Phosphorothioate-linked guanine/cytosine-based stem-loop oligonucleotides induce the extracellular release of...

It has been previously demonstrated that phosphorothioate-linked GpC-based stem-loop oligonucleotides (GC-SL ODN) induce the release of mitochondrial DNA (mtDNA) from chronic lymphocytic leukemia (CLL) B cells. Although CLL B cells are believed to originate from CD5⁺ B cells because of their phenotypic similarities, it remains unclear whether GC-SL ODN can stimulate CD5⁺ B1 cells to secrete mtDNA. To explore this possibility, we compared the frequency of the mtDNA-producing population among peritoneal cells after GC-SL ODN treatment. We found that mtDNA-releasing cells are enriched for peritoneal CD19⁺ B cells upon GC-SL ODN challenge. Among peritoneal CD19⁺ B cells, the CD5⁺ B1a subpopulation was a primary cellular source of mtDNA secretion in GC-SL ODN-elicited immune responses. GC-SL ODN-stimulated mtDNA release by B1a cells was positively regulated by MyD88 and TRIF signaling pathways. In vivo GC-SL ODN treatment increased lipopolysaccharide-induced activation of innate immune cells such as NK cells, suggesting the immune-enhancing effects of mtDNA secretion. Furthermore, the loop size formed by GC-SL ODNs was a critical factor in inducing mtDNA release by B1a cells. Taken together, our results identified GC-SL ODN as promising biomaterials for enhancing immune responses.