A CD8+ T cell clone specific for the MDM-2_53-61 peptide recognizes HLA-A2+ tumor target cell lines that overexpress MDM-2 and natural peptides contained in HPLC fraction 48 derived from the PCI-13 HNC line. Clone 1A1 was harvested from a well seeded at 0.3 cell/well in limiting dilution culture from a normal HLA-A2+ donor stimulated in vitro with MDM-2_53-61 peptide-pulsed autologous DCs. After expanding the clone to sufficient numbers, it was analyzed against a range of target cell lines that vary in their constitutive levels of HLA-A2 and MDM-2 protein using the IFN-gamma ELISPOT assay (panel A). The PCI-13 and PCI-1 HNC and the MEL526 melanoma lines are HLA-A2+ and overexpress the MDM-2 protein (as determined by Western Blot and IHC analyses, Figure 3 and data not shown). The T2 cell line is HLA-A2+, but fails to overexpress MDM-2, while the SCC-74 HNC cell line is HLA-A2-negative. T cell recognition was blocked by addition of the MA2.1 anti-HLA-A2 mAb. The CD8+ T cell clone also selectively recognized T2 targets loaded with the synthetic MDM-2_53-61 peptide or peptides found in HPLC fraction 48 derived from PCI-13 target cells (panel B). Data are reported as the mean ± SD from triplicate determinations and are representative of two assays performed.