Toxic shock syndrome toxin-1 (TSST-1) is a 22 kilodalton S. aureus exotoxin implicated in the pathogenesis of TSS. TSST-1 activates monocytes to release interleukin 1 and tumor necrosis factor and causes the proliferation of peripheral blood mononuclear cells. We have preliminary data which indicates that TSST-1 binds to Class II major histocompatibility complex (MHC) molecules expressed on B cells and monocytes. We propose to analyze the nature of this interaction. (1) We will ascertain the identity of the TSST-1 binding heterodimer with HLA-DR, HLADP or HLADQ by comparing peptide maps, two dimensional gel electrophoresis patterns and N terminal amino acid (a.a.) sequences. Cells from MHC Class II deficient patients will be used to ascertain that no other cell surface molecules bind TSST-1. We will examine TSST-1 binding to MHC Class II molecules expressed by L cell transfectants and to MHC Class II molecules incorporated into liposomes to ascertain that no other molecules contribute to the binding of TSST-1 to MHC Class II molecules. 2) The determinants on MHC Class II molecules involved in TSST-1 binding will be studied by examining the contribution of sugar residues and of isolated MHC Class II alpha and beta chains to TSST-1 binding, by performing mAb inhibition studies, and by examining TSST-1 binding to L cell transfectants which express isotype mismatched alpha and beta chains or chains with single a.a. substitutions. 3) The TSST-1 site involved in binding to MHC Class II molecules will be investigated by examining the activity of truncated TSST-1 peptides and of TSST-1 mutants generated by oligonucleotide site directed mutagenesis. 4) A mouse model of TSST-1 MHC Class II interaction will be developed because of the availability of better reagents in the murine system and for future in vivo studies. The role of IA vs. IE molecules will be assessed using deletion mutants and the determinants on these molecules involved in TSST-1 binding will be mapped using L cells transfected with exon shuffled alpha and beta chains and chains with point mutations and in phase insertions. The proposed studies define a new function for MHC Class II molecules as receptors for bacterial toxins and will enhance our understanding of diseases caused by TSST-1 and by related bacterial toxins. The potential of TSST-1 antagonists to disrupt the quaternary complex of MHC Class II molecule - peptide antigen - T cell receptor - CD4 has important implications for therapeutic intervention in diseases associated with chronic T cell activation e.g. autoimmune, graft versus host and allergic diseases.
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