Hattori M, Sudo T, Iizuka M, Kobayashi S, Nishio S, Kano S, Minato N
Basic Research Laboratories, Toray Industries, Inc., Kanagawa, Japan.
J Exp Med. 1987 Oct 1;166(4):833-49. doi: 10.1084/jem.166.4.833.
Continuous cell lines could be reproducibly established by culturing spleen cells from adult mice injected with MLV-producer cells or directly infected with Mo-MLV with rIL-2, whereas the culture of normal splenic cells with rIL-2 induced only transient and limited proliferation resulting in no such lines. All of the lines showed morphological characteristics as LGL with Thy-1+,Lyt-1-,L3T4-,Lyt-2-,AsGM1+,FcR gamma+ phenotype without exception, and most of them exhibited typical NK-patterned cytotoxicity. Analysis of reverse transcriptase activity of the culture supernatants as well as Southern hybridization of the DNA from the lines using an Mo-MLV-specific cDNA probe indicated no evidence of retroviral replication or proviral integration, suggesting that the generation of cell lines reflected a reactive process and viral infection was not directly responsible. It was subsequently revealed that Thy-1+,Lyt-1+,Lyt-2- spleen cells from mice infected with Mo-MLV in vivo spontaneously produced surprising amounts of IL-3 in vitro, leading to the possibility that IL-3 was responsible for the generation of lines. The possibility was directly supported by the observation that continuous lines with identical characteristics could be generated completely in vitro by sequential stimulation with rIL-3 and rIL-2 from normal spleen cells without any involvement of Mo-MLV. The C beta gene of TCR was shown to be rearranged in all the lines examined, indicating the LGL lines were all genetically committed to T cell lineage. Unlike the situation in normal splenic populations expanded by rIL-2, where the expression of IL-2-R was progressively lost, constitutive expression of high-affinity-IL-2-R was observed in all the lines and thus, this was considered to explain the unlimited proliferation of them in response to rIL-2 alone. These results suggested the probable role of IL-3 in the regulation of growth and differentiation of a set of LGL committed to T cell lineage. The possible implications of the phenomenon in the regulation of hematopoiesis as well as in the control of Mo-MLV-induced leukemogenesis were discussed.
通过培养来自注射了莫洛尼鼠白血病病毒（Mo-MLV）产生细胞的成年小鼠的脾细胞，或直接用重组白细胞介素-2（rIL-2）感染莫洛尼鼠白血病病毒（Mo-MLV）的脾细胞，可重复性地建立连续细胞系，而用rIL-2培养正常脾细胞仅诱导短暂且有限的增殖，不会产生此类细胞系。所有细胞系均无一例外地表现出具有Thy-1+、Lyt-1-、L3T4-、Lyt-2-、AsGM1+、FcRγ+表型的大颗粒淋巴细胞（LGL）的形态特征，且大多数细胞系表现出典型的自然杀伤（NK）模式的细胞毒性。对培养上清液的逆转录酶活性分析以及使用Mo-MLV特异性cDNA探针进行的细胞系DNA的Southern杂交表明，没有逆转录病毒复制或前病毒整合的证据，这表明细胞系的产生反映了一个反应过程，病毒感染并非直接原因。随后发现，体内感染Mo-MLV的小鼠的Thy-1+、Lyt-1+、Lyt-2-脾细胞在体外自发产生了惊人数量的白细胞介素-3（IL-3），这使得IL-3可能是细胞系产生的原因。用rIL-3和rIL-2对正常脾细胞进行连续刺激，在完全没有Mo-MLV参与的情况下，能在体外完全产生具有相同特征的连续细胞系，这一观察结果直接支持了这一可能性。在所检测的所有细胞系中，T细胞受体（TCR）的Cβ基因均发生了重排，表明LGL细胞系均在基因上定向于T细胞谱系。与用rIL-2扩增的正常脾细胞群体不同，在正常脾细胞群体中IL-2受体（IL-2-R）的表达会逐渐丧失，而在所有细胞系中均观察到高亲和力IL-2-R的组成性表达，因此，这被认为解释了它们仅对rIL-2产生无限增殖的原因。这些结果表明IL-3可能在调控一组定向于T细胞谱系的LGL的生长和分化中发挥作用。讨论了该现象在造血调控以及Mo-MLV诱导的白血病发生控制中的可能意义。
