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Generation of lytic natural killer 1.1+, Ly-49- cells from multipotential murine bone marrow progenitors in a stroma-free culture: definition of cytokine requirements and developmental intermediates.

Williams NS, Moore TA, Schatzle JD, Puzanov IJ, Sivakumar PV, Zlotnik A, Bennett M, Kumar V - J. Exp. Med. (1997)

Bottom Line: Preculture in IL-6, IL-7, SCF, and flt3-L was necessary for inducing IL-15 responsiveness in the progenitors because the cells failed to significantly expand when cultured in IL-15 alone from the outset.Similar results were obtained with Lin-, CD44+, CD25-, c-kit+ lymphoid progenitors obtained from adult thymus.However, despite the apparent lack of these inhibitory MHC receptors, the NK cells generated could distinguish MHC class I+ from class I- syngeneic targets, suggesting the existence of novel class I receptors.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9072, USA. williams.n@pathology.swmed.edu

ABSTRACT
We have developed a stroma-free culture system in which mouse marrow or thymus cells, known to be enriched for lymphoid progenitors, can be driven to generate natural killer (NK) cells. Culture of lineage marker (Lin)-, c-kit+, Sca2+, interleukin (IL)-2/15Rbeta (CD122)- marrow cells in IL-6, IL-7, stem cell factor (SCF), and flt3 ligand (flt3-L) for 5-6 d followed by IL-15 alone for an additional 4-5 d expanded the starting population 30-40-fold and gave rise to a virtually pure population of NK1.1+, CD3- cells. Preculture in IL-6, IL-7, SCF, and flt3-L was necessary for inducing IL-15 responsiveness in the progenitors because the cells failed to significantly expand when cultured in IL-15 alone from the outset. Although culture of the sorted progenitors in IL-6, IL-7, SCF, and flt3-L for the entire 9-11-d culture period caused significant expansion, no lytic NK1.1+ cells were generated if IL-15 was not added, demonstrating a critical role for IL-15 in NK differentiation. Thus, two distinct populations of NK progenitors, IL-15 unresponsive and IL-15 responsive, have been defined. Similar results were obtained with Lin-, CD44+, CD25-, c-kit+ lymphoid progenitors obtained from adult thymus. The NK cells generated by this protocol lysed the NK-sensitive target YAC-1 and expressed markers of mature NK cells with the notable absence of Ly-49 major histocompatibility complex (MHC) receptors. However, despite the apparent lack of these inhibitory MHC receptors, the NK cells generated could distinguish MHC class I+ from class I- syngeneic targets, suggesting the existence of novel class I receptors.

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Expression of IL-15Rα messenger RNA and IL-2/ 15Rβ on Lin−, c-kit+, Sca2+ progenitors and an IL-15–responsive  population derived by culture of  the progenitors in IL-6, IL-7, SCF,  and flt3-L. (A) IL-15Rα message  levels were determined with RNA  isolated from sorted Lin−, c-kit+,  Sca2+ progenitors in two experiments (lanes 1 and 2) and from a  population of splenic NK cells (45%  NK1.1+ by flow cytometry) derived by culture of spleen cells in  500 U/ml of recombinant human  IL-2 for 4 d (lane 3). (B) IL-2/ 15Rβ expression was examined by  flow cytometry on gated Lin−,  c-kit+, Sca2+ cells using PE-labeled  anti–IL-2/15Rβ mAb TM-β1.  The data are representative of three  experiments. (C) Sorted Lin−,  c-kit+, Sca2+ cells, cultured for 5 d  in IL-6, IL-7, SCF, and flt3-L,  were stained for IL-2/15Rβ expression using FITC-labeled TM-β1. Shaded curves represent staining with TM-β1, whereas open  curves represent staining with isotype control mAbs. The data are  representative of two experiments.
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Figure 2: Expression of IL-15Rα messenger RNA and IL-2/ 15Rβ on Lin−, c-kit+, Sca2+ progenitors and an IL-15–responsive population derived by culture of the progenitors in IL-6, IL-7, SCF, and flt3-L. (A) IL-15Rα message levels were determined with RNA isolated from sorted Lin−, c-kit+, Sca2+ progenitors in two experiments (lanes 1 and 2) and from a population of splenic NK cells (45% NK1.1+ by flow cytometry) derived by culture of spleen cells in 500 U/ml of recombinant human IL-2 for 4 d (lane 3). (B) IL-2/ 15Rβ expression was examined by flow cytometry on gated Lin−, c-kit+, Sca2+ cells using PE-labeled anti–IL-2/15Rβ mAb TM-β1. The data are representative of three experiments. (C) Sorted Lin−, c-kit+, Sca2+ cells, cultured for 5 d in IL-6, IL-7, SCF, and flt3-L, were stained for IL-2/15Rβ expression using FITC-labeled TM-β1. Shaded curves represent staining with TM-β1, whereas open curves represent staining with isotype control mAbs. The data are representative of two experiments.

Mentions: To determine the ability of this “lymphoid-enriched” progenitor population to give rise to NK cells, sorted Lin−, c-kit+, Sca2+ cells were cultured with IL-15 for 9–11 d. However, these cells failed to expand significantly in IL-15 alone (Table 1). Cell recovery on average (n = 6) was only 1.8-fold over the input numbers of cells, but these cells did lyse the NK-sensitive tumor YAC-1 (Fig. 1). It is possible that a small number of the progenitors are truly capable of responding to IL-15 alone. We do not favor this hypothesis because cell surface expression of IL-2/15Rβ was not detected on the sorted population (Fig. 2 B), and the β chain is usually required for signal transduction (24). Alternatively, this growth may represent contamination by rare mature NK cells.


Generation of lytic natural killer 1.1+, Ly-49- cells from multipotential murine bone marrow progenitors in a stroma-free culture: definition of cytokine requirements and developmental intermediates.

Williams NS, Moore TA, Schatzle JD, Puzanov IJ, Sivakumar PV, Zlotnik A, Bennett M, Kumar V - J. Exp. Med. (1997)

Expression of IL-15Rα messenger RNA and IL-2/ 15Rβ on Lin−, c-kit+, Sca2+ progenitors and an IL-15–responsive  population derived by culture of  the progenitors in IL-6, IL-7, SCF,  and flt3-L. (A) IL-15Rα message  levels were determined with RNA  isolated from sorted Lin−, c-kit+,  Sca2+ progenitors in two experiments (lanes 1 and 2) and from a  population of splenic NK cells (45%  NK1.1+ by flow cytometry) derived by culture of spleen cells in  500 U/ml of recombinant human  IL-2 for 4 d (lane 3). (B) IL-2/ 15Rβ expression was examined by  flow cytometry on gated Lin−,  c-kit+, Sca2+ cells using PE-labeled  anti–IL-2/15Rβ mAb TM-β1.  The data are representative of three  experiments. (C) Sorted Lin−,  c-kit+, Sca2+ cells, cultured for 5 d  in IL-6, IL-7, SCF, and flt3-L,  were stained for IL-2/15Rβ expression using FITC-labeled TM-β1. Shaded curves represent staining with TM-β1, whereas open  curves represent staining with isotype control mAbs. The data are  representative of two experiments.
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Related In: Results  -  Collection

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Figure 2: Expression of IL-15Rα messenger RNA and IL-2/ 15Rβ on Lin−, c-kit+, Sca2+ progenitors and an IL-15–responsive population derived by culture of the progenitors in IL-6, IL-7, SCF, and flt3-L. (A) IL-15Rα message levels were determined with RNA isolated from sorted Lin−, c-kit+, Sca2+ progenitors in two experiments (lanes 1 and 2) and from a population of splenic NK cells (45% NK1.1+ by flow cytometry) derived by culture of spleen cells in 500 U/ml of recombinant human IL-2 for 4 d (lane 3). (B) IL-2/ 15Rβ expression was examined by flow cytometry on gated Lin−, c-kit+, Sca2+ cells using PE-labeled anti–IL-2/15Rβ mAb TM-β1. The data are representative of three experiments. (C) Sorted Lin−, c-kit+, Sca2+ cells, cultured for 5 d in IL-6, IL-7, SCF, and flt3-L, were stained for IL-2/15Rβ expression using FITC-labeled TM-β1. Shaded curves represent staining with TM-β1, whereas open curves represent staining with isotype control mAbs. The data are representative of two experiments.
Mentions: To determine the ability of this “lymphoid-enriched” progenitor population to give rise to NK cells, sorted Lin−, c-kit+, Sca2+ cells were cultured with IL-15 for 9–11 d. However, these cells failed to expand significantly in IL-15 alone (Table 1). Cell recovery on average (n = 6) was only 1.8-fold over the input numbers of cells, but these cells did lyse the NK-sensitive tumor YAC-1 (Fig. 1). It is possible that a small number of the progenitors are truly capable of responding to IL-15 alone. We do not favor this hypothesis because cell surface expression of IL-2/15Rβ was not detected on the sorted population (Fig. 2 B), and the β chain is usually required for signal transduction (24). Alternatively, this growth may represent contamination by rare mature NK cells.

Bottom Line: Preculture in IL-6, IL-7, SCF, and flt3-L was necessary for inducing IL-15 responsiveness in the progenitors because the cells failed to significantly expand when cultured in IL-15 alone from the outset.Similar results were obtained with Lin-, CD44+, CD25-, c-kit+ lymphoid progenitors obtained from adult thymus.However, despite the apparent lack of these inhibitory MHC receptors, the NK cells generated could distinguish MHC class I+ from class I- syngeneic targets, suggesting the existence of novel class I receptors.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9072, USA. williams.n@pathology.swmed.edu

ABSTRACT
We have developed a stroma-free culture system in which mouse marrow or thymus cells, known to be enriched for lymphoid progenitors, can be driven to generate natural killer (NK) cells. Culture of lineage marker (Lin)-, c-kit+, Sca2+, interleukin (IL)-2/15Rbeta (CD122)- marrow cells in IL-6, IL-7, stem cell factor (SCF), and flt3 ligand (flt3-L) for 5-6 d followed by IL-15 alone for an additional 4-5 d expanded the starting population 30-40-fold and gave rise to a virtually pure population of NK1.1+, CD3- cells. Preculture in IL-6, IL-7, SCF, and flt3-L was necessary for inducing IL-15 responsiveness in the progenitors because the cells failed to significantly expand when cultured in IL-15 alone from the outset. Although culture of the sorted progenitors in IL-6, IL-7, SCF, and flt3-L for the entire 9-11-d culture period caused significant expansion, no lytic NK1.1+ cells were generated if IL-15 was not added, demonstrating a critical role for IL-15 in NK differentiation. Thus, two distinct populations of NK progenitors, IL-15 unresponsive and IL-15 responsive, have been defined. Similar results were obtained with Lin-, CD44+, CD25-, c-kit+ lymphoid progenitors obtained from adult thymus. The NK cells generated by this protocol lysed the NK-sensitive target YAC-1 and expressed markers of mature NK cells with the notable absence of Ly-49 major histocompatibility complex (MHC) receptors. However, despite the apparent lack of these inhibitory MHC receptors, the NK cells generated could distinguish MHC class I+ from class I- syngeneic targets, suggesting the existence of novel class I receptors.

Show MeSH
Related in: MedlinePlus