of regulatory cells that suppress the induction, effector and memory phases of anti-tumor immune response. The importance of these tumor-driven negative regulations is evident in mice bearing slow growing autochthonous cancer as well as in patients with a clinically evident tumor. Almost paradigmatically BALB-neuT mice show that during the slow progression of their mammary lesions immature myeloid cells,67 suppressive NKT cells68 and natural Treg cells42 expand. As the availability of anti-CD25 mAb allows a simple even if gross interference with Treg cells, we assessed how a temporary interference with Treg cells extend vaccine efficacy. In BALB- neuT mice central tolerance activated by ErbB-2 expression in the thymus and its overexpression in the mammary lesions lead to the deletion of CD8+ T cell clones reacting at high affinity with dominant ErbB-2 epitopes. Because of this deletion, anti-ErbB-2 DNA vaccination elicits solely CD4+ cell expansion and anti- ErbB-2 antibodies.23,69,70 However, the combination of DNA vaccination with the depletion of Treg cells triggers a higher antibody response combined with the onset of a CD8+ cytotoxic response against the ErbB-2 immunodominant epitope. This unexpected cytotoxic response is due to the freeing of a latent pool of low-avidity CD8+ T cells bearing cryptic TCR from Treg constraints. The stronger and composite response elicited inhibits cancer progression at stages in which the vaccine alone is ineffective. While dramatic, this extension of vaccine efficacy is no longer able to secure mice survival when diffuse and invasive microscopic cancers become palpable.43 These results suggest that a more sophisticated interference with tumor-borne regulatory and suppressor mechanisms may serve to endow oncoantigen vaccines with therapeutic efficacy. The exploitation of the plasmids both coding for the antigen and expressing shRNA able to silence molecules that negatively control the immune response may offer new ways to extend oncoantigen vaccine efficacy.36 Conclusions: First Fulfillments of the Promise The antibody and cell mediated response elicited by the electroporation of HuRTErbB-2 plasmid in healthy dogs and in pet dogs after the surgical removal of mammary cancer proves the efficacy of DNA vaccines electroporation in higher animals (Cavallo F, unpublished results). Moving from this evidence, we started a clinical trial in pet dogs with advanced mucosal malignant melanoma to assess the potential of the electroporation of a plasmid coding for the human chondroitin sulfate proteoglycan-4 (CSPG4), a molecule highly expressed by human and dog melanomas whereas it has a restricted distribution in normal tissues.71 Dogs with biopsy-confirmed CSPG4+ malignant melanoma and absence of metastases beyond the first regional lymph nodes were subjected to surgery and then electroporated at monthly intervals with a plasmid coding for human CSPG4. In all dogs the vaccine elicited antibodies to human CSPG4. Their cross-reactivity with the canine ortholog shows that the plasmid coding for this heterologous molecule effectively breaks immune tolerance to dog CSPG4. So far, the intensity of the elicited immune response appears to go along with the extension of the time of tumor recurrence and dog survival (Cavallo F et al., in preparation). Preliminary data from ongoing trials with cancer patients are showing that DNA vaccines induce immune responses that goes along with an inhibition of tumor progression.52,72 When plasmids are electroporated, the response elicited in patients with recurrent prostate cancer is much higher than that triggered by the sole injection of the plasmid.20 Italian authorities have recently approved a phase I/II clinical trial based on repeated electropora- tions of the chimeric RHuTErbB-2 plasmid in patients in whom a primary advanced ErbB-2+ squamous carcinomas of the oral cavity, oropharynx, hypopharynx has been surgically removed (EudraCT, 2011-001104-34). Both the implemented dog trial with CSPG4 and the incipient trial with RHuTErbB-2 plasmids are aimed to exploit the immune response not to cure an existing cancer, but to extend the tumor free survival and delay tumor relapse. 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