We have established a clonal cell culture system that supports the proliferation of committed natural killer (NK) cell progenitors of mice to investigate the pathway and cytokine regulation of NK cell development. Day 14 fetal thymocytes cultured in methylcellulose with interleukin-7 (IL-7), IL-15, and steel factor (SF ) formed diffuse colonies that could not be classified to known colony types. Single-cell origin of the colonies was established by micromanipulation of the colony-forming cells. Cells in the colonies are very blastic, showing no cytoplasmic differentiation, and express Ly5, Thy-1, and CD25 but not myeloid, B, mature T, or NK cell markers. The cells lack T, B, and myeloid potentials but can differentiate to mature NK cells in fetal thymus organ culture, suggesting that the colonies consist of NK committed progenitors. Examination of the minimal cytokine requirement for the NK colony formation showed that IL-7 and SF are indispensable for the formation of immature NK cell colonies. Both IL-2 and IL-15 increased the frequency of colonies. In contrast to IL-2, IL-7, and IL-15, IL-4 strongly inhibited the formation of the colonies. This quantitative clonal culture will provide a useful means to examine the mechanism of NK cell development.

NATURAL KILLER (NK) cells are large granular lymphocytes that appear to be distinct from T or B lymphocytes in both morphology and function. NK cells mediate the killing of certain tumor cells and virally infected cells in non–major histocompatability complex (MHC)-restricted manners.1,2 They are also known to mediate the rejection of donor cells in bone marrow transplantation.3 Although the importance of NK cells has been established in various immune responses, their ontogeny and regulation of development are largely unknown.

NK cells and T lymphocytes share a number of features, including surface markers, growth factor requirements, and functions.4-6 Recently several groups of investigators reported that NK cells are also related to T cells in their developmental pathways. In humans, NK/T common precursors have been reported to be present in fetal thymus.7 In mice, a certain population of thymocytes has been shown to have a potential to develop into both NK and T cells, suggesting that NK/T common precursors also exist in murine thymus.8 9 However, it still remains to be clarified how NK or NK/T common progenitors develop from multipotential hematopoietic progenitor cells and how and when NK/T common progenitors diverge to form separate NK and T-cell lineages.

The cytokine regulation of the growth and survival of early NK precursor cells also needs to be elucidated. Recently, mice lacking the common γ-chain (the common receptor component of interleukin-2 [IL-2], IL-4, IL-7, IL-9, and IL-15 receptors)10-16 were established and found to lack NK cells.17 18 This observation strongly suggested that cytokines which use the common γ-chain as a receptor component play important roles in regulating the growth and differentiation of NK cells. However, it still remains to be determined which cytokines are critical for NK cell development and how these cytokines interact to regulate the growth of NK cell progenitors. The roles of cytokines that do not use the common γ-chain also remain to be clarified.

To examine the developmental pathway and cytokine requirement of NK cells in more detail, it is necessary to establish a clonal culture system that supports the proliferation of NK cell progenitors without stromal cells. Here we report that murine fetal thymocytes plated in methylcellulose culture form immature NK cell colonies in the presence of IL-7, IL-15, and steel factor (SF, c-kit ligand). Both IL-7 and SF were found to be indispensable, and IL-2 and IL-15 increased the frequency of the immature NK cell colonies. IL-4, which also uses the common γ-chain as a receptor component, strongly inhibited the formation of immature NK colonies. We believe that this quantitative and clonal cell culture system is useful for analyzing the developmental pathway and cytokine regulation of NK cell progenitors.

Mice and cell preparation.Female C57BL/6-Ly5.2 and male DBA/2 mice were purchased from Charles River (Raleigh, NC). Male C57BL/6-Ly5.1 congenic mice were purchased from Jackson Laboratories (Bar Harbor, ME). To obtain fetal thymocytes, female C57BL/6-Ly5.2 mice were mated with male DBA/2 or C57BL/6-Ly5.1 congenic mice for 18 hours. The day of vaginal plugging was designated as day 0 of gestation. Cell suspension of fetal thymocytes was prepared by gently pressing the thymus lobes between two slide glasses and by repeated pipetting.

Cytokines.Purified human recombinant IL-2, murine recombinant IL-13, and murine recombinant IL-3 were purchased from R&D system (Minneapolis, MN). Murine IL-4 and human IL-7 were provided by Sanofi Winthorp, Inc (Malven, PA). Purified simian recombinant IL-15 and recombinant murine SF were gifts from Elaine K. Thomas of Immunex (Seattle, WA). Purified recombinant human IL-11 was provided by P. Schendel of Genetic Institute (Cambridge, MA). Purified human erythropoietin (Epo) was a gift from Genetic Institute (Cambridge, MA). Unless otherwise specified, concentrations of cytokines used were as follows: IL-2, 100 ng/mL; IL-3, 10 ng/mL; IL-4, 10 ng/mL; IL-7, 200 U/mL; IL-13, 10 ng/mL or 50 ng/mL; IL-11, 100 ng/mL; IL-15, 100 ng/mL; SF, 100 ng/mL; Epo; 2 U/mL.

Monoclonal antibodies (MoAbs).The following MoAbs were used for flow cytometric analysis and cell sorting: biotin-conjugated-anti-CD3 (clone; 145-2C11) (Pharmingen, San Diego, CA),19 fluorescein isothiocyanate (FITC)-conjugated-anti-CD4 (YTS 191.1) (Caltag Laboratories, South San Francisco, CA), phycoerythrin (PE)-conjugated-anti-CD8 (YTS 169.4) (Caltag), PE-conjugated-anti-CD25 (PC61.5.3) (Caltag),20 FITC-conjugated-anti-Thy1.2 (30-H12) (Pharmingen),21 biotin-conjugated-anti-NK1.1 (PK136) (Pharmingen),22 PE-conjugated-anti-B220 (RA3-6B2) (Pharmingen),23 biotin-conjugated-anti-c-kit (ACK4),24 biotin-conjugated-anti-Mac-1 (M1/70),25 biotin-conjugated-anti-Gr-1 (RB6-8C5) (Pharmingen),26 biotin-conjugated-anti-TER (TER119) (Pharmingen),27 FITC-conjugated-anti-Ly5.1 (A20-1.7; kindly provided by Dr H. Fleming of Emory University, Atlanta, GA), FITC-conjugated-anti-Ly5 (30F11.1).28 

Flow cytometric analysis and cell sorting.For direct staining of cells with FITC or PE-conjugated antibodies, 2 × 105 cells were incubated with appropriate dilutions of MoAbs for 20 minutes on ice. For indirect staining of cells with biotin-conjugated antibodies, 2 × 105 cells were first incubated with biotin-conjugated antibodies on ice for 20 minutes, then followed by staining with FITC-conjugated streptavidin (Caltag) for 15 minutes on ice. In all experiments, cells stained with appropriate isotype-matched control Igs were also prepared as negative controls. Cells were washed once with phosphate-buffered saline (PBS) supplemented with 1% deionized fraction-V bovine serum albumin (BSA) (Sigma, St Louis, MO), and analyzed and/or sorted using FACS Vantage (Becton Dickinson, Mountain View, CA).

Fetal thymus organ culture.Fetal thymic organ culture was performed as described by Jenkinson et al29 and Kingston et al30 with slight modifications. Thymic lobes from day 14 BDF1 fetuses were individually plated in the wells of a Terasaki plate (Nunc, Kamstrup, Denmark). Thirty microliters of medium containing 1 × 105 test cells was added to each well. The plate was then inverted to allow a lobe and cells to contact at the bottom of a hanging drop. After 20 hours of incubation at 37°C, the lobes were transferred individually onto filter membranes (Costar, Cambridge, MA; pore size, 8 μm) floating on the surface of medium using a pasteur pipette and cultured for 8 days. Cells were recovered from the lobes, stained with antibodies, and analyzed by flow cytometry. In some experiments, NK1.1+ Ly5.1+ cells were prepared from fetal thymus organ culture by sorting using FACS Vantage. Medium used for fetal thymic organ culture was RPMI 1640 (Sigma) supplemented with L-glutamine (2 mmol/L), nonessential amino acids (1 mmol/L; GIBCO Laboratories, Grand Island, NY), 2-mercaptoethanol (2-ME, 0.1 mmol/L; Sigma), and 10% fetal calf serum (FCS; Intergen, Purchase, NY).

Clonal culture for immature NK cell colonies.Methylcellulose culture was performed using 35-mm Falcon suspension culture dishes (Becton Dickinson Labware, Lincoln Park, NJ). Whole or fractionated fetal thymocytes were cultured in the medium consisting of α-medium (Flow Laboratories, Rockville, MD), 1.2% 1,500-cp methylcellulose (Shinetsu Chemical, Tokyo, Japan), 5% FCS, 1% deionized fraction-V BSA, and 0.1 mmol/L 2-ME in the presence of designated cytokines. Dishes were incubated at 37°C in a humidified atmosphere flushed with 5% CO2 for 14 days. In some experiments, c-kit+CD25 cells were individually plated in culture by micromanipulation as described previously.31 

Clonal culture for myeloid and pre-B cells.Basic methods of culture for myeloid and pre-B cells have been described previously.31 32 Briefly, cells were cultured in 35-mm Falcon suspension culture dishes containing α-medium, 1.2% 1,500-cp methylcellulose, 30% FCS, 1% deionized fraction-V BSA, and 0.1 mmol/L 2-ME. For culture of myeloid lineage cells, IL-3, IL-11, and Epo were added to the medium. For pre-B cell culture, IL-7, IL-11, and SF were added to the medium. Dishes were incubated at 37°C in a humidified atmosphere flushed with 5% CO2 for 14 days.

Cytolytic assay.Ly5.1+NK1.1+ cells were prepared by sorting with FACS Vantage from the fetal thymus organ culture and cultured in the presence of IL-2 (500 ng/mL; 500 U/mL) for 7 days. Cells were washed twice and used as effector cells. As control effector cells, immature NK cell colonies were individually picked, pooled, and washed twice. NK-sensitive YAC-1 cells were obtained from American Type Culture Collection (Rockville, MD). YAC-1 cells, 1 × 106, were labeled with 100 μCi of sodium chromate (51Cr) for 2 hours at 37°C. Cells were washed three times and plated in the wells of round-bottomed 96-well plates at the concentration of 2,000 cells/100 μL. Varying numbers of effector cells in a volume of 100 μL were added to the target cells and the plates were incubated at 37°C for 4 hours. The plates were then centrifuged once and the radioactivity of 100 mL of determined using a gamma counter. Percent specific lysis was calculated as follows: (ER-SR)/(MR-SR), where ER is the experimental 51Cr release in the presence of effector cells, SR is a spontaneous release of 51Cr from culturing target cells alone, and MR the maximum 51Cr release in the presence of 0.5% Triton-X.

Establishment of a clonal culture system that supports the proliferation of immature NK cells.We chose day 14 murine fetal thymocytes as the material for growing candidate NK cell colonies because a relatively high percentage of the cells had been shown to have the potential of developing into NK cells.8 After trying several cytokine combinations, we found that fetal thymocytes cultured in methylcellulose with IL-7, IL-15, and SF form very diffuse colonies that could not be classified to known colony types (Fig 1A). Most of the cells in the colonies were small- to medium-sized round cells. May-Grünwald Giemsa staining of the colonies demonstrated that all cells in the colonies are blastic, showing no signs of cytoplasmic differentiation (Fig 1B). The nuclei often showed prominent nucleoli and mitotic cells were also common. The colony-forming efficiencies of the fetal thymocytes were about 1%. To further characterize the nature of cells in the colonies, we analyzed the expression of surface molecules by flow cytometry. As shown in Fig 2, cells in the colonies expressed Ly5, Thy-1, and CD25, but not myeloid markers (Mac-1, Gr-1), erythloid markers (TER), B-cell markers (B220), or mature NK- and T-cell markers (NK1.1, CD3, CD4, and CD8). These results suggested that the cells in the colonies are immature hematopoietic cells. Because the cells expressed Thy-1 and CD25, it was likely that these cells are immature NK or T cells. Therefore, we next examined T-cell and NK cell potentials using fetal thymus organ culture. This technique had been shown to support both NK and T-cell development.33 As shown in Fig 3, the cells converted to NK1.1+ but remained CD3, indicating that the cells in the colonies are committed to NK cell lineage. We also examined the functional status of the NK1.1+ cells present in the fetal thymus organ culture. We used the standard 51Cr release assay and NK sensitive YAC-1 cells as targets. As shown in Fig 4, the NK1.1+ cells expressed cyotoxic activity toward YAC-1 cells. Figure 4 also shows that the cells in the immature NK cell colonies are functionally incompetent. These results confirmed that the colonies derived from the fetal thymocytes are immature NK cell colonies.

Fig. 1.

Photomicrographs of a immature NK cell colony. (A) One quarter of a representative immature NK cell colony seen on an inverted microscope. Original magnification (OM) × 40. (B) A portion of a May-Grünwald Giemsa–stained smear of the colony. OM × 100.

Fig. 1.

Photomicrographs of a immature NK cell colony. (A) One quarter of a representative immature NK cell colony seen on an inverted microscope. Original magnification (OM) × 40. (B) A portion of a May-Grünwald Giemsa–stained smear of the colony. OM × 100.

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Fig. 2.

Surface phenotype of cells constituting the immature NK cell colonies. Thymocytes obtained from day 14 fetus were plated in methylcellulose culture with SF, IL-7, and IL-15. After 14 days of culture, immature NK cell colonies were individually picked, pooled, and stained with MoAbs specific for molecules indicated in each FACS profiles. Stained cells were analyzed by FACS Vantage using CellQuest software. Dotted and solid lines in each profiles represent cell stained with isotype-matched Ig and specific MoAbs, respectively.

Fig. 2.

Surface phenotype of cells constituting the immature NK cell colonies. Thymocytes obtained from day 14 fetus were plated in methylcellulose culture with SF, IL-7, and IL-15. After 14 days of culture, immature NK cell colonies were individually picked, pooled, and stained with MoAbs specific for molecules indicated in each FACS profiles. Stained cells were analyzed by FACS Vantage using CellQuest software. Dotted and solid lines in each profiles represent cell stained with isotype-matched Ig and specific MoAbs, respectively.

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Fig. 3.

Surface phenotype of cells developing in the fetal thymus lobe culture. Fetal thymocytes from C57BL/6-Ly5.1/C57BL/6-Ly5.2 F1 mice (Ly5.1/Ly5.2) were plated in methylcellulose culture with SF, IL-7, and IL-15. After 14 days of culture, immature NK cell colonies were individually picked, pooled, and incubated in hanging drops for 20 hours with fetal thymus lobes from BDF1 (Ly5.2) mice. The lobes were cultured on filter membranes for an additional 8 days. Cells recovered from the lobes were stained with MoAbs specific for molecules indicated in each FACS profiles. (A) Expression of Ly5.1 (donor marker) by the cells from cultured lobes. (B) Expression of NK1.1 and CD3 by donor-derived cells. Only the gated cell population (R1) is shown in each FACS profile. Dotted and solid lines represent cells stained with isotype-matched Ig and specific MoAbs, respectively.

Fig. 3.

Surface phenotype of cells developing in the fetal thymus lobe culture. Fetal thymocytes from C57BL/6-Ly5.1/C57BL/6-Ly5.2 F1 mice (Ly5.1/Ly5.2) were plated in methylcellulose culture with SF, IL-7, and IL-15. After 14 days of culture, immature NK cell colonies were individually picked, pooled, and incubated in hanging drops for 20 hours with fetal thymus lobes from BDF1 (Ly5.2) mice. The lobes were cultured on filter membranes for an additional 8 days. Cells recovered from the lobes were stained with MoAbs specific for molecules indicated in each FACS profiles. (A) Expression of Ly5.1 (donor marker) by the cells from cultured lobes. (B) Expression of NK1.1 and CD3 by donor-derived cells. Only the gated cell population (R1) is shown in each FACS profile. Dotted and solid lines represent cells stained with isotype-matched Ig and specific MoAbs, respectively.

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Fig. 4.

Cytolytic activity of NK1.1+ cells derived from immature NK cell colonies. Fetal thymocytes from C57Bl/6-Ly5.1/C57Bl/6-Ly-5.2 F1 mice were cultured under the conditions described in the legend for Fig 3. After 8 days of fetal thymus organ culture, Ly5.1+NK1.1+ cells were prepared by FACS sorting from the cells in the fetal thymus lobe culture, cultured for an additional 7 days with IL-2 (500 ng/mL), and assayed for cytolytic activity as described in Materials and Methods. As a control, the NK1.1 cells from the immature NK cell colonies were assayed for cytolytic activity. (○), NK1.1+ cells from the fetal thymus culture; (•), cells from the immature NK cell colonies.

Fig. 4.

Cytolytic activity of NK1.1+ cells derived from immature NK cell colonies. Fetal thymocytes from C57Bl/6-Ly5.1/C57Bl/6-Ly-5.2 F1 mice were cultured under the conditions described in the legend for Fig 3. After 8 days of fetal thymus organ culture, Ly5.1+NK1.1+ cells were prepared by FACS sorting from the cells in the fetal thymus lobe culture, cultured for an additional 7 days with IL-2 (500 ng/mL), and assayed for cytolytic activity as described in Materials and Methods. As a control, the NK1.1 cells from the immature NK cell colonies were assayed for cytolytic activity. (○), NK1.1+ cells from the fetal thymus culture; (•), cells from the immature NK cell colonies.

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Myeloid and B-cell potentials of the cells from immature NK cell colonies.We next tested the cells in the immature NK cell colonies for possible possession of myeloid and B-cell potentials. Thymocytes obtained from day 14 fetuses were cultured in methylcellulose in the presence of IL-7, IL-15, and SF. Fourteen days later, immature NK cell colonies were individually picked, pooled, and recultured in the presence of IL-3, IL-11, and Epo for analysis of myeloid potential, or in the presence of IL-7, IL-11, and SF for B-cell potential. The cells in the colonies were unable to form myeloid or pre-B cell colonies whereas bone marrow cells formed myeloid and pre-B cell colonies under the same culture conditions (data not shown). These results confirmed that the colonies we identified contained NK committed progenitors.

Linearity studies of the immature NK cell colony formation.To test if the observed colony formation could be used as an assay for quantifying NK progenitors, we plated 0.3 × 103, 1 × 103, and 3 × 103 fetal thymocytes in methylcellulose with IL-7, IL-15, and SF. The number of immature NK cell colonies were scored after 14 days of culture. As shown in Fig 5, a linear relationship was observed between the number of cells plated and the number of immature NK cell colonies. This result indicated that the methylcellulose culture may be used as an assay for NK progenitors.

Fig. 5.

A linearity study of immature NK cell colony formation from fetal thymocytes. 0.3 × 103, 1 × 103, and 3 × 103 of day 14 fetal thymocytes were cultured with IL-7, IL-15, and SF in methylcellulose and colonies were scored on day 14 of culture. Data represent mean ± SD of quadruplicate cultures.

Fig. 5.

A linearity study of immature NK cell colony formation from fetal thymocytes. 0.3 × 103, 1 × 103, and 3 × 103 of day 14 fetal thymocytes were cultured with IL-7, IL-15, and SF in methylcellulose and colonies were scored on day 14 of culture. Data represent mean ± SD of quadruplicate cultures.

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Clonality of immature NK cell colonies.To confirm the single-cell origin of the immature NK cell colonies, we next plated individual cells into methylcellulose by use of micromanipulation. As described in detail later, progenitors for the immature NK cell are enriched most in the c-kit+CD25 fraction of fetal thymocytes. Therefore, we used c-kit+CD25 cells for confirmation of clonality by micromanipulation. We plated a total of 154 cells individually by using a micromanipulater into cultures containing IL-7, IL-15, and SF. Eight immature NK cell colonies developed. Flow cytometric analyses of the pooled colonies showed that the cells in the colonies express Thy-1 and CD25 but not B220, Gr-1, Mac-1, NK1.1, or CD3. These results established the clonal origin of the immature NK cell colonies.

Maturational stages of the progenitors for the immature NK cell colonies.Fetal thymocytes contain precursor cells at various maturational stages of T-cell and NK cell lineages.33-35 Although the majority of the precursor cells are committed to T-cell lineage, it has been suggested that earlier precursors have a potential to develop cells in other lineages cells including NK cells.8,9 Therefore, it was important to determine the maturational stages of the fetal thymocytes that form the immature NK cell colonies. CD25 and c-kit have been shown to be useful markers for separating different stages of intrathymic precursor cells.33 35 Therefore, we examined colony formation from thymocytes separated on the basis of CD25/c-kit expression. As shown in Table 1, most of the cells forming the immature NK cell colonies are c-kit+. Both CD25 and CD25+c-kit+ cells formed the immature NK cell colonies, although the frequency of colony forming cells in CD25 populations was slightly higher than that of CD25+ population.

Table 1.

Colony Formation From Subpopulation of Fetal Thymocytes Separated on the Basis of CD25/c-kit Expression

Cells PlatedNo. of Colonies
TypeNo./DishGMMastNK
CD25c-kit+ 300 1 ± 1 1 ± 1 20 ± 2 (79)* 
CD25+c-kit+ 500 0 ± 1 1 ± 1 19 ± 1 (75) 
CD25c-kit 1,500 1 ± 1 2 ± 1 (8) 
CD25+c-kit 4,000 0 ± 1 3 ± 1 (11) 
Cells PlatedNo. of Colonies
TypeNo./DishGMMastNK
CD25c-kit+ 300 1 ± 1 1 ± 1 20 ± 2 (79)* 
CD25+c-kit+ 500 0 ± 1 1 ± 1 19 ± 1 (75) 
CD25c-kit 1,500 1 ± 1 2 ± 1 (8) 
CD25+c-kit 4,000 0 ± 1 3 ± 1 (11) 

Designated populations of cells were prepared by FACS sorting from day 14 fetal thymocytes of BDF1 mice and cultured with IL-7, IL-15, and SF in methylcellulose. Colonies were counted on day 14 of culture. Data represent mean ± SD of quadruplicate cultures.

*

Total number of immature NK cell colonies in four plates is shown in parentheses.

Minimal cytokine requirements for formation of the immature NK cell colonies.The immature NK cell colonies were initially identified in culture containing IL-7, IL-15, and SF. We next examined the minimal cytokine requirement for the development of the immature NK cell colonies. As shown in Table 2, neither IL-7, IL-15, nor SF alone could support the formation of the colonies. A combination of IL-7 and SF supported formation of the immature NK cell colonies from both CD25 and CD25+c-kit+ cells. IL-15 was not essential for the colony formation, but increased the frequency of colonies. These results indicate that IL-7 and SF act synergisticaly, and are indispensable for supporting immature NK cell colonies.

Table 2.

Minimal Cytokine Requirement of Immature NK Colonies

Cells PlatedCytokinesNo. of Colonies
TypeNo./DishGMMastNK
CD25c-kit+ 300 IL-7 0 (0)* 
  IL-15 0 (0) 
  SF 0 (0) 
  IL-7, IL-15 0 (0) 
  IL-7, SF 0 ± 1 1 ± 1 11 ± 2 (45) 
  IL-15, SF 1 ± 1 0 (0) 
  IL-7, IL-15, SF 1 ± 1 0 ± 1 20 ± 2 (79) 
CD25+c-kit+ 500 IL-7 0 (0) 
  IL-15 0 (0) 
  SF 0 (0) 
  IL-7, IL-15 0 (0) 
  IL-7, SF 0 ± 1 0 ± 1 12 ± 3 (46) 
  IL-15, SF 0 (0) 
  IL-7, IL-15, SF 1 ± 1 1 ± 1 16 ± 1 (64) 
Cells PlatedCytokinesNo. of Colonies
TypeNo./DishGMMastNK
CD25c-kit+ 300 IL-7 0 (0)* 
  IL-15 0 (0) 
  SF 0 (0) 
  IL-7, IL-15 0 (0) 
  IL-7, SF 0 ± 1 1 ± 1 11 ± 2 (45) 
  IL-15, SF 1 ± 1 0 (0) 
  IL-7, IL-15, SF 1 ± 1 0 ± 1 20 ± 2 (79) 
CD25+c-kit+ 500 IL-7 0 (0) 
  IL-15 0 (0) 
  SF 0 (0) 
  IL-7, IL-15 0 (0) 
  IL-7, SF 0 ± 1 0 ± 1 12 ± 3 (46) 
  IL-15, SF 0 (0) 
  IL-7, IL-15, SF 1 ± 1 1 ± 1 16 ± 1 (64) 

CD25c-kit+ or CD25+c-kit+ cells were prepared by FACS sorting from day 14 fetal thymocytes. Culture was performed in the presence of designated cytokines. Colonies were scored on day 14 of culture. Data represent mean ± SD of quadruplicate culture.

*

Total number of immature NK cell colonies in 4 dishes is shown in parentheses.

The effect of IL-2.The results shown in Table 2 indicated that IL-15 is a growth factor for immature NK cells. We next examined the effects of IL-2 because it has been shown that IL-2 and IL-15 share many biologic activities, including the ability to promote the growth of mature NK cells.6,36 As shown in Table 3, both IL-2 and IL-15 increased the frequencies of immature NK cell colonies in the presence of IL-7 and SF. Table 3 also shows that there are neither synergistic nor additive effects between IL-2 and IL-15. These results indicate that IL-2 and IL-15, previously shown to be growth factors for mature NK cells,6 36 are also growth factors for the NK cells at earlier stages of development.

Table 3.

Effects of IL-2 and IL-15 on the Formation of Immature NK Cell Colonies

Cells PlatedCytokinesNo. of Colonies
TypeNo./DishGMMastNK
CD25c-kit+ 300 IL-7, SF 1 ± 1 11 ± 1 (42)3-150 
  IL-2, IL-7, SF 0 ± 1 1 ± 1 19 ± 1 (76) 
  IL-15, IL-7, SF 18 ± 2 (71) 
  IL-2, IL-15, IL-7, SF 1 ± 1 19 ± 2 (75) 
CD25+c-kit+ 500 IL-7, SF 1 ± 1 12 ± 1 (49) 
  IL-2, IL-7, SF 0 ± 1 1 ± 1 24 ± 1 (94) 
  IL-15, IL-7, SF 1 ± 1 19 ± 2 (76) 
  IL-2, IL-15, IL-7, SF 1 ± 1 24 ± 1 (95) 
Cells PlatedCytokinesNo. of Colonies
TypeNo./DishGMMastNK
CD25c-kit+ 300 IL-7, SF 1 ± 1 11 ± 1 (42)3-150 
  IL-2, IL-7, SF 0 ± 1 1 ± 1 19 ± 1 (76) 
  IL-15, IL-7, SF 18 ± 2 (71) 
  IL-2, IL-15, IL-7, SF 1 ± 1 19 ± 2 (75) 
CD25+c-kit+ 500 IL-7, SF 1 ± 1 12 ± 1 (49) 
  IL-2, IL-7, SF 0 ± 1 1 ± 1 24 ± 1 (94) 
  IL-15, IL-7, SF 1 ± 1 19 ± 2 (76) 
  IL-2, IL-15, IL-7, SF 1 ± 1 24 ± 1 (95) 

CD25c-kit+ or CD25+c-kit+ cells from day 14 fetal thymocytes were cultured in the presence of designated cytokines. Colonies were scored on day 14 of culture. Data represent mean ± SD of quadruplicate cultures.

F3-150

Total number of immature NK cell colonies in 4 plates is shown in parentheses.

Effects of IL-4 and IL-13.IL-7, IL-2, and IL-15 use common γ-chain as a receptor component.10,13,14,16 We next examined the effects of IL-4 on the formation of immature NK cell colonies, because IL-4 also uses the common γ-chain as a receptor component11,12 and shares several biologic activities with IL-2.37,38 We also examined the effects of IL-13 along with IL-4, because many biologic activities of IL-4 have been shown to be substituted by IL-13.39 The results are presented in Table 4. Surprisingly, addition of 10 ng/mL IL-4 to the culture completely abolished the formation of immature NK cell colonies. Although 50 ng/mL of IL-13 slightly reduced the frequencies of immature NK cell colonies, 10 ng/mL IL-13 did not abrogate the formation of immature NK cell colonies.

Table 4.

Effects of IL-4 and IL-13 on Immature NK Cell Colonies

Cells PlatedCytokinesNo. of Colonies
TypeNo./DishGMMastNK
CD25c-kit+ 300 IL-7, IL-15, SF 1 ± 1 1 ± 1 24 ± 2 (97)4-150 
  IL-4 (10 ng/mL), IL-7, IL-15, SF 1 ± 1 2 ± 1 0 (0) 
  IL-13 (10 ng/mL), IL-7, IL-15, SF 1 ± 1 2 ± 1 22 ± 1 (86) 
  IL-13 (50 ng/mL), IL-7, IL-15, SF 0 ± 1 1 ± 1 16 ± 2 (65) 
CD25+c-kit+ 500 IL-7, IL-15, SF 27 ± 1 (107) 
  IL-4 (10 ng/mL), IL-7, IL-15, SF 0 ± 1 1 ± 1 0 (0) 
  IL-13 (10 ng/mL), IL-7, IL-15, SF 1 ± 1 26 ± 1 (104) 
  IL-13 (50 ng/mL), IL-7, IL-15, SF 18 ± 2 (80) 
Cells PlatedCytokinesNo. of Colonies
TypeNo./DishGMMastNK
CD25c-kit+ 300 IL-7, IL-15, SF 1 ± 1 1 ± 1 24 ± 2 (97)4-150 
  IL-4 (10 ng/mL), IL-7, IL-15, SF 1 ± 1 2 ± 1 0 (0) 
  IL-13 (10 ng/mL), IL-7, IL-15, SF 1 ± 1 2 ± 1 22 ± 1 (86) 
  IL-13 (50 ng/mL), IL-7, IL-15, SF 0 ± 1 1 ± 1 16 ± 2 (65) 
CD25+c-kit+ 500 IL-7, IL-15, SF 27 ± 1 (107) 
  IL-4 (10 ng/mL), IL-7, IL-15, SF 0 ± 1 1 ± 1 0 (0) 
  IL-13 (10 ng/mL), IL-7, IL-15, SF 1 ± 1 26 ± 1 (104) 
  IL-13 (50 ng/mL), IL-7, IL-15, SF 18 ± 2 (80) 

CD25c-kit+ and CD25+c-kit+ cells sorted from day 14 fetal thymocytes were cultured in the presence of cytokines. Colonies were scored on day 14 of culture. Data represent mean ± SD of quadruplicate cultures.

F4-150

Total number of colonies in 4 plates is shown in parentheses.

To examine the regulation of NK cell development, we established a clonal culture system that supports the proliferation of immature NK cells. c-kit+ fetal thymocytes plated in methylcellulose media containing IL-7, IL-15, and SF formed colonies consisting of committed NK progenitor cells. IL-7 and SF were found to be indispensable for supporting the formation of immature NK cell colonies and IL-15 and IL-2 increased the frequency of the colonies. IL-2, IL-7, and IL-15 use the common γ-chain and JAK-3 kinase as signal transduction molecules.40-42 Therefore, our results are consistent with the recent observations that mice lacking common γ-chain and JAK-3 kinase are devoid of mature NK cells.17,18,43 In our culture system, IL-7 appeared to be an important cytokine because no colonies formed without IL-7. However, it has been reported that mice lacking IL-7 receptor α-chain have normal numbers of functional NK cells.44,45 This indicates that other cytokines can compensate for the absence of IL-7 in NK cell development. Alternatively, there may be receptors for IL-7 that do not use IL-7 receptor α-chain as a component. SF and its receptor, c-kit, have been shown to be important for the growth of human immature and mature NK cells.46 47 Here we showed that SF also functions as a growth factor for murine immature NK cells. It appears that, in addition to common γ-chain–JAK-3 kinase signal transduction pathway, signaling through a transmembrane tyrosine kinase receptor is important for the growth of immature NK cells.

Surprisingly, IL-4 revealed strong inhibitory effects on the formation of the immature NK cell colonies. IL-4, like IL-2, IL-7, and IL-15, uses a common γ-chain10,13,14,16 and JAK-3 kinase as signal transduction molecules.40-42 IL-4 and IL-2 also share JAK-1 kinase as a signal transduction molecule.48,49 It is unlikely that IL-4 competes with IL-2, IL-7, or IL-15 for binding to common γ-chain because very low concentrations (10 ng/mL) of IL-4 are sufficient to abolish immature NK cell colony formation and IL-2, IL-7, and IL-15 at concentrations as high as 1 mg/mL did not affect the inhibitory effect of IL-4 (data not shown). Therefore, the inhibitory effects of IL-4 may be due to activation of IL-4 specific signaling pathways. It has been reported that IL-4 evokes specific responses, including tyrosine phosphorylation of 4PS/IRS-2 and activation of Stat6.48,50 51 Whether these molecules are involved in the IL-4–mediated growth inhibition of immature NK cells needs to be clarified.

Other investigators have reported that fetal thymus organ culture supplemented by cytokines supports the development of cytolytic cells. Widmer et al52 have shown that IL-7 and IL-2 increase the cytolytic activities of cultured fetal thymocytes while IL-4 inhibits development of cytolytic cells. More recently, Leclercq et al53 have reported that IL-15 also can induce the development of cytolytic cells in fetal thymus organ culture. In these reports, however, it was not clear what developmental stages of the cytolytic cells are regulated by these cytokines. In addition, the nature of cytolytic cells was not defined clearly. Our results presented in this report pertain to cytokine regulation of NK cells at the progenitor level.

Accumulating data suggest that NK and T cells share their developmental pathways. CD16+ or c-kit+ cells in murine thymus have been reported to have T and NK lineage potentials.8,9 More recently, Zúñiga-Pflücker et al35 have reported that c-kit+CD25 cells in murine fetal thymus have NK/T potentials whereas c-kit+CD25+ cells contain only T-cell committed progenitors. However, we found that c-kit+CD25+ cells are able to form immature NK cell colonies. The apparent discrepancy may be due to differences in the sensitivities of their in vivo and our cell culture assays. Regardless, our results suggest that the expression of CD25 is not an absolute marker of commitment to T-cell lineage. We are not certain whether the NK cell colonies we identified are derived solely from NK committed progenitors or both committed and NK/T common progenitors because our culture conditions may not be permissive to proliferation of T-cell progenitors. To clarify this point, a culture system that supports clonal proliferation of T-cell progenitors needs to be established.

Although this appears to be the first report of a culture system which supports proliferation of murine immature NK cell growth without stromal cell elements, cytokine-based culture systems for human NK cells and progenitors have already been reported. Sánchez et al7 have shown that certain populations of human fetal thymocytes cultured with IL-2, IL-7, and SF give rise to mature NK cells. Shibuya et al47 and Mrozek et al54 have shown generation of mature NK cells from human CD34+lin cells in culture with IL-2 and SF or IL-15 and SF, respectively. A major difference exists between the human and our murine culture system in that our murine assay is directed to early stages of NK cell development only (Figs 2 and 3).

At the present time we are not certain why NK progenitors are present in the thymus. It has been shown that athymic nude mice have a normal number of mature NK cells.55 It is possible that there are several pathways of NK cell development, including the one occurring in thymus. It is unlikely that the colony formation described in this report applies only to the intrathymic NK progenitors, because we recently observed the immature NK cell colony formation from fetal liver cells similar to that from fetal thymocytes (unpublished observation). The clonal assay for immature NK cell progenitors described in this report will be useful in the studies of the origins of NK cells and characterization of the cytokine regulation of the early stages of NK cell development.

We thank Dr Haiqun Zeng for assistance in cell sorting and Dr Pamela N. Pharr, Dr Stewart D. Lyman, and Anne G. Leary for assistance in preparation of this manuscript.

Supported by National Institutes of Health Grants No. DK32294 and DK/HL48714, Office of Research and Development, Medical Research Service, Department of Veterans Affairs.

Address reprinte requests to Makio Ogawa, MD, PhD, Ralph H. Johnson Medical Center, 109 Bee St, Charleston SC 29401-5799.

1
Trinchieri G: Biology of natural killer cells: Adv Immunol 47:187, 1989
2
Ritz
J
Schmidt
RE
Michon
J
Hercend
T
Schlossman
SF
Characterization of functional surface structures on human natural killer cells.
Adv Immunol
42
1988
181
3
Murphy
WJ
Kumar
V
Bennett
M
Rejection of bone marrow allografts by mice with severe combined immune deficiency (SCID).
J Exp Med
165
1987
1212
4
Lanier
LL
Spits
H
Phillips
JH
The developmental relationship between NK cells and T cells.
Immunol Today
13
1992
392
5
Herberman
RB
Reynolds
CW
Ortaldo
JR
Mechanism of cytotoxicity by natural killer (NK) cells.
Annu Rev Immunol
4
1986
651
6
Grabstein
KH
Eisenman
J
Shanebeck
K
Rauch
C
Srinivasan
S
Fung
V
Beers
C
Richardson
J
Schoenborn
MA
Ahdieh
M
Johnson
L
Alderson
MR
Watson
JD
Anderson
DM
Giri
JG
Cloning of a T cell growth factor that interacts with the β chain of the interleukin-2 receptor.
Science
264
1994
965
7
Sánchez
MJ
Muench
MO
Roncarolo
MG
Lanier
LL
Phillips
JH
Identification of a common T/natural killer cell progenitor in human fetal thymus.
J Exp Med
180
1994
569
8
Rodewald
HR
Moingeon
P
Lucich
JL
Dosiou
C
Lopez
P
Reinherz
EL
A population of early fetal thymocytes expressing FcγRII/III contains precursors of T lymphocytes and natural killer cells.
Cell
69
1992
139
9
Matsuzaki
Y
Gyotoku
JI
Ogawa
M
Nishikawa
SI
Katsura
Y
Gachelin
G
Nakauchi
H
Characterization of c-kit positive intrathymic stem cells that are restricted to lymphoid differentiation.
J Exp Med
178
1993
1283
10
Takeshita
T
Asao
H
Ohtani
K
Ishii
N
Kumaki
S
Tanaka
N
Munakata
H
Nakamura
M
Sugamura
K
Cloning of the γ chain of the human IL-2 receptor.
Science
257
1992
379
11
Kondo
M
Takeshita
T
Ishii
N
Nakamura
M
Watanabe
S
Arai
K
Sugamura
K
Sharing of the interleukin-2 (IL-2) receptor gamma chain between receptors for IL-2 and IL-4.
Science
262
1993
1874
12
Russel
SM
Keegan
AD
Harada
N
Nakamura
Y
Noguchi
M
Leland
P
Friedmann
MC
Miyajima
A
Puri
RK
Paul
WE
Leonard
WJ
Interleukin-2 receptor gamma chain: A functional component of the interleukin-4 receptor.
Science
262
1993
1880
13
Kondo
M
Takeshita
T
Higuchi
M
Nakamura
M
Sudo
T
Nishikawa
S
Sugamura
K
Functional participation of the IL-2 receptor gamma chain in IL-7 receptor complexes.
Science
263
1994
1453
14
Noguchi
M
Nakamura
Y
Russell
SM
Ziegler
SF
Tsang
M
Cao
X
Leonard
WJ
Interleukin-2 receptor gamma chain: A functional component of the interleukin-7 receptor.
Science
262
1993
1877
15
Kimura
Y
Takeshita
T
Kondo
M
Ishii
N
Nakamura
M
Van SJ
Sugamura K
Sharing of IL-2 receptor gamma chain with the functional IL-9 receptor complex.
Int Immunol
7
1995
115
16
Giri
JG
Ahdieh
M
Eisenman
J
Shanebeck
K
Grabstein
K
Kumaki
S
Namen
A
Park
LS
Cosman
D
Anderson
D
Utilization of the beta and gamma chain of the IL-2 receptor by the novel cytokine IL-15.
EMBO J
13
1994
2822
17
Disanto
JP
Muller
W
Guy-Grand
D
Fischer
A
Rajewsky
K
Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor γ chain.
Proc Natl Acad Sci USA
92
1995
377
18
Cao
XE
Shores
W
Hu-Li
J
Anver
MR
Keisall
BL
Russell
SM
Drago
J
Noguchi
M
Grinberg
A
Bloom
ET
Paul
WE
Katz
SI
Love
PE
Leonard
WJ
Defective lymphoid development in mice lacking expression of the common cytokine receptor γ chain.
Immunity
2
1995
223
19
Leo
O
Foo
M
Sach
DH
Samelson
L
Bluestone
JA
Identification of monoclonal antibody specific for a murine T3 polypeptide.
Proc Natl Acad Sci USA
84
1987
1374
20
Lowenthal
JW
Corthesy
P
Tougne
C
Lees
R
MacDonald
HR
Nabholz
M
High and low affinity IL-2 receptors: Analysis by IL-2 dissociation rate and reactivity with monoclonal anti-receptor antibody PC61.
J Immunol
135
1985
3988
21
Ledbetter
JA
Herzenberg
LA
Xenogenic monoclonal antibodies to mouse lymphoid differentiation antigens.
Immunol Rev
47
1979
63
22
Koo
G
Dumont
F
Tutt
M
Hackett
J
Kumar
V
The NK1.1(-) mouse: A model to study differentiation of murine NK cells.
J Immunol
137
1986
3742
23
Coffman
B
Surface antigen expression and immunoglobulin rearrangement during pre-B cell development.
Immunol Rev
69
1982
5
24
Nishikawa
S
Kusakabe
M
Yoshida
K
Ogawa
M
Hayashi
S
Kunisada
T
Era
T
Sakakura
T
Nishikawa
SI
In utero manipulation of coat color fomation by a monoclonal anti-c-kit antibody: Two distinct waves of c-kit-dependency during melanocyte development.
EMBO J
10
1991
2111
25
Springer
T
Galfre
G
Secher
DS
Milstein
C
Mac-1: A macrophage differentiation antigen identified by monoclonal antibody.
Eur J Immunol
9
1979
301
26
Holmes
KL
Langdon
WY
Fredrickson
TN
Coffman
RL
Hoffman
PM
Hartely
JW
Morse
III MC
Analysis of neoplasms induced by Cas-Br-M MuLV tumor extracts.
J Immunol
137
1986
679
27
Ikuta
K
Kina
T
MacNeil
I
Uchida
N
Peault
B
Chien
YH
Weissman
IL
A developmental switch in thymic lymphocyte maturation potential occurs at the level of hematopoietic stem cells.
Cell
62
1990
863
28
Ewijk
W
van Soest
P
van den Engh
G
Fluorescence analysis and anatomic distribution of mouse T lymphocyte subset defined by monoclonal antibodies to antigens Thy-1, Lyt-1, Lyt-2, and T-200.
J Immunol
127
1990
2594
29
Jenkinson
EJ
Franchi
LL
Kingston
R
Owen
JJT
Effect of deoxyguanosine on lymphopoiesis in the developing thymus rudiment in vitro: Application in the production chimeric thymus rudiments.
Eur J Immunol
12
1982
583
30
Kingston
R
Jenkinson
EJ
Owen
JJT
A single stem cell can recolonize an embryonic thymus, producing phenotypically distinct T-cell populations.
Nature
317
1985
811
31
Hirayama
F
Shih
JP
Awgulewitsch
A
Warr
GW
Clark
SC
Ogawa
M
Clonal proliferation of murine lymphohemopoietic progenitors in culture.
Proc Natl Acad Sci USA
89
1992
5907
32
Hirayama
F
Ogawa
M
Negative regulation of early T lymphopoiesis by interleukin-3 and interleukin-1α.
Blood
86
1995
4527
33
Moore
TA
Zlotnik
A
T-cell lineage commitment and cytokine responses of thymic progenitors.
Blood
86
1995
1850
34
Zlotnik
A
Moore
TA
Cytokine production and requirements during T-cell development.
Curr Opin Immunol
7
1995
206
35
Zúñiga-Pflücker
JC
Jiang
D
Lenardo
MJ
Requirement of TNF-α and IL-1α in fetal thymocytes commitment and differentiation.
Science
268
1995
1906
36
Carson
WE
Giri
JG
Lindemann
MJ
Linett
ML
Ahdieh
M
Paxton
R
Anderson
D
Eisenmann
J
Grabstein
K
Caligiuri
MA
Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor.
J Exp Med
180
1994
1395
37
Fernandez-Botran
R
Sanders
VM
Oliver
KG
Chen
YW
Krammer
PH
Uhr
JW
Vitetta
ES
Interleukin-4 mediates autocrine growth of helper T cells after antigenic stimulation.
Proc Natl Acad Sci USA
83
1986
9689
38
Mule
JJ
Kronsnick
A
Rosenberg
SA
IL-4 regulation of murine lymphokine-activated killer activity in vitro. Effects on the IL-2-induced expansion, cytoxicity, and phenotype of lymphokine-activated killer effects.
J Immunol
142
1989
726
39
Callard
RE
Matthews
DJ
Hibbert
L
IL-4 and IL-13 receptors: Are they one and the same?
Immunol Today
17
1996
108
40
Ihle
JN
Cytokine receptor signaling.
Nature
377
1995
591
41
Russell
SM
Johmston
JA
Noguchi
M
Kawamura
M
Bacon
CM
Friedmann
M
Berg
M
McVicar
DW
Witthuhn
BA
Silvennoinen
O
Goldman
AS
Schmalstieg
FC
Ihle
JN
O'Shea
JJ
Leonard
WJ
Interaction of IL-2Rβ and γc chains with jak1 and jak3: Implications for XSCID and XCID.
Science
266
1994
1042
42
Miyazaki
T
Kawahara
A
Fuji
H
Nakagawa
Y
Minami
Y
Lui
ZJ
Oishi
I
Silvennoinen
O
Witthuhn
BA
Ihle
JN
Taniguchi
T
Functional activation of Jak1 and Jak3 by selective association with IL-2 receptor subunits.
Science
266
1994
1045
43
Park
SY
Saijo
K
Takahashi
T
Osawa
M
Arase
H
Hirayama
N
Miyake
K
Nakauchi
H
Shirasawa
T
Saito
T
Developmental defects of lymphoid cells in Jak3 kinase-deficient mice.
Immunity
3
1995
771
44
He
YW
Malek
TR
Interleukin-7 receptor α is essential for the development of γδ T cells, but not natural killer cells.
J Exp Med
184
1996
289
45
Maki
K
Sunaga
S
Komagata
Y
Kodaira
Y
Mabuchi
A
Karasuyama
H
Yokomuro
K
Miyazaki
JI
Ikuta
K
Interleukin 7 receptor-deficient mice lack γδ T cells.
Proc Natl Acad Sci USA
93
1996
7172
46
Matos
ME
Schnier
GS
Beecher
MS
Ashman
LK
Williams
DE
Caligiuri
MA
Expression of a functional c-kit receptor on a subset of natural killer cells.
J Exp Med
178
1993
1079
47
Shibuya
A
Nagayoshi
K
Nakamura
K
Nakauchi
H
Lymphokine requirement for the generation of natural killer cells from CD34+ hematopoietic progenitor cells.
Blood
85
1995
3538
48
Yin
T
Tsang
ML-S
Yang
YC
JAK1 kinase forms complexes with interleukin-4 receptor and 4PS/insulin receptor substrate-1-like protein and is activated by interleukin-4 and interleukin-9 in T lymphocytes.
J Biol Chem
269
1994
26614
49
Morla
AO
Schreurs
J
Myajima
A
Wang
JYJ
Hematopoietic growth factors activate the tyrosine phosphorylation of distinct sets of proteins in interleukin-3-dependent murine cell lines.
Mol Cell Biol
8
1988
2214
50
Quelle
FW
Shimoda
K
Thierfelder
W
Fischer
C
Kim
A
Reuben
SM
Cleveland
JL
Pierce
JH
Keegan
AD
Nelms
K
Paul
WE
Ihle
JN
Cloning of Stat6, Stat proteins that are tyrosine phosphorylated in response to IL-4 and IL-3 but are not required for mitogenesis.
Mol Cell Biol
15
1995
3336
51
Lin
JX
Migone
TS
Tsang
M
Friedmann
M
Weatherbee
JA
Zhou
L
Yamauchi
A
Bloom
ET
Mietz
J
John
S
Leonard
WJ
The role of shared receptor motifs and common Stat proteins in the generation of cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13 and IL-15.
Immunity
2
1995
331
52
Widmer
MB
Morrissey
PJ
Namen
AE
Voice
RF
Watson
JD
Interleukin 7 stimulates growth of fetal thymic precursors of cytolytic cells: Induction of effector function by interleukin 2 and inhibition by interleukin 4.
Int Immunol
2
1990
1055
53
Leclercq
G
Debacker
V
De Smedt
M
Plum
J
Differential effect of interleukin-15 and interleukin-2 on differentiation of bipotential T/natural killer progenitor cells.
J Exp Med
184
1996
325
54
Mrozek
E
Anderson
P
Caligiuri
MA
Role of interleukin-15 in the development of human CD56+ natural killer cells from CD34+ hematopoietic progenitor cells.
Blood
87
1996
2632
55
Herberman
RB
Nunn
ME
Larvin
DH
Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogenic tumors. I. Distribution reactivity and specificity.
Int J Cancer
16
1975
216
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