Optimized Tetramer Analysis Reveals Ly49 Promiscuity for MHC Ligands

Emily McFall, Megan M. Tu, Nuha Al-Khattabi, Lee-Hwa Tai, Aaron S. St.-Laurent, Velina Tzankova, Clayton W. Hall, Simon Belanger, Angela D. Troke, Andrew Wight, Ahmad Bakur Mahmoud, Haggag S. Zein, Mir Munir A. Rahim, James R. Carlyle and Andrew P. Makrigiannis
J Immunol December 1, 2013, 191 (11) 5722-5729; DOI: https://doi.org/10.4049/jimmunol.1300726

Abstract

Murine Ly49 receptors, which are expressed mainly on NK and NKT cells, interact with MHC class I (MHC-I) molecules with varying specificity. Differing reports of Ly49/MHC binding affinities may be affected by multiple factors, including cis versus trans competition and species origin of the MHC-I L chain (β2-microglobulin). To determine the contribution of each of these factors, Ly49G, Ly49I, Ly49O, Ly49V, and Ly49Q receptors from the 129 mouse strain were expressed individually on human 293T cells or the mouse cell lines MHC-I–deficient C1498, H-2b–expressing MC57G, and H-2k–expressing L929. The capacity to bind to H-2Db– and H-2Kb–soluble MHC-I tetramers containing either human or murine β2-microglobulin L chains was tested for all five Ly49 receptors in all four cell lines. We found that most of these five inhibitory Ly49 receptors show binding for one or both self–MHC-I molecules in soluble tetramer binding assays when three conditions are fulfilled: 1) lack of competing cis interactions, 2) tetramer L chain is of mouse origin, and 3) Ly49 is expressed in mouse and not human cell lines. Furthermore, Ly49Q, the single known MHC-I receptor on plasmacytoid dendritic cells, was shown to bind H-2Db in addition to H-2Kb when the above conditions were met, suggesting that Ly49Q functions as a pan–MHC-Ia receptor on plasmacytoid dendritic cells. In this study, we have optimized the parameters for soluble tetramer binding analyses to enhance future Ly49 ligand identification and to better evaluate specific contributions by different Ly49/MHC-I pairs to NK cell education and function.

Introduction

Natural killer cells are lymphocytes that play a major role in the innate immune system as illustrated by individuals with NK cell deficiency, who suffer from otherwise benign viral infections (1). NK cells travel throughout the body via the blood and lymphatics and kill abnormal cells, including cancerous cells and pathogen-infected cells. NK cell killing is regulated via receptor binding to constitutively expressed or induced surface proteins on host cells (2). Unlike T and B cells, NK cells do not undergo gene rearrangement during development to acquire receptor diversity and specificity. These innate responses are regulated by a large array of surface receptors, some of which are stochastically expressed (3). NK cells have different types of receptors that can be either activating or inhibitory. Ligand binding by activating receptors will lead to killing of target cells through the release of cytotoxic granules containing granzymes and perforin, whereas inhibitory receptors prevent granule release, leading to host cell survival. Cytokine secretion is similarly regulated by such receptors. Because multiple receptors with opposing function are expressed simultaneously, the action of NK cells depends on the balance between these activating and inhibiting signals. These receptors include CD94/NKG2, NKR-P1, PIR, killer cell Ig-like receptor (KIR), Ly49, and others. Certain receptors are particular to specific species such as KIRs found in humans and Ly49, the functional analog found in mice. KIRs are type I transmembrane glycoproteins composed of Ig-like domains encoded by genes located in the leukocyte receptor complex.

In contrast to KIR, Ly49 are type II transmembrane glycoproteins and are expressed as homodimers linked by four disulfide bonds. Ly49 genes are composed of seven exons and encode the following domains: exon 1 is noncoding, exon 2 forms the cytoplasmic domain, exons 3 and 4 form the transmembrane domain and α-helix stalk region, respectively, wheres exons 5, 6, and 7 form the C-type lectin-like extracellular domain (NK recognition domain). The NK recognition domain is composed of two α-helixes and two antiparallel β-sheets. It is the main region of the Ly49 receptor that interacts with ligand; however, the extracellular stalk may also be involved in binding (4, 5). The stalk in some Ly49 has conserved glycosylation sites, which when glycosylated lead to steric hindrance and decreased binding to MHC-I (5).

Ly49 are expressed mainly on NK cells, NKT cells, and a small percentage of activated CD8+ T cells. Additionally, two Ly49, Ly49B and Ly49Q, are expressed on myeloid-derived cells, including macrophages, neutrophils, and DCs (6, 7). Initial cDNA screening of inbred mouse strains revealed extreme genetic diversity and polymorphism in different mouse strains (8), which was later revealed to be a result of genomic plasticity within the Ly49 region resulting in cluster sizes ranging from 8 to 22 Ly49 genes (9, 10). Most Ly49 receptors identified to date are inhibitory and contain a cytoplasmic ITIM domain encoded in exon 2 that becomes phosphorylated during ligand binding. Src homology region 2 domain-containing phosphatase 1 is recruited, activated, and ultimately dephosphorylates tyrosine residues in kinases, leading to signal termination (11). Dampening of NK cell function ultimately results in target cell survival. Paradoxically, NK cell licensing and functional potential depend on the expression of self–MHC-I receptors during development (12, 13). In agreement with these education models, the absence of Ly49 expression by NK cells in gene-targeted mice results in lack of self–MHC-I education and the loss of the ability to kill MHC-I–deficient cells (14).

Crystallography studies revealed that Ly49A interacts with the side of the MHC-I molecule, H-2Dd, at the α1 and α2 domains and also associates with the β2-microglobulin (β2m) L chain, but it binds beneath the peptide-binding platform itself (15, 16). Because NK cells also express MHC-I molecules it is possible for Ly49 to interact with MHC-I on the same cell, as has been shown for Ly49A and H-2Dd (17). This is referred to as cis interaction, whereas binding to MHC-I on another cell is a trans interaction. Cis and trans interactions have two distinct receptor conformations made possible due to the flexible stalk; the stalk is able to bend into a back-fold conformation allowing for trans binding and straightens into an extended conformation for cis interactions (18). Cis interactions are stable and sequester Ly49A so that most receptors are not available for MHC-I binding in trans (19). The end result is that the NK cell is more sensitive to small changes in MHC-I levels on target cells and ultimately lowers the activation threshold (17). Finally, using a Ly49A mutant with an inflexible stalk domain, it was shown that cis binding is essential for the ability of Ly49A to educate NK cells (20).

The objective of this study was to determine the full potential of binding between Ly49 from 129 strain inbred mice and self–MHC-I. The putative functional inhibitory Ly49 in the 129 strain mice includes Ly49E, Ly49EC2, Ly49G, Ly49I1, Ly49O, Ly49S, Ly49T, Ly49Q1, and Ly49V. No mAbs have been produced for Ly49EC2 and Ly49S, although the open reading frames are intact and the proteins are likely expressed, and Ly49ec1, Ly49i2, Ly49q2, and Ly49q3 are pseudogenes in 129 strain mice due to early stop codons and/or missing exons (21). Previously, the Ly49 from 129 strain mice were tested for binding to seven different MHC-I molecules using 293T as the host cell type expressing the Ly49 proteins and soluble mouse MHC-I tetramers containing human β2m (22). Binding was observed for Ly49G:Dd, Ly49I:Kd, Ly49O:Dd/Ld, and Ly49V:Db/Dd/Dk/Ld (22). Similarly, using lacZ-based reporter cell assays, as well as soluble MHC-I tetramers, it was previously reported that the plasmacytoid dendritic cell (pDC)–expressed Ly49Q displays strong H-2Kb binding, but apparently no binding to H-2Db or MHC-I alleles expressed in cells from mice possessing H-2a,q,k,d MHC haplotypes (23). However, since these initial studies there have been new advances in our knowledge of Ly49/MHC-I interactions in the B6 inbred mouse strain, including the role of cis versus trans competitive binding and loss of binding observed when the whole MHC-I tetramer is not mouse derived (24).

In light of new knowledge concerning B6-derived Ly49/MHC-I interactions, two questions have been raised. First, are 129-derived Ly49/MHC-I interactions also affected when the MHC-I tetramer is composed of a human β2m L chain compared with a mouse β2m? Second, what is the contribution of cis interactions to 129-derived Ly49/MHC-I binding? In the present study five inhibitory receptors (Ly49G, Ly49I, Ly49O, Ly49Q, and Ly49V) derived from 129 strain inbred mice were tested for binding to the self–MHC-I molecules in 129 strain mice (H-2Db and H-2Kb) using MHC-I tetramers containing either human or mouse β2m. Additionally, Ly49 were expressed in four different cell lines varying in their expression of MHC-I, as well as species origin, to resolve the above questions.

Materials and Methods

Mice

H2Db−/− and H2Kb−/− mice on a B6 background were purchased from Taconic Farms (Albany, NY). All breeding and manipulations performed on animals were in accordance with university guidelines and approved by the University of Ottawa Animal Ethics Committee.

Cells

BWZ.36 cells were obtained from Dr. N. Shastri (University of California, Berkeley, CA). Construction of the CD3ζ/NKR-P1B/Ly49Q chimeric receptor and BWZ assay was described previously (23). L929 was developed from s.c. areolar and adipose tissue fibroblasts of a C3H/An mouse and is MHC-I+ for the H-2k haplotype. YB2/0 (rat hybridoma) and YB.Dd, YB.Db, YB.Dk, YB.Kk, and YB.Ld stable transfectants were provided by Dr. Stephen Anderson (SAIC-Frederick, Frederick, MD). YB.Kb/Dd was a gift of Dr. Kevin Kane (University of Alberta, Edmonton, AB, Canada). The coding region of H-2Kb and H-2Db was amplified and cloned into the pEF6-TOPO vector (Invitrogen) and then used to make stable transfectants of L929 via lipofectamine (Invitrogen). MC57G cells are MHC-I+ (H-2b) and are derived from B6 fibroblasts. MC57G was obtained from Dr. W.-K. Suh (Institut de Recherches Cliniques de Montréal, Montreal, QC, Canada) and 293T cells (human embryonic kidney epithelial) were obtained from Dr. M.-A. Langlois (University of Ottawa). DCs were isolated from collagenase (Roche Diagnostics, Laval, QC, Canada)-treated splenocytes using anti-CD11c–conjugated microbeads (Miltenyi Biotec, Auburn, CA). MHC-I–deficient C1498 cells are a derivative of the C1498 acute myeloid leukemia cells of B6 origin and were previously produced in our laboratory (14).

Acid treatment

Cells were acid-treated as previously described with minor modifications (17). Cells were washed twice with PBS and resuspended at room temperature in citrate buffer (0.133 M citric acid and 0.066 M Na2HPO4 [pH 3.3]) at a density of 2 × 106 cells/ml. Acid treatment was stopped after 4 min with 40 ml 5% heat-inactivated FBS in PBS. Cells were washed with PBS once more and subsequently stained with tetramers as described below. Cell viability was not adversely affected by acid treatment as assessed by trypan blue exclusion and forward and side scatter analysis.

Abs and soluble MHC-I tetramers

mAb staining was carried out using FITC-labeled 4D11 (binds to Ly49G in 129 strain mice) (eBioscience), FITC-labeled 4E5 (Ly49O and Ly49V) (BD Pharmingen), PE-labeled 14B11 (Ly49I) (eBioscience), and purified or biotinylated NS-34 (Ly49Q), which was a gift from Dr. Noriko Toyama-Sorimachi (International Medical Center, Tokyo, Japan). Secondary mAbs used for flow cytometry included PE-labeled goat anti-rat IgG or PE-labeled streptavidin. All Ab testing was carried out with 0.25 μg of the given Ab. Streptavidin-PE–conjugated murine class I MHC tetramers H-2Db and H-2Kb were provided by the National Institutes of Health Tetramer Core Facility at Emory University (Emory University Vaccine Center, Atlanta, GA). Tetramers were refolded in the presence of murine β2m L chain and a peptide previously shown to form a stable tetrameric structure, and are as follows: H-2Db, gp33–41 (KAVYNFATC) of lymphocytic choriomeningitis virus; H-2Kb, OVA257–264 (SIINFEKL) of chicken OVA. PE-conjugated Db and Kb tetramers containing human β2m L chain were purchased from Beckman Coulter (Fullerton, CA). Staining reactions included 20% normal rat/mouse serum (Sigma-Aldrich, Oakville, ON, Canada) or 10 μg/ml 2.4G2 (anti-CD16/32; BD Pharmingen) mAb for blocking nonspecific binding. No differences in staining were seen when either 2.4G2, rat, or mouse serum was used, as judged by the mean fluorescence intensity (MFI) of the staining (data not shown). Cells (n = 250,000) were counted for each cell line and incubated at 4°C with 2.4G2. Staining was then carried out with the mAb or tetramers listed above for 20 min at 4°C or 37°C, respectively. Washing (2 ml FACS buffer [0.5% BSA and 0.2% sodium azide in PBS]) and centrifugation at 500 × g for 5 min was performed at the beginning, between mAb stainings, and at the end of the experiment. Ten thousand events were collected (excluding dead cells) during flow cytometry, and experiments were repeated a minimum of three times. Flow cytometry was performed on a CyAN-ADP using Summit software (Beckman Coulter). Data were analyzed with Kaluza software.

Results

BWZ.Ly49Q reporter cells are stimulated by cells expressing the α1α2 domains of H-2Kb, but not H-2Db

We have previously shown via BWZ reporter cell assays and soluble tetramer analyses that H-2Kb is a strong ligand for Ly49Q (23), and via gene-knockout mice that this interaction is key for the production of IFN-α by pDCs (25). In particular, BWZ.Ly49Q but not BWZ.36 parental reporter cells were stimulated by cells of the H-2b haplotype, as well as immobilized recombinant H-2Kb. However, BWZ.Ly49Q reporter cells did not respond to cells derived from non–H-2b mice, B2m−/−, H-2Kb−/−H-2Db−/− mice, nor to recombinant H-2Db (23). To further test the selective binding of Ly49Q we decided to “rescue” a nonstimulating tumor cell, specifically L929 fibroblasts, which bear H-2k MHC-I molecules, by transfection of H-2Kb or H-2Db. As previously reported, L929 tumor cells are unable to stimulate lacZ production by BWZ.Ly49QBALB or BWZ.Ly49QB6 reporter cells (23). However, stable expression of H-2Kb, but not H-2Db, in L929 was able to stimulate both types of BWZ.Ly49Q reporter cells, but not parental BWZ.36 parental cells, in line with previous findings (Fig. 1A). Two different clones of each transfectant were tested and showed similar results. PMA plus ionomycin treatment of all reporter cell lines showed that each was functional.

FIGURE 1.
FIGURE 1.

Ly49Q-CD3ζ fusion receptor-bearing BWZ cells are stimulated by cells expressing the α1α2 domain of H-2Kb, but not H-2Db. (A) BWZ.36 parental cells or transductants expressing a chimeric protein (introduced by a retrovirus containing IRES-GFP) with the extracellular domain of either B6 or BALB/c Ly49Q proteins were incubated with parental L929 mouse fibroblasts or two separate stable transfectant clones expressing either H-2Kb or H-2Db. (B) CD11c+ DCs isolated from the indicated gene-knockout mouse strains were coincubated with BWZ reporter cells. (C) Parental and Ly49Q-CD3ζ fusion receptor–transduced BWZ reporter cells were incubated with parental rat YB2/0 cell line or stable transfectants expressing the indicated mouse H-2 molecules. The YB.Kb/Dd cell line expresses an H-2 fusion molecule composed of the α1α2 domains of H-2Kb and the α3/transmembrane/cytosolic domains of H-2Dd. After overnight incubation, lacZ production was revealed by addition of chlorophenol-red-β-D-galactopyranoside substrate and OD measurements at the indicated wavelengths. Maximum lacZ production capability by BWZ cells is shown by PMA/ionomycin treatment. (D) BWZ.36 parental cells and transductants expressing either B6 or BALB/c Ly49Q proteins were briefly treated with mild acid to disrupt class I MHC/peptide interactions and then stained with specific PE-conjugated mAb or PE-conjugated MHC-I tetramers (H-2Kb or H-2Db) containing mouse β2m L chain. After mAb or tetramer staining, cells were analyzed by flow cytometry. BWZ.36 parental staining levels are shown in black; specific staining of stable Ly49 transfectants before and after acid treatment are shown in red and blue, respectively. Average MFI fold increase in the non–acid-treated transfectants over the parentals is indicated. Data are representative of three independent experiments.

Ligand-bearing DCs were previously shown to be good stimulators of the T cell–derived BWZ cells (23). In agreement with L929 transfectants, we found that DCs from H-2Db−/− mice were able to stimulate BWZ.Ly49Q reporter cells, but H-2Kb−/− DCs were lacking in this respect (Fig. 1B). Finally, to test a larger panel of MHC-I transfectants and to localize the domains necessary for Ly49Q binding, a panel of mouse MHC-I stable transfectants of the rat hybridoma YB2/0 were used as stimulator cells. H-2Db, H-2Dd, H-2Dk, H-2Kk, and H-2Ld were not able to stimulate lacZ production by Ly49Q reporter cells; however, a fusion MHC-I composed of the α1α2 domains of H-2Kb and the α3, transmembrane, and cytosolic domains of H-2Dd efficiently stimulated both types of BWZ.Ly49Q reporter cells (Fig. 1C). These results reinforce our previous findings that in this assay Ly49Q appears to be exquisitely specific for a single type of MHC-I, but also extend our findings and show that α1α2, but not α3, domains are sufficient for Ly49Q binding.

Two troubling matters were perceived: 1) it is not intuitive that pDCs would express a receptor that is vital for their function, but that only binds a ligand found in some mouse strains, and 2) the reporter cells themselves express H-2Kk and H-2Dk molecules that may interfere with the assay via cis competition, as has been shown for Ly49Q and other Ly49 (17, 23), or this may cause selection of reporter cells that only respond to very strong receptor/ligand binding, assuming that H-2Kk/Dk/Db binding is less strong than Ly49Q binding to H-2Kb. We made several attempts to produce MHC− variants of BWZ.36 cells to test this hypothesis by chemical mutagenesis followed by anti–MHC-I staining and cell sorting, but each time the resulting MHC-I− BWZ.36 variants did not have functional lacZ responses to PMA/ionomycin. We next tried to resolve these questions using soluble MHC-I tetramers.

Ly49Q binds to H-2Db tetramers containing mouse β2m in the absence of cis interactions

We previously reported that H-2Kb tetramers refolded with human β2m L chain bound to mouse pDCs and that this binding could be blocked with anti-Ly49Q mAb (23). Furthermore, tetramer binding was significantly increased on pDCs from H-2Kb−/−H-2Db−/− mice, suggesting that Ly49Q on pDCs can bind to H-2Kb in both cis and trans (23). Thus, it may be possible that cis interactions with MHC-I inhibit trans binding of Ly49Q to MHC-I, as reported for other Ly49 (26), and possibly explaining the inability of Ly49Q to bind H-2Db in BWZ reporter cell assays. To investigate this possibility, we treated BWZ cells expressing Ly49Q with mild acid to eliminate possible cis interactions and tested binding of H-2Kb and H-2Db tetramers containing mouse β2m. In accordance with the reporter assay data, H-2Kb but not H-2Db tetramer bound to Ly49Q. However, upon mild acid treatment to disrupt cis interactions, H-2Db tetramer was also able to bind to Ly49Q, suggesting that both H-2Kb and H-2Db are able to bind Ly49Q in the absence of any cis interactions (Fig. 1D). As well, we generated Ly49Q129 stable transfectants of an MHC-I− variant of the mouse B cell line, C1498, and then stained them with soluble H-2Kb and H-2Db tetramers containing human β2m. The lack of MHC-I expression in this C1498 derivative will not allow for cis interactions. As previously reported, H-2Db/human β2m tetramers failed to interact with Ly49Q129 even in the absence of possible cis interactions, whereas H-2Kb/human β2m tetramers bound strongly to C1498.Ly49Q cells compared with parental C1498 cells (Fig. 2A, middle panel). There was no binding of either tetramer to the parental C1498 line. It has been previously reported that the species origin of the β2m L chain affects Ly49/MHC-I binding (24, 26). To determine whether this was a factor in Ly49Q/MHC-I binding, the experiment was repeated but with tetramers containing mouse β2m. This time, both H-2Kb/mouse β2m and H-2Db/mouse β2m tetramers showed positive staining of C1498.Ly49Q129 cells (Fig. 2A, right panel), and, additionally, H-2Kb tetramer binding was increased. As expected, owing to the absence of cis interactions in the MHC-I–deficient variant of C1498, acid treatment did not affect tetramer staining levels as judged by the overlapping histograms for nontreated and acid-treated cells (Fig. 2A, right panel), with negligible quantified differences in MFI of acid-treated relative to non–acid-treated (Fig. 2B). Overall, these results show that under optimized conditions, Ly49Q can bind to MHC-I molecules other than H-2Kb.

FIGURE 2.
FIGURE 2.

Enhanced Ly49 binding to MHC-I molecules is observed in the absence of cis interactions using tetramers containing mouse β2m L chain. An MHC-I–deficient derivative of the C1498 cell line was stably transfected with the indicated Ly49 from 129 strain mice, treated with mild acid to disrupt class I MHC/peptide interactions, and then stained with specific PE- or FITC-conjugated mAb or PE-conjugated MHC-I tetramers (H-2Kb or H-2Db) containing human (middle panel) or mouse (right panel) β2m L chain. After mAb or tetramer staining, cells were analyzed by flow cytometry. (A) C1498 parental staining levels are shown as black histograms, and specific staining of stable Ly49 transfectants before and after acid treatment are shown as red and blue histograms, respectively. Average MFI fold increase in the non–acid-treated transfectants over the parentals is indicated. (B) Quantification of binding of MHC-I tetramers (H-2Kb or H-2Db) containing mouse β2m L chain to acid-treated C1498-MHC-I–deficient cells transfected with the indicated Ly49. Relative MFI indicates the fold increase in binding following acid treatment compared with no treatment (dotted line). Means ± SEM of two to three independent experiments are shown.

We previously tested the MHC-I specificity of the 129 strain repertoire of Ly49 molecules using tetramers containing human β2m (22). To determine whether some interactions were previously missed because of this factor, a simultaneous comparison was performed of the ability of human versus mouse β2m-containing H-2Kb and H-2Db tetramers to bind to MHC-I–deficient C1498 cells stably transfected with Ly49G129, Ly49I129, Ly49O129, and Ly49V129. Expression levels of the Ly49 molecules are shown with specific mAbs (Fig. 2A). Positive staining with human β2m-containing tetramers was only observed for Ly49Q (H-2Kb) and Ly49V (H-2Kb and H-2Db), as previously reported (22, 23). In contrast, a larger number of interactions were identified with tetramers containing mouse β2m: Ly49G (H-2Db), Ly49I (H-2Kb), Ly49O (H-2Db), Ly49Q (H-2Kb and H-2Db), and Ly49V (H-2Kb and H-2Db). Furthermore, the binding of MHC-I/mouse β2m tetramers to Ly49V, similar to Ly49Q, was greater than that seen with MHC-I/human β2m tetramers, and weak but reproducible binding of Ly49O:Kb and Ly49I:Db was observed. The binding ability of MHC-I tetramers containing either human or mouse β2m to Ly49-transfected MHC-I–deficient C1498 cells is summarized in Table I. These results confirm and extend prior reports that the presence of human β2m in mouse MHC-I molecules inhibits binding by Ly49. In particular, these results demonstrate that pDCs have the ability to bind to MHC-I alleles other than H-2Kb.

View this table:
Table I. Enhanced Ly49 binding with MHC-I tetramers containing mouse β2m

Cis interactions with H-2b and H-2k MHC-I molecules inhibit Ly49 trans binding to soluble MHC-I tetramers

Testing the MHC-I tetramer binding capacity of Ly49 expressed on an MHC-I–deficient cell line in Fig. 2 represented a “best case scenario,” as it has been previously reported that cis Ly49/MHC-I interactions inhibit trans MHC-I binding by Ly49AB6, Ly49CB6, and Ly49IB6 (26). To determine whether cis interactions can also inhibit MHC-I binding by Ly49 from 129 strain mice, the tetramer binding experiments shown in Fig. 2 were repeated in the MHC-I-sufficient MC57G fibrosarcoma cell line isolated from B6 mice, which expresses H-2Kb and H-2Db (data not shown). Overall, the binding of MHC-I tetramers to MC57G-expressing Ly49 was absent or very low compared with MHC-I–deficient C1498 cells (Fig. 3A). Tetramers containing human β2m only showed weak binding for Ly49Q (H-2Kb) and Ly49V (H-2Kb) (Fig. 3A, middle panel). In comparison, tetramers containing mouse β2m displayed only slightly stronger binding of the same Ly49 and, additionally, Ly49V binding of H-2Db could be detected (Fig. 3A, right panel). These results are summarized in Table II. This observed reduction in binding of the MHC-I tetramers in MC57G was confirmed to be due to Ly49/MHC-I cis interactions because acid treatment of the Ly49 transgenic MC57G cells resulted in increased binding (Fig. 3A, middle panel, 3B). Notably, major increases were seen for Ly49G (H-2Db), Ly49O (H-2Db), Ly49Q (H-2Db), and Ly49V (H-2Kb and H-2Db). Therefore, the ability of 129 strain Ly49 to bind MHC-I (containing either human or mouse β2m) in trans is inhibited by cis interactions with self–MHC-I.

FIGURE 3.
FIGURE 3.

MHC-I coexpression in an H-2b cell line drastically reduces soluble MHC-I tetramer binding to Ly49 molecules. (A) The MHC-I–sufficient MC57G cell line was stably transfected with the indicated Ly49 from 129 strain mice, treated with mild acid to disrupt class I MHC/peptide interactions, and then stained with specific PE- or FITC-conjugated mAb or PE-conjugated MHC-I tetramers (H-2Kb or H-2Db) containing human (middle panel) or mouse (right panel) β2m L chain. After mAb or tetramer staining, cells were analyzed by flow cytometry. MC57G parental staining levels are shown in black; specific staining of stable Ly49 transfectants before and after acid treatment are shown in red and blue, respectively. Average MFI fold increase in the non–acid-treated transfectants over the parentals is indicated. (B) Quantification of binding of MHC-I tetramers (H-2Kb or H-2Db) containing mouse β2m L chain to acid-treated MC57G transfected with the indicated Ly49. Relative MFI indicates the fold increase in binding following acid treatment compared with no treatment (dotted line). Means ± SEM of two to three independent experiments are shown.

View this table:
Table II. Endogenous MHC-I expression results in weaker binding of Ly49 to MHC-I tetramers

The inability of BWZ.Ly49Q reporter cells to be stimulated by H-2Db is suggested to be due to inhibition of binding via cis interactions with H-2Kk and/or H-2Dk (Fig. 1D). To test this hypothesis, the tetramer binding analysis was repeated with the C3H.He-derived (H-2k) L929 fibroblast cell line. As observed with MC57G, tetramers containing human β2m bound to Ly49Q (H-2Kb) and to Ly49V (H-2Kb and H-2Db), in agreement with BWZ reporter cell results (Fig. 4A, middle panel). Tetramers containing mouse β2m showed increased strength of these bindings plus the detectable binding of H-2Kb to Ly49I and a slight affinity in H-2Db for all Ly49 (Fig. 4A, right panel). However, similar to MC57G, stable Ly49 expression on L929 showed less affinity for soluble MHC-I compared with the same Ly49 expressed on the MHC-I–deficient C1498 cell line (Fig. 2). MHC-I tetramer binding to Ly49 expressed on L929 cells is summarized in Table II. Acid treatment of Ly49-expressing L929 resulted in increased binding of soluble tetramers to Ly49G (H-2Kb), Ly49I (H-2Db), Ly49O (H-2Kb and H-2Db), Ly49Q (H-2Kb and H-2Db), and Ly49V (H-2Kb and H-2Db) (Fig. 4A, right panel, 4B). These results suggest that H-2Kk and/or H-2Dk cis binding to Ly49Q inhibits H-2Db and reduces H-2Kb binding in trans. This cis-mediated inhibition is not Ly49Q-specific and is seen to affect trans binding of soluble MHC-I tetramers by other Ly49 as well.

FIGURE 4.
FIGURE 4.

MHC-I coexpression in an H-2k cell line also reduces soluble MHC-I tetramer binding to Ly49 molecules. (A) MHC-I–sufficient L929 cells were stably transfected with the indicated Ly49 from 129 strain mice, treated with mild acid to disrupt class I MHC/peptide interactions, and then stained with specific PE- or FITC-conjugated mAb or PE-conjugated MHC-I tetramers (H-2Kb or H-2Db) containing human (middle panel) or mouse (right panel) β2m L chain. After mAb or tetramer staining, cells were analyzed by flow cytometry. L929 parental staining levels are shown as a black histogram and specific staining of stable Ly49 transfectants before and after acid treatment are shown as red and blue histograms, respectively. Average MFI fold increase of non–acid-treated transfectants over parentals is indicated. In cases marked by an asterisk, fold increase was calculated either as the ratio of tetramer staining in the transfectants to the parentals or as the ratio of specific tetramer staining to control PE-conjugated streptavidin staining. (B) Quantification of binding of MHC-I tetramers (H-2Kb or H-2Db) containing mouse β2m L chain to acid-treated L929 transfected with the indicated Ly49. Relative MFI indicates the fold increase in binding following acid treatment compared with no treatment (dotted line). Means ± SEM of two to three independent experiments are shown.

Ly49 expressed on human 293T cells do not show optimal binding of soluble MHC-I tetramers due to cross-species cis interactions with human MHC-I

Many previous studies, including our own, have used easily transfectable primate cell lines such as human kidney epithelial 293T or green monkey COS7 cells to study Ly49/MHC-I binding (22, 26, 27). Because 293T cells lack mouse MHC-I, we predicted that soluble MHC-I tetramer binding to Ly49 expressed on these cells would be efficient, as there are no competing cis interactions, and that binding efficiency would be similar to that seen with MHC-I–deficient C1498 Ly49 stable transfectants. However, soluble MHC-I tetramer staining of human 293T cells was very similar to that observed with MHC-I–sufficient MC57G or L929 cell lines. Specifically, tetramers bound strongly to Ly49I (H-2Kb), Ly49Q (H-2Kb), and Ly49V (H-2Kb and H-2Db) (Fig. 5A). The binding of Ly49O and Ly49Q to H-2Db observed using C1498 cells was greatly reduced in transfected 293T cells. The comparison of MHC-I tetramer binding ability to Ly49 expressed by 293T versus C1498 cells is summarized in Table III.

FIGURE 5.
FIGURE 5.

Lack of mouse MHC-I coexpression in human cells still results in low MHC-I tetramer binding by Ly49. (A) The human 293T cell line was stably transfected with the indicated Ly49 from 129 strain mice, treated with mild acid to disrupt class I MHC/peptide interactions, and then stained with specific PE- or FITC-conjugated mAb or PE-conjugated MHC-I tetramers (H-2Kb or H-2Db) containing human (middle panel) or mouse (right panel) β2m L chain. After mAb or tetramer staining, cells were analyzed by flow cytometry. 293T parental staining levels are shown as black histograms, and specific staining of stable Ly49 transfectants before and after acid treatment are shown as red and blue histograms, respectively. Average MFI fold increase of non–acid-treated transfectants over parentals is indicated. In cases marked by an asterisk, fold increase was calculated either as the ratio of tetramer staining in the transfectants to the parentals or as the ratio of specific tetramer staining to control PE-conjugated streptavidin staining. (B) Quantification of binding of MHC-I tetramers (H-2Kb or H-2Db) containing mouse β2m L chain to acid-treated MC57G transfected with the indicated Ly49. Relative MFI indicates the fold increase in binding following acid treatment compared with no treatment (dotted line). Means ± SEM of two to three independent experiments are shown.

View this table:
Table III. Increased MHC-I tetramer binding by Ly49 expressed on mouse cell lines

The surprising nonoptimal binding of Ly49 expressed on human 293T cells with soluble MHC-I tetramers suggests that there may be interference preventing binding of the tetramer to the Ly49 receptor expressed on the surface. Although 293T cells do not express mouse MHC-I due to their origin, it was of interest to determine whether cross-species cis interactions between human MHC-I and mouse Ly49 could be the cause of this observed low binding of MHC-I tetramer. Acid treatment of the Ly49-transfected 293T cells resulted in notably increased tetramer binding for Ly49G (H-2Db), Ly49I (H-2Db), Ly49O (H-2Kb and H-2Db), Ly49Q (H-2Db), and Ly49V (H-2Kb) (Fig, 5A, right panel, 5B). These results strongly suggest that human MHC-I and mouse Ly49 are able to bind.

Discussion

We previously reported that Ly49Q binds to H-2Kb, but not H-2Db, in BWZ reporter cell experiments (23). In the present study, we show that Ly49Q does in fact bind to H-2Db and that the likely reason for the lack of interaction in the BWZ assay was the expression of H-2Kk and/or H-2Dk on the BWZ cells themselves causing cis interactions with the chimeric Ly49Q leading to competition for trans binding to H-2Db. In BWZ reporter cell and tetramer assays H-2Kb was able to overcome cis interactions, but H-2Db was not, suggesting it has a weaker affinity than H-2Kb for Ly49Q. Furthermore, tetramer binding analyses showed H-2Db binding to Ly49Q only when the tetramers were refolded with mouse β2m L chain; a lack of competing cis interactions alone was not enough to allow H-2Db/human β2m to bind to Ly49Q. Because cis competition and species origin of the L chain were not taken into account when the 129 strain Ly49/MHC-I binding characteristics were first studied (22), tetramer analyses were repeated in the present study using stable transfectants of MHC-I–deficient and MHC-I–sufficient mouse and human cell lines and novel interactions were uncovered.

Specifically, we found that in addition to Ly49Q, both Ly49G and Ly49O bound to H-2Db/mouse β2m tetramers and that Ly49I was able to bind H-2Kb/mouse β2m tetramers. Most or all of these interactions were lost when cis MHC-I interactions from either H-2b or H-2k haplotypes were introduced via Ly49 expression in MC57G or L929 cell lines, respectively, or when MHC-I tetramers containing human β2m were used to stain Ly49 transfectants. Our present observations are similar to previous studies showing Ly49A binds to H-2Db/mouse β2m tetramers (28), but no interaction of Ly49A was observed with H-2Db/human β2m tetramers (27). In line with these observations, Ly49D binding to H-2Dd is only observed when the tetramer contains mouse L chain (5).

We have reported that NK cells expressing Ly49I129 and Ly49O/V129 on an H-2b background show evidence of licensing or self–MHC-I education in that they make more IFN-γ than the corresponding negative subset upon activation via NKG2D or Nkp46 stimulatory receptors (29). Our present study confirms the possibility of a licensing effect of H-2b for Ly49I129 and Ly49O/V129, as H-2Kb or H-2Db tetramers bound to these Ly49 under optimal conditions. We were unable to detect any difference in IFN-γ production upon stimulation of Ly49G129-positive versus -negative NK cells, although we show in the present study that Ly49G129 can bind to H-2Db. It is possible that the lack of licensing of Ly49G129 seen previously is due to H-2Db being a weak self-educator as shown in splenocyte rejection assays using H-2Kb−/−, H-2Db−/−, or H-2Kb−/−H-2Db−/− mice as donors (14, 29). Furthermore, this hypothesis agrees with the observations that Ly49A can bind to H-2Db when the L chain is of mouse origin (24), but that Ly49A+ NK cell subsets in H-2b background mice produce little if any IFN-γ upon stimulation, compared with Ly49A+ subsets in H-2d background mice (30); H-2Dd is a strong ligand for Ly49A (31).

Additionally, when human β2m- and mouse β2m-containing tetramers both showed binding to the same Ly49, the mouse β2m-containing tetramers showed higher binding as measured by increased MFI. In no case was it observed that tetramers made with human β2m L chain showed a specificity that was not also observed with MHC-I/mouse β2m tetramers. The β2m sequence is 70% homologous between humans and mice and therefore relatively conserved through evolution. The peptide, H chain, and L chain are held together solely by noncovalent bonds, and for this reason MHC-I tetramers are unstable. However, tetramers produced with human β2m possess enhanced stability compared with those made with mouse β2m. Mouse MHC-I/human β2m tetramers are used for T cell clonal expansion assays, as the L chain does not significantly affect TCR binding to the MHC-I groove/peptide complex.

The soluble tetramer assay, although greatly improved in the present study, is still prone to false-negative results for the following reasons. First, non–mouse peptides were used to produce the tetramers used in the present study. However, it has been long known that peptide sequence greatly affects Ly49 affinity for MHC-I (27, 28). Chicken OVA peptide (H-2Kb) and lymphocytic choriomeningitis virus gp33 peptide (H-2Db) were chosen for the present study for the sake of comparison with previous studies and for their ability to stabilize tetramers. Ly49 mediate their function in mice by binding MHC-I molecules presenting endogenous peptides. Second, tetramer H chains are produced in bacteria and so will lack posttranslational glycosylation and perhaps other modifications. Glycosylation of MHC-I, specifically on the α2 domain of H-2Dd, is necessary for optimal binding by Ly49A and Ly49C (4). Receptor downmodulation on NK cells from mice expressing single MHC-Ia molecules, as previously reported (32), may still be the best way to determine Ly49/MHC-I specificity for the above reasons. This assay shows that Ly49A surface expression on NK cells from H-2Db single MHC-I transgenic mice is significantly lower than NK cells from B2m−/− mice (32), in agreement with mouse β2m tetramer studies (24, 28). However, it is possible that binding affinity and surface downmodulation do not always go hand in hand. For example, Ly49C was clearly downmodulated by the presence of H-2Kb, but Ly49I was not despite reports of Ly49I interacting with soluble H-2Kb tetramers containing either mouse or human β2m (26, 27, 32).

The data presented in the present study suggest that the lesser MHC-I tetramer binding of Ly49 transiently transfected into 293T cells in our 2001 study was due in large part to binding of mouse Ly49 to human MHC-I molecules in cis. Ly49-expressing 293T cells stained with mouse β2m-containing tetramers still showed a low level of tetramer specificity, especially as compared with C1498-MHC-I–deficient cells expressing the same Ly49. This was unexpected because 293T cells are human-derived and so cis interactions, which can inhibit tetramer binding, were not thought to take place. Following acid treatment, however, MHC-I tetramer binding to Ly49-transfected 293T cells notably increased, suggesting possible cross-species cis interaction between human MHC-I and mouse Ly49 on 293T cells, thus leading to the observed low MHC-I tetramer binding. Other groups have also shown the ability of receptor/ligand interactions across different species, particularly between humans and mice. The MHC-I–related neonatal Fc receptor, FcRn, whose role is to regulate the serum half-life of IgG and albumin, exhibits cross-reactive species binding based on ELISA and surface plasmon resonance results. Mouse FcRn is able to bind human IgG1 and human serum albumin, as well as IgG from other species, including guinea pig, rat, bovine, and sheep (33, 34). Furthermore, species cross-reactivity has been reported for human and mouse ligand/receptor interactions from the TNF superfamily based on flow cytometry analysis (35).

Interestingly, the greatest increases in binding after acid treatment were observed with H-2Db tetramer binding for human 293T cells (Fig. 5B) as well as the mouse-derived cells MC57G (Fig. 3B) and L929 (Fig. 4C). The low H-2Db tetramer binding prior to acid treatment suggests that cis interactions on the cell surface were a greater hindrance toward H-2Db tetramer binding than to that of H-2Kb. This may be due to the previously reported ability of H-2Db to bind a greater number of 129-derived Ly49 receptors, specifically Ly49V, G2, and O, than that of H-2Kb to only Ly49I (22). The diversified binding capacity of H-2Db may lead to H-2Db being more likely to be involved in cis interactions with the various Ly49 receptors transfected in the respective human and mouse cell lines.

In summary, the data presented in the present study suggest caution in the use of non-mouse cell lines for the identification of ligands for mouse Ly49, as they may lead to false-negative results. Furthermore, we show that the affinity for different MHC-I molecules by 129-derived Ly49 is greater than previously appreciated owing to cis interactions with native MHC-I. Finally, we confirm that MHC-I tetramers show the greatest affinity for 129-Ly49 when they have been produced with mouse-derived L chain.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Drs. Lionel Filion and Gina Graziani (University of Ottawa) for helpful discussions.

Footnotes

  • This work was supported by Canadian Institutes of Health Research Operating Grant 62841 (to A.P.M.). A.P.M. holds a Canada Research Chair in Innate Pathogen Resistance.

  • Abbreviations used in this article:

    DC
    dendritic cell
    KIR
    killer cell Ig-like receptor
    β2m
    β2-microglobulin
    MFI
    mean fluorescence intensity
    MHC-I
    MHC class I
    pDC
    plasmacytoid dendritic cell.

  • Received March 25, 2013.
  • Accepted September 29, 2013.

References

  1. ↵
    1. Orange J. S.
    2006. Human natural killer cell deficiencies. Curr. Opin. Allergy Clin. Immunol. 6: 399–409.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Lanier L. L.
    2005. NK cell recognition. Annu. Rev. Immunol. 23: 225–274.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Raulet D. H.,
    2. R. E. Vance,
    3. C. W. McMahon
    . 2001. Regulation of the natural killer cell receptor repertoire. Annu. Rev. Immunol. 19: 291–330.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Lian R. H.,
    2. J. D. Freeman,
    3. D. L. Mager,
    4. F. Takei
    . 1998. Role of conserved glycosylation site unique to murine class I MHC in recognition by Ly-49 NK cell receptor. J. Immunol. 161: 2301–2306.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Mason L. H.,
    2. J. Willette-Brown,
    3. S. K. Anderson,
    4. W. G. Alvord,
    5. R. L. Klabansky,
    6. H. A. Young,
    7. J. R. Ortaldo
    . 2003. Receptor glycosylation regulates Ly-49 binding to MHC class I. J. Immunol. 171: 4235–4242.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Toyama-Sorimachi N.,
    2. Y. Tsujimura,
    3. M. Maruya,
    4. A. Onoda,
    5. T. Kubota,
    6. S. Koyasu,
    7. K. Inaba,
    8. H. Karasuyama
    . 2004. Ly49Q, a member of the Ly49 family that is selectively expressed on myeloid lineage cells and involved in regulation of cytoskeletal architecture. Proc. Natl. Acad. Sci. USA 101: 1016–1021.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Gays F.,
    2. J. G. Aust,
    3. D. M. Reid,
    4. J. Falconer,
    5. N. Toyama-Sorimachi,
    6. P. R. Taylor,
    7. C. G. Brooks
    . 2006. Ly49B is expressed on multiple subpopulations of myeloid cells. J. Immunol. 177: 5840–5851.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Makrigiannis A. P.,
    2. S. K. Anderson
    . 2000. Ly49 gene expression in different inbred mouse strains. Immunol. Res. 21: 39–47.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Anderson S. K.,
    2. K. Dewar,
    3. M. L. Goulet,
    4. G. Leveque,
    5. A. P. Makrigiannis
    . 2005. Complete elucidation of a minimal class I MHC natural killer cell receptor haplotype. Genes Immun. 6: 481–492.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Belanger S.,
    2. L. H. Tai,
    3. S. K. Anderson,
    4. A. P. Makrigiannis
    . 2008. Ly49 cluster sequence analysis in a mouse model of diabetes: an expanded repertoire of activating receptors in the NOD genome. Genes Immun. 9: 509–521.
    OpenUrlCrossRefPubMed
  11. ↵
    1. McVicar D. W.,
    2. D. N. Burshtyn
    . 2001. Intracellular signaling by the killer immunoglobulin-like receptors and Ly49. Sci. STKE. 2001: re1.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Fernandez N. C.,
    2. E. Treiner,
    3. R. E. Vance,
    4. A. M. Jamieson,
    5. S. Lemieux,
    6. D. H. Raulet
    . 2005. A subset of natural killer cells achieves self-tolerance without expressing inhibitory receptors specific for self-MHC molecules. Blood 105: 4416–4423.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Kim S.,
    2. J. Poursine-Laurent,
    3. S. M. Truscott,
    4. L. Lybarger,
    5. Y. J. Song,
    6. L. Yang,
    7. A. R. French,
    8. J. B. Sunwoo,
    9. S. Lemieux,
    10. T. H. Hansen,
    11. W. M. Yokoyama
    . 2005. Licensing of natural killer cells by host major histocompatibility complex class I molecules. Nature 436: 709–713.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Bélanger S.,
    2. M. M. Tu,
    3. M. M. Rahim,
    4. A. B. Mahmoud,
    5. R. Patel,
    6. L. H. Tai,
    7. A. D. Troke,
    8. B. T. Wilhelm,
    9. J. R. Landry,
    10. Q. Zhu,
    11. et al
    . 2012. Impaired natural killer cell self-education and “missing-self” responses in Ly49-deficient mice. Blood 120: 592–602.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Tormo J.,
    2. K. Natarajan,
    3. D. H. Margulies,
    4. R. A. Mariuzza
    . 1999. Crystal structure of a lectin-like natural killer cell receptor bound to its MHC class I ligand. Nature 402: 623–631.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Matsumoto N.,
    2. M. Mitsuki,
    3. K. Tajima,
    4. W. M. Yokoyama,
    5. K. Yamamoto
    . 2001. The functional binding site for the C-type lectin-like natural killer cell receptor Ly49A spans three domains of its major histocompatibility complex class I ligand. J. Exp. Med. 193: 147–158.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Doucey M. A.,
    2. L. Scarpellino,
    3. J. Zimmer,
    4. P. Guillaume,
    5. I. F. Luescher,
    6. C. Bron,
    7. W. Held
    . 2004. Cis association of Ly49A with MHC class I restricts natural killer cell inhibition. Nat. Immunol. 5: 328–336.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Back J.,
    2. E. L. Malchiodi,
    3. S. Cho,
    4. L. Scarpellino,
    5. P. Schneider,
    6. M. C. Kerzic,
    7. R. A. Mariuzza,
    8. W. Held
    . 2009. Distinct conformations of Ly49 natural killer cell receptors mediate MHC class I recognition in trans and cis. Immunity 31: 598–608.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Back J.,
    2. A. Chalifour,
    3. L. Scarpellino,
    4. W. Held
    . 2007. Stable masking by H-2Dd cis ligand limits Ly49A relocalization to the site of NK cell/target cell contact. Proc. Natl. Acad. Sci. USA 104: 3978–3983.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Chalifour A.,
    2. L. Scarpellino,
    3. J. Back,
    4. P. Brodin,
    5. E. Devèvre,
    6. F. Gros,
    7. F. Lévy,
    8. G. Leclercq,
    9. P. Höglund,
    10. F. Beermann,
    11. W. Held
    . 2009. A role for cis interaction between the inhibitory Ly49A receptor and MHC class I for natural killer cell education. Immunity 30: 337–347.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Makrigiannis A. P.,
    2. D. Patel,
    3. M. L. Goulet,
    4. K. Dewar,
    5. S. K. Anderson
    . 2005. Direct sequence comparison of two divergent class I MHC natural killer cell receptor haplotypes. Genes Immun. 6: 71–83.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Makrigiannis A. P.,
    2. A. T. Pau,
    3. A. Saleh,
    4. R. Winkler-Pickett,
    5. J. R. Ortaldo,
    6. S. K. Anderson
    . 2001. Class I MHC-binding characteristics of the 129/J Ly49 repertoire. J. Immunol. 166: 5034–5043.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Tai L. H.,
    2. M. L. Goulet,
    3. S. Belanger,
    4. A. D. Troke,
    5. A. G. St-Laurent,
    6. A. Mesci,
    7. N. Toyama-Sorimachi,
    8. J. R. Carlyle,
    9. A. P. Makrigiannis
    . 2007. Recognition of H-2Kb by Ly49Q suggests a role for class Ia MHC regulation of plasmacytoid dendritic cell function. Mol. Immunol. 44: 2638–2646.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Michaëlsson J.,
    2. A. Achour,
    3. A. Rölle,
    4. K. Kärre
    . 2001. MHC class I recognition by NK receptors in the Ly49 family is strongly influenced by the β2-microglobulin subunit. J. Immunol. 166: 7327–7334.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Tai L. H.,
    2. M. L. Goulet,
    3. S. Belanger,
    4. N. Toyama-Sorimachi,
    5. N. Fodil-Cornu,
    6. S. M. Vidal,
    7. A. D. Troke,
    8. D. W. McVicar,
    9. A. P. Makrigiannis
    . 2008. Positive regulation of plasmacytoid dendritic cell function via Ly49Q recognition of class I MHC. J. Exp. Med. 205: 3187–3199.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Scarpellino L.,
    2. F. Oeschger,
    3. P. Guillaume,
    4. J. D. Coudert,
    5. F. Lévy,
    6. G. Leclercq,
    7. W. Held
    . 2007. Interactions of Ly49 family receptors with MHC class I ligands in trans and cis. J. Immunol. 178: 1277–1284.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Hanke T.,
    2. H. Takizawa,
    3. C. W. McMahon,
    4. D. H. Busch,
    5. E. G. Pamer,
    6. J. D. Miller,
    7. J. D. Altman,
    8. Y. Liu,
    9. D. Cado,
    10. F. A. Lemonnier,
    11. et al
    . 1999. Direct assessment of MHC class I binding by seven Ly49 inhibitory NK cell receptors. Immunity 11: 67–77.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Michaëlsson J.,
    2. A. Achour,
    3. M. Salcedo,
    4. A. Kâse-Sjöström,
    5. J. Sundbäck,
    6. R. A. Harris,
    7. K. Kärre
    . 2000. Visualization of inhibitory Ly49 receptor specificity with soluble major histocompatibility complex class I tetramers. Eur. J. Immunol. 30: 300–307.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Patel R.,
    2. S. Bélanger,
    3. L. H. Tai,
    4. A. D. Troke,
    5. A. P. Makrigiannis
    . 2010. Effect of Ly49 haplotype variance on NK cell function and education. J. Immunol. 185: 4783–4792.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Jonsson A. H.,
    2. L. Yang,
    3. S. Kim,
    4. S. M. Taffner,
    5. W. M. Yokoyama
    . 2010. Effects of MHC class I alleles on licensing of Ly49A+ NK cells. J. Immunol. 184: 3424–3432.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Karlhofer F. M.,
    2. R. K. Ribaudo,
    3. W. M. Yokoyama
    . 1992. MHC class I alloantigen specificity of Ly-49+ IL-2-activated natural killer cells. Nature 358: 66–70.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Johansson S.,
    2. M. Johansson,
    3. E. Rosmaraki,
    4. G. Vahlne,
    5. R. Mehr,
    6. M. Salmon-Divon,
    7. F. Lemonnier,
    8. K. Kärre,
    9. P. Höglund
    . 2005. Natural killer cell education in mice with single or multiple major histocompatibility complex class I molecules. J. Exp. Med. 201: 1145–1155.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Ober R. J.,
    2. C. G. Radu,
    3. V. Ghetie,
    4. E. S. Ward
    . 2001. Differences in promiscuity for antibody-FcRn interactions across species: implications for therapeutic antibodies. Int. Immunol. 13: 1551–1559.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Andersen J. T.,
    2. M. B. Daba,
    3. G. Berntzen,
    4. T. E. Michaelsen,
    5. I. Sandlie
    . 2010. Cross-species binding analyses of mouse and human neonatal Fc receptor show dramatic differences in immunoglobulin G and albumin binding. J. Biol. Chem. 285: 4826–4836.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    1. Bossen C.,
    2. K. Ingold,
    3. A. Tardivel,
    4. J. L. Bodmer,
    5. O. Gaide,
    6. S. Hertig,
    7. C. Ambrose,
    8. J. Tschopp,
    9. P. Schneider
    . 2006. Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human. J. Biol. Chem. 281: 13964–13971.
    OpenUrlAbstract/FREE Full Text
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