Limits...
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.

Show MeSH

Related in: MedlinePlus

Lytic activity of Lin−, c-kit+, Sca2+ cells cultured with a  cocktail of early acting cytokines ± IL-15. Sorted cells were cultured in  the indicated cytokines as described in Materials and Methods. Cytotoxic  activity was assessed on YAC-1 tumor cells. The data are representative of  six experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199105&req=5

Figure 1: Lytic activity of Lin−, c-kit+, Sca2+ cells cultured with a cocktail of early acting cytokines ± IL-15. Sorted cells were cultured in the indicated cytokines as described in Materials and Methods. Cytotoxic activity was assessed on YAC-1 tumor cells. The data are representative of six 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)

Lytic activity of Lin−, c-kit+, Sca2+ cells cultured with a  cocktail of early acting cytokines ± IL-15. Sorted cells were cultured in  the indicated cytokines as described in Materials and Methods. Cytotoxic  activity was assessed on YAC-1 tumor cells. The data are representative of  six experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199105&req=5

Figure 1: Lytic activity of Lin−, c-kit+, Sca2+ cells cultured with a cocktail of early acting cytokines ± IL-15. Sorted cells were cultured in the indicated cytokines as described in Materials and Methods. Cytotoxic activity was assessed on YAC-1 tumor cells. The data are representative of six 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