Peptide-specific T cell responses measured by gamma interferon ELISPOT assays. (A and B) The NQM induces peptide-specific T cell response, comparable to the peptide YMF, an analog peptide for RMF epitope. CD3 T cells from a healthy HLA-A*02:01+ donor were stimulated with either NQM or YMF peptide for three (A) or five (B) rounds. The peptide-specific response was measured by the IFN-gamma Elispot assay by pulsing autologous CD14+ cells (antigen-presenting cells, APC) with individual peptide. Each data point represents a mean +/− SD from triplicate cultures. (C and D) The NLM peptide induced strong peptide-specific T cell responses which cross-reacted to the native sequence NQM in a HLA-A*02:01 homozygous donor. CD3 T cells from a healthy HLA-A*02:01 homozygous donor were stimulated with either NQM or NLM peptide three times (C). Similarly, T cells from a HLA-A*02:01 heterozygous donor were stimulated with NQM, NLM, or NYM three times (D) and the peptide-specific response was measured by the IFN-gamma secretion upon challenging with individual peptide. Each data point represents the mean +/− SD from triplicate cultures. Data are representative of results from ten experiments from six donors.