Engineering T cell receptors to examine the role of affinity and kinetics in T helper cell function

This thesis describes the engineering of class II restricted T cell receptors (TCRs) and their subsequent structural and functional characterization. Engineering high affinity class II TCRs provides novel reagents to examine the relationship between increased affinity and T cell activation, peptide specificity, and energetic effects of peptide recognition. The stability and affinity mutations enabled crystal growth and diffraction of the highest affinity TCR complexed with peptide-MHC. High affinity TCRs could be useful diagnostic tools or therapeutics.; In Chapter two a process of directed evolution using yeast display was used to select for TCRs properly folded on the surface of yeast with increased resistance to thermal denaturation. The stability clones were soluble at higher levels than wild type and enabled kinetic and structural analysis of the TCR. The location of the stability mutations provides insights into possible mechanisms of protein stabilization.; Chapter three describes the engineering of a panel of TCRs with higher affinity using site directed mutagenesis and yeast display. A total of six TCRs were isolated, all with different mutations in peptide binding regions. The relative affinities of the clones were characterized in yeast and the possible structural effects of the affinity mutations described.; In Chapter four the relationship of TCR affinity and function is explored. Kinetic and affinity measurements of the engineered TCRs showed that the highest affinity clone has an 800-fold increase in affinity compared to wild type. T cells transfected with these TCRs function as well as wild type, have broadened fine peptide specificity, but retain exquisite antigen specificity. Quantitative analysis using the high affinity TCRs has revealed multiple hot spots of peptide recognition that account for the remarkable antigen specificity of T cells.; Chapter five details the engineering of a class II restricted human TCR isolated from a patient with multiple sclerosis called 3A6. Yeast display of TCRs has previously relied on the use of an antibody specific for a properly folded TCR, here various strategies were used to generate stabilized 3A6 TCRs. Helpful strategies included: alteration of error-prone PCR dNTP concentrations, increasing induction media galactose concentrations, and generation of three-domain TCR constructs (Valpha-VbetaCbeta).