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

描述(申请人提供)：microRNAs(MiRNAs)是一种小RNA(~22个核苷酸)，通过抑制蛋白质翻译或降解mRNA转录本来调节基因表达。MiRNA基因调控途径在进化上高度保守，通过控制细胞信号、增殖和凋亡等一系列生物学过程，正在成为免疫细胞发育和功能的主要参与者。我们最近发现，CD8T细胞的miRNA谱系是动态的，感染后迅速变化，幼稚细胞被激活并分化为效应细胞，然后分化为记忆细胞。 特别是，我们发现miR-17~92簇在快速扩张的效应细胞中高度上调，在记忆命运型(记忆前体效应细胞，MPECs)中的表达相对较低，而在短期效应细胞(SLECs)中的表达相对较低。MiR-17~92的缺失或结构性表达揭示了miR-17~92在促进终末效应器分化中的CD8T细胞内在关键作用，这与我们的假设miR-17~92可能是调节效应器和记忆命运的假设相一致。这些研究为研究miR-17~92调节效应器和记忆命运的分子机制奠定了坚实的框架。利用CD8T细胞特异性miR-17~92的表达增减，我们有令人信服的初步证据支持我们的假设，即miR-17~92降低了抗原激活T细胞的阈值，从而通过过度刺激和增殖促进终末效应器的分化：(I)miR-17~92的缺失以一种依赖于系统抗原水平的方式影响CTL的扩张和存活；(Ii)T细胞信号的抑制物(肿瘤抑制蛋白PTEN和抑制性受体PD-1)在miR-17~92过表达时减少；(Iii)当miR-17~92过表达时，抗原对T细胞的激活作用增强。在R03资助机制下，这个自给自足的小型研究项目的目标是进行深入的实验，最终评估miR-17~92对抗原特异性CD8T细胞中T细胞信号阈值的作用，并确定受miR-17~92调控的关键信号通路和蛋白质。我们将使用次优抗原肽剂量和不同亲和力的改变的多肽配体，在miR-17~92表达增强或缺失的双重模型中测量TCR的敏感性；我们将量化miR-17~92过度表达或丢失时TCR刺激下游的信号转导级联；我们将对激活早期缺乏或过度表达miR-17~92的效应者CD8 T细胞进行基因图谱分析，以潜在地寻找参与调节T细胞刺激、增殖和终末效应器分化的新的miR-17~92靶点。强效记忆CD8 T细胞的诱导 细胞是疫苗接种的重要目标。因此，更好地理解效应器和记忆谱系规范的分子调控，对于在肿瘤生长、感染和疫苗接种过程中操纵CD8 T细胞免疫具有直接的相关性。

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.