CD8 T Cell Intrinsic Role of MicroRNA-17~92 in TCR Signaling

描述（由申请人提供）：微小RNA（miRNA）是通过抑制蛋白质翻译或降解mRNA转录物来调节基因表达的小RNA（~22个核苷酸）。miRNA基因调控途径在进化上高度保守，并且通过控制多种生物学过程如细胞信号传导、增殖和凋亡而成为免疫细胞发育和功能的主要参与者。我们最近已经表明，CD 8 T细胞的miRNA库是动态的，并且在感染后迅速变化，因为幼稚细胞被激活并分化为效应细胞，然后是记忆细胞。 特别是，我们发现miR- 17~92簇在快速扩增的效应细胞中高度上调，在记忆命运的（记忆前体效应细胞，MPECs）中的表达相对低于短寿命效应细胞（SLEC）亚群。miR-17~ 92的缺失或组成型表达揭示了miR-17 ~ 92在通过促进增殖驱动终末效应细胞分化中的关键CD 8 T细胞内在作用，这与我们的假设一致，即miR-17~92可能调节效应细胞和记忆命运。这些研究为探讨miR-17~92调控效应子和记忆命运的分子机制奠定了坚实的基础。 使用miR-17~92表达的CD 8 T细胞特异性增益或损失，我们有令人信服的初步证据支持我们的假设，即miR-17~92降低抗原激活T细胞的阈值，从而通过过度刺激和增殖促进末端效应分化：（i）miR- 17~92的缺失以依赖于抗原的全身水平的方式影响CTL扩增和存活;（ii）T细胞信号传导抑制剂miR-17~92过表达后，肿瘤抑制蛋白PTEN和抑制性受体PD-1表达减少;（iii）在miR- 17~92过表达时，抗原对T细胞的活化增强。在R 03资助机制下，该小型独立研究项目的目标是进行深入的实验，明确评估miR-17~92对抗原特异性CD 8 T细胞中T细胞信号转导阈值的作用，并确定miR- 17~92调控的关键信号通路和蛋白质。我们将在我们的miR-17~92表达获得或丧失的双重模型中使用次优抗原肽剂量以及不同亲和力的改变的肽配体来测量TCR敏感性;我们将在miR-17~92过表达或丧失时量化TCR刺激下游的信号转导级联;我们将在活化的早期阶段对缺乏或过表达miR-17~92的效应CD 8 T细胞进行基因分析，以潜在地鉴定新的miR-17~92。92个靶点参与调节T细胞刺激、增殖和终末效应分化的强度。 诱导强效记忆性CD 8 T细胞 细胞是疫苗接种的重要目标。因此，更好地理解效应子和记忆谱系特化的分子调控，如从所提出的研究中得到的，与在肿瘤生长、感染和疫苗接种期间操纵CD 8 T细胞免疫直接相关。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are small RNAs (~22 nucleotides) that regulate gene expression by inhibiting protein translation or by degrading mRNA transcripts. The miRNA gene regulatory pathway is highly evolutionarily conserved and is emerging as a major player in immune cell development and function, by controlling a diverse array of biological processes such as cell signaling, proliferation and apoptosis. We have recently shown that the miRNA repertoire of CD8 T cells is dynamic and rapidly changes after infection as naive cells get activated and differentiate into effector cells and then memory cells. In particular, we found that the miR- 17~92 cluster is highly upregulated in rapidly expanding effector cells, with relatively lower expression in memory-fated (memory precursor effector cells, MPECs) than short-lived effector cell (SLECs) subsets. Consistent with our hypothesis that miR-17~92 may be regulating effector and memory fates, deletion or constitutive expression of miR-17~92 revealed a critical CD8 T cell-intrinsic role of miR-17~92 in driving terminal effector differentiation by promoting proliferation. These studies lay a strong framework for investigating the molecular mechanisms by which miR-17~92 regulates effector and memory fates. Using CD8 T cell-specific gain or loss of expression of miR-17~92, we have compelling preliminary evidence supporting our hypothesis that miR-17~92 decreases the threshold of T cell activation by antigen, thus promoting terminal effector differentiation through excessive stimulation and proliferation: (i) Loss of miR- 17~92 impacts CTL expansion and survival, in a manner that is dependent on systemic levels of antigen; (ii) Inhibitors of T cell signaling (the tumor suppressor protein, PTEN, and inhibitory receptor PD-1) are decreased upon overexpression of miR-17~92; (iii) T cell activation by antigen is enhanced upon overexpression of miR- 17~92. Under the R03 funding mechanism, the goal of this small self-contained research project is to conduct incisive experiments that will definitively evaluate the role of miR-17~92 on T cell signaling threshold in antigen-specific CD8 T cells, and also identify key signaling pathways and proteins that are regulated by miR- 17~92. We will measure TCR sensitivity in our dual model of gain or loss of expression of miR-17~92 using suboptimal antigenic peptide dose as well as altered peptide ligands of varying affinity; we will quantify signal transduction cascades downstream of TCR stimulation upon overexpression or loss of miR-17~92; and we will conduct gene profiling of effector CD8 T cells lacking or overexpressing miR-17~92 during early stages of activation towards potentially identifying novel miR-17~92 targets involved in regulating strength of T cell stimulation, proliferation and terminal effector differentiation. Induction of potent memory CD8 T cells is an important goal of vaccination. Thus, greater understanding of molecular regulation of effector and memory lineage specification, as resulting from proposed studies, has direct relevance to manipulation of CD8 T cell immunity during tumor outgrowth, infection and vaccination.