Double-stranded DNA (dsDNA) in the cytoplasm of eukaryotic cells is abnormal and typically indicates the presence of pathogens or mislocalized self-DNA. Multiple sensors detect cytosolic dsDNA and trigger robust immune responses via activation of type I interferons. Several cancer immunotherapy treatments also activate cytosolic nucleic acid sensing pathways, including oncolytic viruses, nucleic acid-based cancer vaccines, and pharmacological agonists. We report here that cytosolic dsDNA introduced into malignant cells can robustly upregulate expression of CCL22, a chemokine responsible for the recruitment of regulatory T cells (Tregs). Tregs in the tumor microenvironment are thought to repress anti-tumor immune responses and contribute to tumor immune evasion. Surprisingly, we found that CCL22 upregulation by dsDNA was mediated primarily by interferon regulatory factor 3 (IRF3), a key transcription factor that activates type I interferons. This finding was unexpected given previous reports that type I interferon alpha (IFN-α) inhibits CCL22 and that IRF3 is associated with strong anti-tumor immune responses, not Treg recruitment. We also found that CCL22 upregulation by dsDNA occurred concurrently with type I interferon beta (IFN-β) upregulation. IRF3 is one of two transcription factors downstream of the STimulator of INterferon Genes (STING), a hub adaptor protein through which multiple dsDNA sensors transmit their signals. The other transcription factor downstream of STING, NF-κB, has been reported to regulate CCL22 expression in other contexts, and NF-κB has also been associated with multiple pro-tumor functions, including Treg recruitment. However, we found that NF-κB in the context of activation by cytosolic dsDNA contributed minimally to CCL22 upregulation compared with IRF3. Lastly, we observed that two strains of the same cell line differed profoundly in their capacity to upregulate CCL22 and IFN-β in response to dsDNA, despite apparent STING activation in both cell lines. This finding suggests that during tumor evolution, cells can acquire, or lose, the ability to upregulate CCL22. This study adds to our understanding of factors that may modulate immune activation in response to cytosolic DNA and has implications for immunotherapy strategies that activate DNA sensing pathways in cancer cells.

真核细胞细胞质中的双链 DNA (dsDNA) 是异常的，通常表明存在病原体或错误定位的自身 DNA。多个传感器检测胞质 dsDNA 并通过激活 I 型干扰素触发强大的免疫反应。几种癌症免疫疗法还激活胞质核酸传感途径，包括溶瘤病毒、基于核酸的癌症疫苗和药理学激动剂。我们在此报告，引入恶性细胞的胞质 dsDNA 可以强力上调 CCL22 的表达，CCL22 是一种负责招募调节性 T 细胞 (Treg) 的趋化因子。肿瘤微环境中的Treg被认为可以抑制抗肿瘤免疫反应并有助于肿瘤免疫逃避。令人惊讶的是，我们发现双链DNA对CCL22的上调主要是由干扰素调节因子3（IRF3）介导的，IRF3是激活I型干扰素的关键转录因子。这一发现是出乎意料的，因为之前有报道称 I 型干扰素 α (IFN-α) 抑制 CCL22，并且 IRF3 与强抗肿瘤免疫反应相关，而不是与 Treg 招募相关。我们还发现 dsDNA 上调 CCL22 与 I 型干扰素 β (IFN-β) 上调同时发生。 IRF3 是干扰素基因ST刺激器 (STING) 下游的两个转录因子之一，干扰素基因 ST 刺激器是一种中枢接头蛋白，多个 dsDNA 传感器通过它传输信号。据报道，STING 下游的另一个转录因子 NF-κB 可在其他情况下调节 CCL22 表达，并且 NF-κB 还与多种促肿瘤功能相关，包括 Treg 募集。 然而，我们发现与 IRF3 相比，在胞质 dsDNA 激活的情况下，NF-κB 对 CCL22 上调的贡献微乎其微。最后，我们观察到同一细胞系的两个菌株响应 dsDNA 上调 CCL22 和 IFN-β 的能力存在显着差异，尽管两种细胞系中都有明显的 STING 激活。这一发现表明，在肿瘤进化过程中，细胞可以获得或失去上调 CCL22 的能力。这项研究增加了我们对可能调节细胞质 DNA 免疫激活的因素的理解，并对激活癌细胞 DNA 传感途径的免疫治疗策略具有影响。