Minato N, Hattori M, Sudo T, Kano S, Miura Y, Suda J, Suda T
Department of Medicine, Jichi Medical School, Tochigi, Japan.
J Exp Med. 1988 Mar 1;167(3):762-76. doi: 10.1084/jem.167.3.762.
Blast colonies were developed by rIL-3 from the spleen cells of mice pretreated with 5-fluorouracil (5-FU) in the methylcellulose cultures. When such IL-3-induced blast colonies were individually lifted up and recultured in the presence of rIL-3 and recombinant erythropoietin (rEpo), a variety of hematopoietic colonies developed from every single colony, including neutrophils, macrophages, eosinophils, megakaryocytes, mast cells, and erythroblasts. The results indicated that IL-3-induced blast colonies consisted of multipotential hematopoietic progenitor cells. By culturing individual IL-3-induced blast colonies in the presence of rIL-2 and irradiated peritoneal macrophages, on the other hand, the proliferation of homogeneous lymphoid cells was observed in 5 of 24 wells, each of which received a single blast colony. Morphologically, they were typical large granular lymphocytes (LGL), and thus it was indicated that LGL could be differentiated directly from the hematopoietic progenitor cells utterly in vitro by rIL-2 and accessory macrophages. From one of these culture wells, a continuous LGL line, IL3B1, was successfully obtained. The proliferation of IL3B1 was dependent on IL-2 in the presence of accessory macrophages, but they no longer responded to IL-3, nor to another T cell growth factor, IL-4. Flow cytometric analysis indicated that the phenotype of IL3B1 was Thy-1+,T3+,L3T4-,Lyt-2-,T200+ Asialo GM1+, whereas that of original IL-3-induced blast cells was Thy-1+,T3-,L3T4-,Lyt-2-,B220-. The results suggested that the expression of T3 molecules was induced in the process of LGL differentiation from the hematopoietic progenitor cells in vitro. Conforming to this, it was revealed that both gamma and beta chain genes of the TCR were rearranged in IL3B1. Northern blot analysis indicated that IL3B1 had abundant mRNA for gamma chain, while mRNA for beta chain was rather faint. Functionally, IL3B1 exhibited typical NK-patterned cytotoxic activity against a panel of tumor cell targets. In addition, they showed significant cytotoxic activity against normal bone marrow cells, as well as various factor-dependent myelogenous progenitor cell lines, all of which were resistant to endogenous NK activity of the fresh spleen cells. These results indicated that at least a set of T3+ LGL with rearranged TCR genes could be directly differentiated from isolated hematopoietic progenitor cells in vitro. Results also suggested that such a prethymically differentiated subset of T-lineage lymphocytes, namely T3+ double-negative LGL, had particular cytotoxic activity in addition to conventional NK activity, which might well contribute to feedback regulation of hematopoiesis.
在甲基纤维素培养体系中，用5-氟尿嘧啶（5-FU）预处理的小鼠脾细胞在重组白细胞介素-3（rIL-3）作用下形成了原始细胞集落。当将这些由IL-3诱导形成的原始细胞集落单独挑出，并在rIL-3和重组促红细胞生成素（rEpo）存在的情况下进行再培养时，每个集落都形成了多种造血集落，包括中性粒细胞、巨噬细胞、嗜酸性粒细胞、巨核细胞、肥大细胞和成红细胞。结果表明，IL-3诱导的原始细胞集落由多能造血祖细胞组成。另一方面，通过在rIL-2和经辐照的腹腔巨噬细胞存在的情况下培养单个IL-3诱导的原始细胞集落，在24个孔中的5个孔中观察到了均匀淋巴样细胞的增殖，每个孔接种了单个原始细胞集落。从形态学上看，它们是典型的大颗粒淋巴细胞（LGL），因此表明LGL可以在体外通过rIL-2和辅助巨噬细胞直接从造血祖细胞中分化出来。从其中一个培养孔中成功获得了连续的LGL系IL3B1。在有辅助巨噬细胞存在的情况下，IL3B1的增殖依赖于IL-2，但它们不再对IL-3以及另一种T细胞生长因子IL-4产生反应。流式细胞术分析表明，IL3B1的表型为Thy-1 +、T3 +、L3T4 -、Lyt-2 -、T200 +、无唾液酸GM1 +，而原始的IL-3诱导的原始细胞的表型为Thy-1 +、T3 -、L3T4 -、Lyt-2 -、B220 -。结果表明在体外从造血祖细胞分化为LGL的过程中诱导了T3分子的表达。与此相符的是，发现在IL3B1中TCR的γ链和β链基因均发生了重排。Northern印迹分析表明，IL3B1有丰富的γ链mRNA，而β链mRNA则相当微弱。在功能上，IL3B1对一组肿瘤细胞靶标表现出典型的NK模式的细胞毒活性。此外，它们对正常骨髓细胞以及各种因子依赖性髓系祖细胞系也表现出显著的细胞毒活性，而这些细胞对新鲜脾细胞的内源性NK活性具有抗性。这些结果表明，至少一组具有重排TCR基因的T3 + LGL可以在体外直接从分离的造血祖细胞中分化出来。结果还表明，这种胸腺前分化的T系淋巴细胞亚群，即T3 +双阴性LGL，除了具有传统的NK活性外，还具有特殊的细胞毒活性，这很可能有助于造血的反馈调节。
