Immunomodulation of dendritic cells through signaling pathways

The idea of cancer immunotherapy is supported by the potential to harness the potency and specificity of the immune system. One approach is the use of dendritic cells (DCs) to present tumor antigens to T-cells and thus generate tumor-specific immunity. Therefore, DCs need to be mature. DC maturation is correlated with activation of transcription factors, which in turn direct alterations in gene expression thus regulating DC properties.
We focus our research on the transcription factors nuclear factor-kB (NF-kB) and signal transducer and activator of transcription-3 (STAT3) of which the upregulation has been described to have opposing effects with regard to T-cell stimulation.
NF-kB has been described to enhance antigen-presentation by DCs. Since A20, a zinc finger protein with ubiquitine-modifying activity, has been described to negatively regulate NF-kB in a number of cell types, including mouse DCs, we evaluated the presence of A20 in human DCs and whether downregulation of A20 results in more potent DCs. We report that DCs activated with poly(I:C) upregulate A20. Downregulation of A20 results in a higher activation of NF-kB and activator protein-1, which in turn results in increased and sustained IL-6, IL-10 and IL-12 secretion. Silencing the immunosuppressive IL-10, demonstrated that IL-10 inhibits T-cell proliferation. We demonstrated that A20 downregulated DCs skew naive CD4+ T-cells towards IFN-g producing T helper 1 cells, a process dependent on IL-12. Furthermore, A20/IL-10 downregulated DCs had an enhanced capacity to prime MelanA-specific CD8+ T-cells. Finally, we demonstrated that potent T-cell stimulatory DCs are generated upon electroporation with poly(I:C12U), A20/IL-10 siRNA and antigen-encoding mRNA, introducing a one step approach to improve DC-based vaccines.
Another target in DCs that is linked to NF-kB is STAT3. STAT3 expression in DCs has been correlated with suppression of NF-kB (after TLR4 stimulation), low production of IL-12 and high expression of the tryptophan-catabolising enzyme indoleamine-2,3-dioxygenase, factors that hamper the development of effector T-cells. Although it has been described that STAT3 expression in DCs is induced by the IL-6 family as well as by IL-10, we observed that DCs activated through electroporation of 2 mRNAs encoding CD40L and constitutive active TLR4, have high levels of activated STAT3. In future experiments we will evaluate whether inhibition of STAT3 results in DCs with an enhanced T-cell stimulatory capacity. To this end, different approaches will be evaluated. These include the co-electroporation of DCs with the activating mRNAs and 1. IL-10 siRNA, since autocrine IL-10 induces pSTAT3, 2. STAT3 siRNA to delete STAT3 mRNA and 3. different STAT3 variants with dominant negative properties.
In conclusion, the data presented here have important implications in DC-based immunotherapy, not only for the stimulation of T-cells against cancer or infectious diseases, but also for the tolerization of T-cells for the treatment of autoimmune diseases and in transplantation.