Immunostimulatory activity of Y-shaped DNA nanostructures mediated through the activation of TLR9

Abstract

Immunostimulatory oligodeoxynucleotides (DNAs) have been widely studied in pharmaceutical and biomedical research fields for applications in cancer immunotherapy and vaccination. Toll-like receptors (TLRs) are critical for the instruction and orchestration of the host immune system composed of innate and adaptive immunity. In particular, TLR9 responds to DNAs with unmethylated deoxycytosine-deoxyguanosine (CpG) motifs, thereby inducing the activation of innate immune cells, such as dendritic cells, and consequently, adaptive immune cells. In this study, we developed two kinds of Y-shaped double-stranded DNA nanostructures (Y-DNAs), including a single unit composed of three DNA strands (YS-DNA) and a ligated multiunit complex formed by crosslinking each YS-DNA (YL-DNA), and investigated whether they have immunostimulatory activity in innate immune cells. YS-DNA and YL-DNA induced the production of immune cytokines such as IL-12 and TNF-α and the expression of costimulatory molecules such as CD80 and CD86 in primary mouse dendritic cells and macrophage cells (RAW264.7 cells). A Coprecipitation study demonstrated that YL-DNA was directly associated with TLR9. The induction of immune cytokines by YS-DNA and YL-DNA was abolished in TLR9-deficient primary mouse dendritic cells. The results demonstrated that Y-DNAs induced the activation of dendritic cells and macrophages mediated by the activation of TLR9, as shown by the expression of immune cytokines and costimulatory molecules. The results suggest that Y-DNA nanostructures provide a beneficial strategy for immunotherapy by modulating the immune system.