Institute of Immunotherapy, Fujian Medical University, Fuzhou, 350122, China.
The School of Basic Medical Sciences, Fujian Medical University, Fuzhou, 350122, China.
Cancer Immunol Immunother. 2024 Nov 2;74(1):4. doi: 10.1007/s00262-024-03851-x.
Lymphocyte antigen 6 complex, locus E (Ly6E) has been initially demonstrated to involve in T cell activity and impair viral infectivity. Recently, high expression levels of Ly6E have been reported in tumor microenvironment (TME) of various types of cancers. However, the immunoregulatory mechanism of Ly6E manipulating TME remains unknown.
TCGA database and Kaplan-Meier plotter database were used to evaluate the correlation between Ly6E expression levels and cancer patient survival. After analyzing Ly6E expression levels in human breast cancer tissues and tumor cell lines, we generated Ly6E knockout (KO) and overexpression (OE) mouse cell lines. Cell proliferation ability in vitro and the ability of growth and metastasis in mouse tumor models were compared between KO/OE and wild-type tumor cells. On day 7 after tumor implantation, tumor tissues were separated for flow cytometric assay, bulk RNA sequencing and single-cell RNA sequencing (ScRNA-seq). The role of Ly6E-expressing tumor cell on macrophage was analyzed in vitro.
Our result surprisingly found that high Ly6E expression levels were associated with CD8 T cell exclusion in tumor tissues and resistance to immunotherapy. Our data showed that knockout of Ly6E in tumor cells prompts tumor regression and inhibits tumor metastases, and Ly6E-OE tumor cells vice versa. The enhanced anti-tumor effect of Ly6E knockout in tumor cells was dependent on T cell response and formed long-lasting memory. The increase in the CD8 T-cell infiltration into the tumor islet of Ly6E-KO tumors confirmed the role of Ly6E on T cell exclusion. ScRNA-seq analysis showed that M2 macrophages are particularly abundant in the Ly6E-expressing tumor tissues, especially M2-4 macrophage cluster identified by high levels of Arg-1, indicates that Ly6E-expressing tumor cells might restrict T cell infiltration via M2 macrophages. Moreover, in vitro assay showed that cell culture media derived from Ly6E-positive tumor cells promoted macrophage migration and M2 polarization.
Our study illuminated that Ly6E-expressing tumor cells facilitated the accumulation of M2 macrophages in TME, which contributes to CD8 T cell exclusion and provides new insights for improving efficacy of cancer immunotherapy.
淋巴细胞抗原 6 复合物，基因座 E（Ly6E）最初被证明参与 T 细胞活性并损害病毒感染力。最近，已报道 Ly6E 在各种类型癌症的肿瘤微环境（TME）中的高表达水平。然而，Ly6E 调节 TME 的免疫调节机制尚不清楚。
TCGA 数据库和 Kaplan-Meier plotter 数据库用于评估 Ly6E 表达水平与癌症患者生存的相关性。在分析人类乳腺癌组织和肿瘤细胞系中的 Ly6E 表达水平后，我们生成了 Ly6E 敲除（KO）和过表达（OE）小鼠细胞系。比较 KO/OE 和野生型肿瘤细胞在体外的增殖能力以及在小鼠肿瘤模型中的生长和转移能力。在肿瘤植入后第 7 天，分离肿瘤组织进行流式细胞术分析、批量 RNA 测序和单细胞 RNA 测序（ScRNA-seq）。在体外分析表达 Ly6E 的肿瘤细胞对巨噬细胞的作用。
我们的结果令人惊讶地发现，高 Ly6E 表达水平与肿瘤组织中 CD8 T 细胞排斥和免疫治疗抵抗有关。我们的数据表明，肿瘤细胞中 Ly6E 的敲除会促使肿瘤消退并抑制肿瘤转移，而 Ly6E-OE 肿瘤细胞则相反。肿瘤细胞中 Ly6E 敲除增强的抗肿瘤作用依赖于 T 细胞反应并形成持久的记忆。Ly6E-KO 肿瘤中 CD8 T 细胞浸润肿瘤胰岛的增加证实了 Ly6E 对 T 细胞排斥的作用。ScRNA-seq 分析表明，M2 巨噬细胞在表达 Ly6E 的肿瘤组织中特别丰富，特别是通过高水平 Arg-1 鉴定的 M2-4 巨噬细胞簇，表明 Ly6E 表达的肿瘤细胞可能通过 M2 巨噬细胞限制 T 细胞浸润。此外，体外实验表明，来自 Ly6E 阳性肿瘤细胞的细胞培养上清液促进了巨噬细胞的迁移和 M2 极化。
本研究表明，表达 Ly6E 的肿瘤细胞促进了 TME 中 M2 巨噬细胞的积累，这有助于 CD8 T 细胞排斥，并为提高癌症免疫治疗的疗效提供了新的见解。
