Physical Basis of T Cell Activation by Superantigens

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DESCRIPTION (provided by applicant): Superantigens (SAGs) are proteins of bacterial or viral origins that stimulate T cells by cross-linking T cell receptors (TCRs) and major histocompatibility (MHC) class II molecules. Stimulation leads to hyperactive responses associated with the massive release of pyrogenic and inflammatory cytokines, usually followed by T cell anergy or deletion. Two classes of SAGs have been identified: exogenous soluble proteins secreted by bacteria, such as the staphylococcal enterotoxins (SEs), and endogenous retroviral-encoded transmembrane proteins, such as the SAGs of mouse mammary tumor viruses (Mtv SAGs). Our goal is to elucidate the physical basis of T cell activation by bacterial and viral SAGs through determination of the three-dimensional structure of TCR-SAG and SAG-MHC complexes by X-ray crystallographic techniques and to correlate this information with affinity measurements of TCR-SAG and SAG-MHC interactions. We will determine the crystal structures of representative TCR f3 chain-SAG and SAG-MHC complexes in order to define the diverse strategies that SAGs have evolved for binding TCR and MHC. We previously determined the structures of a mouse TCR f3 chain complexed with SEB. We will now determine the structures of a human TCR B chain complexed with toxic shock syndrome toxin-1 (TSST-1) and streptococcal pyrogenic exotoxin C (SPEC). To identify the high-affinity, Zn2+ dependent SAG binding site on MHC class II, we recently solved the structure of SPEC bound to HLA-DR2a bearing a self-peptide from myelin basic protein (MBP). We will now extend this study to SEA and SED, which cross-link class II molecules on APCs. The structure of the SPEC-DR2a/MBP complex reveals that SPEC makes extensive contacts with the bound MBP peptide, suggesting that peptide can directly influence SAG binding and presentation. The affinity of SPEC for HLA-DR2a molecules bearing analogs of the MBP peptide will be determined. The ability of defined MHC/peptide complexes to present SPEC to T cells will be tested by expressing DR molecules with covalently-linked single peptides on the surface of class Il-negative DAP-3 cells. We also propose to define the kinetic and affinity parameters governing T cell activation by bacterial SAGs by engineering mutants of SPEC and TSST-1 with both higher and lower affinities for TCR and MHC than the wild type toxins. Finally, in order to establish the basis for MHC recognition by viral SAGs, we will express soluble forms of Mtv7 SAG for use in direct binding and co-crystallization experiments with the I Ed class H molecule.