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The Journal of Immunology, 1998, 160: 744-753.
Copyright © 1998 by The American Association of Immunologists

Natural Killer Cell Development and Function Precede {alpha}ß T Cell Differentiation in Mouse Fetal Thymic Ontogeny1

James R. Carlyle, Alison M. Michie, Sarah K. Cho and Juan Carlos Zúñiga-Pflücker2

Department of Immunology, University of Toronto, Toronto, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Natural killer (NK) cells mediate MHC-unrestricted cytolysis of virus-infected cells and tumor cells. In the adult mouse, NK cells are bone marrow-derived lymphocytes that mature predominantly in extrathymic locations but have also been suggested to share a common intrathymic progenitor with T lymphocytes. However, mature NK cells are thought to be absent in mouse fetal ontogeny. We report the existence of thymocytes with a mature NK cell phenotype (NK1.1+/CD117-) as early as day 13 of gestation, approximately 3 days before the appearance of CD4+/CD8+ cells in T lymphocyte development. These mature fetal thymic NK cells express genes associated with NK cell effector function and, when freshly isolated, display MHC-unrestricted cytolytic activity in vitro. Moreover, the capacity of fetal thymic NK cells for sustained growth both in vitro and in vivo, in addition to their close phenotypic resemblance to early precursor thymocytes, confounds previous assessments of NK lineage precursor function. Thus, mature NK cells may have been inadvertently included in previous attempts to identify multipotent and bipotent precursor thymocytes. These results provide the first evidence of functional NK lymphocytes in mouse fetal ontogeny and demonstrate that NK cell maturation precedes {alpha}ß T cell development in the fetal thymus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Migration of fetal liver-derived hemopoietic precursors to the early fetal thymic rudiment occurs by day 12 of mouse gestation. The earliest hemopoietic cells to colonize the thymus, thymic lymphoid progenitors (TLPs),3 are multipotent lymphoid-committed precursors capable of giving rise to the B, T, thymic dendritic, and NK cell lineages but lack significant potential for myeloid and other hemopoietic cells (1, 2). Soon after exposure to the thymic microenvironment, these precursors rapidly lose B lymphoid potential and become committed to the T/NK lineages (3). Subsequently, a wave of thymocyte differentiation is established in the fetal thymus, marked by the ordered appearance of various developmental stages along the pathway to mature T cells (1, 2, 4). In fetal thymic ontogeny, the development of a defined subset of {gamma}{delta} T cells precedes that of conventional {alpha}ß T cells (5). However, the ordered appearance of NK cells remains unknown within the context of thymocyte development, and functional NK cells are thought to be absent in mouse fetal ontogeny.

NK cells are responsible for mounting MHC-unrestricted cytolysis of virus-infected and transformed cells (6, 7, 8, 9). The development of mature peripheral {alpha}ß and {gamma}{delta} T cells is thymus-dependent and does not occur efficiently in mice that fail to develop a proper thymic epithelium (nu/nu, or nude mice) due to a defect in expression of the winged-helix nude (whn) gene (10, 11). However, the development of NK cells is thymus independent, and these cells are present at normal to elevated levels in athymic nude mice as well as in mice defective in the ability to rearrange genes encoding the Ag receptors (severe combined immune deficiency (SCID) and RAG-deficient mice) (12, 13, 14, 15). Nonetheless, in addition to peripheral sites for NK lymphopoiesis, NK cell development can occur within the thymus, and these cells have been suggested to share a common thymic progenitor with T lymphocytes within the TLP population (1, 2, 16, 17, 18, 19). Previous studies provided evidence for, but failed to define, a proposed bipotent thymic progenitor for T and NK cells (16, 20, 21, 22); additionally, these reports did not outline the earliest stages of NK cell development in fetal ontogeny. Instead, these investigations demonstrated that various purified populations of thymocytes can give rise to either T or NK cells under different in vitro or in vivo conditions (16, 20, 21, 22). Importantly, none of these studies addressed the possibility that NK cells derived from populations of precursor thymocytes, upon i.v. injection or in vitro culture, represented an outgrowth of an already existent subset of mature NK cells.

To investigate these questions, we analyzed day 13 to 15 mouse fetal thymocytes, which contain precursors for all lymphoid lineages, but no mature {alpha}ß T or B lymphocytes, and have an overall CD3-/CD4-/CD8- triple-negative (TN) phenotype (1, 2, 4). Recently, we reported the identification of a novel population of thymocytes that serve as common committed progenitors for T and NK lymphocytes (3). These precursors display both the NK1.1 molecule (NKR-P1C) of NK cells (23, 24) as well as the CD117 (c-kit) molecule characteristic of hemopoietic precursors (17, 25, 26, 27). We now report the identification of NK1.1+ thymocytes with a mature NK cell phenotype, lacking expression of CD117. These fetal thymic NK cells are evident as early as day 13 to 14 of gestation, express genes associated with NK cell effector function, and display MHC-unrestricted cytolytic activity directly ex vivo. Strikingly, despite the above functional characteristics and lack of CD117 surface expression, these mature NK cells possess a composite phenotype similar to early precursor thymocytes, including a CD44+ (Pgp-1), CD25- (IL-2R{alpha}), CD16/32+ (Fc{gamma}RIII/II), CD24low (HSA), CD90+/- (Thy-1), CD122+ (IL-2Rß), CD2+/- (LFA-2), CD5- (Ly-1), and TN phenotype. Many of these characteristics have been previously used in an attempt to define bipotent T/NK precursor cells as well as early T lineage precursors (16, 22). We now demonstrate directly that fetal thymic NK cells are capable of substantial growth, both in vitro and in vivo, contributing significantly to the NK cell reconstitution potential of precursor-phenotype thymocytes upon adoptive transfer. Thus, NK cell progenitor activity reported in previous studies that failed to exclude fetal thymic NK cells may have stemmed from an outgrowth of pre-existing mature NK cells, in addition to bona fide NK cell precursor activity. These results indicate that the early fetal thymus is completely capable of supporting NK lineage development and that NK cell maturation and function precede {alpha}ß T cell differentiation in mouse fetal ontogeny.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Mice

Timed-pregnant C57BL/6 and Swiss.NIH mice were obtained from the National Cancer Institute, Frederick Cancer Research and Development Center (Frederick, MD). RAG-2-/- mice were bred and maintained in our animal facility.

Flow cytometric analysis and cell sorting

Single-cell suspensions were stained for surface expression of various markers using FITC-, Cychrome-, APC-, phycoerythrin (PE)-, or Red-613-conjugated mAbs obtained from PharMingen (San Diego, CA) or Life Technologies (Bethesda, MD), respectively, in staining buffer (Hank’s-buffered saline (HBS) with 1% BSA and 0.05% NaN3). Cells were stained in 100 µl for 30 min on ice and washed twice before analysis. Stained cells were analyzed with FACScan or FacsCalibur flow cytometers using Lysis II or CellQuest software (Becton Dickinson; Mountain View, CA); data was live-gated by size and lack of propidium iodide uptake. All plots display 10,000 events contoured at 50% (log), except in Figure 5Go, where dot plots show 20,000 events. Events contained in each quadrant are given as percentages in the upper right corner.



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  		FIGURE 5. Precursor-phenotype fetal thymocytes sorted according to NK1.1 expression show distinct reconstitution potential upon in vivo adoptive transfer. a, FACS of CD24/CD25-depleted day 15 Sw (H-2q) FT for NK1.1-/CD44+ (R1, 27%) and NK1.1+/CD44+ (R2, 12%) cells. Cells (1 x 105) were injected i.v. into sublethally irradiated (750 cGy) adult RAG-2-/- mice (H-2b). b, Spleen cells from reconstituted or nonreconstituted (Control) mice were analyzed 3 wk after i.v. injection of sorted NK1.1+ and NK1.1- subsets of CD44+/CD25- thymocytes. Flow cytometric analysis of NK1.1 vs H-2Kq and CD45R (B220) vs H-2Kq reveals that both populations of precursor-phenotype thymocytes give rise to donor-derived (H-2Kq+) NK cells upon adoptive transfer, yet only the multipotent NK1.1- subset is capable of generating B lymphocytes, as revealed by CD45R expression on NK1.1- donor cells.

 
CD24low/CD25- day 15 fetal thymocytes, fetal liver, and adult RAG-2-/- thymocytes were obtained by Ab/complement-mediated lysis. Single-cell suspensions were incubated on ice with 50 to 100 µl of J11d.2 (anti-CD24) and 100 to 500 µl of 7D4 (anti-CD25) culture supernatant for 15 min, followed by the addition of medium plus a 1/10 dilution of Low-Tox rabbit complement (Cedar Lane, Hornby, ON) to a final volume of 3 ml, and cells were incubated at 37°C for 30 min. After complement-mediated lysis, viable cells were recovered by discontinuous density gradient centrifugation over Lympholyte-M (Cedar Lane). CD24low/CD25- cells represented ~4% of total day 15 fetal thymocytes. For cell sorting, fetal thymus single-cell suspensions were prepared and stained for FACS as described above, except that no NaN3 was added to staining buffer. Cells were sorted using a Coulter Elite cytometer (Hialeah, FL); sorted cells were 98 to 99% pure, as determined by postsort analysis.

RT-PCR analysis

CD24low/CD25- cell suspensions from day 15 fetal thymocytes, day 15 fetal liver, and adult RAG-2-/- mice were prepared as described above. Total RNA was obtained from cell pellets using the Trizol RNA isolation protocol (Life Technologies). RNA was resuspended in 25 µl diethyl-pyrocarbonate (DEPC)-treated (0.1%) dH2O, and residual genomic DNA was digested using RNase-free DNase (Boehringer Mannheim, Indianapolis, IN). RNA was re-extracted using the Trizol protocol and resuspended in 25 µl DEPC-treated dH2O. cDNA was prepared from 1 µg of each RNA using random hexamer primers and the cDNA Cycle kit (Invitrogen, San Diego, CA). Subsequent PCR analysis was performed using titrations of cDNA in a 1/5 dilution series in dH2O. dH2O and RT reactions done in the absence of avian myeloblastosis virus (AMV) reverse transcriptase were included as negative controls. PCR was performed for 30 s at 94°C, 45 s at 50 to 55°C (depending on primer Tm’s), and 30 s at 72°C for 32 cycles, with a hot start at 94°C for 2 min and a final extension at 72°C for 3 min, using annealing temperatures specific for primer pairs as determined using the OLIGO program (NBI Software, Plymouth, MN). All PCR reactions for each group were performed using the same cDNA batches as shown for ß-actin, and all PCR products correspond to the expected molecular size. Gene-specific primers used for PCR are as follows (5'->3'): ß-actin 5', GAT GAC GAT ATC GCT GCG CTG; ß-actin 3', GTA CGA CCA GAG GCA TAC AGG; NKR-P1 (genes 2, 34, 40) 5', AAG GTA CAC ATT GCC AGA CAT; NKR-P1A (gene 2) 3', GTA GAC ATG GCT CAG TGA TTG; NKR-P1B (gene 34) 3', GGA CAG GGG AGA GAT GGA GAT; NKR-P1C (gene 40, NK1.1) 3', GAG TCA ACG AAT GGA AAG GAA; Ly-49A 5', TTC TGC TTC CTT CTT CTG GTA; Ly-49A 3', TGT GTT CAA GGC AAG TTT AGA; Ly-49C 5', AGA CCA GAA AAA CGC CAA CTT; Ly-49C 3', TTC ACT GTT CCA TCT GTC CTG; perforin 5', ATG TTC CCC AGT CGT GAG AGG; perforin 3', AAG GTG GAG TGG AGG TTT TTG; CD95L (Fas-ligand) 5', AAG AGA ACA GGA GAA ATG GTG; CD95L 3', AGA TTT GTG TTG TGG TCC TTC.

51Cr-release cell-mediated cytotoxicity assay

Single-cell suspensions from freshly isolated day 15 fetal thymocytes from timed-pregnant C57BL/6 mice and adult RAG-2-/- mice were sorted for a CD3-/CD90+ (Thy-1) phenotype with or without NK1.1 expression. Sorted cells were assayed for cytolytic activity using a standard 51Cr-release assay (28). Sorted NK1.1+/CD90+/CD3- or NK1.1-/CD90+/CD3- cells were washed twice and aliquoted at different effector to target ratios in 100 µl of culture medium (DMEM medium supplemented with 12% FCS, 2 mM glutamine, 10 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml gentamicin, 110 µg/ml sodium pyruvate, 50 µM 2-ME, and 10 mM HEPES, pH 7.4). Target YAC-1 or EL4 cells were labeled with 51Cr for 1 h and used at 3 x 103 cells in 100 µl per well (U-bottom, 96-well plates). Cells were mixed at the indicated E:T ratios; then plates were centrifuged for 30 s and placed in culture for 4 h at 37°C. An amount equal to 100 µl of culture supernatant was collected and measured in a gamma counter. Supernatant from target cells cultured alone or target cells plus 1% SDS gave the spontaneous or maximal release counts, respectively. Spontaneous release was <10% of maximal release. Counts obtained from culture supernatants at different E:T ratios (experimental release) were used to determine percent specific lysis, as previously described (28).

In vivo adoptive transfer

CD24/CD25-depleted day 15 fetal thymocytes from Sw mice were sorted for NK1.1-/CD44+ (CD117+) and NK1.1+/CD44+ (CD117-) cells. Sorted cells (105 of each) were washed twice and resuspended in 300 µl of culture medium, then injected into the tail vein of sublethally irradiated (750 cGy) adult RAG-2-/- mice. Mice were killed by cervical dislocation 3 wk later and tissues were harvested for analysis. Single-cell suspensions of spleen, thymus, lymph node, and bone marrow were analyzed by flow cytometry.

FTOC reconstitution

CD24/CD25-depleted day 15 fetal thymocytes were sorted for NK1.1-/CD117+ and NK1.1+/CD117- cells. Lymphocyte-depleted thymic lobes were prepared by culturing day 15 fetal thymic lobes from timed-pregnant Sw mice in FTOC medium containing 1.35 mM dGuo, as previously described (29, 30). After 5 to 6 days, dGuo-containing medium was replaced with FTOC medium for one day; then lobes were rinsed twice, resuspended in 10 µl medium, and placed in Terasaki plates at two lobes (one thymus) per well. Sorted donor cells (1–3 x 103 of each) were washed twice with medium before reconstitution, resuspended in 20 µl medium, and added to dGuo-treated alymphoid fetal thymic lobes in Terasaki plates. After adding donor cells or medium alone, the plates were inverted ("hanging drop"), and cultures were incubated at 37°C in a humidified incubator containing 5% CO2 in air for 24 to 48 h. Lobes were then transferred to standard FTOC for 10 to 12 days. Cell suspensions from reconstituted thymic lobes were analyzed by flow cytometry.

OP9 stromal cell line coculture

In parallel with FTOC reconstitutions, CD24/CD25-depleted day 15 fetal thymocytes were sorted for NK1.1-/CD117+ and NK1.1+/CD117- cells. Sorted cells (1–3 x 103 of each) were cocultured for 11 days on confluent monolayers of OP9 cells (31, 32) in medium containing IL-3, IL-6, IL-7, and SCF (50 ng/ml of each cytokine), then stimulated with LPS (10 µg/ml) and IL-7 for 4 to 6 days before harvesting for flow cytometry.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Mature NK cells (NK1.1+/CD117-) are present early in mouse fetal thymic ontogeny

To outline the developmental appearance of the NK cell lineage in fetal thymic ontogeny, we analyzed expression of the NK cell marker, NK1.1 (NKR-P1C) (23, 24), on day 13 to 16 fetal thymocytes and fetal liver-derived hemopoietic cells. The existence of NK1.1+ cells during fetal ontogeny has been controversial because NK1.1 expression was reported to be absent in the fetal thymus (16, 17, 33, 34), although earlier investigations had suggested that this was not the case (28, 35). Figure 1Go shows that a significant percentage of total thymocytes display detectable NK1.1 expression as early as day 13 of gestation. We recently reported the identification of a novel precursor phenotype that marks a developmental stage of thymocyte lineage commitment to the T and NK cell fates in early fetal thymic ontogeny (3). These progenitors coexpress NK1.1 and the receptor for SCF, CD117 (c-kit), which is characteristic of hemopoietic precursors in the fetal liver, bone marrow, and thymus (17, 25, 26, 27). As shown in Figure 1Go, NK1.1+/CD117+ cells represent the majority of NK1.1+ cells in the fetal thymus by day 13 of gestation. However, between days 13 and 14 of gestation, approximately one day after the NK1.1+/CD117+ stage is first observed, there is an emergence of NK1.1+ thymocytes lacking expression of CD117 (Fig. 1Go), corresponding to a mature NK cell phenotype. By days 14 to 15, the NK1.1+/CD117- population predominates, and the percentage of cells coexpressing NK1.1 and CD117 diminishes thereafter. Significant expression of NK1.1 was not detected on day 13 to 16 fetal liver cells (Fig. 1Go, day 15, and data not shown), suggesting that the efficient generation of NK1.1+ cells in fetal ontogeny occurs during or after migration of hemopoietic precursors from the fetal liver to the thymus.



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  		FIGURE 1. Identification of lymphocytes with an NK1.1+/CD117- (c-kit) mature NK cell phenotype during fetal thymic ontogeny. Two-parameter flow cytometric analysis of cell surface expression of NK1.1 vs CD117 on fetal thymocytes from timed-pregnant C57BL/6 mice (days 13, 14, 15, 16 of gestation), fetal liver cells (day 15 of gestation), and thymocytes from adult mice (8 wk old). NK1.1+ cells lacking CD117 expression are first detectable in the fetal thymus between days 13 and 14 of gestation.

 
Fetal thymic NK cells resemble early precursor thymocytes

CD24 (HSA) and CD25 (IL-2R{alpha}) are two markers frequently used to discriminate later T lineage differentiation stages from TLPs, which are CD24low/CD25-. Figure 2Go shows day 15 fetal thymocytes and fetal liver cells before and after anti-CD24 (J11d.2) and anti-CD25 (7D4) depletion. Day 15 fetal thymocytes were chosen because these cells contain no mature {alpha}ß T or B lymphocytes, possess an overall CD3-/CD4-/CD8- TN phenotype (36, 37), and contain a significant population of mature NK cells (NK1.1+/CD117-, Fig. 1Go). Postdepletion analysis verified that populations were >98% depleted of both CD24high and CD25+ cells (M1/69, anti-CD24; 3C7, anti-CD25; Fig. 2Goa and data not shown); CD24low/CD25- cells represented ~4% of total day 15 fetal thymocytes. Although CD24/CD25 depletion of fetal thymocytes and fetal liver cells enriched for CD24low cells expressing high levels of CD117 (Fig. 2Goa), we noticed a significant increase in CD117-/CD24- cells among depleted fetal thymocytes but not fetal liver cells (Fig. 2Goa). Therefore, depleted cells were analyzed further for expression of various lymphocyte differentiation markers.



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  		FIGURE 2. Fetal thymic NK cells phenotypically resemble early precursor thymocytes. a, Flow cytometric analysis of cell surface expression of CD117 vs CD24 on day 15 fetal thymocytes and fetal liver cells before and after Ab/complement-mediated depletion of CD24high (HSA, J11d.2) and CD25+ (IL-2R{alpha}, 7D4) cells. b, Two-parameter analysis of CD24low/CD25- fetal thymocytes and fetal liver cells for expression of various lymphocyte differentiation markers vs CD117. The majority of CD24/CD25-depleted fetal thymocytes consist of CD117+ multipotent progenitors and CD117- mature NK cells, the latter population being absent in the fetal liver.

 
Early investigations into thymocyte differentiation revealed that precursor potential resides in a TN fraction, a designation that originally included a CD5-/low (Ly-1) phenotype (38). Recent reports have determined that the earliest precursor thymocytes are CD5low, while fetal liver-derived hemopoietic precursors possess a CD5- phenotype (39). Analysis of CD24low/CD25- day 15 fetal thymocytes and fetal liver cells for expression of CD5 vs CD117 confirmed that among the CD117+ and CD24low populations, which have been shown to possess multilineage precursor potential, fetal thymocytes express higher levels of CD5 than fetal liver cells (Fig. 2Gob and data not shown). However, a significant population of CD5- cells lacking expression of both CD117 and CD24 was present among fetal thymocytes but absent in fetal liver cells (Fig. 2Gob and data not shown). Remarkably, this CD5-/CD117-/CD24- thymocyte population accounted for approximately 30 to 40% of CD24/CD25-depleted day 15 fetal thymocytes (Fig. 2Gob).

Previous studies have shown that early progenitor thymocytes express CD44 (Pgp-1), CD16/32 (Fc{gamma}RIII/II), and low levels of CD90 (Thy-1) (16, 17). Although the roles for these markers in lymphocyte development remain to be elucidated, they provide useful tools for the developmental staging of distinct precursor thymocytes: CD44 is present on precursor thymocytes up to and including the pro-T cell stage, when CD25 is up-regulated and commitment to the T lineage occurs (4, 18, 19, 36); high level expression of CD16/32 has been associated with a putative bipotent T/NK precursor stage, with diminishing expression after the pro-T cell stage (16, 33, 40); and the earliest precursor thymocytes bear low levels of CD90 until the pro-T cell stage, when high level expression of CD90 is attained (4). Further analysis of CD24/CD25-depleted day 15 fetal thymocytes revealed that a significant population of CD117- cells expressed both CD44 and CD16/32 (Fig. 2Gob); again, these cells were absent in fetal liver cell preparations (Fig. 2Gob). Analysis of CD90 expression demonstrated that these CD117- thymocytes were predominantly CD90high, while CD117+ precursors were CD90low to CD90- among fetal thymocyte and fetal liver cells, respectively.

Another marker that has recently been correlated to a subset of early fetal thymocytes is CD122 (IL-2Rß) (22, 41). CD122 expression has been suggested to be expressed on a population of putative bipotent T/NK precursors, as well as on the earliest thymic immigrant cells (22, 41). A subset of fetal liver cells, including the earliest committed B lineage precursors (Fraction A, CD45R(B220)+/CD43+/CD24-) (42), also express CD122 (41). Flow cytometric analysis of CD24/CD25-depleted thymocytes revealed that the majority of CD117- cells expressed CD122, while expression of CD122 was virtually absent on fetal liver cell preparations (Fig. 2Gob). Because CD122 is also present on all mature resting NK cells in the adult mouse, and the overall phenotype of the CD117-/CD24- thymocyte population resembled that of NK cells, we analyzed these cells for expression of NK1.1. As shown in Figure 2Gob, the majority (>80%) of CD117- cells among CD24/CD25-depleted thymocytes expressed NK1.1, while significant expression of NK1.1 on depleted fetal liver cells could not be detected. These NK1.1+/CD117- fetal thymocytes also express the novel pan-NK cell marker, DX5 (43), while fetal liver cells and CD117+ fetal thymocytes, including our recently described NK1.1+/CD117+ fetal thymic NK1.1+ progenitor (FTNK) cells, lack expression of DX5 (data not shown; J. R. Carlyle et al., manuscript in preparation). Further characterization of the overall phenotype of these cells was performed by multiparameter flow cytometric analysis of sorted NK1.1+ day 15 fetal thymocytes. A detailed summary of the composite phenotype of the NK1.1+/CD117- fetal thymic NK cell population is outlined in Table IGo, in comparison to our recently described fetal thymic NK1.1+/CD117+ (FTNK) T/NK-committed progenitors, and NK1.1-/CD117+ fetal TLPs (3).


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  		Table I. Phenotypic characterization of fetal thymocyte subsets grouped according to expression of NK1.1 and CD117a

 
Taken together, our results show that mature (NK1.1+/CD117-) NK cells are highly enriched among precursor-phenotype thymocyte populations in which CD117 expression is not determined (Fig. 2Go). Thus, the fetal thymus contains a subset of mature NK lymphocytes that displays some markers typical of precursor thymocytes, such as CD44 and CD16/32, yet lacks expression of numerous differentiation markers, including CD3, CD4, CD8 (i.e., TN), CD5, CD24, CD25, and other lineage (Lin) markers, such as B220, Mac-1, Gr-1, and Ter-119. To determine whether fetal thymic NK cells were indeed functionally mature, we characterized their properties further.

Fetal thymic NK cells express genes associated with NK cell effector function

The NK1.1 molecule (NKR-P1C) is a member of the NKR-P1 gene family (23, 24) and forms part of a proposed NK receptor gene complex that identifies TN lymphocytes with NK cell function (28, 44, 45, 46). To determine whether the mature NK cell phenotype of NK1.1+/CD117- fetal thymocytes was indicative of NK cell function, we assessed the expression of various genes associated with NK cell effector function by performing RT-PCR on RNA isolated from total and CD24/CD25-depleted (NK-enriched) day 15 fetal thymocytes and fetal liver cells. As a positive control, RNA was also isolated from CD24/CD25-depleted adult RAG-2-/- thymuses, which lack thymocytes beyond the early pre-T cell stage (CD44-/CD25+), yet contain normal to elevated numbers of mature NK cells (15).

Consistent with the finding that cells with a mature NK phenotype are virtually absent among fetal liver suspensions (Figs. 1Go and 2Go), no significant expression of these genes could be detected by RT-PCR on RNA isolated from day 15 fetal liver cells (Fig. 3Goa, fetal liver (FL)). In contrast, among total day 15 fetal thymocytes, low level expression of NK-related genes could be detected (Fig. 3Goa, FT), including products of the NKR-P1 gene family (NKR-P1A; NKR-P1B; and NKR-P1C, NK1.1) (23, 24), the Ly-49 gene family (Ly-49A; Ly-49C) (44, 47), Fas ligand (CD95L) (48, 49), and the cytolytic pore-forming molecule, perforin (50, 51, 52). In addition, it has been previously demonstrated that immature fetal thymocytes express the cell death-associated protease/caspase (53), granzyme B, at an early stage in ontogeny (54). To further characterize expression of these NK-related gene products, day 15 thymocyte suspensions from two unrelated (albeit NK1.1-expressing) strains of mice, Sw and C57BL/6 (B6), were isolated and enriched for mature NK cells by depleting for CD24/CD25 before RNA isolation. Consistent with the degree of NK cell enrichment observed phenotypically in Figures 1Go and 2Go, expression of NK-related genes was dramatically enhanced by CD24/CD25 depletion (Fig. 3Goa vs Fig. 3Gob, respectively, FT). As expected, CD24/CD25-depleted adult RAG-2-/- thymocytes (Fig. 3Gob, adult thymocytes (AT)) and splenocytes (data not shown) also express these NK function-associated genes. Among CD24/CD25-depleted fetal liver cells, only background expression was detectable for any of the NK-related gene products tested (data not shown); the inability to phenotypically identify significant numbers of NK cells among fetal liver suspensions limits further attempts to enrich for such cells or their immediate precursors, without employing in vitro culture techniques. Nevertheless, day 15 fetal thymocytes express numerous gene products typically associated with NK cell effector function.



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  		FIGURE 3. NK-enriched day 15 fetal thymocytes express characteristic gene products of functional NK cells. RT-PCR analysis for the expression of NK cell effector function-associated genes on RNA isolated from (a) total day 15 Sw fetal thymocytes (FT) and fetal liver (FL) cells, and (b) CD24/CD25-depleted Sw and C57BL/6 (B6) day 15 fetal thymocytes and RAG-2-/- adult thymocytes. CD24/CD25-depleted (NK-enriched) day 15 fetal thymocytes express members of the NKR-P1 and Ly-49 gene families, perforin, and Fas ligand (CD95L).

 
RT-PCR analysis of NK-enriched cell populations, although not quantitative nor conclusive, revealed a number of significant findings. In comparison to NKR-P1A and NKR-P1C levels, NKR-P1B expression was quite high among Sw fetal thymocytes, while remaining very low in fetal B6 and adult RAG-2-/- thymocytes (Fig. 3Gob). This difference appears to be strain-specific because the same trend was observed for adult spleen cells from each of the strains (data not shown). These data suggest that strain-specific expression of NKR-P1 gene family members may be controlled at the transcriptional level. In contrast, expression of Ly-49 gene family members appears to be developmentally regulated in the thymus. Expression levels of both Ly-49A and Ly-49C, in comparison with the other genes tested, were higher in adult (RAG-2-/-) thymocytes than in fetal (Sw and B6) thymocytes. This does not appear to be due to strain-specific differences, because Ly-49A/C expressions were comparable in all three strains among adult splenocytes (data not shown). Thus, it appears that in the fetal thymus, expression of Ly-49 genes may be developmentally delayed, while NKR-P1 molecules appear quite early in ontogeny. This may have important functional consequences because these two families of molecules are postulated to possess opposing regulatory roles in NK cell effector function, with Ly-49 acting as an inhibitory receptor (44, 47, 55), and NKR-P1 acting as a positive modulator of NK cell activity and lytic function (45, 55).

Freshly isolated fetal thymic NK cells display MHC-unrestricted cytotoxicity in vitro

To determine whether fetal thymic NK cells are functional, we tested the ability of freshly sorted NK1.1+ (CD117-) fetal thymocytes to perform MHC-unrestricted cytolysis of 51Cr-labeled YAC-1 target cells. Thymocytes obtained at day 15 of fetal gestation were sorted by FACS for a CD3-/CD90+ phenotype, with or without NK1.1 expression. As shown in Figure 4Go, freshly sorted NK1.1+ day 15 fetal thymocytes, without a requirement for pre-exposure to cytokines such as IFN-{gamma}, IL-2, IL-12, or IL-15, were capable of lysing NK-sensitive YAC-1 target cells but failed to lyse the NK-insensitive EL-4 cell line. As expected, freshly isolated NK1.1+ thymocytes from adult RAG-2-/- mice also lysed YAC-1 targets, while failing to lyse EL-4 cells (Fig. 4Go). In contrast, fetal thymocytes lacking expression of the NK1.1 marker failed to lyse either target, as did total (unsorted) fetal thymocytes (Fig. 4Go). The latter observation is consistent with previous attempts to detect NK cell function in freshly isolated fetal thymocytes (16, 28, 34, 35); in the absence of purification, the high frequency of NK1.1- thymocytes could inhibit NK cell cytotoxic function. Importantly, we show that freshly isolated fetal thymic NK cells possess cytolytic function at a developmental stage before the appearance of CD4+/CD8+ DP cells in fetal thymic ontogeny. Thus, functional NK cell development precedes {alpha}ß T cell differentiation in mouse fetal thymic ontogeny.



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  		FIGURE 4. Freshly isolated fetal thymic NK cells mediate MHC-unrestricted cytotoxicity in vitro. Fresh day 15 fetal thymocytes (B6) and RAG-2-/- adult thymocytes were sorted for CD3-/CD90+ (Thy-1) cells, with or without NK1.1 expression, and tested for cytotoxicity against NK-sensitive YAC-1 cells or the NK-insensitive cell line, EL-4. Sorted NK1.1+ day 15 fetal thymocytes mediate MHC-unrestricted cytotoxicity of YAC-1 targets ex vivo, in the absence of exogenous cytokine treatment, while sorted NK1.1- or total (unsorted) day 15 fetal thymocytes fail to lyse either target.

 
In vivo adoptive transfer of precursor-phenotype thymocytes

The early developmental maturity of fetal thymic NK cells, combined with their close phenotypic resemblance to early progenitor thymocytes (Fig. 2Go and Table IGo), implies that previous descriptions of purported multipotent, bipotent T/NK, or unipotent NK lineage "precursor" thymocytes, in particular those involving populations not defined according to CD117 or NK1.1 expression, may have inadvertently included pre-existing mature NK cells (16, 17, 22). To address this issue directly, we used in vivo adoptive transfers. CD44+/CD25- cells, which have been thought to contain multipotent precursors for the T, B, and NK lineages (4), were sorted from CD24/CD25-depleted day 15 fetal thymocytes (Sw mice, H-2q) and subdivided according to NK1.1 expression (Fig. 5Goa). Three weeks after i.v. injection into sublethally irradiated (750 cGy) RAG-2-/- (H-2b) host mice, tissues were examined for evidence of donor-derived (H-2Kq+) progeny. As shown in Figure 5Go, both the NK1.1+ and NK1.1- subsets of CD44+/CD25- thymocytes were capable of giving rise to donor-derived NK cells (Fig. 5Gob, NK1.1+/H-2Kq+) in the spleen. However, only the multipotent NK1.1- subset (CD117+, Fig. 2Gob) was capable of generating B cells, as determined by CD45R (B220) expression on NK1.1- donor-derived progeny (Fig. 5Gob, CD45R+/H-2Kq+). These donor-derived CD45R+ cells also expressed surface IgM (data not shown). Cells from nonreconstituted (Control) mice showed no background staining for donor class I (H-2Kq+) expression (Fig. 5Gob). The reconstitution potential of the NK1.1- subset is not limited only to B and NK cell lineages because these cells are also capable of giving rise to T cells in FTOC reconstitutions (3). However, we could find no evidence of T lineage reconstitution in the thymus upon in vivo adoptive transfer of either subset. This observation is consistent with previous studies assessing the precursor potential of fetal thymocytes upon adoptive transfer into adult host mice (18, 56). It has been suggested that this may be due to a developmental stage difference; fetal thymocytes exhibit a reduction in thymic reconstitution potential compared with their analogous "CD4low" adult counterpart upon i.v. adoptive transfer and may have difficulty homing to the adult thymus microenvironment (16, 40, 56). Therefore, we employed an in vitro FTOC reconstitution assay for T and NK cell potential. Additionally, for detecting B and NK cell potential, we employed a sensitive in vitro coculture assay using the bone marrow-derived stromal cell line, OP9 (3).

Fetal thymic NK cells are capable of sustained growth in vitro

To address the growth potential of fetal thymic NK cells in a T lineage assay, we assessed their ability to reconstitute dGuo-depleted FTOCs. CD24/CD25-depleted day 15 fetal thymocytes were sorted for NK1.1-/CD117+ (FTLP) and NK1.1+/CD117- (NK) cells, and 1 to 3 x 103 donor cells were used for FTOC reconstitution. As previously demonstrated (3), sorted NK1.1-/CD117+ (FTLP) cells gave rise to immature CD4/CD8 double-positive and mature CD4 and CD8 single-positive cells (Fig. 6Goa). In addition, FTLP cells were capable of generating mature T cells, as determined by high-level expression of {alpha}ßTCR on NK1.1- cells (Fig. 6Goa {alpha}ßTCR+/NK1.1-), as well as a few NK cells (Fig. 6Goa, {alpha}ßTCR-/NK1.1+) (3). In contrast, NK1.1+/CD117- (NK) cells remained double-negative for both CD4 and CD8 and exclusively gave rise to an outgrowth of NK cells (Fig. 6Goa). These in vitro-cultured NK cells are large granular lymphocytes. The low level staining observed for {alpha}ßTCR is due to increased background staining because we failed to detect DJß rearrangement on their DNA by PCR (data not shown). Cell yields from FTOCs indicated that fetal thymic NK cells have the capacity to expand at least 10-fold in this assay, depending on the length of the culture period.



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  		FIGURE 6. Sustained growth of fetal thymic NK cells upon in vitro culture. CD24/CD25-depleted day 15 fetal thymocytes were sorted for NK1.1-/CD117+ and NK1.1+/CD117- cells, which were cocultured under differential conditions. a, dGuo-depleted fetal thymic lobes were reconstituted with 1 to 3 x 103 cells, either NK1.1-/CD117+ or NK1.1+/CD117-, and cultured for 12 days in FTOC. Flow cytometric analysis for CD4 vs CD8 and NK1.1 vs {alpha}ßTCR expression reveals that both subsets give rise to NK cells, yet only the NK1.1- subset is capable of generating conventional {alpha}ß T cells, as indicated by high level expression of {alpha}ßTCR on NK1.1- cells. b, In parallel with FTOC reconstitutions, 1 to 3 x 103 sorted NK1.1-/CD117+ or NK1.1+/CD117- cells were cocultured for 11 days on confluent monolayers of OP9 bone marrow-derived stromal cells in the presence of cytokines (IL-3, -6, -7, and SCF), then stimulated with LPS and IL-7 for an additional 6 days before analysis. Flow cytometric analysis of CD45R (B220) vs IgM and NK1.1 vs CD90 reveals that both populations give rise to NK cells, yet only the NK1.1- subset is capable of generating B lymphocytes, as revealed by IgM expression on CD45R+ cells.

 
To examine the growth potential of fetal thymic NK cells further in a B cell assay, we seeded sorted cells onto OP9 bone marrow-derived stromal cells. In parallel with FTOC reconstitutions, 1 to 3 x 103 sorted NK1.1-/CD117+ (FTLP) and NK1.1+/CD117- (NK) cells were cocultured with confluent OP9 cells, as described previously (3). Again, as recently shown (3), sorted NK1.1-/CD117+ (FTLP) cells gave rise to mature B cells, indicated by expression of surface IgM on CD45R+ cells (Fig. 6Gob, CD45R+/IgM+), and a few NK cells (Fig. 6Gob, NK1.1+/CD90+/-). However, NK1.1+/CD117- (NK) cells remained negative for both CD45R and IgM and again gave rise to an outgrowth of mature NK cells, the majority of which expressed CD90 (Fig. 6Gob, NK1.1+/CD90+/-); these NK cells also stained positive for the DX5 mAb (data not shown). Although we have not quantitated the extent of growth potential exhibited by fetal thymic NK cells in this assay (at least 50-fold), they continue to divide in culture and can be maintained for at least 1 to 2 mo in vitro. However, they must be continually passed onto fresh OP9 cells because the OP9 cells are lysed due to the cytolytic activity of the NK cells. In addition, the growth of fetal thymic NK cells is enhanced with exogenous IL-2. Therefore, the reported existence of NK cell precursors in the fetal thymus that can grow out in vitro in the presence of IL-2 was most likely due to an expansion of this pre-existing subset of mature NK cells (22, 34, 57).

The in vivo and in vitro data shown in Figures 5Go and 6Go, together with our previous evidence (3), suggest that the NK1.1- (CD117+) subset of CD44+/CD25- thymocytes represents multipotent lymphoid-restricted precursors, while the NK1.1+ population contains mature NK cells (CD117-) that are capable of sustained growth after adoptive transfer in vivo or in vitro. Thus, there is a population of pre-existing NK1.1+ cells among precursor-phenotype fetal thymocytes that contain mature and functional NK cells capable of confounding lineage potential assays if not properly distinguished from multipotent CD117+/CD44+/CD25-/NK1.1- precursors. In light of these findings, the purported discovery of bipotent T/NK precursors requires reassessment.

Conclusions

Our data provide the first evidence of the development of mature and functional NK cells in mouse fetal ontogeny. The fact that NK cell maturation initially occurs within the early fetal thymus, together with the recent description of NK1.1+ {alpha}ß T cells (58) and the NK1.1+/CD117+ (FTNK) bipotent T/NK progenitor stage of thymocyte development (3), further reinforces the developmental and lineage relationships between T and NK cells. Moreover, we show that NK cells are phenotypically present in the fetal thymus by day 13 of gestation, before the onset of VDJ rearrangement of the TCRß locus; as well, NK cell function is detectable by day 15, before the appearance of CD4+/CD8+ cells in fetal thymic ontogeny. This indicates that NK cell development precedes that of {alpha}ß T lymphocytes. Although an analogous subset of NK cells (CD56+/CD5-) was observed in the human fetal thymus (59, 60), the earliest stages of NK cell development were not outlined, and it remains unknown during human fetal thymic ontogeny whether NK cells are present and/or functional before {alpha}ß T cell differentiation. Thus, our data are the first to show that the maturation of functional NK cells, like that of the canonical V{gamma}3+ {gamma}{delta} T cells, precedes {alpha}ß T cell development (5).

Our identification of fetal thymic NK cells, together with the inability to detect significant NK1.1 expression in the fetal liver (Figs. 1Go, 2Go, and 4Go), suggests that fetal NK cell differentiation may be restricted to the thymus until the establishment of peripheral sites of NK lymphopoiesis. This could explain why NK cells do not reach significant levels in the circulation until the neonatal stage (28, 35, 46, 61), when hemopoietic function shifts from the fetal liver to the neonatal/adult bone marrow, a site that is known to be capable of supporting NK lineage maturation (62, 63). It may be that the bone marrow is primarily responsible for peripheral NK cell production whereas the fetal liver may be incapable of supporting NK lineage differentiation, possibly due to the absence of particular cytokines or stromal microenvironments (64, 65). Although the fetal thymus is capable of supporting complete NK cell maturation, thymus-derived NK cells may be locally involved in regulation of thymopoiesis (66) and may not reach the periphery. Consistent with this, we have failed to detect significant numbers of NK cells in the fetal blood and spleen until day 16 of gestation (J. R. Carlyle, manuscript in preparation). Nonetheless, mature NK cells differentiate early during fetal thymic ontogeny, exhibit gene expression patterns consistent with NK cell effector function, and display MHC-unrestricted cytotoxicity ex vivo, without a requirement for pre-exposure to cytokines.

Importantly, the close phenotypic resemblance of fetal thymic NK cells to early precursor thymocytes implies that previous descriptions of purported NK precursor and bipotent T/NK precursor potentials may have been contaminated with these pre-existing mature NK cells. Indeed, our in vivo transfer experiments provide direct evidence that the NK1.1+ subset of CD44+/CD25- fetal thymocytes (which also expresses CD16/32) can reconstitute NK cells upon adoptive transfer. Therefore, previous findings that have used CD44, CD16/32, and/or CD122 to identify progenitor thymocytes, in particular where characterization of NK1.1 and/or CD117 is lacking, may have inadvertently included mature NK cells within a putative precursor population. Fetal thymic NK cells are capable of sustained outgrowth, both in vitro and in vivo, potentially obscuring bona fide multipotent, bipotent, and unipotent NK lineage precursor activity. Hence, investigations that failed to exclude pre-existing NK cells before assessing NK lineage potential (16, 17, 22, 33), including the purported discovery of bipotent T/NK precursors, must now be re-evaluated in light of our observations. Indeed, the early developmental expression of NK1.1 and other members of the NKR-P1 gene family suggest that such NK cell molecules might be included as lineage (Lin) differentiation markers for future hemopoietic precursor evaluations, both intrathymic and extrathymic. Our identification of mature and functional NK cells in fetal ontogeny sheds new light on our understanding of NK lineage development and function and could aid in the derivation of long-lived mouse NK cell lines.


   Acknowledgments
 
We thank Drs. Michael Julius, Michael Lenardo, Richard G. Miller, Philippe Poussier, and Hergen Spits for discussions and for critically reading the manuscript, and Cheryl Smith for technical assistance with cell sorting.


   Footnotes
 
1 This work was supported by grants from the Medical Research Council of Canada (MRC) and the National Cancer Institute of Canada (to J.C.Z.-P.), and by an MRC studentship (to J.R.C) and an MRC scholarship (to J.C.Z.-P.). Back

2 Address correspondence and reprint requests to Dr. J.C. Zúñiga-Pflücker, Department of Immunology, University of Toronto, Medical Sciences Building, Toronto, Ontario, M5S 1A8 Canada. E-mail address: Back

3 Abbreviations used in this paper: TLP, thymic lymphoid progenitor; FTLP, fetal TLP; FTNK: fetal thymic NK1.1+ progenitor; FTOC: fetal thymic organ culture; HSA: heat stable antigen; RAG: recombination-activating gene; SCF: stem cell factor; TN, triple negative; FT, fetal thymocytes; Sw, Swiss.NIH; dGuo, deoxyguanosine. Back

Received for publication August 27, 1997. Accepted for publication October 3, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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