Hussey R E, Clayton L K, Diener A, McConkey D J, Howard F D, Rodewald H R, D'Adamio L, Dallenbach F, Stein H, Schmidt E V
Laboratory of Immunobiology, Dana-Farber Cancer Institute, Boston, MA 02115.
J Immunol. 1993 Feb 15;150(4):1183-94.
To elucidate the role of CD3 eta in thymic development and to determine whether CD3 eta is involved in the negative selection process, CD3 eta was overexpressed > 100 fold in transgenic (tg) mice using a Thy-1 promoter and regulatory elements. CD3 eta was readily observed in the majority of cortical thymocytes and in a fraction of medullary thymocytes in tg mice by immunohistochemical staining with an anti-CD3 eta-specific mAb. In contrast, endogenous CD3 eta levels were too low to detect in normal littermates. Flow cytometric analysis demonstrated an increased level of TCR on thymocytes with intermediate TCR density in tg animals and parallel biochemical studies showed a marked increased in TCR-associated CD3 zeta-eta heterodimers and CD3 eta-eta homodimers relative to controls. Despite this change in surface TCR phenotype, there was no significant alteration in the total numbers or proportion of CD4+CD8+ double-positive or CD4+CD8- or CD4-CD8+ single-positive thymocytes or peripheral T cells. Percentages of SP V beta 5, V beta 6, and V beta 8 thymocytes in tg animals were unaltered compared to normal littermates when backcrossed either to C57BL/6 (H-2b) or DBA/2 (H-2d) backgrounds. Furthermore, induction of DNA fragmentation with anti-CD3 epsilon mAb treatment in vivo was not significantly different for tg and normal littermates. Collectively, these data imply that CD3 eta is not a limiting component of the negative selection process.
为了阐明CD3 η在胸腺发育中的作用，并确定CD3 η是否参与阴性选择过程，利用Thy-1启动子和调控元件在转基因（tg）小鼠中使CD3 η过表达超过100倍。通过用抗CD3 η特异性单克隆抗体进行免疫组织化学染色，在tg小鼠的大多数皮质胸腺细胞和一部分髓质胸腺细胞中很容易观察到CD3 η。相比之下，正常同窝小鼠体内内源性CD3 η水平过低而无法检测到。流式细胞术分析表明，tg动物中具有中等TCR密度的胸腺细胞上TCR水平升高，同时平行的生化研究显示，相对于对照组，与TCR相关的CD3 ζ-η异二聚体和CD3 η-η同二聚体显著增加。尽管表面TCR表型发生了这种变化，但CD4+CD8+双阳性或CD4+CD8-或CD4-CD8+单阳性胸腺细胞或外周T细胞的总数或比例没有显著改变。当回交到C57BL/6（H-2b）或DBA/2（H-2d）背景时，tg动物中SP Vβ5、Vβ6和Vβ8胸腺细胞的百分比与正常同窝小鼠相比没有改变。此外，在体内用抗CD3 ε单克隆抗体处理诱导DNA片段化，tg小鼠和正常同窝小鼠之间没有显著差异。总体而言，这些数据表明CD3 η不是阴性选择过程的限制因素。
