An essential role for miR-15/16 in Treg suppression and restriction of proliferation

The miR-15/16 family is a highly expressed group of tumor suppressor miRNAs that target a large network of genes in T cells to restrict their cell cycle, memory formation and survival. Upon T cell activation, miR-15/16 are downregulated, allowing rapid expansion of differentiated effector T cells to mediate a sustained immune response. Here, using conditional deletion of miR-15/16 in immunosuppressive regulatory T cells (Tregs) that express FOXP3, we identify new functions of the miR-15/16 family in T cell immunity. miR-15/16 are indispensable to maintain peripheral tolerance by securing efficient suppression by a limited number of Tregs. miR-15/16-deficiency alters Treg expression of critical functional proteins including FOXP3, IL2Ra/CD25, CTLA4, PD-1 and IL7Ra/CD127, and results in accumulation of functionally impaired FOXP3loCD25loCD127hi Tregs. Excessive proliferation in the absence of miR-15/16 inhibition of cell cycle programs shifts Treg diversity and produces an effector Treg phenotype characterized by low expression of TCF1, CD25 and CD62L, and high expression of CD44. These Tregs fail to control immune activation of CD4+ effector T cells, leading to spontaneous multi-organ inflammation and increased allergic airway inflammation in a mouse model of asthma. Together, our results demonstrate that miR-15/16 expression in Tregs is essential to maintain immune tolerance. Overall design: Live CD4+FOXP3+ Tregs (bulk cKO, bulk WT and WT CD25hi and CD25lo bin) were isolated from murine spleen and lymph nodes by FACS. cKO Tregs refer to miR-15/16 deficient Tregs. Samples are biological replicates of 2-5 mice per group.

miR-15/16家族是一类高表达的肿瘤抑制性微小RNA（miRNA），可靶向T细胞内庞大的基因调控网络，进而限制T细胞的细胞周期进程、记忆形成与存活过程。当T细胞活化时，miR-15/16的表达水平会下调，使得分化后的效应T细胞得以快速扩增，以介导持续的免疫应答。本研究通过在表达FOXP3的免疫抑制性调节性T细胞（regulatory T cells, Tregs）中条件性敲除miR-15/16，揭示了该家族在T细胞免疫中的全新功能。miR-15/16对于通过有限数量的Tregs实现高效免疫抑制以维持外周免疫耐受至关重要。miR-15/16缺失会改变Tregs中关键功能蛋白的表达谱，包括FOXP3、IL2Ra/CD25、CTLA4、PD-1以及IL7Ra/CD127，并导致功能受损的FOXP3loCD25loCD127hi Tregs的积累。在缺失miR-15/16对细胞周期程序的抑制作用后，Tregs会出现过度增殖，这一改变会重塑Treg的细胞多样性，并产生以低表达TCF1、CD25和CD62L、高表达CD44为特征的效应性Treg表型。这类Tregs无法有效调控CD4+效应T细胞的免疫活化，进而在哮喘小鼠模型中引发自发性多器官炎症，并加剧气道变应性炎症。综上，本研究结果证实，Tregs中miR-15/16的表达对于维持免疫耐受具有不可或缺的作用。

整体实验设计：通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）从小鼠脾脏及淋巴结中分离活的CD4+FOXP3+ Tregs，涵盖批量cKO Tregs、批量野生型（wild type, WT）Tregs，以及野生型CD25hi和CD25lo亚群。其中cKO Tregs特指miR-15/16缺陷型Tregs。每组设置2~5只小鼠的生物学重复。