Current perspectives of natural killer cell education by MHC class I molecules

Natural killer (NK) cells are immune cells that medi-ate their effector functions efficiently without prior exposure to antigen and depend on germline-encoded receptors for their activation. NK cells have highly spe-cific target cell recognition mechanisms, which trigger cytotoxicity and the secretion of cytokines. NK cells have been traditionally classified as innate immune cells; however, recent reports suggest that similar to B and T cells, NK cells may also display immuno-logical memory1–3, which blurs the distinction between the properties of NK cells and adaptive lymphocytes. Furthermore, NK cells may have an important role in early protection against viral infection4. Genetic evi-dence also indicates associations between the genes and alleles of killer cell immunoglobulin-like receptors (KIRs) (a major family of NK cell receptors) and the development of autoimmune diseases, and the out-come of pregnancy and infections, suggesting a role for NK cells in many different diseases and fundamental biological processes5. Although NK cells can mediate cell killing, they can also influence other immune cells by secreting cytokines6.

Dominant control of NK cell activation is mediated by inhibitory receptors that bind to MHC class I mol-ecules on surrounding cells. Because of the abundant expression pattern of MHC class I molecules on normal cells as markers of ‘self ’, these cells can resist NK cell attack. However, when cells lose expression of one or more MHC class I alleles, which can occur during certain viral infections or neoplastic transformations, they often, but not always, become NK cell targets. This process is known as missing-self recognition and has been observed

in allogeneic stem cell transplantation, in which it can contribute to a potent graft-versus-leukaemia effect and improve patient survival7–9.

The phenomenon of ‘hybrid resistance’ against parental haematopoietic bone marrow grafts, which was discovered more than 50 years ago in mice, laid the foundations for the work on NK cell education that is discussed in this Review. Hybrid resistance refers to a situation in which parental haematopoietic bone marrow grafts, but not solid tissue grafts, are rejected by F1 hybrid mice10. This rejection was not predicted by the laws of transplantation at that time, which stated that graft rejection required the presence of foreign MHC molecules on the graft compared to the host. Hybrid resistance was shown to be thymus-independent11, excluding a role for conventional T cells; however, mis-matches in the MHC complex between recipients and donors were crucial determinants of the outcome12. The involvement of NK cells in hybrid resistance was shown in the mid-1970s13. The finding that the introduction of an H-2Dd transgene onto an H-2b background resulted in NK cell-mediated natural resistance to grafts that expressed H-2b (REFS 14,15) showed that MHC class I molecules themselves affected the specificity of graft rejection by NK cells in a hybrid resistance-like situ-ation. The subsequent discovery of inhibitory Ly49 receptors16, which are specific for MHC class I mol-ecules, was an important landmark that explained how NK cells interacted with MHC molecules. Interactions between inhibitory receptors and MHC class I mole-cules are now known to be required for the development of fully functional NK cells17–20.

Department of Microbiology, Tumour and Cell Biology, Karolinska Institutet, Box 280, SE‑171 77, Stockholm, Sweden.Correspondence to P.H.e‑mail: [email protected] doi:10.1038/nri2835Published online 6 September 2010

Killer cell immunoglobulin-like receptors (KIRs). These receptors, which are encoded on human chromosome 19, are expressed by natural killer cell subsets and by a minor population of T cells. Inhibitory KIRs have locus and allele specificity for MHC class I molecules.

Missing-self recognitionThe recognition and attack of cells that do not express MHC class I molecules — in other words, that are ‘missing self’ — by natural killer cells. This provides a surveillance mechanism to detect virally infected or transformed cells that downregulate MHC class I expression.

Current perspectives of natural killer cell education by MHC class I moleculesPetter Höglund and Petter Brodin

Abstract | From the early days of natural killer (NK) cell research, it was clear that MHC genes controlled the specificity of mouse NK cell-dependent responses, such as the ability to reject transplanted allogeneic bone marrow and to kill tumour cells. Although several mechanisms that are involved in this ‘education’ process have been clarified, most of the mechanisms have still to be identified. Here, we review the current understanding of the processes that are involved in NK cell education, including how the host MHC class I molecules regulate responsiveness and receptor repertoire formation in NK cells and the signalling pathways that are involved.

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© 20 Macmillan Publishers Limited. All rights reserved10

mailto:[email protected]

Graft-versus-leukaemia An immune response that is mounted by the transplanted cells against the tumour cells of the host. This response is one of the reasons why allogeneic transplants can be curative for cancer.

In this Review, we discuss current models on how MHC class I molecules control the functional develop-ment of NK cells and use the term education to describe this process. We summarize the read-outs that are used to measure NK cell education and review the studies that have investigated the influence of activating and inhibi-tory signals on this process. Finally, we discuss how signals that are induced by MHC class I molecules in mice lead to the formation of a unique NK cell repertoire, which is composed of NK cell subsets that are optimally tuned for efficient missing-self recognition and that together determine the global NK cell function in an individual.

MHC class I receptors and NK cell educationThe most extensively studied MHC class I-specific inhibitory receptors that are expressed by NK cells are the Ly49 receptors (in mice)16 and KIRs (in humans)21,22. Both Ly49 receptors and KIRs are encoded in polygenic and polymorphic gene families. In humans, 17 KIR-encoding genes have been identified and are clustered in combinations that produce more than 40 haplotypes23. A limited comparison of the Ly49 locus in four inbred mouse strains showed great variation in the number of genes and alleles between the strains24, suggesting that the Ly49 locus in mice may be as complex as the human KIR locus. In addition to Ly49 receptors and KIRs, the conserved NK group 2, member A (NKG2A) recep-tor binds to the non-classical MHC class I molecules Qa-1b (in mice) and HLA-E (in humans)25,26. Human NK cells also express immunoglobulin-like transcript 2 (ILT2), which recognizes the non-classical MHC class I molecule HLA-G27.

Until recently, it was thought that all NK cells expressed at least one inhibitory receptor for self-MHC class I mol-ecules; this model was supported by early work using human NK cell clones28,29 and provided a satisfactory explanation for self tolerance and for the involvement of NK cells in missing-self recognition. In parallel, studies of humans and mice that lacked MHC class I molecules had shown that although these individuals had normal numbers of NK cells, they were hypo responsive30–33. These NK cells were thought to be unique to MHC class I-deficient individuals, but work from several lab-oratories has now shown that hyporesponsive NK cells exist in normal humans and mice17,18,20,34. It has also become clear that active engagement of inhibitory recep-tors on NK cells by self-MHC class I molecules is the key event that determines whether an NK cell will be functionally capable of mediating missing-self recogni-tion (education), or whether it will be hyporesponsive in terms of cytotoxicity and cytokine secretion following stimulation. Currently, because no marker for educated NK cells exists, the distinction between an educated and an uneducated NK cell can only be made by using functional assays. BOX 1 and FIG. 1 summarize four types of read-outs that are most frequently used to measure NK cell education, and highlight the advantages and disadvantages of each read-out.

Regarding the functional consequences of NK cell education, it is clear that binding to self-MHC class I molecules results in the ability of NK cells to respond to tumour cells, MHC-deficient cells and antibody-mediated crosslinking of activating receptors in vitro17,18,20,34. The ability of NK cells to reject haematopoietic bone marrow

Box 1 | Measures of NK cell education

In vivo rejection studiesA classical read-out for natural killer (NK) cell education in vivo is the rate of rejection of long-term transplanted cells, including bone marrow or tumour cells. More quantitative in vivo assays that use fluorescently labelled target cells have recently been developed and have made in vivo rejection studies much more useful for these studies (FIG. 1a). A drawback of in vivo studies in general is that it is not possible to directly investigate the responses of individual NK cells or NK cell subsets.

Cell–cell interaction assaysAssays that are based on cellular interactions in vitro (FIG. 1b) are easier to control in terms of kinetics and specificity, and the response of individual cells can be measured through the use of flow cytometry. However, the potential impact of inhibitory receptors or of other unknown receptor–ligand combinations on the interaction under study is difficult to control.

Stimulation of activating receptorsThe cleanest way to measure the intrinsic responsiveness in individual NK cells is to directly stimulate a given activating receptor, such as NK cell-associated antigen 1.1 (NK1.1), NK group 2, member D (NKG2D) or NK cell protein 46 (NKp46), through the use of antibodies, and to quantify the strength of the effector response that is induced by that receptor through the use of flow cytometry (FIG. 1c). Using this technique, it is possible to quantify the responsiveness of an individual NK cell to a particular activation pathway without the involvement of inhibitory receptors. How well the strength of the signal that is induced by the antibody stimulation relates to that of the natural ligand is unknown, which is a weakness of this technique.

Measures of the inhibitory receptor repertoire Changes in the inhibitory receptor repertoire can potentially be used as a measure of NK cell education (FIG. 1d), at least in mice. In humans, the influence of MHC class I molecules on the NK cell repertoire is less clear and is more difficult to study owing to the higher complexity of human studies compared with studies in mice.

Conclusion and future directionsIn reality, it is necessary to perform all of these assay types in parallel, as they allow for different types of conclusions. As a future development, a direct in vitro assay for inhibitory receptor function would be useful to facilitate quantitative measures of NK cell education in complex interactions.

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Nature Reviews | Immunology

a In vivo rejection b Cell–cell interactions

c Stimulation of activating receptor d NK cell inhibitory receptor repertoire

High dose CFSE

Low dose CFSE

Mix

Read-outs: Killing, cytokine secretion, activation, adhesion

Advantages: Physiological, allows measures of adhesion

Disadvantages: Net outcome of several receptor–ligand interactions

Read-outs: Degranulation, cytokine secretion, cell activation

Advantages: Specific, reductionistic, quantitative

Disadvantages: Less physiological

Read-outs: The frequencies of NK cell subsets that express variouscombinations of inhibitory receptors

Advantages: Physiological, ex vivo

Disadvantages: Not possible to assess functional relevance

MHC–

MHC+

Inoculation

No rejection

RejectionNK cell

Target cell

NK cellLy49D only(activating receptor)

Activating receptor

Antibody

Ly49A only(inhibitoryreceptor)

Read-outs: Rejection of MHC– compared with MHC+ cells

Advantages: Physiological, in vivo, quantitative, rapid

Disadvantages: Difficult to identify NK cell receptors involved in rejection

L2

L1

L3

R2

R1

R3

β2-microglobulin (β2m). A single immunoglobulin-like domain that non-covalently associates with the main polypeptide chain of MHC class I molecules. In the absence of β2m, MHC class I molecules are unstable and are therefore found at very low levels at the cell surface.

grafts in vivo is also tightly coupled to NK cell educa-tion events, although this is more difficult to study at the single cell level14,15,19,31,32. However, the role of NK cell education in other complex functions in vivo, such as the ability to control viral infection, is less clear. Nevertheless, a recent study has shown that NK cells that lack self-specific Ly49 receptors (the so-called ‘uneducated’ NK cells) responded more strongly when stimulated with a murine cytomegalovirus (MCMV)-derived antigen35. In addition, following adoptive transfer, these NK cells pro-vided better protection against MCMV infection than cells that expressed the self-specific Ly49 receptors35. Some caution should be taken when interpreting these findings because the possible roles of the self-specific NKG2A receptor, which is known to be sufficient for NK cell education34, were not taken into consideration. Nevertheless, this study is similar to previous studies that used β2-microglobulin (β2m)-deficient mice, which

lack MHC class I molecules, and, consequently, also lack educated NK cells. These mice were shown to control MCMV infections as efficiently as normal mice did36. More work is required to investigate the link between NK cell education and the various functional properties of NK cells in vivo.

Finally, it is interesting to discuss NK cell educa-tion in relation to the theory of NK cell ‘priming’. This theory proposes that NK cells need a priming signal, possibly interleukin-15 (IL-15) that is presented in trans by dendritic cells37, or secreted IL-18 (REF. 38), to be able to mediate their effector functions. It is not currently known how NK cell priming and NK cell education are related; priming is required in MHC-expressing animals, in which NK cell education by MHC class I molecules is also operational, but whether priming is required in MHC-deficient animals, which contain only uneducated NK cells, has not yet been examined.

Figure 1 | Measures of NK cell education. a | In vivo rejection of 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled cells. This method allows quick and quantitative measurements of natural killer (NK) cell-mediated rejection rates over time. b | In vitro assays based on NK cell–target cell interactions that are mediated through different receptors (R) and ligands (L), the effects of which can be determined, for example, by classical chromium release. c | Direct stimulation of activating receptors with antibodies allows NK cell responsiveness to specific stimuli to be measured directly without the interference of other receptors. d | Measurements of the expression levels of inhibitory receptors by NK cells using multicolour flow cytometry allow for the estimation of the size of NK cell subsets that express various combinations of inhibitory receptors.

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a Arming model b Disarming model

MHC class I

WeakMHC class Iligand

StrongMHC class Iligand

Inhibitoryreceptor

NK cell

Educating cell

Activatingreceptor

Activatingligand

Unarmed(hyporesponsive)

Armed(responsive)

Disarmed(hyporesponsive)

Balanced(responsive)

+/–? + + –

–

c Cis-interaction model d Rheostat modelNo cis interaction(hyporesponsive)

Cis interaction(responsive)

Weakly responsive

Stronglyresponsive

+– +–

Models for NK cell educationSeveral models have been proposed to describe various aspects of NK cell education by MHC class I molecules (FIG. 2). Although each model contains unique features, they also share several key aspects and are, therefore, best seen as complementary to stimulate further work and debate, and not as mutually exclusive models of the same biological process.

Arming model. In the arming model (FIG. 2a), NK cells acquire functional competence after ligation of inhibitory receptors by self-MHC class I molecules. Signalling from inhibitory receptors thus promotes functional maturation in an active way. The difficulty in conceiving an ‘instruc-tive’ role for inhibitory receptors has led to criticism of the arming model. However, recent studies have shown that signalling via immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptors can lead to phosphorylation of signalling substrates downstream of ITIM phosporylation39, giving credence to this model40. The term NK cell ‘licensing’, which was coined by Kim and Yokoyama17, is commonly used synonymously with arming, but could also imply other mechanisms41.

Disarming model. The disarming model postulates that NK cells that lack inhibitory receptors for self-MHC class I molecules are ‘disarmed’ to retain self-tolerance, resulting in a form of NK cell anergy42 (FIG. 2b). Interactions with MHC class I molecules prevent this process and the NK cell matures normally, making this model easier to reconcile with a more conventional role for inhibitory receptor signalling. The main distinc-tions between the arming and disarming models are thus twofold: in the arming model, NK cells are hypo-responsive by default, whereas the disarming model sug-gests that NK cells are responsive in their initial state. Furthermore, arming implies a new instructive role for inhibitory receptors, whereas disarming implies a conventional inhibitory role.

Cis-interaction model. A third model (FIG. 2c) is based on the ability of Ly49 receptors to bind to MHC class I alleles in cis, that is, in the same cell membrane43–47. The basis for this model was the observation that Ly49 recep-tors can transmit inhibitory signals even in the absence of ligand interaction, that is, as ‘unengaged’ receptors, provided that they accumulate in the immunological synapse at the target cell interphase45. According to the model, interactions between MHC class I molecules and Ly49 receptors in cis sequesters Ly49 receptors and pre-vents their relocation to the immunological synapse. As a consequence, the inhibitory influence of unengaged Ly49 receptors on signalling from activating receptors decreases and the NK cell becomes more responsive45. It is unknown whether all Ly49 receptors (or human KIRs) can engage MHC class I molecules in cis, making the generality of the cis-interaction model unclear.

Rheostat model. Both the arming and disarming mod-els propose that NK cell education elicits binary sig-nals, leading to NK cells that are either ‘on’ or ‘off ’. By contrast, NK cells have recently been shown to have increased or decreased responsiveness depending on the strength of the inhibitory signal that is received, sug-gesting that NK cell education operates in a quantitative manner19,48,49. This aspect of NK cell education, termed the rheostat model34 (FIG. 2d), states that NK cell respon-siveness can either be ‘tuned up’ (arming-like) or ‘tuned down’ (disarming-like), but in a quantitative rather than in a binary manner depending on the inhibitory signal

Figure 2 | Models for NK cell education. a | In the arming model, signalling by an inhibitory receptor leads to natural killer (NK) cell functional competence; in the absence of arming, the NK cell remains hyporesponsive. b | In the disarming model, the NK cell is activated by default; however, in the absence of an inhibitory receptor for self-MHC class I molecules, the NK cell becomes anergic or hyporesponsive. The presence of an inhibitory receptor, in contrast, rescues the NK cell from anergy and allows it to be responsive. c | The cis-interaction model is based on the ability of inhibitory receptors to bind to MHC class I molecules in cis, that is, in the same cell membrane. This binding has been suggested to prevent a ligand-independent inhibitory signal and, thus, change a hyporesponsive NK cell to a responsive NK cell; this signal is delivered by the unengaged Ly49 receptors that cluster in the immune synapse. d | The rheostat model represents a dynamic view of NK cell education. Based on the strength of the inhibitory signal during NK cell education, the responding NK cell balances its activation threshold as a rheostat, which allows the maturation of NK cells to be optimally tuned by the inhibitory input.

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P

PP


MHC class I

Inhibitoryreceptor

NK cell

Target cell

SHP1ITIM

ABL1

CBLVAV1

VAV1

Dephosphorylation

Disruption of activation

Phosphorylation

Complexdisruption

CRK

C3G

2

1

3

Anergy A state of T cell unresponsiveness following stimulation with an antigen. It can be induced by stimulation with a large amount of a specific antigen in the absence of engagement of co-stimulatory molecules.

E3 ubiquitin ligase The enzyme that is required to attach the molecular tag ubiquitin to proteins that are destined for degradation by the proteasomal complex.

Motheaten viable mice(mev/mev mice). Mice that have a mutation in the coding region of the catalytic domain of SH2-domain-containing protein tyrosine phosphatase 1 (SHP1), which results in two aberrant loss-of-function proteins (one of 67 kDa and one of 71 kDa). These mice develop severe combined immunodeficiency and systemic autoimmunity.

that is received by individual NK cells48,49. The rheostat model is thus consistent with both the arming and the disarming model, and could also mechanistically be compatible with the cis-interaction model48,50.

Signalling in NK cell educationTo understand how NK cell education is mediated, it is necessary to understand the signalling pathways that are involved in transmitting signals from both inhibitory and activating receptors in NK cells.

Inhibitory signalling. Inhibitory Ly49 receptors and KIRs contain ITIMs in their cytoplasmic tail, which are phospho rylated, probably by a SRC-family kinase, leading to the recruitment of phosphatases: mainly SH2-domain-containing protein tyrosine phosphatase 1 (SHP1), but also SHP2 and SH2-domain-containing inositol-5- phosphatase (SHIP)51. One consequence of this inhibi-tory signalling is the dephosphorylation of the nucleotide exchange factor VAV1, leading to a block in the downstream propagation of activating signals52 (FIG. 3).

However, it was recently shown that ITIM-containing receptors can also mediate other downstream signals in parallel with the dephosphorylation of VAV1, such as the disruption of a signalling complex that is comprised of the E3 ubiquitin ligase Casitas B-lineage lymphoma (CBL; also known as c-Cbl), the adaptor protein CRK and the guanine exchange factor C3G39. This dissocia-tion is associated with the phosphorylation of CRK by the tyrosine-protein kinase ABL1 (also known as c-Abl) and the formation of CRK–ABL1 complexes (FIG. 3). Therefore, this signalling pathway involves an active phosphorylation event that occurs downstream of the ITIM motifs39,40, an event that has not been described previously following ligation of an ITIM-coupled recep-tor. However, the importance of this signalling pathway requires further study and independent confirmation from other groups.

Another recently described mechanism of inhibition, possibly not directly linked to the ligation of inhibitory receptors, acts to adjust the threshold of activation for human NK cells in the steady state. This mechanism involves an inhibitory effect by CBL on phosphorylated VAV1 (FIG. 3), which must be overcome for activation to occur. This model explains why synergistic activation signals are required for resting human NK cells to reach a threshold of sufficient activation to override this inhibi-tion53,54. Thus, signalling from inhibitory receptors and the balance between activation and inhibition is increas-ingly complex with several parallel pathways acting together. Whether the net outcome of these signals will be termination of activating signals, education or both, is likely to be influenced by tissue location, the differentia-tion status of the NK cell and factors that are provided by other immune cells.

Many studies have linked NK cell education with interactions between inhibitory receptors and MHC class I molecules17–20,34,48,49,55,56. Fewer studies, however, have directly addressed the roles of inhibitory signalling molecules in NK cell education. Kim et al. showed that the ITIM of the inhibitory Ly49A receptor was involved

in NK cell education17. However, arming still occurred in motheaten viable mice (mev/mev mice), in which the ITIM phosphatase SHP1 has strongly reduced activity17, suggesting that ITIM-mediated signalling for arming occurs independently of SHP1. A more recent study by Orr et al. showed similar results using mev/mev mice in an MCMV model system35.

By contrast, Lowin-Kropf et al. showed an impor-tant role for SHP1 in NK cell education by using transgenic mice that expressed a dominant-negative mutation of SHP1 (REF. 57). In these mice, killing of MHC class I-deficient target cells was decreased, which was proposed to be due to poor NK cell responsive-ness that was mediated by aberrant inhibitory signalling during NK cell education57. No explanation for the dis-crepancy between these studies has been supported by data in the literature, but one possibility is that different levels of SHP1 expression regulate arming and func-tional activation; low residual levels of SHP1 activity

Figure 3 | Three layers of inhibitory influences in NK cells. Initial signalling from immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing inhibitory receptors involves the recruitment of SH2-domain-containing protein tyrosine phosphatase 1 (SHP1). The classical view of how SHP1 mediates inhibition is by the dephosphorylation of the nucleotide exchange factor VAV1, which leads to the termination of NK cell activating signals (circled 1 in the figure). A second, simultaneous pathway of inhibition is the tyrosine-protein kinase ABL1-mediated phosphorylation of the adaptor protein CRK, which leads to the disruption of an activation complex consisting of the E3 ubiquitin ligase Casitas B-lineage lymphoma (CBL; also known as c-Cbl), CRK and the guanine exchange factor C3G (circled 2 in the figure). A third, recently suggested pathway involves the continuous inhibition of phosphorylated VAV1 by CBL, which counteracts activating signals (circled 3 in the figure). This pathway does not seem to be directly controlled by inhibitory receptors, but is nevertheless important as it involves molecules that are involved in inhibitory receptor function. It is not known whether these pathways can occur independently or function in an integrated manner.

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in mev/mev mice but not in mice expressing mutant SHP1 would thus distinguish these two facets of ITIM-mediated signalling. Indeed, different thresholds for arming and functional activation mediated by MHC class I molecules have been suggested55,58.

A role for another ITIM-associated phosphatase, SHP2 (REF. 59), has been proposed to account for the ITIM-dependent but SHP1-independent nature of NK cell education. It is also possible that the impaired NK cell responsiveness in mice that express mutant SHP1 (REF. 57) is caused by the binding of the mutant SHP1 molecules to the Ly49 ITIMs, which blocks the bind-ing of other, unknown molecules that might be impor-tant for transmitting educating signals following MHC class I binding.

SHIP is another molecule that is involved in inhibi-tory NK cell receptor signalling. When SHIP expression was disrupted in mice, unexpected alterations in the inhibitory Ly49 receptor repertoire were seen, including an increased frequency of NK cells expressing Ly49A, Ly49C and Ly49I and a decreased frequency of NK cells expressing Ly49G2 and CD94 (REF. 60). Interestingly, a recent study from the same group showed that this effect was dependent on the MHC haplotype because the increase in the number of Ly49A-expressing NK cells was only seen in mice that expressed a strong MHC class I ligand for the H-2Dd receptor and not in mice expressing a weak ligand61. This intriguing result suggests that SHIP may be involved in regulating the frequencies of self-spe-cific inhibitory receptors during MHC class I-mediated NK cell education. The mechanism responsible for this effect remains to be investigated, but it may involve a direct role for SHIP in signalling from Ly49A and Ly49C, receptors that SHIP has been shown to directly associate with60 or result from indirect effects that are triggered by loss of function of other known SHIP-binding receptors, such as the 2B4 (also known as CD244) receptor61.

Phosphatases have key functions in various immune cells and therefore other immune cells, in addition to NK cells, are affected in mouse models of phosphatase defi-ciency62,63. Thus, data from these models must be inter-preted with caution64. More work is needed in alternative settings, possibly using conditional and NK cell-specific mutants, to more precisely test the differences that are outlined above in relation to NK cell education.

Activating signals. NK cells express a wide array of acti-vating receptors. Some of these are expressed by most or all NK cells, such as 2B4 (which has both an activating and an inhibitory function), NK cell protein 46 (NKp46), NK cell-associated antigen 1.1 (NK1.1), NKG2D and CD16, whereas others, including activating KIRs, Ly49D, Ly49H, DNAX accessory molecule 1 (DNAM1) and NKG2C, are only expressed by subsets of NK cells. The signalling pathways that are triggered by these receptors can be divided into at least three groups: immunoreceptor tyrosine-based activation motif (ITAM)-mediated signal-ling pathways51, DAP10-mediated signalling pathways51

and the specific 2B4-mediated signalling pathway51, which belongs to the signalling lymphocytic activation molecule (SLAM) family of receptors.

The roles of activating receptor signalling in NK cell education are poorly understood and different models propose different requirements for them (FIG. 2). To look for such effects we collected studies that examined the effects of various signalling molecule mutations on NK cell function in vivo (Supplementary information S1 table). A major problem we experienced when trying to summarize these studies was the difficulty in sepa-rating the effects of a given mutation on NK cell educa-tion from the effects on effector functions in mature NK cells. The reason for this is the use of the same read-out in both situations, that is, functional competence. A future challenge for the field will be to identify markers that can distinguish educated from uneducated NK cells and that are independent from NK cell function.

Nevertheless, one conclusion that could be drawn from these studies was that no single mutation could reproduce the broadly hyporesponsive phenotype of NK cells from MHC class I-deficient mice, suggesting that the regulation of hyporesponsiveness by MHC class I molecules may involve several pathways. Other types of important information were also revealed, such as the presence of marked redundancies, interrelations between adaptor and intermediate molecules downstream of the various activating receptors and the notion that different effector functions, such as production of cytokines (for example, interferon-γ) and cytotoxicity, are controlled by distinct signalling pathways51. The fact that different functions are controlled by different pathways suggests that there is functional heterogeneity in the NK cell pop-ulation; this is supported by the recent hypothesis that NK cells with potentially different functional properties occupy distinct niches in the body65.

One potential marker for education, which is differ-ent from function, would be changes in the frequencies of inhibitory receptors in the NK cell repertoire (FIG. 1d). The expression of Ly49 receptors and KIRs results from an incompletely understood process that is based on the stochastic use of bidirectional promoters for the genes that encode Ly49 molecules66 or KIRs67 that either tran-scribe forward (switched ‘on’) or in reverse (switched ‘off ’). The direction of this on/off switch depends on which one of the several competing transcription fac-tors binds to the promoter. Together this provides the molecular explanation for the selective gene expression in the Ly49 or KIR locus that occurs in individual NK cells66,67. Therefore, each receptor has an independent probability of expression that results in the formation of a complex NK cell repertoire of many different subsets, which express anywhere from zero to five or more KIR or Ly49 receptors55,68,69.

In addition to this transcriptional regulation of recep-tor genes, MHC class I molecules have also been shown to influence the frequencies of NK cells that express Ly49 receptors in mice69–72 and KIRs in humans55, although the latter has been more controversial68. We argue below that in mice at least, this process is based on both the enrichment of some subsets and the contraction of other subsets and that this mechanism serves to tune the rep-ertoire quantitatively to self-MHC class I molecules to secure optimal missing-self recognition in the NK cell

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Table 1 | Genetic modulation of molecules that are involved in activating receptor signalling in mouse NK cells

Targeted molecule

NKG2D pathway

Missing-self recognition

ADCC Ly49D pathway

Stimulation of NK1.1

Stimulation of Ly49D

Polyclonal stimulation

Inhibitory receptor repertoire

Refs

VAV1, VAV2 and VAV3

↓ → ↓ → ND ND ND Normal frequencies of NKG2A and Ly49A

86

BCL-10 → → ND → ↓ ↓ ↓ Higher frequencies of NKG2A and Ly49A, which is reversed by IL-2 stimulation

87–89

CARMA1 → ND ND ND ↓ ↓ ↓ Normal frequencies of NKG2A, Ly49A and Ly49G2

88,89

LAT1 ND ND ND ND → ND → Normal frequency of NKG2A and reduced frequencies of Ly49A, Ly49C, Ly49G2 and Ly49I

74

LAT2 ND ND ND ND ↑ ND → Normal frequency of NKG2A and reduced frequencies of Ly49A, Ly49C, Ly49G2 and Ly49I

74

LAT1 and LAT2

→ → ND ND ↓ ↓ → Unaltered frequencies of NKG2A, NKG2C, NKG2E and Ly49G2, but reduced frequencies of Ly49A, Ly49C and Ly49I

74,90

FYN → ↓ ↓ → ND → ND Reduced frequency of Ly49A, but unaltered frequencies of Ly49C, Ly49G2 and Ly49I

73,91,92

LCK → → ND → ND ND ND Unaltered frequencies of Ly49A, Ly49C, Ly49I and Ly49G2

73,92

LFA1 →, ↓ ↓ ND ND ND ND ND No effect 93–95

CD3ζ, FcγR and DAP12

→ ↓ ND ND ND ND ND Unaltered frequencies of NKG2A, NKG2C and NKG2E; suspected lower frequencies of Ly49A and Ly49G2

90

DAP10 ↓,→ ND ND → → → → Unaltered frequencies of NKG2A, NKG2C and NKG2E

96–98

DAP12 ↓, → → ND ↓ → ↓ ND Unaltered frequencies of NKG2A, NKG2C, Ly49C, Ly49G2 and Ly49I; suspected lower frequency of Ly49A

90, 98–100,

116

CD45 → → → → →, ↓ ↓ → Unaltered frequencies of Ly49A, Ly49C, Ly49F, Ly49G2, Ly49I, NKG2A, NKG2C and NKG2E; reduced frequency of Ly49A

92, 101–103

CRACC ↓ ↓ ND ↓ ND → → Unaltered frequencies of Ly49G2 and NKG2A, NKG2C and NKG2E

104

SAP ↓ ↓ ND → ND ND → Unaltered frequencies of Ly49A, Ly49C, Ly49G2 and Ly49I

91,105

SAP, EAT and ERT

↓ ↓ ND ↑ ND ND → Unaltered frequencies of NKG2A, Ly49C, Ly49F, Ly49G2 and Ly49I

106

NKG2D ↓ → ND ND →, ↑ → → Unaltered frequencies of Ly49A, Ly49C, Ly49F, Ly49G2, Ly49I and NKG2A

107,108

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Antibody-dependent cell-mediated cytotoxicity A mechanism by which natural killer (NK) cells kill other cells, such as virus-infected target cells that are coated with antibodies. The Fc portions of the coating antibodies interact with the Fc receptor (FcγRIII; also known as CD16) that is expressed by NK cells, thereby initiating a signalling cascade that results in the release of cytotoxic granules (containing perforin and granzyme B), which induce apoptosis of the antibody-coated cell.

system. Similarly, changes in the Ly49 receptor reper-toire in mice with targeted mutations in specific compo-nents of activating signalling pathways may thus reflect effects on the process of NK cell education, which can be determined without the complication of having to study effector responses.

Several of the studies that we reviewed identified changes to the Ly49 receptor repertoire following tar-geted mutations of the signalling pathways of NK cells (TABLE 1). For example, deficiency of the SRC-family kinase FYN leads to alterations in the Ly49 receptor repertoire that was interpreted by the authors as effects of an activating signalling pathway on NK cell educa-tion73. FYN expression was also shown to be important for the targeting of cells that have downregulated MHC class I expression. Whether this targeting was because of altered NK cell education or of FYN signalling from activating receptors that are involved in missing-self recognition cannot be discerned. Another example is mice that are deficient for linker for activation of T cells (LAT) and linker for activation of B cells (LAB); these molecules are co-expressed by NK cells and their deficiencies also produce skewed Ly49 receptor rep-ertoires74. Interestingly, although both LAT and LAB contribute to the ITAM-induced signalling pathway downstream of DAP12, they mediate different changes in the Ly49 receptor repertoire after their ablation74,

which suggests that they may affect distinct pathways in NK cell education. Based on the current literature, it is not possible to exclude any of the postulated models for NK cell education (FIG. 2). Our opinion is that sev-eral signalling pathways probably have roles in NK cell education, but clearly much more work is needed before definitive conclusions can be made about the specific signals that are involved.

Induced hyporesponsiveness in NK cells. NK cell responsiveness can be affected in mature genetically unaltered NK cells following overstimulation by acti-vating receptors. Co-culture of NK cells with tumour cells that expressed ligands for the activating NKG2D receptor resulted in hyporesponsiveness of the NK cells not only to NKG2D triggering but also to other unre-lated stimuli, such as missing-self recognition and anti-body-dependent cell-mediated cytotoxicity75. Signalling by pathways that were unrelated to NKG2D did not result in similar hyporesponsiveness, implying that certain receptor systems might be preferentially used for the induction of hyporesponsiveness by overstimulation75. Conversely, continuous NKG2D engagement did not induce global hyporesponsiveness as Ly49D trigger-ing remained intact. This is in contrast with MHC class I-deficient NK cells, which are hyporesponsive even to Ly49D triggering34,75. Nevertheless, induced

Table 1 (cont.) | Genetic modulation of molecules that are involved in activating receptor signalling in mouse NK cells

Targeted molecule

NKG2D pathway

Missing-self recognition

ADCC Ly49D pathway

Stimulation of NK1.1

Stimulation of Ly49D

Polyclonal stimulation

Inhibitory receptor repertoire

Refs

SYK and ZAP70

→ → ↓ ↓ → ↓ → Reduced frequency of Ly49G2, but normal frequencies of Ly49C and Ly49I

99,109

PLCγ2 ↓ ↓ ND ND ↓ ND → Reduced frequencies of Ly49A, Ly49C, Ly49G2 and Ly49I, but normal frequencies of NKG2A, NKG2C and NKG2E

110,111

p110δ (PI3K)

→, ↓ →, ↓ ND ↓ ↓, → ↓ → Increased frequencies of Ly49C, Ly49G2 and Ly49I; normal frequencies of Ly49A and Ly49G2; reduced frequencies of Ly49C and Ly49I

112–114

p110γ (PI3K)

→, ↓ → ND ND ↓ ND → Increased frequency of Ly49G2 and reduced frequencies of Ly49C and Ly49I

112,113

p110γ and p110δ

↓ ND ND ↓ ↓ ND ND Increased frequencies of Ly49C and Ly49I; normal frequency of Ly49G2; reduced frequencies of Ly49A, Ly49C, Ly49F and Ly49I

112,113

p85α (PI3K)

↓ ↓ ND ND ↓ ND ↓ Reduced frequency of Ly49A

115

↑, increase; ↓, decrease; →, no effect; ADCC, antibody-dependent cell-mediated cytotoxicity; BCL-10, B-cell lymphoma 10; CARD9, caspase-recruitment domain 9; CARMA1, CARD–MAGUK protein 1; CRACC, CD2-like-receptor activating cytotoxic cells; DNAM1, DNAX accessory molecule 1; EAT, Ewing’s sarcoma-associated transcript; ERT, EAT2-related transducer; FcγR, Fc receptor for IgG; IL-2, interleukin-2; LAT, linker for activation of T cells; LFA1, lymphocyte function-associated antigen 1; NK, natural killer; NK1.1, NK cell-associated antigen 1.1; NKG2D, NK group 2, member D; NKp46, NK cell protein p46; PI3K, phosphoinositide 3-kinase; ND, not determined; PLCγ2, phospholipase Cγ2; SAP, SLAM-associated protein; SYK, spleen tyrosine kinase; ZAP70, ξ-chain-associated protein kinase of 70 kDa.

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hyporesponsiveness of mature NK cells by chronic exposure to activating stimuli is compatible with the disarming and the rheostat model of continuous tun-ing of responsiveness (FIG. 2d) and could represent one aspect of NK cell education.

Transgenic expression of ligands for activating NK cell receptors in vivo has also provided interesting informa-tion. In one such study, Oppenheim et al. expressed the ligand for the NKG2D receptor retinoic acid early tran-script 1ε (RAE1ε) in epithelial cells or in all cells of the body. This resulted in impaired NKG2D-mediated NK cell function and a reduced ability to reject missing-self target cells in vivo76; similar data were also published by Wiemann et al.77. Together these results suggest that over-stimulation via NKG2D can lead to hyporesponsiveness of other unrelated signalling pathways. Interestingly, the inability to reject β2m-deficient target cells by RAE1ε-exposed NK cells was reversible following general NK cell stimulation by administration of polyinosinic–polycyti-dylic acid (polyI:C)76, suggesting that the down-tuning of responsiveness can be reversed, which is in line with the rheostat model of NK cell education48.

In a later in vivo study, ectopic expression of m157, the MCMV-encoded ligand for the activating Ly49H receptor, resulted in hyporesponsiveness not only to m157 stimulation but also to NK1.1 crosslinking with antibodies, Ly49D stimulation (by Chinese hamster ovary cells) and NKG2D stimulation (by YAC-1 cells)78. Thus, consistent with the data presented above for NKG2D-induced hyporesponsiveness, Ly49H stimu-lation can also affect other signalling pathways. Also in this system, hyporesponsiveness could be reversed following in vitro culture of cells with IL-2, but not with polyI:C78. In a parallel study, Sun et al. expressed m157 using a retroviral system and following repeated m157-mediated stimulation reported similar hypore-sponsiveness of Ly49H+ NK cells. However, in this par-allel study, no evidence of global hyporesponsiveness was obtained, as stimulation with antibodies that were specific for NK1.1 or Ly49D elicited similar effector responses by Ly49H+ NK cells, irrespective of whether they were exposed first to m157 (REF. 79).

In line with these studies, a recent study of human NK cells showed that NK cells that expressed an activat-ing receptor but no inhibitory receptors for self-MHC class I molecules exhibited a hyporesponsive phenotype56. Together, these studies show that the constitutive pres-ence of an activating ligand in vivo downregulates NK cell responsiveness in subsets that express an activating recep-tor for this ligand. More work is needed to determine the signalling pathways that can induce global hyporespon-siveness. Furthermore, it will be important to determine whether induced hyporesponsiveness is distinct from NK cell education, or whether it is a tuning event that is an integrated part of the NK cell.

The NK cell repertoire and NK cell educationAs alluded to above and described in more detail in sev-eral recent reviews42,48,80, interactions with self-MHC class I molecules during NK cell education render indi-vidual NK cell subsets ‘useful’ in terms of their ability

to mediate missing-self recognition17,18,20,34,55,81. A major unresolved question is in what way, if any, NK cell edu-cation results in changes in the distribution of inhibitory receptors? In other words, is there an MHC class I- controlled mechanism that leads to the accumulation of useful (optimally educated) NK cell subsets, and that limits the presence of less useful (uneducated or subopti-mally educated) subsets? Such a link would seem natural and could also, if it exists, be clinically useful in terms of screening for an individual’s ‘optimally tuned’ NK cells by using flow cytometry of the KIR repertoire.

At first sight there does not seem to be a mechanism to limit the number of uneducated NK cell subsets, as NK cells that lack inhibitory receptors are readily identi-fied in both mice and in humans17,18,20,34. These cells are present but rendered hyporesponsive by unknown mech-anisms17,18,20,34. By contrast, a decrease in the frequency of NK cells that express too many inhibitory receptors has been observed in mice70,71,82 and has been proposed to eliminate those NK cell subsets that would be too easily inhibited and therefore less useful83. In humans, no such reduction in expression of two different KIRs by NK cells has been seen so far55,68. When it comes to the possibility of positive enrichment of useful subsets, recent evidence has shown an accumulation of NK cells that express single Ly49 receptors in response to self-MHC class I-mediated stimulation69. This effect becomes less clear as the number of inhibitory receptors expressed by NK cells increases (P.B. and P.H., unpublished observa-tions), implying that the complexity that is imposed by the co-expression of several inhibitory receptors may obscure the biological effects that determine NK cell education. A corresponding influence on the KIR repertoire by human MHC class I molecules has yet to be identified.

It remains to be established why the influence of self-MHC molecules on the human NK cell repertoire seems marginal, given that such effects are obvious in mice; this could have several explanations. Through inbreed-ing and transgenesis, allelic variations of both Ly49 receptors and MHC class I molecules can be prevented in mice, which leads to an increased predictability of specificities and affinities between inhibitory receptors and the MHC molecules. For similar reasons, other factors of potential importance in NK cell education, such as signalling from activating receptors, responses to the NK cell activating cytokine IL-15 and sensitivity to apoptosis, may unpredictably vary in the human population, contributing to greater variability and dif-ficulties in detecting small differences. In humans, the dependence on an outbred study population has high-lighted the fact that the KIR locus itself is a strong pre-dictor of the KIR repertoire, and MHC-guided effects may be obscured by this effect. Research in mice has not suffered from this bias as the impact of the Ly49 locus on the Ly49 repertoire can be easily avoided by comparing mice with different MHC class I haplotypes on a fixed genetic background. However, major differ-ences in the Ly49 gene complex are seen between those mouse strains that have been compared, which implies that the mouse Ly49 locus may have a similar impact on the Ly49 repertoire24.

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In summary, numerous studies support an MHC class I-mediated functional education for NK cells, which may also be quantitative in nature19,34,48,49,55,56,84. These functional effects are accompanied by changes in the NK cell repertoire, which may also be significant for the overall NK cell response in vivo (P.B. and P.H., unpublished observations).

NK cell education: future directionsSeveral models have been proposed to explain how signal-ling through the inhibitory receptor controls the thres hold for activation in mature NK cells41,42,48. The important question to resolve now is how inhibitory and activating signalling pathways are functionally linked during NK cell

education and how this link may result in the dynamic thresholds that have been postulated34,48,49,55. To under-stand this, we must learn more about the molecular events that occur during inhibitory signalling and advances in this area have been made recently39,54. The roles of various activating signalling pathways in NK cell education also require further study. In addition, the link between NK cell education and repertoire formation implies a connection between signalling from activating and inhibitory recep-tors to pathways, such as those of IL-15 and transforming growth factor-β85, that control proliferation and death in NK cells. By exploring these avenues, new clues may be found that could shed light on some of the remaining mysteries of MHC class I-guided NK cell education.

1. Sun, J. C., Beilke, J. N. & Lanier, L. L. Adaptive immune features of natural killer cells. Nature 457, 557–561 (2009).

2. O’Leary, J. G., Goodarzi, M., Drayton, D. L. & von Andrian, U. H. T cell- and B cell-independent adaptive immunity mediated by natural killer cells. Nature Immunol. 7, 507–516 (2006).

3. Cooper, M. A. et al. Cytokine-induced memory-like natural killer cells. Proc. Natl Acad. Sci. USA 106, 1915–1919 (2009).

4. Orange, J. S. Human natural killer cell deficiencies. Curr. Opin. Allergy Clin. Immunol. 6, 399–409 (2006).

5. Khakoo, S. I. & Carrington, M. KIR and disease: a model system or system of models? Immunol. Rev. 214, 186–201 (2006).

6. Johansson, S., Berg, L., Hall, H. & Höglund, P. NK cells: elusive players in autoimmunity. Trends Immunol. 26, 613–618 (2005).

7. Karre, K., Ljunggren, H. G., Piontek, G. & Kiessling, R. Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature 319, 675–678 (1986).

8. Ljunggren, H. G. et al. Empty MHC class I molecules come out in the cold. Nature 346, 476–480 (1990).

9. Ruggeri, L. et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 295, 2097–2100 (2002).

10. Cudkowicz, G. & Stimpfling, J. H. Induction of immunity and of unresponsiveness to parental marrow grafts in adult F-1 hybrid mice. Nature 204, 450–453 (1964).

11. Cudkowicz, G. Genetic control of bone marrow graft rejection. I. Determinant-specific difference of reactivity in two pairs of inbred mouse strains. J. Exp. Med. 134, 281–293 (1971).

12. Cudkowicz, G. & Stimpfling, J. H. Deficient growth of C57BL marrow cells transplanted in F1 hybrid mice. Association with the histocompatibility-2 locus. Immunology 7, 291–306 (1964).

13. Kiessling, R. et al. Evidence for a similar or common mechanism for natural killer cell activity and resistance to hemopoietic grafts. Eur. J. Immunol. 7, 655–663 (1977).

14. Höglund, P. et al. Natural resistance against lymphoma grafts conveyed by H-2Dd transgene to C57BL mice. J. Exp. Med. 168, 1469–1474 (1988).

15. Ohlen, C. et al. Prevention of allogeneic bone marrow graft rejection by H-2 transgene in donor mice. Science 246, 666–668 (1989).

16. Karlhofer, F. M., Ribaudo, R. K. & Yokoyama, W. M. MHC class I alloantigen specificity of Ly-49+ IL-2-activated natural killer cells. Nature 358, 66–70 (1992).

17. Kim, S. et al. Licensing of natural killer cells by host major histocompatibility complex class I molecules. Nature 436, 709–713 (2005).

18. Anfossi, N. et al. Human NK cell education by inhibitory receptors for MHC class, I. Immunity 25, 331–342 (2006).

19. Johansson, S. et al. Natural killer cell education in mice with single or multiple major histocompatibility complex class I molecules. J. Exp. Med. 201, 1145–1155 (2005). This study was the first to show that different MHC class I alleles have a unique ‘educating impact’ on the NK cell system, suggesting that NK cell education may be a quantitative process.

20. Fernandez, N. C. et al. A subset of natural killer cells achieves self-tolerance without expressing inhibitory receptors specific for self-MHC molecules. Blood 105, 4416–4423 (2005). References 17, 18 and 20 were the first to link MHC class I‑dependent functional maturation with inhibitory receptors and to demonstrate the presence of uneducated NK cells in normal individuals.

21. Moretta, L. & Moretta, A. Unravelling natural killer cell function: triggering and inhibitory human NK receptors. EMBO J. 23, 255–259 (2004).

22. Lanier, L. L. NK cell recognition. Annu. Rev. Immunol. 23, 225–274 (2005).

23. Parham, P. MHC class I molecules and KIRs in human history, health and survival. Nature Rev. Immunol. 5, 201–214 (2005).

24. Carlyle, J. R. et al. Evolution of the Ly49 and Nkrp1 recognition systems. Semin. Immunol. 20, 321–330 (2008).

25. Braud, V. M. et al. HLA-E binds to natural killer cell receptors CD94/NKG2A, B and, C. Nature 391, 795–799 (1998).

26. Vance, R. E., Kraft, J. R., Altman, J. D., Jensen, P. E. & Raulet, D. H. Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Qa-1b. J. Exp. Med. 188, 1841–1848 (1998).

27. Lopez-Botet, M., Angulo, A. & Guma, M. Natural killer cell receptors for major histocompatibility complex class I and related molecules in cytomegalovirus infection. Tissue Antigens 63, 195–203 (2004).

28. Ciccone, E. et al. Self class I molecules protect normal cells from lysis mediated by autologous natural killer cells. Eur. J. Immunol. 24, 1003–1006 (1994).

29. Valiante, N. M. et al. Functionally and structurally distinct NK cell receptor repertoires in the peripheral blood of two human donors. Immunity 7, 739–751 (1997).

30. Bix, M. et al. Rejection of class I MHC-deficient haemopoietic cells by irradiated MHC-matched mice. Nature 349, 329–331 (1991).

31. Höglund, P. et al. Recognition of beta 2-microglobulin-negative (beta 2m-) T-cell blasts by natural killer cells from normal but not from beta 2m– mice: nonresponsiveness controlled by beta 2m– bone marrow in chimeric mice. Proc. Natl Acad. Sci. USA 88, 10332–10336 (1991).

32. Liao, N. S., Bix, M., Zijlstra, M., Jaenisch, R. & Raulet, D. MHC class I deficiency: susceptibility to natural killer (NK) cells and impaired NK activity. Science 253, 199–202 (1991).

33. Zimmer, J. et al. Activity and phenotype of natural killer cells in peptide transporter (TAP)-deficient patients (type I bare lymphocyte syndrome). J. Exp. Med. 187, 117–122 (1998).

34. Brodin, P., Lakshmikanth, T., Johansson, S., Karre, K. & Höglund, P. The strength of inhibitory input during education quantitatively tunes the functional responsiveness of individual natural killer cells. Blood 113, 2434–2441 (2009). References 34 and 49 were the first to show that the strength of inhibitory input by MHC class I molecules through inhibitory Ly49 receptors determines the threshold for activation of NK cells.

35. Orr, M. T., Murphy, W. J. & Lanier, L. L. ‘Unlicensed’ natural killer cells dominate the response to cytomegalovirus infection. Nature Immunol. 11, 321–327 (2010).

36. Tay, C. H., Welsh, R. M. & Brutkiewicz, R. R. NK cell response to viral infections in beta 2-microglobulin- deficient mice. J. Immunol. 154, 780–789 (1995).References 35 and 36 showed that ‘uneducated’ NK cells can clear MCMV infection, questioning the importance of education for some NK cell‑mediated functions in vivo.

37. Lucas, M., Schachterle, W., Oberle, K., Aichele, P. & Diefenbach, A. Dendritic cells prime natural killer cells by trans-presenting interleukin 15. Immunity 26, 503–517 (2007).

38. Chaix, J. et al. Cutting edge: priming of NK cells by IL-18. J. Immunol. 181, 1627–1631 (2008).

39. Peterson, M. E. & Long, E. O. Inhibitory receptor signaling via tyrosine phosphorylation of the adaptor Crk. Immunity 29, 578–588 (2008).This study provided new insights into inhibitory signalling and showed that CRK was phosphorylated by the kinase ABL1 downstream of inhibitory receptors, identifying potential ways by which inhibitory receptor signalling might affect the activating signals that are needed for NK cell education.

40. Yokoyama, W. M. Inhibitory receptors signal activation. Immunity 29, 515–517 (2008).

41. Yokoyama, W. M. & Kim, S. Licensing of natural killer cells by self-major histocompatibility complex class, I. Immunol. Rev. 214, 143–154 (2006).

42. Raulet, D. H. & Vance, R. E. Self-tolerance of natural killer cells. Nature Rev. Immunol. 6, 520–531 (2006).

43. Doucey, M. A. et al. Cis association of Ly49A with MHC class I restricts natural killer cell inhibition. Nature Immunol. 5, 328–336 (2004).

44. Zimmer, J., Ioannidis, V. & Held, W. H-2D ligand expression by Ly49A+ natural killer (NK) cells precludes ligand uptake from environmental cells: implications for NK cell function. J. Exp. Med. 194, 1531–1539 (2001).

45. Chalifour, A. et al. A Role for cis interaction between the inhibitory Ly49A receptor and MHC class I for natural killer cell education. Immunity 30, 337–347 (2009). This study presented a new model for NK cell education based on cis interactions between Ly49 receptors and MHC class I molecules on the NK cell membrane.

46. Andersson, K. E., Williams, G. S., Davis, D. M. & Höglund, P. Quantifying the reduction in accessibility of the inhibitory NK cell receptor Ly49A caused by binding MHC class I proteins in cis. Eur. J. Immunol. 37, 516–527 (2007).

47. Back, J. et al. Distinct conformations of Ly49 natural killer cell receptors mediate MHC class I recognition in trans and cis. Immunity 31, 598–608 (2009).

48. Brodin, P., Karre, K. & Höglund, P. NK cell education: not an on–off switch but a tunable rheostat. Trends Immunol. 30, 143–149 (2009).

49. Joncker, N. T., Fernandez, N. C., Treiner, E., Vivier, E. & Raulet, D. H. NK cell responsiveness is tuned commensurate with the number of inhibitory receptors for self-MHC class I: the rheostat model. J. Immunol. 182, 4572–4580 (2009).

50. Brodin, P. & Höglund, P. Beyond licensing and disarming: a quantitative view on NK-cell education. Eur. J. Immunol. 38, 2934–2937 (2008).

51. Lanier, L. L. Up on the tightrope: natural killer cell activation and inhibition. Nature Immunol. 9, 495–502 (2008).

R E V I E W S



52. Stebbins, C. C. et al. Vav1 dephosphorylation by the tyrosine phosphatase SHP-1 as a mechanism for inhibition of cellular cytotoxicity. Mol. Cell Biol. 23, 6291–6299 (2003).This study provided the first evidence that inhibitory receptor signalling could terminate activating signals by dephosphorylating VAV1, a common factor in both inhibitory and activating pathways.

53. Bryceson, Y. T., March, M. E., Ljunggren, H. G. & Long, E. O. Activation, coactivation, and costimulation of resting human natural killer cells. Immunol. Rev. 214, 73–91 (2006).

54. Kim, H. S., Das, A., Gross, C. C., Bryceson, Y. T. & Long, E. O. Synergistic signals for natural cytotoxicity are required to overcome inhibition by c-Cbl ubiquitin ligase. Immunity 32, 175–186 (2010).

55. Yawata, M. et al. MHC class I-specific inhibitory receptors and their ligands structure diverse human NK-cell repertoires toward a balance of missing self-response. Blood 112, 2369–2380 (2008).

56. Fauriat, C., Ivarsson, M. A., Ljunggren, H. G., Malmberg, K. J. & Michaelsson, J. Education of human natural killer cells by activating killer cell immunoglobulin- like receptors. Blood 115, 1166–1174 (2010).

57. Lowin-Kropf, B., Kunz, B., Beermann, F. & Held, W. Impaired natural killing of MHC class I-deficient targets by NK cells expressing a catalytically inactive form of SHP-1. J. Immunol. 165, 1314–1321 (2000).

58. Jonsson, A. H., Yang, L., Kim, S., Taffner, S. M. & Yokoyama, W. M. Effects of MHC class I alleles on licensing of Ly49A+ NK cells. J. Immunol. 184, 3424–3432 (2010).

59. Yusa, S. & Campbell, K. S. Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) can play a direct role in the inhibitory function of killer cell Ig-like receptors in human NK cells. J. Immunol. 170, 4539–4547 (2003).

60. Wang, J. W. et al. Influence of SHIP on the NK repertoire and allogeneic bone marrow transplantation. Science 295, 2094–2097 (2002).

61. Fortenbery, N. R. et al. SHIP influences signals from CD48 and MHC class I ligands that regulate NK cell homeostasis, effector function, and repertoire formation. J. Immunol. 184, 5065–5074 (2010).

62. Shultz, L. D., Rajan, T. V. & Greiner, D. L. Severe defects in immunity and hematopoiesis caused by SHP-1 protein-tyrosine-phosphatase deficiency. Trends Biotechnol. 15, 302–307 (1997).

63. Tsui, F. W., Martin, A., Wang, J. & Tsui, H. W. Investigations into the regulation and function of the SH2 domain-containing protein-tyrosine phosphatase, SHP-1. Immunol. Res. 35, 127–136 (2006).

64. Jonsson, A. H. & Yokoyama, W. M. Natural killer cell tolerance licensing and other mechanisms. Adv. Immunol. 101, 27–79 (2009).

65. Di Santo, J. P. Natural killer cells: diversity in search of a niche. Nature Immunol. 9, 473–475 (2008).

66. Saleh, A. et al. Identification of probabilistic transcriptional switches in the Ly49 gene cluster: a eukaryotic mechanism for selective gene activation. Immunity 21, 55–66 (2004).

67. Davies, G. E. et al. Identification of bidirectional promoters in the human KIR genes. Genes Immun. 8, 245–253 (2007).

68. Andersson, S., Fauriat, C., Malmberg, J. A., Ljunggren, H. G. & Malmberg, K. J. KIR acquisition probabilities are independent of self-HLA class I ligands and increase with cellular KIR expression. Blood 114, 95–104 (2009).

69. Johansson, S. et al. Probing natural killer cell education by Ly49 receptor expression analysis and computational modelling in single MHC class I mice. PLoS One 4, e6046 (2009).

70. Held, W., Dorfman, J. R., Wu, M. F. & Raulet, D. H. Major histocompatibility complex class I-dependent skewing of the natural killer cell Ly49 receptor repertoire. Eur. J. Immunol. 26, 2286–2292 (1996).

71. Salcedo, M. et al. Altered expression of Ly49 inhibitory receptors on natural killer cells from MHC class I-deficient mice. J. Immunol. 158, 3174–3180 (1997). References 70 and 71 showed a role for MHC class I molecules in the shaping of the inhibitory receptor repertoire.

72. Hanke, T., Takizawa, H. & Raulet, D. H. MHC-dependent shaping of the inhibitory Ly49 receptor repertoire on NK cells: evidence for a regulated sequential model. Eur. J. Immunol. 31, 3370–3379 (2001).

73. Lowin-Kropf, B., Kunz, B., Schneider, P. & Held, W. A role for the src family kinase Fyn in NK cell activation and the formation of the repertoire of Ly49 receptors. Eur. J. Immunol. 32, 773–782 (2002).

74. Whittaker, G. C. et al. Analysis of the linker for activation of T cells and the linker for activation of

B cells in natural killer cells reveals a novel signaling cassette, dual usage in ITAM signaling, and influence on development of the Ly49 repertoire. Blood 112, 2869–2877 (2008).

75. Coudert, J. D., Scarpellino, L., Gros, F., Vivier, E. & Held, W. Sustained NKG2D engagement induces cross-tolerance of multiple distinct NK cell activation pathways. Blood 111, 3571–3578 (2008).

76. Oppenheim, D. E. et al. Sustained localized expression of ligand for the activating NKG2D receptor impairs natural cytotoxicity in vivo and reduces tumor immunosurveillance. Nature Immunol. 6, 928–937 (2005).

77. Wiemann, K. et al. Systemic NKG2D down-regulation impairs NK and CD8 T cell responses in vivo. J. Immunol. 175, 720–729 (2005).

78. Tripathy, S. K. et al. Continuous engagement of a self-specific activation receptor induces NK cell tolerance. J. Exp. Med. 205, 1829–1841 (2008).

79. Sun, J. C. & Lanier, L. L. Tolerance of NK cells encountering their viral ligand during development. J. Exp. Med. 205, 1819–1828 (2008).References 76–79 provided evidence for induced hyporesponsiveness following continuous stimulation of activating NK cell receptors by a transgenically expressed ligand in vivo.

80. Yokoyama, W. M. & Kim, S. How do natural killer cells find self to achieve tolerance? Immunity 24, 249–257 (2006).

81. Fauriat, C. et al. Estimation of the size of the alloreactive NK cell repertoire: studies in individuals homozygous for the group A KIR haplotype. J. Immunol. 181, 6010–6019 (2008).

82. Fahlen, L., Lendahl, U. & Sentman, C. L. MHC class I-Ly49 interactions shape the Ly49 repertoire on murine NK cells. J. Immunol. 166, 6585–6592 (2001).

83. Raulet, D. H. et al. Specificity, tolerance and developmental regulation of natural killer cells defined by expression of class I-specific Ly49 receptors. Immunol. Rev. 155, 41–52 (1997).

84. Kim, S. et al. HLA alleles determine differences in human natural killer cell responsiveness and potency. Proc. Natl Acad. Sci. USA 105, 3053–3058 (2008).

85. Di Santo, J. P. Natural killer cell developmental pathways: a question of balance. Annu. Rev. Immunol. 24, 257–286 (2006).

86. Cella, M. et al. Differential requirements for Vav proteins in DAP10- and ITAM-mediated NK cell cytotoxicity. J. Exp. Med. 200, 817–823 (2004).

87. Malarkannan, S. et al. Bcl10 plays a divergent role in NK cell-mediated cytotoxicity and cytokine generation. J. Immunol. 179, 3752–3762 (2007).

88. Gross, O. et al. Multiple ITAM-coupled NK-cell receptors engage the Bcl10/Malt1 complex via Carma1 for NF-κB and MAPK activation to selectively control cytokine production. Blood 112, 2421–2428 (2008).

89. Hara, H. et al. Cell type-specific regulation of ITAM-mediated NF-κB activation by the adaptors, CARMA1 and CARD9. J. Immunol. 181, 918–930 (2008).

90. Chiesa, S. et al. Multiplicity and plasticity of natural killer cell signaling pathways. Blood 107, 2364–2372 (2006).

91. Bloch-Queyrat, C. et al. Regulation of natural cytotoxicity by the adaptor SAP and the Src-related kinase Fyn. J. Exp. Med. 202, 181–192 (2005).

92. Mason, L. H., Willette-Brown, J., Taylor, L. S. & McVicar, D. W. Regulation of Ly49D/DAP12 signal transduction by Src-family kinases and CD45. J. Immunol. 176, 6615–6623 (2006).

93. Shier, P. et al. Impaired immune responses toward alloantigens and tumor cells but normal thymic selection in mice deficient in the β2 integrin leukocyte function-associated antigen-1. J. Immunol. 157, 5375–5386 (1996).

94. Schmits, R. et al. LFA-1-deficient mice show normal CTL responses to virus but fail to reject immunogenic tumor. J. Exp. Med. 183, 1415–1426 (1996).

95. Matsumoto, G., Nghiem, M. P., Nozaki, N., Schmits, R. & Penninger, J. M. Cooperation between CD44 and LFA-1/CD11a adhesion receptors in lymphokine-activated killer cell cytotoxicity. J. Immunol. 160, 5781–5789 (1998).

96. Gilfillan, S., Ho, E. L., Cella, M., Yokoyama, W. M. & Colonna, M. NKG2D recruits two distinct adapters to trigger NK cell activation and costimulation. Nature Immunol. 3, 1150–1155 (2002).

97. Horng, T., Bezbradica, J. S. & Medzhitov, R. NKG2D signaling is coupled to the interleukin 15 receptor signaling pathway. Nature Immunol. 8, 1345–1352 (2007).

98. Tassi, I. et al. DAP10 associates with Ly49 receptors but contributes minimally to their expression and function in vivo. Eur. J. Immunol. 39, 1129–1135 (2009).

99. Zompi, S. et al. NKG2D triggers cytotoxicity in mouse NK cells lacking DAP12 or Syk family kinases. Nature Immunol. 4, 565–572 (2003).

100. Bakker, A. B. et al. DAP12-deficient mice fail to develop autoimmunity due to impaired antigen priming. Immunity 13, 345–353 (2000).

101. Huntington, N. D., Xu, Y., Nutt, S. L. & Tarlinton, D. M. A requirement for CD45 distinguishes Ly49D-mediated cytokine and chemokine production from killing in primary natural killer cells. J. Exp. Med. 201, 1421–1433 (2005).

102. Hesslein, D. G., Takaki, R., Hermiston, M. L., Weiss, A. & Lanier, L. L. Dysregulation of signaling pathways in CD45-deficient NK cells leads to differentially regulated cytotoxicity and cytokine production. Proc. Natl Acad. Sci. USA 103, 7012–7017 (2006).

103. Yamada, H., Kishihara, K., Kong, Y. Y. & Nomoto, K. Enhanced generation of NK cells with intact cytotoxic function in CD45 exon 6-deficient mice. J. Immunol. 157, 1523–1528 (1996).

104. Cruz-Munoz, M. E., Dong, Z., Shi, X., Zhang, S. & Veillette, A. Influence of CRACC, a SLAM family receptor coupled to the adaptor EAT-2, on natural killer cell function. Nature Immunol. 10, 297–305 (2009).

105. Roncagalli, R. et al. Negative regulation of natural killer cell function by EAT-2, a SAP-related adaptor. Nature Immunol. 6, 1002–1010 (2005).

106. Dong, Z. et al. Essential function for SAP family adaptors in the surveillance of hematopoietic cells by natural killer cells. Nature Immunol. 10, 973–980 (2009).

107. Guerra, N. et al. NKG2D-deficient mice are defective in tumor surveillance in models of spontaneous malignancy. Immunity 28, 571–580 (2008).

108. Zafirova, B. et al. Altered NK cell development and enhanced NK cell-mediated resistance to mouse cytomegalovirus in NKG2D-deficient mice. Immunity 31, 270–282 (2009).

109. Colucci, F. et al. Natural cytotoxicity uncoupled from the Syk and ZAP-70 intracellular kinases. Nature Immunol. 3, 288–294 (2002).

110. Regunathan, J. et al. Differential and nonredundant roles of phospholipase Cγ2 and phospholipase Cγ1 in the terminal maturation of NK cells. J. Immunol. 177, 5365–5376 (2006).

111. Caraux, A. et al. Phospholipase C-γ2 is essential for NK cell cytotoxicity and innate immunity to malignant and virally infected cells. Blood 107, 994–1002 (2006).

112. Kim, N. et al. The p110δ catalytic isoform of PI3K is a key player in NK-cell development and cytokine secretion. Blood 110, 3202–3208 (2007).

113. Tassi, I. et al. p110γ and p110δ phosphoinositide 3-kinase signaling pathways synergize to control development and functions of murine NK cells. Immunity 27, 214–227 (2007).

114. Guo, H., Samarakoon, A., Vanhaesebroeck, B. & Malarkannan, S. The p110 δ of PI3K plays a critical role in NK cell terminal maturation and cytokine/chemokine generation. J. Exp. Med. 205, 2419–2435 (2008).

115. Awasthi, A. et al. Deletion of PI3K‑p85α gene impairs lineage commitment, terminal maturation, cytokine generation and cytotoxicity of NK cells. Genes Immun. 9, 522–535 (2008).

116. Tomasello, E. et al. Combined natural killer cell and dendritic cell functional deficiency in KARAP/DAP12 loss-of-function mutant mice. Immunity 13, 355–364 (2000).

AcknowledgementsThe authors would like to express their thanks to all mem-bers of the Höglund group for important scientific input, to E. Long for critical reading of the manuscript and to K. Kärre and all members of his group for stimulating discussions. Work in our group is supported by grants from the Swedish Research Council, the Swedish Cancer Society, the Karolinska Institutet, the Swedish Foundation for International Cooperation in Research and Higher Education (STINT), the Marianne and Marcus Wallenberg Foundation and the Mary Bevé Foundation.

Competing interests statementThe authors declare no competing financial interests.

FURTHER INFORMATIONPetter HÖglund’s homepage: http://ki.se/ki/jsp/polopoly.jsp?d=23678&a=36415&l=en

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