Benveniste P, Chadwick B S, Miller R G, Reimann J
Ontario Cancer Institute, University of Toronto, Canada.
J Immunol. 1990 Jan 15;144(2):411-9.
Evidence is presented that early steps in T cell differentiation, to the point of TCR expression, can occur in athymic nude bone marrow (BM) and perhaps also in normal BM. Low density nylon wool nonadherent BM from 5- to 8-wk-old nude and normal mice was found to contain dim CD3+ CD4- CD8- cells. This subpopulation was not found in scid/scid low density nylon wool nonadherent BM but was present at similar frequency in both nude and normal BM and expressed comparable fluorescence intensity. With the use of additional markers, a difference between nude and normal dim CD3+ CD4- CD8- cells was found: the ratio of J11d+/J11d- cells was at least threefold higher in nude than in normal BM. Whereas 60% of nude J11d+ CD3+ cells expressed a TCR-alpha/beta and 40% expressed a gamma/delta TCR, almost no TCR-gamma/delta expressing cells were detected in dim CD3+ cells from normal mice in either the J11d+ or J11d- subpopulation. The majority of those cells express a TCR-alpha/beta. We established that nude dim CD3+ CD4- CD8- cells were distinguishable from mature recirculating T cells and developed an in vitro clonal culture system that supported the growth of these cells. When these cells were cocultured in vitro in a limiting dilution system with inactivated anti-CD3 mAb-secreting cells, in the presence of rIL-2 and rIL-4, one in 5 x 10(2) to 1 x 10(3) cells were growth inducible. Most progeny cells expressed a CD3+ CD8+ CD4- surface phenotype. Cultures displayed nonspecific cytolytic reactivity. With the use of a sensitive dot-blot technique, transcripts coding for C alpha, C beta, VC gamma, and C delta TCR genes were detected in these cultures. Rehybridization experiments demonstrated mutually exclusive expression of either C alpha or C delta mRNA transcripts in the majority of clones selected for a high probability of clonality. Most (greater than 95%) of T cell clones derived from normal BM expressed the C alpha transcript with approximately 5% expressing the C delta transcript. In contrast, about 40% of T cell clones derived from nude BM expressed the C delta transcript whereas the remaining 60% expressed 10-fold lower C alpha message than normal BM-derived clones.
有证据表明，T细胞分化到TCR表达阶段的早期步骤可发生在无胸腺裸鼠的骨髓（BM）中，或许也可发生在正常骨髓中。发现5至8周龄裸鼠和正常小鼠的低密度尼龙毛非黏附性骨髓中含有CD3+ CD4- CD8-弱阳性细胞。在重度联合免疫缺陷（scid/scid）小鼠的低密度尼龙毛非黏附性骨髓中未发现该亚群，但在裸鼠和正常小鼠的骨髓中以相似频率存在，且表达相当的荧光强度。使用其他标志物后，发现裸鼠和正常小鼠的CD3+ CD4- CD8-弱阳性细胞存在差异：裸鼠骨髓中J11d+/J11d-细胞的比例至少比正常骨髓高3倍。60%的裸鼠J11d+ CD3+细胞表达TCR-α/β，40%表达γ/δ TCR，而在正常小鼠的CD3+弱阳性细胞中，无论是J11d+还是J11d-亚群，几乎都未检测到表达γ/δ TCR的细胞。这些细胞大多数表达TCR-α/β。我们确定裸鼠的CD3+ CD4- CD8-弱阳性细胞与成熟的循环T细胞不同，并建立了一种支持这些细胞生长的体外克隆培养系统。当这些细胞在有限稀释系统中与分泌失活抗CD3单克隆抗体的细胞进行体外共培养时，在rIL-2和rIL-4存在的情况下，5×10²至1×10³个细胞中有1个细胞具有生长诱导性。大多数子代细胞表达CD3+ CD8+ CD4-表面表型。培养物表现出非特异性细胞溶解反应性。使用灵敏的斑点印迹技术，在这些培养物中检测到编码Cα、Cβ、VCγ和Cδ TCR基因的转录本。再杂交实验表明，在大多数被选作具有高克隆性可能性的克隆中，Cα或Cδ mRNA转录本相互排斥表达。源自正常骨髓的大多数（>95%）T细胞克隆表达Cα转录本，约5%表达Cδ转录本。相比之下，源自裸鼠骨髓的T细胞克隆中约40%表达Cδ转录本，而其余60%表达的Cα信息比源自正常骨髓的克隆低10倍。
