Molecular Analysis Of Human Naive And Memory T Cells

摘要：一组在记忆性CD4+ T细胞中差异表达的cDNA克隆的鉴定。为了确定记忆性CD4+ T细胞的分子特征，我们利用cDNA微阵列测量了100多个供者外周血中新鲜分离的人类记忆性CD4+ T细胞和初始CD4+ T细胞的基因表达。cDNA微阵列方法允许我们同时在基因组尺度上评估基因，为基因表达谱的比较分析和识别某些细胞群体中的差异表达基因提供了强大的工具。在对来自商业来源（包括超过50,000个cDNA克隆）的cDNA微阵列过滤器和来自商业来源和本实验室（约2,800个独特克隆）的定制选择克隆进行序列分析后，我们首次在基因组尺度上了解记忆和幼稚CD4+ T细胞的基因表达模式。我们发现，新分离的记忆细胞和初始细胞CD4+ T细胞表达的基因数量相似，并且14个cDNA克隆在记忆细胞中表达的转录本水平高于初始细胞。记忆性CD4+ T细胞活化诱导下调和上调基因的鉴定。在抗cd3 +抗cd28体外刺激16小时后，我们在记忆CD4+ T中鉴定出135个（130个已知基因和5个ESTs）上调cDNA克隆，68个（42个已知基因和26个ESTs）下调cDNA克隆。有趣的是，在体外刺激后，记忆细胞中上调基因mRNA水平的增加高于初始CD4+ T细胞，并且在记忆细胞和初始CD4+ T细胞中，抗cd3 +抗cd28比单独抗cd3更高。然后，我们通过Northern分析证实了通过cDNA微阵列鉴定的肌动蛋白和细胞因子基因的表达变化。此外，我们将mRNA分析扩展到蛋白质表达，发现细胞因子和肌动蛋白的mRNA和蛋白质水平是相关的。总之，我们已经鉴定了大约200个cDNA克隆，其表达水平在激活后发生变化，并提示上调基因的表达水平是区分记忆反应与初始CD4+ T细胞的分子机制。

Summary: Identification of a set of cDNA clones that are differentially expressed in memory CD4+ T cells. In an attempt to determine the molecular features of memory CD4+ T cells, we have utilized cDNA microarrays to measure gene expression of freshly isolated human memory and naive CD4+ T cells from peripheral blood over 100 donors. cDNA microarray method allows us to simultaneously assess genes at genome scales, providing a powerful tool for comparative analysis of gene expression profiles and for identifying differentially expressed genes in certain cell populations. After a sequential analyses of cDNA microarray filters from commercial source (consisting of over 50,000 cDNA clones) and custom-made selected clones from the commercial source and this laboratory (~2,800 unique clones), we provide the first glimpse into gene expression patterns of memory and naive CD4+ T cells at the genome-scale. We found that freshly isolated memory and naive CD4+ T cells expressed similar numbers of genes, and that 14 cDNA clones expressed higher levels of transcripts in memory cells than in naive cells. Identification of activation induced down- and up-regulated genes in memory CD4+ T cells. We have identified 135 (130 known genes and 5 ESTs) up-regulated and 68 (42 known genes and 26 ESTs) down-regulated cDNA clones in memory CD4+ T after 16 hours of in vitro stimulation with anti-CD3 plus anti-CD28. Interestingly, the increase in mRNA levels of up-regulated genes was greater in memory than in naive CD4+ T cells after in vitro stimulation, and was higher with anti-CD3 plus anti-CD28 than with anti-CD3 alone in both memory and naive CD4+ T cells. We then confirmed the changes in expression of actin and cytokine genes identified by cDNA microarrays by Northern analysis. Furthermore, we extended mRNA analysis to protein expression and found that the levels of mRNA and protein of cytokines and actins were correlated. Together, we have identified approximately 200 cDNA clones whose expression levels changed after activation, and suggest that the level of expression of up-regulated genes is a molecular mechanism that differentiates the response of memory from naive CD4+ T cells.