The overall goal of the proposed research is to determine if helper T-cell epitopes can be predicted on the basis of local antigen instability. The mechanism of immunodominance and the properties of immunodominant epitopes are largely unknown. However, a survey of fine specificity studies reveals a correlation between helper T-cell epitopes and unstable domains in proteins analyzed by hydrogen-deuterium exchange and magnetic resonance. In addition, highly flexible loops are associated with immunodominant epitopes in certain autoantigens including the HSP10 heat shock protein. The novel hypothesis that local instability directs presentation of nearby antigen sequences by favoring proteolytic processing will be tested in the proposed work. The correlation of immunodominant T-cell epitopes with unstable antigen segments will be examined using published T-cell epitope maps and profiles of local instability based on amide protection factors obtained by hydrogen-deuterium exchange NMR and/or crystallographic B factors. T-cell epitopes in T4 lysozyme have been predicted on the basis of local instability. The accuracy of this prediction will be tested by generating a mouse T-cell epitope map for T4 lysozyme using antigen restimulation assays. The influence of HSP10 flexible segments on patterns of immunodominance will be assessed using antigen restimulation assays with T cells primed against HSP10 proteins from divergent sources and HSP10s having modified loop structures. T4 lysozyme and HSP10 are well suited for studies on immunodominance because they are small proteins, and their 3-dimensional structures are available. Moreover, mounting evidence suggest that autoimmune responses to HSPs such as HSP10 have immunomodulatory functions as well as possible role in pathogenic autoimmune disorders. Preliminary results suggest that local antigen instability focuses antigen processing and therefore helper T cell responses on a rather small fraction of the antigen sequence. Results of the proposed research may suggest strategies to improve vaccine design by considering the influence of local antigen instability. This work also may contribute to understanding the initiation and propagation of autoimmune disorders since locally unstable antigen segments are preferred targets of immune attack.
