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CTLs specific to p53 were previously shown to efficiently eradicate p53-overexpressing tumor cells in vitro as well as in vivo. In this report, we demonstrate that these CTLs can also eliminate tumors that display moderate or even low levels of p53. Neither high steady-state levels of p53 nor elevated p53 synthesis is a prerequisite for recognition of tumors by p53-specific CTLs. Instead, our data show that a high p53 turnover rate is an important factor in determining the sensitivity of tumor cells to p53-specific CTLs. Our data suggest that p53 turnover is related to the MHC class I-restricted presentation of p53-derived epitopes at the tumor cell surface and indicate that CTL-mediated immunotherapy that targets p53 can be applied to a wider range of tumors than has thus far been anticipated.

The tumor suppressor protein p53 is overexpressed in up to 50% of all human malignancies, both in solid tumors as well as hematological malignancies, and is therefore an attractive target for immunotherapy. We have recently shown that cytotoxic T lymphocytes (CTL), raised in p53 gene deficient (p53 -/-) mice and recognizing a murine wild-type (wt) p53 peptide, were able to eradicate a mutant p53-induced and overexpressing tumor in p53 +/+ nude mice. These CTL also prevented the outgrowth of a more aggressive p53-overexpressing tumor in immunocompetent C57BL/6 mice. Importantly, this occurred in the absence of demonstrable damage to normal tissue. Possibly due to the aggressive nature of the latter tumor, adoptive transfer of wtp53-specific CTL did not result in the eradication of established tumors, either in nude or immunocompetent mice. Therefore, we explored whether the cytotoxic drug cyclophosphamide (CY) could potentiate the therapeutic activity of wtp53-specific CTL. We show here that CY acts synergistically with adoptively transferred wtp53-specific CTL in controlling the growth of an aggressive mutant p53-induced and overexpressing tumor. Previously described mechanisms underlying the synergism between CY and immune T cells were evaluated, but were not found to be operational in this model.

Murine tumor cells obtained through transfection of expression plasmids carrying activated cellular and/or viral oncogenes constitute formidable tools for immunological tumor research. As reported previously, mouse embryo cells of C57BL/6 origin, transformed by mutated p53 or human papilloma virus type 16 (HPV16), present, at their surface, MHC-bound peptides that are derived from the p53 and the HPV16 E7 oncoproteins, respectively, which can serve as a target for a highly effective antitumor T-cell response. Here, we describe the identification, through molecular cloning, of an additional, highly immunodominant peptide that is presented by the aforementioned HPV16- and p53-transformed cells. This peptide is encoded by a cryptic open reading frame in the backbone sequences of the plasmids that had been used to generate these cells. Considerable amounts of transcripts encompassing this open reading frame were detected in the cells concerned. These transcripts were the result of the bidirectional nature of the retroviral long terminal repeat (LTR) present in the expression plasmids used for transfection, which resulted in transcription of the gene of interest, as well as in transcription of the vector sequences positioned at the other side of the LTR. Due to this mechanism, all tumor cells harboring LTR-driven expression plasmids expressed the highly immunogenic peptide, whereas cells containing plasmids driven by more unidirectional promoters exhibited lower levels of this peptide. LTR-driven expression plasmids were also shown to encode this peptide epitope when used for DNA vaccination, as mice vaccinated with such a plasmid developed a CTL response against this peptide. Our data show that awareness of plasmid backbone-derived epitopes is of crucial importance for the correct interpretation of preclinical experiments and for the design of DNA vaccines.

Human papillomavirus (HPV) E6 and E7 oncoproteins are attractive targets for T-cell-based immunotherapy of cervical cancer. In this study, we demonstrate that dendritic cells (DCs) pulsed with HPV16 E7 protein are not only recognized in vitro by E7-specific CTLs but also elicit E7-specific CTL responses in vivo, associated with protection against a challenge with syngeneic HPV16-induced tumor cells. Vaccination with soluble E7 protein in incomplete Freund's adjuvant likewise induces E7-specific CTL responses associated with tumor protection. The presence of HPV16 E7-specific CTLs in vivo and the observation that depletion of CD8+ cells completely abolishes tumor protection demonstrate that CTLs are the major effector cells in mediating antitumor activity. The in vivo involvement of DCs in the activation of protective CTLs is suggested by the surface display of E7 peptide-loaded MHC class I molecules on these cells after E7 protein immunization. These data show that HPV16 E7 protein-pulsed DCs, as well as the administration of E7 protein antigen in adjuvant, can effectively stimulate tumor-specific MHC class I-restricted CD8+ T-cell-mediated protective immunity to HPV16-induced cancers.

We previously described a motif prediction of major histocompatibility complex allele-specific peptides and an in vitro assay for actual measurement of peptide binding to human leukocyte antigen HLA-A2.1 molecules. Using this method we have identified candidate cytotoxic T lymphocyte (CTL) epitopes derived from a non-self-protein (influenza matrix) and self-protein (p53). We now show that results of binding assays performed over a range of peptide concentrations indicate that distinct differences in HLA-A2.1 peptide binding affinities exist between the influenza matrix and p53 protein. The results for the influenza matrix protein indicate that the peptide that shows the highest binding affinity to HLA-A2.1 is identical to the known immunodominant peptide recognized by influenza virus-specific CTLs. The results for p53 indicate that one of the peptides with a low binding affinity is capable of inducing specific CTL responses, but CTLs recognizing the highest affinity binding peptides were not obtained. These findings are discussed in terms of the distinct implications for induction of cellular immune responses directed against peptides with different binding affinities for HLA-A2.1 of proteins that constitute attractive targets for tumor immunotherapy.