title: CD4+ T-cell epitope determination using unexposed human donor peripheral blood mononuclear cells.
abstract: The engineering of protein therapeutics to improve their stability, their efficacy, 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 rely on peripheral blood lymphocytes from environmentally exposed or disease-bearing donors. This severely limits the opportunity to confirm T-cell epitopes in novel proteins, because exposed donors are not available to novel or engineered proteins. Other methods for determining T-cell epitopes are either computer-modeled predictions based on potential binding to HLA molecules or the identification of peptides presented by HLA molecules removed from the surface of tumor cells or protein-pulsed antigen-presenting cells. Because HLA binding is necessary, but not sufficient, for T-cell responses, these methods must be validated by in vitro presentation assays. The authors describe a dendritic cell-based assay that identifies CD4+ T-cell epitopes in novel proteins using unexposed donors. Predicted T-cell epitopes in the protein of interest were confirmed using cells from two verified exposed 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 reflects de novo priming in vitro, and it accurately identifies primary T-cell epitopes. This assay is a powerful tool for determining relevant immunostimulatory T-cell epitopes for all types of immunoregulatory applications.
authors.affiliation: Genencor International, Palo Alto, California 94304, USA.
authors.forename: M M
authors.initials: MM
authors.lastname: Stickler
authors.forename: D A
authors.initials: DA
authors.lastname: Estell
authors.forename: F A
authors.initials: FA
authors.lastname: Harding
mesh_terms: D017951:Antigen Presentation; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D003713:Dendritic Cells / Q000276:immunology; D018604:Epitope Mapping / Q000379:methods*; D006684:HLA-DR Antigens / Q000276:immunology; D059811:HLA-DRB1 Chains; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D008213:Lymphocyte Activation; D010455:Peptides / Q000276:immunology; D020860:Subtilisin / Q000276:immunology*
chemical_list: D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains; D010455:Peptides; D020860:Subtilisin