The immunosuppresssive role of interleukin 9 in lung cancer

肺癌是全世界癌症死亡的主要原因。最近的研究表明，免疫治疗可能是一种有希望的方法来改善这种疾病的预后。肿瘤浸润性CD8+ T细胞和T辅助（Th）1极化CD4+ T细胞目前被认为是多种免疫治疗药物驱动的抗癌免疫反应的启动和执行的核心参与者。在最后的资助期，我们发现肺肿瘤细胞诱导免疫抑制因子如TGFβ（转化生长因子β），其关闭由Th1和细胞毒性T淋巴细胞介导的抗肿瘤免疫反应，并促进由表达IL-9、IL-10和PD1的Tregs和T细胞主导的免疫抑制反应。我们发现非小细胞肺癌患者的Treg细胞产生IL-9增加。在非小细胞肺癌实验模型中靶向删除IL-9 （IL-9-/-）可导致小鼠成功的肺肿瘤排斥反应，这与诱导的CTL、Foxp3+ Tregs的减少和PD-1+CD4+ T细胞的增加有关。用抗pd -1抗体治疗肺荷瘤小鼠对肺肿瘤生长没有显著影响，这表明需要优化抗pd -1免疫疗法。此外，抗pd -1抗体治疗导致IL-9的诱导。因此，我们接下来用抗IL-9抗体在小鼠肺癌模型中阻断IL-9，从而抑制肿瘤的发展。我们最终检测了IL-9R在肺肿瘤细胞和肿瘤浸润淋巴细胞（TIL）中的表达。总之，我们的研究结果导致了以下新的假设（图8）：肺肿瘤细胞通过产生TGFβ来支持免疫抑制Tregs来逃避宿主的免疫反应。Tregs利用IL-4和tgf - β产生IL-9。IL-9支持肿瘤细胞存活并诱导PD-1在T细胞上的表达，从而抑制抗肿瘤免疫反应。此外，抗pd -1抗体治疗诱导IL-9，表明需要优化抗pd -1免疫治疗。因此，我们下一步想要改进目前的免疫疗法，并在我们的小鼠肺癌模型中应用抗pd -1和抗il -9抗体的组合。此外，将对从非小细胞肺癌患者分离的培养肺细胞和TILs进行抗pd -1和/或抗il -9治疗的功能分析。体外抗pd1 /IL-9抗体的分析将有助于我们更好地建立临床前条件，以改善非小细胞肺癌的免疫治疗。在进一步的研究中，我们将确定在非小细胞肺癌中诱导IL-9的机制，并将确定IL-9对实验性非小细胞肺癌和非小细胞肺癌患者细胞的TIL和肿瘤细胞的功能影响。最后，我们将对实验性非小细胞肺癌的CD4+和CD8+ TIL进行RNA测序分析，以揭示IL-9控制的基因通路。因此，该项目将为IL-9在非小细胞肺癌TIL功能和肿瘤生长中的功能作用提供新的见解，并可能为肺癌的免疫治疗开辟新的途径。

Lung cancer represents the leading cause of cancer deaths worldwide. Recent studies suggest that immunotherapy could be a promising approach to improve the prognosis for this disease. Tumour-infiltrating CD8+ T cells and T helper (Th)1-polarized CD4+ T cells are currently considered to be central players in the initiation and execution of anti-cancer immune responses driven by multiple immunotherapeutic agents. In the last grant period we identified that lung tumour cells induce immunosuppressive cytokines like TGFβ (Transforming growth factor β) that turn off anti-tumour immune-responses mediated by Th1 and cytotoxic T lymphocytes and promote immunosuppressive responses dominated by Tregs and T cells expressing IL-9, IL-10 and PD1. We discovered increased IL-9 production by Treg cells in NSCLC patients. Targeted deletion of IL-9 (IL-9-/-) in an experimental model of NSCLC resulted in successful lung tumour rejection in mice which was associated with induced CTL, reduced Foxp3+ Tregs and increased PD-1+CD4+ T cells. Treatment of lung tumour-bearing mice with anti-PD-1 antibodies did not significantly affect lung tumor growth demonstrating the need to optimize anti-PD-1 immunotherapy. Moreover, anti-PD-1 antibody treatment resulted in an induction of IL-9. For this reason, we next blocked IL-9 with anti-IL-9 antibodies in our murine lung carcinoma model, resulting in inhibition of tumor development. We finally detected IL-9R expression in lung tumour cells and in tumour-infiltrating lymphocytes (TIL). In conclusion, our findings led to the following new grant hypothesis (Fig 8): The lung tumour cells escape the immune response of the host by producing TGFβ to support immunosuppressive Tregs. Tregs use IL-4 together with TGFβ to produce IL-9. IL-9 supports tumour cell survival and induces the expression of PD-1 on T cells thereby inhibiting anti-tumour immune-responses. Moreover, anti-PD-1 antibody treatment induced IL-9 demonstrating the need to optimize anti-PD-1 immunotherapy. Therefore, we next want to improve current immunotherapies and apply a combination of anti-PD-1 and anti-IL-9 antibodies in our murine model of lung cancer. Furthermore, functional analysis will be performed upon anti-PD-1 and/or anti-IL-9 treatment in cultured lung cells and TILs isolated from patients with NSCLC. Analysis of anti-PD1/IL-9 antibodies ex vivo will help us to better set up pre-clinical conditions to improve immunotherapy against NSCLC. In additional studies, we will determine the mechanisms inducing IL-9 in NSCLC and will determine the functional effects of IL-9 on TIL and tumour cells in experimental NSCLC and cells from NSCLC patients. Finally, we will perform RNA seq analyses of CD4+ and CD8+ TIL in experimental NSCLC to uncover gene pathways controlled by IL-9. This project will therefore allow new insights into the functional role of IL-9 on TIL function and tumour growth in NSCLC and may open new avenues for immunotherapy in lung cancer.