CD4(+) T-cell epitope determination using unexposed human donor peripheralblood mononuclear cells

The engineering of protein therapeutics to improve their stability, their e fficacy, or to create "humanized" versions introduces changes to the amino acid sequence that are potential T-cell epitopes. Until now, there has been no available assay to detect primary T-cell responses to novel epitopes in humans. Currently available in vitro protocols for epitope determination r ely on peripheral blood lymphocytes from environmentally exposed or disease -bearing donors. This severely limits the opportunity to confirm T-cell epi topes in novel proteins, because exposed donors are not available to novel or engineered proteins. Other methods for determining T-cell epitopes are e ither computer-modeled predictions based on potential binding to HLA molecu les or the identification of peptides presented by HLA molecules removed fr om the surface of tumor cells or protein-pulsed antigen-presenting cells. B ecause HLA binding is necessary, but not sufficient, for T-cell responses, these methods must be validated by in vitro presentation assays. The author s describe a dendritic cell-based assay that identifies CD4(+) T-cell epito pes in novel proteins using unexposed donors. predicted T-cell epitopes in the protein of interest were confirmed using cells from two verified expose d donors. The major CD4(+) T-cell epitope of the novel protein examined in this study associated with the expression of HLA DRb1*15. This assay reflec ts de novo priming in vitro, and it accurately identifies primary T-cell ep itopes. This assay is a powerful tool for determining relevant immunostimul atory T-cell epitopes for all types of immunoregulatory applications.