dendritic cells and nk cells take centre stage - Nature

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CLOSE ENCOUNTERS OF DIFFERENT KINDS: DENDRITIC CELLS AND NK CELLS TAKE CENTRE STAGE Mariapia A. Degli-Esposti*‡ and Mark J. Smyth§ Abstract | Immune responses are generally divided into innate and adaptive responses, and the efficacy of one is thought to be independent of the other. The regulation of immune responses, however, is complex, and accumulating evidence indicates that multiple interactions between immune effector cells are common and are crucial for the initiation, as well as the outcome, of these responses. Dendritic cells, long recognized as key initiators of primary adaptive immunity, are now also seen as crucial regulators of aspects of innate immunity, in particular natural-killercell function. Reciprocally, natural killer cells can influence the activity of dendritic cells. Here, we review recent exciting progress in this field, and we highlight the impact of this cellular crosstalk on the design of immune-based therapies for control of infection and cancer.

*Immunology and Virology Program, Centre for Ophthalmology and Visual Science, The University of Western Australia, Western Australia 6009, Australia. ‡ Centre for Experimental Immunology, Lions Eye Institute, 2 Verdun Street, Nedlands, Western Australia 6009, Australia. § Cancer Immunology Program, Sir Donald and Lady Trescowthick Laboratories, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia. Correspondence to M.A.D.E. e-mail: mariapia@ cyllene.uwa.edu.au doi:10.1038/nri1549

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Although the immune system has classically been thought to consist of two distinct arms, the innate arm and the adaptive arm, it is becoming increasingly clear that these two arms are tightly interwoven. The cellular components of the innate immune system include neutrophils, granulocytes, monocytes, macrophages, dendritic cells (DCs) and natural killer (NK) cells. The adaptive immune system consists of B cells and αβ T cells. The characterization of γδ T cells provided the first evidence of a lymphocyte subset that could interface both innate and adaptive immunity, a property that can now also be ascribed to other cells, such as NK1.1+ T cells (known as NKT CELLS) and CD4+CD25+ regulatory T cells. In the past four years, much evidence has accumulated to show that the functions of NK cells and DCs are interrelated, a possibility first highlighted by a study published in 1985 (REF. 1). This report was the first to indicate that NK cells might be capable of regulating adaptive T-cell responses by eliminating DCs that have interacted with antigen. In 1999, a study by Fernandez, Zitvogel and colleagues showed that DCs could trigger NK-cell-mediated antitumour responses2, and since 2001, an increasing number of reports have provided further evidence to indicate that

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there is CROSSTALK between DCs and NK cells in the context of immune surveillance for pathogens and tumours. In a sophisticated balance of directing and choreographing, DCs and NK cells set the stage and determine the finale of the ensuing immune responses. In this review, we consider the evidence that supports the ability of NK cells and DCs not only to effect innate and adaptive immune responses but also to regulate these responses. The implications of DC–NK-cell crosstalk for immunotherapy and for immune surveillance for viruses and tumours are also discussed. A focus on the key players

DCs. The activation of specific and effective immune responses requires a system that can survey, decipher and quickly respond to insults such as infection in an appropriate manner. DCs can participate in all of these important activities. Immature DCs are located in peripheral organs and at mucosal surfaces, where they continuously sample the environment for foreign antigens3. After antigen uptake has occurred, DCs process antigen for presentation by cell-surface MHC molecules3. However, before DCs can activate naive T cells, they must complete a maturation programme that is initiated either by direct

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Table 1 | Toll-like-receptor expression by mouse and human dendritic-cell subsets* DC subset

TLR1

TLR2

TLR3

TLR4

TLR5

TLR6

TLR7

TLR8

TLR9

TLR10

CD11c+

+

+‡

+‡

+§

+

CD11c+CD8+

+

+‡

++‡

+‡

+/–

+

+

+

+‡

ND

+

+ or – ||

+

+‡

‡

+

+

‡

++

+

++

ND

+

+

+‡

+

–‡

+

Mouse

Plasmacytoid DCs

+

+/–

+/–

–

++

+‡

++‡

+¶

ND ‡

Human Blood DCs Monocyte-derived DCs (GM-CSF)

‡

‡

ND

+

+

ND

ND

+

ND

ND

ND

Monocyte-derived DCs (GM-CSF + IL-4) +

++‡

+

+/–‡

+

+

+/–

+

–‡

+

Blood plasmacytoid DCs

+

+/–

–‡

–‡

+/–

+

++‡

–

++‡

+/–

+

‡

‡

‡

–

‡

+/–

Plasmacytoid DCs (IL-3)

ND

‡

–

+

–

–

+

++

++

*These data were compiled from several reviews (REFS 4,5,11,147,148) and from the numerous studies investigating Toll-like receptor (TLR) expression and function in dendritic cell (DC) subsets in mice and humans. Cells were either directly isolated or generated in vitro. The cytokines used to generate the latter populations are indicated in parentheses. ‡Values indicate that the expression of the TLR was confirmed by a functional response to the relevant ligands. §The spleen population did not respond to TLR4 ligands, but bone-marrow-derived DCs cultured with granulocyte/macrophage colony-stimulating factor (GM-CSF ) did18. ||Although this subset of DCs did not either produce interleukin-12 (IL-12) or mature in response to R-848, a TLR7 ligand85, a recent report shows that these DCs produce CC-chemokine ligand 5 (CCL5; also known as RANTES) in response to R-848 (REF. 149). ¶Blood DCs do not seem to express cell-surface TLR4, and TLR2 might mediate their responses to lipopolysaccharide150. ND, not determined.

NKT CELLS

(Natural killer T cells). A heterogeneous subset of T cells, most of which express semiinvariant T-cell receptors. In mice, NKT cells were first identified by their expression of the cell-surface molecule NK1.1 (also known as NKR-P1C). CROSSTALK

The bidirectional interactions between two cell types. Crosstalk refers to the delivery of information and signals from cell population one to cell population two and vice versa. It might involve signals that are mediated by cell–cell contact or by soluble factors, such as cytokines. The signals that are received by each population affect its functions. TOLL-LIKE RECEPTOR

(TLR). A member of a family of receptors that recognize conserved molecular patterns that are unique to microorganisms. The lipopolysaccharide component of bacterial cell walls is one such component. Stimulation through TLRs induces the maturation of dendritic cells, leading to the optimal activation of the adaptive immune response. TLR-mediated events signal to the host that a microbial pathogen has been encountered.

exposure to pathogens or by specific interactions with other immune cells. The key to the initiation of immune responses to pathogens is the recognition of pathogenassociated ligands that are not normally expressed by host cells. This recognition is mediated by members of the TOLL-LIKE RECEPTOR (TLR) family (TABLE 1), several of which are expressed by immature DCs4,5. All of the responses of DCs that are considered to be crucial for their functions — that is, survival, proliferation, migration, secretion of cytokines and chemokines, and expression of cell-surface molecules (including co-stimulatory ligands) — are influenced by signalling through TLRs5–11. After pathogen encounter and recognition, DCs become very efficient at stimulating naive T cells, a process that is coincident with increased expression of antigenpresenting molecules (that is, MHC class I and class II) and co-stimulatory molecules (that is, CD40, CD80 and CD86)3. In the absence of appropriate signals, interactions between T cells and immature DCs can result in the induction of tolerance12. Therefore, the signals that are received by DCs decide not only whether a response is warranted but also what type of response is induced. DC ontogeny and DC subsets have recently been reviewed13–15. For the purpose of our discussion, mouse splenic DCs can be broadly categorized into three subsets. Two of these subsets comprise conventional DCs, which are characterized by the expression of high levels of CD11c and the differential expression of CD8α and CD11b. CD11c+CD8α–CD11b+ DCs are mainly located in marginal zones of the spleen. By contrast, CD11c+CD8α+ DCs, which are characterized by the expression of high levels of CD205 (also known as DEC205) and the absence of CD11b, localize to the T-cell areas of the spleen15. CD8α+ DCs cross-present exogenous antigens to CD8+ T cells16 and are required to prime cytotoxic T lymphocyte (CTL) immunity to several viruses17. Contrary to what was originally proposed, the capacity to polarize specific T helper (TH)-cell responses does not seem to be an intrinsic property of specific DC subsets but, instead, the outcome of flexible

NATURE REVIEWS | IMMUNOLOGY

responses that depend on both the signals that are received by the DCs18 and the microenvironment where these signals are received. Plasmacytoid DCs (pDCs), which constitute the third main subset of DCs, are distinguished by the expression of B220, LY6C, CD45RA and intermediate levels of CD11c. The main feature that distinguishes the three mouse DC subsets is their capacity to respond to specific TLR ligands, with pDCs preferentially responding to signals from TLR7 and TLR9, and conventional DCs to signals from TLR3 and TLR4 (TABLE 1). The expression pattern of TLRs and the ability to respond to specific TLR ligands can differ between in vitro-generated and ex vivo-purified DCs, so care must be taken when extrapolating in vitro findings to in vivo biology. pDCs have been functionally distinguished on the basis of their capacity to produce type I interferons (IFNs; that is, IFN-α and IFN-β) in response to viral infection and their reduced ability to activate naive T cells19–21. Recent data indicate that conventional DCs, under appropriate circumstances, secrete high levels of type I IFNs22. Furthermore, pDCs can produce interleukin-12 (IL-12) and induce polarization towards a T 1-CELL response18,23–25. Human DC subsets are less well defined. Human DCs do not express CD8α, but splenic and tonsillar DCs differentially express CD4 and CD11b and, similar to mouse DC subsets, show functional heterogeneity15. The best-characterized human DC populations are monocyte-derived DCs and pDCs. CD11c+CD1a+ CD14– monocyte-derived DCs can be isolated from the blood, or they can be grown in culture in the presence of granulocyte/macrophage colony-stimulating factor and IL-4, from precursors isolated from the blood or the bone marrow. pDCs, which are phenotypically characterized as CD11c–CD45RA+CD123+blood DC antigen 2 (BDCA2)+, are found in the blood and can be generated in vitro from IL-3-supplemented cultures of CD11c–CD4+IL-3 receptor (IL-3R)+ precursors. In humans, responsiveness to TLR3, TLR4, TLR8 and TLR9 distinguishes the two main DC subsets: signalling H

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REVIEWS through TLR3, TLR4 and TLR8 occurs in monocytederived DCs but not in pDCs, and signalling through TLR9 occurs in pDCs but not in monocyte-derived DCs5,25–28 (TABLE 1). Both subsets can elicit TH1 and TH2 responses depending on their environment and the stimuli that they received29–32. Although it is clear that distinct DC subsets have unique functions, the plasticity that exists during DC development and maturation indicates that functional specificity is not imparted during ontogeny but is acquired through the process of stimulation. Evolutionary survival is unlikely to favour a system in which responses that are required for survival advantage are pre-programmed. Plasticity enables appropriate responses to be quickly and safely generated, irrespective of the challenge being faced.

TH1 CELL

(T helper 1 cell). At least two distinct subsets of activated CD4+ T cells have been described. TH1 cells produce interferon-γ and tumour-necrosis factor and support cell-mediated immunity. TH2 cells produce interleukin-4 (IL-4), IL-5 and IL-13, support humoral immunity and downregulate TH1-cell responses. ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY

(ADCC). A mechanism by which natural killer (NK) cells are targeted to IgG-coated cells, resulting in the lysis of the antibody-coated cells. A specific receptor for the constant region of IgG, known as FcγRIII (CD16), is expressed at the surface of NK cells and mediates ADCC. MURINE CYTOMEGALOVIRUS

(MCMV). A member of the herpesvirus family that is often used as a model of a persistent viral infection. It causes an immune response that limits viral replication; however, the pathogen is not completely eliminated, and it establishes life-long persistence within its host. MCMV has considerable sequence homology and shares biological features with human CMV, and it provides a unique model to study in vivo infection in a natural host.

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NK cells. NK cells are specialized lymphocytes that provide a first line of defence through their ability to eliminate pathogen-infected cells and tumours. Unlike T cells, NK cells cannot mount a recall or memory response when re-exposed to the same antigenic challenge. They generally comprise ∼5–20% of peripheral lymphocytes (that is, those present in the spleen, liver and peripheral blood) and are present at lower frequencies in the thymus, lymph nodes and bone marrow, the latter being the main site of NK-cell development in adults. A working model of NK-cell development in mice and humans has recently been reviewed33; however, at present, the proposed pathways of NK-cell differentiation are based on in vitro data. Throughout their development, NK cells are heterogeneous in their cell-surface phenotype, proliferative capacity and function34,35. Mature NK cells can also be heterogeneous, and much remains to be learned about the specific subsets that are present in locations such as the liver, periphery and lymph nodes36. In humans, CD56hi NK cells, which produce large amounts of cytokines, comprise ∼10% of NK cells, whereas CD56low NK cells, which express high levels of the low-affinity Fc receptor for IgG (FcγRIII; also known as CD16) and are powerfully cytotoxic, comprise almost 90% of the NK-cell population. Greater attention should be paid to determining whether there are similar NK-cell populations in mice, particularly to their localization and to the cellular context in which they appear and interact. The function of NK cells is tightly regulated by a fine balance of inhibitory and activating signals that are delivered through a diverse array of cell-surface receptors belonging to the immunoglobulin-like receptor and C-type-lectin receptor families37–39, as well as mediated by pro-inflammatory cytokines. NK-cell inhibitory receptors bind self-MHC class I molecules. Combinatorial expression of inhibitory receptors generates a repertoire of NK cells that can independently survey the expression of almost every MHC molecule, a feature that enables them to limit the spread of pathogens and tumours that subvert innate and adaptive immune defences by selectively downregulating certain MHC class I molecules. However, reduced expression of MHC class I molecules is not a prerequisite for the activation of NK cells, which


requires activating signals from receptors that engage ligands specifically expressed by stressed, transformed or virus-infected cells40,41. NK-cell receptors, and their role in determining the outcome of NK-cell responses, have recently been reviewed37,42,43. NK-cell receptors not only mediate the recognition of appropriate targets but also regulate the effector function of NK cells44,45. Further complexity is generated by cytokine-mediated signals; in addition to driving important effector functions (for example, IFN-γ-mediated antiviral and antiangiogenic effects), cytokines can modify the signals that are induced by receptor-mediated interactions, the expression of specific NK-cell receptors and/or ligands, and the activation and proliferation of specific NK-cell subsets. NK cells, particularly in the periphery, become activated and proliferate in response to stimulation with various cytokines, including many that are produced by DCs. In trying to understand what drives, maintains and defines the outcomes of DC–NK-cell crosstalk, it is important to remember that NK cells are capable of potent cytotoxicity and cytokine production. NK cells use two main pathways to induce the apoptosis of target cells: granule exocytosis46 and death-receptor engagement47–49. NK cells can also be directly activated to induce ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY (ADCC). After activation, NK cells can secrete various cytokines and chemokines that can directly affect the survival of pathogens (for example, IFN-γ) and the growth and spread of tumours (for example, IFN-γ and tumournecrosis factor, TNF), as well as the regulation of haematopoietic-cell differentiation and the priming of immune effectors that are crucial for subsequent adaptive immunity. Therefore, similar to DCs, NK cells can have an essential role in dynamically defining the environment in which immune interactions occur and immune responses are elicited. Unlike DCs, which are known to participate in the instigation of immune responses, the role of NK cells as initiators of immunity remains to be established. Recent data provide unequivocal evidence of the participation of NK cells in coordinating immunity, through their ability to activate and eliminate DCs; however, the initial signals seem to originate from DCs. It is possible that NK cells have a role as initiators of immunity, and the reports that NK cells express TLRs provide some support for this idea28,50,51. The capacity of NK cells to become efficiently activated in response to direct recognition of pathogenencoded proteins (for example, through the interaction of the NK-cell receptor LY49H with the MURINE CYTOMEGALOVIRUS (MCMV)-encoded viral glycoprotein m157)52,53 indicates an alternative scenario in which NK cells might again have a role in the initiation of immune responses independently of TLR-mediated signals. Furthermore, NK cells and other innate cells might detect the first signs of cellular transformation by recognizing ‘stress’ ligands or other similar ligands. The possibility that NK cells initiate immune responses requires urgent evaluation. Next, we discuss evidence that supports a role for NK cells in the generation and maintenance of antigen-specific immune responses.



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DC–NK-cell crosstalk

Historically, the principal function of DCs was defined by their ability to prime antigen-specific immune responses, but their role in innate immunity is now recognized (FIG. 1). Interactions between DCs and NK cells

Immature DC

TLR PAMP and/or cytokines (IFN-α, IFN-β, TNF)

Mature DC

Mature plasmacytoid DC

CD40 CD80 CD86 IL-2 IL-12 IFN-γ

IL-12

NKG2D–MICA or MICB IFN-α, IFN-β, IL-2, IL-12, IL-15, IL-18

IFN-γ

IL-2

B cell

Immature plasmacytoid DC

Antigen, pathogen or non-self molecule

CD40– CD40L

IFN-γ NKT cell

IL-2 NK cell

T cell

CD1d– α-GalCer

NKp30 activating signal acquired

IFN-γ TNF

CD94–NKG2A inhibitory signal lost Lysis of targets, including immature DCs

Plasma cell DC maturation

DC

TH

CTL

HLA-E

IL-2, IL-12, CD40, CD80, CD86

TH1

TH2

Figure 1 | Interplay between dendritic cells and other immune effectors. Dendritic cells (DCs) affect the functions of T cells, B cells, natural killer (NK) cells and natural killer T (NKT) cells. DCs process antigens and present them in the context of peptide–MHC complexes to T cells. In the presence of appropriate signals from co-stimulatory molecules (CD40, CD80 and CD86) and cytokines (interferon-γ (IFN-γ), interleukin-2 (IL-2) and IL-12), DCs induce the activation of naive T cells and the generation of CD4+ T helper (TH ) cells or CD8+ cytotoxic T lymphocytes (CTLs). TH-cell responses can have either a TH1-cell or TH2-cell bias depending on the environmental signals that are received by the DCs, including those mediated by the recognition of microbial components (pathogen-associated molecular patterns, PAMPs) by Toll-like receptors (TLRs). DCs can also affect the functions of B cells, generally by complex interactions that involve signals originating from T cells (CD40–CD40 ligand (CD40L) interactions and IL-2 secretion) that have interacted with activated DCs. Interplay between DCs and NKT cells also occurs. Functional NKT-cell ligands, such as α-galactosylceramide (α-GalCer), are presented to NKT cells by CD1d molecules at the cell surface of DCs. Selective targeting of α-GalCer to DCs induces a stronger and more prolonged NKT-cell response. Reciprocally, NKT cells can promote the maturation of DCs. DCs affect the functions of NK cells and can induce their activation through pathways that require both cell–cell contact (NKG2D (NK group 2, member D)–MICA (MHC-class-I-polypeptide-related sequence A) and/or MICB) and cytokines (IFN-α, IFN-β, IL-2, IL-12, IL-15 and IL-18). DC-derived signals elicit NK-cell-mediated cytolysis, as well as NK-cell-mediated cytokine production. Reciprocally, NK cells can affect DC functions by being involved in DC maturation and DC elimination. Dashed lines represent pathways of cellular differentiation, and solid lines show interplay between DCs and other immune cells. NKp30, NK-cell protein 30; TNF, tumour-necrosis factor.


have been documented in a variety of settings, with evidence emerging of complex bidirectional crosstalk between the two cell types. This exciting scenario and its implications in the generation, maintenance and outcome of immune responses is discussed. The evidence supports the idea that these interactions not only set the stage for immune responses but also define the outcomes. Impact of DC-derived signals. In vitro studies support a role for both cytokines and cell–cell contact in the enhancement of NK-cell function (FIG. 2a) and proliferation (FIG. 2b) by DCs, and these studies provide useful insights into the underlying molecular interactions. Cytokines such as IL-12 and IL-18 promote optimal cytokine production by NK cells. IL-12 promotes the production of IFN-γ by NK cells. In humans, IFN-γ production mediated by DC-derived IL-12 requires the formation of an IMMUNOLOGICAL SYNAPSE and the polarization of pre-formed IL-12 at the DC–NK-cell interface54. NK cells can also be activated by DCs independently of IL-12 (REFS 55–57). In response to bacterial insults, the IL-2 secreted by DCs seems to be more important than IL-12 or IL-18 for eliciting IFN-γ production by NK cells, at least in mice57. The relevance of activities that are mediated by DC-derived IL-2 might need to be considered in the context of IL-15, because at least in humans, the capacity of monocyte-derived DCs to produce IL-2 depends on IL-15 (REF. 58). Type I IFNs are crucial for DC-induced NK-cell activation and have an important role in the induction of NK-cell cytotoxicity56,57, either directly or through the induction of autocrine IL-15. Both type I IFNs and IL-15 can affect NK-cell activation by modulating the expression of ligands for the NK-cell receptor NKG2D (natural-killer group 2, member D)59. A paracrine role in NK-cell proliferation and survival has recently been reported for membrane-bound IL-15 on DCs60. It is worth noting that the relevance of specific cytokines in the DC-mediated activation of NK cells depends on the initial stimulus that is received by the DCs. So, in the presence of IFN-α, NK-cell activation does not depend on IL-12, whereas IL-12 is required for the NK-cell stimulatory capacity of lipopolysaccharide (LPS)-activated DCs56. Furthermore, DCs that are generated in the presence of IL-4 lose the capacity to produce IL-2 in response to LPS61,62, but they can induce NK-cell activation through the engagement of KARAP (killer-cell activating-receptor-associated protein)dependent receptors62. Importantly, IL-4 can promote DC-mediated activation in the lymph nodes62. In addition to inducing NK-cell activation, mature DCs induce the recruitment of NK cells to the lymph nodes in a CXC-chemokine receptor 3 (CXCR3)-dependent manner63. The role of cell–cell contact with DCs in the induction of NK-cell activities is clear55,57,64,65, but the nature of the relevant molecules remains poorly defined. Immature DCs have been reported to induce the activation of NK cells. Although they are poor cytokine producers, immature DCs constitutively express CD48 and CD70, which are ligands for the NK-cell



REVIEWS activating receptors 2B4 and CD27, respectively. These molecules can enhance NK-cell function and might participate in NK-cell activation that is induced by immature DCs. Mature DCs, in addition to producing most of the NK-cell activating cytokines, markedly upregulate cell-surface expression of co-stimulatory molecules and, depending on the stimuli received, might also express ligands for NK-cell activating receptors. The expression of the NKG2D ligands MICA (MHC-class-I-polypeptide-related sequence A) and/or MICB is upregulated by human DCs after exposure to type I IFNs56. Interestingly, mature DCs downregulate the expression of CLRB (C-type-lectin-related B), the

IMMUNOLOGICAL SYNAPSE

A region that can form between two cells of the immune system in close contact, so named because of its similarities to the synapses that occur in the nervous system. The immunological synapse originally referred to the interaction between a T cell and an antigen-presenting cell. It involves adhesion molecules, as well as antigen receptors and cytokine receptors.

a DC-mediated NK-cell activation Cell-contact-dependent signals: NKG2D ligand–NKG2D LFA1–ICAM1

Pathogen TLR TLR-mediated signals

CLRB–NKR-P1B or -P1D CD70–CD27 CD48–2B4 Soluble signals: IFN-α, IFN-β, IL-2, IL-12, IL-15 and IL-18

DC

NK-cell activation

IFN-γ Cytolysis

NK cell

b DC-mediated NK-cell proliferation IL-12 and IL-18

NK-cell proliferation

IL-15

NK-cell survival

c NK-cell-mediated DC activation DC maturation

IFN-γ and TNF Immature DC

d NK-cell-mediated DC elimination NKp30- and TRAILmediated signals

DC lysis

CD94–NKG2Amediated signals

Figure 2 | Outcomes of interactions between dendritic cells and natural killer cells. Dendritic cells (DCs) can affect natural killer (NK)-cell functions by inducing the activation (a) and/or proliferation (b) of NK cells. DC-mediated activation of NK cells, which results in increased NK-cell cytolytic activity and/or interferon-γ (IFN-γ) production, can be induced by both resting and activated DCs, although the latter are more potent activators. Both cellcontact-dependent interactions and soluble cytokine signals are involved. NKG2D (NK group 2, member D) ligands that are expressed by DCs in response to appropriate stimuli have been implicated. A role for adhesion molecules has also been indicated by the finding that LFA1 (lymphocyte function-associated antigen 1)–ICAM1 (intercellular adhesion molecule 1) interactions are important for DC-mediated activation of NK cells. The relevance of interactions mediated by CD70–CD27, CD48–2B4 and CLRB (C-type-lectin-related B)–NKR-P1B (NK-cell receptor protein 1B) or NKR-P1D requires assessment. Cytokine signals are essential for NKcell activation, and several cytokines are involved (a). DC-derived cytokines have also been implicated in DC-mediated proliferation of NK cells (b). Reciprocally, NK cells can affect DC functions and lead to DC activation (c) or DC elimination (d). NK-cell-mediated activation of DCs seems to depend mainly on cytokines, principally tumour-necrosis factor (TNF) and IFN-γ (c), whereas cell-contact-dependent interactions are required for DC elimination (d). IL, interleukin; NKp30, NK-cell protein 30; TLR, Toll-like receptor; TRAIL, TNF-related apoptosis-inducing ligand.

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ligand for the inhibitory receptors NKR-P1B (NK-cell receptor protein 1B) and NKR-P1D66. It remains possible, and in fact probable, that the expression of additional ligands for NK-cell receptors might change during DC maturation, adding further complexity to the already multifarious interactions between DCs and NK cells. Adhesion molecules, which are crucial for cell–cell interactions, help to initiate the establishment of immune synapses and might affect the crosstalk between DCs and T cells. A recent report indicated that the activation of NK cells by DCs involves lymphocyte functionassociated antigen 1 (LFA1)67. The relevance of adhesion molecules in DC–NK-cell crosstalk requires further evaluation, because the strength of the synapse formed between DCs and NK cells might dictate the type of response that is elicited. In general, killing only requires a weak synapse68, whereas a strong synapse might be required for activities that involve cytokine secretion. Impact of NK-cell-derived signals. The capacity of DCs to mediate the activation of NK cells is clear, but NK cells can reciprocally regulate the function of DCs (FIG. 2c,d). One of the more intriguing aspects of DC–NK-cell interactions is that, unlike most normal healthy cells, immature DCs are uniquely susceptible to NK-cell-mediated cytolysis (FIG. 2d), whereas mature DCs are protected69–71. These differences have been attributed to differences in the level of MHC class I expression, specifically of HLA-E, by immature and mature DCs, a concept that is supported by the involvement of signals delivered by the inhibitory receptor complex CD94–NKG2A72. Signals delivered by activating receptors, such as NK-cell protein 30 (NKp30), are also important73 (FIG. 2d), and other NK-cell activating receptors might be required under appropriate circumstances. Interestingly, a small proportion (5–10%) of NK-cell clones, which are characterized by an NKG2Alow phenotype, can also kill mature DCs72. Unlike the destruction of tumour targets, elimination of immature DCs by mouse NK cells seems to be largely dependent on killing that is mediated by death receptors rather than by granule exocytosis74. Notably, the expression of relevant effector molecules by NK cells is modulated by cytokines. TRAIL (TNF-related apoptosisinducing ligand) expression is absent in IL-2-activated NK cells but high in IL-15-activated NK cells75. IFN-β can also induce TRAIL expression by NK cells76, and NK cells treated with IL-18 upregulate the expression of CD95 ligand (also known as FAS ligand)77. The finding that TRAIL is upregulated by NK cells cultured with IL-15 or IFN-β indicates that it might be the interaction with mature DCs that are producing these cytokines that enables NK cells to kill immature DCs. In addition to the resistance that is conferred by nonclassical MHC class I molecules, such as HLA-E, it is worth considering that mature DCs might become resistant to NK-cell-mediated death by mechanisms that are intrinsic to DCs themselves. The expression of protease inhibitor 9 (PI9), an intracellular inhibitor of granzyme B, is upregulated by DCs after maturation78.



REVIEWS

NKG2D

(Natural-killer group 2, member D). A primary activation receptor encoded by the natural killer (NK)-cell gene complex and expressed by all mature NK cells. It recognizes distinct families of ligands that are generally expressed only by infected, stressed or transformed cells. CD94–NKG2A

(CD94–natural-killer group 2, member A). A natural killer (NK)-cell receptor complex that consists of the invariant molecule CD94 and the C-type lectin receptor NKG2A. This complex delivers inhibitory signals to NK cells after recognition of the non-classical MHC molecule Qa-1b, in mice, or HLA-E, in humans.

Expression of PI9 has been proposed to prevent the elimination of DCs by activated CTLs, but it might also protect against NK-cell-mediated lysis. Both mature and immature DCs express the TRAIL receptor DR5 (death receptor 5); however, only immature DCs are sensitive to TRAIL-mediated apoptosis, and this is not always the case74. It remains to be shown which factors (such as, FLIP (caspase-8 (FLICE)-like inhibitory protein) levels and MYC expression79) might be important in determining the sensitivity of DCs to TRAIL-mediated cell death. Although the elimination of mature DCs by NK cells could be a negative-feedback mechanism involved in terminating ongoing immune responses and consequently limiting immunopathology70,80, the relevance of the killing of immature DCs is more intriguing. It has been postulated that the elimination of immature DCs by NK cells ensures maximal activation of adaptive immunity by allowing only mature DCs to be involved in antigen presentation80,81; however, this hypothesis remains to be validated in physiological settings. As well as defining the capacity of NK cells to eliminate DCs, in vitro co-culture of DCs and NK cells has revealed that the death of immature DCs is not an obligatory outcome of their interaction with NK cells. Indeed, under appropriate conditions, NK cells can induce, or at least augment, the maturation of DCs64,65,82 (FIG. 2c). Soluble factors, principally TNF and IFN-γ, and cell–cell contacts are required for effective maturation of DCs64,65. Recently, 4-1BB has been proposed to affect the proliferation and activation of DCs that is mediated by NK cells83. The relative importance of cytokines compared with contact-mediated signals differs depending on the context in which DC–NK-cell interactions occur. For example, DC activation induced by NK cells that have interacted with a target in the presence of type I IFNs is independent of cell–cell contact but requires TNF and IFN-γ 82. An additional factor that determines the outcome of DC–NK-cell interactions is the ratio of the interacting partners: low NK cell to DC ratios favour DC survival and maturation, whereas high NK cell to DC ratios result in elimination of DCs and inhibition of DC maturation64. These findings further highlight the complexity of DC–NK-cell interactions and the sophistication in the level at which control is exerted to ensure appropriate immunological outcomes. Relevance of TLR signals. Emerging evidence indicates that the outcome of DC–NK-cell interactions depends on the initial stimulus and the DC and NK-cell populations that are involved. The most effective DC–NK-cell interactions involve DCs that have been activated through TLR-mediated pathogen recognition. Ten TLRs with specialized recognition properties have been defined4, and the repertoire expressed by DCs varies according to the DC subset and the mouse strain84,85. Importantly, a single pathogen can activate multiple TLRs. TLR4 was thought to be specifically involved in the detection of Gram-negative bacteria through the recognition of LPS, a component of the


bacterial cell wall. Although Tlr4 gene-knockout mice do not respond to LPS, they still mount a response to Gram-negative bacteria86. This promiscuity has important implications because the cytokine-secretion profiles of DCs differ markedly depending on which TLRs are activated87,88 (FIG. 3a). The relevance of signals from TLRs in the context of DC–NK-cell crosstalk still requires careful assessment. Intriguingly, human NK cells express at least some members of the TLR family28,50,51. Although the capacity of NK cells to respond to signals from TLRs requires further analysis, available data indicate that NK cells that are activated directly by signalling through TLRs can produce IFN-γ and TNF and can acquire cytolytic activity against immature DCs89, showing that further complexity exists. Impact of site of encounter. The first contacts between DCs and NK cells probably occur at sites of infection. Indeed, direct contact between these cells has been observed in Malassezia sympodialis-induced skin lesions90 and in sites of imatinib (Gleevec)-induced lichenoid dermatitis91. The lymph nodes are another important site where NK cells and DCs might come in contact. Upregulation of expression of CC-chemokine receptor 7 (CCR7) by maturing DCs favours their migration to lymph nodes. Cytokines that are produced by mature DCs entering lymph nodes might activate resident NK cells and promote their proliferation. CD16–CD56hi NK cells, which are found in human lymph nodes and secondary lymphoid tissues, proliferate in response to membrane-bound IL-15 on DCs60. Interestingly, however, the ability to kill immature DCs is restricted to the CD16+NKG2Alowkiller-cell immunoglobulin-like receptor (KIR)– subset72, which does not proliferate in response to signals from DCs. In mice, NK cells that express TRAIL seem to be the population of NK cells that best eliminates immature DCs (M. E. Wallace and M.J.S., unpublished observations). The localization of these NK cells in lymph nodes needs to be evaluated. The phenotypic and functional differences between NK cells that migrate to tissues and those that are present in the lymph nodes indicate differential immunoregulatory roles for DC–NK-cell interactions at these sites. A recent study has shown that, in mice, recruitment of NK cells to the lymph nodes is effectively mediated through CXCR3-dependent signals by DCs activated by TLR4 ligands63. Importantly, in the lymph nodes, NK cells are an early source of IFN-γ, and they promote proliferation of T cells and polarization towards a TH1 response63. Crosstalk in viral infection. Remarkably, and perhaps somewhat surprisingly, most of the information relating to the complexity of DC–NK-cell interactions has been generated in human systems, and only a limited number of mouse studies have been carried out. Because it is clear that the outcome of DC–NK-cell crosstalk largely depends on the physical and cytokine environments, it is imperative to analyse these interactions in vivo during the course of immune responses elicited by infection, tumour challenge or autoimmunity.



REVIEWS

b Early interactions between DCs and NK cells in MCMV infection

a TLR-mediated activation of DCs

TLR9

TLR

MCMV

Pathogen TLR3 m157 DC

Plasmacytoid DC

IFN-α, IFN-β, IL-2, IL-12, IL-15, IL-18

RAE1 or H60 NKG2D

LY49H

CCL3

IFN-α IFN-β

Kill virus

NK-cell recruitment IFN-α, IFN-β, IL-6, IL-12

IFN-α, IFN-β, IL-2, IL-6, IL-12, IL-15, TNF

NK cell NK-cell activation

c Late interactions between DCs and NK cells IFN-γ TNF GM-CSF

in MCMV infection

Cytolysis of infected targets IL-12 IL-18

DC maturation

IFN-γ

NK-cell proliferation

Cytolysis of infected targets

Figure 3 | Crosstalk between dendritic cells and natural killer cells during viral infection. Interactions between dendritic cells (DCs) and natural killer (NK) cells occur during various stages of the immune responses that are elicited by viral pathogens. a | Recognition of viral components by Toll-like receptors (TLRs) induces the secretion of cytokines by both conventional DCs and plasmacytoid DCs. Several pro-inflammatory cytokines can be secreted. Different types of DC express different sets of TLRs, and TLR-expression patterns also depend on the environment in which DCs are generated. b | Cytokines such as interferon-α (IFN-α) and IFN-β (type I interferons) can mediate direct antiviral effects, but they can also activate NK-cell functions. Early TLRmediated signals might be required for effective activation of NK cells through pathways that also involve interactions with activating NK-cell receptors that recognize viral proteins (such as the murine cytomegalovirus (MCMV) glycoprotein m157) or stress ligands (such as RAE1 (retinoic acid early transcript 1) or H60, which are functional homologues of MICA (MHC-class-I-polypeptide-related sequence A) and MICB). These early interactions result in the recruitment and activation of NK cells. Activated NK cells can efficiently eliminate virally infected targets by cytolytic and cytokine-mediated pathways. Cytokines (such as tumour-necrosis factor (TNF), GM-CSF (granulocyte/macrophage colony-stimulating factor) and IFN-γ) that are produced by activated NK cells could also impact on further DC activation and maturation. (For simplicity, TLR3 and TLR9 are depicted at the cell surface, but they reside in intracellular compartments.) c | DCs — after activation either directly by viral encounter or indirectly by cytokines that are released by NK cells or other immune effectors — produce cytokines (interleukin-12 (IL-12) and IL-18) that affect the proliferation of LY49H+ NK cells late in viral infection. CCL3, CC-chemokine ligand 3; NKG2D, NK group 2, member D.

The role of NK cells during viral infection has been studied in detail for several pathogens, including herpesviruses such as CMV. Compelling evidence of a crucial role for NK cells has been provided by infection of mice with MCMV 92,93. Similarly, studies in humans have implicated NK cells in the control of the severity of infection with herpesvirus94, hepatitis B virus and HIV93. The antiviral activities of NK cells involve both the direct lysis of infected targets and the release of antiviral cytokines95,96. The NK-cell-mediated control of MCMV infection is genetically determined, and effective NK-cell responses result in resistance, which is typified by low levels of virus replication in the spleen. This response is associated with a single dominant locus, Cmv1, which mainly controls viral replication in the spleen97,98. Cmv1 encodes the NK-cell activating receptor LY49H99–101, which was recently shown to activate NK cells after engagement of the MCMV glycoprotein m157 (REFS 52,53). One of the characteristics of the

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LY49H-mediated response during infection with MCMV is the specific proliferation of this NK-cell subset late in infection102. In mouse strains that lack LY49H, which are therefore classified as being susceptible to infection with MCMV, NK cells do not effectively limit viral replication. The virus therefore replicates to high titres in visceral organs, but it is eventually controlled through the activities of CD8+ T cells103. Our studies identified DCs as a principal target of MCMV and showed that infection results in functional impairment of DCs104. More recently, we have used MCMV to study DC–NK-cell crosstalk in response to viral challenge and have defined a functional interaction between LY49H+ NK cells and CD8α+ DCs, in which the presence of LY49H+ NK cells results in maintenance of CD8α+ DCs in the spleen during acute infection with MCMV. Reciprocally, CD8α+ DCs are essential for the proliferation of LY49H+ NK cells by a mechanism that involves IL-12 and IL-18 (REF. 105).



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DANGER SIGNALS

Cell-wall components and other products of pathogens that alert the innate immune system to the presence of potentially harmful invaders, usually by interacting with Toll-like receptors and other pattern-recognition receptors that are expressed by tissue cells and dendritic cells.

The loss of DCs that is observed in the context of the suboptimal NK-cell activation elicited in susceptible mouse strains is unlikely to be the result of DC elimination. There is no evidence of apoptosis of DCs (D. M. Andrews and M.A.D.E., unpublished observations), and virus-mediated downregulation of CD8α expression is unlikely to be occurring, because these cells are poorly infected by MCMV104. Indeed, CD11c+ CD8α–CD11b+ DCs, which are the principal target of MCMV, become resistant to apoptosis as a result of viral interference with activation of the BCL-2 (B-cell lymphoma 2) pathway106. We postulate that the loss of CD8α+ DCs results from viral interference with the generation of DC-lineage-specific populations and/or their full maturation, a phenomenon also described for infection with lymphocytic choriomeningitis virus107. MCMV is known to infect haematopoietic progenitors108–111, and it interferes with the maturation of DCs104. Importantly, our studies showed that efficient longterm activation of NK cells during infection with MCMV requires input from DCs. So, the MCMVspecific proliferation of LY49H+ NK cells that is observed between day 6 and day 8 after infection102 depends on the presence of CD8α+ DCs and requires IL-12 and IL-18 (REF. 105) (FIG. 3c). The requirement for signals in addition to those delivered by IL-12 and IL-18 remains unknown, and contact-dependent interactions, including those involving LY49H and the m157 viral glycoprotein, might also be important. Whether infected DCs express m157 (FIG. 3b) remains to be determined, but the production of appropriate detection reagents, together with the recently generated m157-mutant viruses112–114, will be useful in addressing this important issue. The relevance of LY49H+ NK-cell proliferation to the generation of adaptive immunity and memory and to the long-term outcome of infection with MCMV is not fully understood. Emerging evidence in tumour models indicates that DC–NK-cell interactions affect the outcome of adaptive immune responses, including memory responses to tumour challenge. This might also be true of infection, and DC–NK-cell interactions might be crucial not only during the early phase of innate immunity but also during the development of adaptive immunity, including the memory responses that are essential to keep persistent viruses, such as MCMV, in check. A better understanding of these complex processes will ultimately provide insights that are crucial for improved control of viral infections. pDCs are thought to have a crucial role in viral infection, including infection with MCMV 19. In response to infection with MCMV, pDCs produce cytokines, including IFN-α, IL-12 and TNF, and can activate NK-cell cytotoxicity and IFN-γ production ex vivo115. However, in vivo depletion of pDCs before infection does not completely abrogate IFN-α secretion, does not affect19 (or indeed increases116) IL-12 levels and does not alter splenic MCMV titres116. So, the relevance of pDCs in viral infection requires careful further evaluation.


The role of TLR signalling in the generation of effective antiviral responses has been investigated116,117 (FIG. 3b). pDCs from TLR9-deficient mice showed impaired production of IFN-α and IL-12 in response to MCMV infection, which resulted in reduced NK-cell proliferation and activation, typified by decreased CD69 expression and reduced IFN-γ production116 (FIG. 3b). However, a cytokine response, albeit diminished and delayed, was detected during MCMV infection in the absence of signalling through TLR9 (REFS 116,117). TLR3 has also been implicated, although to a lesser extent, in innate immune responses to MCMV 117, which is indicative of the complexity of the system. The ability of TLR9deficient DCs to activate NK-cell cytolysis is unclear116. The lack of signalling through TLR9 resulted in increased viral titres in the spleen of C57BL/6 mice116, which are usually resistant to infection at this site. Spleen titres were not significantly altered in BALB/c mice (which are usually susceptible to infection) that lacked TLR9. Importantly, the removal of pDCs before infection did not lead to increased viral titres116. From these studies, it was concluded that efficient viral control in a model in which NK-cell activation is generated by LY49H–m157 interactions seems to require signals from TLR9 (REF. 116) (FIG. 3b). Because the LY49H-mediated resistance to MCMV infection is perforin dependent (M.A.D.E., A. A. Scalzo and M.J.S., unpublished observations), it is imperative to establish that NK cells that are activated in the absence of TLR9 signalling lack lytic effector function. The available data provide evidence of early interactions between DCs and NK cells; however, the precise role of DC subsets (including pDCs) and TLR signalling (including through TLR9) in the generation of effective innate antiviral immunity requires further evaluation. Furthermore, TLR9-deficient BALB/c mice were reported to have lower proportions of virus-specific CD8+ T cells116, indicating that further analysis is also required to address the relevance of TLR-mediated DC–NK-cell interactions in the generation and maintenance of adaptive T-cell-mediated antiviral responses. On balance, the available data indicate that DC activation is essential to elicit effective innate immunity to viral pathogens, such as CMV, in which DCs direct effective early activation116 and late proliferation105 of NK cells (FIG. 3b,c). Crosstalk in innate and adaptive immunity to tumours. One of the main difficulties in generating immunity to tumours is that, being derived from self-tissues, they are usually poorly immunogenic and do not display the DANGER SIGNALS that are required for the activation of DCs. NK cells are unique in their ability to detect changes in tumour cells in the absence of inflammatory stimuli, and as such, they can be responsible for rejecting tumours by directly killing tumour cells118,119. Alternatively, they can stimulate components of the adaptive immune system to eliminate tumours45. NK cells can facilitate adaptive antitumour immunity by producing IFN-γ and other cytokines that recruit and activate DCs, T cells and B cells120–122. IFN-γ can directly stimulate the development



REVIEWS

IFN-α, IFN-β, IL-2 NK cell IFN-γ DC

NK-cell inhibitory receptor

NKG2D RAE1 or H60

Peptide– MHC class Ilow

IL-12 HSP gp96

Tumour cell CTL

Perforin IFN–γ

Apoptotic tumour cell

Perforin IFN–γ

IFN–γ Impaired angiogenesis and reduced metastatic capacity of tumour

Figure 4 | Impact of crosstalk between dendritic cells and natural killer cells on immunity to tumours. Dendritic cell (DC)-mediated activation of natural killer (NK) cells can affect their ability to lyse tumours (through perforin-dependent mechanisms) or can reduce the growth and metastatic capacity of tumours (through interferon-γ (IFN-γ)-dependent mechanisms). Interactions between NK cells and tumour cells, which involve both NK-cell activating and inhibitory receptors, are essential. NK cells that have interacted with tumour targets can secrete cytokines that affect the maturation of DCs, so they might participate in the generation and/or maintenance of tumourspecific T-cell-mediated responses. In addition, antigens that are generated in the process of tumour killing by activated NK cells can provide DCs with increased access to tumour antigens. Tumours that secrete certain proteins, such as the heat-shock protein (HSP) glycoprotein 96 (gp96), are rejected by cytotoxic T lymphocyte (CTL)-mediated antitumour responses, the generation of which relies on the presence of NK cells. IL, interleukin; NKG2D, NK group 2, member D; RAE1, retinoic acid early transcript 1.

of CTLs and, consequently, lead to the development of a memory response specific for a tumour45,123. NK cells that mediate primary rejection through the recognition of the CD27 ligand CD70 (REF. 45) or through NKG2D ligands124 expressed by tumour cells evoke T-cell immunity to secondary MHC-class-I-sufficient tumours. Given the requirement for DCs in the activation of naive T cells, it is probable that DC–NK-cell interactions are central to this process. In the case of NK-cell-mediated rejection of tumours that have downregulated MHC class I expression, the involvement of DC–NK-cell communication has been shown more directly, with activated IFN-γ-producing NK cells priming DCs to induce protective CD8+ T-cell responses125 (FIG. 4). The recently described capacity of activated DCs to induce the recruitment of NK cells to the lymph nodes, where they function as an early source of IFN-γ 63, indicates that DC–NK-cell interactions in secondary lymphoid organs might be common and could be exploited therapeutically. IFN-γ secreted during primary tumour rejection that is mediated by NK cells is crucial for the development of CTLs, particularly when tumours express the co-stimulatory molecules CD70 or CD80. By contrast, primary tumour rejection that is mediated by NK cells through NKG2D can prime secondary CTL

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responses in the absence of IFN-γ but requires the priming of CD4 + T cells44. Finally, recent evidence indicates that activated NK cells can also stimulate the maturation of DCs, ‘licensing’ DCs to present antigen to CTLs in lymph nodes65,126. Clearly, much remains to be learned about the immune networks that operate during NK-cell-mediated tumour rejection. It is worth noting that NK cells might enhance adaptive immunity to tumours in a more indirect way. The killing of tumour targets by NK cells could provide DCs with increased access to tumour antigens (FIG. 4). Immature DCs efficiently phagocytose apoptotic and necrotic cells, process antigens for presentation to T cells and mature after exposure to necrotic (but not apoptotic) cells, most probably through the release of heat-shock proteins127,128. Therefore, NK cells might kill targets in a way that recruits DCs, facilitates antigen uptake by DCs and triggers their maturation. Although the links between NK cells and adaptive immunity remain poorly defined at a molecular level, these links are clearly involved in the generation of effective antitumour responses. A recent report showed a correlation between the activation of NK cells and the clonal expansion of CTLs in response to challenge with tumour cells that secrete the heat-shock protein glycoprotein 96 (gp96). gp96-secreting tumours are effectively rejected by CD8+ T cells. However, in the absence of NK-cell activation, or when NK cells are perforin-deficient, CTL responses are not effectively elicited129, indicating that a perforin-dependent positive-feedback loop between NK cells and DCs is required for the generation of effective antitumour activity (FIG. 4). Notably, inflammation can bypass this reliance on perforin, indicating that tumour challenges that do not lead to inflammation, or NK-cell activation, will not generate effective CTL responses. This information must be considered in the design of improved therapeutic applications. Therapeutic implications

Therapeutic approaches that stimulate DCs to produce NK-cell-activating cytokines might provide a precise route of NK-cell activation, minimizing the toxic sideeffects of systemic cytokine administration, with the added advantage that combined activation of NK cells and DCs can augment adaptive immune responses. Therapeutic approaches indicate that NK cells might have a role in controlling human malignancy, particularly when cytokines (for example, IL-2, IL-12, IL-15, IL-18, IL-21 and type I IFNs) are used to induce NK-cell differentiation and activation130–133. Therapies that combine antibodies and cytokines, such as type I IFNs and IL-2, have already been successfully used for the treatment of chronic myeloid leukaemia (CML), melanoma and renal-cell cancer134,135. In particular, emerging evidence indicates that NK cells are crucial for the effectiveness of responses that are induced by IFN-α and rituximab (Rituxin)-antibody therapy of human CML136,137. An attractive proposition under therapeutic consideration is the use of IL-21, which is closely related to IL-2 (REF. 138); IL-21 induces a terminal-differentiation programme in



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CpG SEQUENCES

Oligodeoxynucleotide sequences that include a cytosine-guanosine sequence and certain flanking nucleotides. These have been found to induce innate immune responses through interaction with Toll-like receptor 9.

NK cells that leads to enhanced cytotoxicity, increased IFN-γ production133,139 and potent antitumour effects mediated by the activation of NK cells and CD8+ T cells133,140. Adoptive transfer of NK cells has clearly shown the ability of these cells to mount a therapeutic antitumour response141, and modulation of NK-cell receptors in allogeneic bone-marrow transplants might promote improved graft-versus-leukaemia effects142. Imatinib (Gleevec), a specific inhibitor of the protein tyrosine kinases ABL and cKIT, has been successfully used in the treatment of several malignancies, including CML and gastrointestinal stromal tumours. The possibility that imatinib might exert some of its antitumour effects by promoting activation of NK cells was tested in a mouse model. When imatinib was administered with FLT3 ligand (fms-related tyrosine kinase 3 ligand), it acted on DCs and induced NK-cell activation91. In patients with gastrointestinal stromal tumours, imatinib induced NK-cell activation in 49% of cases. Notably, progression to disease was slower in these patients compared with patients who did not show NK-cell activation after treatment with imatinib91. Despite these promising advances indicating that NK cells might be harnessed to reject mouse and human tumours, the systemic administration of cytokines has been associated with considerable toxicity, particularly for cytokines (such as IL-2 and IFN-α) that non-specifically activate a broad range of immune effectors. Selective activation of DCs might be a better way of inducing both innate and adaptive immune mechanisms to promote effective immune responses. Such activation might be achieved using compounds that mimic pathogen-induced danger signals, resulting in the selective activation of pDCs to produce type I IFNs or conventional DCs to produce IL-12, IL-18 and/or TNF. The administration of synthetic oligodeoxynucleotides containing unmethylated CpG SEQUENCES, which mimic bacterial DNA, has shown considerable promise as an antitumour therapy143,144. These compounds specifically trigger the maturation of DCs and other innate immune cells that express TLR9 (REF. 145). In mice, TLR9 is expressed by all DC subsets, and its engagement results in the induction of both IFN-α and IL-12. In humans, TLR9 seems to be exclusively expressed by pDCs, and its stimulation selectively induces the production of type I IFNs. These important biological differences must be considered when the findings from mouse models are extrapolated to possible human settings. Furthermore, despite these studies having focused on the ability of CpG-containing oligodeoxynucleotides to enhance antitumour T-cell responses, they might also elicit NK-cell functions. Reports of the expression of TLR9 by NK cells describe conflicting results; however, it is possible that some NK-cell subsets might express the appropriate amount of TLR9 for them to be directly activated by exposure to its ligands. Direct activation of NK cells through other TLRs, including TLR2, TLR3, TLR5 (REF. 51) and TLR9 (REF. 89) has been reported. In these studies, direct activation of NK cells through TLR signalling is optimal in the presence of DCderived cytokines, such as IL-2, IL-12, IL-15 and IFN-α,


indicating that DC–NK-cell crosstalk might facilitate TLR-mediated activation of NK cells and that dual activation of NK cells and DCs might greatly contribute to the success of therapies that use TLR ligands to augment immunity. Therapies that exploit DC–NK-cell interactions might also be useful for improving the outcome of allogeneic transplants. NK-cell alloreactivity that is generated in haploidentical, but KIR mismatched, transplants abolishes graft rejection and tumour relapse, and reduces graft-versus-host disease146. The relevance of DC–NK-cell crosstalk and its clinical implications remain to be investigated. Concluding remarks

In the past four years, evidence has accumulated that effective immunity is elicited by a delicate balance of intricate interactions between multiple immune effector cells. NK cells, which were historically considered to be primitive and non-specific executioners, have proved to be highly evolved and sophisticated assassins that not only directly execute immune responses but also regulate their outcome through complex interplay with DCs. DCs have long been recognized as highly versatile components of the immune system that can affect adaptive immunity at multiple levels. Interactions of DCs with T cells, B cells and NKT cells have been described. Studies of the interactions between DCs and NK cells have now revealed a role for DCs in innate immunity. Importantly, DC–NK-cell interactions can also affect adaptive immune responses. The outcome of DC–NK-cell interactions is crucially dependent on the interacting partners, with various DC and NK-cell subsets mediating differing immune responses. DC–NK-cell crosstalk is a dynamic process, and the stimuli received, the site where the interactions occur, and the physical and cytokine milieux have a considerable impact on the significance of the crosstalk and its immunological outcomes. Remarkably, most data regarding DC–NK-cell crosstalk have been generated using in vitro studies of human cells, and at present, the number of studies addressing the relevance of these interactions in the context of in vivo situations is limited. Owing to the ability of DCs to initiate immune responses, DC-immunotherapy protocols are increasingly being tested in clinical trials. Most of these are aimed at eliciting T-cell responses. Because NK cells have established roles in suppressing viral replication and in mediating tumour surveillance, therapies that are designed to elicit both NK-cell and T-cell responses should prove superior to those currently being evaluated. Although it is clear that there is crosstalk between DCs and NK cells, the intricacies of these DC–NK-cell interactions remain to be unravelled. The relevant mechanisms require further clarification, and importantly, their relative significance needs to be addressed in vivo. The coming years will be exciting because understanding DC–NK-cell interactions is sure to bring us a step closer to the development of more effective and specific immunotherapies that will provide long-term protective immunity.



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7.

8. 9.

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122

Shah, P. D., Gilbertson, S. M. & Rowley, D. A. Dendritic cells that have interacted with antigen are targets for natural killer cells. J. Exp. Med. 162, 625–636 (1985). Fernandez, N. C. et al. Dendritic cells directly trigger NK cell functions: cross-talk relevant in innate anti-tumor immune responses in vivo. Nature Med. 5, 405–411 (1999). This was the first study to show that DCs could affect NK-cell functions that are relevant to immune surveillance for tumours in vivo. Steinman, R. M. Some interfaces of dendritic cell biology. APMIS 111, 675–697 (2003). Kaisho, T. & Akira, S. Regulation of dendritic cell function through Toll-like receptors. Curr. Mol. Med. 3, 373–385 (2003). Iwasaki, A. & Medzhitov, R. Toll-like receptor control of the adaptive immune responses. Nature Immunol. 5, 987–995 (2004). Shi, G. X., Harrison, K., Han, S. B., Moratz, C. & Kehrl, J. H. Toll-like receptor signaling alters the expression of regulator of G protein signaling proteins in dendritic cells: implications for G protein-coupled receptor signaling. J. Immunol. 172, 5175–5184 (2004). Hou, W. S. & Van Parijs, L. A Bcl-2-dependent molecular timer regulates the lifespan and immunogenicity of dendritic cells. Nature Immunol. 5, 583–589 (2004). Akira, S. Mammalian Toll-like receptors. Curr. Opin. Immunol. 15, 5–11 (2003). Gordon, S. Pattern recognition receptors: doubling up for the innate immune response. Cell 111, 927–930 (2002). Park, Y., Lee, S. W. & Sung, Y. C. CpG DNA inhibits dendritic cell apoptosis by up-regulating cellular inhibitor of apoptosis proteins through the phosphatidylinositide-3′-OH kinase pathway. J. Immunol. 168, 5–8 (2002). Reis e Sousa, C. Toll-like receptors and dendritic cells: for whom the bug tolls. Semin. Immunol. 16, 27–34 (2004). Steinman, R. M., Hawiger, D. & Nussenzweig, M. C. Tolerogenic dendritic cells. Annu. Rev. Immunol. 21, 685–711 (2003). Heath, W. R. et al. Cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens. Immunol. Rev. 199, 9–26 (2004). Ardavin, C. Origin, precursors and differentiation of mouse dendritic cells. Nature Rev. Immunol. 3, 582–590 (2003). Shortman, K. & Liu, Y. J. Mouse and human dendritic cell subtypes. Nature Rev. Immunol. 2, 151–161 (2002). den Haan, J. M. M., Lehar, S. M. & Bevan, M. J. CD8+ but not CD8– dendritic cells cross-prime cytotoxic T cells in vivo. J. Exp. Med. 192, 1685–1695 (2001). Belz, G. T. et al. Conventional CD8α+ dendritic cells are generally involved in priming CTL immunity to viruses. J. Immunol. 172, 1996–2000 (2004). Boonstra, A. et al. Flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing T helper type 1 and 2 cell development: dependency on antigen dose and differential Toll-like receptor ligation. J. Exp. Med. 197, 101–109 (2003). This study shows that the distinct functions of DC subsets are not intrinsic but, instead, are dictated by environmental signals. Asselin-Paturel, C. et al. Mouse type I IFN-producing cells are immature APCs with plasmacytoid morphology. Nature Immunol. 2, 1144–1150 (2001). This paper identifies plasmacytoid DCs as the cells that produce high amounts of type I IFNs in response to viral challenge. Nakano, H., Yanagita, M. & Gunn, M. D. CD11c+B220+Gr-1+ cells in mouse lymph nodes and spleen display characteristics of plasmacytoid dendritic cells. J. Exp. Med. 194, 1171–1178 (2001). Krug, A. et al. Interferon-producing cells fail to induce proliferation of naive T cells but can promote expansion and T helper 1 differentiation of antigen-experienced unpolarized T cells. J. Exp. Med. 197, 899–906 (2003). Diebold, S. S. et al. Viral infection switches nonplasmacytoid dendritic cells into high interferon producers. Nature 424, 324–328 (2003). Cella, M., Facchetti, F., Lanzavecchia, A. & Colonna, M. Plasmacytoid dendritic cells activated by influenza virus and CD40L drive a potent TH1 polarization. Nature Immunol. 1, 305–310 (2000). Kadowaki, N., Antonenko, S., Lau, J. Y. & Liu, Y. J. Natural interferon α/β-producing cells link innate and adaptive immunity. J. Exp. Med. 192, 219–226 (2000). Krug, A. et al. Toll-like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL-12. Eur. J. Immunol. 31, 3026–3037 (2001).

26. Kadowaki, N. et al. Subsets of human dendritic cell precursors express different Toll-like receptors and respond to different microbial antigens. J. Exp. Med. 194, 863–870 (2001). 27. Jarrossay, D., Napolitani, G., Colonna, M., Sallusto, F. & Lanzavecchia, A. Specialization and complementarity in microbial molecule recognition by human myeloid and plasmacytoid dendritic cells. Eur. J. Immunol. 31, 3388–3393 (2001). 28. Hornung, V. et al. Quantitative expression of Toll-like receptor 1–10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J. Immunol. 168, 4531–4537 (2002). 29. Kalinski, P., Schuitemaker, J. H. N., Hilkens, C. M. U., Wierenga, E. A. & Kapsenberg, M. L. Final maturation of dendritic cells is associated with impaired responsiveness to IFN-γ and to bacterial IL-12 inducers: decreased ability of mature dendritic cells to produce IL-12 during the interaction with TH cells. J. Immunol. 162, 3231–3236 (1999). 30. Vieira, P. L., de Jong, E. C., Wierenga, E. A., Kapsenberg, M. L. & Kalinski, P. Development of TH1-inducing capacity in myeloid dendritic cells requires environmental instruction. J. Immunol. 164, 4507–4512 (2000). 31. Tanaka, H., Demeure, C. E., Rubio, M., Delespesse, G. & Sarfati, M. Human monocyte-derived dendritic cells induce naive T cell differentiation into T helper cell type 2 (TH2) or TH1/TH2 effectors: role of stimulator/responder ratio. J. Exp. Med. 192, 405–411 (2000). 32. Nagai, T. et al. Timing of IFN-β exposure during human dendritic cell maturation and naive TH cell stimulation has contrasting effects on TH1 subset generation: a role for IFN-βmediated regulation of IL-12 family cytokines and IL-18 in naive TH cell differentiation. J. Immunol. 171, 5233–5243 (2003). 33. Colucci, F., Caligiuri, M. A. & Di Santo, J. P. What does it take to make a natural killer? Nature Rev. Immunol. 3, 413–425 (2003). 34. Kim, S. et al. In vivo developmental stages in murine natural killer cell maturation. Nature Immunol. 3, 523–528 (2002). 35. Loza, M. J. & Perussia, B. Final steps of natural killer cell maturation: a model for type 1–type 2 differentiation? Nature Immunol. 2, 917–924 (2001). 36. Cooper, M. A. et al. Human natural killer cells: a unique innate immunoregulatory role for the CD56bright subset. Blood 97, 3146–3151 (2001). 37. Moretta, A. et al. Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu. Rev. Immunol. 19, 197–223 (2001). 38. Raulet, D. H., Vance, R. E. & McMahon, C. W. Regulation of the natural killer cell receptor repertoire. Annu. Rev. Immunol. 19, 291–330 (2001). 39. Natarajan, K., Dimasi, N., Wang, J., Mariuzza, R. A. & Margulies, D. H. Structure and function of natural killer cell receptors: multiple molecular solutions to self, nonself discrimination. Annu. Rev. Immunol. 20, 853–885 (2002). 40. Bauer, S. et al. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285, 727–729 (1999). 41. Cerwenka, A. & Lanier, L. L. Natural killer cells, viruses and cancer. Nature Rev. Immunol. 1, 41–49 (2001). 42. Diefenbach, A. & Raulet, D. H. Strategies for target cell recognition by natural killer cells. Immunol. Rev. 181, 170–184 (2001). 43. Lanier, L. L. The origin and functions of natural killer cells. Clin. Immunol. 95, S14–S18 (2000). 44. Hayakawa, Y. et al. Tumor rejection mediated by NKG2D receptor–ligand interaction is dependent upon perforin. J. Immunol. 169, 5377–5381 (2002). 45. Kelly, J. M. et al. Induction of tumor-specific T cell memory by NK cell-mediated tumor rejection. Nature Immunol. 3, 83–90 (2002). This paper shows that NK cells participate in the generation of specific adaptive immune responses and defines CD27–CD70 interactions as a crucial link between innate and adaptive immunity. 46. Trapani, J. A. & Smyth, M. J. Functional significance of the perforin/granzyme cell death pathway. Nature Rev. Immunol. 2, 735–747 (2002). 47. Smyth, M. J. et al. Nature’s TRAIL — on a path to cancer immunotherapy. Immunity 18, 1–6 (2003). 48. Smyth, M. J. et al. Tumor necrosis factor-related apoptosisinducing ligand (TRAIL) contributes to interferon γdependent natural killer cell protection from tumor metastasis. J. Exp. Med. 193, 661–670 (2001). 49. Takeda, K. et al. Involvement of tumor necrosis factorrelated apoptosis-inducing ligand in surveillance of tumor metastasis by liver natural killer cells. Nature Med. 7, 94–100 (2001).


50. Muzio, M. et al. Differential expression and regulation of Tolllike receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells. J. Immunol. 164, 5998–6004 (2000). 51. Chalifour, A. et al. Direct bacterial protein PAMP recognition by human NK cells involves TLRs and triggers α-defensin production. Blood 104, 1778–1783 (2004). 52. Arase, H., Mocarski, E. S., Campbell, A. E., Hill, A. B. & Lanier, L. L. Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors. Science 296, 1323–1326 (2002). 53. Smith, H. R. et al. Recognition of a virus-encoded ligand by a natural killer cell activation receptor. Proc. Natl Acad. Sci. USA 99, 8826–8831 (2002). 54. Borg, C. et al. NK cell activation by dendritic cells (DCs) requires the formation of a synapse leading to IL-12 polarization in DCs. Blood 104, 3267–3275 (2004). 55. Fernandez, N. C. et al. Dendritic cells (DC) promote natural killer (NK) cell functions: dynamics of the human DC/NK cell cross talk. Eur. Cytokine Netw. 13, 17–27 (2002). 56. Jinushi, M. et al. Critical role of MHC class I-related chain A and B expression on IFN-α-stimulated dendritic cells in NK cell activation: impairment in chronic hepatitis C virus infection. J. Immunol. 170, 1249–1256 (2003). 57. Granucci, F. et al. A contribution of mouse dendritic cellderived IL-2 for NK cell activation. J. Exp. Med. 200, 287–295 (2004). This is a recent report that shows that the IL-2 derived from DCs in response to bacterial encounter contributes to the activation of NK-cell functions. 58. Feau, S. et al. Dendritic cell-derived IL2 production is regulated by IL15 both in humans and mice. Blood 7 Sep 2004 (doi:10.1182/blood-2004-03-1059). 59. Jinushi, M. et al. Autocrine/paracrine IL-15 that is required for type I IFN-mediated dendritic cell expression of MHC class I-related chain A and B is impaired in hepatitis C virus infection. J. Immunol. 171, 5423–5429 (2003). 60. Ferlazzo, G. et al. Distinct roles of IL-12 and IL-15 in human natural killer cell activation by dendritic cells from secondary lymphoid organs. Proc. Natl Acad. Sci. USA 101, 16606–16611 (2004). 61. Sauma, D. et al. Interleukin-4 selectively inhibits interleukin-2 secretion by lipopolysaccharide-activated dendritic cells. Scand. J. Immunol. 59, 183–189 (2004). 62. Terme, M. et al. IL-4 confers NK stimulatory capacity to murine dendritic cells: a signaling pathway involving KARAP/DAP12-triggering receptor expressed on myeloid cell 2 molecules. J. Immunol. 172, 5957–5966 (2004). 63. Martin-Fontecha, A. et al. Induced recruitment of NK cells to the lymph nodes provides IFN-γ for TH1 priming. Nature Immunol. 5, 1260–1265 (2004). This recent publication shows that NK cells are actively recruited to the lymph nodes in a CXCR3dependent manner after injection of mature DCs. Importantly, in the lymph nodes, NK-cell-derived IFN-γ has a key role in polarization towards TH1 responses. 64. Piccioli, D., Sbrana, S., Melandri, E. & Valiante, N. M. Contact-dependent stimulation and inhibition of dendritic cells by natural killer cells. J. Exp. Med. 195, 335–341 (2002). 65. Gerosa, F. et al. Reciprocal activating interaction between natural killer cells and dendritic cells. J. Exp. Med. 195, 327–333 (2002). References 64 and 65, together with reference 73, show that reciprocal interactions occur between DCs and NK cells. They also detail some of the relevant molecular mechanisms and define the impact of such interactions on both DC and NK-cell functions. 66. Iizuka, K., Naidenko, O. V., Plougastel, B. F., Fremont, D. H. & Yokoyama, W. M. Genetically linked C-type lectin-related ligands for the NKRP1 family of natural killer cell receptors. Nature Immunol. 4, 801–807 (2003). 67. Poggi, A. et al. NK cell activation by dendritic cells is dependent on LFA-1-mediated induction of calcium– calmodulin kinase II: inhibition by HIV-1 Tat C-terminal domain. J. Immunol. 168, 95–101 (2002). 68. Purbhoo, M. A., Irvine, D. J., Huppa, J. B. & Davis, M. M. T cell killing does not require the formation of a stable mature immunological synapse. Nature Immunol. 5, 524–530 (2004). 69. Wilson, J. L. et al. Targeting of human dendritic cells by autologous NK cells. J. Immunol. 163, 6365–6370 (1999). 70. Carbone, E. et al. Recognition of autologous dendritic cells by human NK cells. Eur. J. Immunol. 29, 4022–4029 (1999). 71. Ferlazzo, G. et al. The interaction between NK cells and dendritic cells in bacterial infections results in rapid induction of NK cell activation and in the lysis of uninfected dendritic cells. Eur. J. Immunol. 33, 306–313 (2003).



REVIEWS 72. Della Chiesa, M. et al. The natural killer cell-mediated killing of autologous dendritic cells is confined to a cell subset expressing CD94/NKG2A, but lacking inhibitory killer Ig-like receptors. Eur. J. Immunol. 33, 1657–1666 (2003). This study shows that a specific NK-cell subset is involved in killing immature DCs. Although most mature DCs are resistant to NK-cell-mediated death, a small population of NK cells was shown to be capable of killing mature DCs. The results imply that NK-cell subsets can differentially affect DC functions. 73. Ferlazzo, G. et al. Human dendritic cells activate resting natural killer (NK) cells and are recognized via the NKp30 receptor by activated NK cells. J. Exp. Med. 195, 343–351 (2002). 74. Hayakawa, Y. et al. NK cell TRAIL eliminates immature dendritic cells in vivo and limits dendritic cell vaccination efficacy. J. Immunol. 172, 123–129 (2004). 75. Kayagaki, N. et al. Expression and function of TNF-related apoptosis-inducing ligand on murine activated NK cells. J. Immunol. 163, 1906–1913 (1999). 76. Sato, K. et al. Antiviral response by natural killer cells through TRAIL gene induction by IFN-α/β. Eur. J. Immunol. 31, 3138–3146 (2001). 77. Yu, Y. et al. Enhancement of human cord blood CD34+ cellderived NK cell cytotoxicity by dendritic cells. J. Immunol. 166, 1590–1600 (2001). 78. Hirst, C. E. et al. The intracellular granzyme B inhibitor, proteinase inhibitor 9, is up-regulated during accessory cell maturation and effector cell degranulation, and its overexpression enhances CTL potency. J. Immunol. 170, 805–815 (2003). 79. Ricci, M. S. et al. Direct repression of FLIP expression by c-myc is a major determinant of TRAIL sensitivity. Mol. Cell. Biol. 24, 8541–8555 (2004). 80. Moretta, L., Ferlazzo, G., Mingari, M. C., Melioli, G. & Moretta, A. Human natural killer cell function and their interactions with dendritic cells. Vaccine 21 (Suppl. 2), S38–S42 (2003). 81. Ferlazzo, G. & Munz, C. NK cell compartments and their activation by dendritic cells. J. Immunol. 172, 1333–1339 (2004). 82. Mailliard, R. B. et al. Complementary dendritic cell-activating function of CD8+ and CD4+ T cells: helper role of CD8+ T cells in the development of T helper type 1 responses. J. Exp. Med. 195, 473–483 (2002). 83. Pan, P. Y. et al. Regulation of dendritic cell function by NK cells: mechanisms underlying the synergism in the combination therapy of IL-12 and 4-1BB activation. J. Immunol. 172, 4779–4789 (2004). 84. Liu, K. et al. Immune tolerance after delivery of dying cells to dendritic cells in situ. J. Exp. Med. 196, 1091–1097 (2002). 85. Edwards, A. D. et al. Toll-like receptor expression in murine DC subsets: lack of TLR7 expression by CD8α+ DC correlates with unresponsiveness to imidazoquinolines. Eur. J. Immunol. 33, 827–833 (2003). 86. Rescigno, M. et al. Toll-like receptor 4 is not required for the full maturation of dendritic cells or for the degradation of Gram-negative bacteria. Eur. J. Immunol. 32, 2800–2806 (2002). 87. Ozinsky, A. et al. The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between Toll-like receptors. Proc. Natl Acad. Sci. USA 97, 13766–13771 (2000). 88. Re, F. & Strominger, J. L. Toll-like receptor 2 (TLR2) and TLR4 differentially activate human dendritic cells. J. Biol. Chem. 276, 37692–37699 (2001). 89. Sivori, S. et al. CpG and double-stranded RNA trigger human NK cells by Toll-like receptors: induction of cytokine release and cytotoxicity against tumors and dendritic cells. Proc. Natl Acad. Sci. USA 101, 10116–10121 (2004). 90. Buentke, E., D’Amato, M. & Scheynius, A. Malassezia enhances natural killer cell-induced dendritic cell maturation. Scand. J. Immunol. 59, 511–516 (2004). 91. Borg, C. et al. Novel mode of action of c-kit tyrosine kinase inhibitors leading to NK cell-dependent antitumor effects. J. Clin. Invest. 114, 379–388 (2004). 92. Bancroft, G. J., Shellam, G. R. & Chalmer, J. E. Genetic influences on the augmentation of natural killer (NK) cells during murine cytomegalovirus infection: correlation with patterns of resistance. J. Immunol. 126, 988–994 (1981). 93. Biron, C. A. Initial and innate responses to viral infections — pattern setting in immunity or disease. Curr. Opin. Microbiol. 2, 374–381 (1999). 94. Biron, C. A., Byron, K. S. & Sullivan, J. L. Severe herpesvirus infections in an adolescent without natural killer cells. N. Engl. J. Med. 320, 1731–1735 (1989). 95. Brutkiewicz, R. R. & Welsh, R. M. Major histocompatibility complex class I antigens and the control of viral infections by natural killer cells. J. Virol. 69, 3967–3971 (1995).

96. Biron, C. A., Nguyen, K. B., Pien, G. C., Cousens, L. P. & Salazar-Mather, T. P. Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu. Rev. Immunol. 17, 189–220 (1999). 97. Scalzo, A. A., Fitzgerald, N. A., Simmons, A., La Vista, A. B. & Shellam, G. R. Cmv1, a genetic locus that controls murine cytomegalovirus replication in the spleen. J. Exp. Med. 171, 1469–1483 (1990). 98. Scalzo, A. A. et al. The effect of the Cmv1 resistance gene, which is linked to the natural killer cell gene complex, is mediated by natural killer cells. J. Immunol. 149, 581–589 (1992). 99. Brown, M. G. et al. Vital involvement of a natural killer cell activation receptor in resistance to viral infection. Science 292, 934–937 (2001). 100. Daniels, K. A. et al. Murine cytomegalovirus is regulated by a discrete subset of natural killer cells reactive with monoclonal antibody to Ly49H. J. Exp. Med. 194, 29–44 (2001). 101. Lee, S. H. et al. Susceptibility to mouse cytomegalovirus is associated with deletion of an activating natural killer cell receptor of the C-type lectin superfamily. Nature Genet. 28, 42–45 (2001). 102. Dokun, A. O. et al. Specific and nonspecific NK cell activation during virus infection. Nature Immunol. 2, 951–956 (2001). 103. Koszinowski, U. H., Del Val, M. & Reddehase, M. J. Cellular and molecular basis of the protective immune response to cytomegalovirus infection. Curr. Top. Microbiol. Immunol. 154, 189–220 (1990). 104. Andrews, D. M., Andoniou, C. E., Granucci, F., Ricciardi-Castagnoli, P. & Degli-Esposti, M. A. Infection of dendritic cells by murine cytomegalovirus induces functional paralysis. Nature Immunol. 2, 1077–1084 (2001). 105. Andrews, D. M., Scalzo, A. A., Yokoyama, W. M., Smyth, M. J. & Degli-Esposti, M. A. Functional interactions between dendritic cells and NK cells during viral infection. Nature Immunol. 4, 175–181 (2003). 106. Andoniou, C. E., Andrews, D. M., Manzur, M., Ricciardi-Castagnoli, P. & Degli-Esposti, M. A. A novel checkpoint in the Bcl-2-regulated apoptotic pathway revealed by murine cytomegalovirus infection of dendritic cells. J. Cell Biol. 166, 827–837 (2004). 107. Sevilla, N., McGavern, D. B., Teng, C., Kunz, S. & Oldstone, M. B. Viral targeting of hematopoietic progenitors and inhibition of DC maturation as a dual strategy for immune subversion. J. Clin. Invest. 113, 737–745 (2004). 108. Simmons, P., Kaushansky, K. & Torok-Storb, B. Mechanisms of cytomegalovirus-mediated myelosuppression: perturbation of stromal cell function versus direct infection of myeloid cells. Proc. Natl Acad. Sci. USA 87, 1386–1390 (1990). 109. Kondo, K., Kaneshima, H. & Mocarski, E. S. Human cytomegalovirus latent infection of granulocyte–macrophage progenitors. Proc. Natl Acad. Sci. USA 91, 11879–11883 (1994). 110. Gibbons, A. E., Price, P. & Shellam, G. R. Analysis of hematopoietic stem and progenitor cell populations in cytomegalovirus-infected mice. Blood 86, 473–481 (1995). 111. Hahn, G., Jores, R. & Mocarski, E. S. Cytomegalovirus remains latent in a common precursor of dendritic and myeloid cells. Proc. Natl Acad. Sci. USA 95, 3937–3942 (1998). 112. Voigt, V. et al. Murine cytomegalovirus m157 mutation and variation leads to immune evasion of natural killer cells. Proc. Natl Acad. Sci. USA 100, 13483–13488 (2003). 113. French, A. R. et al. Escape of mutant double-stranded DNA virus from innate immune control. Immunity 20, 747–756 (2004). 114. Bubi, I. et al. Gain of virulence caused by loss of a gene in murine cytomegalovirus. J. Virol. 78, 7536–7544 (2004). 115. Dalod, M. et al. Dendritic cell responses to early murine cytomegalovirus infection: subset functional specialization and differential regulation by interferon α/β. J. Exp. Med. 197, 885–898 (2003). 116. Krug, A. et al. TLR9-dependent recognition of MCMV by IPC and DC generates coordinated cytokine responses that activate antiviral NK cell function. Immunity 21, 107–119 (2004). 117. Tabeta, K. et al. Toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection. Proc. Natl Acad. Sci. USA 101, 3516–3521 (2004). 118. 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). 119. van den Broek, M. F., Kagi, D., Zinkernagel, R. M. & Hengartner, H. Perforin dependence of natural killer cellmediated tumor control in vivo. Eur. J. Immunol. 25, 3514–3516 (1995).


120. Smyth, M. J., Hayakawa, Y., Takeda, K. & Yagita, H. New aspects of natural-killer-cell surveillance and therapy of cancer. Nature Rev. Cancer 2, 850–861 (2002). 121. Yuan, D., Wilder, J., Dang, T., Bennett, M. & Kumar, V. Activation of B lymphocytes by NK cells. Int. Immunol. 4, 1373–1380 (1992). 122. Yuan, D., Koh, C. Y. & Wilder, J. A. Interactions between B lymphocytes and NK cells. FASEB J. 8, 1012–1018 (1994). 123. Smyth, M. J. & Kelly, J. M. Accessory function for NK1.1+ natural killer cells producing interferon-γ in xenospecific cytotoxic T lymphocyte differentiation. Transplantation 68, 840–843 (1999). 124. Diefenbach, A., Jensen, E. R., Jamieson, A. M. & Raulet, D. H. Rae1 and H60 ligands of the NKG2D receptor stimulate tumour immunity. Nature 413, 165–171 (2001). 125. Mocikat, R. et al. Natural killer cells activated by MHC class Ilow targets prime dendritic cells to induce protective CD8 T cell responses. Immunity 19, 561–569 (2003). This paper shows that targets that express reduced levels of MHC class I molecules are responsible for more than just the efficient activation of NK cells. NK-cell activation is only the first step in a series of events that results in the maturation of DCs and the generation of adaptive T-cell responses. 126. Cooper, M. A., Fehniger, T. A., Fuchs, A., Colonna, M. & Caligiuri, M. A. NK cell and DC interactions. Trends Immunol. 25, 47–52 (2004). 127. Nicchitta, C. V. Re-evaluating the role of heat-shock protein–peptide interactions in tumour immunity. Nature Rev. Immunol. 3, 427–432 (2003). 128. Wan, T. et al. Novel heat shock protein Hsp70L1 activates dendritic cells and acts as a TH1 polarizing adjuvant. Blood 103, 1747–1754 (2004). 129. Strbo, N., Oizumi, S., Sotosek-Tokmadzic, V. & Podack, E. R. Perforin is required for innate and adaptive immunity induced by heat shock protein gp96. Immunity 18, 381–390 (2003). This study identifies a positive-feedback loop between NK cells and DCs that is required for the activation of NK cells and the clonal expansion of CTLs. NK-cell-derived perforin was found to have a crucial role in supporting the clonal expansion of these T cells. 130. Trinchieri, G. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nature Rev. Immunol. 3, 133–146 (2003). This recent review provides a summary of the biochemistry and biology of IL-12. The role of IL-12 in driving and regulating both innate and adaptive immune responses is reviewed in detail. 131. Smyth, M. J., Tanaguchi, M. & Street, S. E. The anti-tumor activity of IL-12: mechanisms of innate immunity that are model and dose dependent. J. Immunol. 165, 2665–2670 (2000). 132. Hashimoto, W. et al. Natural killer, but not natural killer T, cells play a necessary role in the promotion of an innate antitumor response induced by IL-18. Int. J. Cancer 103, 508–513 (2003). 133. Brady, J., Hayakawa, Y., Smyth, M. J. & Nutt, S. L. IL-21 induces the functional maturation of murine NK cells. J. Immunol. 172, 2048–2058 (2004). 134. Silla, L. M., Chen, J., Zhong, R. K., Whiteside, T. L. & Ball, E. D. Potentiation of lysis of leukaemia cells by a bispecific antibody to CD33 and CD16 (FcγRIII) expressed by human natural killer (NK) cells. Br. J. Haematol. 89, 712–718 (1995). 135. Rosenberg, S. A. Interleukin-2 and the development of immunotherapy for the treatment of patients with cancer. Cancer J. Sci. Am. 6 (Suppl. 1), S2–S7 (2000). 136. Pawelec, G. et al. Relative roles of natural killer- and T cellmediated anti-leukemia effects in chronic myelogenous leukemia patients treated with interferon-α. Leuk. Lymphoma 18, 471–478 (1995). 137. O’Hanlon, L. H. Natural born killers: NK cells drafted into the cancer fight. J. Natl Cancer Inst. 96, 651–653 (2004). 138. Sivakumar, P. V., Foster, D. C. & Clegg, C. H. Interleukin-21 is a T-helper cytokine that regulates humoral immunity and cell-mediated anti-tumour responses. Immunology 112, 177–182 (2004). 139. Kasaian, M. T. et al. IL-21 limits NK cell responses and promotes antigen-specific T cell activation: a mediator of the transition from innate to adaptive immunity. Immunity 16, 559–569 (2002). 140. Moroz, A. et al. IL-21 enhances and sustains CD8+ T cell responses to achieve durable tumor immunity: comparative evaluation of IL-2, IL-15, and IL-21. J. Immunol. 173, 900–909 (2004). 141. Tam, Y. K., Miyagawa, B., Ho, V. C. & Klingemann, H. G. Immunotherapy of malignant melanoma in a SCID mouse model using the highly cytotoxic natural killer cell line NK-92. J. Hematother. 8, 281–290 (1999).



REVIEWS 142. Farag, S. S., Fehniger, T. A., Ruggeri, L., Velardi, A. & Caligiuri, M. A. Natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect. Blood 100, 1935–1947 (2002). 143. Lonsdorf, A. S. et al. Intratumor CpG-oligodeoxynucleotide injection induces protective antitumor T cell immunity. J. Immunol. 171, 3941–3946 (2003). 144. Brunner, C. et al. Enhanced dendritic cell maturation by TNF-α or cytidine-phosphate-guanosine DNA drives T cell activation in vitro and therapeutic anti-tumor immune responses in vivo. J. Immunol. 165, 6278–6286 (2000). 145. Ashkar, A. A. & Rosenthal, K. L. Toll-like receptor 9, CpG DNA and innate immunity. Curr. Mol. Med. 2, 545–556 (2002). 146. Ruggeri, L. et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 295, 2097–2100 (2002). 147. Mazzoni, A. & Segal, D. M. Controlling the Toll road to dendritic cell polarization. J. Leukoc. Biol. 75, 721–730 (2004).

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148. Ito, T., Amakawa, R. & Fukuhara, S. Roles of Toll-like receptors in natural interferon-producing cells as sensors in immune surveillance. Hum. Immunol. 63, 1120–1125 (2002). 149. Proietto, A. I. et al. Differential production of inflammatory chemokines by murine dendritic cell subsets. Immunobiology 209, 163–172 (2004). 150. Hertz, C. J. et al. Microbial lipopeptides stimulate dendritic cell maturation via Toll-like receptor 2. J. Immunol. 166, 2444–2450 (2001).

Acknowledgements We are especially grateful to D. Andrews and C. Andoniou from the laboratory of M.A.D.E. for their ongoing contributions to understanding the relevance of DC–NK-cell interactions in viral immune surveillance, and to M. Wallace from the M.J.S. laboratory for discussions concerning DC–NK-cell crosstalk. Other members of our laboratories are also thanked for their contributions and valuable scientific discussions. Research undertaken in our laboratories is supported by grants from the National Health and Medical Research Council (NHMRC) of Australia, the Wellcome Trust


(United Kingdom) and the Human Frontier Science Program (France). M.A.D.E. is supported by a Wellcome Trust Overseas Senior Research Fellowship in Biomedical Science. M.J.S. is supported by a Research Fellowship and Program Grant from the NHMRC. We apologize to those colleagues whose work has only been referenced indirectly through reviews as a result of space limitations.

Competing interests statement The authors declare no competing financial interests.

Online links DATABASES The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene CD40 | CD80 | CD86 | IFN-α | IFN-β | IFN-γ | IL-12 | MICA | MICB | NKp30 | TNF Access to this interactive links box is free online.

