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The Journal of Immunology, 2003, 171: 5277-5286.
Copyright © 2003 by The American Association of Immunologists

Human CD94 Gene Expression: Dual Promoters Differing in Responsiveness to IL-2 or IL-15

Louis D. Lieto, Francisco Borrego, Chi-hyun You and John E. Coligan1

Receptor Cell Biology Section, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD94 is a C-type lectin required for the dimerization of the CD94/NKG2 family of receptors, which are expressed on NK cells and T cell subsets. Little is known about CD94 gene expression and the elements that regulate CD94 transcription. In this study, we report that CD94 gene expression is regulated by distal and proximal promoters that transcribe unique initial exons specific to each promoter. This results in two species of transcripts; the previously described CD94 mRNA and a novel CD94C mRNA. All NK cells and CD94+, CD8+ {alpha}{beta} T cells transcribe CD94 mRNA. Stimulation of NK and CD8+ {alpha}{beta} T cells with IL-2 or IL-15 induced the transcription of CD94C mRNA. The distal and proximal promoters both contain elements with IFN-{gamma}-activated and Ets binding sites, known as GAS/EBS. Additionally, an unknown element, termed site A, was identified in the proximal promoter. EMSA analyses showed that constitutive factors could bind to oligonucleotide probes containing each element. After treatment of primary NK cells with IL-2 or IL-15, separate inducible complexes could be detected with oligonucleotide probes containing either the proximal or distal GAS/EBS elements. These elements are highly conserved between mice and humans, which suggests that both species regulate CD94 gene expression via mechanisms that predate their evolutionary divergence.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Natural killer cells are a class of lymphocytes that express a wide variety of activation receptors that allow them to recognize potential target cells, such as virally infected and tumorigenic cells, without prior sensitization. The consequences of NK cell activation are target cell lysis and/or the production of inflammatory cytokines, such as TNF-{alpha} and IFN-{gamma} (1, 2, 3). Curiously, many ligands recognized by NK cell activation receptors are proteins expressed by normal cells, which poses an obvious danger to the host (4, 5). To prevent destruction of normal cells and unwanted inflammation, NK cells also express inhibitory receptors that recognize MHC class I molecules on potential target cells (6, 7, 8). MHC class I molecules are expressed by almost all normal cells, and inhibitory signals override activation signals by recruiting protein tyrosine phosphatases to the proximity of signal initiation (9, 10). Class I molecules play a sentinel role in protecting cells from the deleterious effects of NK cell recognition. In contrast, once the expression of class I molecules is down-regulated, as is often the case with viral infection or malignant transformation, NK cells are poised to strike by virtue of their recognition of endogenous ligands and virally encoded proteins (5).

In humans, three distinct types of NK cell inhibitory receptors for MHC class I molecules have been defined (6). The first family of receptors are type I transmembrane molecules belonging to the Ig superfamily and are called killer cell Ig-like receptors (KIR) 2 (2, 10). A second class of receptor, named Ig-like transcripts, also belongs to the Ig superfamily (11). The only member of this family that is expressed on some NK cells is Ig-like transcript 2 (12, 13). The third type of inhibitory receptor is a heterodimer composed of CD94 covalently associated with NKG2A (or its isoform NKG2B) (14, 15). The ligand for this receptor is the nonclassical class I molecule HLA-E (16, 17, 18). The fact that human NK cells express three different types of inhibitory receptors specific for class I molecules suggests that it is critical to tightly control the lytic and inflammatory tendencies of these cells.

In addition to its role in regulating NK cell function, evidence is rapidly accumulating that indicates CD94/NKG2A is also involved in regulating certain T cell functions. In HIV-infected individuals, the percentage of T cells that express CD94/NKG2A is significantly elevated (19, 20). In mice, the percentage of CD8+ T cells that express CD94/NKG2 (A) is dramatically increased after infection with Listeria monocytogenes (LM), polyoma virus, and lymphocytic choriomeningitis virus, such that CD94 can be considered a marker of T cell activation (21, 22, 23). In the case of polyoma virus infection, CD94/NKG2 (A) on CD8+ T cells was shown to inhibit effector function; in contrast, no inhibition of effector function was observed for CD8+ T cells in LM- or lymphocytic choriomeningitis virus-infected mice (22, 23, 24). This may indicate that inhibition of CD8+ T cell function only occurs in cases of persistent infection in which chronic inflammation needs to be controlled. In addition to regulating effector function, inhibitory receptors may play a role in T cell survival. For example, the acquisition of KIR molecules has been shown to promote the survival of CD8+ T cells (25, 26). In mice, it was recently shown that CD8+ T cells, which express high levels of CD94/NKG2 (A) as a result of LM infection, are protected from apoptosis (21). Thus, CD94/NKG2A and KIR inhibitory receptors not only play a vital role in regulating the lytic and inflammatory responses of NK cells, but almost certainly play an important role in T cell biology as well.

CD94 expression is vital for expression of all members of the CD94/NKG2 family of receptors. Currently, little data exist on what regulates gene expression of CD94/NKG2 receptors in NK or T cells. The CD94 gene is located within a 2.5-Mb region on chromosome 12p12-p13 known as the NK complex that contains 19 genes coding for C-type lectins, including the NKG2 family (27, 28). It was previously reported that human CD94 had six exons, multiple start sites of transcription, and a TATA-less promoter (29). In this study, we report that dual reporters regulate CD94 gene expression, which leads to the transcription of two types of transcripts that differ in their 5' untranslated regions (UTR). The promoters show differential sensitivity to IL-2 and IL-15, two cytokines that increase cell surface expression of CD94 on NK cells. In freshly isolated primary NK and CD8+ {alpha}{beta} T cells, only the proximal promoter is active. Both promoters contain GAS/EBS elements, which can form constitutive and inducible DNA-protein complexes. This double element contains a potential binding site for STAT family proteins known as an IFN-{gamma}-activated site (GAS), as well as a potential Ets-binding site (EBS). The proximal promoter also contains an element with no homology to known motifs in close upstream proximity to the GAS/EBS site. Because IL-2 and IL-15 can regulate gene expression through the interactions of transcription factors with GAS and EBS motifs, it is likely that these two cytokines influence CD94 gene expression through the formation of DNA-protein complexes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell isolation and culture conditions

Polyclonal NK and CD8+ {alpha}{beta} T cells were isolated by negative selection from peripheral blood using the appropriate cell isolation kit (Miltenyi Biotec, Auburn, CA). The purity of the isolated cell populations was confirmed by flow cytometry. NK and CD8+ {alpha}{beta} T cells were cultured in IMDM (BioWhittaker, Walkersville, MD) supplemented with 500 U/ml of rIL-2 (Biological Resources Branch, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD) or 5 ng/ml IL-15 (R&D Systems, Minneapolis, MN), 10% human AB serum (BioWhittaker), and L-glutamine (BioSource International, Rockville, MD) at 37°C under an atmosphere of 5% CO2. NK and CD8+ {alpha}{beta} T cell clones were generated, as previously described (16). The NKL cell line was grown in RPMI 1640 (BioSource International) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 200 U/ml rIL-2, 5 µg/ml Plasmocin (Invivogen, San Diego, CA), and 10% FBS at 37°C under an atmosphere of 5% CO2. The YT-Indy cell line was grown in RPMI 1640 (BioSource International) supplemented with 2 mM L-glutamine, 10 mM nonessential amino acids, 5 µg/ml of Plasmocin, and 10% FBS at 37°C under an atmosphere of 5% CO2.

Plasmid construction

The distal promoter was subcloned into the XhoI and HindIII sites of the pGL3-basic reporter plasmid (Promega, Madison, WI). The primers used for the distal promoter deletion constructs were: p-4868Fr, 5'-GTGTCAGAAGCACTCCGTCC-3'; p-4278Fr, 5'-TATATCAAGTCCTGAGATCC-3'; p-3930Fr, 5'-TGTGAGATGCAGACAAAACAG-3'; p-3807Fr, 5'-GTTTGTGGCTGCACCGTTTAC-3'; Rv-1, 5'-AGGCATTAGAGTCTCTTG-3'.

A Quick Change XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to change a G to a C at position -3692 and a T to a G at position -3696, creating the mp-3807 construct. The proximal promoter was subcloned into XhoI and HindIII sites of the pGL3-basic reporter plasmid. Primers used were: 5'-CCGCTCGAGGGTAGAGTCAGAAGAACAGAGTGAG-3' and 5'-CCCAAGCTTCCAAGAGCGAAGTAGAGAAGGCAC-3'. The proximal promoter deletion mutants were constructed using a nested deletion kit following the manufacturer’s instructions (Promega). For scanning mutagenesis, a Quick site-directed mutagenesis kit (Stratagene) was used to create a series of promoter constructs in which sequential 6-bp regions were replaced with a KpnI site. Plasmid inserts were verified by sequence analysis.

Transfection and dual luciferase assay

A total of 2 x 106 YT-Indy or 5 x 106 NKL cells was transiently transfected with 1 µg/106 cells of the reporter plasmid, and either 0.5 µg/106 of the pRL-CMV Renilla control vector (Promega) or 1 µg/106 cells of the pSV-{beta}-galactosidase control vector (Promega), as an internal control for the normalization of transfection efficiency. Electroporation used a Bio-Rad (Hercules, CA) GenePulser (YT-Indy, 300 mV, 0.965 mF; NKL, 250 mV, 0.965 mF). Cells were harvested 24 h (NKL) or 48 h (YT-Indy) after transfection and lysed, and activity was measured on a Monolight 3010 (Analytical Luminescence Laboratory, Sparks, MD). Promega supplied the pGL3-basic reporter plasmid and the luciferase double reporter assay system.

Flow cytometry

Flow cytometric analyses were performed on a FACSort cytofluorometer (BD Immunocytochemistry Systems, San Jose, CA). Direct immunofluorescent staining was performed using PE-conjugated anti-CD94 (HP-3B1) or PE-conjugated anti-CD56 (PN-IM2073) mAb (Beckman Coulter, Fullerton, CA). A PE-conjugated isotype-matched control mAb was used to monitor background-staining levels.

RNA isolation, 5' RACE, RT-PCR, and sequence analyses

Total RNA was prepared from fresh primary NK, CD8+ {alpha}{beta} T, cultured primary NK, CD8+ {alpha}{beta} T, or cultured NKL cells using the RNAqueous Midi kit (Ambion, Austin, TX). RNA concentration was estimated by spectrophotometric analysis. The 5' RACE was performed with the FirstChoice RLM-RACE kit (Ambion); two custom CD94-specific antisense primers were used for nested PCR (primer 1, 5'-GAGTAAATGCTGGCTCAATA-3'; primer 2, 5'-CAGAAATTAACCTCCACAGA-3'). RT-PCR was performed using transcript-specific primers and the Superscript One-Step RT-PCR kit (Invitrogen, Carlsbad, CA). The expression of CD94 transcripts containing exon 1A or full-length exon 1B, as well as {beta}-actin transcripts as an internal control, was determined using 1 µg of total RNA. The following primers were used: CD94, exon 1A, 5'-GCTCATCAAGTTCAAGAGAC-3'; CD94, exon 1B, 5'-GCTAAATTTCTTCATACTCAAC-3'; CD94, exon 6, 5'-AGGTGAACGAATTTCATC-3'; {beta}-actin sense, 5'-CGCGAGAAGATGACCCAGATC-3'; {beta}-actin antisense, 5'-TTGCTGATCCACATCTGCTGG-3'. PCR products were electrophoresed on 1x Tris-boric acid EDTA, 1% agarose gels, and visualized with ethidium bromide staining.

cDNA was ligated into the pCR2.1 vector using the TA cloning kit from Invitrogen. The ligation products were used to transform Top10F' cells that were plated on Luria-Bertani agar plates supplemented with 100 µg/ml ampicillin (KD Medical, Columbia, MD). Single colonies were selected, DNA was isolated and PCR amplified using the primer sets described above. Amplification products were isolated and labeled using Big Dye kit v3.0 terminators (Applied Biosystems, Foster City, CA). Sequence analysis was performed by MWG Biotech (Highpoint, NC). Sequences were aligned with the canonical CD94 transcript using the Blast algorithm from National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/).

EMSA

Whole cell extracts from freshly isolated NK cells or freshly isolated NK cells incubated with IL-2 or IL-15 for 24 h were prepared, as described (30). Briefly, synthetic oligonucleotide probes (Table I) were labeled with [{gamma}-32P]ATP, and binding reactions, gel separation, and detection were performed using 8 µl of whole cell extracts equivalent to 3 x 105 cells. For competition experiments, unlabeled double-stranded oligonucleotides were preincubated with cell extracts for 10 min at room temperature before the addition of the labeled probe.


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  		Table I. Oligonucleotide probes and competitors used in EMSAa

 
Analysis of putative regulatory elements

Putative UTR functional elements were searched for using UTRscan (http://bighost.area.ba.cnr.it/BIG/UTRScan/). Putative transcription factor binding sites were identified using TFSearch (http://molsun1.cbrc.aist.go.jp/research/db/TFSEARCH.html). The thermodynamics of RNA structures were analyzed using mfold (http://www.bioinfo.rpi.edu/applications/mfold/old/rna/form1.cgi).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of CD94 transcripts containing an upstream untranslated exon in human NK and CD8+ {alpha}{beta} T cells

Based on primer extension mapping, multiple transcription initiation sites were previously reported for CD94 transcripts (29). Because the actual sequences of the 5' ends of these transcripts were unknown, we performed 5' RACE on total RNA isolated from NKL cells using a system that produces cDNA exclusively from mRNA with capped 5' ends. Sequence analyses verified the existence and location of the previously reported transcription initiation sites. However, several of the transcripts contained an initial sequence of 97 bp, underlined in Fig. 1A, that did not match the sequence of any of the previously reported mRNA isoforms of CD94. Subsequent analysis of the genomic sequence around the CD94 gene revealed that the 97-bp sequence was identical with a sequence of DNA beginning 3628 bp upstream from the translation initiation site and 3419 bp upstream from the consensus start site of the previously identified exon 1 (hereafter referred to as exon 1B). The 97-bp sequence is an untranslated exon that we have termed exon 1A. Transcripts that contain exon 1A make use of an acceptor site within exon 1B located 100 bp upstream from translation initiation site. These transcripts are 63 bp shorter than transcripts initiated from the consensus start site of transcription for exon 1B. A full-length transcript containing exon 1A has been sequenced and reported to GenBank as CD94C, accession number AY227806. All sequenced 5' ends of transcripts containing exon 1A had the same sequence, suggesting that these transcripts were initiated from a single start site of transcription. UTRscan analysis of the 5' UTR of transcripts with (CD94C) and without (CD94) exon 1A did not reveal any functional elements, such as iron response elements or internal ribosome entry sites, that could affect translation rates (31).



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  		FIGURE 1. The 5' sequences of the CD94C (containing exon 1A) and the CD94 (exon 1B) cDNAs, and the genomic structure of the human CD94 gene showing derivation of these transcripts. A, The underlined portion of the CD94C cDNA identifies the sequence of exon 1A. An asterisk in exon 1B in the CD94 cDNA designates the location of the acceptor splice site for exon 1A in CD94C. Vertical bars indicate the exon-exon boundaries, and the translation initiation site is in bold. Boxes indicate the locations of ATGs upstream of the translation initiation start sites. Only the first ATG in exon 1A fits the Kozak consensus sequence ((A/G)xxATGG). B, A schematic of the CD94 gene structure showing the location of the exons that code for each transcript and the two promoters. The relative gene structure is displayed on the lower bar and the CD94 mRNA structure with a boxed coding sequence is displayed above. The dashed lines indicate how exon 1A and the 5' portion of exon 1B are incorporated into the CD94C transcript.

 
Transcript analysis of NK cell clones by RT-PCR shows that both species of CD94 transcripts are present in all clones (Fig. 2A), as well as the NKL leukemia cell line. All of the clones tested and the NKL cell line were positive for CD94 surface expression, as observed by flow cytometry. We also verified that the YT-Indy NK cell line does not express viable CD94 transcripts (data not shown). CD8+ {alpha}{beta} T cell clones show a different pattern of CD94 transcript and protein expression from NK cells (Fig. 2B). Several of the examined clones express both species of CD94 transcript (e.g., T2-03); some express neither (e.g., T2-08); and others only express transcripts with exon 1B (e.g., T2-17). None of the CD8+ {alpha}{beta} T cell clones expressed transcripts that only contained exon 1A. Many of the CD8+ {alpha}{beta} T cell clones that expressed CD94 transcripts do not express CD94 on their surface. The existence of two species of transcripts that contain different 5' initial exons suggested that dual promoters regulate CD94 gene expression.



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  		FIGURE 2. NK and CD8+ {alpha}{beta} T cells express both CD94 and CD94C transcripts. These mRNAs were detected by RT-PCR and gel electrophoresis. The upper band present in the CD94C and CD94 rows contains a full-length coding sequence for CD94. The lower band contains an alternatively spliced coding sequence that lacks exon 2; see accession number NM007334 for CD94 transcript variant 2 (68 ). Transcripts were derived from either NK or CD8+ {alpha}{beta} T cell clones grown for 3 wk in the presence of IL-2 and feeder cells. Surface expression of CD94 was detected by flow cytometry and is indicated by a positive or negative sign. A, All NK clones tested were CD94+/CD94C+. B, Shown are several representatives of each class of CD8+ {alpha}{beta} T cell clone, CD94-/CD94C-, CD94+/CD94C-, and CD94+/CD94C+.

 
The CD94 gene has distal and proximal promoters with GAS/EBS elements

We demonstrated the existence of two promoters by showing that the regions upstream of each exon 1 could induce transcription of a luciferase reporter gene. The minimal promoter size inducing maximal activity was mapped, and important elements were identified. This was first approached by making a series of constructs that had progressive deletions from the 5' end of each promoter.

For the proximal promoter, the p-344 construct was the smallest construct to show maximal activity in both the YT-Indy and the NKL cell line (Fig. 3A). This indicates that the failure of YT-Indy to express CD94 is not due to differences in regulatory factors that interact with the promoter. The luciferase activity in the YT-Indy cells that were transiently transfected with the p-344 construct was on average ~17-fold higher than in these cells transfected with the pGL3-basic vector. The trend in luciferase activity generated by the panel of constructs was the same for the NKL cell line. These results suggest that the -344 to -215 region contains all of the elements necessary for the maximal activity of the proximal promoter. Transient transfections of the p-344 construct into E6-1 Jurkat T cells showed negligible changes in luciferase activity in comparison with cells transfected with the pGL3-basic vector (data not shown). Subsequently, this region was surveyed for functional cis elements by scanning mutagenesis (Fig. 3B). Mutations in two regions lead to a loss of luciferase activity; one beginning at -249 and the other at -273. A search of the proximal promoter using TFSearch identified a GAS/EBS motif from -250 to -241. The oligomerization and phosphorylation of STAT proteins are induced by multiple cytokines and growth factors with subsequent induction of numerous lymphocyte genes (32). The Ets family of transcription factors plays critical roles in regulating a variety of NK, NKT, and T cell processes, including growth control, activation, perforin expression, and transformation (33, 34, 35, 36). An element without homology to a previously identified regulatory element, as assessed by TFSearch, was identified near the -273 position, hereafter referred to as site A for the convenience of this report.



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  		FIGURE 3. Transcriptional analysis of CD94 proximal promoter constructs in the YT-Indy and NKL cell lines. A, Diagrams of the promoter constructs are shown on the left of each graph. Luciferase activities are from independent experiments performed twice with the YT-Indy cell line and three times with the NKL cell line with the same relative trend. The luciferase activities were normalized to that of the positive control (pGL3-basic). B, Measurement of luciferase activity generated by a series of sequential 6-bp mutations in the p-344 proximal promoter construct from -189 to -279 in YT-Indy cells. The values were normalized to the wt p-344 CD94 proximal promoter construct. The relevant sequence is shown below the graph; the GAS motif located at -249 is underlined; the EBS motif is double underlined; and site A located at -273 is underlined with dashes.

 
The distal promoter construct p-3807, which was the smallest promoter construct examined (179 bp), showed a maximal level of luciferase activity in the YT-Indy and NKL cell lines (Fig. 4A). YT-Indy cells that were transiently transfected with p-3807 construct had an average of ~85-fold higher luciferase activity than those transfected with pGL3-basic vector. Although the magnitude of luciferase activity was lower, a similar trend in relative intensity was observed when these constructs were transfected into NKL cells. Transfection of the p-3807 construct into E6-1 Jurkat T cells produced inconsequential changes in luciferase activity in comparison with the cells transfected with the pGL3 basic vector (data not shown). Analysis of the -3807 to -3628 region using TFSearch identified a GAS/EBS motif at -3697 to -3688. This site was mutated (see Materials and Methods) in the p-3807 construct with two single base pair changes within the dGAS/dEBS motif. This mp-3807 construct resulted in a ~50% decrease in luciferase activity in YT and NKL cells compared with the wt p-3807 construct (Fig. 4B).



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  		FIGURE 4. Transcriptional analysis of the CD94 distal promoter constructs by luciferase assay in the YT-Indy and NKL cell lines. Diagrams of the recombinant promoter constructs are shown on the left of each graph. Luciferase activities are from three independent experiments performed in each cell line with the same relative trend. The luciferase activities were normalized to that of the positive control (pGL3-basic). A, A series of deletion constructs mapping maximal luciferase activity. B, Comparison of luciferase levels between the wt p-3807 distal promoter construct and the mp-3807 distal promoter construct, which has a mutated dGAS/dEBS element.

 
Differential sensitivity of the distal and proximal promoters to IL-2 or IL-15 stimulation

A variety of cytokines are known to activate STAT transcription factors via the Janus kinase-STAT pathway (32). Among these cytokines are IL-2 and IL-15, which have been shown to activate and induce proliferation of human NK cells (37, 38). In agreement with previously reported results, we show that treatment with IL-2 or IL-15 up-regulates CD94 surface expression in primary NK cells (Fig. 5A) (39, 40, 41). To determine whether transcription of CD94 and/or CD94C was regulated by these cytokines, primary NK cells were cultured with IL-2 or IL-15 for various time points and harvested, and total RNA was isolated. RT-PCR analyses show that CD94C is not transcribed in fresh, primary, resting NK cells or CD8+ {alpha}{beta} T cells, which indicates that the distal promoter is not active in these cells (Fig. 5, B and C). After treatment with either IL-2 or IL-15, CD94C transcripts were detectable at 1 h in primary NK cells and showed a steady increase in abundance up to 24 h later. This indicates that either IL-2 or IL-15 can induce transcription from the distal promoter in primary NK cells. CD8+ {alpha}{beta} T cells cultured with IL-2 can also express CD94C (Fig. 2). The proximal promoter is active in fresh NK cells and, under all conditions studied, the CD94 transcript appears to be the most abundant, although these results were not quantitated.



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  		FIGURE 5. Analysis of CD94 protein and transcript expression in primary NK cells isolated from peripheral blood after treatment with IL-2 or IL-15, and transcript expression in freshly isolated primary CD8+ {alpha}{beta} T cells. A, Cell surface expression of CD94 after treatment with IL-2 or IL-15, as measured by FACS analysis; numbers indicate mean fluorescence intensity. B, Changes in CD94 and CD94C mRNA expression after treatment with IL-2 or IL-15, as detected by RT-PCR and gel electrophoresis. C, CD94 and CD94C transcripts in fresh CD8+ {alpha}{beta} T cells freshly isolated from peripheral blood.

 
Constitutive and inducible factors bind to CD94 distal and proximal elements in vitro

The ability of the distal and proximal elements, containing GAS/EBS motifs and the proximal unknown motif (site A), to form protein-DNA complexes was tested by EMSA. Whole-cell extracts from fresh or IL-2-activated primary NK cells were incubated with oligonucleotide probes containing either the dGAS/dEBS, pGAS/pEBS, or site A elements (Table I). One constitutive (dC1) and one IL-2-inducible (dI1) DNA-protein complex were revealed with the dGAS/dEBS probe (Fig. 6A). Similarly, two complexes were observed with the pGAS/pEBS probe, one constitutive (pC1) and one IL-2 inducible (pI1) (Fig. 6B). Two constitutive (SAC1, SAC2) DNA-protein complexes were identified with the site A probe (Fig. 6C). EMSA analysis of IL-15-stimulated NK cells indicated that the DNA-protein complexes were identical with those from the IL-2-stimulated cells for all three probes (data not shown).



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  		FIGURE 6. In vitro complexes formed by oligonucleotide probes containing dGAS/dEBS, pGAS/pEBS, and site A elements, detected by EMSA. Freshly isolated primary NK cells were stimulated with IL-2 and harvested at 24 h. Whole cell extracts were incubated with each probe and resolved by nondenaturing polyacrylamide gel electrophoresis. A, B, and C, Identification of the DNA-protein complexes in the absence and presence of competitor probes. Whole NK cell extracts were either unstimulated (lane 1) or stimulated (A and B, lanes 2–7; C, lanes 2–5) with IL-2 for 24 h before lysis. Competitions (A and B, lanes 3–7; C, lanes 3–5) were performed with 100x unlabeled competitor before incubation with labeled probe. See Table I for sequence of probes and competitors.

 
Proteins forming complexes with wild-type (wt) probes were further characterized by testing the ability of unlabeled competitor probes containing STAT and Ets binding sites (Table I) to inhibit binding of transcription factors to the wt probes. Competition with an excess of unlabeled wt-dGAS/dEBS eliminated both the dC1 and the dI1 complexes (Fig. 6B). Addition of a competitor containing a known GAS motif, Fc{gamma}RI-GAS, effectively competed with the wt-dGAS/dEBS probe for formation of the dC1 and dI1 complexes. The mFc{gamma}RI-GAS competitor, which has a mutated GAS element, was unable to interfere with formation of these complexes. This suggests that the dC1 and dI1 complexes contain transcription factors that recognize the GAS motif common to both the dGAS/dEBS probe and the Fc{gamma}RI-GAS competitor. Neither the Ets-1-PEA3 (polyomavirus enhancer activator 3) nor the mEts-1-PEA3 competitors containing a known and mutated EBS motif, respectively, were able to compete with the probe in forming these complexes.

The pC1 and pI1 complexes formed with the pGAS/pEBS probe were eliminated by competition with unlabeled wt competitor (Fig. 6B). Addition of the Fc{gamma}RI-GAS competitor was able to compete for binding with the wt probe in formation of the pI1 complex, but not the pC1 complex. This suggests that the pI1 complex contains transcription factors that recognize the GAS motif present in the pGAS/pEBS probe and Fc{gamma}RI-GAS competitor. The Ets-1-PEA3 competitors were ineffective at competing for either complex. This shows that the pC1 complex contains transcription factors specific for the pGAS/pEBS element that do not interact with the Fc{gamma}RI-GAS or the Ets-1-PEA3 competitor.

The two constitutive complexes bound to the site A probe were removed by competition with the unlabeled wt site A competitor (Fig. 6C). Located near site A within the probe used for EMSA assays was a putative EBS motif (Table I). However, competition assays performed with Ets-1-PEA3 competitors failed to inhibit complexes formed with the wt probe.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have identified a novel transcript of the CD94 gene, designated CD94C, which contains an untranslated exon 1A located ~3.5 kb upstream from the initiation of translation (Fig. 1B). The previously described CD94 transcript contains a partially untranslated exon at the 5' end, now referred to as exon 1B. CD94C and CD94 transcripts maintain the same start site for translation because exon 1A splices to a donor splice site within exon 1B (Fig. 1A). Distal and proximal promoters regulate transcription of CD94C and CD94 transcripts, respectively. Freshly isolated, primary NK cells express CD94 transcripts, but not CD94C transcripts. This is also the case for freshly isolated CD8+ {alpha}{beta} T cells that are positive for CD94 gene expression. Stimulation with either IL-2 or IL-15 invariably initiates expression of CD94C transcripts in NK cells. In contrast, CD94 transcript-positive CD8+ {alpha}{beta} T cell clones grown in IL-2 can also initiate expression of CD94C transcripts, but do not always do so. These differences in CD94 gene expression patterns may be due to variations in methylation, histone modification, or the binding of repressor factors. The apparent lack of increase in CD94 transcript abundance after IL-2 or IL-15 stimulation, as detected by RT-PCR, may be due to the inherent insensitivities of this method for measuring anything but gross differences in transcript amounts (Fig. 5B).

Analysis of the proximal promoter by scanning mutagenesis identified two sites important for promoter function. The DNA sequence encompassing one of the sites (-249 to -244) was shown to contain a GAS/EBS (pGAS/pEBS) motif, but the sequence encompassing the other site (-273 to -266) could not be related to any previously described transcription factor binding site and was designated site A. A search program revealed the presence of a GAS/EBS (dGAS/dEBS) motif at positions -3697 to -3688 in the distal promoter. Analysis by site-directed mutagenesis indicated that this site was involved in distal promoter function. Both IL-2 and IL-15 induce transcription of the human CD94 gene by activating the distal promoter. IL-2 or IL-15 stimulation of NK or T cells induces phosphorylation and activation of STAT3, STAT5a, and STAT5b in the signal transduction pathway (42, 43, 44, 45, 46). IL-2 treatment of activated T cells has been shown to induce the formation of functional STAT5/ETS complexes (47). The two GAS binding sites, one in each promoter, were identical with the consensus STAT DNA binding site (Fig. 7A) (48, 49).



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  		FIGURE 7. Analysis of the promoter elements. A, Comparison of the human GAS elements with the consensus sequence. B, Sequence comparison of the mouse and human CD94 promoter regions. Identical nucleotides are boxed with a black background. Consensus motifs for regulatory elements are underlined. For each species, the numbers indicate the distance from the start of translation of CD94 (+1).

 
Results obtained by RT-PCR analysis demonstrate that the proximal promoter is constitutively active in all NK cells and CD8+ {alpha}{beta} T cells that express CD94 receptors, while the distal promoter function is inducible. We speculate that the proximal promoter, which has two transcription factor binding sites detectable by scanning mutagenesis, may maintain the basal level of transcription through the SAC1 and/or SAC2 complexes. Site A is unique to the proximal promoter, and EMSA results indicate that all complexes bind to this element in a constitutive fashion. We have been unable to define the motif(s) that binds these complexes. Further EMSA experiments with a series of mutated site A competitors are planned.

Alternatively, or conjointly, the constitutive activity of the proximal promoter may be due to the factors present in the pC1 complex that bind the pGAS/pEBS oligonucleotide probe. Two complexes are formed with the pGAS/pEBS probe: one is constitutively present (pC1), and one is induced after IL-2 or IL-15 treatment (pI1). Similar to the dI1 complex, the pI1 complex binds to the Fc{gamma}RI-GAS competitor. This led us to speculate that the pI1 complex, at least, contains STAT proteins, because these have been previously shown to interact with the Fc{gamma}RI-GAS element (50). The factors that form the pC1 complex can be distinguished from those forming the dC1 complex by their failure to bind to the Fc{gamma}RI-GAS competitor.

The oligonucleotide probe containing the distal promoter dGAS/dEBS element forms both a constitutive (dC1) and an inducible (dI1) complex. The Fc{gamma}RI-GAS element interferes with the formation of both of these complexes. Because the transcription of CD94C occurs only after IL-2 or IL-15 treatment, the constitutively bound factor(s) is apparently not sufficient to induce transcription. The Fc{gamma}RI-GAS element can form complexes with several STAT proteins, including IL-2- or IL-15-activated STAT5a or STAT5b (50). Because the Fc{gamma}RI-GAS competitor probe abolishes the formation of the IL-2- or IL-15-induced dI1 complex, as well as the dC1 complex, it is probable that both of these complexes contain STAT proteins. However, the failure of the dC1 or dI1 complex to shift after incubation with anti-STAT or Ets Abs (data not shown) suggests that if members of these classes of transcription factors are present in these complexes, their epitopes are not accessible or that the conditions tested were not optimum for binding. Elucidation of the transcription factors present in these complexes is the subject of ongoing studies.

The dissimilarities in the behavior of the pC1 and dC1 complexes in response to competition with the Fc{gamma}RI-GAS oligonucleotide competitor (Fig. 6, A and B), mentioned above, may be due to differences in the sequence surrounding each GAS/EBS motif and/or to the difference in the central nucleotide of the GAS motif (Fig. 7A). In the dGAS/dEBS element, the central adenine nucleotide begins a sense-orientated EBS motif. However, in the pGAS/pEBS element, the central thymine motif ends an antisense-orientated EBS motif (Table I). This opposite orientation of the EBS motif between the two elements may lead to differences in the transcription factor complexes that recognize each GAS/EBS element. Such differences may be why the transcription factors in the dC1, dI1, and pI1 complexes bind to the Fc{gamma}RI-GAS element, while the factors that constitute the pC1 complex do not. This implies that the pC1 transcription factors do not interact with the GAS motif in the pGAS/pEBS element and instead most likely bind with the EBS motif. These transcription factors probably recognize an element larger than the central EBS motif because they do not bind with the Ets-1-PEA3 competitor.

Evidence suggests that CD94 receptors originated early in the evolution of the chordates, of which vertebrates are a member. A receptor with homology to human CD94, termed BsCD94-1, was recently identified in Botryllus schlosseri, a urochordate (51). BcCD94-1 gene expression changes in response to allorecognition by B. schlosseri and has been proposed to be a marker of ancestral NK cells. It is suggested that urochordates and vertebrates diverged over 750 million years ago (52). This would indicate that CD94 is an ancient receptor component.

Considering this, it is not surprising that CD94/NKG2 receptors are also found in both primates and rodents (53, 54). Examination of the murine CD94 gene has identified dual promoters that produce two species of transcripts analogous to human CD94 and CD94C (55). Comparison of the human and mouse distal and proximal promoter sequences reveals that the GAS/EBS elements are highly conserved and site A is partially conserved (Fig. 7B). In addition, comparison of the distal promoters revealed that a putative TATA box downstream from the dGAS/dEBS element is also conserved. Because all of the human CD94C cDNAs sequenced had the same 5' UTR, it is possible that transcription is initiated from this conserved TATA box. Comparison of the MHC I receptors of mouse and human NK cells indicates that CD94 and the NKG2 families existed before the divergence of the rodent and primate lineages ~100 million years ago (56). The presence of functional dual promoters in both species and the conservation of elements within these promoters indicate that they most likely play an important role in the regulation of CD94 gene expression.

Why would regulation of the CD94 gene require two promoters? One possibility is to differentially regulate expression in different tissues. For example, distinct promoters have been shown to regulate the expression of the protein kinase DYRK1B gene in different tissues (57). Because CD94 is expressed only by lymphocytes and our data indicate that, at least under the conditions examined, the two promoters do not distinguish expression between NK and T lymphocytes, it is unlikely that the two CD94 promoters exist to regulate cell type expression.

Another possibility is that the dual promoters exist to insure expression of the CD94/NKG2A receptor, which may be particularly vital during early stages of development when KIR receptors are not yet expressed. One promoter may require transcription factors that are not present or active early in development, leaving the remaining promoter as the sole regulator of CD94 transcription. A recent study examining human NK cell receptor ontogeny in umbilical cord-derived stem cells showed that contact-dependent ligand stimulation, along with the presence of IL-2 and IL-15, was important for CD94 and KIR expression (39). After stimulation of single progenitor cells, the frequency of CD94+ NK cells was greater than KIR+ NK cells and CD94 expression occurred earlier. In adults, the majority of peripheral blood NK cells express CD94/NKG2A and at least one KIR receptor; however, the majority of the NK cells in synovial fluid in patients with rheumatoid arthritis are CD94+ and KIR2DL1/L2/L3- (58). Because CD94/NKG2A are likely the only inhibitory receptors expressed by such NK cells, failure to express CD94 in these cells would likely have dire consequences for the host. Thus, dual promoters may exist to insure CD94 expression in cells in which no other inhibitory receptors are expressed. The use of dual promoters to regulate gene expression at different stages during development has been observed for the mouse µ-opioid receptor (59).

The possibility that the dual promoters exist to insure expression of a vital regulatory receptor can be extended to NK cells in general. Analysis of transcription in NK and CD8+ {alpha}{beta} T cells clearly shows that initiation of transcription from the CD94 distal promoter becomes active in cells that are stimulated by exogenous cytokines, such as IL-2 or IL-15. Therefore, the purpose of the distal promoter may be solely to insure CD94 gene expression upon activation of mature cells to guarantee the sensitivity of these cells for detection of MHC class I expression to prevent the targeting of healthy cells.

It is likely that CD94 regulation occurs at several levels. The cell surface expression of CD94 may be regulated at the protein level through sequestering or receptor recycling (60, 61). It is clear that a sophisticated mechanism of translational regulation exists, especially in T cells in which the presence of CD94 transcripts does not guarantee protein expression (Fig. 2B). CD94 expression is probably influenced by the rate of transcription, mRNA stability, and translation efficiency, all of which may be affected by sequences in the 5' and 3' UTRs.

Analysis of the 5' UTR regions of the CD94 and CD94C transcripts, using predicative algorithms, was unable to identify any functional elements such as iron response elements or internal ribosome entry sites (31). Both CD94 and CD94C have relatively long 5' UTRs with AUGs upstream of the translation initiation start site (Fig. 1A). The presence of these 5' UTRs would seem to violate the first AUG rule, stating that translation is initiated at the AUG codon closest to the 5' end (62). However, in both CD94 and CD94C, the first AUG is followed shortly by an in-frame terminator codon. In this situation, the posttermination ribosomes generally resume scanning and can reinitiate at a downstream AUG site (62, 63, 64). Reinitiation is typically inefficient and can reduce translation productivity. It is likely that any issues related to premature initiation of translation affect the processing of the CD94 and CD94C transcripts equally. Moreover, examination of the thermodynamic stability of the 5' UTRs of both transcripts by analyzing their predicted Gibbs free energy indicates that they both have the same relative propensity to form a stable structure (65, 66). The formation of stable structures can impair the movement of the ribosomal 40S subunits as they scan downstream (67).

In summary, we have identified the core promoters of human CD94, which contain conserved GAS/EBS elements that can form protein-DNA complexes. The two promoters differentially regulate expression of the CD94 gene in response to treatment with IL-2 or IL-15. Clarification of the precise roles of these two promoters and the factors that regulate them is under active investigation.


   Acknowledgments
 
We thank Dr. Kerima Maasho and Robert Valas for technical assistance, and Dr. Brian Wilhelm, Dr. Jean Imbert, and Dr. Konstantin Khalturin for helpful discussion.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. John E. Coligan, Receptor Cell Biology Section, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook II, Room 205, 12441 Parklawn Drive, Rockville, MD 20852-1742. E-mail address: jcoligan{at}niaid.nih.gov Back

2 Abbreviations used in this paper: KIR, killer cell Ig-like receptor; EBS, Ets binding site; GAS, IFN-{gamma}-activated sequence; LM, Listeria monocytogenes; UTR, untranslated region; wt, wild type; m, mutant. Back

Received for publication June 6, 2003. Accepted for publication September 11, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Biron, C. A., L. Brossay. 2001. NK cells and NKT cells in innate defense against viral infections. Curr. Opin. Immunol. 13:458.[Medline]
  2. Lanier, L. L.. 1998. NK cell receptors. Annu. Rev. Immunol. 16:359.[Medline]
  3. Soloski, M. J.. 2001. Recognition of tumor cells by the innate immune system. Curr. Opin. Immunol. 13:154.[Medline]
  4. Colucci, F., J. P. Di Santo, P. J. Leibson. 2002. Natural killer cell activation in mice and men: different triggers for similar weapons?. Nat. Immun. 3:807.
  5. Moretta, A., C. Bottino, M. Vitale, D. Pende, C. Cantoni, M. C. Mingari, R. Biassoni, L. Moretta. 2001. Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu. Rev. Immunol. 19:197.[Medline]
  6. Borrego, F., J. Kabat, D. K. Kim, L. Lieto, K. Maasho, J. Pena, R. Solana, J. E. Coligan. 2002. Structure and function of major histocompatibility complex (MHC) class I specific receptors expressed on human natural killer (NK) cells. Mol. Immunol. 38:637.[Medline]
  7. Lanier, L. L.. 2001. Face off: the interplay between activating and inhibitory immune receptors. Curr. Opin. Immunol. 13:326.[Medline]
  8. Raulet, D. H., V. E. Vance, C. W. McMahon. 2001. Regulation of the natural killer cell receptor repertoire. Annu. Rev. Immunol. 19:291.[Medline]
  9. Kabat, J., F. Borrego, A. Brooks, J. E. Coligan. 2002. Role that each NKG2A immunoreceptor tyrosine-based inhibitory motif plays in mediating the human CD94/NKG2A inhibitory signal. J. Immunol. 169:1948.[Abstract/Free Full Text]
  10. Long, E. O.. 1999. Regulation of immune responses through inhibitory receptors. Annu. Rev. Immunol. 17:875.[Medline]
  11. Samaridis, J., M. Colonna. 1997. Cloning of novel immunoglobulin superfamily receptors expressed on human myeloid and lymphoid cells: structural evidence for new stimulatory and inhibitory pathways. Eur. J. Immunol. 2:660.
  12. Colonna, M., F. Navarro, T. Bellon, M. Llano, P. Garcia, J. Samaridis, L. Angman, M. Cella, M. López-Botet. 1997. A common inhibitory receptor for major histocompatibility complex class I molecules on human lymphoid and myelomonocytic cells. J. Exp. Med. 186:1809.[Abstract/Free Full Text]
  13. Cosman, D., N. Fanger, L. Borges, M. Kubin, W. Chin, L. Peterson, M. L. Hsu. 1997. A novel immunoglobulin superfamily receptor for cellular and viral MHC class I molecules. Immunity 7:273.[Medline]
  14. Brooks, A. G., P. E. Posch, C. J. Scorzelli, F. Borrego, J. E. Coligan. 1997. NKG2A complexed with CD94 defines a novel inhibitory natural killer cell receptor. J. Exp. Med. 185:795.[Abstract/Free Full Text]
  15. Lazetic, S., C. Chang, J. P. Houchins, L. L. Lanier, J. H. Phillips. 1996. Human natural killer cell receptors involved in MHC class I recognition are disulfide-linked heterodimers of CD94 and NKG2 subunits. J. Immunol. 157:4741.[Abstract]
  16. Borrego, F., M. Ulbrecht, E. H. Weiss, J. E. Coligan, A. G. Brooks. 1998. Recognition of human histocompatibility leukocyte antigen (HLA)-E complexed with HLA class I signal sequence-derived peptides by CD94/NKG2 confers protection from natural killer cell-mediated lysis. J. Exp. Med. 187:813.[Abstract/Free Full Text]
  17. Braud, V. M., D. S. Allan, C. A. O’Callaghan, K. Soderstrom, A. D’Andrea, G. S. Ogg, S. Lazetic, N. T. Young, J. I. Bell, J. H. Phillips, et al 1998. HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 391:795.[Medline]
  18. Lee, N., M. Llano, M. Carretero, A. Ishitani, F. Navarro, M. Lopez-Botet, D. E. Geraghty. 1998. HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A. Proc. Natl. Acad. Sci. USA 95:5199.[Abstract/Free Full Text]
  19. Costa, P., S. Rusconi, D. Mavilio, M. Fogli, G. Murdaca, D. Pende, M. C. Mingari, M. Galli, L. Moretta, A De Maria. 2001. Differential disappearance of inhibitory natural killer cell receptors during HAART and possible impairment of HIV-1-specific CD8 cytotoxic T lymphocytes. AIDS 15:965.[Medline]
  20. Galiani, M. D., E. Aguado, R. Tarazona, P. Romero, I. Molina, M. Santamaria, R. Solana, J. Pena. 1999. Expression of killer inhibitory receptors on cytotoxic cells from HIV-1-infected individuals. Clin. Exp. Immunol. 115:472.[Medline]
  21. Gunturi, A., R. E. Berg, J. Forman. 2003. Preferential survival of CD8 T and NK cells expressing high levels of CD94. J. Immunol. 170:1737.[Abstract/Free Full Text]
  22. McMahon, C. W., A. J. Zajac, A. M. Jamieson, L. Corral, G. E. Hammer, R. Ahmed, D. H. Raulet. 2002. Viral and bacterial infections induce expression of multiple NK cell receptors in responding CD8+ T cells. J. Immunol. 169:1444.[Abstract/Free Full Text]
  23. Moser, J. M., J. Gibbs, P. E. Jensen, A. E. Lukacher. 2002. CD94-NKG2A receptors regulate antiviral CD8+ T cell responses. Nat. Immun. 3:189.
  24. Miller, J. D., M. Peters, A. E. Oran, G. W. Beresford, L. Harrington, J. M. Boss, J. D. Altman. 2002. CD94/NKG2 expression does not inhibit cytotoxic function of lymphocytic choriomeningitis virus-specific CD8+ T cells. J. Immunol. 169:693.[Abstract/Free Full Text]
  25. Ugolini, S., C. Arpin, N. Anfossi, T. Walzer, A. Cambiaggi, R. Forster, M. Lipp, R. E. Toes, C. J. Melief, J. Marvel, E. Vivier. 2001. Involvement of inhibitory NKRs in the survival of a subset of memory-phenotype CD8+ T cells. Nat. Immun. 2:430.
  26. Young, N. T., M. Uhrberg, J. H. Phillips, L. L. Lanier, P. Parham. 2001. Differential expression of leukocyte receptor complex-encoded Ig-like receptors correlates with the transition from effector to memory CTL. J. Immunol. 166:3933.[Abstract/Free Full Text]
  27. Hofer, E., Y. Sobanov, C. Brostjan, H. Lehrach, M. Duchler. 2001. The centromeric part of the human natural killer (NK) receptor complex: lectin-like receptor genes expressed in NK, dendritic and endothelial cells. Immunol. Rev. 181:5.[Medline]
  28. Renedo, M., I. Arce, K. Montgomery, P. Roda-Navarro, E. Lee, R. Kucherlapati, E. Fernandez-Ruiz. 2000. A sequence-ready physical map of the region containing the human natural killer gene complex on chromosome 12p12.3-p13.2. Genomics 65:129.[Medline]
  29. Rodriguez, A., M. Carretero, J. Glienke, T. Bellon, A. Ramirez, H. Lehrach, F. Francis, M. Lopez-Botet. 1998. Structure of the human CD94 C-type lectin gene. Immunogenetics 47:305.[Medline]
  30. Lecine, P., M. Algarte, P. Rameil, C. Beadling, P. Bucher, M. Nabholz, J. Imbert. 1996. Elf-1 and Stat5 bind to a critical element in a new enhancer of the human interleukin-2 receptor {alpha} gene. Mol. Cell. Biol. 16:6829.[Abstract]
  31. Pesole, G., S. Liuni. 1999. Internet resources for the functional analysis of 5' and 3' untranslated regions of eukaryotic mRNA. Trends Genet. 15:378.[Medline]
  32. Leonard, W. J.. 2001. Cytokines and immunodeficiency diseases. Nat. Rev. Immunol. 1:200.[Medline]
  33. Anderson, M. K., G. Hernandez-Hoyos, R. A. Diamond, E. V. Rothenberg. 1999. Precise developmental regulation of Ets family transcription factors during specification and commitment to the T cell lineage. Development 126:3131.[Abstract]
  34. Barton, K., N. Muthusamy, C. Fischer, C. N. Ting, T. L. Walunas, L. L. Lanier, J. M. Leiden. 1998. The Ets-1 transcription factor is required for the development of natural killer cells in mice. Immunity 9:555.[Medline]
  35. Lacorazza, H. D., Y. Miyazaki, A. Di Cristofano, A. Deblasio, C. Hedvat, J. Zhang, C. Cordon-Cardo, S. Mao, P. P. Pandolfi, S. D. Nimer. 2002. The ETS protein MEF plays a critical role in perforin gene expression and the development of natural killer and NK-T cells. Immunity 17:437.[Medline]
  36. Walunas, T. L., B. Wang, C. R. Wang, J. M. Leiden. 2000. The Ets1 transcription factor is required for the development of NK T cells in mice. J. Immunol. 164:2857.[Abstract/Free Full Text]
  37. Carson, W. E., J. G. Giri, M. J. Lindemann, M. L. Linett, M. Ahdieh, R. Paxton, D. Anderson, J. Eisenmann, K. Grabstein, M. A. Caligiuri. 1994. Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor. J. Exp. Med. 180:1395.[Abstract/Free Full Text]
  38. Domzig, W., B. M. Stadler, R. B. Herberman. 1983. Interleukin 2 dependence of human natural killer (NK) cell activity. J. Immunol. 130:1970.[Abstract]
  39. Miller, J. S., V. McCullar. 2001. Human natural killer cells with polyclonal lectin and immunoglobulin like receptors develop from single hematopoietic stem cells with preferential expression of NKG2A and KIR2DL2/L3/S2. Blood 98:705.[Abstract/Free Full Text]
  40. Mingari, M. C., C. Vitale, C. Cantoni, R. Bellomo, M. Ponte, F. Schiavetti, S. Bertone, A. Moretta, L. Moretta. 1997. Interleukin-15-induced maturation of human natural killer cells from early thymic precursors: selective expression of CD94/NKG2-A as the only HLA class I-specific inhibitory receptor. Eur. J. Immunol. 27:1374.[Medline]
  41. Mingari, M. C., M. Ponte, S. Bertone, F. Schiavetti, C. Vitale, R. Bellomo, A. Moretta, L. Moretta. 1998. HLA class I-specific inhibitory receptors in human T lymphocytes: interleukin 15-induced expression of CD94/NKG2A in superantigen- or alloantigen-activated CD8+ T cells. Proc. Natl. Acad. Sci. USA 95:1172.[Abstract/Free Full Text]
  42. Brunn, G. J., E. L. Falls, A. E. Nilson, R. T. Abraham. 1995. Protein-tyrosine kinase-dependent activation of Stat transcription factors in interleukin-2- or interleukin-4-stimulated T lymphocytes. J. Biol. Chem. 270:11628.[Abstract/Free Full Text]
  43. Johnston, J. A., C. M. Bacon, D. S. Finbloom, R. C. Rees, D. Kaplan, K. Shibuya, J. R. Ortaldo, S. Gupta, Y. Q. Chen, J. D. Giri. 1995. Tyrosine phosphorylation and activation of Stat5, Stat3, and Janus kinases by interleukins 2 and 15. Proc. Natl. Acad. Sci. USA 92:8705.[Abstract/Free Full Text]
  44. Lin, J. X., T. S. Migone, M. Tsang, M. Friedmann, J. A. Weatherbee, L. Zhou, A. Yamauchi, E. T. Bloom, J. Mietz, S. John. 1995. The role of shared receptor motifs and common Stat proteins in the generation of cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13, and IL-15. Immunity 2:331.[Medline]
  45. Nosaka, T., J. M. van Deursen, R. A. Tripp, W. E. Thierfelder, B. A. Witthuhn, A. P. McMickle, P. C. Doherty, G. C. Grosveld, J. N. Ihle. 1995. Defective lymphoid development in mice lacking Jak3. Science 270:800.[Abstract/Free Full Text]
  46. Thomis, D. C., L. J. Berg. 1997. The role of Jak3 in lymphoid development, activation, and signaling. Curr. Opin. Immunol. 9:541.[Medline]
  47. Rameil, P., P. Lecine, J. Ghysdael, F. Gouilleux, B. Kahn-Perles, J. Imbert. 2000. IL-2 and long-term T cell activation induce physical and functional interaction between Stat5 and ETS transcription factors in human T cells. Oncogene 19:2086.[Medline]
  48. Ihle, J. N.. 1996. Stats: signal transducers and activators of transcription. Cell 84:331.[Medline]
  49. Shindler, C., J. E. Darnell. 1995. Transcriptional responses to polypeptide ligands: the JAK-Stat pathway. Annu. Rev. Biochem. 64:621.[Medline]
  50. Yu, C. R., H. A. Young, J. R. Ortaldo. 1998. Characterization of cytokine differential induction of Stat complexes in primary human T and NK cells. J. Leukocyte Biol. 64:245.[Abstract]
  51. Khalturin, K., M. Becker, B. Rinkevich, T. C. Bosch. 2003. Urochordates and the origin of natural killer cells: identification of a CD94/NKR-P1-related receptor in blood cells of Botryllus. Proc. Natl. Acad. Sci. USA 100:622.[Abstract/Free Full Text]
  52. Hedges, S. B.. 2001. Molecular evidence for the early history of living vertebrates. P. E. Ahlberg, ed. Major Events in Early Vertebrate Evolution: Paleontology, Phylogeny, Genetics and Development 119. Taylor & Francis, London.
  53. Khakoo, S. I., R. Rajalingam, B. P. Shum, K. Weidenbach, L. Flodin, D. G. Muir, F. Canavez, S. L. Cooper, N. M. Valiante, L. L. Lanier, P. Parham. 2000. Rapid evolution of NK cell receptor systems demonstrated by comparison of chimpanzees and humans. Immunity 12:687.[Medline]
  54. Vance, R. E., D. M. Tanamachi, T. Hanke, D. H. Raulet. 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.[Medline]
  55. Wilhelm, B. T., J.-R. Landry, F. Takei, D. L. Mager. 2003. Transcriptional control of murine CD94 gene: differential usage of dual promoters by lymphoid cell types. J. Immunol. 171:4219.[Abstract/Free Full Text]
  56. Kumar, S., S. B. Hedges. 1998. A molecular timescale for vertebrate evolution. Nature 392:917.
  57. Leder, S., H. Czajkowska, B. Maenz, K. De Graaf, A. Barthel, H. G. Joost, W. Becker. 2003. Alternative splicing variants of dual specificity tyrosine phosphorylated and regulated kinase 1B exhibit distinct patterns of expression and functional properties. Biochem. J. 372:881.[Medline]
  58. Pridgeon, C., G. P. Lennon, L. Pazmany, R. N. Thompson, S. E. Christmas, R. J. Moots. 2003. Natural killer cells in the synovial fluid of rheumatoid arthritis patients exhibit a CD56bright, CD94bright, CD158 negative phenotype. Rheumatology 42:870.[Abstract/Free Full Text]
  59. Ko, J. L., H. C. Chen, H. H. Loh. 2002. Differential promoter usage of mouse µ-opioid receptor gene during development. Brain Res. Mol. Brain Res. 104:184.[Medline]
  60. Borrego, F., J. Kabat, T. B. Sanni, J. E. Coligan. 2002. NK cell CD94/NKG2A inhibitory receptors are internalized and recycle independently of inhibitory signaling processes. J. Immunol. 169:6102.[Abstract/Free Full Text]
  61. Boullier, S., Y. Poquet, F. Halary, M. Bonneville, J. J. Fournie, M. L. Gougeon. 1998. Phosphoantigen activation induces surface translocation of intracellular CD94/NKG2A class I receptor on CD94- peripheral V{gamma}9 V{delta}2 T cells but not on CD94- thymic or mature T {gamma}{delta} cell clones. Eur. J. Immunol. 28:3399.[Medline]
  62. Kozak, M.. 1999. Initiation of translation in prokaryotes and eukaryotes. Gene 234:187.[Medline]
  63. McCarthy, J. E.. 1998. Posttranscriptional control of gene expression in yeast. Microbiol. Mol. Biol. Rev. 62:1492.[Abstract/Free Full Text]
  64. Morris, D. R., A. P. Geballe. 2000. Upstream open reading frames as regulators of mRNA translation. Mol. Cell. Biol. 20:8635.[Free Full Text]
  65. Mathews, D. H., J. Sabina, M. Zuker, D. H. Turner. 1999. Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 288:911.[Medline]
  66. Zuker, M., D. H. Mathews, D. H. Turner. 1999. Algorithms and thermodynamics for RNA secondary structure prediction: a practical guide. J. Barciszewski, and B. F. C. Clark, eds. RNA Biochemistry and Biotechnology 11. NATO ASI Series, Kluwer Academic Publishers, Dordrecht, Holland.
  67. Kozak, M.. 1989. The scanning model for translation: an update. J. Cell Biol. 108:229.[Abstract/Free Full Text]
  68. Furukawa, H., T. Yabe, K. Watanabe, R. Miyamoto, T. Akaza, K. Tadokoro, S. Tohma, T. Inoue, K. Yamamoto, T. Juji. 1998. An alternatively spliced form of the human CD94 gene. Immunogenetics 148:87.



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