肝脏NKT9细胞在mTORC1-PPARγ介导的脂质代谢调控下抑制大肠癌肝转移的机制研究

Immunotherapy is an emerging therapeutic approach for colorectal cancer liver metastasis. However, there was no clinical benefit for patients with BRAF wild-type and MSS （Microsatellite stability）. In the preliminary study, heterogeneity of NKT cell infiltration was found in metastatic lesions concerning KRAS status (mutation/wild type). NKT9 cell was a newfound subset of NKT cells and little was known concerning its function since it was discovered in 2013. Additionally, IL-9 released by NKT cells was significantly elevated in the core of tumor comparing to the invasive margin and adjacent normal liver tissue. It was identified that the majority of these cells were NKT9 cells. Intriguingly, the cytotoxicity of most immune cells against tumor cells was impaired due to the metabolic pressure under the low glucose condition within tumor lesion. However, NKT9 cells maintained a significantly higher granzyme B induced cytotoxicity than other subtypes of NKT cells. We supposed that hepatic NKT9 cells orchestrate the inhibition of colorectal liver metastasis under the regulation of lipid metabolism mediated by mTORC1-PPARγ. In this study, we would explore the regulation of the fatty acids uptake by PPARγ and study the modulation of immune microenvironment by NKT9 cells through the recruitment of Treg cells by CCL22-CCR4. In this study, the NKT9 cells associated immune scoring system would also be established. Taken together, our data would show that hepatic NKT cells retain the capacity of shaping their function in response to environmental cues, through mTORC1-PPARγ mediated lipid metabolism, adopting an IL-9–producing NKT cell phenotype (namely NKT9) able to mediate tumor control.

免疫治疗是转移性结直肠癌治疗的新策略，但对MSS或BRAF野生型的效果并不明显。本研究首次发现肝转移中NKT（自然杀伤T）细胞的浸润并不因KRAS的突变/野生型而异。同时转移灶内部NKT细胞释放IL-9显著高于癌旁和侵袭边缘，而这类细胞多为NKT9细胞。NKT9是NKT细胞的一种亚型，自2013年被发现以来，在肿瘤中的研究尚属空白。有趣的是，本研究发现在肿瘤内部的低糖条件下，其它免疫细胞因代谢异常致杀伤能力受损，而NKT9却保持着较强的、显著高于其它亚型的颗粒酶B介导的肿瘤杀伤能力。本研究将阐明NKT9细胞在转移灶中的优势分布及功能与mTORC1-PPARγ通路的关联，探讨PPARγ调控NKT9细胞对脂肪酸的摄入和代谢、以及NKT9细胞分泌CCL22募集CCR4+Treg细胞从而调控免疫微环境的作用机制；建立与预后及治疗策略相关的NKT9细胞免疫评分体系，为肿瘤免疫治疗提供新思路。

本研究基本按照原定计划执行，基本完成了预期目标。研究发现肝转移中NKT细胞的浸润并不因KRAS的突变/野生型而异。同时转移灶内部NKT细胞释放IL-9显著高于癌旁和侵袭边缘，而这类细胞多为NKT9细胞。研究发现在肿瘤内部的低糖条件下，其它免疫细胞因代谢异常致杀伤能力受损，而NKT9却保持着较强的、显著高于其它亚型的颗粒酶B介导的肿瘤杀伤能力。研究发现IL9在肿瘤发生发展中的作用，有助于深入地理解IL-9/IL-9R轴和分泌IL-9的淋巴细胞在肿瘤免疫微环境中的作用。.研究完善了Cytof对肠癌肝转移的免疫细胞分布差异及亚型分析：确证Tumor组NKT细胞明显少于Margin和Normal组。发现2个差异NKT细胞亚群： CD3+ CD56+ AMPKlow/-CD45RA+和 CD3+CD56+CD38+NKT cells。IL9的表达在各区域均不明显，这主要是由于IL9表达量的相对差异。课题组通过单细胞测序对免疫细胞亚群进行了进一步分析，发现NKT9细胞的亚群分布。肝内Clec4f+Kupffer细胞和DC是NKT细胞激活的重要抗原提呈细胞。建立Clec4f-iCre+/-: R26-tdTomato+/-小鼠 tdTomato和DC细胞GFP表达的杂交鼠。发现Kupffer cells中 tdTomato 表达较强，DC细胞表达绿色荧光。.依托本课题，研究者还取得如下成果：在肝脏移植免疫及预后风险研究方面：①揭示影响ACLF肝移植术后长期生存的危险因素。提供了亚洲ACLF肝移植的最大规模长期随访数据，对EASL和APASL分级差异的患者分级提供证据；②提出影响儿童肝移植术后远期预后的关键因素，指出免疫细胞移植和肝细胞移植可对儿童遗传代谢性肝病提供新选择；③提出肝动脉的CRL分型，将分型覆盖率提高至99.6%，显著降低血管并发症；④发现2D-SWE可提高儿童移植后肝纤维化的诊断准确率。在原发性肝肿瘤可视化及综合诊疗方面：①制备结合AIE优点、MRI成像高分辨率、光学成像高灵敏度和定量跟踪的Sur@T7-AIE-Gd NPs，创新可视化及siRNA治疗手段。②率先证明肝癌肝移植新辅助应用PD1治疗的有效性和安全性。发现ICI可在术前降低肿瘤负荷，合理选择洗脱期可有效降低排斥发生率。③发现IL9在肿瘤免疫微环境重塑中的作用。培养研究生3名，发表SCI论文12篇