Cellular and peptide requirements for in vitro clonal deletion of immature thymocytes.

Thymocytes from DO10 T-cell-receptor transgenic mice undergo apoptosis, or programmed cell death, when chicken ovalbumin-(323-339) peptide is administered in vivo. Using DO10 mice thymocytes, we have now developed a simple in vitro model system that recapitulates the in vivo clonal-deletion process. When transgenic thymocytes were cocultured with fibroblasts, B cells, or thymic nurse cell lines (all bearing I-Ad) in the presence of chicken ovalbumin-(323-339), deletion of the transgenic TCR+CD4+CD8+ thymocytes was seen within 8-20 hr. Thymocytes designed to bear I-Ad on their surface could mediate the deletion themselves. Thus, thymocyte clonal deletion entirely depends on the stage at which the thymocytes are vulnerable to the onset of apoptosis, rather than on the nature of the peptide antigen-presenting cells. Furthermore, thymic nurse cell line TNC-R3.1 could cause deletion, strongly suggesting that some thymic epithelial/stromal components are potentially capable of participating in negative selection. In all cases examined, little deletion could be induced at a peptide concentration less than 10 nM, thus defining the minimum amount of peptide antigen required for negative selection. The peptide-dependent in vitro negative-selection system will allow further dissection of the molecular and cellular processes involved in clonal deletion due to apoptosis in the thymus. IMAGES:

来自DO10 T细胞受体（T-cell-receptor）转基因小鼠的胸腺细胞，在体内给予鸡卵清蛋白-(323-339)肽时会发生凋亡（apoptosis，又称编程性细胞死亡）。本研究以DO10小鼠胸腺细胞为材料，构建了一套可重现体内克隆清除过程的简易体外模型系统。当转基因胸腺细胞与携带I-Ad的成纤维细胞、B细胞或胸腺滋养细胞系共培养，并加入鸡卵清蛋白-(323-339)肽时，可在8~20小时内观察到转基因TCR+CD4+CD8+胸腺细胞的清除现象。表面表达I-Ad的胸腺细胞可自行介导该清除过程。由此可见，胸腺细胞的克隆清除完全取决于其对凋亡启动的易感阶段，而非呈递该肽类抗原的抗原呈递细胞的属性。此外，胸腺滋养细胞系TNC-R3.1可介导该清除过程，这强烈提示部分胸腺上皮/基质成分具备参与阴性选择（negative selection）的潜力。在所有检测的实验条件中，当肽浓度低于10 nM时几乎无法诱导清除，由此明确了阴性选择所需的最低肽类抗原剂量。这种依赖于肽类的体外阴性选择系统，将有助于进一步解析胸腺中因凋亡引发的克隆清除所涉及的分子与细胞过程。图片：