Differentiation in vitro of T3+ large granular lymphocytes with characteristic cytotoxic activity from an isolated hematopoietic progenitor colony

N. Minato, M. Hattori, T. Sudo, S. Kano, Y. Miura, J. Suda, T. Suda

Research output: Contribution to journalArticle

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Abstract

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 homogenous 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. Flowcytometric 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 γ and β chain genes of the TCR were rearranged in IL3B1. Northern blot analysis indicated that IL3B1 had abundant mRNA for γ chain, while mRNA for β chain was rather faint. Functionally, IL3B1 exhibited typical NK-patterned cytotoxic activity against a panel for 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.

Original languageEnglish
Pages (from-to)762-776
Number of pages15
JournalJournal of Experimental Medicine
Volume167
Issue number3
Publication statusPublished - 1988

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Interleukin-3
Lymphocytes
Hematopoietic Stem Cells
Macrophages
Interleukin-2
Spleen
Erythroblasts
Messenger RNA
Methylcellulose
Megakaryocytes
Hematopoiesis
Peritoneal Macrophages
T-Lymphocyte Subsets
Erythropoietin
In Vitro Techniques
Eosinophils
Mast Cells
Fluorouracil
Bone Marrow Cells
Interleukin-4

ASJC Scopus subject areas

  • Immunology

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Differentiation in vitro of T3+ large granular lymphocytes with characteristic cytotoxic activity from an isolated hematopoietic progenitor colony. / Minato, N.; Hattori, M.; Sudo, T.; Kano, S.; Miura, Y.; Suda, J.; Suda, T.

In: Journal of Experimental Medicine, Vol. 167, No. 3, 1988, p. 762-776.

Research output: Contribution to journalArticle

Minato, N. ; Hattori, M. ; Sudo, T. ; Kano, S. ; Miura, Y. ; Suda, J. ; Suda, T. / Differentiation in vitro of T3+ large granular lymphocytes with characteristic cytotoxic activity from an isolated hematopoietic progenitor colony. In: Journal of Experimental Medicine. 1988 ; Vol. 167, No. 3. pp. 762-776.
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abstract = "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 homogenous 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. Flowcytometric 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 γ and β chain genes of the TCR were rearranged in IL3B1. Northern blot analysis indicated that IL3B1 had abundant mRNA for γ chain, while mRNA for β chain was rather faint. Functionally, IL3B1 exhibited typical NK-patterned cytotoxic activity against a panel for 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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T1 - Differentiation in vitro of T3+ large granular lymphocytes with characteristic cytotoxic activity from an isolated hematopoietic progenitor colony

AU - Minato, N.

AU - Hattori, M.

AU - Sudo, T.

AU - Kano, S.

AU - Miura, Y.

AU - Suda, J.

AU - Suda, T.

PY - 1988

Y1 - 1988

N2 - 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 homogenous 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. Flowcytometric 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 γ and β chain genes of the TCR were rearranged in IL3B1. Northern blot analysis indicated that IL3B1 had abundant mRNA for γ chain, while mRNA for β chain was rather faint. Functionally, IL3B1 exhibited typical NK-patterned cytotoxic activity against a panel for 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.

AB - 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 homogenous 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. Flowcytometric 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 γ and β chain genes of the TCR were rearranged in IL3B1. Northern blot analysis indicated that IL3B1 had abundant mRNA for γ chain, while mRNA for β chain was rather faint. Functionally, IL3B1 exhibited typical NK-patterned cytotoxic activity against a panel for 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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