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Cutting Edge: Expression of Functional CD94/NKG2A Inhibitory Receptors on Fetal NK1.1⁺Ly-49^- Cells: A Possible Mechanism of Tolerance During NK Cell Development¹

P. V. Sivakumar^2,*, A. Gunturi, M. Salcedo, J. D. Schatzle^*, W. C. Lai^*, Z. Kurepa, L. Pitcher, M. S. Seaman, F. A. Lemonnier, M. Bennett^*, J. Forman and V. Kumar^*

^* Department of Pathology and Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75235; and Unité de Biologie Moléculaire du Gène, Institut National de la Santé et de la Recherche Médicale U277, Institut Pasteur, Paris, France

	Abstract

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Fetal liver- and thymus-derived NK1.1⁺ cells do not express known Ly-49 receptors. Despite the absence of Ly-49 inhibitory receptors, fetal and neonatal NK1.1⁺Ly-49^- cells can distinguish between class I^high and class I^low target cells, suggesting the existence of other class I-specific inhibitory receptors. We demonstrate that fetal NK1.1⁺Ly-49^- cell lysates contain CD94 protein and that a significant proportion of fetal NK cells are bound by Qa1^b tetramers. Fetal and adult NK cells efficiently lyse lymphoblasts from K^b-/-D^b-/- mice. Qa1^b-specific peptides Qdm and HLA-CW4 leader peptide specifically inhibited the lysis of these blasts by adult and fetal NK cells. Qdm peptide also inhibited the lysis of Qa1^b-transfected human 721.221 cells by fetal NK cells. Taken together, these results suggest that the CD94/NKG2A receptor complex is the major known inhibitory receptor for class I (Qa1^b) molecules on developing fetal NK cells.

	Introduction

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Murine NK cell effector function is controlled by both positive and negative signaling receptors. These include Ly-49, NKR-P1, and the CD94/NKG2 receptors (1). In the mouse, the ability of NK cells to lyse class I-deficient or class I-allogeneic targets has been largely attributed to the presence or absence of positive and negative signaling receptors belonging to the Ly-49 family (2). Thus, NK cells lyse those targets that do not express an inhibitory ligand for the Ly-49 molecules expressed on effector cells.

We earlier described the generation of NK1.1⁺ cells from murine fetal liver and thymus (3, 4, 5). These cells do not express detectable levels of known Ly-49 receptors (A, C, G2, D, H, and I) on the surface. However, they do contain transcripts of certain Ly-49 molecules, especially Ly-49E (6). Whether these are translated and are expressed on the cell surface is unknown. During ontogeny, Ly-49 receptor expression is first detected on splenic NK1.1⁺ cells on days 3–5 after birth and reaches adult frequencies by days 18–21 (5). Despite the absence of known Ly-49 molecules, fetal- and neonatal-derived NK1.1⁺Ly-49^- cells distinguish between class I^high- and class I^low-expressing cells (5, 6). This suggested that immature NK cells express class I inhibitory receptors that are different from the known Ly-49 molecules. In an effort to identify the inhibitory receptor(s), we report here that a majority of fetal NK1.1⁺Ly-49^- cells express the CD94/NKG2 receptor complex. Further, lysis of targets by fetal NK1.1⁺Ly-49^- cells, as well as mature splenic NK1.1⁺Ly-49^- cells, is inhibited by the nonclassical class I molecule Qa1^b. More importantly, we demonstrate that, during NK cell development, the CD94/NKG2A receptors are expressed earlier than Ly-49 molecules and may serve to maintain tolerance to self in the absence of Ly-49 receptors.

	Materials and Methods

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PCR

CD94 was cloned from fetal and adult NK cells using primers designed from sequences found in the EST database. NKG2A primers were designed from sequences kindly provided by Dr. Fumio Takei (Terry Fox Laboratory, Vancouver, Canada). PCR products were TA cloned into pGEM T-Easy vector (Invitrogen, San Diego, CA) and sequenced using T7 primers. The following primers were used: CD94-1 (F), 5'-GGATCCCTTCTCATGGCAGTTTCTAGG-3'; CD94-3 (R), 5'-GGATCCTTAAATAGGCAGTTTCTTACA-3'; NKG2A-1 (F), 5'-GGATCCATGAGTAATGAACGCGTC-3'; NKG2A-2 (R), 5'-GAATTCCTACTTGTCATCGTCGTCCTTGTAATCGATGGGGAATTTACA-3'

Effector cells

Mouse fetal thymic- and liver-derived NK populations were derived by culture in 500 U/ml IL-2 for 10–14 days as previously described (3, 4, 5). Cells were then phenotyped for expression of NK1.1, Ly-49 (A, C and I, G2 and D) and their ability to bind Qa1^b tetramers. These cells were used as effectors in cytotoxicity assays or for Western analysis as described. Lymphokine-activated killers were generated by culturing C57BL/6 spleen cells in 500 U/ml IL-2 for 4 days.

Target cells

T cell blasts from K^b-/-D^b-/- mice (7) were generated as previously described (8). The classical class I-deficient human B cell tumor 721.221 was a kind gift from Dr L. Lanier (DNAX, Palo Alto, CA). 721.Qa1^b cells were obtained by transfecting 721.221 cells with a mQa1^b construct (pCDNA3.1 expression vector; Invitrogen), selected in 1 mg/ml Geneticin (Life Technologies, Gaithersburg, MD) and analyzed by cell staining as specified below.

Abs, cell staining, and Western analysis

Anti-CD94 polyclonal sera were generated, by immunizing rabbits with keyhole limpet hemocyanin (KLH)-conjugated peptides CD94–2(CIVYSPSKSVSAESCENKNRYICKK) (aa 152–176) or CD94–3 (CSKEEKSWKRSRDFC) (aa 76–89). Rabbit sera were then analyzed for specificity by ELISA using a CD94-GST fusion protein control and also by Western blot, as described. The CD94-GST fusion protein was generated by cloning full length mCD94 into the pESP-1 expression vector (Stratagene, La Jolla, CA). For cell staining, the FcR on the NK cells was blocked using mouse serum (1:10 dilution) for 20 min. Red-670-conjugated Qa1^b tetramers were derived as previously described (9). After blocking, cells were incubated with Qa1^b tetramer, control HLA-A2 tetramer, or Red-670 alone for 45 min, washed once and incubated with PE-conjugated NK1.1 (PharMingen, San Diego, CA) for 30 min. Cells were then washed and analyzed by FACScan (BD Systems, Mountain View, CA). 721.221 or 721.Qa1^b cells were stained with biotinylated anti-Qa1^b or rat isotype control Ab followed by streptavidin-conjugated PE. Anti-Qa1^b Ab was generated by immunizing rats with soluble mouse Qa1^b produced in Drosophila cells (10). This mAb, 910, recognizes plate-bound Qa1^b as well as Qa1^b expressed on transfectants and mouse lymphoblasts (see Figs. 3 and 4; A. Gunturi, Z. Kurepa, and J. Forman, unpublished data). Western analysis was done as described (11).

View larger version (28K): [in this window] [in a new window]   FIGURE 3. The Qa1b-specific peptide Qdm inhibits lysis of 721.Qa1b cells by FL-NKs. A, The human class I-deficient (except HLA-E) 721.221 cells were transfected with mQa1b, selected in Geneticin, and analyzed for Qa1b expression by flow cytometry. Cells were stained with biotinylated isotype control (Rat IgG2) (gray, shaded) or with biotinylated anti-Qa1b Ab (1:50) (black, unshaded), followed by streptavidin-PE. Untransfected cells (left) are not bound by anti-Qa1b Ab whereas 721.Qa1b cells (right) express Qa1b. B, Qdm peptide specifically inhibits lysis of 721.Qa1b but not 721.221 cells by FL-NKs. FL-NK cells cultured in 500 U/ml of IL-2 for 10 days were used as effectors in a standard 4-h chromium release assay against wild-type 721.221 (left panel) and 721.Qa1b (right panel) cells. Target cells were incubated overnight at 26°C with 100 µM of peptide. Cells were labeled with 51Cr for 2 h at room temperature and used as targets. Peptides (500 ng/well) were also added to the wells during the assay.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. Lysis of class I-deficient T cell blasts is inhibited by Qa1b-specific peptides. A, Spleen cells from wild-type C57BL/6 (H2b), TAP-/- (H2b), and Kb-/-Db-/- (H2b) mice were cultured for 2 days in 3 µg/ml Con A. Viable cells were then used for FACS analysis using no Ab (light, unshaded), isotype control (dark, unshaded), or anti-Qa1b mAb 910 (dark, shaded) followed by streptavidin–PE. B, Viable T cell blasts were labeled with 51Cr at 37°C in the presence of 100 µM peptide, washed, and plated at 1000 targets/well with 1000 ng of peptide for 1 h. Effector cells were then added (FT-NK 200:1, adult splenic lymphokine-activated killers 100:1). A standard 4-h chromium release assay was performed.

The Qdm (AMAPRTLLL, Qa1^b-specific), HLA-CW4 (VMAPRTLIL, Qa1^b-specific), FLU (ASNENMETM, D^b-specific), OVA (SIINFEKL, K^b-specific), and HIV-RT (ILKEPVHGV, HLA-A2-specific) peptides were synthesized on an ABS 432A peptide synthesizer and purified using HPLC in our laboratory. Target cells were incubated overnight at 26°C with or without peptides. In some experiments 100 µM peptides were added overnight. In others, specified concentrations of peptides were added to cells for 1–2 h at room temperature before addition to effectors. Standard 4-h chromium release assays were done as described (8).

	Results

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Fetal liver- and thymus-derived NK cells express CD94 and NKG2A transcripts

We considered the murine CD94/NKG2A heterodimer as a candidate inhibitory receptor on fetal and neonatal NK1.1⁺Ly-49^- cells. Using primers designed from the EST database, we cloned murine CD94 from C57BL/6 fetal liver- and thymus-derived NK cells (fetal liver NK (FL-NK)³ and fetal thymic NK (FT-NK), respectively) and also from adult splenic NK cells. FL-NK and FT-NK cells expressed CD94 transcripts, and the sequence obtained was identical to the published sequence (12, 13) (data not shown). We also verified that FL-NK and FT-NK cells expressed mNKG2A by using primers designed from an NKG2A sequence that was kindly shared by Dr. Takei (Terry Fox Lab, Vancouver, BC, Canada)(data not shown).

FL-NK, FT-NK, and adult splenic NK cell lysates express a protein that is identified by an anti-CD94 antiserum

To obtain a rabbit Ab against CD94, we synthesized two peptides from the extracellular domain of mCD94 as described under Materials and Methods. Sera from rabbits immunized with the two different peptides (CD94–2 and CD94–3) reacted against a CD94-GST fusion protein by ELISA (data not shown) and Western analysis (Fig. 1). FL-NK, FT-NK, and adult splenic NK cell lysates were also analyzed for reactivity to anti-CD94 antisera. As shown in Fig. 1, both anti-CD94-2 and anti-CD94-3 antisera reacted with a band that corresponds to the predicted molecular mass of mCD94 (28–30 kDa) (Fig. 1). Anti-CD94-3 also reacted with some smaller size bands (Fig. 1, bottom). Although these bands could have resulted from nonspecific binding or degradation products, we believe that they might be alternatively spliced forms of CD94 (P. V. Sivakumar and V. Kumar, unpublished data). The anti-CD94 antiserum did not cross-react with rat CD94 expressed in the rat leukemia cell line RNK-16 (14). Mouse T cell tumors BW5147 and EL-4 were also CD94 negative (Fig. 1). There was no staining of NK cells as determined by FACs analysis, suggesting that the anti-CD94 antiserum was unable to recognize protein in the native configuration.

View larger version (46K): [in this window] [in a new window]   FIGURE 1. FL-NK, FT-NK, and scid NK cells express CD94 protein. Lysates from 5 million cell equivalents were separated on a 4–20% SDS gel and transferred to nitrocellulose membrane. After blocking in 3% NFDM in TBST, anti-CD94-2 or anti-CD94-3 antisera were added at a 1:200 dilution. Donkey anti-rabbit HRP was used at 1:1000. CD94-GST fusion protein was used as a positive control. Control NKR-P1B-GST and control-GST fusion protein did not react with antisera (data not shown).

The nonclassical class I molecule Qa1^b has been identified recently to be the ligand for mCD94/NKG2A (15). The Qa1^b tetramer binds to the CD94/NKG2A complex but not to CD94 alone (15). Qa1^b tetramers folded in the presence of Qdm peptide (Qa1^b-specific, high affinity D^b, D^d leader peptide) (10) bound to 50–60% of NK1.1⁺Ly-49^- cells derived from fetal liver and thymus (Fig. 2). As previously reported, it also bound to 60% of C57BL/6 splenic NK1.1⁺ cells (9, 15) (Fig. 2). A control HLA-A2 tetramer refolded with the HIV-GAG peptide did not bind to splenic or fetal NK1.1⁺ cells (data not shown). In multiple experiments, between 40 and 70% of IL-2-cultured fetal NK cells were bound by the tetramer. Thus, a significant proportion of fetal liver- and thymus-derived NK1.1⁺Ly-49^- cells express CD94/NKG2 on the surface. The Qa1^b tetramers also bound to 40–60% of NK1.1⁺ cells from fresh day 15 fetal liver and thymus (Fig. 2, and data not shown), suggesting that culture in IL-2 (FL-NK and FT-NK) does not alter the frequencies of NK1.1⁺ cells bound by tetramer. Based on data from Vance et al. (15) and Braud et al. (16), the detection of NKG2A transcripts in FL-NK, and the functional data presented below (Figs. 3 and 4), we believe that CD94 is in a heterodimeric complex with NKG2A or another inhibitory NKG2 receptor.

View larger version (49K): [in this window] [in a new window]   FIGURE 2. Qa1b tetramers bind to fetal and adult NK cells. C57BL/6 fresh fetal liver (a), IL-2-cultured FL-NK (b), and FT-NK (c) cells were stained with Red-670 alone (gray, shaded) or Red-670 conjugated Qa1b tetramers (dark line, unshaded)(1:100 dilution) followed by PE-conjugated NK1.1. Fresh C57BL/6 spleen cells were used as a control. Qa1b tetramer binding is shown on gated NK1.1+ cells in all the panels. A control HLA-A2 tetramer refolded with the HIV-GAG peptide did not bind to murine NK cells (data not shown).

Having established that the Qa1^b tetramer binds to FL-NK and FT-NK cells, we analyzed their ability to receive negative or positive signals from the nonclassical class I molecule Qa1^b. There are no Abs available at present against Qa1^b and CD94/NKG2 that can be used to block interactions. Therefore, we used peptide stability assays to look for the ability of Qa1^b to inhibit NK cell lysis. For these studies, we chose the human classical class Ia-deficient B cell line 721.221, for several reasons. First, a human cell line would not express mouse surface receptors, thereby minimizing cross-reactivity. Second, these cells do not express the dominant leader peptides that bind to Qa1^b since they are deficient for human classical class Ia molecules. Although they do express the human class Ib molecule HLA-E, its leader should not bind Qa1^b. 721.221 cells were transfected with mQa1^b (721.Qa1^b) and analyzed for Qa1^b expression by flow cytometry using an anti-Qa1^b mAb (Fig. 3A). FL-NK cells were used as effectors against these two cell lines (wild-type 721.221 and 721.Qa1^b) in the absence or presence of different peptides. FL-NK cells efficiently lysed untransfected 721.221 cells. Addition of the Qa1^b-binding Qdm peptide (AMAPRTLLL) or control peptides FLU, OVA, and HIV-RT did not have any effect on NK killing (Fig. 3B, left). 721.Qa1^b cells not pulsed with peptides were lysed equally well. However, addition of Qdm (but not FLU, OVA, and HIV-RT) peptides dramatically inhibited lysis of 721.Qa1^b targets (Fig. 3B, right). Similar results were obtained when FT-NK or adult splenic NK cells were used as effectors (data not shown). Another Qa1^b-binding peptide (HLA-CW4 leader) (17) also inhibited lysis (data not shown). These data provide strong evidence that the predominant class Ib-recognizing inhibitory receptor on these fetal NK cells was CD94/NKG2A. Similar results were obtained using human T2 cells transfected with murine Qa1^b (data not shown).

Qa1^b-specific peptides inhibit lysis of T cell blasts by FT-NK and adult splenic NK cells

To further establish that FL-NK and FT-NK cells can be inhibited by Qa1^b, we used T cell blasts as targets. For this, we chose cells from K^b-/-D^b-/- mice (7). These mice offer two advantages. First, they lack classical class I molecules and, therefore, are lysed efficiently by adult splenic NK cells as well as fetal NK cells (Fig. 4B, and data not shown). Second, they lack the dominant Qa1^b leader peptide (D^b leader, Qdm). We also infer this from our observations that Qdm-specific T cell clones do not lyse blasts from K^b-/-D^b-/- mice unless Qdm is added to the assay (M. S. Seaman and J. Forman, unpublished data). Qdm peptide can therefore be loaded on the Qa1^b heavy chain molecules, presumably by displacing other lower affinity peptides. We first analyzed cell surface expression of Qa1^b on LPS blasts from K^b-/-D^b-/- (H2^b) mice. As shown in Fig. 4A, wild-type C57BL/6 (H2^b) mice expressed Qa1^b but TAP^-/- (H2^b) mice do not. Even in the absence of the dominant leader peptide (Qdm), K^b-/-D^b-/- lymphoblasts express Qa1^b on the cell surface (Fig. 4A), confirming observations of Perarnau et al. (7). Because mAb 910 recognizes a peptide-independent epitope on Qa1^b, this result is not surprising. Further, preliminary evidence indicates that Qa1^b is associated with other self peptides on these cells, which could account for the surface expression of this molecule (M. Seaman and J. Forman, unpublished data). Addition of Qdm peptide did not significantly alter Qa1^b cell surface levels (data not shown). Fetal thymic and adult splenic NK cells lysed blasts from K^b-/-D^b-/- mice. Addition of Qdm or HLA-CW4 leader peptides greatly inhibited killing of the blasts (Fig. 4B). These results suggest that the dominant Qdm peptide could displace other self peptides bound to Qa1^b on these cells and inhibit target cell lysis. The HLA-CW4 peptide that can bind Qa1^b with high affinity (17) could also inhibit NK cell lysis. This demonstrates that mouse NK cells can be inhibited by peptides from other species. Control FLU, OVA, and HIV-RT peptides have no effect.

Similar results were obtained when B cell blasts were used as targets (data not shown). In similar assays, there was no effect on the lysis of TAP^-/- blasts by fetal or adult splenic NK cells by addition of different peptides (data not shown). The inhibitory effects of the Qdm and the HLA-CW4 peptides could also be titrated in this K^b-/-D^b-/- system (data not shown). This provides conclusive evidence that fetal NK cells can be inhibited by Qa1^b and that the CD94/NKG2–Qa1^b interaction plays an important role in preventing killing by Ly-49^- NK cells.

	Discussion

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Fetal and neonatal NK1.1⁺Ly-49^- cells can distinguish between class I^high and class I^low targets. They are unable to lyse RMA cells but lyse the TAP-deficient derivative RMA-S cells efficiently. They are also unable to lyse wild-type syngeneic (H2^b) or allogeneic (H2^d) class I-positive lymphoblasts but lyse congenic class I^low T cell blasts from TAP^-/- (H2^b) mice. This strongly suggests the existence of non-Ly-49 receptors that can receive inhibitory signals from class I on these cells. Several pieces of evidence indicate that the CD94/NKG2 receptor complex acts as the major inhibitory receptor complex for class I on these NK cells. First, fetal liver- and thymus-derived NK cells express CD94 protein (Fig. 1), and a significant proportion (between 40–70%) of IL-2-activated fetal NK cells as well as 60% of fresh fetal NK1.1⁺ cells (Fig. 2) are bound by Qa1^b tetramers. Second, we can detect NKG2A transcripts in these cells. Based on this as well as the data of Vance et al. (15), we suggest that CD94 is expressed in a complex with NKG2A on the surface. However, it is possible that other inhibitory NKG2 receptors may also be expressed. Third, we found using two different systems (tumor cell lines as well as blasts) that Qa1^b-specific peptides greatly inhibit lytic activity of fetal NK cells. Lysis of the Qa1^b transfectant 721.Qa1^b is reduced by Qdm but not control peptides. Additionally, lysis of K^b-/-D^b-/- blasts was almost completely abrogated when pulsed with Qdm and HLA-CW4 peptides. It would therefore seem that in the absence of any known Ly-49-class I-mediated inhibition, the Qa1^b-CD94/NKG2 receptor complex can mediate strong negative signals to NK cells. This has implications not only for NK cell function but also for the ontogeny of NK cell receptors.

During development, the CD94/NKG2 receptor complex is expressed well before the Ly-49 receptor system. We and others have demonstrated that Ly-49 receptor expression can be first detected only after birth and reaches adult levels by days 18–21 (5, 18). We have also shown that fetal and neonatal NK1.1⁺Ly-49^- cells are functional and can lyse class I-deficient targets (5, 6). It would therefore be necessary to have a tolerance mechanism in the absence of Ly-49-class I interactions. This seems to be provided by the CD94/NKG2A receptor interacting with Qa1^b. Support for this is strengthened by the fact that immature NK1.1⁺Ly-49^- cells derived from marrow progenitors (19) can also distinguish between class I^high and class I^low targets, and these cells are bound by Qa1^b tetramer (N. S. Williams, unpublished data). Because the frequency of cells expressing surface CD94/NKG2 (as measured by Qa1^b tetramer binding) is the same in Ly-49^- fetal NK cells and Ly-49⁺ adult NK cells, there does not seem to be a developmental regulation in the frequency of cells expressing CD94/NKG2. However, the relative contribution of these two receptor systems in inhibition of NK function in adult Ly-49⁺ cells is unknown. Both receptor systems could contribute to self tolerance. Additional experiments are needed to address this. However, in fetal life, in the absence of any known Ly-49 receptors, the CD94/NKG2 inhibitory system could act as the major inhibitory system to maintain tolerance. One other important aspect that needs to be considered is the presence, of other class I-specific inhibitory receptors on fetal NK cells. Approximately 50% of fetal NK cells are Qa1^b tetramer positive. Does that mean that the other 50% do not distinguish between class I expressing cells? This seems unlikely. One possibility is that these cells express CD94 complexed with other NKG2 receptor family members that are not recognized by Qdm-bound Qa1^b tetramers. It should be noted that all tetramer staining done so far has used refolded Qa1^b in the presence of Qdm peptide. It is possible that there are other peptides that are important in the recognition by CD94/NKG2B or CD94/NKG2C in contrast to CD94/NKG2A. Alternatively, the Qa1^b tetramer-negative cells express other class I-specific inhibitory receptors. Potential candidate receptors would include the murine homologue of ILT2 (LIR1) and gp49B1 (20, 21). Another possible candidate could be the Ly-49E receptor. Toomey et al. have reported that fetal NK cells show higher expression of Ly-49E transcripts when compared with adult NK cells (6). It is possible that Ly-49E acts as another class I-specific inhibitory receptor. The absence of Ly-49E Abs prevents testing this possibility at this time.

To summarize, it seems that the CD94/NKG2A receptor complex acts as a dominant inhibitory receptor complex during NK cell development in the mouse. This conclusion is supported by the fact that NK cells derived from immature human fetal thymocytes have been shown to express the CD94/NKG2A complex as the only class I-specific receptor (22). We hypothesize therefore that inhibitory CD94/NKG2 receptors, by interacting with the nonclassical class I molecule Qa1^b, play an important role in maintaining self tolerance in developing NK cells.

	Acknowledgments

 
We thank Dr. Brian Gordon for providing SCID mice and Dr. Colin Brooks for sharing unpublished data. We also thank Ying Zhang for technical assistance and Maria and Sylvio Pena for breeding mice. We appreciate the helpful discussions of Drs. Dorothy Yuan and Noelle Williams.

	Footnotes

 
¹ This work was supported by Grants AI20451, AI38938, CA36922, and CA70134 to V.K. and M.B and by Grants AI37942 and AI37818 to J.F.

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

³ Abbreviations used in this paper: FL-NK, fetal liver NK; FT-NK, fetal thymic NK; NFDM, nonfat dry milk.

Received for publication March 17, 1999. Accepted for publication April 20, 1999.

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