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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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Ly-49 profile of NK cells generated from culture of Lin−,  c-kit+, Sca2+ progenitors. Expression of NK1.1 was analyzed by setting a  “live” gate based on forward and side scatter of the cells, whereas expression of the remainder of the markers was analyzed on NK1.1+, live cells.  Shaded curves represent staining with the NK1.1 or Ly49 mAbs, whereas  open curves represent staining with isotype control mAbs. The data are  representative of four separate experiments.
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Figure 3: Ly-49 profile of NK cells generated from culture of Lin−, c-kit+, Sca2+ progenitors. Expression of NK1.1 was analyzed by setting a “live” gate based on forward and side scatter of the cells, whereas expression of the remainder of the markers was analyzed on NK1.1+, live cells. Shaded curves represent staining with the NK1.1 or Ly49 mAbs, whereas open curves represent staining with isotype control mAbs. The data are representative of four separate experiments.

Mentions: The NK cells generated by culture of marrow progenitor cells were phenotypically quite similar to mature IL-2–activated splenic NK cells (12, 26, 27). They were CD3−, CD11b+, FcγRIII/II+, IL-2/15Rβ+, c-kit−, and 2B4+ (data not shown) and lysed the NK-sensitive target YAC-1 (Fig. 1). Interestingly, they also expressed gp49B1, an inhibitory receptor of the Ig superfamily shared by mast cells (28). However, they failed to express the Ly-49 family of MHC receptors (Fig. 3). In four experiments, expression of Ly-49A, C/I, and D was undetectable or <3% over isotype controls, whereas mature C57BL/6 splenic NK1.1+ cells are 20% Ly-49A+, 50% Ly-49C/I+, and 50% Ly-49D+. Expression of Ly-49G2 was slightly more variable ranging from 2–8% over isotype controls, whereas nearly 50% of splenic NK cells express this receptor. We have been unable to ascertain whether the in vitro–derived NK cells truly express low levels of Ly-49G2 or whether this antibody simply shows nonspecific binding. In any case, these data suggest that NK cells acquire expression of NK1.1 before expression of Ly-49 receptors. The factors required for induction of Ly-49 molecules are unknown, but it is clear that IL-15 alone is not sufficient.


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)

Ly-49 profile of NK cells generated from culture of Lin−,  c-kit+, Sca2+ progenitors. Expression of NK1.1 was analyzed by setting a  “live” gate based on forward and side scatter of the cells, whereas expression of the remainder of the markers was analyzed on NK1.1+, live cells.  Shaded curves represent staining with the NK1.1 or Ly49 mAbs, whereas  open curves represent staining with isotype control mAbs. The data are  representative of four separate experiments.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Ly-49 profile of NK cells generated from culture of Lin−, c-kit+, Sca2+ progenitors. Expression of NK1.1 was analyzed by setting a “live” gate based on forward and side scatter of the cells, whereas expression of the remainder of the markers was analyzed on NK1.1+, live cells. Shaded curves represent staining with the NK1.1 or Ly49 mAbs, whereas open curves represent staining with isotype control mAbs. The data are representative of four separate experiments.
Mentions: The NK cells generated by culture of marrow progenitor cells were phenotypically quite similar to mature IL-2–activated splenic NK cells (12, 26, 27). They were CD3−, CD11b+, FcγRIII/II+, IL-2/15Rβ+, c-kit−, and 2B4+ (data not shown) and lysed the NK-sensitive target YAC-1 (Fig. 1). Interestingly, they also expressed gp49B1, an inhibitory receptor of the Ig superfamily shared by mast cells (28). However, they failed to express the Ly-49 family of MHC receptors (Fig. 3). In four experiments, expression of Ly-49A, C/I, and D was undetectable or <3% over isotype controls, whereas mature C57BL/6 splenic NK1.1+ cells are 20% Ly-49A+, 50% Ly-49C/I+, and 50% Ly-49D+. Expression of Ly-49G2 was slightly more variable ranging from 2–8% over isotype controls, whereas nearly 50% of splenic NK cells express this receptor. We have been unable to ascertain whether the in vitro–derived NK cells truly express low levels of Ly-49G2 or whether this antibody simply shows nonspecific binding. In any case, these data suggest that NK cells acquire expression of NK1.1 before expression of Ly-49 receptors. The factors required for induction of Ly-49 molecules are unknown, but it is clear that IL-15 alone is not sufficient.

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