探討含溴結構域抑制劑對肺癌反應T細胞的免疫調節作用

Abstract

肺癌為全球癌症死亡之首因，因其難以診斷的特性，使得許多病人確診時皆已為癌症轉移階段，此時化學療法或放射線治療的效果十分有限。過去許多研究證實，腫瘤旁邊常有許多白血球浸潤，尤其是CD8+ T細胞，這些白血球對於腫瘤的治療具有關鍵性的影響。因長期接受腫瘤抗原刺激，多數腫瘤浸潤淋巴球 (tumor-infiltrating lymphocytes, TILs) 處於耗竭 (exhausted) 狀態，分泌較少的細胞激素、表現較多的抑制性免疫檢查點 (immune checkpoints)，以及遇到刺激時呈現低下的分化能力。失去功能的耗竭CD8 T細胞 (Exhausted T cell, TEX) 最終無法有效的清除癌細胞。免疫檢查點阻抗療法 (Immune checkpoint blockade therapy, ICB) 即是利用阻抗免疫檢查點以降低免疫負調控反應，增加T細胞對癌細胞的毒殺能力。免疫檢查點阻抗療法並非對每一位患者都有良好的治療效果。近年的研究指出，耗竭T細胞存在多樣性，可區分為先驅耗竭T細胞 (progenitor TEX)及末端耗竭T細胞 (terminally TEX)，其中前驅耗竭T細胞雖表現高量的免疫檢查點，依然具有高度分化的能力，並且對免疫檢查點阻抗療法的反應較良好。然而，研究發現，使用免疫檢查點阻抗療法後，耗竭T細胞雖然短暫恢復功能，但無法回復成記憶T細胞 (memory T cells) 或功能T細胞的表觀遺傳基因體 (epigenetic landscapes)。因此，我們假設表觀遺傳藥物可藉由重整表觀遺傳調控的方式改善T細胞的功能。實驗室先前的研究中，透過藥物篩選，從145個表觀遺傳藥物中，發現含溴結構域抑制劑 (bromodomain inhibitors, BETi)，可以提升健康人的周邊血液中CD4及CD8 T細胞的多功能性 (T cell polyfunctionality)。 接續先前的研究，我的研究目標是進一步探討含溴結構域抑制劑對於肺癌中失能性T細胞 (dysfunctional T cell) 的免疫調控並進行功能性實驗，我利用肺癌病人惡性胸水 (malignant pleural effusions, MPE)來進行研究，惡性胸水內含有肺癌細胞與免疫細胞，適合用來研究腫瘤微環境內的細胞交互作用。首先，我們使用與耗竭T細胞有關的轉錄因子—Eomes及T-bet來評估胸水中T細胞的免疫功能，發現雖然胸水內部份T細胞處於活化的狀態，可分泌細胞激素並有高度的分化能力，但是末端耗竭T細胞佔所有T細胞的比例明顯較健康受試者高，我們並發現含溴結構域抑制劑可提升胸水中CD8與CD4 T 細胞的多功能性、降低免疫檢查點的表現量、並增加CD8 T細胞的比例。此外，含溴結構域抑制劑可增加惡性胸水中先驅耗竭T細胞 (progenitor exhausted T cell)，同時減少末端耗竭T細胞。 我們更進一步將含溴結構域抑制劑應用在動物實驗上。我們使用小鼠之同源原位肺癌模型，等腫瘤生成後，使用低劑量的含溴結構域抑制劑JQ1治療四週，發現JQ1顯著抑制腫瘤的生長並提升小鼠的存活率。綜合上述體外細胞與活體動物實驗結果，我們證明含溴結構域抑制劑對於耗竭T細胞的具有免疫調控的作用，並可有效的抑制老鼠腫瘤。未來可運用於開發新的免疫療法，造福更多肺癌病人。

Lung cancer is the leading cause of cancer deaths worldwide. Detection of lung cancer at early stages can be difficult since patients tend to be asymptomatic at this stage. Thus, many lung cancer patients present with metastatic diseases at first diagnosis when the efficacy of chemotherapy or radiotherapy is limited. It has been shown that tumor tissues are infiltrated by many immune cells, in particular CD8+ T cells, which play crucial roles in the fight against cancer. On the other hand, due to continuous stimulation of tumor antigens, many tumor-infiltrating lymphocytes (TILs) exhibit the exhausted phenotype, characterized by decreased secretion of effector cytokines, increased expression of immune checkpoints, and defective proliferative capabilities upon stimulation. These exhausted T cells (TEX) are unable to eliminate cancer cells effectively. Clinically, immune checkpoint blockade therapy (ICB) counteracts the inhibitory signaling triggered by immune checkpoints thereby reinvigorating exhausted T cells and enhancing their cytotoxicity. Notably, not every patient receiving immune checkpoint blockade has favorable clinical responses. Further studies showed exhausted T cells are a heterogeneous population composed of progenitor and terminally exhausted subsets. Progenitor exhausted T cells, while expressing high levels of immune checkpoints, have stem-cell properties with further differentiation capacity and respond better to checkpoint blockade therapy as opposed to terminally exhausted T cells. Nevertheless, even though checkpoint blockade therapy may temporarily restore the functions of exhausted T cells, the therapy can not reverse the intrinsic epigenetic landscape of the exhausted phenotype back to that of memory or effector phenotypes of T cells. Therefore, we hypothesized that epigenetic drugs could restore the functions of T cells by reprogramming epigenetic regulations. Our laboratory has previously conducted a screen for T cell poly-functionality using an epigenetic compound library, and found that inhibitors of bromodomain and extraterminal domain (BETi) can enhance the polyfunctionality of CD4+ and CD8+ T cells from the peripheral blood of healthy volunteers. Based on these observations, I aimed to investigate the immunomodulatory effects of BETi on dysfunctional T cells from malignant pleural effusions (MPE) in lung cancer patients, which represent a unique tumor microenvironment where cancer cells and various immune cells interact. First, I investigated the functional states of malignant pleural effusion T cells based on the expression levels of two exhaustion-related transcription factors — Eomes and T-bet. I found that, compared to the circulating T cells from healthy donors, the percentages of terminally exhausted T cells (EomeshiT-Betlo) in malignant pleural effusion were significantly higher. Furthermore, BETi treatment significantly enhanced the polyfunctionality of CD4+ and CD8+ T cells from MPE and decreased the expression of immune checkpoints. Besides, BETi treatment decreased the terminally exhausted T cell subset while increasing the progenitor exhausted T cell subset. Most importantly, we investigated the therapeutic potential of BETi in a syngeneic orthotopic model of mouse lung adenocarcinoma — Lewis lung carcinoma. We showed that JQ1 treatment significantly diminished the tumor growth and improved the overall survivals. In conclusion, through the in vitro and in vivo experiments, we demonstrated the immunomodulatory effects of BETi on exhausted T cells, which paves way to the development of novel immunotherapeutic strategies in treating lung cancer patients.