Molecular Analysis of Human Naive and Memory T Cells

摘要：本项目的目的是表征记忆淋巴细胞的分子特征，阐明免疫记忆的机制。我们研究人类外周血记忆和幼稚CD4+ T细胞作为模型，因为它们在表型和功能上都有很好的特征。记忆性CD4+ T细胞表达CD45R0，初始性CD4+ T细胞表达CD45RA，以此作为分离这两个亚群的基础。评估静止和激活后记忆细胞的整体基因表达。利用cDNA微阵列技术，我们分析了超过58,000个独特的人类cDNA克隆，发现记忆和幼稚CD4+ T细胞表达了约22-27%的克隆。总的来说，记忆和初始CD4+ T细胞在静止和激活后表达相似的基因数量。抑制细胞增殖的基因在静息细胞中高度表达，但在记忆和初始CD4+ T细胞中受到刺激后一致下调。相比之下，激活诱导基因的表达呈现梯度模式，mRNA水平与激活程度相关，并区分记忆与原始细胞。总之，我们的研究结果定义了记忆CD4+ T细胞在静止和激活后的转录性质，并确定了区分记忆CD4+ T细胞和初始CD4+ T细胞反应的分子机制。在全局分析的过程中，我们已经鉴定出170多个已知基因，它们的表达在激活后发生了显著变化。最近，我们将cDNA芯片分析mRNA水平的研究扩展到传统的Northern和RT-PCR，以及选定基因的蛋白质水平。目前，我们正在研究这些激活相关基因的时间变化及其在记忆和初始CD4+ T细胞激活中的功能。

Summary: The goals of this project are to characterize the molecular identity of memory lymphocytes and to elucidate the mechanisms underlying immunological memory. We study human peripheral blood memory and naive CD4+ T cells as a model because they are phenotypically and functionally well characterized. Memory CD4+ T cells express CD45R0 while naive CD4+ T cells express CD45RA, which was used as the basis for isolation of these two subsets. To assess the global gene expression of memory cells at rest and after activation in comparison with na?ve cells using cDNA microarray, we analyzed over 58,000 unique human cDNA clones and found that memory and naive CD4+ T cells expressed about 22-27% of total clones. Overall, memory and naive CD4+ T cells express similar number of genes at rest and after activation. Genes that inhibit cellular proliferation are highly expressed in resting cells but are uniformly down-regulated after stimulation in both memory and naive CD4+ T cells. In contrast, the expression of activation-induced genes displays a gradient pattern in which the mRNA levels correlate with the degree of activation, and differentiate memory from naive cells. Together, our results defines the transcriptional nature of memory CD4+ T cells at rest and after activation, and identify a molecular mechanism that differentiates the response of memory CD4+ T cells from naive CD4+ T cells. In the process of global analysis, we have identified over 170 known genes whose expression was significantly changes after activation. Recently, we have extended the study of mRNA levels analyzed by cDNA microarray to conventional Northern and RT-PCR, as well as protein levels of selected genes. Currently, we are studying the temporal changes of these activation-related genes and their function in memory and naive CD4+ T cell activation.