Department of Microbiology & Immunology, University of California San Francisco, San Francisco, United States.
Sandler Asthma Basic Research Program, University of California, San Francisco, San Francisco, United States.
Elife. 2023 Dec 21;12:RP87900. doi: 10.7554/eLife.87900.
Proper activation of cytotoxic T cells via the T cell receptor and the costimulatory receptor CD28 is essential for adaptive immunity against viruses, intracellular bacteria, and cancers. Through biochemical analysis of RNA:protein interactions, we uncovered a non-coding RNA circuit regulating activation and differentiation of cytotoxic T cells composed of the long non-coding RNA (Metastasis Associated Lung Adenocarcinoma Transcript 1) and the microRNA family miR-15/16. miR-15/16 is a widely and highly expressed tumor suppressor miRNA family important for cell proliferation and survival. miR-15/16 play important roles in T cell responses to viral infection, including the regulation of antigen-specific T cell expansion and memory. Comparative Argonaute-2 high-throughput sequencing of crosslinking immunoprecipitation (AHC) combined with gene expression profiling in normal and miR-15/16-deficient mouse T cells revealed a large network of hundreds of direct miR-15/16 target mRNAs, many with functional relevance for T cell activation, survival and memory formation. Among these targets, contained the largest absolute magnitude miR-15/16-dependent AHC peak. This binding site was among the strongest lncRNA:miRNA interactions detected in the T cell transcriptome. We used CRISPR targeting with homology directed repair to generate mice with a 5-nucleotide mutation in the miR-15/16-binding site in . This mutation interrupted :miR-15/16 interaction, and enhanced the repression of other miR-15/16 target genes, including CD28. Interrupting interaction with miR-15/16 decreased cytotoxic T cell activation, including the expression of interleukin 2 (IL-2) and a broader CD28-responsive gene program. Accordingly, mutation diminished memory cell persistence in mice following LCMV Armstrong and infection. This study marks a significant advance in the study of long non-coding RNAs in the immune system by ascribing cell-intrinsic, sequence-specific in vivo function to . These findings have implications for T cell-mediated autoimmune diseases, antiviral and anti-tumor immunity, as well as lung adenocarcinoma and other malignancies where is overexpressed.
通过对 RNA-蛋白质相互作用的生化分析，我们发现了一个非编码 RNA 回路，该回路由长非编码 RNA (转移相关肺腺癌转录本 1) 和 microRNA 家族 miR-15/16 组成，调节细胞毒性 T 细胞的激活和分化。miR-15/16 是一种广泛且高度表达的肿瘤抑制 miRNA 家族，对细胞增殖和存活很重要。miR-15/16 在 T 细胞对病毒感染的反应中发挥重要作用，包括调节抗原特异性 T 细胞的扩增和记忆。正常和 miR-15/16 缺陷型小鼠 T 细胞的交联免疫沉淀 (AHC) 结合基因表达谱的比较 Argonaute-2 高通量测序显示，数百个直接 miR-15/16 靶 mRNA 的大型网络，许多与 T 细胞激活、存活和记忆形成的功能相关。在这些靶标中， 包含最大的绝对幅度 miR-15/16 依赖的 AHC 峰。该结合位点是在 T 细胞转录组中检测到的最强的 lncRNA:miRNA 相互作用之一。我们使用带有同源定向修复的 CRISPR 靶向技术，生成 miR-15/16 结合位点在 中发生 5 个核苷酸突变的小鼠。该突变中断了 :miR-15/16 相互作用，并增强了对其他 miR-15/16 靶基因，包括 CD28 的抑制。中断 与 miR-15/16 的相互作用降低了细胞毒性 T 细胞的激活，包括白细胞介素 2 (IL-2) 的表达和更广泛的 CD28 反应性基因程序。因此， 突变减少了 LCMV Armstrong 和 感染后小鼠记忆细胞的持久性。这项研究通过将细胞内、序列特异性的体内功能归因于 ，在免疫系统中长非编码 RNA 的研究方面取得了重大进展。这些发现对 T 细胞介导的自身免疫性疾病、抗病毒和抗肿瘤免疫，以及肺腺癌和其他 过度表达的恶性肿瘤具有重要意义。
