demonstrate in this study that naive T cells stimulated by anti-CD3 mAbs both in vivo and in vitro do not respond to the superantigen staphylococcal enterotoxin B nor to soluble forms of anti-CD3 mAbs and APC, but express increased reactivity to plastic-coated forms of the same anti-CD3 mAbs and to their nominal Ag/class II MHC, a finding that is difficult to rationalize with the concept of anergy. Phenotypic and detailed kinetic studies further suggest that a strong signal 1 delivered by anti-CD3 mAbs in the absence of costimulatory molecules does not lead to anergy, but rather induces naive T cells to change their mitogen responsiveness and acquire features of memory T cells. In marked contrast, Ag-experienced T cells are sensitive to anergy induction under the same experimental settings. Collectively, these studies demonstrate that exposure of naive T cells in vivo and in vitro to a strong TCR stimulus does not induce Ag unresponsiveness, indicating that sensitivity to negative signaling through TCR/CD3 triggering is developmentally regulated in CD4(+) T cells.

Effects of activated T cells on osteoclastogenesis depend on how they are activated

Introduction: Activated T cells are emerging as important regulators of osteoclast function in inflammatory diseases. Both pro- and anti-resorptive properties have been described. We reasoned that this reported variability of the effects of T cells on osteoclast formation depends on how T cells are activated in vitro. Methods: We harvested T lymphocytes from 5-week-old C57BL/6 mouse spleens. Activation was performed with anti-CD3ε and -CD28 Ab (Abs), concanavalin A (Con A), phytohemagglutinin (PHA), or the superantigen Staphylococcal enterotoxin A (SEA). Osteoclastogenesis was induced by receptor activator of NF-κB ligand (RANKL) in the mouse macrophage cell line RAW 264.7 cells, or primary macrophage CD11b + cells from mouse spleen. Cells were cultured with T cells or with their conditioned medium. Results: Co-culture of activated T lymphocytes with RAW 264.7 cells inhibited osteoclastogenesis but only when activated by Abs. This effect was CD4 +-dependent. Conditioned medium from activated T lymphocytes with Abs consistently blocked osteoclastogenesis in RAW 264.7 and CD11b + cells. T cells activated with SEA, Con A, and PHA had inconsistent effects on osteoclastogenesis. We then tested the role of interferon (IFN)-γ, a known inhibitor of osteoclastogenesis, in the effects of T cells on osteoclast formation. IFN-γ neutralizing antibody blocked the inhibitory effect of T-cell conditioned medium on osteoclastogenesis. Osteoclast precursors from IFN-γ receptor-null mice treated with 0.1% medium from activated T cells formed osteoclasts. However, higher doses of medium inhibited osteoclastogenesis, so that we cannot exclude that other factors besides IFN-γ may be involved. Conclusions: Available methods to activate T lymphocytes result in variable effects on osteoclastogenesis. IFN-γ is the main factor responsible for the inhibitory effects of activated T cells on osteoclast formation.

Lymphocyte Activation Gene-3 (CD223) Regulates the Size of the Expanding T Cell Population Following Antigen Activation In Vivo

Lymphocyte activation gene-3 (LAG-3) is a CD4-related, activation-induced cell surface molecule that binds to MHC class II with high affinity. In this study, we used four experimental systems to reevaluate previous suggestions that LAG-3(-/-) mice had no T cell defect. First, LAG-3(-/-) T cells exhibited a delay in cell cycle arrest following in vivo stimulation with the superantigen staphylococcal enterotoxin B resulting in increased T cell expansion and splenomegaly. Second, increased T cell expansion was also observed in adoptive recipients of LAG-3(-/-) OT-II TCR transgenic T cells following in vivo Ag stimulation. Third, infection of LAG-3(-/-) mice with Sendai virus resulted in increased numbers of memory CD4(+) and CD8(+) T cells. Fourth, CD4(+) T cells exhibited a delayed expansion in LAG-3(-/-) mice infected with murine gammaherpesvirus. In summary, these data suggest that LAG-3 negatively regulates T cell expansion and controls the size of the memory T cell pool.

Individualized Patient Dosing in Phase I Clinical Trials: The Role of Escalation With Overdose Control in PNU-214936

A patient-specific dose-escalation scheme using a Bayesian model of Escalation with Overdose Control (EWOC) was conducted to establish the maximum tolerated dose (MTD) of PNU-214936 in advanced non-small-cell lung cancer (NSCLC). PNU-214936 is a murine Fab fragment of the monoclonal antibody 5T4 fused to a mutated superantigen staphylococcal enterotoxin A (SEA). Seventy-eight patients with NSCLC were treated with an individualized dose of PNU-214936 calculated using EWOC, based on their anti-SEA antibody level, and given as a 3-hour infusion on 4 consecutive days. Fever (82%; grade 3 to 4, 2.6%) and hypotension (57%; grade 3 to 4, 9%) were the most common toxicities. Eight dose-limiting toxicities occurred, as defined as any grade 4 toxicity occurring within the first 5 days. The MTD was defined as a function of pretreatment anti-SEA antibody level. MTD ranged from 103 ng/kg for patients with anti-SEA concentrations < or = 10 pmol/mL, to 601 ng/kg for patients with anti-SEA concentrations of 91 to 150 pmol/mL. A minor tumor response was demonstrated in five of 66 assessable patients. EWOC determined phase I doses of PNU-214936 that were adjusted for patient anti-SEA antibody level, while safeguarding against overdose. Furthermore, the method permitted the construction of a dosing algorithm that would allow patients in subsequent clinical investigations to be treated with a dose of PNU-214936 that is tailored to their specific tolerance for the agent, as reflected by their pretreatment anti-SEA.

Vbeta-restricted T cell adherence to endothelial cells: a mechanism for superantigen-dependent vascular injury.

To investigate the potential for endothelial cells to operate as superantigen-presenting cells for T cells and the potential for such an interaction to cause endothelial cell activation and injury. Class II major histocompatibility complex (MHC)-positive human umbilical vein endothelial cells (HUVECs) were cocultured for 4 hours with purified T cells and the superantigens staphylococcal enterotoxin B (SEB) or toxic shock syndrome toxin 1 (TSST-1). After staining with fluorescence-conjugated monoclonal antibodies, flow cytometric analysis was performed on the HUVECs and T cells to examine V(beta)-restricted T cell adherence to the endothelial cell monolayer, V(beta)-restricted T cell activation (CD69 up-regulation), surface expression of endothelial cell activation markers, and generation of endothelial microparticles (EMPs). Coculture of purified T cells with class II MHC-positive HUVECs and either TSST-1 or SEB resulted in V(beta)-restricted CD69 up-regulation by CD4 and CD8 cells (V(beta)2 activation for TSST-1; V(beta)3, V(beta)5.1, and V(beta)12 activation for SEB). Additionally, there was CD4 and CD8 T cell V(beta)-restricted adherence to the HUVEC monolayer at 4 hours. Expression of intercellular adhesion molecule 1, E-selectin, and vascular cell adhesion molecule 1 was up-regulated on the class II MHC-positive HUVECs following exposure to superantigen in the presence of T cells, and there was increased EMP release from activated HUVECs, which occurred earlier and was of greater magnitude than that observed in response to tumor necrosis factor alpha. Class II MHC-positive endothelial cells operate as competent superantigen-presenting cells for CD4 and CD8 lymphocytes in vitro. Dual signaling between endothelial cells and T cells results in V(beta)-restricted activation and adherence to endothelial monolayers and endothelial cell activation and release of EMPs expressing inducible cell adhesion molecules. It is proposed that this mechanism could account in part for the vascular injury associated with superantigen-mediated diseases including Kawasaki disease.

Peptide antagonists of superantigen toxins.

Superantigens produced by Staphylococcus aureus and Streptococcus pyogenes are among the most lethal of toxins. Toxins in this large family trigger an excessive cellular immune response leading to toxic shock. Superantigens are secreted by the bacteria as diverse natural mixtures, a complexity that demands development of broad-spectrum countermeasures. We used a rational approach to design short peptides with homology to various domains in a typical superantigen (staphylococcal enterotoxin B) and screened each peptide for its ability to antagonize, in human peripheral blood mononuclear cells, superantigen-mediated induction of the genes encoding T helper 1 cytokines that mediate shock: interleukin-2, interferon-gamma and tumor necrosis factor. A dodecamer peptide proved a potent antagonist against widely different superantigens. This peptide protected mice from killing by superantigens and it was able to rescue mice undergoing toxic shock. The antagonist peptide shows homology to a beta-strand-hinge-alpha-helix domain that is structurally conserved among superantigens, yet currently of unknown function and remote from the binding sites for the known ligands essential for T cell activation, the major histocompatibility complex class II molecule and T cell receptor. The antagonist activity of this peptide thus identifies a novel domain in superantigens that is critical for their toxic action. The antagonist peptide provides a new tool for understanding the mechanism of excessive human immune response activation by superantigens that occurs during toxic shock and for identification of a novel target ligand that may interact with this superantigen domain.

Identification of a Secondary Zinc-binding Site in Staphylococcal Enterotoxin C2: IMPLICATIONS FOR SUPERANTIGEN RECOGNITION

The previously determined crystal structure of the superantigen staphylococcal enterotoxin C2 (SEC2) showed binding of a single zinc ion located between the N- and C-terminal domains. Here we present the crystal structure of SEC2 determined to 2.0 A resolution in the presence of additional zinc. The structure revealed the presence of a secondary zinc-binding site close to the major histocompatibility complex (MHC)-binding site of the toxin and some 28 A away from the primary zinc-binding site of the toxin found in previous studies. T cell stimulation assays showed that varying the concentration of zinc ions present affected the activity of the toxin and we observed that high zinc concentrations considerably inhibited T cell responses. This indicates that SEC2 may have multiple modes of interaction with the immune system that are dependent on serum zinc levels. The potential role of the secondary zinc-binding site and that of the primary one in the formation of the TCR.SEC2.MHC complex are considered, and the possibility that zinc may regulate the activity of SEC2 as a toxin facilitating different T cell responses is discussed.

Cytokine profiles of peripheral blood mononuclear cells from pigs with postweaning multisystemic wasting syndrome in response to mitogen, superantigen or recall viral antigens.

In vitro cytokine profiles of peripheral blood mononuclear cells (PBMC) from pigs with postweaning multisystemic wasting syndrome (PMWS) and healthy pigs were determined in response to recall viral antigens (porcine circovirus type 2; PCV2), mitogens (phytohaemagglutinin) or superantigens (staphylococcal enterotoxin B). PBMC from PMWS-affected pigs, in contrast to those from healthy pigs, responded to recall PCV2 antigen by releasing IL-10 and IFN-gamma, but they were less able or even unable to produce IL-4, IL-2 or IFN-gamma upon challenge with mitogen or superantigen. Moreover, only PCV2 had the ability to downregulate or suppress the release of IL-4 and IL-2 from PBMC from both healthy and diseased animals, and to stimulate the production of pro-inflammatory cytokines (IL-1beta, IL-8). In conclusion, the immune system cells of PMWS pigs have a diminished ability to perform their immunological functions upon viral or immunostimulatory molecules. In addition, PCV2 can alter the functionality of PBMC in both healthy and PMWS pigs.