In autoimmune disease, a breakdown of self-tolerance leads to the generation of an immune response to a specific target antigen or antigens. Clinical and epidemiological evidence indicates that infections play an important role in the induction of autoimmune disorders such as autoimmune myocarditis and diabetes mellitus. One mechanism by which this may occur is through the activation of autoreactive T cells by epitopes on microbial antigens that cross-react with antigens on target organs. It has also been proposed that so- called 'superantigens' (SAGs) from bacteria, mycoplasma, or viruses may initiate autoimmune disease by activating specific anti-self T cell clones. Superantigens are extremely potent T cell stimulants that act by cross- linking T cell antigen receptors (TCRs) and major histocompatibility complex (MHC) molecules. Our goal is to elucidate the physical basis of T cell activation by microbial SAGs through determination of the three-dimensional structure of TCR-SAG complexes by X-ray crystallographic techniques and to correlate this information with affinity measurements of TCR-SAG and SAG-MHC interactions. Thus, we have determined the crystal structure, to 3.5 A resolution, of a TCR beta chain complexed with a bacterial SAG, staphylococcal enterotoxin C3 (SEC3). This work enabled us to understand, for the first time, how SAGs circumvent the normal mechanism for T cell activation by specific peptide/MHC complexes. We now propose to extend our crystallographic studies of the TCR beta- SEC3 complex to a complex between the beta chain and SEB, for which crystals which diffract to high resolution (2.5 A) have been obtained. We also propose to define the relative contributions of TCR-SAG and SAG- MHC interactions to T cell stimulation by engineering a panel of mutants of SEB and SEC3 with both higher and lower affinities for TCR and MHC than the wild type toxin. We will investigate the possibility of cooperativity in the binding of SAGs to TCR and MHC by generating covalently linked SAG-MHC complexes. Finally, we propose to express a recombinant form of the Mycoplasma arthritidis SAG MAM, to measure its binding to TCR and MHC, and to determine the three-dimensional structure of MAM bound to the TCR beta chain and to MHC class II. The results of these studies will define the rules governing T cell activation by microbial SAGs and help lay the groundwork for understanding how SAGs can activate autoreactive T cell clones in autoimmune disease.
