Monoclonal antibodies defining functional sites on the toxin superantigen staphylococcal enterotoxin B.

Abstract

SummaryFour monoclonal antibodies (mAbs) were produced binding to four nonoverlapping epitopes on the superantigen staphylococcal enterotoxin B (SEB). The mAbs were tested for their ability to detect SEB bound to major histocompatibility complex (MHC) class II, to inhibit SEB binding to MHC class II, to inhibit SEB stimulation of T cell hybridomas, to bind to various nonfunctional mutants of SEB, and to capture and present SEB and its mutants to T cells in the absence of MHC class II. We concluded that two mAbs, B344 and B327, bound to epitopes not required for superantigen function, one mAb, 2B33, blocked an MHC interaction site on SEB, and the fourth mAb, B87, blocked the T cell recognition site on SEB. Moreover, two mAbs (B344 and 2B33) were capable of presenting SEB, although much less efficiently than APC, to CD4-but not CD4 § T cell hybridomas. The results confirm the functional domains on SEB originally defined by mutation and show that MHC class II is not always an essential component of the superantigen ligand.S taphylococcus aureus produces a set of exotoxin superantigens that cause food poisoning and toxic shock in man and rapid weight loss in mice sometimes leading to death (1-5). As superantigens these proteins bind to MHC class II molecules and activate a large set ofT cells in a V/3-specific fashion (6-12). This T cell stimulation and the accompanying massive release of lymphokines appear to play an essential role in the toxicity of these proteins (13,14).Although the amino acid sequences of the staphylococcal toxins are related, each has a unique V~ specificity. For example, in mice staphylococcal enterotoxin B (SEB) 1 stimulates T cells bearing V~7, 8.1, 8.2, and 8.3 (6, 10, 11), whereas SEA stimulates T cells bearing V/31, 3, 11, and 17a (10). We have identified mutants of SEB impaired either for binding to MHC or for interaction with ol/3 TCR-VBs (14). These mutations in SEB also eliminated in vivo toxicity in mice. When mapped on the structure of SEB, these mutations indicated different sites on the SEB surface for orb TCR versus MHC interaction (15).In the current study we have confirmed these conclusions using a set of four anti-SEB mAbs specific for four nonoveri Abbreviations used in this paper: CTM, complete tissue culture medium; FBS, fetal bovine serum; SEB, staphylococcal enterotoxin B; F-SEB, fluoresceinated SEB. lapping epitopes on SEB. Two of these antibodies define an MHC and an c~/3 TCR-interaction site on SEB, whereas, the other two react with sites not involved in SEB superantigen activity. Moreover, we found that two of these mAbs could substitute for MHC class II in presentation of SEB to T cell hybridomas. Materials and MethodsCell Lines. Three T cell hybridomas were used in these studies, all derived from B10.BR mice. KS-6.1 (VB8.2+) and

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TCR recognition of peptide/MHC class II complexes and superantigens

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