Apoptosis is a highly regulated form of programmed cell death essential for tissue homeostasis and immune regulation (Sun and Peng, 2009). Apoptosis is characterized by distinct morphological and biochemical features, such as chromatin condensation, DNA fragmentation, and membrane blebbing, and the process is typically non-inflammatory due to the efficient clearance of apoptotic cells by phagocytes (Doonan and Cotter, 2008; Gordon and Plüddemann, 2018). Two major pathways mediate apoptosis: the intrinsic (mitochondrial) pathway, which involves cytochrome c release and apoptosome formation, and the extrinsic (death receptor–mediated) pathway, which is driven by caspase-8 activation (Xu and Shi, 2007; Sayers, 2011; Mustafa et al., 2024).

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated cytosine-phosphate-guanine motifs (CpG-ODNs) have been extensively studied as potential immunostimulatory agents for activating Toll-like receptor 9 (TLR9), an endosomal pattern recognition receptor expressed in a variety of immune cells (Gupta and Agrawal, 2010; Moya et al., 2022). TLR9 engagement initiates MyD88-dependent signaling cascades, leading to nuclear factor–κB and interferon (IFN) regulatory factor activation, thereby promoting cytokine and type I IFN production (O'Neill et al., 2013; Zheng et al., 2019). CpG-ODNs are categorized into three classes (A, B, and C), each differing in sequence, structure, and immunological activity (Hanagata, 2012). Class A CpG-ODNs stimulate plasmacytoid dendritic cells to produce type I IFN, whereas class B CpG-ODNs primarily activate B cells and induce secretion of inflammatory cytokines (Poeck et al., 2004; Jurk and Vollmer, 2007). Class C CpG-ODNs exhibit properties associated with both classes A and B (Gray et al., 2007). To provide enhanced resistance to nucleases, CpG-ODNs are often synthesized with a phosphorothioate backbone, although this modification can affect biological activity and specificity (Scheule, 2000; Clavé et al., 2021).

Accumulating evidence suggests that CpG-ODNs can also induce apoptosis in immune and tumor cells via either TLR9-dependent or -independent mechanisms. For instance, certain CpG-ODNs trigger apoptosis in chronic lymphocytic leukemia and hepatocellular carcinoma cells, which highlights the potential for using CpG-ODNs for therapeutic applications beyond immune stimulation (Liang et al., 2010; Zhang et al., 2014). However, understanding of the molecular mechanisms and sequence-specific determinants of CpG-induced apoptosis remains incomplete. Previously, our group identified MsST, a novel class B CpG-ODN derived from the Streptococcus thermophilus lacZ gene, which demonstrated potent immunomodulatory activity, including the induction of interleukin (IL)-33, a known damage-associated molecular pattern (Shimosato et al., 2010). Administration of MsST to atopic dermatitis model mice exacerbated skin inflammation, a response likely mediated by IL-33–driven activation of type 2 innate lymphoid cells and Th2 cells (Wang et al., 2015; Kotsiou et al., 2018). Notably, IL-33 also reportedly modulates apoptosis-related molecules in a variety of other cell types (Liu et al., 2025), raising the possibility that the immunological effects of MsST include the regulation of apoptotic pathways. These observations prompted us to investigate whether MsST exerts pro-apoptotic activity, potentially linking its immunostimulatory and pathogenic roles via caspase-mediated mechanisms.

In this study, we systematically evaluated the apoptotic potential of MsST using mouse splenocytes. Actinomycin D (ActD) was used as a reference for comparing representative CpG-ODNs from classes A, B, and C, and induction of apoptosis was assessed using DNA fragmentation assays, annexin V/7-aminoactinomycin D (7-AAD) staining, and monitoring of caspase-8 activation. Our aim was to establish a reproducible experimental platform to assess CpG-ODN-induced apoptosis and evaluate the implications regarding the development of CpG-based tools for immunological research and therapeutic interventions.