A “Chimeric” C57L-Derived Ly49 Inhibitory Receptor Resembling the Ly49D Activation Receptor

Cellular Immunology 209, 29 – 41 (2001) doi:10.1006/cimm.2001.1786, available online at http://www.idealibrary.com on

A “Chimeric” C57L-Derived Ly49 Inhibitory Receptor Resembling the Ly49D Activation Receptor Indira K. Mehta, Hamish R. C. Smith, Jian Wang,* David H. Margulies,* and Wayne M. Yokoyama Immunology Program and Rheumatology Division, Department of Medicine and Department of Pathology, Center for Arthritis and Related Diseases, Washington University School of Medicine and Howard Hughes Medical Institute, St. Louis, Missouri 63110; and *Molecular Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892 Received January 17, 2001; accepted March 14, 2001

transmit intracellular biochemical signals (1–3). These receptors are structurally related to either the immunoglobulin (Ig) or the C-type lectin protein superfamilies. Murine NK cells predominately express several distinct C-type lectin-like molecules, including the Ly49 family, that are encoded within the NK gene complex (NKC) on distal mouse chromosome 6 (4, 5). The prototypical Ly49 family member, Ly49A, is a MHC class I-specific receptor that is expressed as a type II transmembrane disulfide-linked homodimer on 20% of C57BL/6 NK cells. Ly49A ⫹ NK cells do not lyse H-2D d-expressing targets, and addition of anti-Ly49A or anti-H-2D d monoclonal antibodies (mAbs) can reverse this inhibitory influence (6). Cell– cell and MHC class I tetramer binding assays as well as surface plasmon resonance studies (7–11) demonstrate a physical interaction between Ly49A and H-2D d that can be blocked with anti-Ly49A or anti-H-2D d mAbs. Definitive evidence of a physical interaction between Ly49A and H-2D d was provided by the recent crystallization of the Ly49A–H-2D d complex (12). Like the inhibitory killer Ig-like receptors (KIR) on human NK cells, Ly49A contains an ITIM (immunoreceptor tyrosinebased inhibitory motif) within its cytoplasmic domain. Ligand binding appears to result in phosphorylation of the ITIM, which then recruits and activates the intracellular tyrosine phosphatase SHP-1 that inhibits NK cell function (13–15). Several other Ly49 family members also contain ITIMs, such as Ly49C, Ly49G, and Ly49I, and inhibition of lysis of MHC class I-expressing targets indicates that they also act as inhibitory receptors for MHC class I (16, 17). In contrast, other Ly49 family members, such as Ly49D and Ly49H, are activation receptors (18 –23). These Ly49 receptors lack cytoplasmic ITIMs and in-

Ly49D is a natural killer (NK) cell activation receptor that is responsible for differential mouse inbred strain-determined lysis of Chinese hamster ovary (CHO) cells. Whereas C57BL/6 NK cells kill CHO, BALB/c-derived NK cells cannot kill because they lack expression of Ly49D. Furthermore, the expression of Ly49D, as detected by monoclonal antibody 4E4, correlates well with CHO lysis by NK cells from different inbred strains. However, one discordant mouse strain was identified; C57L NK cells express the mAb 4E4 epitope but fail to lyse CHO cells. Herein we describe a Ly49 molecule isolated from C57L mice that is recognized by mAb 4E4 (anti-Ly49D). Interestingly, this molecule shares extensive similarity to Ly49D B6 in its extracellular domain, but its cytoplasmic and transmembrane domains are identical to the inhibitory receptor Ly49A B6, including a cytoplasmic ITIM. This molecule bears substantial overall homology to the previously cloned Ly49O molecule from 129 mice the serologic reactivity and function of which were undefined. Cytotoxicity experiments revealed that 4E4 ⴙ LAK cells from C57L mice failed to lyse CHO cells and inhibited NK cell function in redirected inhibition assays. MHC class I tetramer staining revealed that the Ly49O C57Lbound H-2D d and lysis by 4E4 ⴙ C57L LAK cells is inhibited by target H-2D d. The structural basis for ligand binding was also examined in the context of the recent crystallization of a Ly49A–H-2D d complex. Therefore, this apparently “chimeric” Ly49 molecule serologically resembles an NK cell activation receptor but functions as an inhibitory receptor. © 2001 Academic Press Key Words: NK cells; cell surface molecules; FACS (sorting or staining).

INTRODUCTION Natural killer (NK) 1 cell recognition and function are regulated by inhibitory and activation receptors that

verse transcription–polymerase chain reaction; KIR, killer cell inhibitory receptor; B6, C57BL/6; CHO, Chinese hamster ovary; ITIM, immunoreceptor tyrosine-based inhibitory motif; GAM, goat antimouse; ITAM, tyrosine-based activation motif.

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Abbreviations used: NK, natural killer; NKC, NK gene complex; Ig, immunoglobulin; mAb, monoclonal antibody; NKD, N-terminal region of the NK receptor domain; FcR, Fc receptor; RT–PCR, re29

0008-8749/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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stead contain an arginine residue in the transmembrane domain, which allows for association with the disulfide-linked DAP-12 transmembrane signaling protein (24). DAP12 contains an immunoreceptor tyrosine-based activation motif (ITAM) that triggers activation of Syk family tyrosine kinases and activation of NK cell activity (24 –26). Furthermore, cross-linking of Ly49D and Ly49H with their respective antibodies stimulates NK cells, indicating that Ly49D and Ly49H are activation receptors. Functional evidence suggests that Ly49D recognizes allogeneic (19, 22, 27) H-2Dd, as well as xenogeneic ligands (20, 21). Specifically, transfected NK cells expressing Ly49D gain the ability to lyse H-2Ddexpressing target cells as well as CHO (Chinese hamster ovary) cells. This cytotoxicity of allogeneic and xenogeneic targets can be blocked with anti-Ly49D mAbs (clones 12A8 or 4E4, respectively). However, binding assays have not yet verified that Ly49D physically interacts with H-2D d (10). Nevertheless, Ly49D is one of the first NK cell activation receptors determined to be required for killing of a specific target cell (CHO). Although the CHO cell ligand is not yet known, the capacity of Ly49D on C57BL/6 NK cells to activate CHO killing explained the mouse strain-dependent ability of freshly isolated and IL-2-activated (LAK) NK cells to lyse CHO cells. In particular, CHO cells are susceptible to lysis by C57BL/6 NK cells and resistant to lysis by BALB/c NK cells (20, 28). This capacity for CHO cell lysis was mapped to a genetic locus termed Chok in the NKC on mouse chromosome 6. BALB/c mice congenic for the C57BL/6 haplotype of the NKC were used to produce a mAb (clone 4E4) that specifically blocked CHO lysis by C57BL/6 NK cells and that was ultimately determined as recognizing an allotypic determinant on Ly49D from C57BL/6 mice (Ly49D B6). Initial studies indicated that killing could be correlated with mAb 4E4 reactivity, in that C57BL/6 NK cells contained a mAb 4E4 subset capable of CHO lysis, whereas this subset was absent in BALB/c cells. Further examination of a panel of inbred mouse strains revealed that the capacity to kill CHO was generally related to mAb 4E4 reactivity on NK cells. Some strains possessed NK cells that reacted with mAb 4E4 and generally could efficiently lyse CHO targets, similar to C57BL/6 cells, while NK cells from other strains did not react with mAb 4E4 and could not kill CHO, similar to BALB/c cells. However, one strain among the panel, C57L, displayed mAb 4E4 reactivity but not CHO cytotoxicity. Thus, NK cells from C57L were discordant for mAb 4E4 reactivity and CHO killing. In this report we show that even when mAb 4E4reactive LAK cells are isolated from C57L mice, they failed to lyse CHO targets. To examine the molecular basis for this defect we performed cDNA expression and functional analyses. A cDNA was isolated that, on transfected cells, encodes a molecule that reacts with mAb 4E4 and other anti-Ly49D mAbs. The cDNA is

most related to the Ly49O molecule previously cloned from 129 mice (29) but was uncharacterized with respect to serologic and functional activities. However, since Ly49O C57L exhibits an apparent chimerism displaying a high degree of identity to Ly49A in its intracellular domain and Ly49D in its extracellular domain, it contains an ITIM and functions as an inhibitory receptor. MHC class I tetramer binding studies indicate that it is a receptor for H-2D d. Finally, amino acid differences between the Ly49 family members and recent structural information on the Ly49A–H-2D d complex permitted an examination of ligand binding sites. Thus, a chimeric NK cell receptor in C57L mice functions as an inhibitory receptor even though it expresses an epitope for an activation receptor. MATERIALS AND METHODS Mice. C57BL/6 (B6) and C57L mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained in a specific pathogen-free facility at Washington University. Cell lines. The CHO cell line was a kind gift from Dr. P. Stanley (Albert Einstein College of Medicine, Bronx, NY). C1498, Daudi, and YAC-1 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). D12 (C1498 transfected with H-2D d) was previously described (6). All cell lines were maintained in R10 medium [RPMI 1640 (Fisher, St. Louis, MO)] supplemented with 10% FCS (Hyclone, Logan, UT), 100 units/ml penicillin, 100 ␮g/ml streptomycin, 300 ␮g/ml L-glutamine (GIBCO BRL, Grand Island, NY), and 50 ␮M 2-mercaptoethanol (Sigma Chemical Co., St. Louis, MO). Antibodies. Monoclonal antibodies used in this study include: MAR18.5 (mouse IgG2a, anti-rat k light chain), 34-5-8S (mouse IgG2a, anti-H-2D d ␣1,␣2 domain), and 28-14-8S (mouse IgG2a, anti-H-2D b) hybridomas obtained from ATCC; A1 (mouse IgG2a, antiLy49A), hybridoma generously provided by Osami Kanagawa (Washington University, St. Louis); JR9.318 (Mus spretus IgG1, anti-Ly49A), hybridoma kindly supplied by J. Roland (Pasteur Institute, Paris, France); YE1/32 (rat IgG2b, anti-Ly49A) and YE1/48 (rat IgG2a, anti-Ly49A), hybridomas generously provided by Fumio Takei (Terry Fox Laboratories, Vancouver, BC); 4E4 (mouse IgG2a, anti-Ly49D), developed in our laboratory (20); 4E5 (rat IgG2a, antiLy49D), hybridoma kindly provided by John Ortaldo (Frederick Cancer Research and Development Center, Frederick, MD); 12A8 (rat IgG2a, anti-Ly49A, and anti-Ly49D); FITC-conjugated goat anti-mouse (GAM) Ig. All antibodies were purified from spent hybridoma culture supernatants by protein A or protein G affinity chromatography, except for 12A8 (Pharmingen, San Diego, CA) and FITC-GAM (Cappel, Durham, NC).

“CHIMERIC” C57L-DERIVED Ly49 INHIBITORY RECEPTOR RESEMBLING Ly49D

IL-2-activated NK cell preparation. IL-2-activated NK (LAK) cells were generated as described previously (28) with minor modifications. On days 4 –5, adherent cells were washed with HBSS/10% FCS and then cultured in R10 medium supplemented with 1000 units/ml of human recombinant IL-2 (Chiron Corp., Emeryville, CA). Nonadherent cells were removed and transferred to a new dish and resulting adherent cells were washed the next day and expanded. Adherent cells were expanded in culture until day 10 or 11 and used in cytotoxicity assays. For mRNA preparation, IL-2-activated NK cells were depleted of T cells on day 3 as described previously (28) and expanded until day 9. Flow cytometric analysis. On days 5–7 of NK cell preparation, adherent LAKS were harvested with PBS/ 0.2% EDTA, washed with HBSS/10%FCS, stained with directly conjugated mAbs (FITC-4E4 and PE-12A8), and sorted with the MoFlo Cytomation Cell Sorter (Fort Collins, CO). Postsorted cells were washed twice and replated at 0.5–1 ⫻ 10 6 cells/ml overnight. The next day, postsorted LAKS were expanded in culture for 2– 4 days in R10 medium supplemented with 1000 units/ml of IL-2. For stable CHO transfectants, cells were harvested with PBS/0.2% EDTA, washed, and resuspended in sorter buffer (PBS/3% FCS/0.1% sodium azide). Approximately 2 ⫻ 10 5 cells/sample were stained with 1° mAb followed by 2° mAb FITC–GAM or PE-conjugated tetramers. Cells were stained on ice for 30 min. Cells were analyzed on FACScan or FACSCalibur (Becton Dickinson, Sunnyvale, CA) with CellQuest Software. Cytotoxicity. IL-2-activated NK cells from C57BL/6 and C57L mice were used in a standard chromium-51 release assay as effectors. 51Cr-labeled target cells (104) were incubated with effector cells in a final volume of 200 ␮l for 4 h at 37°C. Supernatant (50 ␮l) was collected and assayed for 51Cr content. For redirected inhibition or mAb blocking experiments, effector cells were incubated at various E:T ratios with or without Con A (Pharmacia Biotech, Piscataway, NJ), with or without mAbs in a round-bottom 96-well plate for 15 min prior to addition of labeled targets. Specific cytotoxicity was calculated according to the standard formulas: % specific lysis ⫽ 100 ⫻ (exp ⫺ spont)/(max ⫺ spont), where experimental release (exp) represents radioactive counts from experimental wells, maximum release (max) represents radioactive counts from detergent-lysed target wells, and spontaneous release (spont) represents background radioactive counts from wells with targets alone. Cytotoxicity experiments shown are representative results of average counts calculated from duplicate or triplicate wells from 2– 4 reproducible experiments. DNA transfection. Ly49D B6 CHO transfectants were generated as reported previously (23). For all other transfectants, CHO cells were transfected using Lipofectamine (GIBCO BRL) according to the manu-

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facturer’s instructions. Briefly, 10 5 cells were plated in R10 medium overnight at 37°C. The next day, CHO cells were rinsed with RPMI and overlaid with 2 ␮g DNA/0.01 mg lipofectamine/RPMI mixture (incubated at room temperature, 45 min) at 37°C. After 6 days, the transfected cells were selected in G418 for 2–3 weeks and were subsequently analyzed by FACS for surface expression with anti-Ly49 mAbs. RT–PCR analysis. Ly49D B6 was generated as reported previously (23). For Ly49A B6 and Ly49 C57L, mRNA was isolated from C57BL/6 and C57L-derived LAK cells (depleted of T cells) on day 9 of culture using FAST TRACK mRNA Isolation Kit (Invitrogen, San Diego, CA). First-strand cDNA was generated using 0.5 ␮g of oligo(dT) primer and the Superscript RT Kit (GIBCO BRL). Double-stranded cDNAs were generated by PCR using Expand High Fidelity (Boehringher Mannheim, Mannheim, Germany) on a Perkin–Elmer 9600 Thermal Cycler (Foster City, CA) using the following conditions: 94°C for 1 min; 94°C for 22 s, 50°C for 15 s, 74°C for 45 s, for 30 cycles; 74°C for 5 min. The Ly49-specific primers used to generate cDNAs were forward, 5⬘ CAGAACCACTTCTTGCTAG, and reverse, 3⬘ ACCATTGGACTTAGTCTGC. PCR products were directly sequenced and/or cloned into Bluescript SK-II (Stratagene, La Jolla, CA) and sequenced. Sequencing was performed with an ABI automated sequencer. A 1-kb insert encoding the entire Ly49 open reading frame was excised from BscSK-II with HindIII/XbaI. The excised fragment was ligated to a HindIII/XbaIdigested mammalian expression vector, pcDNAI/neo (Invitrogen), and used for transfection. MHC class I tetramer preparation. Briefly, a cDNA construct encoding the extracellular domains of H-2D d under the control of the T7 promoter was engineered to include the BirA biotinylation signal (30) at the carboxyl terminus of the protein. H-2D d/BirA was expressed in Escherichia coli, refolded in vitro in the presence of human ␤2m and a motif peptide, AGPARAAAL, according to previously published methods (31, 32), and purified by size exclusion chromatography on a Sephadex 75 gel filtration column (Pharmacia Biotech). The purified MHC/peptide complexes were then biotinylated using biotin ligase in the presence of free biotin (Avidity, Denver, CO) at 25°C for 20 h. After removal of the free biotin by dialysis against PBS, tetramers were produced by mixing the biotinylated H-2D d/peptide complex with streptavidin-R-phycoerythrin (Biosource International, Camarillo, CA) at an 8:1 molar ratio. Specificity of the H-2D d tetramer was tested on S167, an Ly49A-transfected CHO cell line (8). A control H-2L d/pMCMV tetramer was made by a similar procedure using an H-2L d/BirA encoding vector (provided by Dr. J. Altman), human ␤2m, and a synthetic peptide MCMV, YPHFMPTNL. Specificity of

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FIG. 1. Strain-specific difference in CHO killing by LAK cells from C57BL/6 (B6) and C57L mice. (A) mAb 4E4 staining of unseparated or mAb 4E4-sorted LAK cells from B6 or C57L mice. Specific staining with FITC-conjugated 4E4 is indicated by a dark line while the unstained control is indicated by a dotted line. (B) Standard 4-h 51chromium release assays were performed with sorted LAKS from B6 or C57L mice against CHO cells (open squares), with mAb 4E4 or isotype control mAb at the indicated E/T ratios. 4E4 ⫹ and 4E4 ⫺ LAK cells were tested against YAC-1 targets as indicated.

the H-2L d tetramers was tested on an antigen-specific T-cell hybridoma. RESULTS ⫹

mAb 4E4 NK Cells from C57L Fail to Mediate CHO Cell Lysis Previous studies from our laboratory showed that unlike C57BL/6 LAK cells, C57L LAK cells were incapable of mediating CHO cell lysis (28). Despite this,

approximately 50% of C57L NK cells react with mAb 4E4 (Fig. 1A), which recognizes the C57BL/6 form of Ly49D, an activation receptor shown to mediate CHO killing. To further examine this discordant observation, IL-2-activated NK cells from C57L mice were sorted into 4E4 ⫹ and 4E4 ⫺ populations (Fig. 1A) and used in cytotoxicity assays. The 4E4 ⫹ NK cell population from C57L mice did not lyse CHO cells, in contrast to the 4E4 ⫹ NK cell population from C57BL/6 mice (Fig. 1B and (20)). Furthermore, addition of mAb 4E4

“CHIMERIC” C57L-DERIVED Ly49 INHIBITORY RECEPTOR RESEMBLING Ly49D

had minimal or no effect on CHO cell lysis by the 4E4 ⫹ C57L population. Even though neither C57L NK population could lyse CHO targets, both subsets exhibited lysis of YAC-1 cells (Fig. 1B), indicating intact killing capacity. These data suggest that mAb 4E4 reacts with a molecule in C57L mice that is not capable of CHO cell lysis. cDNA Cloning of a mAb 4E4-Reactive Ly49 Molecule From C57L To isolate the C57L 4E4-reactive molecule, RT–PCR was performed with Ly49-specific primers on mRNA isolated from IL-2 activated NK cells purified from C57L mice. A PCR product of ⬃984 bps was generated independently from different RT–PCR reactions and directly sequenced (data not shown). Additional multiple independent clones were also isolated and sequenced, providing confirmatory sequences and indicating that the cDNA was not derived from a PCR artifact. The deduced protein sequence contains 262 amino acid residues (Fig. 2). While this work was in progress, another group (29) reported a sequence similar to our C57L Ly49 molecule, which they termed Ly49O. This sequence was isolated from 129 mice but no mAb reactivity or function was reported. The Ly49 C57L sequence reported here contains only one amino acid difference to Ly49O at position 174, probably representing an allotypic difference between C57L and 129 mice. Thus, it is likely that the Ly49 molecule isolated in this report is the C57L allele of Ly49O (Ly49O C57L). Interestingly, further comparison of the C57L protein sequence to other Ly49 family members from B6 mice revealed a 96.5% overall identity to Ly49A and an 82.5% overall identity to Ly49D (Fig. 2). In particular, the C57L sequence is identical to the amino-terminal intracellular and transmembrane domains of Ly49A. On the other hand, the C57L molecule is approximately 56% identical to Ly49D from C57BL/6 mice in these domains. Similar to Ly49A, the C57L molecule contains an ITIM and does not contain a charged residue in the transmembrane domain, which is required for the interaction of Ly49D with the DAP-12 signaling chain (24), implying that it functions as an inhibitory receptor. By contrast, the C57L molecule has extensive identity, approximately 98%, to Ly49D in the N-terminal region of the NK receptor domain (NKD) and bears only 91% identity to Ly49A in this region. All other Ly49 members are less homologous in this region (data not shown). The homology of the extracellular domain of the Ly49O C57L molecule to Ly49D suggested that it should react with anti-Ly49D mAbs. To examine this possibility, a stable transfectant expressing the Ly49O C57L was generated (Fig. 3). The C57L transfectant displayed reactivity to mAbs 4E4 and 4E5, two

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anti-Ly49D-specific mAbs, but no reactivity with any of the anti-Ly49A mAbs. Curiously, the C57L transfectant did not react with mAb 12A8, which recognizes both Ly49A and Ly49D. These data demonstrate that monospecific anti-Ly49D mAbs (clones 4E4 and 4E5) also react with the Ly49O C57L molecule. Thus, the sequence isolated from C57L identifies a NK cell receptor that appears to be structurally chimeric, resembling Ly49A intracellularly and Ly49D extracellularly. mAb 4E4 Recognizes an Inhibitory Receptor on C57L NK Cells The sequence comparison (Fig. 2) and CHO transfectant data (Fig. 3) suggest that mAb 4E4 recognizes a molecule with inhibitory function in C57L mice. To test this hypothesis, we examined the ability of mAb 4E4 to mediate redirected inhibition of Daudi, a human Fc receptor (FcR ⫹) target, by sorted 4E4 ⫹/12A8 ⫺ or 4E4 ⫺/ 12A8 ⫺ C57L LAK cells (Fig. 4). Addition of mAb 4E4 resulted in a slight decrease in the low-level cytolysis of Daudi targets by 4E4 ⫹ LAK cells but not by 4E4 ⫺ LAK cells. In addition, mAb 4E4 inhibited Con A-induced (lectin-facilitated) lysis of Daudi. In either case, inhibition was not observed with isotype-matched control mAb or with 4E4 ⫺ LAK cells. In contrast, addition of mAb 4E4 to 4E4 ⫹ C57BL/6 LAK cells induced lysis of Daudi targets, consistent with an activation role for Ly49D (data not shown). These data demonstrate that cross-linking of the mAb 4E4-reactive molecule on the sorted C57L LAK cells results in inhibition of NK cytotoxicity, consistent with an inhibitory functional role for the C57L Ly49 molecule. The Ly49O C57L Molecule Recognizes H-2D d Since Ly49O C57L is highly related to the extracellular domain of Ly49D and recent studies have suggested that Ly49D may recognize H-2D d (19, 22, 27), we hypothesized that Ly49O C57L may also recognize H-2D d. To address this possibility, H-2D d tetramers were tested for their ability to bind stable transfectants expressing Ly49A B6, Ly49D B6, or Ly49O C57L (Fig. 5). Importantly, both Ly49O C57L and Ly49A B6 transfectants bind H-2D d tetramers. Surprisingly, H-2D d tetramers failed to bind Ly49D transfectants in this assay. Increasing the concentration of H-2D d tetramers resulted in increased fluorescence for Ly49A B6 and Ly49O C57L transfectants but not for Ly49D transfectant (data not shown). Furthermore, H-2D d tetramer binding to Ly49O C57L or to Ly49A B6 was abolished in the presence of mAb 4E4 or mAb JR9.318 (anti-Ly49A), respectively. H-2L d tetramers, used as a negative control, failed to bind any transfectant, indicating specific binding for the H-2D d tetramer. These data demonstrate that Ly49O C57L specifically binds H-2D d.

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FIG. 2. Comparison of the deduced amino acid Ly49OC57L sequences. (A) Alignment of Ly49AB6, Ly49O C57L (GenBank Accession No. AY003920), and Ly49D B6 amino acid sequences are shown. Dashes indicate residues that are identical to those of Ly49AB6. Asterisks indicate spaces or gaps induced by the alignment program. The underlined region delineates the transmembrane domain and the three letter underlined sequences represent potential N-linked glycosylation sites. Œ, Ly49A contacts for H-2Dd at site 1; , Ly49A contacts for H-2Dd at site 2 (some residues are involved in both sites) (12). (B) Schematic representation of percentage amino acid identity between the domains of Ly49AB6, Ly49O C57L, and Ly49DB6. The amino acid sequences were aligned using the GAP subprogram of the GCG sequence analysis package.

The tetramer binding assay results suggested that the Ly49O C57L molecule may exhibit functional recognition of H-2D d with resulting inhibition. To test this, 4E4 ⫹ 12A8 ⫺ -sorted C57L LAK cells were incubated with 51 Cr-labeled H-2D d transfected targets, D12, or the parental cell line, C1498 (Fig. 6). A marked reduction in lysis by the 4E4 ⫹ 12A8 ⫺ C57L LAK cells was observed for targets expressing H-2D d in comparison to the parental cell line. No inhibition of target cell lysis was observed by the 4E4 ⫺

C57L LAK cells (12A8 ⫺ ). Furthermore, addition of either mAb 4E4 or F(ab⬘) 2 fragments of mAb 34-5-8S (anti- H-2D d ) resulted in increased lysis of the H-2D d -expressing targets by the 4E4 ⫹ 12A8 ⫺ C57L LAK cells, indicating that a specific interaction occurs between the mAb 4E4-reactive molecule and H-2D d . Thus, the mAb 4E4-reactive molecule on sorted C57L LAK cells can inhibit NK-cell-mediated cytotoxicity upon recognition of H-2D d on the target cell surface.

“CHIMERIC” C57L-DERIVED Ly49 INHIBITORY RECEPTOR RESEMBLING Ly49D

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FIG. 3. Anti-Ly49 receptor mAb reactivity with stable transfectants expressing Ly49O C57L. Stable CHO transfectants were stained with indicated anti-Ly49A or anti-Ly49D mAbs followed by secondary FITC-conjugated goat ⫻ mouse (GAM) Ig Abs or PE-conjugated 12A8. The solid lines represent specific while dashed lines represent staining with secondary FITC-conjugated GAM mAb alone.

DISCUSSION We have characterized a Ly49 family member from the C57L mouse strain that exhibits a high degree of sequence similarity to previously studied inhibitory and activation Ly49 molecules, namely Ly49A and Ly49D, respectively. This Ly49 molecule is expressed on IL-2activated NK cells from C57L mice and reacts with two of three available anti-Ly49D mAbs, but none of the anti-

Ly49A mAbs. Cytotoxicity assays performed with 4E4⫹ LAK cells isolated from C57L mice revealed that this population could not mediate lysis of CHO or Daudi target cells when used in cytotoxicity or redirected inhibition assays, respectively. This is in contrast to C57BL/6 LAK cells, of which the 4E4 ⫹ population lyses CHO and Daudi target cells in these assays. Thus, even though Ly49OC57L reacts with mAbs known to recognize the Ly49D NK cell

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FIG. 4. Redirected lysis assay using 4E4 ⫹-sorted C57L LAK cells. mAb 4E4-sorted C57L LAK cells were incubated with human Daudi target cells at an E/T of 10:1 in the absence (open bars) or presence of Con A, 8 ␮g/ml final concentration (solid bars). Intact mAb 4E4 or mouse anti-rat (isotype control) was added (10 ␮g/ml final concentration) as indicated.

activation receptor, it functions as an inhibitory molecule. Murine NK cells express products of several distinct but structurally similar, C-type lectin-like families encoded within the NKC and including Ly49, NKR-P1, and CD94/NKG2. Each family expresses both activation and inhibitory receptors. Interestingly, in the CD94/NKG2 and NKR-P1 families there are molecules that are serologically similar but have opposing functions. Specifically, murine NKG2A and NKG2C are inhibitory and activation receptors, respectively, that cross-react with the same mAb and appear to recognize the same Qa-1 b ligand (33). The NK1.1 (NKR-P1C) molecule, as recognized by mAb PK136, functions as an activation molecule expressed on NK cells in C57BL/6 mice (34), whereas the same mAb recognizes NKR-P1B in SJL/J (35) and Swiss NIH (36), which functions as an inhibitory molecule. These studies are highly reminiscent of the studies we report here. While the ligands for these NKR-P1 molecules are not known and they do not appear to be chimeric, the data presented here suggest that mAb 4E4 recognizes two Ly49 molecules that have opposing functions, Ly49D, an activation receptor in C57BL/6 mice and the “chimeric” Ly49, an inhibitory receptor in C57L mice. Thus, our studies provide additional data highlighting the difficulties in relying only on serologic analysis to analyze the complexity of NK cell receptors. More importantly, our studies on Ly49O C57L provide new insight into the function and specificity of the Ly49 family of receptors. The Ly49O C57L molecule binds H-2D d in tetramer binding and functional inhibition assays. The high degree of similarity between the extracellular domains of Ly49D and Ly49O C57L suggested

that both molecules should have a related ligand specificity. Indeed, recent studies provide functional evidence that Ly49D can mediate specific lysis of H-2D dexpressing targets, strongly suggesting that the ligand for Ly49D is H-2D d (19, 22, 27). Surprisingly, however, H-2D d tetramers failed to bind Ly49D transfectants even though the tetramers bind Ly49O C57L transfectants. The lack of H-2D d binding to Ly49D B6 observed in this report is consistent with other studies that also failed to detect binding of Ly49D B6 to H-2D d tetramers (10). While these observations suggest that Ly49D may have weaker affinity for H-2D d than Ly49A B6 or Ly49O C57L, they also permit an analysis of ligand binding sites. It is therefore likely that the amino acid differences in the extracellular regions between the three Ly49s may contribute to the differential binding to H-2D d. The binding of Ly49A B6, Ly49D B6, and Ly49O C57L to H-2D d can be examined in the context of recent Ly49A B6–H-2D d crystal structure, which revealed two interfaces between Ly49A and H-2D d, referred to as site 1 and site 2 (12). Amino acid sequence alignments of Ly49A B6 with Ly49D B6 revealed that Ly49D B6 differs at three positions (positions 223, 244, and 248) of the 25 contact residues implicated in the Ly49A–H-2D d interaction. These 3 residues make contact in site 2, and two of the differences (residues 244 and 248) also contact site 1. Interestingly, Ly49O C57L shares the same amino acid substitutions in these three positions with Ly49D B6, yet, in contrast to Ly49D B6, Ly49O C57L transfectants bind H-2D d tetramers. Thus, the differential binding by Ly49D B6 and Ly49O C57L to H-2D d is difficult to reconcile when only considering the amino acid res-

“CHIMERIC” C57L-DERIVED Ly49 INHIBITORY RECEPTOR RESEMBLING Ly49D

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FIG. 5. Tetramer binding to stable CHO transfectants expressing Ly49 molecules. Stable CHO transfectants were stained with streptavidin–PE-conjugated H-2L d or H-2D d tetramers. For mAb blocking of tetramers, CHO transfectants were preincubated with either mAb JR9.318 or mAb 4E4 (10 ␮g/ml final concentration) followed by H-2L d or H-2D d tetramers. For cell surface staining of Ly49, CHO transfectants were stained with mAb JR9.318 or 4E4 followed by FITC-conjugated GAM Ig.

idues comprising the contact sites derived from the Ly49A B6–H-2D d crystal structure. Previous studies attempting to map the critical residues for Ly49C–MHC interaction have indicated that mutagenesis of even a single amino acid residue in the NKD of Ly49C, (37) but outside of the putative MHC I contact sites, can influence ligand binding and specificity. Furthermore, these studies have revealed that residues within the stalk region can also affect ligand binding (38). Despite the extensive similarity observed for the extracellular domains of Ly49D B6 and Ly49O C57L, Ly49O C57L is highly related to Ly49A B6 in the stalk region. The ability of H-2D d to bind both Ly49A B6 and Ly49O C57L but not Ly49D B6 therefore suggests some influence on binding and/or specificity by other parts of the molecule not previously examined in structural studies. Moreover, Ly49O C57L exhibits three amino acid differences in its NKD (positions 173, 212, and 214) in comparison to Ly49D B6 (Fig. 2). It is possible that one or all of these amino acids may play a role in altering ligand binding, specificity, and/or affin-

ity (in comparison to Ly49D B6), despite the fact that these residues are outside the known contact sites. It is provocative that residues 173, 212, and 214 lie on the membrane proximal face of the NK domain and may be accessible to interactions with the distal stalk region (Fig. 7). Also, the glycosylation site at position 124 is conserved between Ly49A B6 and Ly49O C57L but is missing in Ly49D B6, suggesting that structural effects related to the distal stalk/proximal NKD regions may be important in controlling H-2D d interactions. It is noteworthy that the Cys at position 120 of Ly49D (which aligns with Tyr122 of Ly49A B6) is present only in Ly49D and may also be involved in Ly49D unique dimeric homotopic interactions (Fig. 2). On the other hand, the substitution of Thr223 for Arg223 in Ly49D B6 and Ly49O C57L may create a new glycosylation site that falls within the binding region. Ly49D B6 and Ly49O C57L may exhibit differential glycosylation at this site, consequently interfering with MHC class I interaction. Further studies examining the differential binding of the inhibitory Ly49A B6 and Ly49O C57L receptors and

38

MEHTA ET AL.

FIG. 6. Cytotoxicity of H-2D d-expressing target cells by 4E4 ⫹ 12A8 ⫺-sorted C57L LAK cells. (A) Comparison of 4E4 ⫹ and 4E4 ⫺ C57L LAK cells for cytotoxicity against 51Cr-labeled C1498 or D12 (C1498 transfected with H-2D d) targets at an E/T of 10:1. (B) Intact mAb 4E4, intact mAb mouse anti-rat (isotype control), F(ab⬘) 2 mAb 34-5-8S, or F(ab⬘) 2 mAb 28-14-8S (isotype control) was added (10 ␮g/ml final concentration) as indicated.

activating Ly49D B6 receptor to H-2D d should provide insight into the structural basis for binding between MHC and Ly49 and the role for Ly49 receptor glycosylation. The question arises of how Ly49D B6 mediates NK cell cytotoxicity of H-2D d-expressing targets when it fails to physically bind to H-2D d tetramers. It is possible that the affinity of the interaction of Ly49D with H-2D d is too low to be detected by physical binding studies but is still sufficient to activate NK cells. Alternatively, the Ly49D B6–H-2D d interaction may be peptide-specific, such that the peptide presented by H-2D d may play a role in Ly49D B6 ligand recognition. The ability of a Ly49 family molecule to discriminate between specific MHC/peptide complexes has been previously demonstrated (10). Those data suggest that the MHC class I peptide may alter the affinity or ability of Ly49 to interact with MHC class I. In experiments presented in this report only one MHC/peptide complex was tested; other MHC/peptide combinations may display reactiv-

ity to Ly49D B6. Thus, experiments to examine whether Ly49D B6 can discriminate between MHC/peptide complex should help resolve the discrepancy between the functional data and binding data that indicate that Ly49D B6 interacts with H-2D d. Nevertheless, our studies indicate that the molecule of interest in this report, Ly49O C57L, binds H-2D d. To date, the Ly49 family in C57BL/6 mice consists of 10 or more full-length members (39, 40), with evidence for 5 additional molecules, as predicted by genomic sequences (41). In addition, alleles have been identified for some of the Ly49 molecules (42– 45). Comparison of the deduced amino acid sequence of Ly49O C57L to C57BL/6-derived Ly49 family members revealed high amino acid identity to Ly49A B6, suggesting that Ly49O C57L may be an allele of Ly49A. However, in serologic analysis of stable Ly49O C57L CHO transfectants, none of the anti-Ly49A mAbs, but two of three anti-Ly49D mAbs, recognized these cells. Moreover, bulk C57L LAK cells exhibit reactivity with antiLy49A mAbs (data not shown), suggesting that C57L LAK cells may express an Ly49A allelic variant. Amino acid sequence comparisons of the domains of Ly49A B6, Ly49D B6, and Ly49O C57L revealed that Ly49O C57L is identical in the intracellular/transmembrane to Ly49A B6, highly related to the stalk region of Ly49A B6, and highly related to the NKD region of Ly49D B6. The extensive homology in the NKD to Ly49D and reactivity to anti-Ly49D mAbs suggests instead that Ly49O C57L is the product of an Ly49D allele. However, dual staining of bulk C57L LAK cells with mAbs 4E4 and 12A8 revealed a double-positive population (data not shown) that is serologically similar to the Ly49D population in B6 mice, suggesting that the Ly49O C57L is unlikely to be an Ly49D allele. An Ly49O-like molecule has not yet been identified in the C57BL/6 mouse. Thus, with respect to C57BL/6 forms of the Ly49 family, it is not yet clear if Ly49O C57L represents a completely distinct Ly49 molecule or an allele of an already identified Ly49 gene from C57BL/6. The Ly49A gene consists of seven exons in which the second exon encodes the intracellular domain, the third exon encodes the transmembrane, the fourth exon encodes the stalk, and the fifth through the seventh exons encode the NKD region (46). Although the genomic organization of other Ly49 genes is not yet known, it is likely that Ly49D shares an organization similar to that of Ly49A because it is similar in overall size and domain size and is genetically linked. Interestingly, the apparent chimerism exhibited by the Ly49O C57L molecule can be delineated by the same exonic boundaries defined for Ly49A, in that the region in which Ly49O C57L begins to share extensive homology to Ly49D appears to be encoded from the fifth exon onward. The apparent chimerism exhibited by Ly49O C57L is reminiscent of the dm1 MHC class I mutant mouse strain in which the D region has undergone an intra-

“CHIMERIC” C57L-DERIVED Ly49 INHIBITORY RECEPTOR RESEMBLING Ly49D

39

FIG. 7. Structural representation of the location of the amino acid polymorphisms among Ly49A B6 , Ly49O C57L , and Ly49D B6 . Molecular surface representations (A, C, D, E) and a ribbon diagram (B) of the Ly49A B6 subunits of the Ly49A/H-2D d structure (12) were displayed from the protein data bank coordinates of 1QO3 using GRASP 1.3.6 (51). The two subunits of the homodimer are displayed in shades of blue. The novel glycosylation sites of Ly49O C57L and Ly49D B6 are shown in pink. Position 173, which is distinct in all three proteins is shown in lime. Residues unique to Ly49D B6 are shown in magenta, those unique to Ly49O C57L are shown in red, and those shared between Ly49O C57L and Ly49D B6 are in yellow. The projection shown in (A) is en face with the likely position of the stem region being down and the position of the MHC ligand up. (C) A 90° rotation about the x axis, viewing the top or interface region of the molecule. (D) A 90° rotation of (C) about the y axis, allowing visualization of the side, while (E) is a 180° rotation of A about the x axis, showing the bottom of the molecule.

genic recombination between the D d and L d genes (47– 49). As a result, this mutant mouse expresses a single chimeric D region product D dm1, in which the N-terminal portion of the molecule is D d and the C-terminal is L d. Similarly, it is possible that the chimeric Ly49O C57L may be the result of an intragenic recombination within its NKC locus, resulting in the expression of the chimeric molecule. Alternatively, this hybrid sequence may have been generated by an alternative splicing or trans-splicing mechanism. Similar hybrid-like transcripts have been reported to occur for the KIR (50). Lastly, it is possible that a gene duplication and/or conversion event may have occurred that resulted in a gene that encodes this molecule in C57L. Further detailed analysis of the Ly49 gene cluster in C57BL/6 and C57L should resolve these possibilities, which potentially provide mechanisms for diversifying the NK cell receptor repertoire.

ACKNOWLEDGMENTS The authors thank Drs. Osami Kanagawa, John Ortaldo, Jacques Roland, Pamela Stanley, and Fumio Takei for their kind gifts of reagents, Domenic Fenoglio for cell sorting, and Drs. Michael G. Brown, Jon Heusel, and Lawrence Wang for critical reading of the manuscript. This work was supported by NIH grants to W.M.Y. who is an investigator for the Howard Hughes Medical Institute. H.R.C.S. is supported by Training Grant 5T32AI07163.

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