放射治疗 (RT) 经常用于治疗癌症，包括软组织肉瘤。先前的研究表明，Toll 样受体 9 (TLR9​​) 激动剂胞嘧啶-磷酸-鸟嘌呤寡脱氧核苷酸 (CpG) 可增强移植肿瘤对放疗的反应，但这种增强的机制仍不清楚。在这里，我们使用 CRISPR/Cas9 和化学致癌剂 3-甲基胆蒽 (MCA) 来产生具有高肿瘤突变负荷的原地软组织肉瘤。单次 20 Gy RT 和 2 剂 CpG 治疗可显着增强肿瘤反应，但这种反应可通过 CD8 T 细胞的遗传或免疫耗竭而消除。为了表征对 CpG RT 的免疫反应，我们进行了批量 RNA 测序、单细胞 RNA 测序和质谱流式分析。用 20 Gy 和 CpG 处理的肉瘤表现出表达与激活和增殖相关的标记物（例如粒酶 B、Ki-67 和 IFN-γ）的 CD8 T 细胞增加。 CpG RT 还上调骨髓细胞上的抗原呈递途径。此外，在用 CpG RT 治疗的肉瘤中，TCR 克隆性分析表明克隆性 T 细胞优势有所增加。总的来说，这些发现表明 CpG RT 以 CD8 T 细胞依赖性方式显着延迟肿瘤生长。这些结果为评估 CpG 或其他 TLR9 激动剂联合放疗治疗软组织肉瘤患者的临床试验提供了强有力的依据。

Radiation therapy (RT) is frequently used to treat cancers, including soft-tissue sarcomas. Prior studies established that the toll-like receptor 9 (TLR9) agonist cytosine-phosphate-guanine oligodeoxynucleotide (CpG) enhances the response to RT in transplanted tumors, but the mechanisms of this enhancement remain unclear. Here, we used CRISPR/Cas9 and the chemical carcinogen 3-methylcholanthrene (MCA) to generate autochthonous soft-tissue sarcomas with high tumor mutation burden. Treatment with a single fraction of 20 Gy RT and 2 doses of CpG significantly enhanced tumor response, which was abrogated by genetic or immunodepletion of CD8+ T cells. To characterize the immune response to CpG+RT, we performed bulk RNA-Seq, single-cell RNA-Seq, and mass cytometry. Sarcomas treated with 20 Gy and CpG demonstrated increased CD8 T cells expressing markers associated with activation and proliferation, such as Granzyme B, Ki-67, and IFN-γ. CpG+RT also upregulated antigen presentation pathways on myeloid cells. Furthermore, in sarcomas treated with CpG+RT, TCR clonality analysis suggests an increase in clonal T cell dominance. Collectively, these findings demonstrate that CpG+RT significantly delays tumor growth in a CD8 T cell-dependent manner. These results provide a strong rationale for clinical trials evaluating CpG or other TLR9 agonists with RT in patients with soft-tissue sarcoma.