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Originally published online as doi:10.1189/jlb.0902443 on May 22, 2003

Published online before print May 22, 2003
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(Journal of Leukocyte Biology. 2003;73:731-738.)
© 2003 by Society for Leukocyte Biology

Ly49E expression points toward overlapping, but distinct, natural killer (NK) cell differentiation kinetics and potential of fetal versus adult lymphoid progenitors

Frederik Stevenaert, Katrien Van Beneden, An De Creus, Veronique Debacker, Jean Plum and Georges Leclercq

Department of Clinical Chemistry, Microbiology and Immunology, University of Ghent, University Hospital, Belgium

Correspondence: Dr. Georges Leclercq, Department of Clinical Chemistry, Microbiology and Immunology, University of Ghent, University Hospital, Blok A, 4th Floor, De Pintelaan 185, B-9000 Ghent, Belgium. E-mail: georges.leclercq{at}rug.ac.be


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Using a new antibody, we found previously that contrary to adult natural killer (NK) cells, fetal NK cells have a unique phenotype, as they exclusively express Ly49E. This can be explained by an intrinsic different NK differentiation potential of fetal versus adult lymphoid progenitors, by immaturity of fetal NK cells or by instability of Ly49E expression. Here, we show that adult progenitor cells were still capable of differentiating into Ly49E-expressing NK cells but at a much lower frequency. Surprisingly, Ly49E expression in vitro did not require stromal cells. Kinetic analysis in vivo showed that Ly49E was expressed early, together with CD94/NKG2 and Ly49G2, followed by Ly49C, and finally Ly49D. Transfer of sorted Ly49E-positive fetal NK cells showed stable Ly49E expression, and later, part of these cells up-regulated other Ly49 members. These data indicate that although there are intrinsic differences, there is no strict fetal and adult wave of NK cell differentiation.

Key Words: cellular differentiation • repertoire development • cell-surface molecules


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Natural killer (NK) cells are able to kill a variety of tumor cells and virally infected cells without prior sensitization and proliferation [1 ]. The cytotoxicity of NK cells is regulated via expression of activating receptors and of inhibitory receptors, which block the transduction of the activating signal upon cross-linking of major histocompatibility complex (MHC) class I molecules [2 ]. In mice, two families of inhibitory NK cell receptors have been described. The Ly49 receptor family includes lectin-like, disulfide-linked homodimers encoded by 14 (Ly49A–N) highly related genes [3 4 5 ] capable of discriminating, to a limited extent, different classical MHC class I molecules [6 ]. The second family consists of CD94/NKG2 heterodimers, but only the CD94/NKG2A receptor is inhibitory [7 ,8 ]. It has been shown that CD94/NKG2 heterodimers recognize the nonclassical MHC class I molecule Qa-1b [9 ,10 ].

In mice, the different Ly49 receptors are expressed in a variegated, overlapping manner, as a single NK cell can express more than one member of the Ly49 receptor family [11 ,12 ]. It has been suggested in the "sequential expression model" that Ly49 receptors are expressed in a stochastic, successive way and that the accumulation is terminated once the cell expresses receptors with sufficient avidity for self-MHC class I molecules [12 ,13 ]. Consequently, it has been proposed that NK cells limit the number of inhibitory receptors to distinguish target cells that have undergone slight changes in MHC class I expression [14 ]. Recent studies have demonstrated that Ly49 receptors are expressed in an ordered way on developing NK cells [13 ,15 16 17 ]. Although the signals required to induce Ly49 receptor expression are poorly characterized, the absolute requirement of bone marrow (BM) stromal cells for Ly49 expression in vitro [15 16 17 ] and of the BM microenvironment for complete maturation of NK cells in vivo has been demonstrated [18 ,19 ].

Recently, using the Ly49E/C-specific 4D12 and the NKG2-specific 3S9 monoclonal antibodies (mAb) [20 ], both developed in our laboratory, we could demonstrate that fetal NK cells express Ly49E and that it is the only member of the Ly49 receptor family present on these cells [20 ]. In addition, we and others [20 ,21 ] could show that approximately 90% of freshly isolated fetal NK cells express high levels of CD94/NKG2. Starting from 1–2 weeks after birth, expression of other members of the Ly49 family is detected [13 ], and expression of Ly49E [20 ] and CD94/NKG2 [22 ] strongly decreases. At 6–8 weeks, adult Ly49 expression levels are reached [13 ]. Functionally, fetal NK cells are also distinct from adult NK cells, as fetal NK cells shortly cultured in interleukin (IL)-2 or fetal NK clones are not able to discriminate between wild-type and MHC class I-deficient lymphoblasts [23 ,24 ]. However, some wild-type and class I-deficient tumor cells are differentially lysed [24 ]. A possible explanation for these phenotypic and functional differences between NK cells from fetal and adult mice could be the existence of a fetal and adult wave of NK cell development, where the Ly49E receptor can only be expressed by differentiating fetal progenitors and no longer by adult progenitors, analogous to the developmentally regulated differences described for erythroid lineages as well as for T and B lymphoid lineages. Another possibility is that fetal Ly49E+ NK cells are immature NK cells that have not yet expressed other Ly49 receptors but still have the potential to do so or that Ly49E expression is not stable.

In this study, we have addressed these questions by comparing the NK cell differentiation potential of fetal day (FD) 13 liver (FL) and adult BM lymphoid-committed progenitor cells using a previously described in vitro NK cell differentiation model [16 25 ] and in vivo repopulation studies in RAG2/{gamma}c-/- mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
C57BL/6J (B6) mice were originally purchased from Harlan Netherlands (Zeist). Mice were bred in our breeding facility. To obtain dated pregnant mice, B6 mice were mated for 15 h, and fetuses were removed at FD13 (plug date=day 0). RAG2/{gamma}c-/- mice were obtained from the Nederlands Kanker Instituut (Amsterdam).

Antibodies
mAb used for labeling were anti-NK1.1 [fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, and biotin-conjugated, clone PK136], anti-CD3 [FITC-, allophycocyanin (APC)-, and peridinin chlorophyll protein (PerCP)-conjugated, clone 145-2C11], anti-c-kit (CD117; PE-conjugated, clone 2B8), anti-Ly49A (PE-conjugated, clone A1), and anti-Ly49C/I (PE- and biotin-conjugated, clone 5E6), all obtained from PharMingen (San Diego, CA); anti-Ly49E/C (FITC-conjugated, clone 4D12) [20 ] and anti-Ly49C [biotin-conjugated, clone 4LO3311, further referred to as 4LO; kindly provided by Dr. Suzanne Lemieux, Institute Armand-Frappier, Quebec, Canada]; anti-Ly49A/D (biotin-conjugated, clone 12A8; kindly provided by Dr. John R. Ortaldo, National Cancer Institute, Frederick, MD); anti-Ly49G2 [FITC- and biotin-conjugated, clone 4D11; obtained from the American Type Culture Collection (ATCC), Manassas, VA]; anti-NKG2A/C/E (FITC- and biotin-conjugated, clone 3S9) [20 ] and anti-Sca2 (FITC-conjugated, clone MTS-35; kindly provided by Dr. Richard Boyd, Monash University Medical School, Australia); and anti-Fc receptor for immunoglobulin G (IgG; Fc{gamma}R)II/III (unconjugated, clone 2.4G2; kindly provided by Dr. Jay Unkeless, Mount Sinai School of Medicine, New York, NY). Biotin-conjugated mAb used for depletion were anti-B220 (clone RA3-3A1) and anti-CD11b (clone M1/70; obtained from ATCC), anti-CD2 (clone 12.15A; kindly provided by Dr. Bruno Kyewski, FSP Tumor Immunology Program, Heidelberg, Germany), anti-Gr1 (clone Rb6-8C5; kindly provided by Dr. Barbara Fazekas de St. Groth, Centenary Institute of Cell Biology and Cancer Medicine, Sydney, Australia), anti-Ter119 (clone TER119; kindly provided by Dr. B. Fazekas de St. Groth, Centenary Institute of Cell Biology and Cancer Medicine), and anti-CD4 (clone L3T4; kindly provided by Dr. Michel Pierres, Centre d’Immunologie, Marseille, France). Isotype controls used were mouse IgG2a (PE-conjugated, clone X39), rat IgG2a (FITC-conjugated), and rat IgG2b (FITC-conjugated; all obtained from Becton Dickinson, San Jose, CA). Second-step reagents used were streptavidin-APC and -PE (Becton Dickinson, Mountain View, CA) and streptavidin Tri-color conjugate (Caltag Laboratories, Burlingame, CA).

Preparation of cell suspensions
B6 FD13 livers were removed, and cell suspensions were obtained by passage of the tissue through a 26G needle. Adult BM cells were isolated from 8- to 12-week-old B6 mice by irrigation of the femurs and tibias, followed by purification with Lympholyte®-M (Cedarlane, Hornby, Canada). Spleens of RAG2/{gamma}c-/- mice were removed and disrupted, and RAG2/{gamma}c-/- BM cells were obtained as described above. Erythrocytes from spleens and BM were lysed with 0.17 M NH4Cl. Thymuses from FD17 B6 mice were removed and disrupted using a small potter homogenizer.

Sorting
Freshly isolated FL and adult BM cells were incubated with biotinylated mAb Ter119, CD2, B220, Gr1, CD11b, and NK1.1 [lineage (Lin) markers] for 45 min at 4°C. Freshly isolated FD17 thymocytes were incubated with biotinylated mAb CD4 for 45 min at 4°C. Cells were washed twice, and Lin+ cells and CD4+ thymocytes were depleted using Dynabeads M-280 streptavidin (Dynal, Hamburg, Germany). Freshly isolated splenocytes from 11-day-old mice were incubated with MACS anti-NK (DX5) microbeads (Miltenyi Biotec, Auburn, CA) for magnetic separation. To avoid aspecific binding, the Fc{gamma}R was blocked by preincubation of the cells with saturating amounts of anti-Fc{gamma}RII/III mAb. Fetal liver and adult BM cells were stained with c-kit PE, Sca2 FITC, and streptavidin-Tri-color conjugate. Fetal thymocytes were stained with NK1.1 PE, CD3 APC, and 4D12 FITC. The DX5-enriched cells were stained with NK1.1 PE, CD3 PerCP, 4D12 FITC, 5E6 biotin, and streptavidin-APC. The cells labeled with the different mAb were sorted on a FACSVantage (Becton Dickinson Immunocytometry Systems, Mountain View, CA) with the CellQuest software program (Becton Dickinson Immunocytometry Systems).

In vitro culture conditions and flow cytometric analysis
Sorted c-kit+Sca2+Lin- cells were cultured in 96-well U-bottom plates (Falcon, San Jose, CA) at 10,000 cells/well in complete RPMI medium (RPMI-1640 medium supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, 1x nonessential amino acids, and 5x10-5 M 2-mercaptoethanol; all obtained from Life Technologies, Paisley, UK) and a mixture of 0.5 ng/ml human IL-7 (Immunex, Seattle, WA), 30 ng/ml mouse stem cell factor (SCF; R&D Systems, Minneapolis, MN), and 100 ng/ml human Flt3 ligand (Flt3L; R&D Systems). The cells were refed after 3 days with the same medium. On day 5, the cells were harvested, counted, and replated at 50,000 cells/well in complete RPMI medium containing 25 ng/ml human IL-15 (R&D Systems). At different points of time, the cultures were harvested and incubated with combinations of mAb mentioned above. The Fc{gamma}R was blocked by preincubation of cells with saturating amounts of anti-Fc{gamma}RII/III mAb to avoid aspecific binding. The cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems) with the CellQuest software program for data acquisition and analysis.

In vivo repopulation studies
Sixty thousand to 100,000 sorted c-kit+Sca2+Lin- cells from FL and from adult BM and 10,000–20,000 sorted NK1.1+CD3-4D12+ or NK1.1+CD3-4D12- cells from FD17 thymus were injected intravenously (i.v.) in RAG2/{gamma}c-/- mice, aged 2–3 months, which were irradiated (300 cGy) with a cobalt radiation source 24 h before injection. At different points of time thereafter, the mice were killed, cell suspensions were prepared from BM and spleen, and cell suspensions were incubated with various combinations of mAb. The Fc{gamma}R was blocked by preincubation of cells with anti-Fc{gamma}RII/III mAb.

Reverse transcriptase-polymerase chain reaction (RT-PCR)
Trizol (Life Technologies) was added to sorted c-kit+Sca2+Lin- cells and cultured cells, and RNA was extracted according to the manufacturer’s instructions. Before RT, digestion of DNA was performed with DNase I (Life Technologies). cDNA was synthesized with oligo(dT) as primer using the Superscript kit (Life Technologies). For semiquantitative RT-PCR, three threefold dilutions of each cDNA were amplified. Oligonucleotides used for hypoxanthine guanine phosphoribosyl transferase (HPRT), a housekeeping enzyme, CD94, and Ly49E were described before [23 ,24 ]. For NKG2A/C/E, oligonucleotides used were AGA AAT CTT GGA ATG ACA GTT TGG (sense primer) and TCA AGT GGG AGA TTT ACA CTT ACA AAG ATA TGG (antisense primer). PCR amplification was performed using a PTC-200 DNA engine (MJ Research, Boston, MA) with 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min (HPRT); 35 cycles of 94°C for 30 s, 61°C for 30 s, and 72°C for 1 min (Ly49E); 40 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min (CD94); and 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min (NKG2A/C/E).

DNA analysis
Freshly isolated, total splenocytes, sorted 4D12+5E6- NK cells, and 4D12-5E6+ NK cells from 11-day-old mice were submitted to DNA analysis using propidium iodide (PI) as described before [26 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Ly49E and NKG2/CD94 during in vitro differentiation of fetal liver and adult BM progenitor cells
Sorted progenitor cells with a c-kit+Sca2+Lin- phenotype from FL and from adult BM were cultured in medium containing SCF, Flt3L, and IL-7 for 5 days, which led to an eight- to tenfold increase in cell number. This primary culture was followed by 6–7 days culture in medium containing IL-15 alone (secondary culture) as described before [16 ,25 ]. No stromal cells were added to the primary or secondary culture. Semiquantitative RT-PCR for Ly49E, NKG2A/C/E, and CD94 was performed on isolated RNA from freshly sorted progenitor cells and from cells harvested after primary and secondary culture. Freshly sorted progenitor cells were negative for Ly49E, CD94, and NKG2 mRNA (Fig. 1 ) and for Ly49 and NKG2 protein expression on the cell surface (data not shown). After primary culture, however, the cells became positive for CD94 and NKG2 mRNA, and expression levels were similar for both populations. Using the NKG2-specific 3S9 mAb, we consistently found a small population of cells expressing NKG2 molecules in the NK1.1- subpopulation of both primary NK progenitor cultures (Fig. 2 ). We could not detect cell-surface expression of Ly49E (Fig. 2) or of any other Ly49 family member (data not shown). Surprisingly, high expression levels of Ly49E mRNA were found in FL-derived cells, and in contrast, only a very weak expression level could be detected in BM-derived cells. Although the primers used in Figure 1 only amplify the 3' half of Ly49E, we could also show that when primers were used that span the complete coding sequence, a specific transcript of the correct length (830 bp) was obtained. Sequencing this complete transcript showed that it was identical to the sequence of Ly49E (data not shown). This shows that the absence of detectable protein is not a result of alternative splicing.



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  		Figure 1. In vitro-cultured fetal and adult lymphoid progenitor cells express CD94, NKG2, and Ly49E mRNA. c-Kit+Sca2+Lin- progenitor cells were sorted from FL and BM and were cultured for 5 days in IL-7, Flt3L, and SCF (5d pr.c.), followed by 6–7 days of secondary culture (6d sec.c.) in IL-15 alone. RNA was extracted from freshly sorted cells and from cells after primary and secondary culture. Semiquantitative RT-PCR was performed for HPRT, Ly49E, NKG2A/C/E, and CD94. Three threefold dilutions of each cDNA were amplified. H2O and genomic DNA were used as negative controls (data not shown).

 


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  		Figure 2. Cell-surface expression of Ly49E and NKG2/CD94 on in vitro-cultured fetal and adult progenitor cells. Flow cytometric analysis was performed on sorted c-kit+Sca2+Lin- FL and BM cells after 5 days of primary culture and after 6 days of secondary culture. Cells were stained with PE-conjugated anti-NK1.1 and FITC-conjugated 3S9 (NKG2) or FITC-conjugated 4D12 (Ly49E/C) or with FITC-conjugated anti-NK1.1 and PE-conjugated 5E6 (Ly49C/I). Isotype controls for NK1.1 [mouse IgG2a, PE-conjugated], 4D12 [rat IgG2a, FITC-conjugated], and 3S9 (rat IgG2b, FITC-conjugated) were used to determine aspecific staining. The results shown are representative of at least three experiments.

 
After secondary culture, as expected, a high percentage of cells expressing NK1.1 was present. The mRNA expression levels of CD94 and NKG2 increased and were comparable for both populations (Fig. 1) , and NKG2 surface expression was mainly restricted now to NK1.1-positive cells (Fig. 2) . After secondary culture, as expected, as no stromal cells were added [16 ], the resulting NK cells did not express Ly49A, -C, -D, -I, or -G2 (data not shown). In contrast, using the Ly49E/C-specific 4D12 mAb, we clearly found positive cells. As we could not detect any Ly49C+ cells using the 5E6 mAb, all 4D12+NK1.1+-positive cells probably expressed the Ly49E receptor. Similar to the Ly49E mRNA expression after secondary culture (Fig. 1) , the surface expression frequency as well as the expression level of the Ly49E receptor were significantly different between FL- and BM-derived populations (Fig. 2) . BM progenitors only gave rise to a very small proportion (<5%) of 4D12+NK1.1+ cells, which mainly consisted of 4D12low cells. FL-derived progenitors generated a high percentage (>60%) of 4D12+NK1.1+ cells, which contained a considerable 4D12hi subpopulation.

The NKG2 primers used amplify the NKG2A, -C, and -E genes. To distinguish among these three different genes and to determine the expression level of each gene, we used MboI, Eco147I, and PvuII restriction enzymes, which have a unique restriction site in the PCR product of NKG2A, -C, and -E, respectively [10 ]. The PCR product was almost completely digested with MboI in FL and BM progenitor cells after the primary and secondary culture, indicating the preferential expression of NKG2A. After digestion with PvuII, we could also detect in FL progeny a low but significant expression of NKG2E (data not shown).

Orderly acquisition of CD94/NKG2 and Ly49 family members during NK development in vivo
In search of differences in NK differentiation potential in vivo of FL versus BM progenitor cells, both sorted populations were i.v.-injected in RAG2/{gamma}c-/- mice, which lack B, T, and NK cells. The immunodeficient RAG2/{gamma}c-/- mice allow stable hematopoietic engraftment independent of MHC barriers [27 ]. Eleven and 29 days postinjection, flow cytometric analysis was performed on splenocytes and BM cells. As the results from the flow cytometric analysis of splenocytes were comparable with those of BM cells, only the results of splenocytes are shown in Figure 3 . The NKG2 receptor was expressed early during differentiation, and the expression frequencies on gated NK1.1+CD3- cells from fetal and adult progenitors were quite similar. The decrease in the percentage of 3S9hi NK cells as a function of time after i.v. injection of lymphoid progenitor cells seems to be similar to normal development of NK cells during ontogeny, where in FD17 thymus and spleen, ~90% of the NK cells are 3S9hi, whereas in adult spleen, only ~50% of the NK cells have this phenotype [20 ]. When comparing the expression of different Ly49 members on gated NK1.1+CD3- cells, some striking developmental differences between injected populations were observed. Staining with the 4D12 (Ly49E/C) and 5E6 (Ly49C/I) mAb showed that 50% 4D12hi NK cells were present in the progeny of FL progenitor cells at 11 days postinjection, whereas less than 2% 5E6+ NK cells were detectable, indicating that the majority of 4D12+ NK cells was expressing Ly49E. At the same point in time, only 22% 4D12hi NK cells were present in BM-derived NK cells, whereas already more than 16% 5E6+ NK cells were present. At 29 days after injection, the 4D12hi NK cell population disappeared nearly completely, and the expression frequencies appeared similar for fetal and adult populations. It should be noted that at this point in time, the expression percentages of 4D12 were comparable with the 5E6 expression. Although there was a drastic decrease in the percentage of 4D12+ NK cells at 29 days after transfer, the absolute number of Ly49E+Ly49C- seemed to increase (data not shown). Labeling with 4D11 mAb showed that Ly49G2 was also a Ly49 member expressed early during NK cell differentiation.



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  		Figure 3. Acquisition of CD94/NKG2 and Ly49 family members by developing NK cells in vivo starting from fetal versus adult progenitor cells. Sorted FL and BM progenitor cells were injected i.v. in RAG2/{gamma}c-/- mice, and at 11 and 29 days postinjection, flow cytometric analysis was performed on splenocytes. Cells were incubated with combinations of PE-conjugated anti-NK1.1, APC-conjugated anti-CD3, and FITC-conjugated 3S9, 4D12, or 4D11 mAb; with combinations of biotin-conjugated anti-NK1.1, FITC-conjugated anti-CD3, and PE-conjugated A1 or 5E6 mAb; or with PE-conjugated anti-NK1.1, FITC-conjugated anti-CD3, and biotin-conjugated 12A8 mAb. Biotin-conjugated mAb were revealed with streptavidin-APC. Filled histograms show expression of the indicated receptor on gated NK1.1+CD3- cells. Open histograms represent background staining. Results shown are representative of more than three experiments.

 
It seems that BM NK progenitor cells differentiated faster toward the mature NK phenotype than FL progenitors. At 11 days postinjection, a significant percentage of BM-derived NK cells already expressed Ly49C/I and Ly49D receptors, whereas FL-derived NK cells were negative for the given receptors. Overall, the results in Figure 3 show the orderly acquisition of the different members of the Ly49 receptor family, and the following acquisition pattern can be postulated: The Ly49E and Ly49G2 receptors are the first to be expressed, followed by Ly49C/I, Ly49D, and finally Ly49A. The low Ly49A expression level could be a result of the use of the A1 mAb, which stains the NK cells only weakly. In contrast, the Ly49A-specific mAb JR9-318 clearly stained a small but significant fraction (~6%) of the BM-derived NK cells at 29 days postinjection (data not shown).

As in addition to Ly49E, the NKG2 and Ly49G2 receptors were expressed very early during differentiation (Fig. 3) , we investigated whether there was a preferential expression of Ly49G2 and NKG2 by the 4D12+ population. As shown in Figure 4 , for the fetal and the adult progeny, no preferential coexpression of the given receptors could be found at 11 and 29 days postinjection, as the percentages of cells double-positive for 4D11 and 4D12 and for 3S9 and 4D12 were quite similar to the percentages expected by the product of the expression frequencies of the individual receptors.



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  		Figure 4. Expression pattern of Ly49G2 and NKG2 on developing 4D12+ NK cells in vivo. Four-color flow cytometric analysis was performed on splenocytes isolated from RAG2/{gamma}c-/- mice at days 11 and 29 postinjection of c-kit+Sca2+Lin- progenitor cells from FL or BM. Cells were stained with PE-conjugated anti-NK1.1, PerCP-conjugated anti-CD3, FITC-conjugated 4D12 mAb, and biotinylated 3S9 or 4D11 mAb. Biotin-conjugated mAb were revealed with streptavidin-APC. Analysis was performed by gating on NK1.1+CD3- splenocytes. Results shown are representative for three experiments.

 
Stable expression of Ly49E and acquisition of additional Ly49 receptors upon in vivo transfer of fetal NK cells
As shown in Figure 3 , the expression percentage of 4D12+5E6- cells drastically decreased as a function of time. Several mechanisms could attribute to this decrease: down-regulation of the Ly49E receptor or dilution of the Ly49E+ NK cells within the expanding NK cell population. To determine the fate of Ly49E single-positive NK cells, we sorted 4D12+ and 4D12- NK cells from FD17 thymus, as these cells do not yet express any other members of the Ly49 receptor family on the cell surface [11 ,20 ,23 ], and we injected these NK cells i.v. in RAG2/{gamma}c-/- mice. At 19 days postinjection, splenocytes were analyzed. Results in Figure 5 show that the expression of Ly49E was, once initiated, stable for at least 19 days. Remarkably, also, >90% of injected 4D12- NK cells became 4D12+ after transfer. Only ~10% of the progeny of the injected 4D12+ cells stained with the 4LO (Ly49C) mAb.



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  		Figure 5. Stable expression of Ly49E and acquisition of other members of the Ly49 receptor family upon in vivo transfer of fetal NK cells. Sorted FD17 NK1.1+CD3-4D12+ and NK1.1+CD3-4D12- cells were injected i.v. in RAG2/{gamma}c-/- mice. Flow cytometric analysis was performed on isolated splenocytes 19 days postinjection. Cells were stained with combinations of PE-conjugated anti-NK1.1, PerCP-conjugated anti-CD3, FITC-conjugated 4D12, biotin-conjugated 3S9, biotin-conjugated 4LO, or biotin-conjugated A1, 5E6, 12A8, and 4D11 mAb (All). Biotin-conjugated mAb were revealed with streptavidin-APC. By gating on NK1.1+CD3- splenocytes, analysis was performed. Results are representative of two experiments. Open histograms represent background staining.

 
The results in Figure 5 also show that 4D12+ cells were not restricted in the potential to express other members of the Ly49 family, as 35% of their progeny expressed Ly49A, -C, -D, -G2, or -I. The NKG2 expression frequency increased only slightly, as already ~90% of the sorted 4D12+ and 4D12- NK cells expressed NKG2 (data not shown, and ref. [20 ]).

No difference in proliferation of Ly49E- versus Ly49E+ NK cells
To further investigate the decrease of Ly49E receptor expression, we addressed the proliferation of Ly49E- versus Ly49E+ NK cells. We performed DNA analysis, using PI on sorted 4D12+5E6- and 4D12-5E6+ NK cells from the spleen of 11-day-old mice. At this time, the percentage of Ly49E single-positive NK cells in the spleen has significantly decreased, and the percentage of Ly49C/I-positive NK cells starts to increase and reaches a similar value as the Ly49E+Ly49C/I- NK cell percentage (data not shown). This situation is comparable with an intermediate point in time (between 11 and 29 days) of the transfer studies. The results in Figure 6 show that similar fractions of both populations are undergoing division. Therefore, the reduction of Ly49E-expressing NK cells cannot be attributed solely to the preferential proliferation of the 4D12-5E6+ NK cells.



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  		Figure 6. Cell-cycle analysis of 4D12+5E6- versus 4D12-5E6+ NK cells. DNA analysis using PI of freshly isolated splenocytes sorted 4D12+5E6- and 4D12-5E6+ NK cells from 11-day-old mice. The percentage of cells in the S and G2 + M phase is indicated.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The c-kit+Sca2+Lin- lymphoid-committed progenitor population has been shown to generate NK1.1+ cells under appropriate culture conditions (refs. [15 , 16 , 25 ], and Fig. 2 ). Consistent with a study by Williams et al. [17 ], CD94/NKG2 mRNA transcripts and cell-surface expression could be detected in the NK1.1- population after 5 days of primary culture in SCF, IL-7, and Flt3L, indicating that IL-15 is not required in this process. We also show that Ly49E transcripts, which were confirmed by sequencing (data not shown) but not protein, were present after primary culture of FL and BM progenitor cells. However, the expression level was much higher in the progeny of fetal lymphoid-committed progenitors compared with adult progenitors. Contrary to our results, Williams et al. [17 ] could not find Ly49E transcripts in sorted NK1.1-/IL-2Rß+ cells generated from BM progenitors after primary culture but only in the later stages of the secondary culture on the OP9 stromal cell line. The detection of Ly49E transcripts after primary culture in our study could be a result of the fact that we studied Ly49E mRNA expression in the total progeny, whereas Williams et al. [17 ] tested the sorted NK1.1-/IL-2Rß+ subpopulation, which comprises ~10% of these cells. Surprisingly, the secondary in vitro culture of progenitor cells in the presence of IL-15 but without stromal cells induced surface expression of Ly49E on NK1.1+ cells (Fig. 2) . This is in contrast with the expression of other members of the Ly49 receptor family, for which interactions with BM stromal cells are strictly required [17 ]. The surface expression frequency and expression level of Ly49E were, as for the mRNA transcription levels, much higher in the fetal progeny (Fig. 2) . A recent study by Fraser et al. [28 ] has also shown that long-term IL-2 culture of unseparated fetal FD14 thymocytes results in the presence of Ly49E-expressing NK cells. Although there are several arguments that these Ly49E-expressing NK cells originate from lymphoid progenitor cells in the starting population, one cannot exclude the possibility that the small percentage of Ly49E-expressing NK cells, which are already present in freshly isolated FD14 thymuses, expand during the long IL-2 culture afterwards. The present manuscript clearly demonstrates that sorted lymphoid progenitors are able to differentiate into Ly49E-expressing NK cells in the absence of stromal cells in vitro.

Our in vivo studies show that fetal and adult progenitors were capable of generating Ly49E+ NK cells, but similar to the results of the in vitro cultures, a higher percentage of FL-derived NK cells expressed the Ly49E receptor (Fig. 3) . In this study, we demonstrate for the first time the actual expression sequence of the Ly49 members in vivo starting from lymphoid progenitor cells from FL and BM. Other studies by Dorfman and Raulet [13 ], starting from sorted subpopulations of NK cells already expressing a single Ly49 member, have suggested that the expression order of the members of the Ly49 receptor family on developing NK cells is restricted. Their results suggest that the expression sequence of the genes is as follows: Ly49A, followed by Ly49G2, and finally Ly49C/I. In vitro studies performed by Williams et al. [16 , 17 ] starting from lymphoid progenitor cells from BM have pointed out a different sequential acquisition of Ly49 receptors. Using RT-PCR, they have shown that Ly49G2 is the first to be expressed, followed by Ly49C/I, and finally by Ly49A. Our results are quite similar to those of Williams et al. [16 , 17 ], as we show that Ly49E and Ly49G2 are the first Ly49 receptors to be expressed, followed by Ly49C/I and Ly49D, and finally by Ly49A (Fig. 3) . In spite of the difference in expression order, our results and the results of Williams et al. [16 ,17 ] are not necessarily in opposition to those of Raulet and co-workers [13 ,15 ]. Although our results show the actual order in which the different Ly49 genes are expressed on the surface of NK cells during development in vivo, starting from lymphoid progenitor cells, the results of the group of Raulet [13 ,15 ] show the restriction in Ly49 gene expression on different subpopulations of NK cells.

Whereas our results clearly show that there are differences in NK cell differentiation kinetics and potential, they argue against the existence of a strict fetal and adult wave of NK cell differentiation, which has been suggested for B and T cell differentiation and erythropoiesis. Others have shown that T cell receptor V{gamma}3 T lymphocytes [29 ], CD5+ B-1a cells [30 31 32 ], and {gamma}-hemoglobin-containing red blood cells [33 ] are only generated from fetal hematopoietic progenitor cells, whereas adult progenitor cells have lost this potential. However, critical analysis shows that in the case of B and red blood cell differentiation, the developmental restrictions are perhaps not so clear-cut, as a small but significant population (~10%) of adult progenitor-derived B cells and red blood cells expresses CD5 [32 ] and fetal hemoglobin [34 ,35 ], respectively. The results from the present manuscript regarding the in vitro and in vivo NK differentiation potential of fetal and adult lymphoid progenitors incline toward a model comparable with the model proposed for erythropoiesis and B cell development in which NK progenitors within the same lineage gradually, but not completely, lose the potential to generate NK cells expressing the Ly49E receptor.

In Figure 3 , it is shown that the percentage of 4D12+5E6- NK cells sharply decreased in time. Several hypotheses can be formulated to explain this time-dependent decrease. First, the fetal NK cells could lose Ly49E receptor expression. Second, as fetal NK cells are less cytotoxic than adult NK cells, these fetal NK cells could represent an immature stage, which can still initiate, e.g., the expression of Ly49C, which is recognized by 4D12 and 5E6 mAb, leading to the decrease in 4D12 single-positive cells. Finally, the strong decrease could be a result of the dilution of Ly49E single-positive NK cells within the NK cell population, because of the increased generation of NK cells which do not initiate the expression of Ly49E, or to the preferential expansion of NK cells which do not express Ly49E. The results in Figure 5 show that comparable with the expression of Ly49A and Ly49G2 [13 ], Ly49E expression is, once initiated, stable for at least 19 days. Ly49E single-positive NK cells were still capable of expressing other members of the Ly49 family, including Ly49C, which can only account in part for the decrease in Ly49E single-positive cells shown in Figure 3 . We have also shown via DNA analysis that there does not seem to be a preferential proliferation of the Ly49E-negative NK cell population (Fig. 6) , thereby dismissing this hypothesis for the decrease in Ly49E-positive NK cells. Our conclusion is that the decrease in Ly49E single-positive cells during ontogeny is mainly a result of increased generation of Ly49E- NK cells.

In the past, transfer experiments using fetal hematopoietic stem cells showed that the timing of the fetal to adult hemoglobin switch is largely determined by the gestational age of the transplanted cells [36 ], suggesting that a developmental clock inherent in the hematopoietic stem cells regulates the switch [36 ,37 ]. Similarly, the quicker differentiation of BM versus FL NK progenitor cells observed in our study might be explained by developmental differences inherent in the progenitor cells. From the actual Ly49 expression sequence in vivo, combined with our other results, we propose the following model of NK cell differentiation. Part of the progenitor cells quite rapidly generate NK cells, which initiate Ly49E and G expression, and another part might be quiescent or might undergo cell division before differentiating into NK cells. The NK cell progenitors, regulated by a developmental clock, gradually lose the capacity to initiate expression of the Ly49 members, which are found at the earliest points in time. The result is that a smaller percentage will now initiate Ly49E and G2 expression. It should be noted that NK cells expressing one Ly49 member can coexpress other members at later points in time and that the earlier Ly49 members do not have to be expressed to enable the expression of the later ones. This model could then explain the different Ly49E expression potential between fetal and adult lymphoid progenitors, as well as the time-dependent decrease in Ly49E single-positive NK cells. As shown in Figure 3 , adult progenitors differentiate more quickly, meaning that the period of time regulated by the developmental clock, during which these progenitor cells can generate NK cells expressing the Ly49E member, is shorter, consequently restricting the expression to a smaller percentage of NK cells. The expression pattern of CD94/NKG2 can be explained similarly. Fetal and adult progenitors first preferentially generate CD94/NKG2hi NK cells, followed by a time-dependent increase in the number of progenitors, which can only generate CD94/NKG2low NK cells.

In conclusion, this study addressed the developmental differences with regard to Ly49E receptor expression between FL and BM NK progenitor cells. We could demonstrate that fetal and adult progenitors generated Ly49E+ NK cells; however, they differed in Ly49E expression potential, as fetal cells were much more potent in this respect than adult cells. These results argue against the existence of a strict fetal and adult wave of NK differentiation in which one lineage is replaced by another lineage with restricted potential. Fetal Ly49E-positive NK cells seem to be immature cells, which can still express other members of the Ly49 family upon transfer. Remarkably and in contrast to all other members of the Ly49 receptor family, stromal cells were not required to induce Ly49E surface expression in vitro, suggesting a different expression regulation mechanism. We also demonstrated for the first time the actual expression sequence of several Ly49 receptors in vivo. Questions about the functional role of the Ly49E receptor during fetal and adult life still remain. An important feature in the determination of the actual functional role will be the identification of the ligand for this receptor.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Fund for Scientific Research–Flanders (Belgium) and the Research Fund of Ghent University (Belgium). K. V. B. and A. D. C. are Ph.D. students supported by the Research Fund of Ghent University. F. S. is a research assistant of the Fund for Scientific Research–Flanders. We gratefully acknowledge Drs. S. Lemieux, J. R. Ortaldo, R. Boyd, J. Unkeless, B. Kyewski, B. Fazekas, and Pierres for providing us with mAb. We thank K. Hugelier and M. De Smedt for purification of antibodies and C. Collier and G. De Smedt for animal care.

Received September 9, 2002; revised February 10, 2003; accepted February 12, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Trinchieri, G. (1989) Biology of natural killer cells Adv. Immunol. 47,187-376[Medline]
  2. Raulet, D. H., Vance, R. E., McMahon, C. W. (2001) Regulation of the natural killer cell receptor repertoire Annu. Rev. Immunol. 19,291-330[CrossRef][Medline]
  3. Wong, S., Freeman, J. D., Kelleher, C., Mager, D., Takei, F. (1991) Ly-49 multigene family. New members of a superfamily of type II membrane proteins with lectin-like domains J. Immunol. 147,1417-1423[Abstract]
  4. Smith, H. R., Karlhofer, F. M., Yokoyama, W. M. (1994) Ly-49 multigene family expressed by IL-2-activated NK cells J. Immunol. 153,1068-1079[Abstract]
  5. McQueen, K. L., Freeman, J. D., Takei, F., Mager, D. L. (1998) Localization of five new Ly49 genes, including three closely related to Ly49c Immunogenetics 48,174-183[CrossRef][Medline]
  6. Hanke, T., Takizawa, H., McMahon, C. W., Busch, D. H., Pamer, E. G., Miller, J. D., Altman, J. D., Liu, Y., Cado, D., Lemonnier, F. A., Bjorkman, P. J., Raulet, D. H. (1999) Direct assessment of MHC class I binding by seven Ly49 inhibitory NK cell receptors Immunity 11,67-77[CrossRef][Medline]
  7. Vance, R. E., Tanamachi, D. M., Hanke, T., Raulet, D. H. (1997) Cloning of a mouse homolog of CD94 extends the family of C-type lectins on murine natural killer cells Eur. J. Immunol. 27,3236-3241[Medline]
  8. Lohwasser, S., Hande, P., Mager, D. L., Takei, F. (1999) Cloning of murine NKG2A, B and C: second family of C-type lectin receptors on murine NK cells Eur. J. Immunol. 29,755-761[CrossRef][Medline]
  9. Vance, R. E., Kraft, J. R., Altman, J. D., Jensen, P. E., Raulet, D. H. (1998) Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Qa-1(b) J. Exp. Med. 188,1841-1848[Abstract/Free Full Text]
  10. Vance, R. E., Jamieson, A. M., Raulet, D. H. (1999) Recognition of the class Ib molecule Qa-1(b) by putative activating receptors CD94/NKG2C and CD94/NKG2E on mouse natural killer cells J. Exp. Med. 190,1801-1812[Abstract/Free Full Text]
  11. Kubota, A., Kubota, S., Lohwasser, S., Mager, D. L., Takei, F. (1999) Diversity of NK cell receptor repertoire in adult and neonatal mice J. Immunol. 163,212-216[Abstract/Free Full Text]
  12. Raulet, D. H., Held, W., Correa, I., Dorfman, J. R., Wu, M. F., Corral, L. (1997) Specificity, tolerance and developmental regulation of natural killer cells defined by expression of class I-specific Ly49 receptors Immunol. Rev. 155,41-52[CrossRef][Medline]
  13. Dorfman, J. R., Raulet, D. H. (1998) Acquisition of Ly49 receptor expression by developing natural killer cells J. Exp. Med. 187,609-618[Abstract/Free Full Text]
  14. Takei, F., McQueen, K. L., Maeda, M., Wilhelm, B. T., Lohwasser, S., Lian, R. H., Mager, D. L. (2001) Ly49 and CD94/NKG2: developmentally regulated expression and evolution Immunol. Rev. 181,90-103[CrossRef][Medline]
  15. Roth, C., Carlyle, J. R., Takizawa, H., Raulet, D. H. (2000) Clonal acquisition of inhibitory Ly49 receptors on developing NK cells is successively restricted and regulated by stromal class I MHC Immunity 13,143-153[CrossRef][Medline]
  16. Williams, N. S., Klem, J., Puzanov, I. J., Sivakumar, P. V., Bennett, M., Kumar, V. (1999) Differentiation of NK1.1+, Ly49+ NK cells from flt3+ multipotent marrow progenitor cells J. Immunol. 163,2648-2656[Abstract/Free Full Text]
  17. Williams, N. S., Kubota, A., Bennett, M., Kumar, V., Takei, F. (2000) Clonal analysis of NK cell development from bone marrow progenitors in vitro: orderly acquisition of receptor gene expression Eur. J. Immunol. 30,2074-2082[CrossRef][Medline]
  18. Hackett, J., Tutt, M., Lipscomb, M., Bennett, M., Koo, G., Kumar, V. (1986) Origin and differentiation of natural killer cells. II. Functional and morphologic studies of purified NK-1.1+ cells J. Immunol. 136,3124-3131[Abstract]
  19. Puzanov, I. J., Williams, N. S., Schatzle, J., Sivakumar, P. V., Bennett, M., Kumar, V. (1997) Ontogeny of NK cells and the bone marrow microenvironment: where does IL15 fit in? Res. Immunol. 148,195-201[CrossRef][Medline]
  20. Van Beneden, K., Stevenaert, F., De Creus, A., DeBacker, V., De Boever, J., Plum, J., Leclercq, G. (2001) Expression of Ly49E and CD94/NKG2 on fetal and adult NK cells J. Immunol. 166,4302-4311[Abstract/Free Full Text]
  21. Sivakumar, P. V., Gunturi, A., Salcedo, M., Schatzle, J. D., Lai, W. C., Kurepa, Z., Pitcher, L., Seaman, M. S., Lemonnier, F. A., Bennett, M., Forman, J., Kumar, V. (1999) Expression of functional CD94/NKG2A inhibitory receptors on fetal NK1.1+Ly-49- cells: a possible mechanism of tolerance during NK cell development J. Immunol. 162,6976-6980[Abstract/Free Full Text]
  22. Salcedo, M., Colucci, F., Dyson, P. J., Cotterill, L. A., Lemonnier, F. A., Kourilsky, P., Di Santo, J. P., Ljunggren, H. G., Abastado, J. P. (2000) Role of Qa-1b binding receptors in the specificity of developing NK cells Eur. J. Immunol. 30,1094-1101[CrossRef][Medline]
  23. Van Beneden, K., De Creus, A., Debacker, V., De Boever, J., Plum, J., Leclercq, G. (1999) Murine fetal natural killer cells are functionally and structurally distinct from adult natural killer cells J. Leukoc. Biol. 66,625-633[Abstract]
  24. Toomey, J. A., Salcedo, M., Cotterill, L. A., Millrain, M. M., Chrzanowska Lightowlers, Z., Lawry, J., Fraser, K., Gays, F., Robinson, J. H., Shrestha, S., Dyson, P. J., Brooks, C. G. (1999) Stochastic acquisition of Qa1 receptors during the development of fetal NK cells in vitro accounts in part but not in whole for the ability of these cells to distinguish between class I-sufficient and class I-deficient targets J. Immunol. 163,3176-3184[Abstract/Free Full Text]
  25. Williams, N. S., Moore, T. A., Schatzle, J. D., Puzanov, I. J., Sivakumar, P. V., Zlotnik, A., Bennett, M., Kumar, V. (1997) Generation of lytic natural killer 1.1+, Ly-49- cells from multipotential murine bone marrow progenitors in a stroma-free culture: definition of cytokine requirements and developmental intermediates J. Exp. Med. 186,1609-1614[Abstract/Free Full Text]
  26. Leclercq, G., De Smedt, M., Tison, B., Plum, J. (1990) Preferential proliferation of T cell receptor V3-positive cells in IL-2-stimulated fetal thymocytes J. Immunol. 145,3992-3997[Abstract]
  27. Colucci, F., Soudais, C., Rosmaraki, E., Vanes, L., Tybulewicz, V. L., Di Santo, J. P. (1999) Dissecting NK cell development using a novel alymphoid mouse model: investigating the role of the c-abl proto-oncogene in murine NK cell differentiation J. Immunol. 162,2761-2765[Abstract/Free Full Text]
  28. Fraser, K. P., Gays, F., Robinson, J. H., van Beneden, K., Leclercq, G., Vance, R. E., Raulet, D. H., Brooks, C. G. (2002) NK cells developing in vitro from fetal mouse progenitors express at least one member of the Ly49 family that is acquired in a time-dependent and stochastic manner independently of CD94 and NKG2 Eur. J. Immunol. 32,868-878[CrossRef][Medline]
  29. Ikuta, K., Kina, T., Macneil, I., Uchida, N., Peault, B., Chien, Y., Weissman, I. L. (1990) A developmental switch in thymic lymphocyte maturation potential occurs at the level of hematopoietic stem cells Cell 62,863-874[CrossRef][Medline]
  30. Wortis, H. H., Berland, R. (2001) Cutting edge commentary: origins of B-1 cells J. Immunol. 166,2163-2166[Abstract/Free Full Text]
  31. Thomas Vaslin, V., Coutinho, A., Huetz, F. (1992) Origin of CD5+ B cells and natural IgM-secreting cells: reconstitution potential of adult bone marrow, spleen and peritoneal cells Eur. J. Immunol. 22,1243-1251[Medline]
  32. Hardy, R. R., Hayakawa, K. (1991) A developmental switch in B lymphopoiesis Proc. Natl. Acad. Sci. USA 88,11550-11554[Abstract/Free Full Text]
  33. Zanjani, E. D., Lim, G., McGlave, P. B., Clapp, J. F., Mann, L. I., Norwood, T. H., Stamatoyannopoulos, G. (1982) Adult hematopoietic cells transplanted to sheep fetuses continue to produce adult globins Nature 295,244-246[CrossRef][Medline]
  34. Stamatoyannopoulos, G., Nienhuis, A. W. (1987) Hemoglobin switching Stamatoyannopoulos, G. Nienhuis, A. W. Leder, P. eds. The Molecular Basis of Blood Diseases ,66-105 Saunders Philadelphia, PA.
  35. Stamatoyannopoulos, G., Constantoulakis, P., Brice, M., Kurachi, S., Papayannopoulou, T. (1987) Coexpression of embryonic, fetal, and adult globins in erythroid cells of human embryos: relevance to the cell-lineage models of globin switching Dev. Biol. 123,191-197[CrossRef][Medline]
  36. Wood, W. G., Bunch, C., Kelly, S., Gunn, Y., Breckon, G. (1985) Control of haemoglobin switching by a developmental clock? Nature 313,320-323[CrossRef][Medline]
  37. Wood, W. G., Bunch, C., Kelly, S. (1985) The cellular basis of haemoglobin switching: further studies of fetal-to-adult haemopoietic cell transplantation Prog. Clin. Biol. Res. 191,369-382[Medline]



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