The aim of this study was to examine whether PC61 mAb-mediated functional inactivation of Tregs would enhance the potency of a DC/tumor fusion-based vaccine in a murine melanoma model with established pulmonary metastases. In agreement with previous reports, we were able to demonstrate that administration of mAb PC61 causes significant inactivation of Tregs that is maintained for about 3 weeks until the Treg population returns [20, 21, 25]. It is important to note that after PC61 mAb treatment only a small portion of Tregs is actually being depleted by phagocytes expressing the FcγRIII receptor . The majority of Tregs is being functionally inactivated through antibody-mediated blockade of IL-2 signaling . Remarkably, administration of anti-CD25 in murine models and clinical studies has not resulted in treatment related morbidity due to autoimmune disease , and it is tempting to speculate that tissue-residing Tregs might be affected to a lesser degree by antibody treatment than Tregs found in the lymphatic system.
Furthermore, we observed that PC61 treatment enhanced DC/tumor fusion vaccination in a synergistic fashion, and did not abrogate the induction of T-effector cells despite their transient expression of CD25 molecules. These observations are in disagreement with a recent report which examined the ability of the humanized anti-IL-2Rα mAb daclizumab to deplete Tregs in metastatic melanoma patients receiving antitumor vaccination with peptide- and KLH (keyhole limpet hemocyanin)-loaded DCs . The authors demonstrated that while Tregs were effectively depleted and anti-tumor T cells were induced, daclizumab impaired the acquisition of T-effector function in vivo. A different report as well as our own data suggest otherwise. First, Sampson et al.  showed that depletion of Tregs correlated with increased vaccine-stimulated humoral immunity in glioblastoma patients during temozolomide-induced lymphopenia. Second, after mAb PC61-treatment, we observed increased frequencies of vaccine-induced CD4+ T cells that secreted IFN-γ upon stimulation with tumor cells. And last, administration of mAb PC61 greatly enhanced the efficacy of vaccination with fusion cells and systemic IL-12 which clearly indicates that PC61 does not seem to have any immunoinhibitory effects.
In our studies, the vaccine-induced immune response after treatment with PC61 and fusion cells consisted primarily of tumor-specific CD4+ T cells. This is not without precedence and in accordance with several studies, which have demonstrated that CD4+ T cells play a pivotal effector role during tumor rejection . While the mechanisms responsible for the observed antitumor effect are still being explored, possible effector mechanisms may include Fas/FasL-mediated killing , interferon-γ–mediated angiostasis , interferon-γ–mediated restoration of the antigen-presenting pathway , or the recruitment or activation of innate effectors, including macrophages and eosinophils .
Even though, CD4+ T-effector cells may play an important role in anti-tumor responses, the lack of vaccine-induced CD8+ cytotoxic T cells in the presence of a potent Th-1-biased T-helper cell response is unsettling and may indicate that efficient cross-presentation of tumor-associated antigens did not occur in DC/tumor fusions. B16 melanoma cells are poorly immunogenic due to downregulation of transporters associated with antigen processing (TAP-1 and TAP-2) , resulting in reduced MHC class I expression and a diminished ability to present antigens to CD8+ T cells. TAP downregulation has been observed in multiple human malignancies and plays a critical role in the clinical course of malignant melanoma . It is conceivable that factors leading to downregulation of TAPs in B16 melanoma cells may act in a negative trans-dominant fashion in fusion cells, thereby impairing MHC class I presentation and hence the induction of CD8+ T cells by DC/tumor cell hybrids. If this was true, the observed induction of CD8+ T-cell responses by fusion vaccine in the presence of IL-12 would be either a result of IFN-γ induced antigen-presentation by fusion cells  or due to stimulation of tumor-infiltrating lymphocytes and T cells in draining lymph nodes, as has been described [36, 37]. Therefore, strategies aiming to overexpress TAP1 and TAP2 in fusion cells may represent a promising new approach to increase the immunogenicity of fusion vaccines.
Systemic administration of IL-12 in combination with DC-tumor fusion vaccination has demonstrated impressive anti-tumor responses in murine models. In cancer patients, however, IL-12 administration and the concomitant induction of high IFN-γ levels have resulted in severe toxicity including adverse hematopoietic, intestinal, hepatic, and pulmonary side effects, which precludes systemic application of IL-12 at therapeutically relevant levels . Here, we provide proof of principle that combination treatment consisting of mAb PC61 and systemic IL-12 can lower the dose of cytokine necessary to obtain maximal therapeutic efficacy and may therefore represent a viable strategy to reduce IL-12 mediated toxicity in a clinical setting. Further studies will be needed to determine the therapeutic window of this novel approach.ken together, we conclude that PC61-mediated inactivation of Tregs is a viable strategy to improve the efficacy of DC/tumor fusion vaccination and that combining vaccination with Treg depletion and systemic administration of IL-12 may represent a novel approach to unleash the full potential of DC/tumor fusion vaccines.