Aim

The balance between Th17 cells and T regulatory (Treg) cells has emerged as a prominent factor in regulating cancer development. However, the effect of CpG oligodeoxynucleotides (ODNs) on the differentiation of Treg/Th17 cells has not been well studied. We sought here to explore the function of CpG ODNs in the differentiation of Tregs and Th17 cells in vitro and in vivo.

Methods

Mouse spleen cells were cultured with anti-CD3 monoclonal antibodies in vitro. Tregs and Th17 cell differentiation was induced by transforming growth factor (TGF)-β and interleukin (IL)-2, or TGF-β, IL-6, and IL-23, respectively. Then cells were treated with two CpG ODNs, CpG 1982, or CpG 1826. FBL-3-inoculated C57Bl/6 mice were treated with CpG 1826, tumor vaccine, or combination of CpG 1826 and tumor vaccine. After treatment, spleen cells and serum were isolated, and Tregs/Th17 cells were detected by flow cytometry. The expression of forkhead box P3 (Foxp3), retinoid-related orphan receptor gamma-t (RORγt), IL-10, and IL-17 mRNA was measured by real-time PCR, and protein levels were measured by Western blot and enzyme-linked immunosorbent assay.

Results

The frequency of Treg cells increased significantly (p < 0.05) in the FBL-3-inoculated leukemia mouse model compared with control mice, whereas the frequency of Th17 cells did not change. Median survival of mice after treatment with CpG 1826 and tumor vaccine was significantly prolonged compared with that of control mice (p < 0.05). The frequency of induced Treg cells decreased after treatment with CpG 1826, whereas the frequency of Th17 cells induced by cytokines in vitro and in the murine leukemia model increased following treatment with CpG 1826. Furthermore, after treatment with CpG 1826, the mRNA and protein levels of Foxp3 and IL-10 decreased significantly both in vitro and in vivo (p < 0.05), whereas those of RORγt and IL-17 increased significantly (p < 0.05).

Conclusion

CpG 1826 may inhibit the differentiation of Treg cells induced by cytokines, promote the differentiation of Th17 cells in vitro and in murine leukemia models, and prolong the median survival of mice with leukemia.

中文翻译：

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CpG寡脱氧核苷酸对Treg / Th17细胞分化的影响

目标

Th17细胞与T调节（Treg）细胞之间的平衡已成为调节癌症发展的重要因素。但是，尚未对CpG寡脱氧核苷酸（ODN）对Treg / Th17细胞分化的影响进行研究。我们在这里寻找体外和体内CpG ODNs在Tregs和Th17细胞分化中的功能。

方法

用抗CD3单克隆抗体在体外培养小鼠脾细胞。通过分别转化生长因子（TGF）-β和白介素（IL）-2或TGF-β，IL-6和IL-23诱导Tregs和Th17细胞分化。然后，用两种CpG ODN，CpG 1982或CpG 1826处理细胞。用CpG 1826，肿瘤疫苗或CpG 1826和肿瘤疫苗的组合处理接种FBL-3的C57Bl / 6小鼠。处理后，分离脾细胞和血清，并通过流式细胞术检测Tregs / Th17细胞。前叉盒P3（Foxp3），类维生素A相关孤儿受体γ-t（RORγt），IL-10和IL-17的表达。 通过实时PCR测量mRNA，通过蛋白质印迹和酶联免疫吸附测定法测量蛋白质水平。

结果

与对照小鼠相比，FBL-3接种的白血病小鼠模型中Treg细胞的频率显着增加（p <0.05），而Th17细胞的频率没有变化。与对照组相比，CpG 1826和肿瘤疫苗治疗后小鼠的中位生存期显着延长（p <0.05）。CpG 1826处理后，诱导的Treg细胞的频率降低，而CpG 1826处理后，细胞因子在体外和鼠白血病模型中诱导的Th17细胞的频率增加。此外，CpG 1826处理后，mRNA和蛋白质水平Foxp3和IL-10的两个显著降低体外和体内（p<0.05），而RORγt和IL-17则显着增加（p <0.05）。

结论

CpG 1826可能抑制细胞因子诱导的Treg细胞的分化，在体外和鼠类白血病模型中促进Th17细胞的分化，并延长白血病小鼠的中位生存期。