Mm. Stickler et al., CD4(+) T-cell epitope determination using unexposed human donor peripheralblood mononuclear cells, J IMMUNOTH, 23(6), 2000, pp. 654-660
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.