Ehlers Marc, Laule-Kilian Kirsten, Petter Michaela, Aldrian Christine J, Grueter Baerbel, Würch Andreas, Yoshida Naomi, Watanabe Toshio, Satake Masanobu, Steimle Viktor
Hans Spemann Laboratories, Max Planck Institute of Immunology, Freiburg, Germany.
J Immunol. 2003 Oct 1;171(7):3594-604. doi: 10.4049/jimmunol.171.7.3594.
During thymic T cell development, immature CD4(+)/CD8(+) thymocytes develop into either CD4(+)/CD8(-) helper or CD4(-)/CD8(+) CTLs. The molecular mechanisms governing the complex selection and differentiation steps during thymic T cell development are not well understood. Here we developed a novel approach to investigate gene function during thymocyte development. We transfected ex vivo isolated immature thymocytes with gene-specific morpholino antisense oligonucleotides and induced differentiation in cell or organ cultures. A morpholino oligonucleotide specific for CD8alpha strongly reduces CD8 expression. To our knowledge, this is the first demonstrated gene knockdown by morpholino oligonucleotides in primary lymphocytes. Using this approach, we show here that the transcription factor Runx3 is involved in silencing of CD4 expression during CD8 T cell differentiation. Runx3 protein expression appears late in thymocyte differentiation and is confined to mature CD8 single-positive thymocytes, whereas Runx3 mRNA is transcribed in mature CD4 and CD8 thymocytes. Therefore, Runx3 protein expression is regulated at a post-transcriptional level. The knockdown of Runx3 protein expression through morpholino oligonucleotides inhibited the development of CD4(-)/CD8(+) T cells. Instead, mature cells with a CD4(+)/CD8(+) phenotype accumulated. Potential Runx binding sites were identified in the CD4 gene silencer element, which are bound by Runx protein in EMSAs. Mutagenesis of potential Runx binding sites in the CD4 gene silencer abolished silencing activity in a reporter gene assay, indicating that Runx3 is involved in CD4 gene silencing. The experimental approach developed here should be valuable for the functional analysis of other candidate genes in T cell differentiation.
在胸腺T细胞发育过程中，未成熟的CD4(+)/CD8(+)胸腺细胞发育为CD4(+)/CD8(-)辅助性T细胞或CD4(-)/CD8(+)细胞毒性T淋巴细胞（CTL）。目前对于胸腺T细胞发育过程中复杂的选择和分化步骤的分子机制尚不清楚。在此，我们开发了一种新方法来研究胸腺细胞发育过程中的基因功能。我们用基因特异性吗啉代反义寡核苷酸转染离体分离的未成熟胸腺细胞，并在细胞或器官培养中诱导分化。一种针对CD8α的吗啉代寡核苷酸可强烈降低CD8表达。据我们所知，这是首次在原代淋巴细胞中证实吗啉代寡核苷酸可导致基因敲低。利用这种方法，我们在此表明转录因子Runx3参与CD8 T细胞分化过程中CD4表达的沉默。Runx3蛋白表达在胸腺细胞分化后期出现，且局限于成熟的CD8单阳性胸腺细胞，而Runx3 mRNA在成熟的CD4和CD8胸腺细胞中均有转录。因此，Runx3蛋白表达在转录后水平受到调控。通过吗啉代寡核苷酸敲低Runx3蛋白表达可抑制CD4(-)/CD8(+) T细胞的发育。相反，具有CD4(+)/CD8(+)表型的成熟细胞会积累。在CD4基因沉默元件中鉴定出潜在的Runx结合位点，在电泳迁移率变动分析（EMSA）中这些位点可被Runx蛋白结合。在报告基因分析中，CD4基因沉默子中潜在的Runx结合位点发生突变后，沉默活性消失，这表明Runx3参与CD4基因沉默。本文开发的实验方法对于T细胞分化中其他候选基因的功能分析应具有重要价值。
