Staphylococcal enterotoxins are prototype superantigens characterized by their ability to bind to major histocompatibility complex (MHC) class II molecules and subsequently activate a large fraction of T‐lymphocytes. The crystal structure of staphylococcal enterotoxin type A (SEA), a 27 kDa monomeric protein, was determined to 1.9 A resolution with an R‐factor of 19.9% by multiple isomorphous replacement. SEA is a two domain protein composed of a beta‐barrel and a beta‐grasp motif demonstrating the same general structure as staphylococcal enterotoxins SEB and TSST‐1. Unique for SEA, however, is a Zn2+ coordination site involved in MHC class II binding. Four amino acids including Ser1, His187, His225 and Asp227 were found to be involved in direct coordination of the metal ion. SEA is the first Zn2+ binding enterotoxin that has been structurally determined.

Bacterial superantigens (SAgs) are potent activators of T lymphocytes and play a pathophysiological role in Gram‐positive septic shock and food poisoning. To characterize potential MHC class II binding sites of the bacterial SAg staphylococcal enterotoxin (SE) A, we performed alanine substitution mutagenesis throughout the C‐terminus and at selected sites in the N‐terminal domain. Four amino acids in the C‐terminus were shown to be involved in MHC class II binding. Three of these amino acids, H225, D227 and H187, had a major influence on MHC class II binding and appeared to be involved in coordination of a Zn2+ ion. Alanine substitution of H225 and D227 resulted in a 1000‐fold reduction in MHC class II affinity. Mutation at F47, which is equivalent to the F44 previously shown to be central in the MHC class II binding site of the SAg, SEB, resulted in a 10‐fold reduction in MHC class II affinity. The combination of these mutations in the N‐ and C‐terminal sites resulted in a profound loss of activity. The perturbation of MHC class II binding in the various mutants was accompanied by a corresponding loss of ability to induce MHC class II‐dependent T cell proliferation and cytotoxicity. All of the SEA mutants were expressed as Fab‐SEA fusion proteins and found to retain an intact T cell receptor (TCR) epitope, as determined in a mAb targeted MHC class II‐independent T cell cytotoxicity assay.(ABSTRACT TRUNCATED AT 250 WORDS)

The bacterial superantigen staphylococcal enterotoxin A (SEA) is an extremely potent activator of T lymphocytes when presented on major histocompatibility complex (MHC) The therapeutic use of naked monoclonal antibodies (mAbs) in human epithelial cancer has met with limited success (1). The amount of mAb found to accumulate in the tumor is generally low, tumor cells display extensive heterogeneity in antigen expression, and the effector mechanisms responsible for tumor-cell destruction are insufficient (1, 2). Recent progress in the molecular understanding of T-cell recognition has provided insight into the role of T cells in the response against allogeneic and neoplastic tissues. The vigorous and destructive response against an allograft involves a high frequency of T lymphocytes that, via the T-cell receptor (TCR), recognizes peptide antigens presented in the context of major histocompatibility complex (MHC) molecules. In contrast, the frequency of T cells responding to a tumor is generally low and insufficient to interfere with tumor growth. This suggests that an attractive approach for immunotherapy would be to target a high frequency of T cells to the tumor area. We have therefore developed an antibody-based therapy that provides tumor cells with superantigenicity. Superantigens (SAgs) are a family of bacterial and viral proteins that activate a high frequency of T cells to cytokine release and cell-mediated cytotoxicity (3)(4)(5). Bacterial SAgs bind with high affinity to MHC class II molecules and subsequently interact with T cells expressing particular sequences in the variable (V) region of the TCR , / chain (TCR V(3) (3)(4)(5) MATERIALS AND METHODSProtein Reagents. Recombinant SEA was expressed in E. coli and purified to homogeneity. The hybridomas secreting the C215 (IgG2a) and C242 (IgGl) mAbs, reacting with human colon cancer, were produced at Pharmacia (8, 9). E. coli HB101 was used as host during the DNA construction work (10).

Bacterial superantigens, including the staphylococcal enterotoxins, are the most potent activators of T cells known and have been suggested as a causative factor in Gram‐positive shock in humans. Staphylococcal enterotoxin D (SED) is dependent upon Zn2+ for high affinity interactions with MHC class II molecules and thus SED was co‐crystallized with Zn2+. The crystal structure of SED has been determined in two different space groups, at 2.3 and 3.0 A resolution respectively. The three‐dimensional structure of SED is similar to structures of other bacterial superantigens, although this study has revealed that SED has the unique capability of forming dimers in the presence of Zn2+. The high affinity Zn2+ site used in dimer formation is located on the surface of the beta‐sheet in the C‐terminal domain. Two bound metal ions are coordinated by residues from both molecules in the dimer interface and thus contribute directly to formation of the dimer. A second Zn2+ site is located on the surface close to the domain interface of the molecule. The unique feature of SED in forming a Zn2+‐dependent homodimer seems to facilitate novel and biologically relevant multimeric interactions with MHC class II molecules, as shown by the induction of cytokine mRNA in human monocytes when exposed to SED and SED mutants.

SumnlaryStaphylococcal enterotoxin A (SEA) has two distinct binding sites for major histocompatibility complex (MHC) class II molecules. The aspartic acid located at position 227 (D227) in the COOH terminus of SEA is one of the three residues involved in its interaction with the DR[3 chain, whereas the phenylalanine 47 (F47) of the NH 2 terminus is critical for its binding to the DRot chain. Upon interaction with MHC class II molecules, SEA triggers several cellular events leading to cytokine gene expression. In the present study, we have demonstrated that, contrary to wild-type SEA, stimulation of the THP1 monocytic cell line with SEA mutated at position 47 (SEAF4v~ or at position 227 (SEAD227t0 failed to induce interleukin 113 and tumor necrosis factor-o~ messenger RNA expression. Pretreatment of the cells with a 10-fold excess of either SEAFa7A or SEAD227 A prevented the increase in cytokine messenger RNA induced by wild-type SEA. However, cross-linking of SEAF47A or SEAD227 A bound to MHC class II molecules with F(ab')2 anti-SEA mAb leads to cytokine gene expression, whereas cross-linking with F(ab) fragments had no effect. Taken together, these results indicate that cross-linking of two MHC class II molecules by one single SEA molecule is a requirement for cytokine gene expression.

Anergy is a major mechanism to ensure antigen-specific tolerance in T lymphocytes in the adult. In vivo, anergy has mainly been studied at the cellular level. In this study, we used the T-cell-activating superantigen staphylococcal enterotoxin A (SEA) to investigate molecular mechanisms of -T-lymphocyte anergy in vivo. Injection of SEA to adult mice activates CD4+ T cells expressing certain T-cell receptor (TCR) variable region a8-chain families and induces strong and rapid production of interleukin 2 (IL-2). In contrast, repeated injections of SEA cause CD4+ T-cell deletion and anergy in the remaining CD4+ T cells, characterized by reduced expression of IL-2 at mRNA and protein levels. We analyzed expression of AP-1, NF-KcB, NF-AT, and octamer binding transcription factors, which are known to be involved in the regulation of IL-2 gene promoter activity. Large amounts of AP-1 and NF-cB and significant quantities of NF-AT were induced in SEA-activated CD4+ spleen T cells, whereas Oct-1 and Oct-2 DNA binding activity was similar in both resting and activated T cells. In contrast, anergic CD4+ T cells contained severely reduced levels ofAP-1 and Fos/Juncontaining NF-AT complexes but expressed significant amounts of NF-KcB and Oct binding proteins after SEA stimulation. Resolution of the NF-cB complex demonstrated predominant expression of p50-p65 heterodimers in activated CD4+ T cells, while anergic cells mainly expressed the transcriptionally inactive p50 homodimer. These alterations of transcription factors are likely to be responsible for repression of IL-2 in anergic T cells.Staphylococcal enterotoxins (SEs) belong to a family of bacterial proteins denoted as superantigens (SAgs) because of their ability to activate a high frequency of both CD4+ and CD8+ T cells expressing certain T-cell receptor (TCR) variable region f3 chains (Vp) (1,2). Administration of SE to adult mice induces rapid production of a panel of cytokines, including interleukin 2 (IL-2) and tumor necrosis factor (TNF), and subsequent expansion of reactive T-cell populations (3). After the initial phase of SE-induced activation in vivo, part of the reactive CD4+ T cells are deleted and the remaining CD4+ T-cell population fails to proliferate and secrete IL-2 in response to a subsequent SE challenge (4).

The bacterial superantigen staphylococcal enterotoxin (SE) A (SEA) directs cytotoxic T lymphocytes (CTLs) expressing particular sequences of the T-cell receptor (TCR)

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