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.

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