Natural Killer Cell Tolerance Persists Despite Significant Reduction of Self MHC Class I on Normal Target Cells in Mice

Background

A major group of murine inhibitory receptors on Natural Killer (NK) cells belong to the Ly49 receptor family and recognize MHC class I molecules. Infected or transformed target cells frequently downmodulate MHC class I molecules and can thus avoid CD8⁺ T cell attack, but may at the same time develop NK cell sensitivity, due to failure to express inhibitory ligands for Ly49 receptors. The extent of MHC class I downregulation needed on normal cells to trigger NK cell effector functions is not known.

Methodology/Principal Findings

In this study, we show that cells expressing MHC class I to levels well below half of the host level are tolerated in an in vivo assay in mice. Hemizygous expression (expression from only one allele) of MHC class I was sufficient to induce Ly49 receptor downmodulation on NK cells to a similar degree as homozygous expression, despite a strongly reduced cell surface level of MHC class I. Co-expression of weaker MHC class I ligands in the host did not have any further effect on the degree of Ly49 downmodulation. Furthermore, a single MHC class I allele could downmodulate up to three Ly49 receptors on individual NK cells. Only when NK cells simultaneously expressed several Ly49 receptors and hemizygous MHC class I levels, a putative threshold for Ly49 downmodulation was reached.

Conclusion

Collectively, our findings suggest that in interactions between NK cells and normal untransformed cells, MHC class I molecules are in most cases expressed in excess compared to what is functionally needed to ensure self tolerance and to induce maximal Ly49 downmodulation. We speculate that the reason for this is to maintain a safety margin for otherwise normal, autologous cells over a range of MHC class I expression levels, in order to ensure robustness in NK cell tolerance.     

Probing Natural Killer Cell Education by Ly49 Receptor Expression Analysis and Computational Modelling in Single MHC Class I Mice

Murine natural killer (NK) cells express inhibitory Ly49 receptors for MHC class I molecules, which allows for “missing self” recognition of cells that downregulate MHC class I expression. During murine NK cell development, host MHC class I molecules impose an “educating impact” on the NK cell pool. As a result, mice with different MHC class I expression display different frequency distributions of Ly49 receptor combinations on NK cells. Two models have been put forward to explain this impact. The two-step selection model proposes a stochastic Ly49 receptor expression followed by selection for NK cells expressing appropriate receptor combinations. The sequential model, on the other hand, proposes that each NK cell sequentially expresses Ly49 receptors until an interaction of sufficient magnitude with self-class I MHC is reached for the NK cell to mature. With the aim to clarify which one of these models is most likely to reflect the actual biological process, we simulated the two educational schemes by mathematical modelling, and fitted the results to Ly49 expression patterns, which were analyzed in mice expressing single MHC class I molecules. Our results favour the two-step selection model over the sequential model. Furthermore, the MHC class I environment favoured maturation of NK cells expressing one or a few self receptors, suggesting a possible step of positive selection in NK cell education. Based on the predicted Ly49 binding preferences revealed by the model, we also propose, that Ly49 receptors are more promiscuous than previously thought in their interactions with MHC class I molecules, which was supported by functional studies of NK cell subsets expressing individual Ly49 receptors.     

Transgenic expression of Ly49A on T cells impairs a specific antitumor response

Inhibitory MHC receptors determine the reactivity and specificity of NK cells. These receptors can also regulate T cells by modulating TCR-induced effector functions such as cytotoxicity, cytokine production, and proliferation. Here we have assessed the capacity of mouse T cells expressing the inhibitory MHC class I receptor Ly49A to respond to a well-defined tumor Ag in vivo using Ly49A transgenic mice. We find that the presence of Ly49A on the vast majority of lymphocytes prevents the development of a significant Ag-specific CD8+ T cell response and, consequently, the rejection of the tumor. Despite minor alterations in the TCR repertoire of CD8+ T cells in the transgenic lines, precursors of functional tumor-specific CD8+ T cells exist but could not be activated most likely due to a lack of appropriate CD4+ T cell help. Surprisingly, all of these effects are observed in the absence of a known ligand for the Ly49A receptor as defined by its ability to regulate NK cell function. Indeed, we found that the above effects on T cells may be based on a weak interaction of Ly49A with Kb or Db class I molecules. Thus, our data demonstrate that enforced expression of a Ly49A receptor on conventional T cells prevents a specific immune response in vivo and suggest that the functions of T and NK cells are differentially sensitive to the presence of inhibitory MHC class I receptors.     

Ly49C-Dependent Control of MCMV Infection by NK Cells Is Cis-Regulated by MHC Class I Molecules

Natural Killer (NK) cells are crucial in early resistance to murine cytomegalovirus (MCMV) infection. In B6 mice, the activating Ly49H receptor recognizes the viral m157 glycoprotein on infected cells. We previously identified a mutant strain (MCMV^G1F) whose variant m157 also binds the inhibitory Ly49C receptor. Here we show that simultaneous binding of m157 to the two receptors hampers Ly49H-dependent NK cell activation as Ly49C-mediated inhibition destabilizes NK cell conjugation with their targets and prevents the cytoskeleton reorganization that precedes killing. In B6 mice, as most Ly49H⁺ NK cells do not co-express Ly49C, the overall NK cell response remains able to control MCMVm157^G1F infection. However, in B6 Ly49C transgenic mice where all NK cells express the inhibitory receptor, MCMV infection results in altered NK cell activation associated with increased viral replication. Ly49C-mediated inhibition also regulates Ly49H-independent NK cell activation. Most interestingly, MHC class I regulates Ly49C function through cis-interactions that mask the receptor and restricts m157 binding. B6 Ly49C Tg, β2m ko mice, whose Ly49C receptors are unmasked due to MHC class I deficient expression, are highly susceptible to MCMVm157^G1F and are unable to control a low-dose infection. Our study provides novel insights into the mechanisms that regulate NK cell activation during viral infection.     

MHC class I-Ly49 interactions shape the Ly49 repertoire on murine NK cells

This study aims to determine how the interaction of Ly49 receptors with MHC class I molecules shapes the development of the Ly49 repertoire. We have examined the percentage of NK cells that expressed Ly49A, Ly49G2, and Ly49D in single and double Ly49A/C-transgenic mice on four different MHC backgrounds, H-2(b), H-2(d), H-2(b/d), and beta(2)-microglobulin(-/-). The results show that the total numbers of NK cells were not different among the strains. The prior expression of a Ly49 receptor capable of binding to self MHC class I altered the percentage of NK cells expressing endogenous Ly49A, Ly49G2, and Ly49D even in mice in which no MHC ligand was present for the latter receptors. The NK cells in the Ly49-transgenic mice expressed the same level of endogenous Ly49 receptors as wild-type mice of a similar MHC background. In contrast, the number of NK T cells was reduced in mice in which the Ly49 transgene could bind to a MHC class I molecule. The onset of Ly49 receptor expression on NK cells during ontogeny was not altered in the presence of transgenic Ly49 receptors. These data support a sequential model and argue against a selection model for Ly49 repertoire development on NK cells.     

B6 strain Ly49I inhibitory gene expression on T cells in FVB.Ly49IB6 transgenic mice fails to prevent normal T cell functions

Inhibitory Ly49 receptors expressed on NK cells provide a mechanism for tolerance to normal self tissues. The immunoregulatory tyrosine-based inhibitory motifs present in some Ly49s are able to transmit an inhibitory signal upon ligation by MHC class I ligands. In our system, as well as others, mice transgenic for inhibitory Ly49 receptors express these receptors on both NK and T cells. FVB (H2(q)) mice transgenic for the B6 strain Ly49I (Ly49I(B6)) express the inhibitory Ly49 receptor on the surface of both T and NK cells. Although Ly49I functions to prevent NK-mediated rejection of H2(b) donor bone marrow cells in this transgenic mouse strain, the T cells do not appear to be affected by the expression of the Ly49I transgene. FVB.Ly49I T cells have normal proliferative capabilities both in vitro and in vivo in response to the Ly49I ligand, H2(b). In vivo functional T cell assays were also done, showing that transgenic T cells were not functionally affected. T cells in these mice also appear to undergo normal T cell development and activation. Only upon stimulation with suboptimal doses of anti-CD3 in the presence of anti-Ly49I is T cell proliferation inhibited. These data are in contrast with findings in Ly49A, and Ly49G2 receptor transgenic models. Perhaps Ly49I-H2(b) interactions are weaker or of lower avidity than Ly49A-H-2D(d) interactions, especially in T cells.     

Critical residues at the Ly49 natural killer receptor's homodimer interface determine functional recognition of m157, a mouse cytomegalovirus MHC class I-like protein

NK cell function is regulated by Ly49 receptors in mice and killer cell Ig-like receptors in humans. Although inhibitory Ly49 and killer cell Ig-like receptors predominantly ligate classical MHC class I molecules, recent studies suggest that their activating counterparts recognize infection. The quintessential example is resistance to the mouse CMV in C57BL/6 mice, which depends on the functional recognition of m157, a mouse CMV-encoded MHC class I-like molecule, by Ly49H, an activating NK cell receptor. We have taken advantage of the natural variation in closely related members of the Ly49C-like receptors and the availability of Ly49 crystal structures to understand the molecular determinants of the Ly49H-m157 interaction and to identify amino acid residues discriminating between m157 binding and nonbinding receptors. Using a site-directed mutagenesis approach, we have targeted residues conserved in receptors binding to m157 (Ly49H and Ly49I(129)) but different from receptors lacking m157 recognition (Ly49C, Ly49I(B6), and Ly49U). Wild-type and mutant receptors were transfected into reporter cells, and physical binding as well as functional activation by m157 was studied. Our findings suggested that the Ly49 MHC class I contact "site 2," I226, may not be involved in m157 binding. In contrast, residue Y146 and G151, mapping at the receptor homodimer interface, are likely critical for functional recognition of the m157 glycoprotein. Our combined functional and three-dimensional modeling approach suggested that the architecture of the Ly49H dimer is crucial to accessing m157, but not MHC class I. These results link Ly49 homodimerization variability to the direct recognition of pathogen products.     

Interactions of Ly49 family receptors with MHC class I ligands in trans and cis

The Ly49A NK cell receptor interacts with MHC class I (MHC-I) molecules on target cells and negatively regulates NK cell-mediated target cell lysis. We have recently shown that the MHC-I ligand-binding capacity of the Ly49A NK cell receptor is controlled by the NK cells' own MHC-I. To see whether this property was unique to Ly49A, we have investigated the binding of soluble MHC-I multimers to the Ly49 family receptors expressed in MHC-I-deficient and -sufficient C57BL/6 mice. In this study, we confirm the binding of classical MHC-I to the inhibitory Ly49A, C and I receptors, and demonstrate that detectable MHC-I binding to MHC-I-deficient NK cells is exclusively mediated by these three receptors. We did not detect significant multimer binding to stably transfected or NK cell-expressed Ly49D, E, F, G, and H receptors. Yet, we identified the more distantly related Ly49B and Ly49Q, which are not expressed by NK cells, as two novel MHC-I receptors in mice. Furthermore, we show using MHC-I-sufficient mice that the NK cells' own MHC-I significantly masks the Ly49A and Ly49C, but not the Ly49I receptor. Nevertheless, Ly49I was partly masked on transfected tumor cells, suggesting that the structure of Ly49I is compatible in principal with cis binding of MHC-I. Finally, masking of Ly49Q by cis MHC-I was minor, whereas masking of Ly49B was not detected. These data significantly extend the MHC-I specificity of Ly49 family receptors and show that the accessibility of most, but not all, MHC-I-binding Ly49 receptors is modulated by the expression of MHC-I in cis.     

Positive impact of inhibitory Ly49 receptor-MHC class I interaction on NK cell development

NK cells can kill MHC-different or MHC-deficient but not syngeneic MHC-expressing target cells. This MHC class I-specific tolerance is acquired during NK cell development. MHC recognition by murine NK cells largely depends on clonally distributed Ly49 family receptors, which inhibit NK cell function upon ligand engagement. We investigated whether these receptors play a role for the development of NK cells and provide evidence that the expression of a Ly49 receptor transgene on developing NK cells endowed these cells with a significant developmental advantage over NK cells lacking such a receptor, but only if the relevant MHC ligand was present in the environment. The data suggest that the transgenic Ly49 receptor accelerates and/or rescues the development of NK cells which would otherwise fail to acquire sufficient numbers of self-MHC-specific receptors. Interestingly, the positive effect on NK cell development is most prominent when the MHC ligand is simultaneously present on both hemopoietic and nonhemopoietic cells. These findings correlate with functional data showing that MHC class I ligand on all cells is required to generate functionally mature NK cells capable of reacting to cells lacking the respective MHC ligand. We conclude that the engagement of inhibitory MHC receptors during NK cell development provides signals that are important for further NK cell differentiation and/or maturation.     

Diversity of NK cell receptor repertoire in adult and neonatal mice.

Murine NK cytotoxicity is regulated by two families of MHC class I-specific receptors, namely Ly49 and CD94/NKG2. We developed a single-cell RT-PCR method to analyze expression of all known Ly49 and NKG2A genes in individual NK cells and determined the receptor repertoires of NK cells from adult and neonatal (1-wk-old) C57BL/6 mice. In adult mouse NK cells, up to six different receptors were coexpressed in random combinations. Of 62 NK cells examined, 42 different patterns of receptor expression were observed. Most of them expressed at least one Ly49, whereas NKG2A was detected in 32% of the cells. Over 75% of them expressed Ly49C, I, or NKG2A, which are thought to recognize self-class I MHC (H-2b). Coexpression of multiple Ly49 receptors and NKG2A was stochastic. In contrast, very few neonatal NK cells expressed any Ly49, but almost 60% of them expressed NKG2A. These results demonstrate that adult NK cells are quite heterogeneous and have diverse receptor repertoires. They also suggest that the expression of NKG2A precedes Ly49 expression in NK cell ontogeny, and NKG2A is a major inhibitory receptor in neonatal NK cells.     

The activating Ly49W and inhibitory Ly49G NK cell receptors display similar affinities for identical MHC class I ligands

The Ly49 receptor family plays an important role in the regulation of murine natural killer (NK) cell effector function. They recognize cell surface-expressed class I MHC (MHC-I) and are functionally equivalent to the killer Ig-related receptors (KIRs) in human NK cells. Ly49s exist in activating and inhibitory forms with highly homologous extracellular domains, displaying greater variability in the stalk regions. Inhibitory Ly49s can recognize self-MHC-I and therefore mediate tolerance to self. The role of activating Ly49 receptors is less clear. Some activating Ly49 receptors have been shown to recognize MHC-I molecules. The binding affinity of activating Ly49 receptors with MHC-I is currently unknown, and we sought to examine the affinities of two highly related receptors, an activating and an inhibitory Ly49 receptor, for their shared MHC-I ligands. The ectodomain of inhibitory Ly49G of the BALB/c mouse strain is highly similar to the Ly49W activating receptor in the nonobese diabetic (NOD) mouse. Recombinant soluble Ly49G and W were expressed, refolded, and analyzed for binding affinity with MHC-I by surface plasmon resonance. We found that Ly49G and Ly49W bound with similar affinity to the same MHC-I molecules. These results are a first determination of an activating Ly49 receptor affinity for MHC-I and show that, unlike prior results obtained with activating and inhibitory KIR receptors, functional homologues to Ly49 receptors, activating and inhibitory Ly49, can recognize common MHC-I ligands, with similar affinities.     

Cross-species dependence of Ly49 recognition on the supertype defining B-pocket of a class I MHC molecule

Ly49 recognition of MHC class I (MHC I) can be allele specific. However, the site of interaction on MHC I consists of highly conserved solvent-exposed amino acids, leaving it unclear how allele specificity occurs. In examining the specificity of mouse and rat Ly49, we noticed that MHC I ligands for mouse Ly49G and W, and the rat Ly49i2, typically share the HLA-B7 supertype, defined by a B-pocket that prefers a proline at position 2 in bound peptides. Through mutagenesis, we show that the supertype-defining B-pocket of RT1-A1(c) controls its allele-specific recognition by the syngeneic rat Ly49i2 inhibitory receptor and xenogeneic mouse inhibitory Ly49G and activating Ly49W receptors. Single amino acid substitutions in the B-pocket that did not prevent peptide binding disrupted Ly49 recognition. In contrast, single mutations in other regions of the peptide-binding groove had no effect. We provide a model whereby the B-pocket dictates the conformation of conserved residues at the Ly49 interaction site below, defining Ly49 allele specificity for MHC I. Therefore, at least some Ly49 may recognize supertypes, detectable even across species, and are sensitive to polymorphisms in the supertype-defining B-pocket. This would ensure that expression of specific MHC I supertypes capable of Ag presentation to T cells is sensed by NK cells, and if lacking, targets a cell for elimination, suggesting a supertype-mediated link between innate and adaptive immunity.     

The MHC class I molecule H-2Dp inhibits murine NK cells via the inhibitory receptor Ly49A.

MHC class I molecules strongly influence the phenotype and function of mouse NK cells. NK cell-mediated lysis is prevented through the interaction of Ly49 receptors on the effector cell with appropriate MHC class I ligands on the target cell. In addition, host MHC class I molecules have been shown to modulate the in vivo expression of Ly49 receptors. We have previously reported that H-2Dd and H-2Dp MHC class I molecules are able to protect (at the target cell level) from NK cell-mediated lysis and alter the NK cell specificity (at the host level) in a similar manner, although the mechanism behind this was not clear. In this study, we demonstrate that the expression of both H-2Dd and H-2Dp class I molecules in target cells leads to inhibition of B6 (H-2b)-derived Ly49A+ NK cells. This inhibition could in both cases be reversed by anti-Ly49A Abs. Cellular conjugate assays showed that Ly49A-expressing cells indeed bind to cells expressing H-2Dp. The expression of Ly49A and Ly49G2 receptors on NK cells was down-regulated in H-2Dp-transgenic (B6DP) mice compared with nontransgenic B6 mice. However, B6DP mice expressed significantly higher levels of Ly49A compared with H-2Dd-transgenic (D8) mice. We propose that both H-2Dd and H-2Dp MHC class I molecules can act as ligands for Ly49A.     

Activating and inhibitory Ly49 receptors modulate NK cell chemotaxis to CXC chemokine ligand (CXCL) 10 and CXCL12

NK cells can migrate into sites of inflammatory responses or malignancies in response to chemokines. Target killing by rodent NK cells is restricted by opposing signals from inhibitory and activating Ly49 receptors. The rat NK leukemic cell line RNK16 constitutively expresses functional receptors for the inflammatory chemokine CXC chemokine ligand (CXCL)10 (CXCR3) and the homeostatic chemokine CXCL12 (CXCR4). RNK-16 cells transfected with either the activating Ly49D receptor or the inhibitory Ly49A receptor were used to examine the effects of NK receptor ligation on CXCL10- and CXCL12-mediated chemotaxis. Ligation of Ly49A, either with Abs or its MHC class I ligand H2-D(d), led to a decrease in chemotactic responses to either CXCL10 or CXCL12. In contrast, Ly49D ligation with Abs or H2-D(d) led to an increase in migration toward CXCL10, but a decrease in chemotaxis toward CXCL12. Ly49-dependent effects on RNK-16 chemotaxis were not the result of surface modulation of CXCR3 or CXCR4 as demonstrated by flow cytometry. A mutation of the Src homology phosphatase-1 binding motif in Ly49A completely abrogated Ly49-dependent effects on both CXCL10 and CXCL12 chemotaxis, suggesting a role for Src homology phosphatase-1 in Ly49A/chemokine receptor cross-talk. Ly49D-transfected cells were pretreated with the Syk kinase inhibitor Piceatannol before ligation, which abrogated the previously observed changes in migration toward CXCL10 and CXCL12. Piceatannol also abrogated Ly49A-dependent inhibition of chemotaxis toward CXCL10, but not CXCL12. Collectively, these data suggest that Ly49 receptors can influence NK cell chemotaxis within sites of inflammation or tumor growth upon interaction with target cells.     

Cumulative inhibition of NK cells and T cells resulting from engagement of multiple inhibitory Ly49 receptors

Inhibitory receptors specific for MHC class I molecules are expressed on partially overlapping subpopulations of NK cells and memory T cells. A central question pertinent to NK cell development and function is how the combinatorial expression of different receptors with distinct class I specificities affects functional recognition. We therefore studied the quantitative effects resulting from class I engagement of multiple inhibitory Ly49 receptors. We used a transgenic mouse model in which all NK cells and T cells express two different Ly49 receptors with shared class I specificity. Comparisons of cells from these mice with cells from single transgenic mice and wild-type mice revealed that Ly49 receptors cumulatively inhibit lymphocyte effector functions. Multiple Ly49 interactions also had a cumulative impact on NK cell development. The findings suggest that the interactions of inhibitory receptors with class I are interpreted quantitatively rather than as on/off switches. They have intriguing implications concerning NK cell tolerance and reactivity toward cells with extinguished expression of a limited number of class I molecules.     

Functional consequences of natural sequence variation of murine cytomegalovirus m157 for Ly49 receptor specificity and NK cell activation

The Ly49H activating receptor on C57BL/6 (B6) NK cells plays a key role in early resistance to murine cytomegalovirus (MCMV) infection through specific recognition of the MCMV-encoded MHC class I-like molecule m157 expressed on infected cells. The m157 molecule is also recognized by the Ly49I inhibitory receptor from the 129/J mouse strain. The m157 gene is highly sequence variable among MCMV isolates, with many m157 variants unable to bind Ly49H(B6). In this study, we have sought to define if m157 variability leads to a wider spectrum of interactions with other Ly49 molecules and if this modifies host susceptibility to MCMV. We have identified novel m157-Ly49 receptor interactions, involving Ly49C inhibitory receptors from B6, BALB/c, and NZB mice, as well as the Ly49H(NZB) activation receptor. Using an MCMV recombinant virus in which m157(K181) was replaced with m157(G1F), which interacts with both Ly49H(B6) and Ly49C(B6), we show that the m157(G1F)-Ly49C interactions cause no apparent attenuating effect on viral clearance in B6 mice. Hence, when m157 can bind both inhibitory and activation NK cell receptors, the outcome is still activation. Thus, these data indicate that whereas m157 variants predominately interact with inhibitory Ly49 receptors, these interactions do not profoundly interfere with early NK cell responses.     

Ly49-dependent NK cell licensing and effector inhibition involve the same interaction site on MHC ligands

NK cells become functionally competent to be triggered by their activation receptors through the interaction of NK cell inhibitory receptors with their cognate self-MHC ligands, an MHC-dependent educational process termed "licensing." For example, Ly49A(+) NK cells become licensed by the interaction of the Ly49A inhibitory receptor with its MHC class I ligand, H2D(d), whereas Ly49C(+) NK cells are licensed by H2K(b). Structural studies indicate that the Ly49A inhibitory receptor may interact with two sites, termed site 1 and site 2, on its H2D(d) ligand. Site 2 encompasses the α1/α2/α3 domains of the H2D(d) H chain and β(2)-microglobulin (β2m) and is the functional binding site for Ly49A in effector inhibition. Ly49C functionally interacts with a similar site in H2K(b). However, it is currently unknown whether this same site is involved in Ly49A- or Ly49C-dependent licensing. In this study, we produced transgenic C57BL/6 mice expressing wild-type or site 2 mutant H2D(d) molecules and studied whether Ly49A(+) NK cells are licensed. We also investigated Ly49A- and Ly49C-dependent NK licensing in murine β2m-deficient mice that are transgenic for human β2m, which has species-specific amino acid substitutions in β2m. Our data from these transgenic mice indicate that site 2 on self-MHC is critical for Ly49A- and Ly49C-dependent NK cell licensing. Thus, NK cell licensing through Ly49 involves specific interactions with its MHC ligand that are similar to those involved in effector inhibition.     

A modified FCCS procedure applied to Ly49A-MHC class I cis-interaction studies in cell membranes

The activity of natural killer (NK) cells is regulated by a fine-tuned balance between activating and inhibitory receptors. Dual-color fluorescence cross-correlation spectroscopy (FCCS) was used to directly demonstrate a so-called cis-interaction between a member of the inhibitory NK cell receptor family Ly49 (Ly49A), and its ligand, the major histocompatibility complex (MHC) class I, within the plasma membrane of the same cell. By a refined FCCS model, calibrated by positive and negative control experiments on cells from the same lymphoid cell line, concentrations and diffusion coefficients of free and interacting proteins could be determined on a collection of cells. Using the intrinsic intercellular variation of their expression levels for titration, it was found that the fraction of Ly49A receptors bound in cis increase with increasing amounts of MHC class I ligand. This increase shows a tendency to be more abrupt than for a diffusion limited – three dimensional bimolecular reaction, which most likely reflects the two-dimensional confinement of the reaction. For the Ly49A- MHC class I interaction it indicates that within a critical concentration range the local concentration level of MHC class I can provide a distinct regulation mechanism of the NK cell activity.     

The novel inhibitory NKR-P1C receptor and Ly49s3 identify two complementary, functionally distinct NK cell subsets in rats

The proximal region of the NK gene complex encodes the NKR-P1 family of killer cell lectin-like receptors which in mice bind members of the genetically linked C-type lectin-related family, while the distal region encodes Ly49 receptors for polymorphic MHC class I molecules. Although certain members of the NKR-P1 family are expressed by all NK cells, we have identified a novel inhibitory rat NKR-P1 molecule termed NKR-P1C that is selectively expressed by a Ly49-negative NK subset with unique functional characteristics. NKR-P1C(+) NK cells efficiently lyse certain tumor target cells, secrete cytokines upon stimulation, and functionally recognize a nonpolymorphic ligand on Con A-activated lymphoblasts. However, they specifically fail to kill MHC-mismatched lymphoblast target cells. The NKR-P1C(+) NK cell subset also appears earlier during development and shows a tissue distribution distinct from its complementary Ly49s3(+) subset, which expresses a wide range of Ly49 receptors. These data suggest the existence of two major, functionally distinct populations of rat NK cells possessing very different killer cell lectin-like receptor repertoires.     

Structural and functional aspects of the Ly49 natural killer cell receptors

Natural killer cells are part of the first line of innate immune defence against virus-infected cells and cancer cells in the vertebrate immune system. They are called 'natural' killers because, unlike cytotoxic T cells, they do not require a previous challenge and preactivation to become active. The Ly49 NK receptors are type II transmembrane glycoproteins, structurally characterized as disulphide-linked homodimers. They share extensive homology with C-type lectins, and they are encoded by a multigene family that in mice maps on chromosome 6. A fine balance between inhibitory and activating signals regulates the function of NK cells. Inhibitory Ly49 molecules bind primarily MHC class I ligands, whereas the ligands for activating Ly49 molecules may include MHC class I, but also interestingly MHC class I-like molecules expressed by viruses, as is the case for Ly49H, which binds the m157 gene product of murine cytomegalovirus. In this study, we review the function and X-ray crystal structure of the Ly49 NK cell receptors hitherto determined (Ly49A, Ly49C and Ly49I), and the structural features of the Ly49/MHC class I interaction as revealed by the X-ray crystal structures of Ly49A/H-2Dd and the recently determined Ly49C/H-2Kb.