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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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Lysis of syngeneic Tap+/+ and Tap−/− tumor cells by in vitro  generated NK cells. Target cells were class I+ RMA (Tap+/+) and Q11  (Tap+/+) and class Ilo RMA-S (Tap−/−). The data are representative of  three separate experiments.
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Figure 4: Lysis of syngeneic Tap+/+ and Tap−/− tumor cells by in vitro generated NK cells. Target cells were class I+ RMA (Tap+/+) and Q11 (Tap+/+) and class Ilo RMA-S (Tap−/−). The data are representative of three separate experiments.

Mentions: It has been demonstrated that interaction of Ly-49 receptors with their appropriate MHC class I ligands sends an inhibitory signal to NK cells and prevents NK-mediated lysis of the class I+ target (1). However, despite the failure to express significant levels of Ly-49 MHC receptors, these in vitro–derived NK cells differentiated class I+ from class I− syngeneic tumor cells. They lysed class Ilo Tap-deficient RMA-S cells but failed to lyse RMA, the class I+ parent of RMA-S (Fig. 4). That this difference in lysis was due to the absence of class I on the surface of RMA-S is supported by the observation that the culture-derived NK cells failed to lyse Q11, a class I+ Tap-2 transfectant of RMA-S (16).


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)

Lysis of syngeneic Tap+/+ and Tap−/− tumor cells by in vitro  generated NK cells. Target cells were class I+ RMA (Tap+/+) and Q11  (Tap+/+) and class Ilo RMA-S (Tap−/−). The data are representative of  three separate experiments.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Lysis of syngeneic Tap+/+ and Tap−/− tumor cells by in vitro generated NK cells. Target cells were class I+ RMA (Tap+/+) and Q11 (Tap+/+) and class Ilo RMA-S (Tap−/−). The data are representative of three separate experiments.
Mentions: It has been demonstrated that interaction of Ly-49 receptors with their appropriate MHC class I ligands sends an inhibitory signal to NK cells and prevents NK-mediated lysis of the class I+ target (1). However, despite the failure to express significant levels of Ly-49 MHC receptors, these in vitro–derived NK cells differentiated class I+ from class I− syngeneic tumor cells. They lysed class Ilo Tap-deficient RMA-S cells but failed to lyse RMA, the class I+ parent of RMA-S (Fig. 4). That this difference in lysis was due to the absence of class I on the surface of RMA-S is supported by the observation that the culture-derived NK cells failed to lyse Q11, a class I+ Tap-2 transfectant of RMA-S (16).

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