Development of Natural Killer Cells from Lymphohematopoietic Progenitors of Murine Fetal Liver

Development of Natural Killer Cells from Lymphohematopoietic Progenitors of Murine Fetal Liver YUKHI AIBA, MAKIO OGAWA Ralph H. Johnson Department of Veterans Affairs Medical Center, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina, USA Key Words. Natural killer cells Lymphohematopoietic cells lnterleukins Growth factors ABSTRACT We established a clonal culture system which supports the growth of murine immature natural killer (NK) cells. When we plated day 14 fetal thymocytes in methylcellulose media containing interleukin 2 (IL-2), IL-7 and steel factor (SF), we observed diffuse colonies which could not be classified into known colony types. Cells in the colonies were blast-like and expressed Thy-1 and CD25 but not lineage-specific markers. Cells in the colonies developed into NK1.l' cells in fetal thymus organ culture indicating that the colonies consist of immature NK cells. We then examined the colony-forming ability of fetal liver cells. The combination of IL-2, IL- 7 and SF with or without IL-11 supported formation of few immature NK cell colonies from purified progenitors. Interestingly, addition of IL-11 to the culture stimulated formation of mixed colonies consisting of immature NK cells, B cells, macrophages and/or mast cells. The clonal origin of the mixed NK cell colonies was confirmed by micromanipulation of the colony-forming cells. This culture assay should facilitate the analysis of the pathway and cytokine regulation of NK cell development. Stem Cells 1998;16(suppl I): 193-1 98 INTRODUCTION Natural killer (NK) cells comprise a small subset of lymphocytes and are thought to play important roles in various immune responses [I, 21. Although the functions of NK cells are being extensively studied, their ontogeny and regulation of development are largely unknown. It was reported that NK cells are closely related to T lymphocytes in their developmental pathways [3]. Sunchez et al. [4] have shown that NWT common progenitors are present in human fetal thymus. However. it still remains to be clarified how NK or NWT common progenitors develop from multipotential hematopoietic progenitors, and how and when NWT common progenitors diverge to form separate NK and T cell lineages. The lineage relationship of NK cells with B and myeloid cells should also be clarified. To investigate the pathway of NK cell development, it is necessary to establish a clonal culture system which supports the development of NK cells from multipotential hematopoietic progenitors. Here, we report that murine fetal thymocytes and liver cells plated in methylcellulose culture form immature NK cell colonies. We also found that mixed colonies consisting of NK, B and myeloid cells Characteristics and Potentials of Blood Stem Cells STEM CELLS 1998;16(suppl 1):193-198 OAlphaMed Press. All rights reserved

I94 Hematopoietic Origin of Natural Killer Cells can develop from purified hematopoietic progenitors of fetal liver cells in the presence of interleukin 2 (IL-2), IL-7, steel factor (SF) and IL-11. We believe that this quantitative clonal culture is useful for analyzing the developmental pathway and cytokine regulation of NK cell progenitors. MATERIALS AND METHODS Mice and Cell Preparation Female C57BL16-Ly5.2 and male DBA/2 mice were purchased from Charles River (Raleigh, NC). Male C57BL16-Ly5.1 mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were allowed to mate for 18 h. The day of vaginal plugging was designated as day 0 of gestation. On day 14 of gestation, the mice were sacrificed and cell suspensions of fetal thymus and liver were prepared by gently pressing these organs between two slide glasses and by repeated pipetting. Purification of fetal liver progenitors had been described previously [5]. Cytokines Purified murine recombinant IL-2 was purchased from R&D Systems (Minneapolis, MN). Recombinant human IL-7 was provided by Sanofi Winthrop, Inc. (Malvern, PA). Recombinant murine SF was a gift from Immunex (Seattle, WA). Recombinant human IL-11 was provided by Genetics Institute (Cambridge, MA). Concentrations of cytokines used were as follows: IL-2, 20 ng/ml; IL-7, SF, 100 nglml; 200 U/ml(20ng/ml); IL-11, 100 ng/ml. Flow Cytometry and Cell Sorting The following monoclonal antibodies were used for flow cytometric analysis and cell sorting: biotin-conjugated-anti-cd3 (clone;145-2cll) (Pharmingen; San Diego, CA); 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 S.3) (Caltag); FITC-conjugated-anti-Thy1.2 (30-H12) (Pharmingen); biotin-conjugated-anti-nkl.1 (PK 136) (Pharmingen); PE-conjugated-anti-B220 (RA3-6B2) (Pharmingen); biotin-conjugated-anti-ckit (ACK4), biotin-conjugated-anti-mac-1 (M1/70), biotin-conjugated-anti-gr-1 (RB6-8C5) (Pharmingen); biotin-conjugated-anti-ter (TER119) (Pharmingen); FITC-conjugated-anti-Ly5.1 (A20-1.7, kindly provided by Dr. H. Fleming of Emory University, Atlanta, GA), and FITC-conjugated-anti-Ly5 (30F11.1). Basic techniques of cell staining had been described previously [6]. In all experiments, cells stained with appropriate isotype-matched control immunoglobulins were prepared as negative controls. After staining, cells were analyzed and/or sorted by using FACS Vantage (Becton Dickinson; Mountain View, CA). Clonal Culture for Immature NK Cell Colonies Methylcellulose culture was carried out by using 35mm Falcon suspension culture dishes (Becton Dickinson Labware; Lincoln Park, NJ). Fetal thymocytes or purified progenitor cells of fetal liver were cultured in the medium consisting of a-medium (Flow Laboratories; Rockville, MD), 1.2% 1,500-centipoise methylcellulose (Shinetsu Chemical; Tokyo, Japan), 5% fetal bovine serum, 1 % deionized fraction-v bovine serum albium and 0.lmM 2-mercaptoethanol and cytokines. Dishes were incubated at 37 C in a humidified atmosphere flushed with 5% C02 for 14 or 18 days. Single-cell manipulation of colony-forming cells was carried out as described previously [7]. Fetal Thymus Organ Culture Fetal thymus organ culture was performed as described previously [8]. Briefly, fetal thymi were first incubated with 4 X 10' test cells in a hanging drop culture for 20 h. Samples were then

Aiba, Ogawa 195 transferred onto filter membranes (Costar; Cambridge, MA; pore size, 8mm) and cultured for 11 days. Cells were recovered from the thymic lobes, stained with antibodies and analyzed by flow cytometry. RE s u L T s Immature NK Cell Colony Formation from Fetal Thymocytes We chose day 14 murine fetal thymocytes as the material for growing candidate NK cell colonies because relatively high percentages of the cells had been shown to have the potential of developing into NK cells [9]. After testing several cytokine combinations, we found that fetal thymocytes cultured in the methylcellulose media containing IL-2, IL-7 and SF form diffuse colonies which cannot be classified into known colony types (Fig. 1). May-Griinwald Giemsa staining of the colonies revealed that the cells in the colonies are blast-like, showing no signs of cytoplasmic differentiation. Colony-forming frequency of the fetal thymocytes was about 1%. We then examined expression of surface molecules of the cells in the colonies by flow cytometry. As shown in Figure 2, the cells expressed Thy-1 and CD25 but not Mac-1, Gr-1, TER, CD3 or B220. Because the cells in the colonies expressed Thy- 1 and CD25, it was likely that these cells are immature NK or T cells. Therefore, we next examined the NK and T cell potentials by culturing these cells in fetal thymus organ culture. Fetal thymocytes of CS7BL/6-Ly5.1/Ly5.2 Fl mice were cultured in the methylcellulose media in the presence of IL-2, IL-7 and SF. After incubation for 14 days, candidate NK cell colonies were individually picked, pooled and plated in culture with fetal thymic lobes of CS7BL/6-Ly5.2 mice. Ten days later cells recovered from the thymic lobes were analyzed for the expression of Ly5.1, CD3, NK1.l and B220. As shown in Figure 3, most of test LyS. I' cells expressed NK1.l but neither CD3 nor B220. This result indicated that the diffuse colonies derived from the fetal thymocytes are immature NK cell colonies. Mixed NK Cell Colony Formation from Fetal Liver Progenitors We next examined the colonyforming ability of purified hematopoietic progenitors of fetal Figure I. A photomicrograph of an immature NK cell colony. One quarter of a representative immature NK cell colony seen on an inverted microscope. -Mac- 1, Gr- 1, TER - CD25- Figure 2. Surface phenotypes of the cells in the immature NK cell colonies. Fetal thymocytes were cultured in methylcellulose media with IL-2, IL-7 and SF. After 14 days of culture, immature NK cell colonies were individually picked, pooled and stained with monoclonal antibodies specific for molecules indicated in each FACS profile.

~ -~ 196 Hematopoietic Origin of Natural Killer Cells -Ly-5. t- Figure 3. Surface phenotypes of the cells developing in fetal thymus organ culture. Fetal thymocytes from C57BW6-Ly5. I/C57BW- 6-Ly5.2 Fl mice (LyS.l/Ly5.2) were plated in rlzethylcellulose culture with IL-2, 1L-7 and SF. After 14 days of culture, immature NK cell colonies were individually picked, pooled and incubated in a hanging drop culture for 20 h with fetal thymus lobes from BDFl (Ly5.2) mice. The lobes were then cultured on filter membranes for I0 days. Cells harvested from the lobes were stained with monoclonal antibodies specific for molecules indicated in each FACS profile. liver cells. Low density (<1.0770), lineage marker-negative, c-kit'sca- 1' cells were prepared from day 14 murine fetal liver. We have previously shown that colony-forming cells can be enriched by this method [5]. The cells were plated in methylcellulose media with 1L-2. IL-7 and SF. As shown in Table 1, immature NK cell colonies developed although their frequencies were low. In an effort to increase the frequency of the colonies, we found that addition of IL-11 to the culture increases the frequency of colony-forming cells (Table 1). More interestingly, mixed colonies consisting of diffuse round cells characteristic of immature NK cells and other types of cells were also detected (Fig. 4). Table 1 shows the results of a representative study. In the same table, colony-forming ability of the fetal thymocytes is also shown. To confirm the clonality of the mixed colonies, we next plated the c-kit'sca-1' cells individually by micromanipulation into methylcellulose media containing IL-2, IL-7, IL-11 and SF. From 100 individually plated cells, 12 mixed NK cell colonies developed after 18 days of culture. This study established the clonal origin of the mixed NK cell colonies. To characterize the cells in the mixed colonies in more detail, each colony was lifted individually from the culture and divided into two aliquots. Cells in one aliquot were examined for the expression of B220 and Thy-1 by flow cytometry. All colonies contained Thy-1' B220- cells. Eleven out of the 12 colonies contained B220' cells, suggesting that most of the colonies have B lineage cells. Representative fluorescence-activated cell sort profiles were shown in Figure 5. Cells in the other aliquot of the colonies were subjected to May-Grunwald Giemsa staining for examination of myeloid lineage cells. All mixed colonies contained macrophages; two out of the 12 contained mast cells. These results indicated that most of the mixed Table 1. Colun! tornidtion from feial Incr and li'tdl th!mus cells Cells plated # of Colonies Type #/Dish Cytokines Mixed NK PureNK Pre-B M Mast M-mast Sca-l'c-kit'FL 50 IL-2, IL-7, SF 0 1t1 3+1 7+2 1t1 2t1 IL-2, IL-7, L ll, SF 7+1 2+1 4+1 721 1t1 2t1 FT 1,000 IL-2, IL-7, SF 0 1121 0 0 2t1 0 IL-2, IL-7, IL-I 1, SF 0 13t1 0 0 3t2 0 Sca- I'c-kit' FL cells were prepared as described in Materials and Methods. Cells were cultured for 18 days in methylcellulose media in the presence of designated cytokines. Data represent mean + SD of quadruplicate cultures. Abbreviations: Mixed NK: mixed NK colonies; Pure NK: NK cell colonies; Pre-B: Pre-B cell colonies; M: macrophage colony; mast: mast cell colony; M- mast: macrophage-mast cell mixed colony; FL: fetal liver; FT: fetal thymus.

Aiba, Ogawa 197 NK cell colonies contain immature K, B and myeloid cells and suggested that the colonies are derived from lymphohematopoietic progenitors. DISCUSSION Although much effort has been made to clarify the pathway and regulation of NK cell development, there are still questions to be resolved in NK cell ontogeny. This is mainly because of the lack of a clonal culture system which supports the growth and development of NK cells from uncommitted progenitors. Here, we report the establishment of a clonal culture which supports the growth of immature NK cells, B cells, macrophages and/or mast cells from single hematopoietic progenitors of murine fetal liver. Recently, several reports suggested that NK, T and B cells arise from common lymphoid progenitors [lo-141. However, because of the lack of a clonal culture which supports the growth of multiple lymphoid lineage cells simultaneously, the concept of common lymphoid progenitors was not established unequivocally. The culture system reported here will greatly facilitate identification of the common lymphoid progenitors. Our study revealed that fetal thy- Figure 4. A photomicropraph of a mixed NK cell colony. One quarter of a representative mixed NK cell colony seen on an inverted microscope is shown. Thy- 1 Figure 5. Representative FACS profiles of colonies derived from single cells. Sca-l'c-kit' cells were prepared from fetal liver and were plated individually by micromanipulation into methylcellulose media containing IL-2, 1L-7, IL-I1 and SF. After 18 days of culture, colonies were identified in situ and picked individually. Cells from each colony were stained with PE-anti-B220 and FITC-anti-Thy-I and analyzed by FACS Vantage. Left: a mixed NK colony with B lineage cells. Right: a mixed NK colony without B lineage cells. mocytes cannot form mixed colonies containing NK and B lineage cells. This appears to indicate that fetal thymi contain committed NK and B cell progenitors but not common NK/B progenitors. Alternatively, very few thymocytes have the potential to develop to both NK and B cells and our culture is not sensitive enough to detect the few common progenitors. Regardless, our culture system will now allow direct study of the mechanisms regulating commitment of multipotent progenitors to NK cell lineage. ACKNOWLEDGMENT We thank Dr. Haiqun Zeng for assistance in cell sorting and Dr. Pamela N. Pharr and Mrs. Anne G. Leary in preparation of this manuscript.

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