Dendritic Cells Loaded with Tumor Antigens and a Dual Immunostimulatory and Anti-Interleukin 10-Specific Small Interference RNA Prime T Lymphocytes against Leukemic Cells

Abstract

Vaccines using dendritic cells (DCs) harboring leukemic antigens to stimulate T cells is a possible treatment of acute myeloid leukemia (AML). Limitations of breaking tolerance to leukemic cells and lack of specific activation of T cell-mediated cytotoxicity may explain the discouraging clinical results with this approach. To break self-tolerance against AML cells, we loaded DCs with AML antigens and a bifunctional small interference (si) RNA targeting interleukin (IL) 10 and simultaneously activating toll-like receptors (TLRs). In vitro, this active siRNA inhibited (P < .05) IL-10 production by silencing the IL-10 gene in DCs. The active siRNA stimulated production of tumor necrosis factor alpha, implying activation of TLRs. Vaccination in a nonimmunogenic rat model mimicking human AML with the loaded DCs induced a substantial and specific T-cell cytotoxicity. Leukemic rats treated with the active siRNA lived longer and had markedly less leukemic cell mass infiltrating their bone marrow compared with rats given inactive siRNA (P < .05). Furthermore, compared with inactive siRNA treatment, the active siRNA led to significantly less extramedullar leukemic dissemination, evidenced by reduced matrix metalloproteinase activity and smaller spleens. Our data demonstrate that this bifunctional siRNA may work as an immunomodulatory drug with antileukemic properties.

Figure 1

The mRNA expression of IL-10 relative to that GAPDH was markedly reduced in DCs transfected with active compared with inactive siRNA. Values are mean + SEM, n = 4, *P < .05.

Figure 2

The active siRNA induced specific T-cell cytotoxicity. Lysis of autologous leukemic cells were increased at all effector-to-target cell ratios tested when T cells from leukemic rats treated with DCs transfected with active siRNA (squares) were compared with those given inactive siRNA (triangles). The specific lysis of autologous leukemic cells given anti-MCH class I antibodies (circles) was negligible. MHC class I expression did not differ between untransfected DCs or DCs transfected with active or inactive siRNA (data not shown). T cells and target cells were harvested from blood and spleen before further purification with monoclonal antibodies and flow cytometric sorting. *P < .05. Values are mean and SEM, n = 10 per group.

Figure 3

Increased survival on treatment with active siRNA. Leukemic rats treated with DCs transfected with active siRNA (thick filled line) lived longer compared with those given either inactive siRNA (thin solid line) or with untreated rats (dashed line). n = 10 per group.

Figure 4

The bone marrow leukemic cellularity was significantly lower among rats treated with DCs transfected with the active siRNA (triangles) compared with those given inactive siRNA (circles), or with untreated rats (squares). Individual values are shown and are given as percentages of the total number of nucleated cells. The horizontal lines represent median values. n = 10 per group.

Figure 5

Spleen weight was significantly reduced in rats treated with DCs transfected with the active siRNA (triangles) compared with those given inactive siRNA (circles) or with untreated rats (squares). Values are individual weights. n = 10 per group.

Figure 6

MMP-related activity decreased significantly in rats given DCs transfected with the active siRNA (triangles) compared with those given inactive siRNA (circles) or control rats (squares). Individual values are given and expressed as a percentage of the mean value obtained among untreated leukemic rats. n = 10 per group.