The effectiveness of the dendritic cell (DC) vaccination protocols that are currently in use could be improved by providing the DCs with a more potent maturation signal. We therefore investigated whether the T-cell stimulatory capacity of human monocyte-derived DCs could be increased by co-electroporation with different combinations of CD40L, CD70, and constitutively active toll-like receptor 4 (caTLR4) encoding mRNA. We show that immature DCs electroporated with CD40L and/or caTLR4 mRNA, but not those electroporated with CD70 mRNA, acquire a mature phenotype along with an enhanced secretion of several cytokines/chemokines. Moreover, these DCs are very potent in inducing naive CD4(+) T cells to differentiate into interferon-gamma (IFN-gamma)-secreting type 1 T helper (Th1) cells. Further, we assessed the capacity of the electroporated DCs to activate naive HLA-A2-restricted MelanA-specific CD8(+) T cells without the addition of any exogenous cytokines. When all three molecules were combined, a >500-fold increase in MelanA-specific CD8(+) T cells was observed when compared with immature DCs, and a >200-fold increase when compared with cytokine cocktail-matured DCs. In correlation, we found a marked increase in cytolytic and IFN-gamma/tumor necrosis factor-alpha (TNF-alpha) secreting CD8(+) T cells. Our data indicate that immature DCs genetically modified to express stimulating molecules can induce tumor antigen-specific T cells in vitro and could prove to be a significant improvement over DCs matured with the methods currently in use.

IntroductionMultiple myeloma (MM) is a lethal plasma cell malignancy hallmarked by uncontrolled accumulation of monoclonal plasma cells in the bone marrow (BM). 1 There, the MM cells receive signals to survive and proliferate because of the existence of functional, mutual interactions through growth factors and adhesion molecules. 2 Importantly, these interactions also confer resistance to conventional therapies. 2 One of the most important growth factors involved in MM progression is insulin-like growth factor-1 (IGF-1). [3][4][5] This cytokine induces proliferation of both interleukin-6 (IL-6)-independent and -dependent cell lines, acts synergistically with IL-6, and protects MM cells from dexamethasone-induced apoptosis. 3 Moreover, IGF-1 also stimulates homing and production of angiogenic factors. 4,6,7 Binding to its receptor results in the activation of the Ras/Raf/MAPK (mitogen-activated protein kinase)/Erk (mainly triggering proliferation and secretion of vascular endothelial growth factor) and the PI-3K (phosphatidylinositol-3 kinase)/Akt pathways (involved mainly in antiapoptotic stimuli and mediating migration). 6,8 Both IGF-1 serum levels and IGF-1 receptor (IGF-1R) expression by the MM cells are important negative prognostic factors in MM. 9,10 The central role of IGF-1 in the MM pathophysiology is further supported by the encouraging preclinical results observed after targeting the IGF-1R with neutralizing antibodies or small molecule inhibitors. Previously, we demonstrated the efficacy of the IGF-1R tyrosine kinase inhibitor picropodophyllin to block the function of IGF-1R in vitro and in vivo and this both in murine and human MM cells. 11,12 The anti-MM activity of selective IGF-1R inhibitors was also confirmed with the use of the selective kinase inhibitor NVP-ADW742 in an orthotopic xenograft MM model. 4 Clinical trials examining the effect of both blocking antibodies and small molecule inhibitors are currently ongoing. 3 Bim (Bcl2like11) is a member of the BH3-only group of the Bcl-2 protein family and is a mediator of apoptosis. 13,14 Bim is a sensor of cell stress (withdrawal or inhibition of growth factor signaling or treatment with numerous cytotoxic agents) 13,15 and promotes apoptosis either indirectly by antagonizing the inhibitory function of the Bcl-2/Bcl-Xl members and thus liberating the innate cytotoxic Bax-like members or directly by activating the Bax-like members. 13 Three major isoforms of Bim are known: extra-long (BimEL), long (BimL), and short (BimS). All 3 isoforms induce apoptosis; the shortest being the most potent but the longest the most preponderant. 16 The proapoptotic activity of Bim seems to be controlled in various ways. In healthy cells, Bim is sequestered in its inactive form to the microtubular cytoskeleton or exists as inactive heterodimers (with antiapoptotic Bcl-2 family members) sequestered to the mitochondria. 17 In B and MM cells, Bim appears to be constitutively associated with Mcl-1. At induction of apoptosis, Bim is released from Mcl-1, thus activating ...

IV administration offers better repeatability and better sensitivity when compared to IP. In larger tumours, multiple factors may contribute to underestimation of tumour burden. It might, therefore, be beneficial to test novel therapeutics on small tumours to enable an accurate evaluation of tumour burden.

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The WNT/β-catenin signaling pathway is a prominent player in many developmental processes, including gastrulation, anterior–posterior axis specification, organ and tissue development, and homeostasis. Here, we use human pluripotent stem cells (hPSCs) to study the dynamics of the transcriptional response to exogenous activation of the WNT pathway. We describe a mechanism involving the WNT target gene SP5 that leads to termination of the transcriptional program initiated by WNT signaling. Integration of gene expression profiles of wild-type and SP5 mutant cells with genome-wide SP5 binding events reveals that SP5 acts to diminish expression of genes previously activated by the WNT pathway. Furthermore, we show that activation of SP5 by WNT signaling is most robust in cells with developmental potential, such as stem cells. These findings indicate a mechanism by which the developmental WNT signaling pathway reins in expression of transcriptional programs.