Development of effective tumor immunotherapy using a novel dendritic cell-targeting Toll-like receptor ligand.

Although dendritic cell (DC)-based immunotherapy shows little toxicity, improvements should be necessary to obtain satisfactory clinical outcome. Using interferon-gamma injection along with DCs, we previously obtained significant clinical responses against small or early stage malignant tumors in dogs. However, improvement was necessary to be effective to largely developed or metastatic tumors. To obtain effective methods applicable to those tumors, we herein used a DC-targeting Toll-like receptor ligand, h11c, and examined the therapeutic effects in murine subcutaneous and visceral tumor models and also in the clinical treatment of canine cancers. In murine experiments, most and significant inhibition of tumor growth and extended survival was observed in the group treated with the combination of h11c-activated DCs in combination with interferon-gamma and a cyclooxygenase2 inhibitor. Both monocytic and granulocytic myeloid-derived suppressor cells were significantly reduced by the combined treatment. Following the successful results in mice, the combined treatment was examined against canine cancers, which spontaneously generated like as those in human. The combined treatment elicited significant clinical responses against a nonepithelial malignant tumor and a malignant fibrous histiocytoma. The treatment was also successful against a bone-metastasis of squamous cell carcinoma. In the successful cases, the marked increase of tumor-responding T cells and decrease of myeloid-derived suppressor cells and regulatory T cells was observed in their peripheral blood. Although the combined treatment permitted the growth of lung cancer of renal carcinoma-metastasis, the marked elevated and long-term maintaining of the tumor-responding T cells was observed in the patient dog. Overall, the combined treatment gave rise to emphatic amelioration in DC-based cancer therapy.

Effect of ovarian hormones on maturation of dendritic cells from peripheral blood monocytes in dogs.

Previously, we reported that ovarian hormones affect the immune response against E. coli isolated from the dogs affected with pyometra. In order to investigate mechanisms underlying the immune modulation, we examined the effects of ovarian hormones on the generation of dendritic cells (DCs), the most potent antigen presenting cell. DCs were differentiated from peripheral blood monocytes (PBMOs) using a cytokine cocktail. Both estrogen receptor and progesterone receptors were expressed by the PBMOs and immature DCs. When various ovarian hormones were added to the culture for the DC differentiation, progesterone significantly decreased the expression of DC maturation markers, such as CD1a, CD80 and CD86, on mature DCs. Conversely, the addition of estrogen to the cultures increased the expression of CD86, but not other maturation makers. Furthermore, DCs differentiated in the presence of progesterone did not stimulate allogeneic mononuclear cells in PB. Taken together, these results indicate that progesterone diminishes the maturation of DCs, leading to decreased immune responses against invading pathogens.

Enhancement of anti-tumor immune responses by transfection of IFNγ gene into tumor using a novel type synthetic vector.

The existence of Th1 responses in a tumor microenvironment elicits a better prognosis for the patients. Transfection of Th1 polarizing cytokines, such as IFNγ, into tumor cells is an effective way to set up an appropriate microenvironment. Using a novel type synthetic vector composed of polyamidoamine dendrons, we transfected canine IFNγ gene into canine tumor cell lines, and examined direct and indirect effects of dendritic cells (DCs) against tumor growth in vitro. A cloned canine IFNγ gene expressed functional protein that induces maturation of DCs. When the canine IFNγ gene was transfected into canine tumor cell lines using the synthetic vector, most cells secreted canine IFNγ. Secretion of IFNγ reduced with time, but was maintained for 48h. DCs incubated with the IFNγ-transfected tumor cells exhibited greater suppressive activity and induced significantly higher cytotoxic activity against the tumor cells, relative to those incubated with untransfected tumor cells and comparable dose of IFNγ. Successful transfection of IFNγ by the synthetic vector efficiently enhanced the anti-tumor immune function of DCs, and sets up a suitable microenvironment for improvement in tumor therapy.

Production of canine soluble CD40 ligand to induce maturation of monocyte derived dendritic cells for cancer immunotherapy.

CD40 ligand (CD40L) expressed by activated T cells is shown to induce maturation of immature dendritic cells (DCs) and this maturation is a vital part in DC based tumor immunotherapy. We constructed an expression vector by cloning the extracellular domain of canine CD40L fused to the signal sequence of canine IL-12p40. When PBMCs were incubated with canine granulocyte-macrophage (GM) -CSF and IL-4, expression of CD86 was significantly elevated, but the majority of cells displayed the morphology of immature DCs. Following addition of the expressed canine soluble CD40L (csCD40L) to the DC-inducing culture, the cell morphology shifted to that of mature DCs, and expression of CD80, CD86, MHC class II and CD1a was significantly enhanced. This morphological change and enhancement of expression was observed even when the csCD40L was present only in the second half period of the culture. Furthermore, the csCD40L caused a significant increase in IL-12 production from DCs. These results show that the csCD40L significantly promotes the maturation and activation of canine monocyte derived DCs.