Ospedale Policlinico San Martino, IRCCS for Oncology, 16132, Genoa, Italy.
Immunogenetic and Cell Culture Department, Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, 919677-3117, Iran.
J Exp Clin Cancer Res. 2017 Oct 11;36(1):140. doi: 10.1186/s13046-017-0608-z.
Recently, immunotherapy with anti-PD-1 antibodies has shown clinical benefit in recurrent Small Cell Lung Cancer (SCLC). Since anti-PD-1 re-activates anti-tumor Cytotoxic T Lymphocyte (CTL) responses, it is crucial to understand the mechanisms regulating HLA class I, and PD-L1 expression in HLA-negative SCLC. Here we addressed the role of IL-27, a cytokine related to both IL-6 and IL-12 families.
The human SCLC cell lines NCI-N592, -H69, -H146, -H446 and -H82 were treated in vitro with different cytokines (IL-27, IFN-γ, IL-6 or a soluble IL-6R/IL-6 chimera [sIL-6R/IL-6]) at different time points and analyzed for tyrosine-phosphorylated STAT proteins by Western blot, for surface molecule expression by immunofluorescence and FACS analyses or for specific mRNA expression by QRT-PCR. Relative quantification of mRNAs was calculated by the ΔΔCT method. The Student's T test was used for the statistical analysis of experimental replicates.
IL-27 triggered STAT1/3 phosphorylation and up-regulated the expression of surface HLA class I antigen and of TAP1 and TAP2 mRNA in four out of five SCLC cell lines tested. The IL-27-resistant NCI-H146 cells showed up-regulation of HLA class I by IFN-γ. IFN-γ also induced expression of PD-L1 in SCLC cells, while IL-27 was less potent in this respect. IL-27 failed to activate STAT1/3 phosphorylation in NCI-H146 cells, which display a low expression of the IL-27RA and GP130 receptor chains. As GP130 is shared in IL-27R and IL-6R complexes, we assessed its functionality in response to sIL-6R/IL-6. sIL-6R/IL-6 failed to trigger STAT1/3 signaling in NCI-H146 cells, suggesting low GP130 expression or uncoupling from signal transduction. Although both sIL-6R/IL-6 and IL-27 triggered STAT1/3 phosphorylation, sIL-6R/IL-6 failed to up-regulate HLA class I expression, in relationship to the weak activation of STAT1. Finally sIL-6R/IL-6 limited IL-27-effects, particularly in NCI-H69 cells, in a SOCS3-independent manner, but did not modify IFN-γ induced HLA class I up-regulation.
In conclusion, IL-27 is a potentially interesting cytokine for restoring HLA class I expression for SCLC combined immunotherapy purposes. However, the concomitant activation of the IL-6 pathway may limit the IL-27 effect on HLA class I induction but did not significantly alter the responsiveness to IFN-γ.
最近，抗 PD-1 抗体的免疫疗法在复发性小细胞肺癌（SCLC）中显示出了临床益处。由于抗 PD-1 重新激活了抗肿瘤细胞毒性 T 淋巴细胞（CTL）反应，因此了解调节 HLA Ⅰ类和 PD-L1 在 HLA 阴性 SCLC 中表达的机制至关重要。在这里，我们研究了白细胞介素 27（IL-27）的作用，白细胞介素 27 与白细胞介素 6 和白细胞介素 12 家族有关。
将人类 SCLC 细胞系 NCI-N592、-H69、-H146、-H446 和 -H82 在体外分别用不同的细胞因子（IL-27、IFN-γ、IL-6 或可溶性 IL-6R/IL-6 嵌合体 [sIL-6R/IL-6]）在不同时间点处理，并通过 Western blot 分析酪氨酸磷酸化 STAT 蛋白，通过免疫荧光和流式细胞术分析表面分子表达，或通过 QRT-PCR 分析特定 mRNA 表达。通过 ΔΔCT 法计算 mRNA 的相对定量。使用 Student's T 检验对实验重复进行统计学分析。
IL-27 触发 STAT1/3 磷酸化，并上调五种 SCLC 细胞系中的四种细胞表面 HLA Ⅰ类抗原和 TAP1 和 TAP2 mRNA 的表达。IL-27 抗性的 NCI-H146 细胞通过 IFN-γ 上调 HLA Ⅰ类。IFN-γ 也诱导 SCLC 细胞中 PD-L1 的表达，而 IL-27 在这方面的作用较弱。IL-27 不能激活 NCI-H146 细胞的 STAT1/3 磷酸化，该细胞表达低水平的 IL-27RA 和 GP130 受体链。由于 GP130 存在于 IL-27R 和 IL-6R 复合物中，我们评估了其对 sIL-6R/IL-6 的功能。sIL-6R/IL-6 不能在 NCI-H146 细胞中触发 STAT1/3 信号转导，这表明 GP130 表达低或与信号转导脱偶联。尽管 sIL-6R/IL-6 和 IL-27 都能触发 STAT1/3 磷酸化，但 sIL-6R/IL-6 不能上调 HLA Ⅰ类表达，这与 STAT1 的弱激活有关。最后，sIL-6R/IL-6 以 SOCS3 非依赖性方式限制了 IL-27 效应，特别是在 NCI-H69 细胞中，但并没有改变 IFN-γ 诱导的 HLA Ⅰ类上调。
总之，IL-27 是一种具有潜力的细胞因子，可用于恢复 HLA Ⅰ类表达，用于 SCLC 联合免疫治疗目的。然而，IL-6 途径的同时激活可能会限制 IL-27 对 HLA Ⅰ类诱导的作用，但不会显著改变对 IFN-γ 的反应性。
