The immunosuppresssive role of interleukin 9 in lung cancer

肺癌是全球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前途的方法，以改善这种疾病的预后。肿瘤浸润性CD 8 + T细胞和辅助性T细胞（Th）1极化的CD 4 + T细胞目前被认为是由多种免疫调节剂驱动的抗癌免疫应答的启动和执行的核心参与者。在上一个资助期，我们发现肺肿瘤细胞诱导免疫抑制细胞因子，如TGFβ（转化生长因子β），其关闭由Th 1和细胞毒性T淋巴细胞介导的抗肿瘤免疫应答，并促进由表达IL-9、IL-10和PD 1的TcB和T细胞主导的免疫抑制应答。我们发现NSCLC患者中Treg细胞产生的IL-9增加。在NSCLC实验模型中靶向缺失IL-9（IL-9-/-）导致小鼠成功的肺肿瘤排斥反应，这与诱导的CTL、Foxp 3 + T细胞减少和PD-1+ CD 4 + T细胞增加有关。用抗PD-1抗体治疗携带肺肿瘤的小鼠没有显著影响肺肿瘤生长，表明需要优化抗PD-1免疫疗法。此外，抗PD-1抗体处理导致IL-9的诱导。出于这个原因，我们接下来在我们的鼠肺癌模型中用抗IL-9抗体阻断IL-9，导致肿瘤发展的抑制。我们最后检测了IL-9 R在肺肿瘤细胞和肿瘤浸润淋巴细胞（TIL）中的表达。总之，我们的发现导致了以下新的格兰特假说（图8）：肺肿瘤细胞通过产生TGFβ以支持免疫抑制性TGF 3来逃避宿主的免疫应答。TNF α使用IL-4和TGFβ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应答。此外，抗PD-1抗体治疗诱导IL-9，表明需要优化抗PD-1免疫疗法。因此，我们接下来想要改进当前的免疫疗法，并在我们的肺癌鼠模型中应用抗PD-1和抗IL-9抗体的组合。此外，将在培养的肺细胞和从NSCLC患者分离的TIL中进行抗PD-1和/或抗IL-9治疗后的功能分析。抗PD 1/IL-9抗体的离体分析将有助于我们更好地建立临床前条件，以改善针对NSCLC的免疫治疗。在另外的研究中，我们将确定在NSCLC中诱导IL-9的机制，并将确定IL-9对实验性NSCLC中的TIL和肿瘤细胞以及来自NSCLC患者的细胞的功能效应。最后，我们将对实验性NSCLC中的CD 4+和CD 8 + TIL进行RNA测序分析，以揭示IL-9控制的基因通路。因此，该项目将使人们对IL-9在NSCLC中对TIL功能和肿瘤生长的功能作用有新的认识，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.