Spangrude G J, Weissman I L
Department of Pathology, Stanford University School of Medicine, CA 94305.
J Immunol. 1988 Sep 15;141(6):1877-90.
A limiting dilution system for cloning thymic CFU (CFUt) from murine bone marrow has been critically evaluated to test the clonal origin of the thymic colonies. Simultaneous limiting dilution transfer of three populations of bone marrow, each expressing a unique allelic cell surface determinant, resulted in independent segregation of donor-derived thymocyte populations within groups of recipient mice. Statistical analysis of the data allowed an estimate of 1 CFUt/3.3 x 10(4) i.v. transferred bone marrow cells. A pulse-chase experiment was utilized to establish whether CFUt seed directly to the thymus, or whether thymic seeding is secondary to extra-thymic engraftment. The results supported the conclusion that bone marrow CFUt utilize a specific interaction with thymic blood vessel endothelial cells to recognize and enter the thymus, and that this seeding occurs within 4 h of i.v. infusion. A kinetic analysis of emigration of the CFUt progeny into the peripheral blood revealed that, in most cases, an early wave of predominantly CD4+ CD8- lymphocytes emerges from the thymus approximately 4 wk after radiation and reconstitution. In a few cases, the first progeny of CFUt to emerge from the thymus were predominantly CD4- CD8+. Commitment of CFUt to TCR beta-chain rearrangements was assessed by quantitating expression of the V beta 8 family of TCR V region genes. Although some clones expressed a significantly higher or lower percentage of V beta 8+ cells, these differences were not stable with time. Thus, CFUt do not undergo absolute commitment to cell surface phenotype of TCR rearrangement, as reflected by the phenotypes of their progeny. Clones of mature peripheral progeny of CFUt could be expanded in culture in the presence of mitogen and growth factors; approximately 30 to 50% of proliferating clones could mediate cytotoxicity in a lectin-dependent assay, further indicating that CFUt are not absolutely committed to a particular T cell function.
一种用于从小鼠骨髓中克隆胸腺集落形成单位（CFUt）的极限稀释系统已得到严格评估，以测试胸腺集落的克隆起源。对三个表达独特等位基因细胞表面决定簇的骨髓群体进行同步极限稀释转移，导致受体小鼠组内供体来源的胸腺细胞群体独立分离。对数据的统计分析得出，静脉注射转移的骨髓细胞中每3.3×10⁴个有1个CFUt。利用脉冲追踪实验来确定CFUt是直接播种到胸腺，还是胸腺播种继发于胸腺外植入。结果支持以下结论：骨髓CFUt利用与胸腺血管内皮细胞的特异性相互作用来识别并进入胸腺，且这种播种发生在静脉注射后4小时内。对CFUt后代向外周血迁移的动力学分析表明，在大多数情况下，在辐射和重建后约4周，一波主要为CD4⁺CD8⁻淋巴细胞的早期浪潮从胸腺中出现。在少数情况下，从胸腺中出现的CFUt的首批后代主要是CD4⁻CD8⁺。通过定量TCR V区基因的Vβ8家族的表达来评估CFUt对TCRβ链重排的定向。尽管一些克隆表达的Vβ8⁺细胞百分比显著更高或更低，但这些差异随时间并不稳定。因此，CFUt不会像其后代的表型所反映的那样，对TCR重排的细胞表面表型进行绝对定向。CFUt成熟外周后代的克隆可在有丝分裂原和生长因子存在的情况下在培养中扩增；在凝集素依赖性测定中，约30%至50%的增殖克隆可介导细胞毒性，这进一步表明CFUt不会绝对定向于特定的T细胞功能。
