Missense mutation in the p53 tumor suppressor gene is the most common genetic event associated with human cancer. As a result of mutation, two types of HLA class I-restricted cytotoxic T lymphocyte (CTL)-defined tumor epitopes can be derived from a mutated p53 molecule: a tumor specific peptide that would incorporate the missense mutation and multiple tumor associated wild type sequence (wt) peptides derived from the rest of the mutated p53 molecule. Generation of a class I HLA-restricted tumor specific p53 epitope requires that a missense mutation occur within a CTL-defined epitope and not abrogate processing or presentation of the epitope or create a completely novel epitope. Due to the constraints imposed by antigen processing and presentation, p53 missense mutations have a low potential of giving rise to mutant epitopes and vaccines targeting them are considered impractical. In contrast, wt p53 epitopes are considered attractive candidates for developing broad applicable cancer vaccines. Relative to the commonly occurring class I HLA-A*0201 (HLA-A2) restriction element, four CTL-defined wt p53 epitopes, p53[65-73], p53[149-157], p53[217-225], and p53[264-272], have been identified and well characterized for vaccine use. Recently, we have established that 7 of the 19 p53 missense mutations detected in 40 HLA-A2+ squamous cell carcinoma of the head and neck (SCHHN) occurred within or immediately flanking one of three HLA-A2-restricted CTL-defined epitopes. Three p53 missense mutations, S149C, Y220H and Y220C, could be incorporated into 9 mer peptides that were capable of in vitro generation of anti-peptide CTL. Of the three mutant p53 peptides, the availability of the HLA-A2+ SCCHN cell line, UD-SCC 6, which expresses the p53 Y220C mutation, permitted the demonstration that the p53 mutant Y220C peptide represents a naturally presented HLA-A2-restricted, CTL-defined epitope. In the case of the autologous UD-SCC 6 system, the UD-SCC 6 tumor cell line readily processes and presents the p53 Y220C mutation for CTL recognition. In addition, an enriched population of CD8+ T cells isolated from PBMC obtained from the patient was IVS responsive to the mutant peptide. The most important finding of our study is that p53 Y220C mutation can yield a CTL-defined mutant p53 epitope. The high prevalence of this mutation in tumors obtained from HLA-A2+ patients with SCCHN mandates further analyses of sites of genetic alterations in p53 relative to tumors obtained from HLA-A2+ patients. If the prevalence of this mutation is confirmed in larger cohorts of patients with cancer, vaccines incorporating this tumor-specific epitope should have a much broader applicability than previously considered for a given p53 mutation. T cell activation is a critical step in mounting an appropriate immune defense against infectious agents such as viruses and bacteria. Central to understanding how antigen presenting cells mediate the full activation of T cells is knowledge of the interaction of MHC molecules with the T-cell receptor (TCR). We previously described a novel experimental system in order to address how engagement of TCR leads to full T cell activation. We have produced recombinant forms of the mouse AHIII TCR and its MHC ligand, H2-Db-p1058, as well as a human pMHC complex with which it interacts, HLA-A2-p1049. Having demonstrated that we have in possession a functional AHIII cytotoxic T cell line and can produce large quantities of active recombinant protein for use in functional and biochemical analysis, we performed surface plasmon resonance (SPR) experiments in order to distinguish between two models of T cell activation, an affinity-based model and a kinetic-rate model. Using our co-crystal structure of AHIII and HLA-A2-p1049, we selected 11 mutations in the A2-p1049 molecule for SPR studies. To relate these binding constants to T cell activity, we performed cytotoxic lysis assays.
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