Visualization of inhibitory Ly49 receptor specificity with soluble major histocompatibility complex class I tetramers

Murine natural killer (NK) cells are inhibited from killing their targets by the interaction between inhibitory, C-type lectin like Ly49 receptors and major histocompatibility complex (MHC) class I molecules. The receptors have overlapping specificity, and it has been difficult to analyze specific aspects of the interaction between different Ly49 receptors and their respective ligands. We have addressed this problem using tetramers of bacterially expressed, non-glycosylated, MHC class I molecules refolded with different peptides. Our results indicate that this technology is useful for analysis of Ly49 receptor specificity as well as for monitoring of NK cell subsets, with the following major conclusions emerging from this study: (1) tetramers of H-2D(d) bound the Ly49A receptor; the MHC associated glycan, previously suggested to be involved in recognition by this receptor, is thus not required for Ly49A receptor binding; (2) in support and extension of a recent report indicating peptide selectivity in the recognition of H-2K(b) by Ly49C(+) cells, H-2K(b) tetramer binding to Ly49C receptors was strongly influenced by the peptide presented by the MHC class I molecule; (3) tetramer binding allowed visualization of interactions that have not previously been detected in functional studies, such as the recognition of H-2D(b) by Ly49A and Ly49C.     

H-2 allele specificity of the NK cell C-type lectin-like MHC class I receptor Ly49A visualized by soluble Ly49A tetramer

Ly49A is a C-type lectin-like receptor on NK cells that recognizes MHC class I ligands, H-2D(d) and D(k). The engagement of Ly49A with the ligands inhibits activation of NK cells and protects target cells from lysis by NK cells. Here we express the extracellular region of Ly49A with an N-terminal biotinylation tag in Escherichia coli to obtain soluble Ly49A (sLy49A) after refolding. sLy49A is indistinguishable from native Ly49A expressed on NK cells serologically and in the ability to specifically bind H-2D(d) after tetramerization with R-phycoerythrin-coupled streptavidin. The fluorescently labeled tetramer of sLy49A is applied to explore MHC class I haplotype specificity of Ly49A. We demonstrate the hierarchical reactivity of Ly49A with H-2 of various alleles in the order of d > k, r > p > v > q > s > z. Reactivity of sLy49A tetramer to spleen lymphocytes from B10.QBR mice (H-2K(b), I(b), D(q), Qa-1/Tla(b)) but not from C57BL/10 mice (H-2(b)) identifies H-2D(q) and L(q) as candidates for a Ly49A ligand. Binding of sLy49A tetramer to H-2D(q)- or L(q)-transfected cell lines demonstrates that the two highly related MHC class I molecules, H-2D(q) and L(q), are ligands for Ly49A. sLy49A tetramer staining also demonstrates preferential expression of Ly49A ligand on a subset of B cells in P/J mice. These results provide the basis to examine the molecular mechanism by which Ly49A discriminates polymorphic MHC class I molecules.     

Purified MHC class I molecules inhibit activated NK cells in a cell-free system in vitro

Natural killer cells have been shown to interact with MHC class I molecules via inhibitory receptors. However, it is not known whether the inhibition induced by MHC class I molecules requires other NK cell-target cell interactions. Thus, we examined whether purified MHC class I molecules alone were able to inhibit NK cell function. Purified H-2K(b) and H-2D(b) molecules inhibited the release of IFN-gamma from spleen (H-2(b))-derived lymphokine-activated killer (LAK) cell cultures stimulated by anti-NK1.1 antibody in a concentration-dependent manner. LAK cells generated from newborn mice that express low levels of MHC class I binding Ly49 inhibitory receptors were significantly less sensitive to inhibition by H-2K(b) compared to LAK cells from adult mice. Furthermore, LAK cells generated from spleen cells of Ly49C-transgenic mice were significantly more sensitive to inhibition by H-2K(b) compared to non-transgenic littermates. Taken together, the data indicate that MHC class I induced inhibition of NK cell mediated effector functions, as assessed by IFN-gamma release after NK1.1 triggering, does not require additional cell surface molecules other than MHC class I.     

Thymus-dependent modulation of Ly49 inhibitory receptor expression on NK1.1+gamma/delta T cells

Major histocompatibility complex (MHC) class I-specific inhibitory receptors are expressed not only on natural killer (NK) cells but also on some subsets of T cells. We here show Ly49 expression on gamma/delta T cells in the thymus and liver of beta2-microglobulin-deficient (beta2m-/-) and C57BL/6 (beta2m+/+) mice. Ly49C/I or Ly49A receptor was expressed on NK1.1+gamma/delta T cells but not on NK1.1-gamma/delta T cells. The numbers of NK1.1+gamma/delta T cells were significantly smaller in beta2m+/+ mice than in beta2m-/- mice with the same H-2b genetic background. Among NK1.1+gamma/delta T cells, the proportions of Ly49C/I+ cells but not of Ly49A+ cells, were decreased in beta2m+/+ mice, suggesting that cognate interaction between Ly49C/I and H-2Kb is involved in the reduction of the number of Ly49C/I+ gamma/delta T cells in beta2m+/+ mice. The frequency of Ly49C/I+ cells in NK1.1+gamma/delta T cells was lower in both lethally irradiated beta2m+/+ mice transplanted with bone marrow (BM) from beta2m-/- mice and lethally irradiated beta2m-/- mice transplanted with BM from beta2m+/+ mice than those in adult thymectomized BM-transplanted chimera mice. These results suggest that reduction of Ly49C/I+ NK1.1+gamma/delta T cells in beta2m+/+ mice is at least partly due to the down-modulation by MHC class I molecules on BM-derived haematopoietic cells or radioresistant cells in the thymus.     

MHC class I molecules on adenovirus E1A-expressing tumor cells inhibit NK cell killing but not NK cell-mediated tumor rejection

Expression of adenovirus E1A gene products in tumor cells enhances NK cell lysis in vitro and NK-mediated rejection in vivo, despite increasing class I molecules on tumor cells. It is unclear why the increased expression of MHC class I molecules does not appear to confer resistance to killing by NK cells. One possibility is the unique capacity of E1A to sensitize cells to multiple NK cell killing mechanisms including perforin/granzyme, Fas ligand, tumor necrosis factor-alpha and TRAIL. To examine this issue, MCA-102-E1A tumor cells (H-2(b)) that express E1A and are NK sensitive were transfected with H-2D(d), the ligand for the NK inhibitory receptor, Ly49A. Expression of H-2D(d) molecules by MCA-102-E1A cells protected them from lysis by a Ly49A(+) NK cell clone and Ly49A(+) NK cells isolated from C57BL/6 nude mice. In contrast, NK cell-mediated rejection of MCA-102-E1A tumor cells was not inhibited by the expression of H-2D(d) molecules, nor was killing by polyclonal populations of NK cells isolated from C57BL/6-nude mice. H-2D(d) interacts with several inhibitory Ly49 receptors that are non-clonally expressed on NK cells in C57BL/6 mice: Ly49A (20% of NK cells), Ly49G2 (54% of NK cells) and Ly49C/I (47% of NK cells). Our data indicate that while E1A sensitizes cells to NK cell killing, it does not interfere with signal transduction by inhibitory NK receptors. Therefore, a small population of NK cells that do not express Ly49A, Ly49G2 or Ly49C/I inhibitory receptors are likely responsible for the rejection of MCA-102-E1A-D(d) tumor cells in vivo.     

A species-specific determinant on beta2-microglobulin required for Ly49A recognition of its MHC class I ligand

The mouse inhibitory NK cell receptor Ly49A recognizes the mouse MHC class I molecule H-2D(k). The present study focuses on the species specificity of beta(2)-microglobulin (beta(2)m), an invariant component of MHC class I, in the interaction between Ly49A and H-2D(k). Transfection of the beta(2)m-defective mouse cell line R1E/TL8x.1 with human (h) beta(2)m induced cell-surface expression of H-2D(k), but failed to protect the cells from killing by Ly49A(+) NK cells. In contrast, the cells transfected with mouse (m) beta(2)m were protected from killing by Ly49A(+) NK cells. These data indicate that Ly49A distinguishes mbeta(2)m from hbeta(2)m when it recognizes the H-2D(k) complexes. To identify the species-specific determinant of beta(2)m required for Ly49A recognition of H-2D(k), we prepared a panel of mbeta(2)m mutants and tested the H-2D(k) that included each of the beta(2)m mutants for its capacity to engage Ly49A on NK cells. Ly49A failed to functionally recognize the H-2D(k) that included the mbeta(2)m with K3R and Q29G mutations. Moreover, Ly49A was able to recognize the H-2D(k) that included the hbeta(2)m with R3K and G29Q mutations. These data indicate that Lys3 and Gln29 consist of the central part of the species-specific determinant of beta(2)m required for Ly49A recognition of H-2D(k). The two residues are conserved in the mouse and the rat, in which NK cells use Ly49 family molecules as the receptors specific for MHC class I. These results suggest functional importance of beta(2)m in NK cell recognition of target cells.     

Recognition of H-2K(b) by Ly49Q suggests a role for class Ia MHC regulation of plasmacytoid dendritic cell function

Ly49Q is a member of the polymorphic Ly49 family of NK cell receptors that displays both a high degree of conservation and a unique expression pattern restricted to myeloid lineage cells, including plasmacytoid dendritic cells (pDC). The function and ligand specificity of Ly49Q are unknown. Here, we use reporter cell analysis to demonstrate that a high-affinity ligand for Ly49Q is present on H-2(b), but not H-2(d), H-2(k), H-2(q), or H-2(a)-derived tumor cells and normal cells ex vivo. The ligand is peptide-dependent and MHC Ia-like, as revealed by its functional absence on cells deficient in TAP-1, beta(2)m, or H-2K(b)D(b) expression. Furthermore, Ly49Q is specific for H-2K(b), as the receptor binds peptide-loaded H-2K(b) but not H-2D(b) complexes, and Ly49Q recognition can be blocked using anti-K(b) but not anti-D(b) mAb. Greater soluble H-2K(b) binding to ligand-deficient pDC also suggests cis interactions of Ly49Q and H-2K(b). These results demonstrate that Ly49Q efficiently binds H-2K(b) ligand, and suggest that pDC function, like that of NK cells, is regulated by classical MHC Ia molecules. MHC recognition capability by pDC has important implications for the role of this cell type during innate immune responses.     

The Qa-1b molecule binds to a large subpopulation of murine NK cells

Recent studies on human NK cells have demonstrated that the NK cell CD94/NKG2 receptors bind to the nonclassical MHC class I molecule HLA-E. A functional CD94/NKG2 complex has not yet been identified in rodents, but cDNA encoding rat and mouse CD94 and NKG2 have recently been cloned, suggesting that CD94/NKG2 receptors may exist in species other than man. The mouse nonclassical MHC class I molecule Qa-1 shares several features with HLA-E. This suggests that Qa-1 may be similarly recognized by murine NK cells. To study the ability of Qa-1 to bind to murine NK cells, we have produced a soluble tetrameric form of Qa-1^b . In the present study, we demonstrate that Qa-1^b tetramers distinctly bind to a large subset of fresh or IL-2-activated NK1.1⁺ /CD3⁻ splenocytes independently of the expression of Ly49 inhibitory receptors. Binding occurs whether NK cells have evolved in an MHC class I-expressing or in an MHC class I-deficient environment. Our data suggest the existence of a Qa-1-recognizing structure on a large subpopulation of murine NK cells that may be similar to the human CD94/NKG2 heterodimeric complex.     

A simplified procedure for the preparation of MHC/peptide tetramers: chemical biotinylation of an unpaired cysteine engineered at the C-terminus of MHC-I

Recently, a powerful approach for the detection of MHC/peptide-specific T cells has been made possible by the engineering of soluble-tetrameric MHC/peptide complexes, consisting of singly biotinylated MHC/peptide molecules bound to fluorescent-labeled streptavidin. These tetrameric molecules are thought to compensate for the low affinity and relative fast dissociation rate of the TCR/MHC-peptide interaction by increasing the avidity of this interaction, thus allowing the stable binding of MHC/peptide tetramers to TCR expressing cells. Here we describe a new more simplified procedure for obtaining MHC/peptide tetramers using the well-characterized H-2K(b)/VSV system. This procedure consists of the incorporation of an unpaired cysteine residue at the C-terminus of the H-2K(b) molecule, allowing site-specific biotinylation by a -SH-specific biotinylating reagent. The H-2K(b)/VSV tetramers bound only to hybridomas expressing H-2K(b)/VSV-specific TCRs. When coated on a plate, these tetramers were able to induce IL-2 release by those hybridomas. Furthermore, H-2K(b)/VSV tetramers bound to CTL populations obtained from mice immunized with VSV-peptide. The specificity of the binding was further refined by studying cross-recognition of VSV by CTL populations obtained from mice immunized with single amino acid substituted VSV peptide variants. H-2K(b)/VSV tetramers bound only to those CTL populations that cross-reacted with the wild-type VSV peptide. Our method provides a simple, efficient and inexpensive procedure for making MHC/peptide tetramers, a highly specific and very useful reagent with a number of important applications in basic and clinical T cell research.     

Ly49A expression on T cells alters T cell selection

Ly49 receptors are inhibitory receptors expressed on subsets of both NK cells and NK1.1(+) T cells. The function of these receptors on NK cells is believed to be important in maintaining self-tolerance, yet their role on T cells is unclear. In this report we investigated how an Ly49A transgene alters T and NK cell development in an in vivo environment, where a ligand for Ly49A is expressed. Ly49A transgenic mice that co-expressed an MHC ligand for Ly49A, H-2D(d), developed a severe inflammatory disorder that resulted in death within the first weeks of age. T cells expressing forbidden TCR V(beta) chains were found both in the thymus and periphery of transgenic mice, while non-transgenic littermates had successfully deleted these T cell subsets. These data indicate that the expression of Ly49A on T cells could alter T cell selection and allow survival of potentially self-reactive T cells.     

Quantifying the reduction in accessibility of the inhibitory NK cell receptor Ly49A caused by binding MHC class I proteins in cis

Murine natural killer (NK) cells are inhibited by target cell MHC class I molecules via Ly49 receptors. However, Ly49 receptors can be made inaccessible to target cell MHC class I by a cis interaction with its MHC class I ligand within the NK cell membrane. It has recently been demonstrated that MHC class I proteins transfer from the target cells to the NK cell. Here, we establish that the number of transferred MHC class I proteins is proportional to the number of Ly49A receptors at the NK cell surface. Ly49A+ NK cells from mice expressing the Ly49A ligand H-2D(d) showed a 90% reduction in Ly49A accessibility compared to Ly49A+ NK cells from H-2D(d)-negative mice. The reduction was caused both by lower expression of Ly49A and interactions in cis between Ly49A and H-2D(d) at the NK cell surface. Approximately 75% of the Ly49A receptors on H-2D(d)-expressing NK cells were occupied in cis with endogenous H-2D(d) and only 25% were free to interact with H-2D(d) molecules in trans. Thus, H-2D(d) ligands control Ly49A receptor accessibility through interactions both in cis and in trans.     

Natural killing of MHC class I(-) lymphoblasts by NK cells from long-term bone marrow culture requires effector cell expression of Ly49 receptors.

NK cells from long-term bone marrow culture (LTBMC) were compared with IL-2-activated splenic NK cells [short-term spleen cell culture (STSC)] with regard to expression of inhibitory Ly49 receptors and cytotoxic function. In the LTBMC, the total number of NK cells expressing either one of the Ly49 molecules A, C/I and G2 was strongly reduced (10-15% of NK1.1(+) cells) compared to the STSC (80-90% of NK1.1(+) cells). With regard to cytotoxic function, we confirmed that LTBMC-derived NK cells efficiently killed the prototype NK target YAC-1. However, against other targets, killing was more variable. First, while STSC-derived NK cells clearly distinguished MHC class I(-) from MHC class I(+) tumor cell targets, LTBMC-derived NK cells did not; they either killed both targets equally well or not at all. Secondly, LTBMC-derived NK cells were largely incapable of killing lymphoblast targets deficient in MHC class I expression. To test whether this cytotoxic defect was due to the low number of Ly49(+) NK cells in the LTBMC, we separated Ly49(+) and Ly49(-) NK cells by cell sorting and tested them individually. This experiment showed that only Ly49(+) NK cells in the LTBMC were able to kill MHC class I(-) lymphoblasts (and to distinguish them from MHC class I(+)), despite good cytotoxicity against YAC-1 cells in both populations. These data suggest that certain modes of NK cell triggering are dependent on Ly49 receptor expression. From our results, we speculate that inhibitory receptors are expressed before triggering receptors for normal self cells during NK cell development, which may be an important mechanism to preserve self tolerance during the early stages of NK cell maturation.     

Role of interaction between Ly49 inhibitory receptors and cognate MHC I molecules in IL2-induced development of NK cells in murine bone marrow cell cultures

Murine bone marrow (BM) cell preparations lack mature cytotoxic natural killer (NK) cells, but NK cells may be induced in these cell preparations by culturing with interleukin-2 (IL2). Present study was aimed at studying the role of interactions between Ly49 molecules and major histocompatibility complex (MHC) class I molecules during IL2-induced development of mature NK cells in BM cell cultures. Addition of monoclonal antibodies (mabs) specific to class I MHC molecules of H-2b haplotype, to block any interaction of MHC I molecules with their receptors, was found to inhibit NK cell development. Mouse NK cells express several types of Ly49 molecules including Ly49C, which is an inhibitory receptor specific to MHC I molecules of H-2b haplotype. Blocking Ly49-MHC I interaction by using anti-Ly49C mab inhibited the development of cytotoxic NK cells. Addition of anti-Ly49A (no specificity for H-2b MHC I molecules) or anti-Ly49D (activating receptor specific for MHC I molecules of many H-2 haplotypes including H-2b) mabs, however, had no effect on IL2-induced NK cell development in BM cells. Mabs specific to Ly49C molecule and MHC I molecules of H-2b haplotype inhibited the development of mature NK cells from highly purified NK precursor cell population. These results indicate that specific interaction between inhibitory self-reactive Ly49 molecules and MHC I molecules may be crucial for NK cell development. We propose a model in which Ly49-MHC I interaction may have a permissive role in allowing development of only such NK cell clones that expresses at least one self-reactive inhibitory Ly49 molecule so that lysis of autologous healthy cells by mature NK cells may be avoided.     

Fine tuning of natural killer cell specificity and maintenance of self tolerance in MHC class I-deficient mice

TAP1 −/−, β2-microglobulin (β2m) −/− and TAP1/β2m −/− mice all express low but quantitatively different levels of MHC class I molecules. Using these mice, we have addressed questions relating to the fine tuning of natural killer (NK) cell specificity and maintenance of self tolerance in the NK cell system. NK cells from B6 wild-type mice killed target cells from TAP1 −/−, β2m −/− and TAP1/β2m −/− mice in vivo and rejected bone marrow grafts from the same mice in vivo at equivalent levels. NK cells from TAP1 −/−, β2m −/− mice did not kill target cells or reject bone marrow grafts from TAP1/β2m −/− mice. NK cells in all MHC class I-deficient mice were tolerant to autologous MHC class I-deficient cells, as revealed by in vitro cytotoxicity assays using NK cell effectors activated with the interferon-inducing agent Tilorone, or by in vivo bone marrow graft experiments. However, the self-tolerant state of MHC class I-deficient NK cells was broken by in vitro stimulation with IL-2 for 4 days. Under these conditions, NK cells from the MHC class I-deficient mice killed autologous MHC class I-deficient cells while MHC class I-positive targets were spared. The C-type lectin inhibitory receptor Ly49C has a specificity for H-2K^b and is expressed on a subset of NK1.1⁺ cells in B6 mice. Wild-type and all MHC class I-deficient mice had similar numbers of Ly49C-positive NK1.1⁺ cells. However, Ly49C expression was markedly down-regulated on NK1.1⁺ cells from B6 mice, as compared to TAP1 −/−, β2m −/− and TAP1/β2m −/− mice. In vitro stimulation of NK cells with IL-2 for 4 days did not significantly change this pattern. The present results are discussed in relation to the role of MHC class I molecules and Ly49 receptors in shaping the NK cell repertoire and raise new questions about maintenance of self tolerance in the NK cell system.     

Structure of the Ly49 family of natural killer (NK) cell receptors and their interaction with MHC class I molecules

Natural killer (NK) cells are an essential component of the innate immunity toward tumors and virally infected cells. The function of NK cells is regulated by a precise balance between inhibitory and activating signals. These signals are mediated by NK cell receptors that bind either classical MHC class I molecules or their structural relatives such as MICA, ULBP, RAE-1, and H-60. Two separate families of NK cell receptors have been identified: the immunoglobulin-like family (KIR, LIR) and C-type lectin-like family (Ly49, NKG2D, and CD94/NKG2). Here we summarize the structure of Ly49 C-type lectin-like proteins hitherto solved (Ly49A, Ly49C and Ly49I) and their interaction with MHC class I molecules as determined by the co-crystal structure of Ly49A/H-2Dd and Ly49C/H-2Kb.     

Direct assessment of MHC class I binding by seven Ly49 inhibitory NK cell receptors.

Mouse NK cells express at least seven inhibitory Ly49 receptors. Here we employ a semiquantitative cell-cell adhesion assay as well as class I/peptide tetramers to provide a comprehensive analysis of specificities of Ly49 receptors for class I MHC molecules in eight MHC haplotypes. Different Ly49 receptors exhibited diverse binding properties. The degree of class I binding was related to the extent of functional inhibition. The tetramer studies demonstrated that neither glycosylation nor coreceptors were necessary for class I binding to Ly49 receptors and uncovered peptide-specific recognition by a Ly49 receptor. The results provide a foundation for interpreting and integrating many existing functional studies as well as for designing tests of NK cell development and self-tolerance.     

Clonal analysis of NK cell development from bone marrow progenitors in vitro: orderly acquisition of receptor gene expression

In the mouse, two families of MHC class I-specific receptors, namely Ly49 and CD94/NKG2, have been identified on NK cells. Individual NK cells can express several Ly49 molecules as well as members of the CD94/NKG2 family. The expression of multiple receptors with different specificities for MHC class I is thus thought to generate NK cells with diverse recognition patterns. To delineate the mechanism by which NK cells begin to express different patterns of Ly49 and CD94/NKG2 molecules, we developed a clonal assay in which NK1.1(-), IL-2/ IL-15 receptor beta+ NK precursors generated by culture of multipotential Lin(-), c-kit+ progenitors in IL-7, stem cell factor and flt3 ligand are induced to differentiate into NK1.1+ , Ly49+ NK cells. Examination of the clonal populations thus generated revealed heterogeneity in the pattern of Ly49 and CD94/NKG2 gene expression. In addition, a distinct kinetic pattern of expression was observed. CD94, NKG2A, NKG2C and Ly49B were expressed first followed by Ly49G, then Ly49C and I and finally, Ly49A, D, E and F. The data suggest a stochastic but ordered acquisition of class I receptors on NK cells in which developing NK cells become capable of expressing distinct receptors at different times but show no absolute prerequisite to express the receptors that are acquired early in NK development for the expression of those that are acquired later.     

Interaction of the NK cell inhibitory receptor Ly49A with H-2Dd: identification of a site distinct from the TCR site

Natural killer cell function is controlled by interaction of NK receptors with MHC I molecules expressed on target cells. We describe the binding of bacterially expressed Ly49A, the prototype murine NK inhibitory receptor, to similarly engineered H-2Dd. Despite its homology to C-type lectins, Ly49A binds independently of carbohydrate and Ca2+ and shows specificity for MHC I but not bound peptide. The affinity of the Ly49A/H-2Dd interaction as determined by surface plasmon resonance is from 6 to 26 microM at 25 degrees C and is greater by ultracentrifugation at 4 degrees C. Biotinylated Ly49A stains H-2Dd-expressing cells. Competition experiments indicate that the Ly49A and T cell receptor (TCR) binding sites on MHC I are distinct, suggesting complex regulation of cells that bear both TCR and NK cell receptors.     

MHC class Ia molecules alone control NK-mediated bone marrow graft rejection

Mice with functionally deleted genes encoding MHC class I heavy (H-2K(b), H-2D(b)) and light (beta2-microglobulin) chains were used in bone marrow cell transfer experiments to study the role of class Ia and Ib molecules in NK cell function. Absence of H-2K(b) and absence of H-2D(b) on bone marrow cells resulted in complete and in almost complete NK-mediated rejection, respectively. Absence of either H-2 class Ib (at least when expressed in H-2 class Ia-deficient mice) or cell surface class Ia free heavy chains did not result in bone marrow rejection. Thus, in C57BL/6 adult mice, the inactivation of NK cells required for bone marrow cell engraftment relies entirely upon-H-2 class Ia molecules. These results imply the existence of an inhibitory receptor which recognizes either directly or indirectly H-2D(b) molecules and further suggest that in C57BL/6 mice the NK cells which do not express a H-2K(b) specific inhibitory receptor necessarily express an H-2D(b)-specific one.