Activation of murine T cells by toxic shock syndrome toxin-1. The toxin-binding structures expressed on murine accessory cells are MHC class II molecules

Toxic shock syndrome toxin-1 (TSST-1)-binding structures present on murine lymphoid tissues were investigated by using 125I-TSST-1. T-depleted C57BL/6 spleen cells incubated with TSST-1 for 3 h at 0 degree C were mitogenic to splenic T cells, indicating that the former cells bind and present TSST-1 to T cells. TSST-1-binding activity was observed in C57BL/6 splenic B cells and L cells transfected with I-Ab genes, but not in splenic T cells and control L cells. Scatchard plot analysis showed that these B cells and transfectants bound TSST-1 with similar binding affinity. SDS-PAGE analysis showed that lysates of C57BL/6 spleen cells and the I-Ab-positive transfectants contain a single band which bound TSST-1 and comigrated with I-Ab heterodimers. TSST-1-binding activity observed clearly in C57BL/6. BALB/c, and C3H/HeN spleen cells and L cells transfected with I-Ab or I-Ak genes was not reduced by paraformaldehyde fixation. Binding of 125I-TSST-1 to the three spleen cells was markedly reduced by anti-I-A antibodies, but not by anti-I-E antibodies. C57BL/6, C3H/HeN, and (C3H/HeN x C57BL/6) F1 T cells were activated by TSST-1 to proliferate and produce IL-2 in the presence of FT6.2 cells, LT1-30-3 cells and either of them, respectively, but not in the presence of control L cells. These results indicate that I-A molecules function as the structures via that accessory cells directly bind TSST-1 on the cell surface and present a triggering signal of TSST-1 to T cells.     

Molecular requirements for T cell activation by the staphylococcal toxic shock syndrome toxin-1

The activation of Ag-specific, Ia molecule-restricted, TCR V beta 3+ T cell clones by staphylococcal toxic shock syndrome toxin-1 (TSST-1), was investigated. The results show that although Ag- and TSST-1-induced activation of T cell clones both require TCR expression and similar biologic activation signals, the Ia molecule requirement for TSST-1 recognition was much less stringent than that observed for antigenic peptide recognition. In addition, T cell clones recognized TSST-1 without processing by APC. These results suggest that the ability of TSST-1 to polyclonally activate T cells is dependent on TCR recognition of the intact toxin molecule bound to a nonpolymorphic region(s) of the Ia molecule resulting in the same activation events induced by Ag recognition.     

Bacterial heat shock proteins enhance class II MHC antigen processing and presentation of chaperoned peptides to CD4+ T cells

APCs process heat shock protein (HSP):peptide complexes to present HSP-chaperoned peptides on class I MHC molecules, but the ability of HSPs to contribute chaperoned peptides for class II MHC (MHC-II) Ag processing and presentation is unclear. Our studies revealed that exogenous bacterial HSPs (Escherichia coli DnaK and Mycobacterium tuberculosis HSP70) delivered an extended OVA peptide for processing and MHC-II presentation, as detected by T hybridoma cells. Bacterial HSPs enhanced MHC-II presentation only if peptide was complexed to the HSP, suggesting that the key HSP function was enhanced delivery or processing of chaperoned peptide Ag rather than generalized enhancement of APC function. HSP-enhanced processing was intact in MyD88 knockout cells, which lack most TLR signaling, further suggesting the effect was not due to TLR-induced induction of accessory molecules. Bacterial HSPs enhanced uptake of peptide, which may contribute to increased MHC-II presentation. In addition, HSPs enhanced binding of peptide to MHC-II molecules at pH 5.0 (the pH of vacuolar compartments), but not at pH 7.4, indicating another mechanism for enhancement of MHC-II Ag processing. Bacterial HSPs are a potential source of microbial peptide Ags during phagocytic processing of bacteria during infection and could potentially be incorporated in vaccines to enhance presentation of peptides to CD4+ T cells.     

Characterization of T cell receptors engineered for high affinity against toxic shock syndrome toxin-1

Superantigens, including bacterial enterotoxins, are a family of proteins that bind simultaneously to MHC class II molecules and the Vbeta regions of T cell receptors. This cross-linking results in the activation of a large population of T cells that release massive amounts of inflammatory cytokines, ultimately causing a condition known as toxic shock syndrome. The staphylococcal superantigen toxic shock syndrome toxin-1 (TSST-1) is a causative agent of this disease, but its structure in complex with the cognate T cell receptor (human Vbeta2.1) has not been determined. To understand the molecular details of the interaction and to develop high affinity antagonists to TSST-1, we used directed evolution to generate a panel of high affinity receptors for TSST-1. Yeast display libraries of random and site-directed hVbeta2.1 mutants were selected for improved domain stability and for higher affinity binding to TSST-1. Stability mutations allowed the individual Vbeta domains to be expressed in a bacterial expression system. Affinity mutations were generated in CDR2 and FR3 residues, yielding improvements in affinity of greater than 10,000-fold (a K(D) value of 180 pmol). Alanine scanning mutagenesis of hVbeta2.1 wild-type and mutated residues allowed us to generate a map of the binding site for TSST-1 and to construct a docking model for the hVbeta2.1-TSST-1 complex. Our experiments suggest that the energetic importance of a single hVbeta2.1 wild-type residue likely accounts for the restriction of TSST-1 specificity to only this human Vbeta region. The high affinity mutants described here thus provide critical insight into the molecular basis of TSST-1 specificity and serve as potential leads toward the development of therapeutic agents for superantigen-mediated disease.     

A toxic shock syndrome toxin-1 peptide that shows homology to mycobacterial heat shock protein 18 is presented as conventional antigen to T cells by multiple HLA-DR alleles.

We observe that PBMC from most adults (16 of 18 subjects tested) show a small but significant in vitro proliferative response to a 30-amino acid-long peptide (peptide 2, amino acids 34-63) derived from toxic shock syndrome toxin. By contrast, PBMC from newborn blood and thymocytes do not proliferate to this peptide, and furthermore, peptide 2 did not displace the binding of radiolabeled TSST-1 to MHC class II positive cells, nor did it induce IL-1 beta mRNA in monocytes, indicating that this peptide does not behave as a superantigen. Proliferation of PBMC to peptide 2 could be blocked by anti-HLA-DR, but not by anti-HLA-DP or DQ mAb, suggesting that HLA-DR molecules are the restriction elements for the recognition of this peptide by T cells. This premise was further confirmed by demonstrating that mouse L cells transfected with human HLA-DR, but not HLA-DP or DQ molecules, supported the proliferation of purified T cells to peptide 2. Studies with subjects of known HLA-DR types showed that all types tested are capable of responding to this peptide, PBMC from adults exposed to mycobacterial Ag showed significantly better proliferative response to peptide 2 than unexposed adults. Studies with truncations of this peptide suggest that a "core" region of eight amino acids that is conserved between low m.w. heat shock proteins and peptide 2 may be critical to T cell recognition of this peptide. The universal presentation of peptide 2 by HLA-DR molecules may contribute to the widespread natural immunity observed against toxic shock syndrome toxin.     

Functional analysis of the TCR binding domain of toxic shock syndrome toxin-1 predicts further diversity in MHC class II/superantigen/TCR ternary complexes

Superantigens (SAGs) aberrantly alter immune system function through simultaneous interaction with lateral surfaces of MHC class II molecules on APCs and with particular variable regions of the TCR beta-chain (Vbeta). To further define the interface between the bacterial SAG toxic shock syndrome toxin-1 (TSST-1) and the TCR, we performed alanine scanning mutagenesis within the putative TCR binding region of TSST-1 along the central alpha helix adjacent to the N-terminal alpha helix and the beta7-beta9 loop as well as with two universally conserved SAG residues (Leu(137) and Tyr(144) in TSST-1). Mutants were analyzed for multiple functional activities, and various residues appeared to play minor or insignificant roles in the TCR interaction. The locations of six residues (Gly(16), Trp(116), Glu(132), His(135), Gln(136), and Gln(139)), each individually critical for functional activity as well as direct interaction with the human TCR Vbeta2.1-chain, indicate that the interface occurs in a novel region of the SAG molecule. Based on these data, a model of the MHC/TSST-1/TCR ternary complex predicts similarities seen with other characterized SAGs, although the CDR3 loop of Vbeta2.1 is probably involved in direct SAG-TCR molecular interactions, possibly contributing to the TCR Vbeta specificity of TSST-1.     

Induction of murine hemopoietic growth factors by toxic shock syndrome toxin-1

Toxic shock syndrome toxin-1 (TSST-1), an extra-cellular 22 kDa single chain protein produced by most Staphylococcus aureus strains isolated from patients with toxic shock syndrome (TSS), induces modifications of blood cell values similar to those observed during TSS. We therefore analyzed the effects of TSST-1 on the proliferation and differentiation of murine granulocyte-macrophage progenitor cells (CFU-culture) and the eventual role of endotoxin in this response. TSST-1 had no direct effect on the proliferation of CFU-culture and was unable to influence the CSF-induced proliferation and differentiation of these progenitors. In contrast, TSST-1 was a potent inducer in spleen cell cultures of a factor with an ability to induce both colony formation by bone marrow cells and proliferation of an IL-3-dependent cell line. Nanogram amounts of TSST-1 were able to induce the release of CSF activity in spleen cell cultures from both normal and LPS-hyporesponsive mice. Cells from C3H/HeJ mice were as responsive as cells from C3H/He Pas mice. Furthermore, in spleen cell cultures from normal mice, TSST-1 and LPS did not act synergistically to induce CSF activity. Nanogram amounts of TSST-1 were also able to induce CSF activity in vivo but failed to induce IL-3 activity in the serum and organ-conditioned media from TSST-1-treated mice.     

Screening of staphylococci for the toxic shock syndrome toxin-1 (TSST-1) gene

Dot blot hybridization was used to screen 820 staphylococci for the presence of the gene coding for TSST-1. The DNA of 33 strains among 70 Staph. aureus strains isolated from suspected toxic shock syndrome (TSS) cases hybridized with the probe. These results agreed perfectly with those obtained with a phenotypic method (immunodiffusion). Among 608 Staph. aureus strains isolated over a period of one month from hospitalized patients, 66 (11%) hybridized with the probe; of these strains, 64 (97%) were found to produce TSST-1 in vitro. None of 145 coagulase-negative staphylococcal strains harboured DNA hybridizing with the probe. The data indicate that this genotypic assay is suitable for epidemiological studies.     

Superantigenic activity of toxic shock syndrome toxin-1 is resistant to heating and digestive enzymes

Aims: To elucidate the stability of superantigenic activity and pathogenesis of toxic shock syndrome toxin 1 (TSST‐1) and staphylococcal enterotoxin A (SEA) against heating and digestive enzymes. Methods and Results: Purified TSST‐1 and SEA were treated with heating, pepsin and trypsin that are related to food cooking, stomach and intestine conditions. The integrity, superantigenic activity and toxicity of treated TSST‐1 and SEA were analysed by Western blotting, spleen cell culture, cytokine assay and toxic shock models. Both TSST‐1 and SEA showed strong resistance to heating, pepsin and trypsin digestion. Furthermore, the treated TSST‐1 showed significant higher induction of interferon‐γ and toxic shock compared with that of SEA. Pepsin‐ or trypsin‐digested TSST‐1 fragments still showed significant superantigenic and lethal shock toxicities. Conclusions: The superantigenic activity of TSST‐1 was stable to heating and digestive enzymes. Pepsin‐ and trypsin‐digested TSST‐1 fragments still showed superantigenic and lethal shock activities, indicating that digested TSST‐1 could cross epithelial cells and induce systemic toxicity. Significance and Impact of the Study: This study found, for the first time, that pepsin‐ or trypsin‐digested smaller TSST‐1 retained significant superantigenic and lethal shock activities. The different resistance of TSST‐1 and SEA participates in the different pathogenic activities during food poisoning and toxic shock syndrome.     

The nucleotide and partial amino acid sequence of toxic shock syndrome toxin-1

The nucleotide sequence of toxic shock syndrome toxin-1 (TSST-1) has been determined. In addition, one-third of the predicted amino acid sequence was confirmed by amino acid sequence analysis of cyanogen bromide-generated TSST-1 protein fragments. The DNA sequencing results identified a 708-base pair open reading frame starting with an ATG, 7 base pairs downstream from a Shine-Dalgarno sequence, and terminating at a UAA stop codon. Amino acid analysis of the intact protein defined the NH2 terminus of the mature protein and located the cleavage point for the signal peptide (Ala/Ser). The signal peptide contained the first 40 amino acids and had characteristic structural similarities with other bacterial signal peptides. The coding sequence of the mature protein was 585 base pairs (194 amino acids) in length, and the molecular weight of the predicted protein was 22,049. This is in good agreement with the previously reported molecular weight of TSST-1 (22,000), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NH2-terminal amino acid sequence analysis performed on isolated TSST-1 CNBr fragments determined the position of the peptides in the TSST-1 sequence and verified the predicted amino acid sequence in those positions. Computer analyses of the amino acid sequence showed that TSST-1 has little or no sequence homology with biologically related toxins, streptococcal pyrogenic exotoxin A, and staphylococcal enterotoxins B and C.     

Aldehyde modification of peptide immunogen enhances protein-reactive antibody response to toxic shock syndrome toxin-1.

Introduction of aldehyde groups into protein conjugates enhanced the immune response to a coupled peptide without the use of strong adjuvants. Synthetic peptides representing the N-terminal (residues 1-16) and internal (residues 53-65) epitopes of toxic shock syndrome toxin-1 (TSST-1) were coupled to carrier protein, and carbonyl tags were introduced by Amadori reaction with glycolaldehyde. Modified and unmodified antigens in alum were used to immunize rabbits and the reactivities of antisera were compared. Aldehyde modification augmented the response detected by ELISA, which included enhanced binding to peptides and to native TSST-1. In western blot, TSST-1 was detected by antiserum elicited to the N-terminal peptide, but not that generated to the peptide representing the internal sequence. The same antiserum also neutralized TSST-1 activity in a lymphocyte proliferation assay. The circular dichroism spectrum of the N-terminal peptide indicated a propensity for helical conformation, similar to the structure at the corresponding sequence of the native protein. These data suggest that aldehyde modification can boost immunogenicity of peptide-based vaccines, generating epitope-specific immune responses against the cognate protein antigens without using potent adjuvants.     

A toroid model for in vitro investigations of toxic shock syndrome toxin-1 production

Human behaviours and consumer products may affect vaginal microbial ecology, thereby influencing women's health. Relevant experimentation systems are needed to understand such possible links. Here, we describe the development of a practical semi-solid in vitro model to assess the effects of interactions between vaginal environment and the presence of tampons, on bacterial communities, including the production of toxic shock syndrome toxin-1 (TSST-1) by Staphylococcus aureus.     

Bindings of toxic shock syndrome toxin-1 and staphylococcal enterotoxins A, B, and C to rabbit spleen cells

Toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxins (SE) A, B, and C were studied on binding to rabbit spleen cells. The toxins showed remarkable mitogenic effects on the cells. Among them, SEA and TSST-1 had much stronger mitogenic activities than SEB and SEC. Binding study showed that labeled TSST-1 and SEA bound considerably to cells, but that labeled SEB or SEC was not observed to bind at a detectable level under the same conditions as TSST-1 and SEA. Competitive binding analysis between toxins to cells proved that TSST-1 and SEA clearly competed with each other in binding. Scatchard plots for TSST-1 and SEA in binding were linear at the doses used. The Scatchard analysis for TSST-1 and SEA gave a dissociation constant of 2.5 X 10(-9) M and 7.6 X 10(-8) M and the number of binding sites per cell of 5.3 X 10(3) and 1.0 X 10(5), respectively.     

Study of the biological activities of toxic shock syndrome toxin-1. I. Proliferative response and interleukin 2 production by T cells stimulated with the toxin

The mitogenic and interleukin 2 (IL 2) production-inducing effects of toxic shock syndrome toxin-1 (TSST-1) on murine lymphocytes were investigated. TSST-1, an exotoxin produced by Staphylococcus aureus recovered from patients with toxic shock syndrome (TSS), is thought to be a causative agent of the syndrome. TSST-1 was mitogenic for splenic T cells and peanut agglutinin (PNA)-negative thymocytes, but not for T cell-depleted spleen cells, PNA-positive thymocytes or IL 2-dependent CTLL 2-cells. A factor mitogenic for CTCC-2 cells with a molecular weight of 30-35 kdaltons was obtained by stimulating spleen cells with TSST-1 and it was absorbed by CTLL-2 cells, indicating that the factor is IL 2. For substantial amounts of IL 2 to be produced, 10 ng or more of TSST-1 per ml and 48 hr or more of incubation were required. Removal of T cells abrogated the IL 2 production by spleen cells. T cells obtained by the nylon wool column method alone produced IL 2 on TSST-1 stimulation in the presence of either macrophages or a macrophage lysate containing interleukin 1. However, T cells obtained by a combination of the nylon wool column method and anti-Ia antibody treatment produced IL 2 in the presence of macrophages but not of the macrophage lysate, indicating that IL 2 production by TSST-1-stimulated T cells is absolutely dependent on the presence of accessory cells.     

Staphylococcal enterotoxin B and toxic shock syndrome toxin-1 bind to distinct sites on HLA-DR and HLA-DQ molecules

Staphylococcal enterotoxins (SE) activate human T cells in vitro. This requires the presence of Ia+ accessory cells but does not require processing of the toxin by the accessory cell. We and others have recently demonstrated direct binding of SE to human MHC class II molecules. In this study, we have compared in detail the ability of class II molecules to bind two SE, toxic shock syndrome toxin-1 (TSST-1) and SEB. Scatchard analysis of equilibrium binding data indicate that SEB binds to Ia+ human cell lines with a 10-fold lower affinity than TSST-1. Likewise, SEB precipitates HLA-DR alpha- and beta-chains from detergent lysates of Ia+ cells less efficiently than TSST-1. The binding of TSST-1 and SEB to transfected L cells expressing HLA-DR and HLA-DQ gene products was differentially inhibited by anti-HLA-DR mAb. There was virtually no cross-inhibition of TSST-1 and SEB in competitive binding assays. We conclude that TSST-1 and SEB bind to two MHC class II sites which can be distinguished by their relative accessibility to blocking by anti-class II mAb and heterologous toxin.     

Receptors for toxic shock syndrome toxin-1 and staphylococcal enterotoxin A on human blood monocytes.

Staphylococcal toxic shock syndrome toxin-1 (TSST-1) as well as staphylococcal enterotoxin A (SEA) and B (SEB) have recently been shown to bind directly to the class II major histocompatibility antigen, HLA-DR. Whereas others have characterized TSST-1 and SEA binding to HLA-DR on transfected L cells or B lymphoma cell lines, we sought evidence for direct binding of TSST-1 and SEA to HLA-DR on purified human monocytes. A single class of high-affinity receptors was found for both TSST-1 (dissociation constant (Kd) 40 nM, 3.4 x 10(4) receptors per cell) and SEA (Kd 12 nM, 3.2 x 10(4) receptors per cell) on normal human monocytes. Affinity cross-linking of 125I-labeled toxins to monocytes revealed the presence of two membrane protein subunits with molecular masses consistent with the alpha and beta chains of human HLA-DR (35 and 28 kDa, respectively). The anti-HLA-DR monoclonal antibody L243, but not L203 or 2.06, inhibited radiolabeled toxin binding to human monocytes and neutralized the mitogenic response of human T lymphocytes to both toxins. However, L243 was consistently more effective in blocking radiolabeled TSST-1 than SEA binding to human monocytes from the same donors, suggesting that TSST-1 and SEA may be binding to overlapping epitopes rather than to the same epitope on HLA-DR. Because TSST-1 and SEB bind to distinct epitopes on HLA-DR and because SEA cross competes with both TSST-1 and SEB on the HLA-DR receptor, we postulate that SEA occupies a binding site within HLA-DR that overlaps both TSST-1 and SEB.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of protein tyrosine phosphorylation in monokine induction by the staphylococcal superantigen toxic shock syndrome toxin-1.

The staphylococcal superantigen toxic shock syndrome toxin-1 (TSST-1) is a potent inducer of IL-1 beta and TNF-alpha synthesis in human monocytes. As superantigens are high affinity ligands for MHC class II molecules, the induction of monokines by TSST-1 provides a biologically relevant model of MHC class II-mediated transmembrane signaling. In this study, we show that TSST-1 induces cytoplasmic protein tyrosine phosphorylation in the human monocytic cell line THP-1. This induction was greatly enhanced by cross-linking TSST-1 with biotin-avidin. The functional relevance of tyrosine phosphorylation induced by TSST-1 was demonstrated by the finding that three specific inhibitors of protein tyrosine kinases strongly inhibited the induction of IL-1 beta mRNA by TSST-1. These data suggest that protein tyrosine kinase activation plays a critical role in MHC class II-mediated transmembrane signalling by staphylococcal superantigens.     

A tumor-associated glycoprotein that blocks MHC class II-dependent antigen presentation by dendritic cells

Tumors evade immune surveillance despite the frequent expression of tumor-associated Ags (TAA). Tumor cells escape recognition by CD8(+) T cells through several mechanisms, including down-regulation of MHC class I molecules and associated Ag-processing machinery. However, although it is well accepted that optimal anti-tumor immune responses require tumor-reactive CD4(+) T cells, few studies have addressed how tumor cells evade CD4(+) T cell recognition. In this study, we show that a common TAA, GA733-2, and its murine orthologue, mouse epithelial glycoprotein (mEGP), function in blocking MHC class II-restricted Ag presentation by dendritic cells. GA733-2 is a common TAA that is expressed normally at low levels by some epithelial tissues and a subset of dendritic cells, but at high levels on colon, breast, lung, and some nonepithelial tumors. We show that ectopic expression of mEGP or GA733-2, respectively, in dendritic cells derived from murine bone marrow or human monocytes results in a dose-dependent inability to stimulate proliferation of Ag-specific or alloreactive CD4(+) T cells. Dendritic cells exposed to cell debris from tumors expressing mEGP are similarly compromised. Furthermore, mice immunized with dendritic cells expressing mEGP from a recombinant adenovirus vector exhibited a muted anti-adenovirus immune response. The inhibitory effect of mEGP was not due to down-regulation of functional MHC class II molecules or active suppression of T cells, and did not extend to T cell responses to superantigen. These results demonstrate a novel mechanism by which tumors may evade CD4(+) T cell-dependent immune responses through expression of a TAA.