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The angiogenic factor Midkine (MDK) is overexpressed in various human malignant tumors, although its expression is low or undetectable in normal adult tissues. Its expression in tumors and its detection in plasma have been associated with poor disease outcome, whereas its blockade was found to contribute to tumor regression. By weekly stimulation of T lymphocytes harvested in HLA-A2 healthy donors, we derived CD8 T cell lines specific for several MDK peptides. The T cell response was mostly dominated by two nonamer peptides localized in the signal peptide and in the C-terminal part of the protein, as assessed by IFN-gamma ELISPOT and HLA-A2 tetramer labeling. Peptide-specific T cell lines recognized cells transfected with an MDK-encoded plasmid and tumor cell lines naturally expressing the MDK protein, but not untransfected cells. T cell presentation of the two MDK epitopes was found to be TAP dependent. Experiments performed in HLA-A2 transgenic mice demonstrated the capacity of the two identified CD8 T cell epitopes to elicit a cytotoxic response. Altogether, our data show that the secreted MDK protein is a candidate vaccine for multiple cancers.

Because of the wide distribution of the survivin Ag in a variety of tumors, we have investigated the survivin-specific CD4+ T cell response in healthy donors and cancer patients. Screening of the entire sequence of survivin for HLA class II binding led to the identification of seven HLA-DR promiscuous peptides, including four HLA-DP4 peptides. All of the peptides were able to prime in vitro CD4+ T cells of eight different healthy donors. The peptide-specific T cell lines were stimulated by dendritic cells loaded with the recombinant protein or with the lysates of tumor cells. The high frequency of responders (i.e., immunoprevalence) was provided by a wide reactivity of multiple peptides. Six peptides were T cell stimulating in at least half of the donors and were close to CD8+ T cell epitopes. HLA-DR molecules were more frequently involved in T cell stimulation than were HLA-DP4 molecules, and hence immunoprevalence relies mainly on HLA-DR promiscuity in the survivin Ag. In two cancer patients a spontaneous CD4+ T cell response specific for one of these peptides was also observed. Based on these observations, the tumor-shared survivin does not appear to be the target of immune tolerance in healthy donors and cancer patients and is a relevant candidate for cancer vaccine.

The primary goal of cancer vaccines is to induce CD8+ T cells specific for tumor-associated antigens (TAA) but the characterization of these cells has been difficult because of the low sensitivity of ex vivo assays. Here, we focused on TAA-specific CD8+ T-cell responses in melanoma patients after vaccination with autologous dendritic cells loaded with lysates derived from allogeneic tumor-cell lines (Lysate-DC). Out of 40 patients treated, 16 patients developed immune response to tumor-cell lysate and/or CD8+ T cells specific for differentiation and cancer-testis antigens. TAA-specific CD8+ T-cell responses were detected by interferon (IFN)-gamma enzyme-linked immunospot after in vitro sensitization and were, either transient during the treatment period or delayed, that is, observed after completion of all vaccinations. We could not correlate these immune responses to clinical data as none of the patients achieved an overall objective response according to Response Evaluation Criteria in Solid Tumors criteria. Three patients were reported as stable disease and 10 patients presented evidence of antitumor activity. We found that TAA-specific T cells characterized in 4 patients produced perforin ex vivo, but no IFN-gamma in enzyme-linked immunospot. Differential expression of IFN-gamma and perforin was also observed for viral-specific T cells. Altogether, our results show that Lysate-DC therapy elicited tumor-specific CD8+ T cells nonlimited to human leukocyte antigen-A2+ patients, with some T cells secreting perforin ex vivo and IFN-gamma only after restimulation. The differential expression of perforin and IFN-gamma by antitumor and antiviral CD8+ T cells supports that the sole use of IFN-gamma production to monitor T cells overlooks functional T-cell subpopulations triggered by vaccines.

Immunization with peptide or recombinant proteins generally fails to elicit CTL, which are thought to play a key role in the control of virus-infected cells and tumor growth. In this study we show that the nontoxic B subunit of Shiga toxin fused to a tumor peptide derived from the mouse mastocytoma P815 can induce specific CTL in mice without the use of adjuvant. The Shiga B subunit acts as a vector rather than as an adjuvant, because coinjection of the tumor peptide and the B subunit as separate entities does not lead to CTL induction. We also demonstrated that in vitro the B subunit mediates the delivery of various exogenous CD8 T cell epitopes into the conventional MHC class I-restricted pathway, as this process is inhibited by brefeldin A and lactacystin and requires a functional TAP system. In contrast to other nonviral methods for transport of exogenous Ags into the endogenous MHC class I pathway that involve macropinocytosis or phagocytosis, the Shiga B subunit targets this pathway in a receptor-dependent manner, namely via binding to the glycolipid Gb3. Because this receptor is highly expressed on various dendritic cells, it should allow preferential targeting of the Shiga B subunit to these professional APCs. Therefore, the Shiga B subunit appears to represent an attractive vector for vaccine development due to its ability to target dendritic cells and to induce specific CTL without the need for adjuvant.