T cells have also been proposed to recognize Qa-1 in various contexts 3031323334. Although it seems reasonable to presume that recognition of Qa-1 by T cells is mediated by the TCR, it should be noted that ‘NK’ receptors (including CD94 and NKG2 receptors) can be expressed by T cells 3536, and it remains possible that some of the effects of Qa-1 on T cells might be due to T cell expression of CD94/NKG2 heterodimers.

Although previous studies demonstrated that NK cells can bind to soluble Qa-1 tetramers 937, as can heterologous transfectants expressing CD94/NKG2A 9, it has never been formally demonstrated that Qa-1 tetramer binding by NK cells is due to NK expression of CD94/NKG2, and moreover, additional Qa-1 receptors might exist. Here we report the identification and characterization of two putative activating receptors on mouse NK cells, CD94/NKG2C and CD94/NKG2E, which, we demonstrate, bind Qa-1^b. We also provide evidence that CD94/NKG2 receptors are the only Qa-1 receptors on NK cells. Our results have implications for our understanding of NK cells and the Qa-1 molecule and will permit dissection of the role of class I–specific activating receptors in the genetically and experimentally amenable mouse model.

	Materials and Methods

Top Abstract Materials and Methods Results and Discussion References

 
Primers, Probes, PCR, and Sequencing.
Table contains a list of PCR primers used in this study. For PCR, an annealing temperature of 55°C was used throughout. Oligonucleotide probes were hybridized to Southern blots at 50°C and washed twice briefly in 2x SSC/1% SDS at room temperature and then for 30 min at 50°C in 1x SSC/1% SDS before exposure to film. Sequencing reactions were performed using the Big Dye Terminator Ready Mix (PE Biosystems) and were resolved and analyzed using an ABI Prism 310 sequencer (PE Biosystems).

Molecular Cloning of NKG2C and NKG2E.
In the course of amplifying NKG2 cDNA ends (as described previously; reference 9), one clone similar to human NKG2C was obtained but was lacking its 5' end and start codon. Based on this clone, a sequencing primer (NKG2C3'ex2) was designed and used to obtain the sequence of the 5' coding and untranslated regions (UTRs) directly from bacterial artificial chromosome (BAC) B6-3 (clone 91i19). 3' coding and untranslated sequence was obtained by sequencing the end of a genomic fragment, H3.5. Together, these 5' and 3' sequences were used to design two sets of primers to amplify full length NKG2C and -E open reading frames (ORFs). The NKG2C 5' and 3' UTR primers recognized sites just outside of the predicted ORF, whereas the NKG2C5'ATG and NKG2C3'#3 primers bound just within the ORF. Both sets of primers were used to amplify NKG2 sequences from oligo dT–primed cDNA generated by standard methods from IL-2–cultured NK cell RNA. The products, generated with Taq polymerase (Promega Corp.), were cloned directly into the T-tailed T easy vector (Promega Corp.) and sequenced on both strands. Clones generated with the UTR primer pair were designated ‘C-UTR’ or ‘E-UTR,’ whereas clones generated with the ATG primer pair were designated ‘C-ORF’ or ‘E-ORF.’ Two clones, E-UTR4 and C-ORF4, were found to encode full length NKG2E and NKG2C, respectively, and the sequences were judged free of PCR-induced coding errors based on comparisons of multiple independent clones. Clone C-ORF4 is identical to sequences previously reported to GenBank 2627. Clone E-UTR4 contains a silent TC mutation at nucleotide 414.

BACs and Genomic Analysis.
BACs were identified from the Genome Systems C57BL/6 BAC library as previously described 9. Two HindIII fragments of 3.5 and 9 kb that hybridized to an NKG2A exon 5/6 probe were cloned into HindIII-digested pBluescript SKII(+) (Stratagene Inc.) to generate clones H3.5 and H9. These clones were sequenced using T3 and T7 primers, as well as the following primers, to determine the exon/intron boundaries for exons 4–7: NKG25'ex5, NKG23'ex5, NKG25'ex6, NKG23'ex6, NKG25'ex7, and NKG23'#3.

Quantitation of NKG2A, NKG2C, and NKG2E mRNA.
Sorted purified (>98% pure) NK1.1⁺CD3^– IL-2–cultured NK cells or nylon wool–passed, IL-2–cultured NK cells were used as starting material to make total RNA using the Ultraspec II reagent (Biotecx Labs.). Approximately 2 µg of RNA was reverse transcribed using an oligo-dT primer (GIBCO BRL), murine leukemia virus reverse transcriptase (GIBCO BRL), dNTPs (Promega Corp.), and RNasin RNase inhibitor (Promega Corp.) in a 20 µl volume. 1 µl of the resulting cDNA was used as a template for 27 cycles of PCR using the NKG2C 5' 437 and NKG2C3'#3 primers, which bind to identical sites in all three NKG2 genes and generate a product of 298 bp. 0.1 µl of -[³²P]dCTP was added to each 100-µl PCR reaction. 1–5 µl of each PCR reaction was digested with gene-specific restriction enzymes: MboI digests only NKG2A, at nucleotide 541; StuI digests only NKG2C, at nucleotide 562; and PvuII digests only NKG2E, at nucleotide 617. The digests were resolved on 6.5% PAGE, and band intensities were quantified using PhosphorImager analysis (Molecular Dynamics). As controls, plasmids containing each of the cDNAs were used as templates for the identical PCR and digestion protocol.

Stable CHO Transfectants and Qa-1 Tetramer Staining.
The expression vector for NKG2A has been described 9. To generate expression constructs, NKG2C and -E were amplified from clones C-ORF4 and E-UTR4 by PCR using Pfu polymerase (Stratagene Inc.), primer NKG2C5'ATG, and one of either primer NKG2C3'#3 (used for NKG2C) or NKG2AHANot3' (used for NKG2E). The products were digested with XhoI and NotI, and cloned into the pME18S expression vector (sequence available from EMBL/GenBank/DDBJ under accession number AB009864), and confirmed by sequencing. The latter primer added a nine–amino acid HA epitope tag (amino acid sequence YPYDVPDYA) to the COOH terminus (extracellular) of the NKG2E molecule, allowing its detection with an anti-HA mAb. Previous results with NKG2A–HA 9 showed the tag is unlikely to affect ligand binding. For our studies, the tag proved unnecessary, as we were subsequently successful in developing anti-NKG2 mAbs. The expression construct for NKG2C did not encode an HA-tagged molecule. The pME18S expression vectors were stably transfected into CHO cells along with a pIRES vector (Clontech) encoding mouse CD94 and the neo cDNA. NKG2E (but not NKG2C) was also cotransfected with pME18S-DAP12, but resulting clones were not assessed for DAP12 expression. Transfection was with the Lipofectamine reagent (GIBCO BRL) as previously described 38. 48 h after transfection, CHO cells were passaged into RPMI supplemented with 1 mg/ml G418 (GIBCO BRL). Once drug-resistant cells started to grow out, bulk transfectants were sorted for bright surface expression of NKG2A, -C, or -E using the 20d5 anti-NKG2 mAb or Qa-1 tetramer as staining reagent. After sorting, cells were cloned by limiting dilution, and positive clones were expanded without G418. Qa-1 tetramers, complexed with the Qdm peptide (AMAPRTLLL), were generated and used as described previously 939, except that a Superdex200 gel filtration column (Pharmacia) was used in all purification steps.

Sequence Alignment and Analysis.
Nucleotide sequences were aligned with CLUSTAL W 40, using the portion of each sequence corresponding to the carbohydrate recognition domain (CRD) of NKG2A (taken here as the last 363 nucleotides of the mNKG2A ORF). For each pair of aligned sequences, the program DnaSP 3.0 41 was used to calculate the number of synonymous substitutions per synonymous site (Ks) and the number of nonsynonymous substitutions per nonsynonymous site (Ka). This program uses the unweighted algorithm of Nei and Gojobori 42, which can be explained in brief. First, the total number of nonsynonymous sites (N, at which nucleotide changes give rise to amino acid changes) and synonymous sites (S, at which nucleotide changes are silent) are determined for each sequence, and the average values of S and N for each pair of sequences are used. Then, for each pair of sequences, the number of synonymous (S_d) and nonsynonymous (N_d) nucleotide differences observed is determined. For a pair of codons that differ by more than one nucleotide, the different possible evolutionary paths that could give rise to the observed change need to be taken into account (different evolutionary paths can involve different numbers of synonymous and nonsynonymous substitutions). In the unweighted algorithm, all of the different possible pathways are considered equally likely. S/S_d is the proportion of synonymous differences, which is then used to estimate Ks. See Nei and Gojobori 42 for more details.

	Results and Discussion

Top Abstract Materials and Methods Results and Discussion References

 
Molecular Cloning of NKG2C and NKG2E.
In the course of amplifying mouse NKG2A cDNA ends (see Materials and Methods), we obtained the 3' end of a cDNA that exhibited similarity to human NKG2C. Based on genomic sequence obtained from a BAC clone (clone 91i19), we were able to design PCR primers to amplify the entire coding region of an NKG2C cDNA from C57BL/6 mice. The consensus sequence obtained was identical to the sequence also reported recently by others 2627. In addition, we also obtained a novel NKG2C-like cDNA that we termed mouse NKG2E. It must be emphasized that mouse NKG2C is no more related to human NKG2C than to human NKG2E, nor does mouse NKG2E exhibit more similarity to human NKG2E than to human NKG2C (Fig. 1 B). Thus, the mouse NKG2C and NKG2E genes are named in the order of their discovery, rather than on the basis of homology to their human ‘counterparts.’

Mouse NKG2C and NKG2E are 91% identical to each other at the amino acid level and are new members of the rapidly expanding family of lectin-like receptors expressed by NK cells. Mouse NKG2C and -E exhibit greatest homology with the extracellular CRD of mouse NKG2A (95% similar and 93% identical at the amino acid level), but this similarity disappears almost entirely outside of the CRD (Fig. 1). The previously identified mouse NKG2D molecule 3843, on the other hand, differs markedly from the other NKG2 molecules and is in fact no more related to these molecules than it is to CD94 (Fig. 1 B). These comparisons confirm that NKG2D is likely not a bona fide NKG2 family member.

Unlike mouse NKG2A, mouse NKG2C and -E do not contain immunoreceptor tyrosine-based inhibitory motif–like sequences in their cytoplasmic tails. Instead, like human NKG2C and -E, mouse NKG2C and -E appear to contain a positively charged residue (arginine) in their transmembrane domains. Notably, the position of the charged residue within the transmembrane domain is slightly different between the mouse and human molecules (Fig. 1 A). Similar charges have been shown to play an important role in other receptors by mediating associations with activating signaling subunits such as DAP12 44 or FcR 45, but there are no data on the relevant signaling partner for mouse NKG2C or -E.

The cytoplasmic tails of mouse NKG2C and -E do not appear to contain any obvious signaling motifs themselves, although their length (64 amino acids) allows for the possibility that at least some component of the signal transduction by NKG2C/E might be through their own receptor tails. The tails do not share any regions of substantial homology with the human NKG2C/E molecules, with the exception of an invariant four–amino acid motif (PPEK) that is presumed to directly abut the cytoplasmic face of the lipid bilayer. The role of this motif is not known.

NKG2 and CD94 mAbs Establish CD94/NKG2 as the Predominant Qa-1 Receptor on NK Cells.
To study CD94/NKG2 expression by NK cells, we have generated mAbs against the mouse NKG2 and CD94 molecules. The specificity of these antibodies was confirmed by staining COS cells that had been transiently transfected with CD94, NKG2, or control (Ly49A) cDNAs (Fig. 2 A). The 18d3 mAb recognized COS cells transfected with CD94 alone as well as CD94/NKG2 cotransfectants, but it did not recognize COS cells transfected with NKG2A alone. In contrast, the 20d5 mAb recognized COS cells transfected with NKG2A alone as well as CD94/NKG2A cotransfectants. The 20d5 mAb also appears to recognize NKG2C and NKG2E, as 20d5 did not stain COS cells transfected with CD94 alone but did stain CD94/NKG2C/DAP12 and CD94/NKG2E/DAP12 transfectants (Fig. 2 A). NKG2C and NKG2E reach the cell surface inefficiently when transfected alone into COS cells (data not shown). Cotransfection with CD94 improved surface expression, and the further addition of DAP12 had a small additional effect (data not shown). As was previously documented 9, CD94 and NKG2A did not require each other to reach the cell surfaces of COS transfectants.

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Figure 2 (A) The 18d3 mAb recognizes mouse CD94, and the 20d5 mAb recognizes mouse NKG2A, -C, and -E. COS cells were transiently transfected with expression vectors encoding the indicated cDNAs (as described in Materials and Methods). The same batch of transfected cells was stained with either 18d3 or 20d5 hybridoma supernatants, followed by FITC-conjugated anti–rat IgG. Ly49A-transfected COS cells were used as a negative control. (B) CD94/NKG2 receptors are the only detectable Qa-1 receptors on NK cells. Nylon wool–nonadherent splenocytes were preincubated with 2.4G2 (anti–FcII/III receptor) supernatant, and in the center panel with unconjugated 20d5 (anti-NKG2A/C/E) mAb. The cells were then pelleted and stained with PK136 (anti-NK1.1; PE conjugate, left panel; FITC conjugate, center and right panels), streptavidin–PE-complexed Qa-1 tetramers, and/or 20d5 FITC, as indicated. The percentages of NK1.1+ cells is indicated.

NKG2C and -E Recognize Qa-1^b.
Based on the high degree of similarity between the extracellular ligand binding domains of NKG2A, -C, and -E, it is reasonable to hypothesize that CD94/NKGC and CD94/NKG2E heterodimers, like CD94/NKG2A heterodimers, would recognize Qa-1^b. We generated stable transfectants of CHO cells expressing high levels of CD94/NKG2A, CD94/NKG2C, and CD94/NKG2E (Fig. 3). All three stable cell lines, but not untransfected CHO cells, stained brightly with soluble, tetramerized Qa-1/β₂ microglobulin/Qdm complexes. We previously showed that CD94 alone is incapable of binding Qa-1^b 9. The results therefore provide direct evidence that both CD94/NKG2C and CD94/NKG2E recognize Qa-1^b–Qdm complexes. The degree of staining appeared to correlate roughly with the levels of NKG2 and CD94, as independently assessed by staining with anti-NKG2 and anti-CD94 mAbs. In particular, the CD94/NKG2A transfectant stained most brightly with tetramer and with anti-CD94 and anti-NKG2 antibodies, whereas the CD94/NKG2C transfectant stained least brightly with all three staining reagents. Thus, our data do not reveal a gross difference in the avidity of the various CD94/NKG2 subunits for Qa-1^b. Evidence in humans using quantitative surface plasmon resonance techniques suggests that CD94/NKG2A binds HLA-E with higher affinity than does CD94/NKG2C 21. Such differences may also exist for the mouse CD94/NKG2 receptors, as our data is not quantitative. Because the Qa-1 protein was produced in Escherichia coli and is therefore unglycosylated, our results also demonstrate that CD94/NKG2C and CD94/NKG2E, like CD94/NKG2A 9, can recognize carbohydrate-independent epitopes on their ligands.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 3 CD94/NKG2C and CD94/NKG2E are receptors for Qa-1b. CHO cells were stably transfected with expression vectors encoding CD94 and NKG2 cDNAs as described in Materials and Methods. Untransfected CHO cells or stable CHO transfectants were stained with PE-complexed tetramers of soluble Qa-1b/Qdm class I molecules or with streptavidin–PE as a negative control. The same cells were also stained with FITC-conjugated 20d5 mAb (anti-NKG2) or with FITC-conjugated 18d3 mAb (anti-CD94). The mean fluorescence intensity (MFI) is indicated for each histogram.

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Figure 4 The mouse CD94/NKG2 gene cluster. (A) Relative positions of Nkg2c and Nkg2e within the C57BL/6 (B6) NK complex on mouse chromosome six, as inferred in part from references 9 and 48 and the data presented in B. The map is oriented with the centromere to the left. The Genome Systems clone number for each BAC clone is indicated to the right of each clone; the numbers at left are more convenient internal reference numbers. (B) Three BAC clones (B6-1, B6-3, and B6-4) and two HindIII genomic subclones (H3.5 and H9) were digested with HindIII, resolved by agarose gel electrophoresis, and analyzed by Southern blot. The blot was probed with an oligonucleotide probe specific for Nkg2e, stripped, and then reprobed with an oligonucleotide probe specific for Nkg2c and Nkg2a (but not Nkg2e).

NKG2C and NKG2E Are Extensively Alternatively Spliced.
In addition to the cDNA clones encoding full length NKG2C and NKG2E, we obtained multiple clones encoding NKG2C- or NKG2E-like sequences that differed by various insertions and deletions (Fig. 5). The positions of the insertions and/or deletions corresponded precisely to canonical splice acceptors and donors determined from the genomic sequencing (Table ), and so it seems unlikely that these variants were an artifact of PCR. Rather, they probably arose by alternative splicing of primary NKG2C and NKG2E transcripts. The splice variants all involve alternative splice donors or acceptors present in exons 4 and 5, encoding the extracellular stalk and part of the CRD. Alternative splicing of other exons was not observed. Some of the variants produce reading frame shifts that result in premature stop codons and truncated receptors that would not be predicted to bind ligand (Fig. 5). A previously identified NKG2C cDNA called NKG2C2 26 likely arose from alternative use of the splice acceptors and donors identified here. One particularly interesting clone was E-UTR1, which was found to contain a chimeric NKG2C/NKG2E cDNA (Fig. 5). It is possible that this transcript was generated as an artifact of PCR. Alternatively, if NKG2C and -E share the same transcriptional orientation in the genome as they do in humans, it is possible that a single primary message encoding both genes was transcribed and spliced to form the chimeric receptor. Alternative splicing, particularly involving exons encoding the transmembrane and stalk domains, has also been observed for other C-type lectin-like receptors, including mouse Ly49 C, D, G, H, and J, human CD94, and human and mouse NKG2B, a splice variant of NKG2A 274751525354. Despite their apparent prevalence, the function of these alternative splices is not clear.

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Figure 5 Alternative splicing of Nkg2c and Nkg2e genes. Exons found to be involved in alternative splices are shown as boxes at the top, and various cDNA clones sequenced are depicted below. The portions of the cDNA sequences depicted with a solid black line are predicted to be translatable, whereas the portions of the cDNA depicted with a stippled line are predicted to follow an in-frame stop codon. Only clones encoding NKG2E1, -C1, or -C2 are predicted to generate full length protein, and the nucleotide positions of in-frame stop codons are indicated in parentheses. Exon–intron boundaries were confirmed by direct sequencing of genomic clones, but the distances between exons were not determined. Dashed vertical lines within exons represent the presence of cryptic splice acceptors or donors. Based on sequence polymorphisms between NKG2C and -E, clone E-UTR1 appears to be a fusion of transcripts from both genes. This might represent an artifact of PCR, or it may have been generated in vivo. The exons are numbered according to homology with the human NKG2A gene. The C2 sequence was not obtained in this study but was found previously 26. Additional splice variants appear to exist (Fig. 6) but have not yet been cloned and sequenced.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Figure 6 NKG2C and NKG2E are extensively alternatively spliced. (A) PCR primers (5'ex2 and 3'575) flanking the region that is alternatively spliced were used to amplify product from control templates and from various cDNA samples. The PCR products were resolved on a 3% agarose gel and stained with ethidium bromide. The control templates were: –, distilled water; A, NKG2A cDNA; 1, NKG2E clone E-UTR1; 2, NKG2E clone E-UTR2; E1, NKG2E clone E-UTR4, which encodes a full length protein, NKG2E1; 3, NKG2E clone E-ORF3; and C1, NKG2C clone C-ORF4, which encodes a full length protein, NKG2C1. cDNA samples were derived from RNA obtained from: 1, highly purified, IL-2–cultured NK1.1+CD3– NK cells; 2, highly purified, IL-2–cultured NK1.1+CD3– NK cells that had also been sorted for expression of NKG2 using the 20d5 mAb; 3, unsorted IL-2–cultured LAK (lymphokine-activated killer) cells, preparation A; 4, unsorted IL-2–cultured LAK cells, preparation B; and 5, unsorted IL-2–cultured LAK cells, preparation C. A total of eight distinct bands were visible, although some were very weak. Based on Southern blot experiments (B and C), these bands could be assigned to either NKG2E or NKG2C, as indicated on the right side of the gel. (B) The agarose gel shown in A was analyzed by Southern blot and probed with an oligonucleotide probe (E-probe), which recognizes NKG2E but not NKG2C. (C) The blot was then stripped and reprobed with an oligonucleotide probe (C-probe), which recognizes NKG2C but not NKG2E. The right portion of the blot was exposed longer than the left to enhance detection of weaker bands and prevent overexposure of the brighter control bands. It should be noted that not all splice variants detected by this method have been cloned. The NKG2C2 splice variant was only very weakly visible.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Figure 7 NKG2A message represents 95% of the total NKG2 message in NK cells. Primers recognizing NKG2A, -C, and -E equivalently were used to amplify NKG2 cDNA in a PCR reaction containing -[32P]–labeled nucleotides. The primers span a region of the NKG2 transcripts not affected by alternative splicing. PCR products were digested by the indicated enzymes and resolved on 6.5% PAGE. A control reaction with no template did not result in any detectable product (not shown). S+P is a double digest with StuI and PvuII. RNA was made from nylon wool–passed splenocytes cultured for 4 d in IL-2 (unsorted LAK cDNA) or from sorted, purified (>98% pure) IL-2–cultured NK cells (NK1.1+CD3–). Very weak bands (not clearly visible in the figure) were detected by longer exposure for the StuI and PvuII digests of PCR products derived from cDNA. The percentage of PCR product digested in each lane by the indicated restriction enzyme is shown and was quantified using PhosphorImager analysis.

Although it is difficult to adjudicate between the two above evolutionary histories, the second model may be more likely for the following reasons. First, all three species examined contain multiple NKG2 genes, including at least one inhibitory and one activating type, consistent with a primitive duplication event that was transmitted to all lineages. Second, the 5' (cytoplasmic) portions of the inhibitory and activating NKG2 genes tend to vary substantially from each other, even within species, suggesting that the genes have been diverging for some time. Unless gene conversion–like events—which can affect discrete parts of genes—are invoked, it is difficult to reconcile the evolutionary divergence apparent in the 5' portions of mouse NKG2A and -C with the finding that they do not exhibit any synonymous substitutions in their CRDs. Lastly, the remarkable overall similarity seen in the genomic structures of the mouse and human NKG2 loci (Fig. 4 and reference 49) suggests that the gene families did not result from independent duplication events. It is interesting that gene conversion events have also been proposed to play a role in the evolution of MHC class I genes 56.

Our previous work on mouse CD94/NKG2A provided evidence that recognition of class I was a primitive function of NK cells that predated the divergence of mouse and human ancestors. Assuming that NKG2 genes underwent a primitive duplication, as argued above, our work seems to suggest that both activating and inhibitory class I recognition may have been primitive functions of NK cells. This is interesting, as it suggests some fundamental role for activating receptors in the recognition of class I. This notion is further supported by the observation that families of class I–specific receptors, including Ly49, KIR, and leukocyte Ig-like receptors/Ig-like transcript families, invariably consist of both activating and inhibitory paralogs. Indeed, the pairing of inhibitory and activating isoforms extends beyond receptors specific for class I, and includes the paired Ig-like receptors on B cells and myeloid cells 57 and the CD28/CTLA-4 coreceptors on T cells. Although well understood in the case of T cells, the molecular logic behind the existence of paralogous activating and inhibitory receptors on NK cells is still a matter of conjecture. We anticipate that the identification of mouse CD94/NKG2C and CD94/NKG2E as activating receptors will permit the appropriate in vivo experimental manipulations required to dissect their function.