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The Journal of Immunology, 2002, 169: 22-30.
Copyright © 2002 by The American Association of Immunologists

Ly-49s3 Is a Promiscuous Activating Rat NK Cell Receptor for Nonclassical MHC Class I-Encoded Target Ligands1

Christian Naper*,{dagger}, Shigenari Hayashi*, Lise Kveberg{dagger}, Eréne C. Niemi*, Lewis L. Lanier{ddagger}, John T. Vaage2,3,{dagger},§ and James C. Ryan2,*

* Veterans Affairs Medical Center, Northern California Institute for Research and Education, and University of California, San Francisco, CA 94121; {dagger} Department of Anatomy, University of Oslo, Oslo, Norway; {ddagger} Department of Microbiology and Immunology and Cancer Research Institute, University of California, San Francisco, CA 94143; and § Institute of Immunology, Rikshospitalet, University Hospital, Oslo, Norway


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies of the rapid rejection of MHC-disparate lymphocytes in rats, named allogeneic lymphocyte cytotoxicity, have indicated that rat NK cells express activating receptors for nonclassical MHC class I allodeterminants from the RT1-C/E/M region. Using an expression cloning system that identifies activating receptors associated with the transmembrane adapter molecule DAP12, we have cloned a novel rat Ly-49 receptor that we have termed Ly-49 stimulatory receptor 3 (Ly-49s3). A newly generated anti-Ly-49s3 Ab, mAb DAR13, identified subpopulations of resting and IL-2-activated NK cells, but not T or B lymphocytes. Depletion of Ly-49s3-expressing NK cells drastically reduced alloreactivity in vitro, indicating that this subpopulation is responsible for a major part of the observed NK alloreactivity. DAR13-mediated blockade of Ly-49s3 inhibited killing of MHC-congenic target cells from the av1, n, lv1, and c haplotypes, but not from the u or b haplotypes. A putative ligand was mapped to the nonclassical MHC class I region (RT1-C/E/M) using intra-MHC recombinant strains. Relative numbers of Ly-49s3+ NK cells were reduced, and surface levels of Ly-49s3 were lower, in MHC congenic strains expressing the putative Ly-49s3 ligand(s). In conclusion, we have identified a novel Ly-49 receptor that triggers rat NK cell-mediated responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Natural killer cells play an important role in immune defense against intracellular infections (1). Infected cells are eliminated by NK cells without prior sensitization. NK cells also rapidly reject allogeneic bone marrow grafts and lymphoblasts, presumably using complex arrays of activating and inhibitory receptors that recognize MHC class I (MHC-I)4 molecules on target cells (2, 3, 4, 5). In rodent NK cells, inhibitory Ly-49 receptors were the first MHC-I-binding receptors to be characterized (6). In the presence of self MHC-I ligands, target cells are protected from NK cytolysis, consistent with the missing self hypothesis proposed by Kärre and colleagues (7), i.e., that targets cells are killed if they fail to express a full array of self MHC-I ligands. As was predicted by early studies of the rapid rejection of MHC-disparate lymphocytes in rats termed allogeneic lymphocyte cytotoxicity (ALC) (8, 9, 10), NK cells also express stimulatory receptors for target MHC-I ligands (2, 11, 12, 13, 14). These activating MHC receptors stimulate NK cell effector functions through their association with a membrane adapter protein whose cytoplasmic domain contains immunoreceptor tyrosine-based activation motifs. Following receptor ligation, the immunoreceptor tyrosine-based activation motifs in these adapter molecules are tyrosine phosphorylated and recruit stimulatory tyrosine kinases such as Syk and ZAP-70, through which NK cells are triggered. One such adapter molecule is DAP12, which couples to structurally diverse NK receptors including activating members of the lectin (Ly-49, CD94/NKG2) superfamily (15, 16) and of the Ig (killer cell Ig-like receptor) superfamily (reviewed in Ref. 11). DAP12-associated members of the Ly-49, CD94/NKG2, and killer cell Ig-like receptor families have been implicated in the specific recognition of individual MHC-encoded target structures by NK cells.

In vivo studies in mice have shown that the overriding effects of inhibitory receptors largely overshadow activation of NK killing by MHC-binding receptors (17). In contrast, ALC studies in rats provided ample evidence for stimulatory NK allorecognition both in vivo and in vitro (8, 9), and suggested that rat NK cells express a more potent array of activating receptors directed against several distinct target cell allo-MHC determinants encoded within the nonclassical class Ib RT1-C/E/M region (5, 10, 18, 19). We speculated that, like MHC-binding NK receptors in mice and in humans, these rat NK receptors might be functionally associated with DAP12. Using a previously described expression cloning system that identifies cell surface receptors by their ability to associate with DAP12 (20, 21), in this study we describe a novel stimulatory rat Ly-49 receptor (Ly-49s3) that activates the natural killing of allogeneic lymphocytes. Unlike mouse-activating Ly-49 molecules, which in general have narrow specificities for individual MHC-encoded alleles, rat Ly-49s3 recognizes a broad array of MHC-encoded target determinants in rat strains of the c, av1, lv1, and n haplotypes. Using intra-MHC recombinants, we have localized putative Ly-49s3 target ligands in the n and av1 haplotypes near to or within the nonclassical MHC-I region, RT1-C/E/M. Our results suggest that killing of allogeneic lymphocytes is determined in part by an activating Ly-49 receptor that is broadly reactive with polymorphic class Ib ligands from several different MHC haplotypes.


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

Four- to 6-wk-old female BALB/c mice and BALB/c nu/nu mice were from Simonsen (Gilroy, CA) and were reared under conventional conditions in accordance with institutional guidelines. The rat strains and their MHC haplotypes used in these studies are listed in Table IGo. Breeding pairs from PVG (RT1c or rat MHC haplotype c; RT1-Ac-B/Dc-C/E/Mc or c-c-c), PVG.1U (u or u-u-u), PVG.1AV1 (av1 or a-a-av1), PVG.R23 (u-a-av1), and PVG.R8 (a-u-u) rats were obtained from Harlan U.K. Limited (Bicester, U.K.), whereas PVG.1LV1 (lv1) and PVG.1N (n or n-n-n) were from G. W. Butcher (The Babraham Institute, Cambridge, U.K.). These rat strains were reared under conventional conditions in Oslo and screened for common rat pathogens. Buffalo (b or b-b-b) rats were purchased from Harlan (Horst, The Netherlands). BN.1B (b-b-n) were obtained from H. J. Hedrich (Medizinische Hochschule, Hannover, Germany). The animals were housed in compliance with guidelines set by the Experimental Animal Board under the Ministry of Agriculture of Norway.


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  		Table I. MHC constitution of the rat strains used

 
Flow cytometry

For single-color analysis, 50 µl cells (0.2–0.5 x 107 cells/ml) were incubated with primary Ab for 30 min on ice. After three washes, labeled cells were incubated with F(ab') 2 of FITC-conjugated goat anti-mouse Ig (ICN/Cappel, Aurora, OH). For three-color flow cytometry, mononuclear splenocytes from 2- to 4-mo-old male rats were separated by centrifugation on Lymphoprep and depleted of Ig+ cells with sheep anti-rat Ig-coated M450 magnetic Dynabeads (Dynal, Oslo, Norway). Primary labeling was with a combination of FITC-conjugated anti-NKR-P1 (mAb 3.2.3; from J.C. Hiserodt, Pittsburgh, PA; FITC conjugated according to standard methods), PE-conjugated anti-CD3 (mAb G4.18; from BD PharMingen, San Diego, CA), and biotinylated DAR13 (anti-Ly-49s3, see below; mouse IgG1; biotinylated according to standard procedures), followed by R-PE-indodicarbocyanine-conjugated streptavidin (DAKO, Glostrup, Denmark). The cells were analyzed on a FACScan (BD Biosciences, Mountain View, CA).

Generation of effector cells and cytotoxicity assays

IL-2-activated NK cells were generated from mononuclear splenocytes that were depleted of T cells with mAb anti-CD3 G4.18 and rabbit serum as a source of complement, then isolated by positive selection of NKR-P1+ cells using magnetic Dynabeads, as previously described (18). Selected CD3-NKR-P1+ cells were then cultured in RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 1 mM Na pyruvate, 5 x 10-5 M 2-ME, antibiotics, and rat rIL-2, which was obtained from the dialyzed cell culture supernatant of a Chinese hamster ovary (CHO) cell line stably transfected with a rat IL-2 expression construct (22). Ly-49s3-negative cells were obtained from the IL-2 NK cultures by negative selection with rat anti-mouse IgG1-coated M450 magnetic Dynabeads (Dynal) preincubated with DAR13 mAb (mouse IgG1) ascites. Cellular depletion was done immediately before the cytotoxicity assays. The resultant Ly-49s3- NK populations were routinely contaminated with less than 1% Ly-49s3+ cells, as determined by flow cytometry (data not shown).

The generation of Con A-activated lymphoblast target cells and 4-h 51Cr release assay was performed as previously described (10). The P815 mouse mastocytoma line used as target cells in redirected killing assays was from the American Type Culture Collection (ATCC, Manassas, VA). In mAb blocking or redirected killing experiments, 3–5 µg purified DAR13 mAb, or isotype-matched control mAbs Wes42 (anti-human signal regulatory protein (SIRP), a gift from M. C. Nakamura and W. E. Seaman, University of California, San Francisco, CA) or TIB96 (anti-mouse IgD b allotype from ATCC) were added to effectors 20 min before the addition of target cells. Spontaneous release was usually between 5 and 15% of the total cpm in the cells. The results are presented as median values from triplicates for each E:T cell ratio.

Expression cloning of Ly-49s3 cDNA

The cDNA library used to clone Ly-49s3 was generated from an NK cell subset (KLRH1+) from PVG rats, and has been described previously (23). In short, template mRNA from IL-2-activated KLRH1+ NK cells was used for the production of adapted cDNA, which was unidirectionally ligated into the EcoRI (5') and XhoI (3') sites of the expression vector pMET7 (DNAX, Palo Alto, CA). The ligation product was transformed into DH10B ElectroMAX Escherichia coli (Life Technologies, Gaithersburg, MD), titered, and amplified on Luria-Bertrani ampicillin agar plates. Library DNA (complexity 550,000) was purified using Qiagen Tip-500 columns (Qiagen, Chadworth, CA).

A total of 20 µg cDNA library was transiently transfected into a 50% confluent monolayer of 293T cells stably transfected with mouse DAP12 as a fusion protein tagged with the FLAG epitope (293T-DAP12/FLAG) (20) in a 175-cm2 flask using lipofectamine and serum-free OPTI-MEM, according to the manufacturer’s instructions (Life Technologies). At 48 h, transfected cells were incubated for 30 min with anti-FLAG mAb M2 (0.5 µg/106 cells; mouse IgG1; Sigma-Aldrich, St. Louis, MO) in FACS buffer (PBS with 10 mM EDTA, 3% FBS). After washing, cells were incubated for 30 min with FITC-conjugated goat anti-mouse Ig (ICN/Cappel), and then washed extensively. The brightest 0.2% of FLAG+-transfected cells were sorted by flow cytometry and lysed for 30 min at room temperature in Hirt solution (10 mM EDTA, 0.6% SDS). After addition of NaCl to 1 M, the nuclei and proteins were precipitated at 4°C overnight and removed. The episomal pMET7 sublibrary was purified and precipitated from the Hirt supernatant, then transformed by bacterial electroporation into DH10B ElectroMAX E. coli, followed by amplification on Luria-Bertrani ampicillin plates and Qiagen plasmid purification. The resultant plasmid sublibrary was transfected into 293T-DAP12/FLAG cells for two additional rounds of sorting and plasmid recovery. Individual bacterial colonies were thereafter transfected in pools, and finally, as individual clones. Clones that stained positive with the anti-FLAG mAb M2 by flow cytometry were sequenced and analyzed using the Wisconsin Genetics Computer Group Program package (Madison, WI).

Stable transfection of cells

The stable Ly-49s3 transfectant of the RNK-16 rat NK cell line (RNK-16.Ly-49s3) was generated, as previously described (24), using the Ly-49s3 cDNA subcloned into the EMCV-SR{alpha} expression vector, followed by electroporation and selection in RPMI, 10% FCS, L-glutamine, penicillin/streptomycin, and 2-ME supplemented with 1 mg/ml G418 (complete G418 RPMI). The wild-type RNK-16 cell line was from C. Reynolds (National Cancer Institute, Frederick, MD). Using identical settings, 3 x 106 CHO cells (ATCC) were electroporated in the presence of 3 µg pMX-neo-mouse-DAP12/FLAG and 15 µg pMET7-Ly-49s3. After selection in complete G418 RPMI, stable Ly-49s3 and DAP12/FLAG cotransfectants of CHO (CHO.Ly-49s3) were identified by the cell surface expression of the FLAG epitope, as determined by flow cytometry. CHO cells stably cotransfected with human SIRP{beta} and DAP12/FLAG were a gift from M. C. Nakamura and W. E. Seaman (San Francisco, CA).

Production of the anti-Ly-49s3 mAb DAR13

BALB/c mice were immunized four times 2 wk apart with i.p. injections of 5 x 107 CHO.Ly-49s3 cells. Mice were boosted i.p. 3 wk after the last immunization, and splenocytes were harvested 3 days thereafter. A total of 5 x 107 splenocytes was fused with 107 SP2/0 mouse myeloma cells (ATCC) using Hybrimax PEG/DMSO solution (Sigma-Aldrich) and IMDM according to standard methods. After selection in complete hypoxanthine/aminopterin/thymidine medium (IMDM-Iscove’s medium, 20% FBS, pyruvate, penicillin/streptomycin, 2-ME, vitamins, nonessential amino acids, 1x hypoxanthine/aminopterin/thymidine, 10% hybridoma cloning factor (Igen, Gaithersburg, MD)), hybridoma supernatants were screened by flow cytometry for the presence of Abs against 293T-DAP12/FLAG cells transiently transfected with Ly-49s3 cDNA. Following subcloning, the specificity of the resultant anti-Ly-49s3 DAR13 mAb was confirmed against primary PVG rat NK cells and against control transfectants. For large-scale mAb production, 107 DAR13 hybridoma cells were injected i.p. into pristane-primed BALB/c nu/nu mice. Malignant ascites was harvested and purified by solvent-accessible surface precipitation, followed by dialysis against PBS.

Cell surface labeling, immunoprecipitation, and Western blotting

For cell surface labeling, 4 x 107 IL-2-activated NK cells from PVG rats were washed in PBS and resuspended in 30 µl lactoperoxidase solution (1 mg/ml in PBS), 30 µl glucose oxidase solution (150 µg/ml in PBS), and 30 µl glucose solution (50 mg/ml) in the presence of 0.5 mCi Na125I (Amersham Pharmacia, Arlington Heights, IL). After incubation for 20 min at room temperature, cells were washed extensively with cold PBS and subjected to lysis in Triton X-100 buffer (150 mM NaCl, 20 mM HEPES pH 7.4, 1% Triton X-100, 1 mM EDTA, 10% glycerol, aprotinin, leupeptin, and PMSF). Clarified lysates were subjected to overnight immunoprecipitation on protein A-Sepharose beads (Amersham Pharmacia) coated with rabbit anti-mouse Ig (ICN/Cappel) and loaded with DAR13 mAb, or with isotype-matched control mAb Wes42 (anti-human SIRP). After resolution on 8% SDS-PAGE, the gel was dried and subjected to autoradiography.

To demonstrate the association of Ly-49s3 with DAP12, 4 x 107 wild-type RNK-16 cells and an equivalent number of RNK-16.Ly-49s3 stable transfectants were stimulated with pervanadate, a tyrosine phosphatase inhibitor. After washing in cold TBS (20 mM Tris, pH 7.4, 150 mM NaCl), cells were lysed for 2 h at 4°C in digitonin lysis buffer (20 mM triethanolamine, pH 7.8, 150 mM NaCl, 1 mM MgSO4, 2.5 mM CaCl2, 1 mM sodium orthovanadate, 1% digitonin (Calbiochem, San Diego, CA), aprotinin, leupeptin, and PMSF). Clarified lysates were subjected to a 4-h immunoprecipitation on protein A-Sepharose beads coated with rabbit anti-mouse Ig and either DAR13 mAb or control mAb Wes42. After extensive washing in 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) wash buffer (10 mM CHAPS, 150 mM NaCl, 20 mM Tris, pH 7.8, 1 mM sodium orthovanadate, aprotinin/leupeptin/PMSF), immunoprecipitates were resolved on 15% SDS-PAGE under reducing conditions. Following semidry transfer onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, Marlborough, MA), membranes were blocked in TBS with 0.1% Tween 20 and 3% BSA and incubated with mAb anti-phosphotyrosine (4G10; Upstate Biotechnology, Saranac Lake, NY) or with a previously described rabbit anti-mouse DAP12 antiserum (generated in rabbits by immunization with a synthetic peptide corresponding to the cytoplasmic domain of mouse DAP12 (21)). After extensive washing, anti-phosphotyrosine blots were developed using alkaline phosphatase-conjugated anti-mouse IgG2b and colorimetric substrates (nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate; Roche, Indianapolis, IN), according to the manufacturer’s instructions. Anti-DAP12 blots were developed using HRP-conjugated donkey anti-rabbit Ab and Supersignal chemiluminescent substrate (Pierce, Rockford, IL) before exposure to film.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Complementation expression cloning of the Ly-49s3 cDNA

Previous experiments from our laboratory (5, 10, 18) and from others (19) showed that rat NK cells most likely express activating receptors directed against target Ags encoded within the nonclassical rat MHC class Ib region RT1-C/E/M. Since structurally divergent MHC-binding NK receptors in mice and humans have been found to associate with the signaling adapter DAP12 (11), we speculated that rat NK receptors for RT1-C/E/M-encoded ligands might also be functionally associated with DAP12. In an attempt to identify novel DAP12-associated receptors in rat NK cells, we used a complementation cloning approach, as previously described (20, 21). The negatively charged aspartic acid residue in the transmembrane domain of DAP12 precludes its expression on the cell surface of 293T cells unless coupled to an associating receptor. Thus, cDNAs for DAP12-associated receptors can be identified in 293T cells stably transfected with DAP12, by their ability to induce cell surface expression of DAP12. We used this expression requirement of DAP12 to screen a cDNA library derived from a highly alloreactive NK cell subset (KLRH1+) from the PVG rat strain (23). By transiently transfecting the cDNA library into 293T cells stably expressing FLAG-tagged mouse DAP12 (293T-DAP12/FLAG cells), we were able to isolate several clones that induced DAP12/FLAG expression on the cell surface. Sequence analysis of one clone, which induced surface expression of DAP12 at levels similar to mouse Ly-49H (Fig. 1GoA), revealed it to be a novel member of the lectin-like Ly-49 (KLRA) family with structural features of an activating receptor. As this was the third reported rat Ly-49 molecule with stimulatory structural features, we have named it Ly-49 stimulatory receptor 3, or Ly-49s3 (Fig. 1GoB). Like the stimulatory mouse Ly-49D and -H receptors, rat Ly-49s3 does not possess a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM), yet it contains a transmembrane basic residue (R) that is most likely involved in the recruitment of DAP12. Among previously published rat Ly-49 molecules, the extracellular domain of Ly-49s3 shares 53–56% amino acid identity with the corresponding domains of the F344 strain receptors Ly-49i1, -s1, and -s2 (formerly Ly-49.9, .29, and .12, respectively; Fig. 1Go, B and C (25).



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  		FIGURE 1. Isolation of Ly-49s3 cDNA and generation of the anti-Ly-49s3 mAb DAR13. A, The rat Ly-49 stimulatory receptor 3 (Ly-49s3) was cloned by its ability to induce surface expression of a mouse DAP12/FLAG fusion protein in 293T cells. Induction was comparable with that of the activating mouse Ly-49H receptor (MLY-49H). Mock-transfected 293T cells are shown as a control (CTR.). B, Deduced amino acid sequence of the Ly-49s3 receptor, in comparison with the Ly-49 inhibitory receptor 1 (Ly-49i1; previously denoted Ly-49.9), Ly-49s1 (previously Ly-49.29), and Ly-49s2 (previously Ly-49.12). The position of the transmembrane basic residue (R) in Ly-49s1, Ly-49s2, and Ly-49s3 is indicated with an asterisk. A putative ITIM motif in Ly-49i1 is marked with a stippled line, and the predicted transmembrane region is underlined. C, Dendrogram of the different rat Ly-49 receptors based on total amino acid similarity. D, Staining of CHO cells stably cotransfected with Ly-49s3 and a mouse DAP12/FLAG construct (CHO.Ly-49s3) with mAb DAR13 mAb (a) and anti-FLAG mAb M2 (b), but not with the isotype-matched anti-SIRP mAb Wes42 (c). By contrast, mAb DAR13 (a) did not stain CHO cells stably cotransfected with human SIRP{beta} and DAP12/FLAG (CHO.huSIRP{beta}), while anti-FLAG (b) and anti-SIRP (c) mAbs stained positively. Sequence data are available from GenBank/EMBL/DDJB under accession no. AY102036.

 
Generation of the anti-Ly-49s3 mAb DAR13

To generate a mAb against the Ly-49s3 receptor, we cotransfected CHO cells with Ly-49s3 and a mouseDAP12/FLAG construct. These CHO.Ly-49s3 transfectants apparently express the Ly-49s3-mouseDAP12/FLAG complex on their surface, as judged by flow cytometry using the anti-FLAG mAb M2 (Fig. 1GoD). Following immunization of BALB/c mice and fusion with SP2/0 myeloma targets, we isolated the DAR13 mAb that specifically stained the CHO.Ly-49s3 transfectants, but did not stain control CHO cells or CHO cells cotransfected with DAP12/FLAG and another DAP12-associated receptor, human SIRP{beta} (CHO.huSIRP{beta}). As shown in Fig. 1GoD, while both CHO.huSIRP{beta} and CHO.Ly-49s3 transfectants react with anti-FLAG mAb M2, only CHO.Ly-49s3 stains with the DAR13 mAb, while an isotype-matched control mAb Wes42, which recognizes human SIRP, fails to bind CHO.Ly-49s3 (but does bind CHO.huSIRP{beta} cells). These data demonstrate that DAR13 mAb reacts with Ly-49s3 and not with the FLAG epitope or with DAP12 alone. It cannot be excluded that the DAR13 mAb reacts with other uncharacterized rat Ly-49 receptors that might be highly homologous with Ly-49s3 in the extracellular region. But if so, the distinct functional and phenotypic results obtained for DAR13 in different MHC haplotypes (see below) suggest that these Ly-49 receptors most likely also have overlapping functions and specificities for MHC-related ligands.

A major NK cell subset in PVG rats expresses Ly-49s3

As shown in Fig. 2GoA, the DAR13 mAb defines a major NK subset in PVG rats. A total of 64% of IL-2-activated PVG NK cells react with DAR13 mAb and hence are Ly-49s3+, while only 24% label with the STOK2 mAb (26), which we have shown to react with the inhibitory Ly-49 receptor Ly-49i2 (44). Relative numbers of Ly-49s3+ NK cells are somewhat increased as a result of IL-2 culture, as can be deduced from the results described below for fresh PVG NK cells. By contrast, the majority of T and B cells are Ly-49s3 negative; thus, only ~2% of freshly isolated cervical lymph node cells and <1% of thymocytes stain with DAR13 mAb (Fig. 2Go, B and C).



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  		FIGURE 2. A major NK cell subpopulation in PVG rats expresses the Ly-49s3 receptor, as judged by staining with mAb DAR13. A, DAR13 mAb labeling of IL-2-activated PVG NK cells, in comparison with STOK2 mAb reacting with the Ly-49i2 receptor. B and C, Ly-49s3-specific fluorescence of cervical lymph node (LN) cells and thymocytes, respectively; D, reactivity with a subpopulation of freshly isolated CD3-NKR-P1+ splenic NK cells (but not with CD3+NKR-P1+ or CD3+NKR-P1- T lymphocytes) is demonstrated.

 
We have previously shown that the inhibitory Ly-49i2 receptor is expressed by small subsets of NKR-P1+ T cells in addition to NK cells (26), but this is apparently not the case for the stimulatory Ly-49s3 receptor. Triple labeling studies of rat splenocytes with DAR13 mAb in combination with anti-CD3 and anti-NKR-P1 showed that ~30% of the CD3-NKR-P1+ NK cells were Ly-49s3+. In contrast, less than 1% of the CD3+NKR-P1+ T cell population was Ly-49s3 bright positive. It could not be excluded that some NKR-P1+ T cells express Ly-49s3 at very low levels, as there was a shoulder on the negative peak on the indicated fluorescence histogram, but if so, their expression levels were much lower than for NK cells (Fig. 2GoD). Conventional NKR-P1- T cells, in contrast, were clearly Ly-49s3 negative (Fig. 2GoD).

Ly-49s3 stimulates redirected lysis in RNK-16 cells

To aid in functional studies, we transfected the rat NK-like cell line RNK-16 with the Ly-49s3 cDNA and examined the receptor-specific effects in redirected cytotoxicity assays against FcR+ target cells. Transfected RNK-16 cells were screened by flow cytometry for clones stably expressing Ly-49s3, as depicted for one clone (RNK-16.Ly-49s3) in Fig. 3GoA. Neither wild-type RNK-16 cells nor the RNK-16.Ly-49s3 transfectants killed the FcR+ mouse mastocytoma target P815. However, the RNK-16.Ly-49s3 line exhibited brisk redirected lysis of P815 in the presence of stimulating quantities of DAR13 mAb (Fig. 3GoB). Addition of mAb DAR13 had no effect on P815 killing by wild-type RNK-16, as compared with the isotype control mAb Wes42, which had no appreciable effect on P815 killing by either NK effector cell (Fig. 3GoB and data not shown).



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  		FIGURE 3. The Ly-49s3 receptor mediates redirected lysis of FcR+ P815 tumor cells. A, Expression of Ly-49s3 by the NK-like RNK-16 cell line stably transfected with the Ly-49s3 cDNA (RNK-16.Ly-49s3). Flow cytometry using the DAR13 mAb (open curve) and the isotype-matched mAb Wes42 as a control (Ctr.; shaded curve) is demonstrated. B, Cytotoxicity of the Ly-49s3+ RNK-16.Ly-49s3 cell line against P815 tumor target cells in the presence of 3 µg/well DAR13 mAb ({blacksquare}) or the Wes42 mAb control (•).

 
The homodimeric Ly-49s3 receptor associates with rat DAP12

Immunoprecipitation of Ly-49s3 from lysates of 125I-surface-labeled IL-2-activated rat NK cells revealed that Ly-49s3 migrates as a 80- to 100-kDa glycoprotein under nonreducing conditions and as a 45- to 50-kDa glycoprotein under reducing conditions, confirming that, like other Ly-49 molecules, Ly-49s3 is a disulfide-linked homodimer on NK cells (Fig. 4GoA).



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  		FIGURE 4. The Ly-49s3 receptor is a DAP12-associated homodimeric transmembrane protein. A, Immunoprecipitation from Triton X-100 lysates from 125I surface-iodinated IL-2-activated PVG NK cells using the DAR13 mAb and the isotype-matched control Wes42 mAb. Migration of precipitates on 8% SDS-PAGE is shown under nonreducing (NR) and reducing (Red.) conditions. B, Coprecipitation of the Ly-49s3 receptor with endogenous DAP12 in the rat NK-like line RNK-16 NK stably transfected with Ly-49s3 cDNA (RNK-16.Ly-49s3). RNK-16 wild-type cells (RNK-16 wt) are shown as a control. Immunoprecipitations were with DAR13 mAb and the isotype-matched control Wes42 mAb, as indicated. After resolution on reducing 15% SDS-PAGE, the Western blot was developed with the anti-phosphotyrosine 4G10 mAb, showing coprecipitation of a 12- to 14-kDa tyrosine-phosphorylated protein with Ly-49s3. This Ly-49s3-associated protein likely is DAP12, as shown in a parallel Western blot developed with a rabbit anti-DAP12 antiserum (C).

 
The Ly-49s3 receptor was initially identified by its ability to associate with mouse DAP12 on the surface of 293T cells. Coprecipitation with Ly-49s3 of a surface-labeled protein that migrated with a molecular mass of <17 kDa under reducing conditions (Fig. 4GoA) suggested that Ly-49s3 was constitutively associated with this adapter molecule in IL-2-activated rat NK cells. To confirm that the Ly-49s3 receptor associates with rat DAP12, we immunoprecipitated Ly-49s3 from digitonin lysates of pervanadate-stimulated RNK-16.Ly-49s3 transfectants and from untransfected RNK-16 wild-type cells. After resolution of immunoprecipitates on 15% reducing SDS-PAGE, anti-phosphotyrosine Western blotting revealed that Ly-49s3 specifically coprecipitates with a 12- to 14-kDa tyrosine-phosphorylated protein (Fig. 4GoB), which most likely represents DAP12 as judged by anti-DAP12 Western blot analysis (Fig. 4GoC). Thus, Ly-49s3 associates with rat DAP12 in RNK-16 transfectants.

NK reactivity against MHC-mismatched lymphoblast targets is highly enriched in the Ly-49s3+ NK subset

We have shown previously that NK alloreactivity is markedly enriched in the Ly-49i2+ subset (26, 27), but enrichment was even more pronounced in the Ly-49s3+ NK subset. Bulk cultures of IL-2-activated NK cells were depleted of Ly-49s3+ cells using DAR13 mAb and magnetic Dynabeads, and alloreactivity of undepleted and Ly-49s3-negative NK cells was compared against a panel of MHC-mismatched Con A blast targets that were all on the PVG strain background. As shown in Fig. 5Go, undepleted NK cells from PVG.1U (RT1u haplotype or u) rats displayed high cytolytic activity against MHC-mismatched n, av1, lv1, and c haplotype blasts, while syngeneic (u) control targets were spared. Removal of the Ly-49s3+ NK cell subset reduced killing against all targets near to that seen for syngeneic control targets. Similar results were obtained when using NK cells from additional MHC-congenic strains such as PVG (c) and PVG.1AV1 (av1) (data not shown). Based on these findings, we speculated that the Ly-49s3 receptor is an important triggering MHC alloreceptor for rat NK cells.



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  		FIGURE 5. The Ly-49s3+ NK subset is strongly alloreactive. In vitro depletion of Ly-49s3+ NK cells with mAb DAR13 from a culture of IL-2-activated NK cells from the PVG.1U strain (u MHC haplotype) led to a strongly reduced cytolysis of lymphoblast targets expressing the n, av1, lv1, and c MHC haplotypes. Levels of killing of syngeneic (u haplotype) targets (Ctr.) were not affected. Shown are cytolytic activities of depleted (Ly-49s3-, {blacksquare}) and undepleted (CTR., •) NK cells.

 
Ly-49s3 is a broadly reactive receptor for MHC-encoded target determinants in the n, av1, lv1, and c haplotypes; mapping of a putative Ly-49s3 ligand to the MHC class Ib RT1-C/E/M region

Receptor-blocking experiments using DAR13 mAb and alloreactive NK effector cells revealed that Ly-49s3 exhibits an apparent broad specificity for MHC-encoded ligands. Addition of purified DAR13 to the cytotoxic assays markedly inhibited killing of MHC-mismatched n, av1, and lv1 haplotype targets by IL-2-activated PVG (c haplotype) NK cells (Fig. 6GoA). The blocking effect was especially pronounced toward haplotype n allogeneic targets with a reduction of cytotoxicity almost to syngeneic baseline levels, suggesting that the Ly-49s3 receptor is essential for the killing of n haplotype allotargets by PVG NK cells. Addition of DAR13 mAb had no effect on killing of allogeneic blasts from u haplotype rats. These data suggested that the n, av1, and lv1, but not the u MHC haplotypes encode target determinants for the Ly-49s3 receptor. Haplotype c blasts were predictably not killed by syngeneic PVG NK effectors, and Ab blockade experiments using this E:T cell combination were uninformative. Targets from c haplotype rats share a functionally defined activating NK allodeterminant, ALC-2, with blasts from av1 and lv1 haplotype rats (28). To check for additional ligands in c haplotype targets, we used NK cells from allogeneic PVG.1U (u) rats as effectors. DAR13 mAb inhibited killing of c as well as of av1 haplotype allogeneic targets by PVG.1U effectors, confirming the existence of a Ly-49s3 target structure in the c haplotype (Fig. 6GoB).



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  		FIGURE 6. Ly-49s3 is a receptor for target MHC ligands from the n, av1, lv1, and c rat MHC (RT1) haplotypes, but not from the u or b haplotypes: mapping of target ligands to the nonclassical class I region RT1-C/E/M. Blocking of NK cytolytic activities against different Con A blast targets by the addition of DAR13 mAb to the cytotoxic cultures. The MHC constitutions of the target cells are indicated above each graph, specified for the RT1-A (classical class I), -B/D (class II), and -C/E/M (nonclassical class I) regions, respectively. Lymphoblast targets were from PVG.1N (RT1-An-B/Dn-C/E/Mn or n-n-n), PVG.1AV1 (a-a-av1), PVG.1LV1 (l-l-lv1), PVG.1U (u-u-u), PVG (c-c-c), PVG. R8 (a-u-u), PVG. R23 (u-a-av1), Buffalo (b-b-b), and BN.1B (b-b-n). IL-2-activated NK cell effectors were from either PVG (A, C, and D) or PVG.1U rats (B). Cytolysis is shown in the presence of blocking quantities (5 µg/well) of purified DAR13 mAb ({blacksquare}) or of the isotype-matched control mAb TIB-96 (•).

 
Using lymphoblast targets from intra-MHC recombinant strains, the Ly-49s3 target structure(s) was mapped to the class Ib region of the rat MHC (RT1-C/E/M), and not to the class Ia (RT1-A) or class II (RT1-B/D) regions. This MHC class Ib region has been shown previously to contain several functionally defined stimulatory NK allodeterminants (10). DAR13 mAb-mediated blocking of Ly-49s3 on PVG NK cells led to reduced killing of RT1-Au-B/Da-C/E/Mav1 (or u-a-av1) targets from PVG.R23 rats, but not of a-u-u lymphoblasts from PVG.R8 rats, while the isotype-matched control mAb TIB-96 had no appreciable effect on the killing of either target. These data showed that the av1-encoded Ly-49s3 target determinant was located in either the class II (RT1-B/D) or the class Ib (RT1-C/E/M) region. Direct RT1-C/E/M mapping was obtained using targets derived from the BN.1B r37 intra-MHC recombinant (b-b-n) rat. Similar to u-u-u targets (Fig. 6GoA), DAR13 mAb did not affect PVG NK cell lysis of b-b-b targets, but markedly inhibited killing of b-b-n recombinant targets (Fig. 6GoD). These experiments localized the prominent Ly-49s3 ligand in the n haplotype to the nonclassical MHC-I region, RT1-C/E/M, and it seems reasonable to assume that the putative Ly-49s3 ligands in the other MHC haplotypes may also be encoded in this region.

Ly-49s3 expression is reduced in the presence of its putative MHC ligand(s)

Like the inhibitory rat Ly-49i2 receptor (27) and inhibitory Ly-49 receptors in the mouse (29, 30), expression levels of Ly-49s3 on the surface of individual NK cells were decreased in rats bearing its putative MHC ligand(s), as judged by three-color flow cytometric studies of ex vivo isolated spleen NK cells from a panel of MHC-congenic rats on the PVG strain background. These studies showed that Ly-49s3 expression levels on CD3-NKR-P1+ NK cells were 6–10 times higher in Ly-49s3 ligand-negative PVG.1U rats (u-u-u) when compared with levels seen in ligand-positive PVG.1N rats (n-n-n) (Fig. 7GoA). The markedly reduced Ly-49s3 expression in n-n-n haplotype NK cells correlates with the deduced importance of this receptor in inducing NK-mediated lysis of n-n-n allotargets (see above), and suggests that this haplotype encodes a dominant (or multiple) ligand(s) for the Ly-49s3 receptor. Intermediate expression levels were observed for NK cells from ligand-bearing PVG.1AV1 (a-a-av1), PVG.1LV1 (l-l-lv1), and PVG (c-c-c) rats, which displayed staining intensities that were approximately half that seen in NK cells from ligand-deficient PVG.1U (u-u-u) rats (Fig. 7GoA and data not shown). As noted above, these three haplotypes share a common operationally defined NK allodeterminant, ALC-2, which has been previously mapped to the MHC class Ib region, RT1-C/E/M (28). Similarly, the locus in the a-a-av1 haplotype that leads to a reduced Ly-49s3 surface expression level maps close to the RT1-C/E/M region in intra-MHC recombinant strains between the a-a-av1 and u-u-u haplotypes, although we could not formally exclude class II (RT1-B/D)-specific effects due to the lack of informative PVG recombinants. As shown in Fig. 7GoB, NK cells from PVG.R8 (Aa-B/Du-Cu or a-u-u) expressed 2- to 3-fold more Ly-49s3 than did NK cells from PVG.R23 (u-a-av1) rats. Since these data were generated in NK gene complex identical congenic strains differing only in the MHC, the data suggest that activating as well as inhibitory rat Ly-49s may calibrate expression levels in the presence of their corresponding MHC ligands.



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  		FIGURE 7. In vivo reduction of the NK cell surface level of Ly-49s3 in MHC congenic strains expressing putative Ly-49s3 ligand. Freshly isolated CD3-NKR-P1+ splenic NK cells from the PVG.1N (RT1-An-B/Dn-C/E/Mn or n-n-n), PVG.1AV1 (a-a-av1), and PVG.1U (u-u-u) rat strains (A), or from PVG.R23 (u-a-av1) and PVG.R8 (a-u-u) intra-MHC recombinant rats (B) were analyzed by three-color flow cytometry for reactivity with mAb DAR13.

 
Ly-49s3+ NK cells from ligand-bearing rats exhibited not only reduced cell surface receptor levels, their relative numbers were also significantly diminished in the same set of MHC congenic PVG strains. Thus, ligand-negative PVG.1U rats had a significantly higher fraction (38%) of Ly-49s3+ splenic NK cells than did the ligand-positive strains, PVG (29%), PVG.1AV1 (28%), PVG.1LV1 (32%), and PVG.1N (23%) (Table IIGo). Notably, relative numbers of Ly-49s3+ NK cells were lowest in the PVG.1N strain, which again correlates with the presence of an apparent prominent Ly-49s3 ligands in the n haplotype.


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  		Table II. Proportion of Ly-49s3+ NK cells in reduced in strains expressing MHC ligand(s) for the Ly-49s3 receptor1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although NK cells were first identified by their ability to recognize and kill targets in an MHC-unrestricted manner, early experiments in rodents have shown that the MHC haplotype of a target can affect its susceptibility to NK lysis. In mice, the lectin-like Ly-49 molecules are the predominant MHC-binding receptors on NK cells, and the same is likely the case in rats (25, 27). Inhibitory Ly-49 receptors have cytoplasmic ITIM motifs that recruit inhibitory tyrosine phosphatases such as SHP-1, whereas activating receptors lack ITIMs, but have basic transmembrane residues through which they recruit the activating transmembrane adapter protein DAP12. In the current study, we have used a complementation expression cloning technique to isolate a novel member of the DAP12-associated rat Ly-49 family, Ly-49s3, which stimulates the selective NK cell killing of targets expressing MHC-encoded molecules in lv1, av1, c, and n haplotype rats. Fine mapping of the Ly-49s3 target structures in the n and av1 haplotypes localized putative Ly-49s3 ligands telomeric to the class Ia MHC region RT1-A, and within or near to the class Ib region RT1-C/E/M.

These data extend and support previous functional and genetic studies implicating Ly-49-encoded receptors in the killing of MHC-disparate lymphoblast targets by highly alloreactive rat NK cells (25, 26, 27). In these and related studies, allospecific inhibitory functions in rat NK cells are in general restricted by classical RT1-A-encoded ligands (27, 28), while all currently known alloactivating NK functions are restricted by class Ib molecules encoded within the RT1-C/E/M region (5, 10, 31). Although the exact number of MHC class I genes and gene fragments encoded from RT1-C/E/M has yet to be fully determined, it is estimated that this region contains ~45 MHC class I-like genes in the n haplotype (32, 33). Similar to the H2-D/Q/T/M region of the mouse MHC complex, the telomeric RT1-C/E/M region contains four distinct clusters of class I genes that are separated by sets of framework genes conserved between the two species. The last three clusters contain genes that resemble the H2-T and H2-M genes, while the most centromeric of these four clusters harbors genes that are more similar to classical RT1-A genes than to H2-D and -Q. Cell surface density of the RT1-C/E/M-encoded molecules is low, being <10% the density of the classical RT1-A molecules, and little is known about their tissue distribution and regulation. Only one example of T cell restriction by an RT1-C/E/M-encoded molecule has been described (34). The RT1-C/E/M molecules have therefore been regarded as nonclassical MHC-I molecules, and their physiologic functions remained largely obscure. However, previously published functional and genetic data have shown that target ligands that activate alloreactive NK cells reside within the most centromeric class Ib cluster (the RT1-C/E/M cluster), which harbors between 10 and 15 functional class I genes, including alleles of the RT1-C, RT1-E, and RT1-U loci (32, 35), notably the RT1.Cl molecule, which is deleted in the lm1 MHC mutant rat strain, and RT1.Eu (5, 10, 19). It can only be speculated whether Ly-49s3 is cross-reactive with several distinct RT1-C/E-encoded ligands, being the products of one or more loci within this MHC-I cluster, or whether Ly-49s3 reacts with a ligand that is shared between several unrelated MHC haplotypes.

Rat Ly-49s3 shares common structural and functional features with the activating Ly-49 receptors in mice (12, 13, 14). Notably, like the stimulatory mouse Ly-49D, -H, -P, and -W receptors, rat Ly-49s3 is a lectin-like dimer with a putative DAP12-docking transmembrane residue. In addition, like activating mouse Ly-49 receptors, rat Ly-49s3 appears to bind MHC-related ligands. Significant differences between mouse and rat NK allorecognition systems can be seen from a careful evaluation of available data from each species. To date, all known stimulatory MHC targets of rat alloreactive NK cells are encoded within the RT1-C/E/M class Ib region (5, 10, 19), while alloinhibitory rat receptors appear to recognize RT1-A class Ia ligands (27, 28, 31). In the mouse, both inhibitory and activating Ly-49 receptors recognize class Ia molecules. In addition, the most widely studied activating Ly-49 receptor, Ly-49D, recognizes MHC-encoded xenogeneic ligands (36, 37). Unlike the broadly alloreactive rat Ly-49s3 receptor, mouse Ly-49D has a narrow allogeneic specificity for the classical mouse Dd allele of mouse MHC-I (38), whereas the Ly-49P, Ly-49R, and Ly-49W receptors all recognize Dd and Dk (13, 14). Thus, in mice, individual activating Ly-49 receptors display narrow specificities against MHC class Ia ligands, as well as against MHC-related ligands encoded by other mammalian species, whereas Ly-49s3 is broadly reactive with ligands from several MHC haplotypes. Another notable difference between mouse and rat is that whereas mouse-activating Ly-49 receptors generally are specific for allo-MHC-I molecules, the Ly-49s3 receptor apparently also reacts with a self-MHC ligand in the PVG strain from which it was isolated. This suggests that Ly-49s3+ PVG NK cells may be potentially autoreactive. This is in line with previous functional data showing the expression of an activating class Ib NK allodeterminant (ALC-2) in a syngeneic PVG strain setting, in which self-tolerance was ensured by a dominant inhibitory influence from the PVG class Ia region (RT1-Ac) (28) No xenogeneic ligands for rat Ly-49 receptors have yet been identified, but a limited study of different class I-negative target lines and lymphoblasts failed to reveal an influence of mouse H2 on rat NK lysis (39).

The physiologic functions of activating mouse and rat Ly-49 receptors are difficult to discern. Typically, an individual mouse expresses only a limited range of activating Ly-49 receptors (BALB/c expresses only inhibitory functional Ly-49 proteins) with limited specificities for MHC-I. An individual NK cell expressing an activating Ly-49 will often also express an inhibitory Ly-49 against the same MHC-I ligand. For example, the mouse class I molecule H2-Dd is recognized by the stimulatory Ly-49D receptor as well as by the inhibitory Ly-49A and Ly-49G2 receptors, and nearly all Ly-49D+ NK cells coexpress either Ly-49A or -G2 in selected strains of mice (17). Since inhibitory signals are thought to predominate over stimulatory signals, the MHC-dependent activation of natural killing of H2-Dd+ targets by, for instance, Ly-49D, might be easily abrogated by inhibitory signals generated through Ly-49A. In rats, the classical MHC class Ia ligands for inhibitory Ly-49 receptors are distinct from the nonclassical MHC class Ib ligands recognized by activating Ly-49 receptors. Classical and nonclassical MHC-I ligands differ in their tissue-specific expression, and may be differentially regulated in response to cellular stress or inflammation. Inflammation or stress-induced up-regulation of nonclassical class Ib ligands on rat hemopoietic or inflammatory cells might lead to enhanced NK activation, overriding tonic inhibitory effects mediated through inhibitory Ly-49 receptors. In these ways, activating rat Ly-49 receptors may play a role in controlling ongoing cellular immune responses during inflammatory states.

It is also possible that the physiologic significance of activating Ly-49 receptors lies in their recognition of foreign cells or of cells expressing virally encoded xenogeneic ligands. Xenorecognition by Ly-49s3 has not yet been studied, but the activating mouse Ly-49D and Ly-49H receptors each appear to recognize xenogeneic ligands. Mouse Ly-49D binds to mouse H2-Dd, but it also displays xenogeneic cross-reactivity against an MHC-encoded molecule in RT1l and RT1lv1 haplotype rats, as well against a target structure on Chinese hamster cells (36, 37, 38). Mouse Ly-49H does not react with common mouse MHC haplotypes (40), but appears to be involved in the strain-specific resistance to murine CMV (41, 42), presumably through the recognition of a novel virus-encoded MHC-like homologue (m157) on infected cells (43). Xenorecognition of foreign MHC may simply be a fortuitous coincidence, but certainly the recognition of CMV-infected cells by Ly-49H serves an evolutionary advantage in mice. It is intriguing to speculate that many activating Ly-49 receptors might recognize foreign or pathogenic proteins, and, as such, activating Ly-49 receptors may play important physiologic roles in innate immune surveillance against foreign cells or against selected infectious agents. The true physiologic functions of polymorphic activating Ly-49 receptors have yet to be fully defined, however, and are the subject of ongoing investigation.


   Footnotes
 
1 J.C.R., S.H., and E.C.N. were supported by National Institutes of Health RO1 AI 44126 and by the U.S. Veterans Administration. L.L.L. was supported by National Institutes of Health RO1 CA 89294. J.T.V., C.N., and L.K. were supported by the Norwegian Cancer Society and the Research Council of Norway. C.N. is a research fellow of the Norwegian Cancer Society. Back

2 J.T.V. and J.C.R. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. John T. Vaage, Institute of Immunology, Rikshospitalet, N-0027 Oslo, Norway. E-mail address: j.t.vaage{at}basalmed.uio.no Back

4 Abbreviations used in this paper: MHC-I, MHC class I; ALC, allogeneic lymphocyte cytotoxicity; CHO, Chinese hamster ovary; ITIM, immunoreceptor tyrosine-based inhibitory motif; SIRP, signal regulatory protein. Back

Received for publication February 5, 2002. Accepted for publication April 22, 2002.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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C. Naper, K.-Z. Dai, L. Kveberg, B. Rolstad, E. C. Niemi, J. T. Vaage, and J. C. Ryan
Two Structurally Related Rat Ly49 Receptors with Opposing Functions (Ly49 Stimulatory Receptor 5 and Ly49 Inhibitory Receptor 5) Recognize Nonclassical MHC Class Ib-Encoded Target Ligands
J. Immunol., March 1, 2005; 174(5): 2702 - 2711.
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K. J. Lavender, B. J. Ma, E. T. Silver, and K. P. Kane
The Rat RT1-A1c MHC Molecule Is a Xenogeneic Ligand Recognized by the Mouse Activating Ly-49W and Inhibitory Ly-49G Receptors
J. Immunol., March 15, 2004; 172(6): 3518 - 3526.
[Abstract] [Full Text] [PDF]

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