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Differentiation of NK1.1⁺, Ly49⁺ NK Cells from flt3⁺ Multipotent Marrow Progenitor Cells¹

Noelle Sevilir Williams^2,*, Jennifer Klem^*,, Igor J. Puzanov^*,, P. V. Sivakumar^*,, Michael Bennett^* and Vinay Kumar^*

^* Department of Pathology and Graduate Program in Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75235

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
To delineate factors involved in NK cell development, we established an in vitro system in which lineage marker (Lin)^-, c-kit⁺, Sca2⁺ bone marrow cells differentiate into lytic NK1.1⁺ but Ly49^- cells upon culture in IL-7, stem cell factor (SCF), and flt3 ligand (flt3L), followed by IL-15 alone. A comparison of the ability of IL-7, SCF, and flt3L to generate IL-15-responsive precursors suggested that NK progenitors express the receptor for flt3L. In support of this, when Lin^-, c-kit⁺, flt3⁺ or Lin^-, c-kit⁺, flt3^- progenitors were utilized, 3-fold more NK cells arose from the flt3⁺ than from the flt3^- progenitors. Furthermore, NK cells that arose from flt3^- progenitors showed an immature NK1.1^dim, CD2^-, c-kit⁺ phenotype as compared with the more mature NK1.1^bright, CD2^+/-, c-kit^- phenotype displayed by NK cells derived from flt3⁺ progenitors. Both progenitors, however, gave rise to NK cells that were Ly49 negative. To test the hypothesis that additional marrow-derived signals are necessary for Ly49 expression on developing NK cells, flt3⁺ progenitors were grown in IL-7, SCF, and flt3L followed by culture with IL-15 and a marrow-derived stromal cell line. Expression of Ly49 molecules, including those of which the MHC class I ligands were expressed on the stromal or progenitor cells, as well as others of which the known ligands were absent, was induced within 6–13 days. Thus, we have established an in vitro system in which Ly49 expression on developing NK cells can be analyzed and possibly experimentally manipulated.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mature NK cells express a characteristic pattern of cell surface markers, including NK1.1, CD2, CD16, 2B4, and members of the Ly49 family of class I-binding receptors in the mouse (reviewed in Ref. 1) and CD56, CD16, and killer activatory (KAR)³ and killer inhibitory (KIR) class I-binding receptors in the human (2, 3). Class I interactions have been shown to both activate and inhibit NK cells depending on the Ly49 or KIR/KAR molecule engaged (4, 5, 6), although it is likely that other non-MHC receptors also regulate NK functional activity.

Despite strides in the understanding of NK cell specificity and function, the developmental sequence leading to generation of NK cells expressing a mature cell surface phenotype remains to be fully elucidated. Human CD34⁺ hematopoietic progenitor cells have been described that will give rise to CD56⁺ NK cells in vitro under the influence of IL-2 or IL-15 alone (7, 8) or in conjunction with other cytokines (8, 9, 10, 11, 12). Stem cell factor (SCF), for example, generally enhances the expansion of NK cells from CD34⁺ progenitors when used with IL-15 or IL-2, but alone it has no effects on NK differentiation. However, mice with mutations in SCF or its receptor do not show significant defects in generation of NK cells, perhaps because of redundancy with another factor (13, 14). Interestingly, flt3 ligand (flt3L) has strong homology to SCF, and both are produced by stromal cells (15, 16). Like SCF, flt3L can synergize with a number of other growth factors to support proliferation of primitive mouse and human hematopoietic progenitor cells (17, 18, 19, 20), and administration of flt3L in vivo causes expansion of NK cells (21, 22). Knockout mice lacking flt3L exhibit a deficiency with low to absent mature NK cells (23), an observation that warrants further analysis of the role of this cytokine in NK cell development.

We have previously identified a murine progenitor population characterized as lineage marker (Lin)^-, c-kit⁺, Sca2⁺ that is unable to respond efficiently to IL-15 alone (24). Preculture of these cells with IL-6, IL-7, SCF, and flt3L induces IL-15 responsiveness in a fraction of the treated cells, and subsequent culture in IL-15 leads to the generation of large numbers of NK1.1⁺ but Ly49^- cells. In this manuscript, a refined characterization of those cytokines that support NK cell generation in vitro is presented. These studies reveal a critical role for flt3L in NK cell differentiation. Additionally, a stromal cell-based system is presented that is capable of generating Ly49⁺ NK cells from bone marrow-derived progenitors.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals

C57BL/6 or (C57BL/6 x DBA.2)F₁ mice 7–12 wk old, bred at University of Texas Southwestern Medical Center, were used as the source of bone marrow progenitors. CB17.SCID mice obtained from Taconic Farms (Germantown, NY) were used as recipients for in vivo repopulation studies. The SCID mice were irradiated with 175 rad using a Mark I irradiator (J. L. Shepherd and Associates, San Fernando, CA) and were maintained under pathogen-free conditions with acidified water containing antibiotics (200 mg neomycin sulfate, 200 mg sulfamethoxazole, 50 mg trimethoprim, and 5.2 mg polymixin B in 500 ml H₂0, pH 2.5, all from Sigma, St. Louis, MO) for 2 wk followed by acidified water only for the remaining 1–2 wk.

Monoclonal Abs

Except as noted below, all mAbs and their isotype controls were obtained from PharMingen (San Diego, CA). Anti-Ly49G2 (4D11) and anti-Ly49D (4E5) were provided by Dr. J. Ortaldo (National Cancer Institute, Frederick, MD), anti-Ly49A (JR9-318) was the gift of Dr. J. Roland (Institut Pasteur, Paris, France), and anti-Sca2 hybridoma supernatant was provided by Dr. G. Spangrude (University of Utah, Salt Lake City, UT). Goat anti-rat Texas Red (Southern Biotechnology Associates, Birmingham, AL) or streptavidin-Red670 (Life Technologies, Grand Island, NY) was used to detect some primary Abs.

Stromal cells

OP9 stromal cells (25) were the kind gift of Dr. J.C. Zúñiga-Pflücker (University of Toronto, Ontario, Canada). The cells were passaged weekly in MEM (Life Technologies) containing 20% FCS, 200 nM glutamine, 100 µg/ml streptomycin sulfate, and 100 U/ml penicillin.

Cell preparation, isolation, and analysis of precursor cells

Lin^-, c-kit⁺, Sca2⁺ progenitors were isolated by a combination of magnetic bead depletion of Lin⁺ cells and cell sorting as previously described (24). Lin^-, c-kit⁺, flt3⁺ or flt3^- progenitors were isolated in a similar fashion. Analysis of cultured cells was performed by sequential staining with anti-FcIII/II (2.4G2), a biotinylated mAb, and finally streptavidin-Red670 plus a FITC- and/or PE-conjugated mAb.

In vitro culture conditions

Sorted Lin^-, c-kit⁺, Sca2⁺; Lin^-, c-kit⁺, flt3⁺; or Lin^-, c-kit⁺, flt3^- marrow cells were cultured in 96-well U-bottom plates (Falcon, San Jose, CA) at 10,000 cells/well in 0.2 ml of complete RPMI (RPMI 1640 containing 10% FBS, 100 µg/ml streptomycin sulfate, 100 U/ml penicillin, 1 mM sodium pyruvate, 2 mM glutamine, and 1x nonessential amino acids) and a mixture of 0.5 ng/ml murine IL (mIL)-7 (PeproTech, Rocky Hill, NJ), 30 ng/ml mouse SCF (BioSource, Camarillo, CA), and 100 U/ml murine flt3L (a gift from Dr. D. Rennick at DNAX, Palo Alto, CA). The cells were refed with the same media on day 3, and then on day 5 the cultures were harvested, washed, counted, and replated at 15,000/well in complete RPMI containing 30 ng/ml mIL-15 (a gift from Dr. T. Trout, Immunex, Seattle, WA), 150 ng/ml human IL-15 (R&D Systems, Minneapolis, MN), or 5000 IU/ml of recombinant human IL-2 (Chiron, Emeryville, CA). Cells grown on OP9 stroma were plated on day 5 at 200,000–500,000 cells per well in 24-well plates (Falcon) containing a confluent monolayer of OP9 stromal cells in IL-15- or IL-2-containing media. After an additional 3 days, the cultures were refed with the same media, and on day 11 or 12 of total culture time, the cells were harvested for analysis.

In vivo repopulation studies

Lymphocyte- and blast-sized (LB) Lin^-, c-kit⁺, flt3⁺, or flt3^- progenitors were sorted from (C57BL/6 x DBA.2)F₁ bone marrow as described above. The percentage of each population in whole bone marrow was calculated (% = fraction of LB cells x fraction of Lin^- cells in LB gate x fraction of c-kit⁺, flt3⁺ or c-kit⁺, flt3^- cells in LB, Lin^- gate x 100). The cells were washed two times with PBS and resuspended such that the equivalent of 1.4 x 10⁷ whole bone marrow cells for each progenitor was contained in 0.75 ml. CB17.SCID mice were irradiated with 175 rad 0.5–1 h before injection. The mice were then injected with 0.75 ml of cells via the tail vein and maintained as described above. Donor type cells were detected with anti-K^b FITC (PharMingen).

Western blot analysis

IL-2 cultured SCID NK cells (1–2 x 10⁶ cells) or NK cells derived from Lin^-, c-kit⁺, Sca2⁺ progenitor cells (10⁵ cells) were lysed in a HNTG lysis buffer (10% glycerol, 50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1 mM MgCl₂) with 1% Triton X-100 for 30 min on ice. Cleared lysates were separated on a 4–20% SDS-PAGE gel after addition of 2x sample buffer (50 mM Tris, pH 6.8, 2% SDS, 0.1% bromphenol blue, and 5% 2-ME). Separated proteins were transferred onto a nitrocellulose membrane. Western analysis was done after blocking the membrane in TBST (20 mM Tris and 500 mM NaCl) with 3% nonfat dry milk for 30 min. Primary anti-CD94-2 Ab (26) was added at a dilution of 1:200 in TBST for 1 h at room temperature, followed by the secondary Ab (donkey anti-rabbit IgG-HRP conjugate, 1:1000 dilution, Amersham Pharmacia Biotech, Piscataway, NJ) for 1 h. The membrane was developed in enhanced chemiluminescence developing solution (Amersham Pharmacia Biotech). A CD94-GST fusion protein generated in yeast was used as a control. Lysates from T cell lines EL-4 and Bw5147 did not show CD94 expression (data not shown).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Flt3L potently induces IL-15 responsiveness in Lin^–, c-kit⁺, Sca2⁺ progenitors

We previously identified a population of multipotent progenitors in mouse bone marrow characterized as Lin^- (B220, Gr1, Mac1, CD2, Ter119, and NK1.1), c-kit⁺, Sca2⁺, that gives rise to NK1.1⁺ cells in vitro (24). Although culture of these progenitors in mIL-15 alone led to generation of small numbers of lytic NK1.1⁺ cells, primary culture in a mixture of early acting cytokines, including IL-6, IL-7, SCF, and flt3L, for 5 days before secondary culture in mIL-15 significantly enhanced by 10- to 30-fold the number of NK cells generated. We inferred from these data that primary culture of the progenitor cells in early acting cytokines gave rise to a population of IL-15-responsive precursors that subsequently gave rise to NK cells on exposure to IL-15. Indeed, we previously demonstrated that, whereas the starting population of progenitors was IL-2/15Rß^-, 5–15% of the cells following the primary culture were IL-2/15Rß⁺ (24). More recently, we found that when the IL-2/15Rß⁺ and IL-2/15Rß^- cells generated in the primary culture were sorted and placed in culture in IL-15, only the IL-2/15Rß⁺ precursor cells gave rise to NK cells (data not shown). It was not clear from this analysis, however, which of the early acting cytokines was critical for expanding and/or inducing the differentiation of an IL-15-responsive cell that subsequently could give rise to NK1.1⁺ cells on culture in IL-15.

Early experiments in which IL-6 was or was not included indicated that, whereas IL-6 did increase cell yields during the primary culture, the yield of NK cells was lower overall. We hypothesize that this effect was due to the ability of IL-6 to stimulate differentiation and proliferation of non-NK progenitors. Therefore, subsequent cytokine comparisons were performed using only IL-7, SCF, and flt3L.

Progenitors were cultured for 5 days with SCF alone, flt3L alone, IL-7 + SCF, IL-7 + flt3L, or IL-7 + SCF + flt3L. To determine the relative ability of these various cytokine combinations to expand and/or induce the differentiation of an IL-15-responsive precursor, the cells were then washed and replated in mIL-15 for an additional 6 days. The fold expansion in mIL-15 on a per-cell basis was then calculated for each condition (Table I). Neither SCF nor flt3L alone generated cells that responded efficiently to IL-15. Although the addition of IL-7 to SCF did not appear to have an effect, the addition of IL-7 to flt3L enhanced the ability of the generated population to respond to IL-15 (p = 0.10, Mann-Whitney test (27)). This observation suggests that IL-7 may play an important role in the generation of IL-15-responsive NK precursors, at least for cells grown in the absence of SCF. However, cells cultured in IL-7 + flt3L were 2.5-fold better at responding to IL-15 than were cells grown in IL-7 + SCF. This difference, while not highly significant (p = 0.05, Mann-Whitney test), also suggests an important role for flt3L in NK development. In fact, culture in IL-7 + flt3L stimulated generation of a population of cells on day 5 that responded more efficiently to IL-15 than those populations generated by all the other cytokine combinations. Although the addition of SCF to IL-7 + flt3L did not improve the ability of the generated cells to respond to IL-15, it did improve the overall yield of NK cells, largely because of the additional proliferation induced during the primary culture before culture in IL-15 (data not shown). Taken together, these data imply that IL-7 and flt3L specifically expand or induce differentiation of IL-15-responsive NK precursors from Lin^-, c-kit⁺, Sca2⁺ progenitors. SCF has an additive effect on the overall expansion of this population of cells, but it does not affect the frequency with which IL-15-responsive NK precursors are generated.

It has been reported that flt3L^-/- mice lack NK cells, whereas IL-7^-/- or IL-7R^-/- mice show minimal defects in NK development (23, 28, 29). We chose, therefore, to focus initially on the role of flt3L in NK development. The ability of the Lin^-, c-kit⁺, Sca2⁺ population to respond to flt3L suggested that this population may express the receptor for flt3L (flt3). A four-color flow cytometry analysis revealed that 20–30% of Lin^-, c-kit⁺, Sca2⁺ cells express flt3 (Fig. 1). A similar proportion of the total Lin^-, c-kit⁺ population also express flt3. Because of the greater ease with which three-color as compared with four-color sorting can be performed, we chose to analyze the NK potential of LB Lin^-, c-kit⁺, flt3⁺ vs Lin^-, c-kit⁺, flt3^- populations, which represent respectively 0.2% and 1.0–1.5% of whole bone marrow. Subsequent experiments in which Lin^-, c-kit⁺, Sca2⁺, flt3⁺ or flt3^- populations were isolated, however, yielded similar results (data not shown).

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Identification of Lin-, c-kit+, flt3+ and Lin-, c-kit+, flt3- populations in murine bone marrow. Bone marrow cells were isolated from a C57BL/6 mouse and stained as indicated in Materials and Methods in the following order: anti-Sca2 supernatant; goat anti-rat Texas Red; normal rat serum; a mixture of lineage-specific (Lin) Abs, including anti-Gr1 (RB6-8C5), anti-B220 (RA3-6B2), anti-CD11b/Mac1 (M1/70), anti-CD2 (RM2-5), anti-NK1.1 (PK136), and anti-Ly-76 (Ter119); and finally streptavidin Red670 plus anti-c-kit FITC (2B8) and anti-flt3 PE (A2F10.1). The stained cells were analyzed on a FACStarPlus flow cytometer. Flt3 expression was analyzed on gated LB Lin-, c-kit+, Sca2+ cells (R1 gate) or LB Lin-, c-kit+ cells (R2 gate).

Lin^-, c-kit⁺, flt3⁺ and Lin^-, c-kit⁺, flt3^- cells were cultured in IL-7, SCF, and flt3L for 5 days followed by IL-15 alone for an additional 6 days. Significantly, flt3⁺ progenitors gave rise to, on average, 3-fold more NK cells on a per-cell basis than did flt3^- progenitors (Fig. 2A). This was due in large part to the fact that the population of NK precursors generated by culture of flt3⁺ progenitors in IL-7, SCF, and flt3L responded more efficiently to IL-15 on a per-cell basis to generate NK1.1⁺ cells than did the population of NK precursors generated in the same manner from flt3^- progenitors (Fig. 2B). To control for differences in NK generation due to stimulation of the flt3⁺ but not flt3^- progenitors with flt3L itself in the primary culture, we attempted to culture both populations with IL-7 and SCF alone followed by culture in IL-15. The flt3^- population gave rise to similar numbers of NK cells as when IL-7, SCF, and flt3L were utilized (data not shown). Surprisingly, however, the flt3⁺ population failed in two separate experiments to survive with IL-7 and SCF alone (data not shown). This suggests that somehow signals sent through flt3 are critical for the survival of Lin^-, c-kit⁺, flt3⁺ cells. SCF does, however, augment proliferation of these flt3⁺ cells in the presence of flt3L, because SCF + IL-7 + flt3L stimulated 2-fold more proliferation of Lin^-, c-kit⁺, flt3⁺ cells than IL-7 and flt3L alone (data not shown).

View larger version (31K): [in this window] [in a new window]   FIGURE 2. Lin-, c-kit+, flt3+ cells are more efficient at generating NK cells in vitro than Lin-, c-kit+, flt3- cells. Lin-, c-kit+, flt3+ and Lin-, c-kit+, flt3- progenitors were isolated from C57BL/6 bone marrow as indicated in Materials and Methods and plated in IL-7, SCF, and flt3L for 5 days followed by mIL-15 alone for an additional 6 days. In A, the number of NK cells generated per progenitor was calculated by multiplying the total cell yield after culture in IL-7, SCF, and flt3L and mIL-15 by the percentage of NK1.1+ cells in the final population. In B, the number of NK cells generated per NK precursor was then calculated by multiplying the fold increase in cell number in mIL-15 by the percentage of NK1.1+ cells. The results represent the mean ± SEM from four independent experiments.

Mature peripheral NK cells are NK1.1^bright, CD2⁺, and flt3^- (Ref. 30 and J. Klem and V. Kumar, unpublished observations). They are largely c-kit^-, although a small fraction is positive for this receptor (30). Interestingly, not only did flt3⁺ progenitors give rise to more NK cells, the cells derived from these progenitors more closely resembled mature NK cells than did the cells derived from flt3^- progenitors. NK cells derived from flt3^- progenitors lysed the prototypical NK target YAC-1 as well as or better than NK cells derived from flt3⁺ progenitors (data not shown). In addition, NK cells derived from both progenitors expressed NK1.1. However, the level of expression of the NK1.1 receptor on NK cells generated from the flt3^- progenitors was much lower than that on NK cells generated from the flt3⁺ cells (Fig. 3). Furthermore, the NK cells derived from flt3^- progenitors failed to express the lymphoid marker CD2 and continued to express c-kit. A significant fraction of the flt3⁺ progenitor-derived NK cells, on the other hand, did express CD2, and the cells were NK1.1^bright and c-kit^–/dim. Both populations of NK cells were negative for flt3.

View larger version (35K): [in this window] [in a new window]   FIGURE 3. Cell surface phenotype of NK cells generated from Lin-, c-kit+, flt3+ or Lin-, c-kit+, flt3- progenitors. Cells were stained as indicated in Materials and Methods. A, NK cells derived in vitro from Lin-, c-kit+, flt3+ progenitors. B, NK cells derived from Lin-, c-kit+, flt3- progenitors. Shaded curves represent staining with isotype control Abs; open curves represent staining with the indicated Abs. NK1.1 expression was examined on live gated cells, whereas expression of CD2, c-kit, and flt3 was examined on gated NK1.1+ live cells. The data are representative of staining patterns observed in two to six independent experiments.

View larger version (53K): [in this window] [in a new window]   FIGURE 6. Culture of Lin-, c-kit+, flt3+ cells on OP9 stroma induces expression of Ly49 molecules. Lin-, c-kit+, flt3+ or flt3- cells were cultured as described in Fig. 5. Cells were stained as described in Materials and Methods with Abs to NK1.1 (PK136), Ly49G2 (4D11), Ly49C/I/H (1F8), and Ly49C/I (5E6). A and C represent staining of NK cells derived in the presence of OP9 stroma from flt3+ and flt3- progenitors, respectively. B and D represent staining of NK cells derived from flt3+ and flt3- progenitors, respectively, in the absence of stroma. E shows a two-color analysis of 1F8 and 4Dll staining on gated NK1.1+ cells derived from culture of Lin-, c-kit+, flt3+ cells in IL-7, SCF, and flt3L followed by culture with IL-2 on OP9 stroma. The numbers indicated in each quadrant represent the percentage of positive cells minus the percentage of cells staining nonspecifically with isotype control mAbs. The data are representative of four separate experiments.

View larger version (33K): [in this window] [in a new window]   FIGURE 4. NK cells derived from Lin-, c-kit+, Sca2+ progenitors express CD94. IL-2-cultured SCID NK cells (1–2 x 106) or NK cells derived from Lin-, c-kit+, Sca2+ progenitors (105) were lysed, run on an SDS-PAGE gel, and blotted as described in Materials and Methods. CD94 was detected with a polyclonal rabbit anti-mouse CD94 Ab followed by a donkey anti-rabbit IgG-HRP conjugate. The membrane was developed in enhanced chemiluminescence developing solution. A CD94-GST fusion protein generated in yeast was used as a control.

To determine whether the ability of flt3⁺ and flt3^- progenitors to give rise to somewhat distinct NK1.1⁺ cells in vitro could be recapitulated in vivo under more physiologic conditions, Lin^-, c-kit⁺, flt3⁺ or flt3^- bone marrow progenitors were sorted from (C57BL/6 x DBA.2J)F₁ mice (H-2^b/d) and injected into 175 rad-treated CB17.SCID mice (H-2^d). Semiallogeneic donor cells were utilized so that their presence in the host could be detected with Abs to H-2^b MHC. The absence of T cells in the host prevents rejection of the allogeneic marrow cells, and NK-mediated rejection of (H-2^b/d)F₁ bone marrow cells by parental H-2^d NK cells has been shown to be insignificant (32). It should be noted that the numbers of flt3⁺ and flt3^- progenitor cells injected per mouse were 30,000 and 120,000, respectively. The relative number of each population utilized was calculated from the relative ratios of flt3⁺ and flt3^- cells in normal adult marrow (i.e., 1:4). The mice were then sacrificed on day 27 or 36 after cell transfer. Data from these two time points were pooled. Despite the fact that four times as many flt3^- cells were injected, the total number of splenic NK cells generated in mice injected with flt3⁺ cells was 2-fold higher than the number in mice injected with flt3^- cells. Thus, the differences in the NK-generating activity in flt3⁺ vs flt3^- cells in vitro could be reproduced in vivo as well. However, NK cells from both sets of mice expressed a completely mature phenotype: NK1.1^bright, CD2⁺, c-kit^-. Additionally, near-normal percentages of Ly49⁺ cells were detected (Table II). Thus, under more physiologic conditions both types of progenitors give rise to NK cells with a mature phenotype, although less efficiently from the flt3^- population.

Because both flt3⁺ and flt3^- progenitor cells gave rise to Ly49⁺ NK cells in vivo, we reasoned that their inability to generate Ly49⁺ cells in vitro could be due to inadequacies of the culture system. It is known that NK development in vivo is bone marrow dependent (33). We hypothesized, therefore, that additional signals from bone marrow-derived stromal cells may be necessary for complete NK maturation, including Ly49 induction. To provide an environment conducive to lymphoid development, we chose to utilize a stromal cell line, OP9, derived from the calvaria of a B6C3F2-op/op mouse deficient in the production of M-CSF (25). The haplotype of this stromal cell line is H-2^k (data not shown). Lin^-, c-kit⁺, flt3⁺ and Lin^-, c-kit⁺, flt3^- progenitors from C57BL/6 mice, grown for 5 days in IL-7, SCF, and flt3L, were thus plated on confluent monolayers of this OP9 stroma in the presence of human IL-15 or high-dose human IL-2 (5000 IU/ml). IL-2 and IL-15 were used interchangeably in these experiments, because we have noted that 5000 IU/ml human IL-2 gives results similar to those obtained with 150 ng/ml human IL-15 (data not shown). After 6 days of culture on stroma with IL-15 or IL-2, both populations gave rise to NK1.1^bright populations that expressed a high density of CD2, although a small fraction of the flt3^--derived NK cells were still CD2^–/dim at this time point (Fig. 5). Thus, stroma is able to provide the signals necessary to up-regulate CD2 on NK cells derived from flt3^- progenitors. However, using this protocol, only the NK cells derived from the flt3⁺ but not flt3^- population were induced to express Ly49 molecules (Fig. 6, A and C). Cells reactive with the Ab 4D11, known to detect Ly49G2 (34), and with the Ab 5E6, known to detect the C57BL/6 forms of Ly49C and Ly49I (35), were present. In addition, a subset of NK cells derived from flt3⁺ progenitors stained positive with an Ab, 1F8, recently generated in our laboratory and known to detect Ly49C, Ly49I, and Ly49H but not Ly49G2 or Ly49D (T. George and M. Bennett, unpublished data). In some experiments, such as the one shown in Fig. 6, there was some apparent staining of NK1.1^- cells with the 5E6 mAb. Although there is a remote possibility that this represents true expression of Ly49 molecules on NK1.1^- cells, we feel it more likely to be nonspecific sticking of the 5E6 mAb to residual stromal cells in the culture. First and most importantly, if it were true expression of Ly49C or Ly49I, one would expect to see a similar staining pattern with the 1F8 mAb, which is also known to detect Ly49C and Ly49I. Second, the 5E6⁺ NK1.1^- populations are less pronounced when cleaner preparations, having fewer residual stromal cells, are utilized (data not shown).

View larger version (30K): [in this window] [in a new window]   FIGURE 5. Culture of Lin-, c-kit+, flt3+ or flt3- cells on OP9 stroma induces expression of CD2. Lin-, c-kit+, flt3+ or flt3- cells were cultured for 5 days in IL-7, SCF, and flt3L. The cells were then washed and plated at 200,000–500,000 per well in 24-well plates containing a confluent monolayer of OP9 stroma in the presence of 150 ng/ml of human IL-15 or 5000 IU/ml human IL-2. Cells were stained as described in Materials and Methods for NK1.1 and CD2. The numbers indicated in each quadrant represent the percentage of positive cells minus the percentage of cells staining nonspecifically with isotype control mAbs. The data are representative of two separate experiments.

Generation of Ly49⁺ NK1.1⁺ cells from Lin^-, c-kit⁺ progenitors

Although coculture of Lin^-, c-kit⁺, flt3⁺ progenitors with OP9 stroma did yield NK1.1⁺, Ly49⁺ cells, the frequency of Ly49⁺ cells generated in vitro was low. Because in vivo transfer of this population gave rise to near-normal numbers of Ly49⁺ NK cells (Table II), we hypothesized that interactions with other cells, in addition to stroma, may be important for generation of NK1.1⁺, Ly49⁺ cells. Therefore, we decided not to separate flt3⁺ or flt3^- cells from the Lin^-, c-kit⁺ marrow population. Thus, Lin^-, c-kit⁺ cells were cultured with IL-7, SCF, and flt3L for 5 days as usual, followed by culture with IL-2 in the absence or presence of stromal cells. Cultures were harvested at 3, 6, 10, or 13 days after exposure to IL-2, and the frequency of NK1.1⁺ and Ly49⁺ cells was determined (Table III). By day 10, the frequency of NK1.1⁺ cells had reached close to 100% in the presence or absence of stromal cells. As expected, Ly49⁺ cells were generated only when the cells were cultured with stroma. However, as compared with cultures initiated with Lin^-, c-kit⁺, flt3⁺ cells, a substantially greater number of Ly49⁺ cells was generated. As with the expression of NK1.1, Ly49 molecules were first detected 3 days after culture with IL-2 and stroma, and the numbers increased rapidly to peak at 10 days postculture. Ly49 receptors reactive with four distinct Abs, including 4D11, 1F8, JR9-318, and 4E5, could be detected. The time course analysis revealed that some Ly49 molecules could be detected earlier than others. Thus, Ly49 receptors recognized by the mAbs 4D11 and 1F8 were noted as early as 3 days after culture on stroma, whereas Ly49 receptors detected by JR9-318 and 4E5 were not noted until day 6. While the frequency of 4D11⁺ and 1F8⁺ cells at later time points approached that seen on peripheral C57BL/6 NK cells, the frequency of 4E5⁺ and JR9-318⁺ cells did not. The reason for this discrepancy is not yet clear, but possible explanations are explored in Discussion.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We present here a culture system in which NK cell differentiation from multipotent stem cells to fully mature NK cells can be recapitulated. On the basis of these studies, we propose a two-step model of NK cell differentiation (Fig. 7). In the first step, multipotent stem cells containing NK cell progenitors are driven to become IL-2/15-responsive NK precursors under the influence of several early acting cytokines, especially flt3L. We hypothesize that this effect is due to induction of the IL-2/15Rß (CD122) chain on a fraction of the progenitor cells. This step can occur in the absence of stroma (at least in vitro). In the second step, the NK precursors, so generated, give rise to mature Ly49-expressing NK cells on being exposed to IL-2/15. In this step, the induction of Ly49 molecules but not NK1.1 is strictly dependent on the presence of stromal cells. Using this two-part culture system, we have investigated the mechanisms responsible for NK cell differentiation.

View larger version (41K): [in this window] [in a new window]   FIGURE 7. Proposed two-step model of NK differentiation. In the first step, a population of multipotent Lin-, c-kit+, flt3+ cells containing NK progenitors are driven to become IL-2/15Rß+ NK precursors under the influence of several early acting cytokines. The most important of these early acting cytokines is flt3L. In vivo, NK cells do not develop in flt3L-/- mice, and in our in vitro model, flt3+ progenitors die in the absence of flt3L. In the second step, the NK precursors, so generated, give rise to mature Ly49-expressing NK cells on being exposed to IL-15. (High doses of IL-2 can substitute for IL-15.) In this step, the induction of Ly49 molecules but not NK1.1 is strictly dependent on the presence of stromal cells.

Likewise, the importance of flt3L in NK development has been suggested by several recent reports. First, NK1.1^bright cells are missing in flt3L^-/- mice (23). Second, administration of recombinant flt3L induces a striking increase in the absolute number of mature nonactivated NK cells within various tissues (21, 22). This increase in NK cell numbers may provide a possible additional explanation for the potent antitumor effect of in vivo-administered flt3L (41, 42, 43), which was previously attributed solely to an increase in dendritic cells (44).

Although such observations suggest a significant potential for flt3L in therapy of both cancer and viral infection through expansion of NK cells, they do not address the mechanism by which this occurs. The data presented in this paper show that flt3L acts by expanding and/or inducing the differentiation of a progenitor population into IL-15-responsive precursors capable of giving rise to NK cells on in vitro culture in IL-15 (Fig. 7). The progenitor on which flt3L exerts its effect is identified in the mouse as Lin^-, c-kit⁺, flt3⁺. Somewhat surprisingly, in light of data from the flt3L^-/- mouse, the corresponding Lin^-, c-kit⁺, flt3^- population also gave rise to NK, albeit less efficiently than the flt3⁺ population (Fig. 2). An explanation for this discrepancy may lie in the phenotype of NK cells derived from each progenitor. NK cells derived from flt3^- progenitors showed a less mature NK1.1^dim, CD2^-, c-kit⁺ phenotype than NK cells derived from flt3⁺ progenitors, which were NK1.1⁺, CD2^+/-, c-kit^-. It is possible that in flt3L^-/- mice, Lin^-, c-kit⁺, flt3^- cells inefficiently give rise to NK cells. As these NK cells express quite dim levels of NK1.1, it could appear that flt3L^-/- mice lack NK1.1^bright cells as previously reported (23). It would be interesting to further phenotype any NK cells that develop in these mice for expression of CD2 and c-kit.

It should be noted, however, that immature NK1.1^dim, CD2^-, c-kit⁺ NK cells do not exist in the spleens of normal mice in vivo. Such NK cells may represent a developmental artifact that occurred because in vitro the flt3^- progenitors did not progress through a normal developmental pathway important for the up-regulation of NK1.1 and CD2 and down-regulation of c-kit. We therefore assessed the NK potential of flt3⁺ and flt3^- progenitors in vivo. Significantly, both flt3⁺ and flt3^- progenitors gave rise to NK cells with a mature CD2⁺, c-kit⁺, NK1.1^bright phenotype, albeit less efficiently from the flt3^- population. Thus, signals necessary to up-regulate NK1.1 and CD2 and to down-regulate c-kit were provided to the flt3^- progenitors in vivo. One possible explanation for the ability of flt3^- cells to give rise to mature NK cells in vivo but not in vitro is that flt3⁺ and flt3^- cells are in the same lineage pathway, with flt3^- cells being more immature than flt3⁺ cells. Thus, in vivo but not in vitro, flt3^- cells receive sufficient signals to complete their normal developmental pathway. In support of this, preliminary data have suggested that NK1.1⁺ cells are detectable in the peripheral blood of mice injected with flt3⁺ progenitors earlier than they are detectable in mice injected with flt3^- progenitors. Furthermore, flt3^- cells have a greater capacity than flt3⁺ cells to self-renew following serial transfers into irradiated recipients, again suggesting that flt3^- cells are more immature than flt3⁺ cells (N. S. Williams and V. Kumar, unpublished data). Because flt3L^-/- mice lack NK1.1^bright cells, it is possible that flt3^- progenitors normally must differentiate into flt3⁺ cells before entering the NK differentiation pathway and that interaction of flt3L with flt3⁺ cells is essential for the development of mature NK cells. In support of this, we found in vitro that flt3L was absolutely essential for the survival of Lin^-, c-kit⁺, flt3⁺ cells. These cells failed to survive when cultured in IL-7 and SCF alone. Together, these data reinforce the hypothesis that flt3L plays a critical role in NK cell differentiation and suggest an explanation for the NK cell-deficient phenotype of flt3L^-/- mice.

Although we have not pursued the observation here, it was interesting to note that IL-7 also seemed to play an important role in the development of IL-15-responsive precursors from Sca2⁺ progenitors. This is in contrast to the observation that IL-7R^-/- mice show no defects in NK cells. It is possible that in vivo, signaling through this receptor can be compensated for by other cytokines not present in our in vitro system. On the other hand, IL-7^-/- mice were found to have a 3-fold decrease in the absolute number of NK cells. It is therefore a formal possibility that IL-7 can exert its effects, at least on NK cells, independently of the IL-7R receptor. We are currently examining Lin^-, c-kit⁺, flt3⁺ NK progenitors for the presence of the IL-7R-chain in an effort to better understand the role of IL-7 in NK cell development.

In addition to allowing maturation of NK cells from both flt3⁺ and flt3^- populations to a NK1.1^bright, CD2^+, c-kit^- stage, in vivo transplantation also allowed for expression of Ly49 molecules on these NK cells. We show here for the first time that this environment can be recreated in vitro by growing flt3⁺ progenitors in IL-7, SCF, and flt3L and then placing them on a confluent monolayer of the bone marrow stromal cell line, OP9, in the presence of IL-15 or IL-2. The NK cells generated expressed Ly49 molecules reactive with three distinct Ly49 Abs, 4D11 (Ly49G2), 1F8 (Ly49C/I/H), and 5E6 (Ly49C/I). It is tempting to speculate, on the basis of the larger percentage of cells reactive with 1F8 vs 5E6, that Ly49H may be expressed on these cells, but definitive evidence of this awaits more extensive analysis. It is clear, however, that at least three distinct Ly49⁺ subsets are generated, because two-color analysis of 4D11 vs 1F8 revealed the presence of two single-positive populations in addition to a double-positive population.

Because the frequency of Ly49⁺ cells was lower than that observed on mature peripheral NK cells, we speculated that additional cellular interactions may be necessary for inducing the normal frequency of Ly49 molecules. Therefore, we utilized a less refined Lin^-, c-kit⁺ progenitor population to initiate the cultures. Interestingly, this strategy did yield a higher frequency of Ly49⁺ cells for reasons we do not yet understand. Using this protocol, Ly49 molecules reactive with four distinctive Abs were detectable. In addition, the time course analysis performed revealed that Ly49 molecules reactive with the mAbs 4D11 and 1F8 appeared at least 3 days earlier than molecules reactive with the mAbs 4E5 and JR9-318. Such findings mirror observations made of the onset of Ly49 expression on NK cells isolated from neonatal spleens. In these studies, expression of Ly49G2, as detected with 4D11, preceded expression of Ly49A (45, 46). It is tempting to speculate that subsets of Ly49 molecules may be expressed with different kinetics, although definitive evidence of such regulation awaits further analysis of individual Ly49 gene expression in developing NK cells. Finally, it is significant that at the earliest time points examined, individual Ly49⁺ NK cells could express more than one Ly49 molecule (Fig. 6E and data not shown). Several models have been put forth to explain regulation of the repertoire of Ly49 molecules in NK cells. In one model, NK cells stochastically express individual Ly49 molecules one at a time and continue to do so until an interaction of sufficient magnitude between an expressed Ly49 and self-class I MHC occurs (47). In a second model, individual NK cells begin to express one, two, or multiple Ly49 molecules at one time. Those NK cells that express at least one self-MHC-reactive Ly49 molecule but not so many self-reactive receptors as to be insensitive to virus- or tumor-induced changes in class I are then selected for expansion (47). Our data at this point do not allow us to distinguish between these two hypotheses. Neither model seems to adequately explain our observations, because we observe certain Ly49 molecules being expressed in concert (i.e., 4D11- and 1F8-reactive molecules), whereas others require additional time before detection (i.e., 4E5- and JR9-318-reactive molecules). Furthermore, the majority of Ly49⁺ NK cells fail to express Ly49A, the Ly49 molecule known to bind the H-2^k MHC of the OP9 stroma (48). Both models would predict the need for at least one self-reactive Ly49 receptor for survival and/or expansion. In this regard, it is interesting to note that both Ly49C/I (T. George and M. Bennett, unpublished data) and Ly49G2 (N. S. Williams and V. Kumar, unpublished data) can receive weak negative signals from H-2^k. If there are true kinetic differences in the expression pattern of the Ly49 molecules, with Ly49C/I and Ly49G2 being expressed before Ly49A and Ly49D, it is possible that the weak signals sent by H-2^k through Ly49G2 or Ly49C/I are sufficient in many cases to shut off further Ly49 expression. In this regard it is important to note that the progenitors remain in close proximity to the stromal cells during their entire development in vitro. Weak signals may be more effective here than in vivo, where developing NK cells may interact only briefly with stromal cells. These and other issues relating to the development of the Ly49 repertoire can now be addressed by utilizing progenitor and stromal cells of different MHC types and by interrupting contact of the developing NK cells with the stroma in this in vitro culture system.

In conclusion, our experiments have demonstrated a role for flt3L in expanding and/or inducing the differentiation of Lin^-, c-kit⁺, flt3⁺ progenitors into cells capable of responding to IL-15 to give rise to NK cells. The steps of NK differentiation from the flt3⁺ progenitor can be largely mimicked in vitro by culture with IL-7, SCF, and flt3L followed by culture with IL-15. However, induction of Ly49 is critically dependent on additional signals provided by bone marrow-derived stromal cells.

	Acknowledgments

 
We thank Tad George for help with in vivo experiments, Angie Mobley and Bonnie Darnell for all aspects of cell sorting, and Dr. John Schatzle for helpful discussions.

	Footnotes

 
¹ N.S.W. was supported by a postdoctoral fellowship from the Leukemia Research Foundation and by Individual National Research Service Award 1F32 CA76765-01 from the National Cancer Institute. This work was additionally supported by National Institutes of Health Grants AI20451, AI38938, CA36922, and CA70134.

² Address correspondence and reprint requests to Dr. Noelle S. Williams, Department of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9072. E-mail address:

³ Abbreviations used in this paper: KAR, killer activatory receptor; KIR, killer inhibitory receptor; Lin, lineage marker; SCF, stem cell factor; flt3L, flt3 ligand; LB, lymphocyte and blast sized; mIL, murine IL.

Received for publication March 12, 1999. Accepted for publication June 28, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Sivakumar, P. V., I. Puzanov, N. S. Williams, M. Bennett, and V. Kumar. 1998. Ontogeny and differentiation of murine natural killer cells and their receptors. In Specificity, Function, and Development of NK Cells, Vol. 230. K. Kärre and M. Colonna, eds. Springer-Verlag, Berlin. p. 161.
2. Lanier, L. L., C. Chang, M. Azuma, J. J. Ruitenberg, J. J. Hemperly, J. H. Phillips. 1991. Molecular and functional analysis of human natural killer cell-associated neural cell adhesion molecule (N-CAM/CD56). J. Immunol. 91:4421.
3. Lanier, L. L.. 1997. Natural killer cells: from no receptors to too many. Immunity 6:371.[Medline]
4. Karlhofer, F. M., R. Ribaudo, W. M. Yokoyama. 1992. MHC class I alloantigen specificity of Ly-49⁺ IL-2 activated natural killer cells. Nature 358:66.[Medline]
5. Moretta, A., S. Sivori, M. Vitale, D. Pende, L. Morelli, R. Augugliaro, J. C. Bottino, L. Moretta. 1995. Existence of both inhibitory (p58) and activatory (p50) receptors for HLA-C molecules in human natural killer cells. J. Exp. Med. 182:875.[Abstract/Free Full Text]
6. Mason, L. H., S. K. Anderson, W. M. Yokoyama, H. R. C. Smith, R. Winkler-Pickett, J. R. Ortaldo. 1996. The Ly-49D receptor activates murine natural killer cells. J. Exp. Med. 184:2119.[Abstract/Free Full Text]
7. Lotzova, E., C. A. Savary, R. E. Champlin. 1993. Genesis of human oncolytic natural killer cells from primitive CD34⁺CD33^- bone marrow progenitors. J. Immunol. 150:5263.[Abstract]
8. Mrozek, E., P. Anderson, M. A. Caligiuri. 1996. Role of interleukin-15 in the development of human CD56⁺ natural killer cells from CD34⁺ hematopoietic progenitor cells. Blood 87:2632.[Abstract/Free Full Text]
9. Shibuya, A., K. Nagayoshi, K. Nakamura, H. Nakauchi. 1995. Lymphokine requirement for the generation of natural killer cells from CD34⁺ hematopoietic progenitor cells. Blood 85:3538.[Abstract/Free Full Text]
10. Silva, M. R. G., R. Hoffman, E. F. Srour, J. L. Ascensao. 1994. Generation of human natural killer cells from immature progenitors does not require marrow stromal cells. Blood 84:841.[Abstract/Free Full Text]
11. Bennett, I. M., O. Zatsepina, L. Zamai, L. Azzoni, T. Mikheeva, B. Perussia. 1996. Definition of a natural killer NKR-P1A⁺/CD56^-/CD16^- functionally immature human NK cell subset that differentiates in vitro in the presence of interleukin 12. J. Exp. Med. 184:1845.[Abstract/Free Full Text]
12. Yu, H., T. A. Fehniger, P. Fuchshuber, K. S. Thiel, E. Vivier, W. E. Carson, M. A. Caligiuri. 1998. Flt3 ligand promotes the generation of a distinct CD34(+) human natural killer cell progenitor that responds to interleukin-15. Blood 92:3647.[Abstract/Free Full Text]
13. Seaman, W. E., N. Talal. 1981. Natural killing by spleen cells from W/W^v and Sl/Sl^d anemic mice. Exp. Hematol. 9:691.[Medline]
14. Jr Hackett, J., M. Bennett, V. Kumar. 1985. Origin and differentiation of natural killer cells. I. Characteristics of a transplantable NK cell precursor. J. Immunol. 134:3731.[Abstract]
15. Lyman, S. D., L. James, T. Vanden Bos, P. de Vries, K. Brasel, B. Gliniak, L. T. Hollingsworth, K. S. Picha, H. J. McKenna, R. R. Splett, et al 1993. Molecular cloning of a ligand for the flt3/fltk-2 tyrosine kinase receptor: a proliferative factor for primitive hematopoietic cells. Cell 75:1157.[Medline]
16. Lisovsky, M., S. E. Braun, Y. Ge, H. Takahira, L. Lu, V. G. Savchenko, S. D. Lyman, H. E. Broxmeyer. 1996. Flt3-ligand production by human bone marrow stromal cells. Leukemia 10:1012.[Medline]
17. Jacobsen, S. E., C. Okkenhaug, J. Mykleburst, O. P. Veiby, S. D. Lyman. 1995. The flt3 ligand potently and directly stimulates the growth and expansion of primitive murine bone marrow progenitor cells in vitro: synergistic interactions with interleukin (IL)-11, IL-12, and other hematopoietic growth factors. J. Exp. Med. 181:1358.
18. McKenna, H. J., P. de Vries, K. Brasel, S. D. Lyman, D. E. Williams. 1995. Effect of flt3 ligand on the ex vivo expansion of human CD34⁺ hematopoietic progenitor cells. Blood 9:3413.
19. Namikawa, R., M. O. Muench, J. E. de Vries, M.-G. Roncarolo. 1996. The FLK2/FLT3 ligand synergizes with interleukin-7 in promoting stromal-cell-independent expansion and differentiation of human fetal pro-B cells in vitro. Blood 5:1881.
20. Yonemura, Y., H. Ku, S. D. Lyman, M. Ogawa. 1997. In vitro expansion of hematopoietic progenitors and maintenance of stem cells: comparison between FLT3/FLK-2 ligand and KIT ligand. Blood 6:1915.
21. Brasel, K., H. J. McKenna, P. J. Morrissey, K. Charrier, A. E. Morris, C. C. Lee, D. E. Williams, S. D. Lyman. 1996. Hematologic effects of flt3 ligand in vivo in mice. Blood 88:2004.[Abstract/Free Full Text]
22. Shaw, S. G., A. A. Maung, R. J. Steptoe, A. W. Thomson, N. L. Vujanovic. 1998. Expansion of functional NK cells in multiple tissue compartments of mice treated with flt3-ligand: implications for anti-cancer and anti-viral therapy. J. Immunol. 161:2817.[Abstract/Free Full Text]
23. McKenna, H. J., R. E. Miller, K. Brasel, E. Maraskovsky, C. Maliszewski, B. Pulendran, D. Lynch, M. Teepe, E. R. Roux, J. Smith, et al 1996. Targeted disruption of the flt3 ligand gene in mice affects multiple hematopoietic lineages, including natural killer cells, B lymphocytes, and dendritic cells. Blood 88:474a. (Abstr.).
24. Williams, N. S., T. A. Moore, J. D. Schatzle, I. J. Puzanov, P. V. Sivakumar, A. Zlotnik, M. Bennett, V. Kumar. 1997. 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. J. Exp. Med. 186:1609.[Abstract/Free Full Text]
25. Kodama, H., M. Nose, S. Niida, S. Nishikawa, S.-I. Nishikawa. 1994. Involvement of the c-kit receptor in the adhesion of hematopoietic stem cells to stromal cells. Exp. Hematol. 22:979.[Medline]
26. Sivakumar, P. V., A. Gunturi, M. Salcedo, J. D. Schatzle, W. C. Lai, Z. Kurepa, L. Pitcher, M. S. Seaman, F. A. Lemonnier, M. Bennett, et al 1999. Expression of functional CD94/NKG2A Inhibitory receptors on fetal NK1.1⁺Ly-49^- cells: a possible mechanism of tolerance during NK cell development. J. Immunol. 162:6976.[Abstract/Free Full Text]
27. Siegel, S.. 1956. Nonparametric Statistics for the Behavioral Sciences McGraw-Hill Book Co., New York.
28. He, Y.-W., T. Malek. 1996. Interleukin-7 receptor is essential for the development of ⁺ T cells, but not for natural killer cells. J. Exp. Med. 184:289.[Abstract/Free Full Text]
29. Moore, T. A., U. von Freeden-Jeffry, R. Murray, A. Zlotnik. 1996. Inhibition of T cell development and early thymocyte maturation in IL-7^-/- mice. J. Immunol. 157:2366.[Abstract]
30. Williams, N. S., J. Klem, I. J. Puzanov, P. V. Sivakumar, J. D. Schatzle, M. Bennett, V. Kumar. 1998. Natural killer cell differentiation: insights from knockout and transgenic mouse models and in vitro systems. Immunol. Rev. 165:47.[Medline]
31. Mingari, M. C., C. Vitale, C. Cantoni, R. Bellomo, M. Ponte, F. Schiavetti, S. Bertone, A. Moretta, L. Moretta. 1997. Interleukin-15-induced maturation of human natural killer cells from early thymic precursors: selective expression of CD94/NKG2-A as the only HLA class I-specific inhibitory receptor. Eur. J. Immunol. 27:1374.[Medline]
32. Bennett, M.. 1972. Rejection of marrow allografts: importance of H-2 homozygosity of donor cells. Transplantation 14:289.[Medline]
33. Haller, O., H. Wigzell. 1977. Suppression of natural killer cell activity with radioactive strontium: effector cells are marrow dependent. J. Immunol. 118:1503.[Abstract/Free Full Text]
34. Mason, L. H., J. R. Ortaldo, H. A. Young, V. Kumar, M. Bennett, S. K. Anderson. 1995. Cloning and functional characteristics of murine large granular lymphocyte-1: a member of the Ly-49 gene family (Ly-49G2). J. Exp. Med. 182:293.[Abstract/Free Full Text]
35. Brennan, J., S. Lemieux, J. D. Freeman, D. L. Mager, F. Takei. 1996. Heterogeneity among Ly-49C natural killer (NK) cells: characterization of highly related receptors with differing functions and expression patterns. J. Exp. Med. 184:2085.[Abstract/Free Full Text]
36. Roland, J., P. A. Cazenave. 1992. Ly-49 antigen defines an ß TCR population of i-IEL with an extrathymic maturation. Int. Immunol. 4:699.[Abstract/Free Full Text]
37. Mason, L. H., J. Willette-Brown, S. K. Anderson, P. Gosselin, E. W. Shores, P. E. Love, J. R. Ortaldo, D. W. McVicar. 1998. Characterization of an associated 16-kDa tyrosine phosphoprotein required for Ly-49D signal transduction. J. Immunol. 160:4148.[Abstract/Free Full Text]
38. Puzanov, I. J., M. Bennett, V. Kumar. 1996. IL-15 can substitute for the marrow microenvironment in the differentiation of natural killer cells. J. Immunol. 157:4282.[Abstract]
39. Lodolce, J. P., D. L. Boone, S. Chai, R. E. Swain, T. Dassopoulos, S. Trettin, A. Ma. 1998. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity 9:669.[Medline]
40. Kennedy, M. K., M. B. Glaccum, K. A. Brasel, M. E. Embers, S. N. Brown, C. R. Willis, K. Charrier, P. J. Morrissey, S. Joyce, and J. J. Peschon. 1998. Impaired development of natural killer cells and natural T cells in IL-15-deficient mice. Proceedings of the 10th International Congress of Immunology. The Immunologist. Suppl. 1:159(Abstr.).
41. Chen, K., S. Braun, S. Lyman, Y. Fan, C. M. Traycoff, E. A. Wiebke, J. Gaddy, G. Sledge, H. E. Broxmeyer, K. Cornetta. 1997. Antitumor activity and immunotherapeutic properties of flt3-ligand in a murine breast cancer model. Cancer Res. 57:3511.[Abstract/Free Full Text]
42. Esche, C., V. M. Subbotin, C. Maliszewski, M. T. Lotze, M. R. Shurin. 1998. Flt3 ligand administration inhibits tumor growth in murine melanoma and lymphoma. Cancer Res. 58:380.[Abstract/Free Full Text]
43. Péron, J.-M., C. Esche, V. M. Subbotin, C. Maliszewski, M. T. Lotze, M. R. Shurin. 1998. Flt3-ligand administration inhibits liver metastases: role of NK cells. J. Immunol. 161:6164.[Abstract/Free Full Text]
44. Lynch, D. H.. 1998. Induction of dendritic cells (DC) by flt3 ligand (FL) promotes the generation of tumor-specific immune responses in vivo. Crit. Rev. Immunol. 18:99.[Medline]
45. Sivakumar, P. V., M. Bennett, V. Kumar. 1998. Fetal and neonatal NK1.1⁺Ly49^- cells can distinguish the presence and absence of class I on target cells: evidence for a Ly-49-independent negative signaling receptor. Eur. J. Immunol. 27:3100.
46. Dorfman, J. R., D. H. Raulet. 1998. Acquisition of Ly49 receptor expression by developing natural killer cells. J. Exp. Med. 187:609.[Abstract/Free Full Text]
47. Raulet, D. H., W. Held, I. Correa, J. R. Dorfman, M.-F. Wu, L. Corral. 1997. Specificity, tolerance and developmental regulation of natural killer cells defined by expression of class I-specific Ly49 receptors. Immunol. Rev. 155:41.[Medline]
48. Brennan, J., D. Mager, W. Jefferies, F. Takei. 1994. Expression of different members of the Ly-49 family defines distinct natural killer cell subsets and cell adhesion properties. J. Exp. Med. 180:2287.[Abstract/Free Full Text]

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				T. Kouro, V. Kumar, and P. W. Kincade Relationships between early B- and NK-lineage lymphocyte precursors in bone marrow Blood, November 15, 2002; 100(10): 3672 - 3680. [Abstract] [Full Text] [PDF]

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				O. Shimozato, J. R. Ortaldo, K. L. Komschlies, and H. A. Young Impaired NK Cell Development in an IFN-{gamma} Transgenic Mouse: Aberrantly Expressed IFN-{gamma} Enhances Hematopoietic Stem Cell Apoptosis and Affects NK Cell Differentiation J. Immunol., February 15, 2002; 168(4): 1746 - 1752. [Abstract] [Full Text] [PDF]

				B. Kunz and W. Held Positive and Negative Roles of the Trans-Acting T Cell Factor-1 for the Acquisition of Distinct Ly-49 MHC Class I Receptors by NK Cells J. Immunol., May 15, 2001; 166(10): 6181 - 6187. [Abstract] [Full Text] [PDF]

				A. B. Pawlowska, S. Hashino, H. McKenna, B. J. Weigel, P. A. Taylor, and B. R. Blazar In vitro tumor-pulsed or in vivo Flt3 ligand-generated dendritic cells provide protection against acute myelogenous leukemia in nontransplanted or syngeneic bone marrow-transplanted mice Blood, March 1, 2001; 97(5): 1474 - 1482. [Abstract] [Full Text] [PDF]

				Q. Wu, Y. Sun, J. Wang, X. Lin, Y. Wang, L. E. Pegg, A. Futterer, K. Pfeffer, and Y.-X. Fu Signal Via Lymphotoxin-{{beta}}R on Bone Marrow Stromal Cells Is Required for an Early Checkpoint of NK Cell Development J. Immunol., February 1, 2001; 166(3): 1684 - 1689. [Abstract] [Full Text] [PDF]

				T. A. Fehniger and M. A. Caligiuri Interleukin 15: biology and relevance to human disease Blood, January 1, 2001; 97(1): 14 - 32. [Full Text] [PDF]

				J. R. Ortaldo, R. Winkler-Pickett, and G. Wiegand Activating Ly-49D NK receptors: expression and function in relation to ontogeny and Ly-49 inhibitor receptors J. Leukoc. Biol., November 1, 2000; 68(5): 748 - 756. [Abstract] [Full Text]

				M. Lohoff, G. S. Duncan, D. Ferrick, H.-W. Mittrucker, S. Bischof, S. Prechtl, M. Rollinghoff, E. Schmitt, A. Pahl, and T. W. Mak Deficiency in the Transcription Factor Interferon Regulatory Factor (Irf)-2 Leads to Severely Compromised Development of Natural Killer and T Helper Type 1 Cells J. Exp. Med., August 7, 2000; 192(3): 325 - 336. [Abstract] [Full Text] [PDF]

				F. Colucci and J. P. Di Santo The receptor tyrosine kinase c-kit provides a critical signal for survival, expansion, and maturation of mouse natural killer cells Blood, February 1, 2000; 95(3): 984 - 991. [Abstract] [Full Text] [PDF]

				S. Sivori, M. Falco, E. Marcenaro, S. Parolini, R. Biassoni, C. Bottino, L. Moretta, and A. Moretta Early expression of triggering receptors and regulatory role of 2B4 in human natural killer cell precursors undergoing in vitro differentiation PNAS, April 2, 2002; 99(7): 4526 - 4531. [Abstract] [Full Text] [PDF]

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