The lncRNA Malat1 Inhibits miR-15/16 to Enhance Cytotoxic T Cell Activation and Memory Cell Formation [Ago2 HITS-CLIP]

Proper activation of cytotoxic T cells via the T cell receptor and the costimulatory receptor CD28 is essential for adaptive immunity against viruses, many intracellular bacteria and cancers. Through biochemical analysis of RNA:protein interactions, we uncovered a novel non-coding RNA circuit regulating activation and differentiation of cytotoxic T cells composed of the long non-coding RNA Malat1 (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 also play important roles in T cell responses to viral infection, including the regulation of antigen-specific T cell expansion and T cell memory. Comparative Argonaute-2 high throughput sequencing of crosslinking immunoprecipitation (Ago2 HITS-CLIP, or AHC) combined with gene expression profiling in normal and miR-15/16-deficient T cells revealed a large network of several hundred direct miR-15/16 target mRNAs, many with functional relevance for T cell activation, survival and memory formation. Among these targets, the long non-coding RNA Malat1 contained the largest absolute magnitude miR-15/16-dependent AHC peak in T cells. This binding site was also 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 Malat1. This mutation interrupted Malat1:miR-15/16 interaction, and enhanced the repression of other miR-15/16 target genes, including CD28. Interrupting Malat1 interaction with miR-15/16 decreased cytotoxic T cell activation, including the expression of IL-2 and a broader CD28-responsive gene program. Accordingly, Malat1 mutation diminished memory cell persistence following LCMV Armstrong and Listeria monocytogenes infection. This study marks a significant advance in the study of long noncoding RNAs in the immune system by ascribing cell-intrinsic, sequence-specific in vivo function to Malat1. These findings have implications for T cell-mediated autoimmune diseases, antiviral and anti-tumor immunity, as well as lung adenocarcinoma and other malignancies where Malat1 is overexpressed. Comparative Ago2 HITS-CLIP (High Throughput Sequencing following Crosslinking Immuno-Precipitation) was performed on Ago2 HITS-CLIP data from primary mouse CD8+ T cells isolated from the spleens of naive mice. T cells were stimulated for 72 hours with anti-CD3 and anti-CD28 antibodies followed by 2 days resting in IL-2 supplemented media. Cells from mice lacking the miR-15/16 binding site in Malat1, Malat1scr/scr; Bl6/J WT; and mir-15/16 deleted by CD4-cre, miR-15/16Δ/Δ were used.

通过T细胞受体与共刺激受体CD28实现细胞毒性T细胞的正确激活，是针对病毒、多数胞内细菌与癌症的适应性免疫所必需的。本研究通过对RNA-蛋白质相互作用进行生化分析，发现了一条调控细胞毒性T细胞激活与分化的新型非编码RNA通路，该通路由长链非编码RNA Malat1（转移相关肺腺癌转录本1，Metastasis Associated Lung Adenocarcinoma Transcript 1）与miR-15/16微小RNA家族构成。miR-15/16是一类广泛且高表达的肿瘤抑制性微小RNA家族，在细胞增殖与存活过程中发挥重要调控作用。此外，该家族在T细胞针对病毒感染的免疫应答中也扮演关键角色，包括调控抗原特异性T细胞扩增与T细胞记忆形成。
通过对正常与miR-15/16缺陷型T细胞开展交联免疫沉淀结合高通量测序的Argonaute-2分析（又称Ago2 HITS-CLIP，简称AHC），并结合基因表达谱分析，我们揭示了一个包含数百个miR-15/16直接靶mRNA的庞大调控网络，其中多数靶分子与T细胞激活、存活及记忆形成的功能密切相关。在这些靶标中，长链非编码RNA Malat1的miR-15/16依赖性AHC信号峰在T细胞中具有最大的绝对强度。该结合位点亦是在T细胞转录组中检测到的最强的长链非编码RNA-微小RNA相互作用之一。
我们利用CRISPR靶向结合同源定向修复（HDR）技术，构建了在Malat1基因的miR-15/16结合位点处携带5个核苷酸突变的小鼠模型。该突变打断了Malat1与miR-15/16的相互作用，并增强了对其他miR-15/16靶基因的抑制作用，其中包括CD28。阻断Malat1与miR-15/16的相互作用会抑制细胞毒性T细胞的激活，包括白细胞介素-2（IL-2）的表达以及更广泛的CD28应答基因程序。相应地，在淋巴细胞脉络丛脑膜炎病毒阿姆斯特丹株（LCMV Armstrong）与单核细胞增生李斯特菌（Listeria monocytogenes）感染后，Malat1突变会减弱记忆细胞的存活能力。
本研究通过赋予Malat1细胞自主性、序列特异性的体内功能，在免疫系统长链非编码RNA研究领域取得了重要进展。上述发现对于T细胞介导的自身免疫病、抗病毒与抗肿瘤免疫，以及Malat1过表达的肺腺癌与其他恶性肿瘤的研究均具有重要参考价值。
本研究的Ago2 HITS-CLIP（交联免疫沉淀高通量测序）数据源自从野生型小鼠脾脏分离的原代小鼠CD8+ T细胞。T细胞经抗CD3与抗CD28抗体刺激72小时后，在添加白细胞介素-2的培养基中静置培养2天。实验分别使用了三类小鼠来源的细胞：Malat1基因miR-15/16结合位点缺失的小鼠（Malat1scr/scr）、C57BL/6J野生型小鼠（Bl6/J WT），以及经CD4-cre重组酶介导删除miR-15/16的小鼠（miR-15/16Δ/Δ）。