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.     

Skewing of the NK cell repertoire by MHC class I via quantitatively controlled enrichment and contraction of specific Ly49 subsets

A major task for the immune system is to secure powerful immune reactions while preserving self-tolerance. This process is particularly challenging for NK cells, for which tolerizing inhibitory receptors for self-MHC class I is both cross-reactive and expressed in an overlapping fashion between NK cells. We show in this study that during an education process, self-MHC class I molecules enrich for potentially useful and contract potentially dangerous NK cell subsets. These processes were quantitatively controlled by the expression level of the educating MHC class I allele, correlated with susceptibility to IL-15 and sensitivity to apoptosis in relevant NK cell subsets, and were linked to their functional education. Controlling the size of NK cell subsets with unique compositions of inhibitory receptors may represent one mechanism by which self-MHC class I molecules generate a population of tolerant NK cells optimally suited for efficient missing self-recognition.     

Recruitment of activation receptors at inhibitory NK cell immune synapses

Natural killer (NK) cell activation receptors accumulate by an actin-dependent process at cytotoxic immune synapses where they provide synergistic signals that trigger NK cell effector functions. In contrast, NK cell inhibitory receptors, including members of the MHC class I-specific killer cell Ig-like receptor (KIR) family, accumulate at inhibitory immune synapses, block actin dynamics, and prevent actin-dependent phosphorylation of activation receptors. Therefore, one would predict inhibition of actin-dependent accumulation of activation receptors when inhibitory receptors are engaged. By confocal imaging of primary human NK cells in contact with target cells expressing physiological ligands of NK cell receptors, we show here that this prediction is incorrect. Target cells included a human cell line and transfected Drosophila insect cells that expressed ligands of NK cell activation receptors in combination with an MHC class I ligand of inhibitory KIR. The two NK cell activation receptors CD2 and 2B4 accumulated and co-localized with KIR at inhibitory immune synapses. In fact, KIR promoted CD2 and 2B4 clustering, as CD2 and 2B4 accumulated more efficiently at inhibitory synapses. In contrast, accumulation of KIR and of activation receptors at inhibitory synapses correlated with reduced density of the integrin LFA-1. These results imply that inhibitory KIR does not prevent CD2 and 2B4 signaling by blocking their accumulation at NK cell immune synapses, but by blocking their ability to signal within inhibitory synapses.     

Unravelling natural killer cell function: triggering and inhibitory human NK receptors

Natural killer (NK) cells represent a highly specialized lymphoid population characterized by a potent cytolytic activity against tumor or virally infected cells. Their function is finely regulated by a series of inhibitory or activating receptors. The inhibitory receptors, specific for major histocompatibility complex (MHC) class I molecules, allow NK cells to discriminate between normal cells and cells that have lost the expression of MHC class I (e.g., tumor cells). The major receptors responsible for NK cell triggering are NKp46, NKp30, NKp44 and NKG2D. The NK-mediated lysis of tumor cells involves several such receptors, while killing of dendritic cells involves only NKp30. The target-cell ligands recognized by some receptors have been identified, but those to which major receptors bind are not yet known. Nevertheless, functional data suggest that they are primarily expressed on cells upon activation, proliferation or tumor transformation. Thus, the ability of NK cells to lyse target cells requires both the lack of surface MHC class I molecules and the expression of appropriate ligands that trigger NK receptors.     

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.     

Paired NK cell receptors controlling NK cytotoxicity

Human natural killer (NK) cells possess an arsenal of receptors programmed to regulate the NK cell functions, once encountering a target cell. In general, the activating receptors mediate cytotoxicity when engaged by their tumor specific, stress induced, virally encoded, or rarely, self ligands. Whereas, the inhibitory receptors bind self molecules, mostly MHC class I, presented on all normal and healthy nucleated cells. However, NK cells also possess numerous, highly homologous, pairs of receptors that sometimes even share the same ligands but display divergent functions. In this review we describe the NK cell repertoire of paired receptors and discuss questions regarding their function and mode of action. We focus primarily on the three PVR-binding receptors; the co-stimulatory DNAM1 and CD96 and the inhibitory TIGIT.     

Enhanced recognition of human NK receptors after influenza virus infection

The NK cell cytotoxic activity is regulated by both inhibitory and activating NK receptors. Thus, changes in the expression levels and in the affinity or avidity of those receptors will have a major effect on the killing of target cells. In this study, we demonstrate that the binding of NK-inhibitory receptors is enhanced after influenza virus infection. Surprisingly, however, no change in the level of class I MHC protein expression was observed on the surface of the infected cells. The increased binding was general, because it was observed in both the killer cell Ig-like receptor 2 domain long tail 1 and leukocyte Ig-like receptor-1. The increased binding was functional, was not dependent on the interaction with viral hemagglutinin-neuraminidase, was not dependent on the glycosylation site, and was not abolished after mutating the transmembrane or cytosolic portions of the class I MHC proteins. Confocal microscopy experiments showed increased binding of NK receptor-coated beads to infected cells expressing the appropriate class I MHC proteins. In addition, specific cell-free bead aggregates covered with class I MHC proteins were observed only in infected cells. We therefore suggest that the influenza virus use a novel mechanism for the inhibition of NK cell activity. This mechanism probably involves the generation of class I MHC complexes in infected cells that cause increased recognition of NK receptors.     

A central role for death receptor-mediated apoptosis in the rejection of tumors by NK cells

NK cells provide a line of defense against tumors and virus-infected cells that have lost the expression of one or more MHC class I isoforms. Here, we investigate whether inhibitors of apoptosis can block the rejection of tumors mediated by NK cells, by introducing the long form of Fas-associated death domain-like IL-1beta-converting enzyme-associated inhibitory protein (FLIP(L)) and poxvirus cytokine response modifier A (CrmA) into the MHC class I-deficient T lymphoma cell line RMA-S. RMA-S cells do not normally express Fas in vitro, and it was previously postulated that the rejection of these tumors by NK cells is strictly perforin dependent. We show that perforin-deficient NK cells directly mediate Fas up-regulation on RMA-S cells and thereafter kill the cells in a Fas-dependent manner, and that RMA-S FLIP(L) and RMA-S CrmA are protected from such killing. When injected in immunocompetent recipients, RMA-S cells up-regulate Fas, rendering in vivo-passed mock-transduced cells sensitive to Fas-mediated apoptosis. Moreover, RMA-S FLIP(L) and RMA-S CrmA cells establish aggressive tumors, in contrast to RMA-S mock cells that are rejected. These results demonstrate that FLIP(L) and CrmA function as tumor progression factors by protecting MHC class I-deficient tumors from rejection mediated by NK cells. Moreover, our data indicate that death receptor-mediated apoptosis has a more prominent role in the clearance of NK-sensitive tumors than previously suggested.     

Influenza virus infection augments NK cell inhibition through reorganization of major histocompatibility complex class I proteins

The killing by natural killer (NK) cells is regulated by inhibitory, costimulatory, and activating receptors. The inhibitory receptors recognize mainly major histocompatibility complex (MHC) class I molecules, while the activating NK receptors recognize stress-induced ligands and viral products. Thus, changes in the expression of the various inhibitory and activating ligands will determine whether target cells will be killed or protected. Here, we demonstrate that after influenza virus infection the binding of the two NK inhibitory receptors, KIR2DL1 and the LIR1, to the infected cells is specifically increased. The increased binding occurs shortly after the influenza virus infection, prior to the increased recognition of the infected cells by the NK activating receptor, NKp46. We also elucidate the mechanism responsible for this effect and demonstrate that, after influenza virus infection, MHC class I proteins redistribute on the cell surface and accumulate in the lipid raft microdomains. Such redistribution allows better recognition by the NK inhibitory receptors and consequently increases resistance to NK cell attack. In contrast, T-cell activity was not influenced by the redistribution of MHC class I proteins. Thus, we present here a novel mechanism, developed by the influenza virus, of inhibition of NK cell cytotoxicity, through the reorganization of MHC class I proteins on the cell surface.     

Recognition of HLA-Cw4 but not HLA-Cw6 by the NK cell receptor killer cell Ig-like receptor two-domain short tail number 4

NK cells are cytotoxic to virus-infected and tumor cells that have lost surface expression of class I MHC proteins. Target cell expression of class I MHC proteins inhibits NK cytotoxicity through binding to inhibitory NK receptors. In contrast, a similar family of activating NK receptors, characterized by the presence of a charged residue in their transmembrane portion and a truncated cytoplasmic tail, augment lysis by NK cells when ligated by an appropriate class I MHC protein. However, the class I MHC specificity of many of these activating NK receptors is still unknown. Here, we show enhanced lysis of HLA-Cw4 but not HLA-Cw6-expressing cells, by a subset of NK clones. This subset may express killer cell Ig-like receptor two-domain short tail number 4 (KIR2DS4), as suggested by staining with various mAb. It is still possible, however, that these clones may express receptors other than KIR2DS4 that might recognize HLA-Cw4. Binding of KIR2DS4-Ig fusion protein to cells expressing HLA-Cw4 but not to those expressing HLA-Cw6 was also observed. The binding of KIR2DS4-Ig to HLA-Cw4 is weaker than that of killer cell Ig-like receptor two-domain long tail number 1 (KIR2DL1)-Ig fusion protein; however, such weak recognition is capable of inhibiting lysis by an NK transfectant expressing a chimeric molecule of KIR2DS4 fused to the transmembrane and cytoplasmic portion of KIR2DL1. Residue alpha14 is shown to be important in the KIR2DS4 binding to HLA-Cw4. Implications of the role of the activating NK receptors in immunosurveillance are discussed.     

Using carboxyfluorescein diacetate succinimidyl ester to monitor human NK cell division: analysis of the effect of activating and inhibitory class I MHC receptors

Human NK cells labelled intracellularly with the fluorescent dye 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) were used to assess the effect of ligating class I MHC receptors on NK cell division. The NK cell lines used in these studies expressed a selection of the killer immunoglobulin-like receptors CD158b and CD158a and the CD94/NKG2 family of C-type lectin receptors. The NK cells were cultured in medium containing recombinant (r)IL-2 and receptors were ligated using plastic bound mAb or using soluble murine IgG mAb and FcRII+ gamma-irradiated murine P815 cells. The results obtained show that ligating class I MHC-activating receptors in either culture system stimulates NK cells to divide. Quantitative analysis of cell division reveals that a substantial loss of NK progenitor cells occurs when NK cell-activating receptors are ligated using plastic bound mAb, consistent with concomitant activation-induced cell death. By contrast, progenitor cell loss is prevented when activating receptors are ligated using soluble mAb and P815 cells, suggesting a role for cellular costimulation in cell survival. When inhibitory receptors are coligated with activating receptors using soluble mAb and P815 cells, NK cell division is inhibited. These results demonstrate the potential importance of the activating and inhibitory class I MHC receptors in regulating NK cell proliferation.     

Ly49I NK cell receptor transgene inhibition of rejection of H2b mouse bone marrow transplants

The Ly49 family of genes encode NK cell receptors that bind class I MHC Ags and transmit negative signals if the cytoplasmic domains have immunoregulatory tyrosine-based inhibitory motifs (ITIMs). 5E6 mAbs recognize Ly49C and Ly49I receptors and depletion of 5E6+ NK cells prevents rejection of allogeneic or parental-strain H2d bone marrow cell (BMC) grafts. To determine the function of the Ly49I gene in the rejection of BMC grafts, we transfected fertilized eggs of FVB mice with a vector containing DNA for B6 strain Ly49I (Ly49IB6). Ly49IB6 is ITIM+ and is recognized by 5E6 as well as Ly49I-specific 8H7 mAbs. Normal FVB H2q mice reject H2b but not H2d BMC allografts, and the rejection of H2b BMC was inhibited partially by anti-NK1.1 and completely by anti-asialo GM1, but not by anti-CD8, Abs. In FVB mice, NK1.1 is expressed on only 60% NK cells. FVB. Ly49IB6 hosts failed to reject H2d or H2b BMC, but did reject class I-deficient TAP-1-/- BMC, indicating that NK cells were functional. Nondepleting doses of anti-Ly49I Abs reversed the acceptance of H2b BMC by FVB.Ly49IB6 mice. FVB.Ly49IB6+/- mice were crossed and back-crossed with 129 mice-H2b, 5E6-, poor responders to H2d BMC grafts. While transgene-negative H2b/q F1 or first-generation back-crossed mice rejected H2b marrow grafts (hybrid resistance), transgene-positive mice did not. Thus B6 strain Ly49I receptors transmit inhibitory signals from H2b MHC class I molecules. Moreover, Ly49IB6 has no positive influence on the rejection of H2d allografts.     

Intercellular transfer of carcinoembryonic antigen from tumor cells to NK cells

The inhibition of NK cell killing is mainly mediated via the interaction of NK inhibitory receptors with MHC class I proteins. In addition, we have previously demonstrated that NK cells are inhibited in a class I MHC-independent manner via homophilic carcinoembryonic Ag (CEA) cell adhesion molecules (CEACAM1)-CEACAM1 and heterophilic CEACAM1-CEA interactions. However, the cross-talk between immune effector cells and their target cells is not limited to cell interactions per se, but also involves a specific exchange of proteins. The reasons for these molecular exchanges and the functional outcome of this phenomenon are still mostly unknown. In this study, we show that NK cells rapidly and specifically acquire CEA molecules from target cells. We evaluated the role of cytotoxicity in the acquisition of CEA and demonstrated it to be mostly killing independent. We further demonstrate that CEA transfer requires a specific interaction with an unknown putative NK cell receptor and that carbohydrates are probably involved in CEA recognition and acquisition by NK cells. Functionally, the killing of bulk NK cultures was inhibited by CEA-expressing cells, suggesting that this putative receptor is an inhibitory receptor.     

CD66a interactions between human melanoma and NK cells: a novel class I MHC-independent inhibitory mechanism of cytotoxicity

NK cells are able to kill virus-infected and tumor cells via a panel of lysis receptors. Cells expressing class I MHC proteins are protected from lysis primarily due to the interactions of several families of NK receptors with both classical and nonclassical class I MHC proteins. In this study we show that a class I MHC-deficient melanoma cell line (1106mel) is stained with several Ig-fused lysis receptors, suggesting the expression of the appropriate lysis ligands. Surprisingly, however, this melanoma line was not killed by CD16-negative NK clones. The lack of killing is shown to be the result of homotypic CD66a interactions between the melanoma line and the NK cells. Furthermore, 721.221 cells expressing the CD66a protein were protected from lysis by YTS cells and by NK cells expressing the CD66a protein. Redirected lysis experiments demonstrated that the strength of the inhibitory effect is correlated with the levels of CD66a expression. Finally, the expression of CD66a protein was observed on NK cells derived from patients with malignant melanoma. These findings suggest the existence of a novel class I MHC-independent inhibitory mechanism of human NK cell cytotoxicity. This may be a mechanism that is used by some of the class I MHC-negative melanoma cells to evade attack by CD66a-positive NK cells.     

Human NK cells differ more in their KIR2DL1-dependent thresholds for HLA-Cw6-mediated inhibition than in their maximal killing capacity

In this study we have addressed the question of how activation and inhibition of human NK cells is regulated by the expression level of MHC class I protein on target cells. Using target cell transfectants sorted to stably express different levels of the MHC class I protein HLA-Cw6, we show that induction of degranulation and that of IFN-γ secretion are not correlated. In contrast, the inhibition of these two processes by MHC class-I occurs at the same level of class I MHC protein. Primary human NK cell clones were found to differ in the amount of target MHC class I protein required for their inhibition, rather than in their maximum killing capacity. Importantly, we show that KIR2DL1 expression determines the thresholds (in terms of MHC I protein levels) required for NK cell inhibition, while the expression of other receptors such as LIR1 is less important. Furthermore, using mathematical models to explore the dynamics of target cell killing, we found that the observed delay in target cell killing is exhibited by a model in which NK cells require some activation or priming, such that each cell can lyse a target cell only after being activated by a first encounter with the same or a different target cell, but not by models which lack this feature.     

HSV-2 Specifically Down Regulates HLA-C Expression to Render HSV-2-Infected DCs Susceptible to NK Cell Killing

Both NK cells and CTLs kill virus-infected and tumor cells. However, the ways by which these killer cells recognize the infected or the tumorigenic cells are different, in fact almost opposite. CTLs are activated through the interaction of the TCR with MHC class I proteins. In contrast, NK cells are inhibited by MHC class I molecules. The inhibitory NK receptors recognize mainly MHC class I proteins and in this regard practically all of the HLA-C proteins are recognized by inhibitory NK cell receptors, while only certain HLA-A and HLA-B proteins interact with these receptors. Sophisticated viruses developed mechanisms to avoid the attack of both NK cells and CTLs through, for example, down regulation of HLA-A and HLA-B molecules to avoid CTL recognition, leaving HLA-C proteins on the cell surface to inhibit NK cell response. Here we provide the first example of a virus that through specific down regulation of HLA-C, harness the NK cells for its own benefit. We initially demonstrated that none of the tested HSV-2 derived microRNAs affect NK cell activity. Then we show that surprisingly upon HSV-2 infection, HLA-C proteins are specifically down regulated, rendering the infected cells susceptible to NK cell attack. We identified a motif in the tail of HLA-C that is responsible for the HSV-2-meduiated HLA-C down regulation and we show that the HLA-C down regulation is mediated by the viral protein ICP47. Finally we show that HLA-C proteins are down regulated from the surface of HSV-2 infected dendritic cells (DCs) and that this leads to the killing of DC by NK cells. Thus, we propose that HSV-2 had developed this unique and surprising NK cell-mediated killing strategy of infected DC to prevent the activation of the adaptive immunity.     

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.     

NK cells promote islet allograft tolerance via a perforin-dependent mechanism

Although major histocompatibility complex (MHC) class II-restricted CD4 T cells are well appreciated for their contribution to peripheral tolerance to tissue allografts, little is known regarding MHC class I-dependent reactivity in this process. Here we show a crucial role for host MHC class I-dependent NK cell reactivity for allograft tolerance in mice induced through either costimulation blockade using CD154-specific antibody therapy or by targeting LFA-1 (also known as CD11a). Tolerance induction absolutely required host expression of MHC class I, but was independent of CD8 T cell-dependent immunity. Rather, tolerance required innate immunity involving NK1.1(+) cells, but was independent of CD1d-restricted NKT cells. Therefore, NK cells seem to be generally required for induction of tolerance to islet allografts. Additional studies indicate that CD154-specific antibody-induced allograft tolerance is perforin dependent. Notably, NK cells that are perforin competent are sufficient to restore allograft tolerance in perforin-deficient recipients. Together, these results show an obligatory role for NK cells, through perforin, for induction of tolerance to islet allografts.     

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.