Engineering the T Cell Receptor to Protect Host Cells in Superantigen-Induced Septic Shock

134 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2006.Superantigens (SAgs) are potent pathogenic agents. Produced by bacteria and viruses, these proteins target a subset of white blood cells, namely T cells, which are responsible for the cell-mediated responses of the immune system. SAgs stimulate T cell populations by crosslinking a T cell receptor (TCR) with a class II major histocompatability complex (pMHC) product on the host cell surface, thus circumventing the normal recognition of intracellularly-processed antigens. Because SAgs can bind to many different TCRs, SAg engagement of T cells leads to polyclonal expansion of up to 20% of all T cells. In contrast, normal pMHC recognition might involve the stimulation of one in many thousands of T cells. Since this specificity is the hallmark of T cell immunity, bacteria and viruses that produce SAgs appear to derive some benefit from massive T cell activation. A class of SAgs is the enterotoxins of the bacterium Staphylococcus aureus, including B and C3 (SEB and SEC3, respectively). These toxins maintain an ability to bind many different variable domains of the TCR beta chain (Vbeta), resulting in stimulation of large subsets of T cells. A crystal structure of the SEC3:TCR complex suggested that SAgs might bind many TCR Vbeta regions by recognizing TCR conformations that are conserved in SE-reactive T cells. Here, the functional interaction of the 2C ValphaVbeta TCR with a high-affinity variant of SEC3 was characterized by alanine scanning, revealing a binding hotspot involving Vbeta TCR CDR2. Additionally, the Valpha CDR2 region of the TCR was shown to be critical for the stabilization of the proposed TCR:SAg:pMHC ternary complex. Guided by these results, TCR mutants were engineered by directed mutation and yeast surface display to bind SEB and SEC3 with subnanomolar affinities. Soluble forms of the Vbeta TCR that retained high affinity and inhibited SE-mediated T cell activity in vitro were produced. As a result of this work, high-affinity Vbeta TCR may be evaluated as potential antagonists to T cells in SE-induced septic shock.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD