Tumor peptide-based vaccines are more effective when they include tumor-specific Th cell-defined as well as CTL-defined peptides. Presently, two overlapping wild-type sequences (wt) p53 helper peptides, p53(108-122) and p53(110-124), have been identified as HLA-DR1- and/or HLA-DR4-restricted epitopes. These HLA-DR alleles are expressed by approximately 35% of subjects with cancer. To identify Th cell-defined wt p53 peptides suitable for use on the remaining subject population, a dendritic cell (DC)-based coculture system was developed. CD4(+) T cells isolated from PBMC obtained from HLA-DR4(-) normal donors were stimulated ex vivo with autologous DC transfected with wt p53 or mutant p53 cDNA. Reactivity of T cells was tested in ELISPOT IFN-gamma assays against DC pulsed individually with a panel of algorithm-predicted, multiple HLA-DR-binding wt p53 peptides. The wt p53(25-35) peptide was identified as capable of inducing and being recognized by CD4(+) T cells in association, at a minimum, with HLA-DR7 and -DR11 molecules, each of which is expressed by approximately 15% of the population. In addition, the presence of anti-p53(25-35) CD4(+) Th cells was shown to enhance the in vitro generation/expansion of HLA-A2-restricted, anti-wt p53(264-272) CD8(+) T cells, which from one donor were initially "nonresponsive" to the wt p53(264-272) peptide. The wt p53(25-35) peptide has attributes of a naturally presented Th cell-defined peptide, which could be incorporated into antitumor vaccines applicable to a broader population of subjects for whom a wt p53 helper peptide is presently unavailable, as well as used for monitoring anti-p53 Th cell activity in cancer subjects receiving p53-based immunotherapy.