Marcus Altfeld, Partners AIDS Research Center, Massachusetts General Hospital, Bldg. 149, 13th Street, 6th floor, Charlestown, MA 02129, USA. (fax: 617 724 8586; e-mail: email@example.com).
Increasing evidence supports the notion that the innate immune response, and in particular, natural killer cells play a central role in determining the quality of the host immune response to infection. In this review we highlight recent evidence that suggests that NK cells influence the clinical fate of HIV-infected individuals.
Immune control of HIV-1 infection – beyond CD8+ T cells
The design of an effective vaccine against human immunodeficiency virus (HIV) relies on our ability to define the correlates of protection against infection and disease progression. Over the past two decades, significant advances have been achieved in our understanding of the pathogenesis of HIV-1 disease and in characterizing the immune responses generated following infection. A number of observations strongly suggest that CD8+ T cells play an important role in the containment of HIV-1 infection. These include evidence of (i) the temporal association between the appearance of HIV-specific CD8+ T cell responses following acute infection and the reduction in viral replication to set-point ; (ii) the selection for escape mutations in viral epitopes targeted by CD8+ T cells ; (iii) the significant association of particular MHC-class I alleles with protection from HIV-1 disease progression ; and (iv) the increase in viral replication following depletion of CD8+ cells in the macaque model of AIDS virus infection . However, HIV-1-specific CD8+ T cell immunity alone is not sufficient to explain the large heterogeneity observed in the clinical manifestation of HIV-1 disease. More recent advances in our understanding of the immune response to viral infections support the involvement of additional components of the immune system in the control of HIV-1 disease, and might help to identify the mechanisms underlying protective immunity in HIV-1 infection.
Viral infections typically induce very similar patterns of immune responses. These include an early induction of type 1 interferons secreted by dendritic cells, increased expression of interleukin-15, and a proliferation of natural killer (NK) cells. Subsequently, large quantities of Th1 type 1 cytokines (IFN-γ, tumour necrosis factor-α, and several chemokines) are released, which drive a strong Th1-type adaptive immune response, followed by a rapid proliferation of T cells. This host response to infection results in the clearance of the viral infection or a reduction in viral replication in persistent viral infections (Fig. 1) . This characteristic evolution of the antiviral immune response is also observed in HIV-1 infection, demonstrating that this infection does not alter the kinetics of the innate or adaptive immune response. The described temporal association  between the induction of HIV-specific immune responses and the reduction of the initial viral replication might therefore serve rather as a reflection of the normal kinetics of antiviral activity, than an indication that a particular subset of immune cells plays a unique role in controlling the infection. Several lines of evidence now suggest that the earliest viral and immunological events occurring during primary HIV-1 infection have an important impact on determining the set-point of HIV-1 replication, and the rate of disease progression . Whether these are related to responses elicited by the innate immune response, the adaptive immune response, or both, is still unclear. Furthermore, increasing evidence suggests that the correct interplay of these components of the antiviral immune response might hold the key to protective immunity in HIV-1 infection.
The strongest evidence for a role of the immune system in controlling HIV-1 disease comes from a number of epidemiological studies demonstrating a strong influence of individual HLA class I alleles on determining the rate of HIV-1 disease progression . The precise mechanism underlying HLA class I associations in HIV-1 disease are however not understood. Several subsets of cells belonging to the hematopoietic lineage express receptors that bind to HLA class I molecules, including CD8+ T cells, monocytes, dendritic cells, and NK cells. There has been a great ease at demonstrating that CD8+ T cells target viral epitopes presented by HLA class I molecules on infected cells, and that these epitope-specific CD8+ T cells lose their capacity to recognize and lyse target cells following alteration in those presented epitopes following the selection of sequence mutations. Nevertheless, few of these sequence mutations within HLA class I presented epitopes have been shown to have a significant impact on the level of viral replication in vivo. One of the few cases pertains to amino acid changes within the HLA-B27-restricted epitope KRWIIMGLNK (KK10) in p24 Gag, that have been associated with a subsequent increase in viral replication, suggesting that recognition of this epitope by the immune system is vital for control over viral replication . Initial studies demonstrated that CD8+ T cells directed against the KK10 epitope are highly immunodominant in HIV-1-infected individuals expressing HLA-B27, and lose their ability to recognize HIV-1-infected cells following sequence mutations within this epitope, suggesting an important role of these epitope-specific CD8+ T cells in mediating control of viral replication.
More recent studies demonstrate that sequence mutations within the same KK10 epitope that occur during primary infection also facilitate the interaction of HLA class I binding receptors on dendritic cells (DCs), immunoglobulin-like transcript-4 (ILT-4), and the peptide/HLA class I complex . The interaction of ILT-4 with the HLA/peptide complex delivers inhibitory signals to DCs resulting in toleragenic antigen-presentation to newly developing virus-specific CD8+ T cells. In addition to CD8+ T cells and DCs, NK cells also express a variety of different receptors that bind to HLA class I molecules that are critically involved in modulating their function. Interestingly, KIR3DL1, an inhibitory NK cell receptor associated with slower HIV-1 disease progression, can bind to the HLA-B27+ KK10 epitope complex . Whilst the functional consequences of this interaction between KIR3DL1 and the HLA-B27/peptide complex still need to be elucidated, these novel data exemplify the promiscuity of protective HLA-peptide interactions with receptors expressed on DCs, NKs and T cells (Fig. 2), suggesting that the strong protective effect of specific HLA class I molecules in HIV-1 disease might be mediated by multiple mechanisms.
Antibody-inducing and more recently T cell based vaccines have failed to induce protective immunity thus far, emphasizing the need to gain a more holistic understanding of HIV-1-specific immunity, including a better understanding of the role of innate immunity. In this review we summarize the current knowledge of NK cell biology and how it relates to HIV-1 associated pathogenesis and potential new therapeutic approaches.
Impact of HIV-1 infection on NK cell subsets
Natural killer cells are large bone marrow derived granular lymphocytes [11, 12]. They were first termed null cells, as they were characterized by the lack of a T cell receptor or B cell receptor. Their natural capacity to lyse malignant cells without the need for antigen sensitization endowed these cells with their name, natural killer cells. As NK cells did not possess an antigen specific receptor, they were placed into the innate arm of the immune system, and were initially considered to be nonspecific in their interactions with tumour cells or virally infected cells. However over the past decades, the field has come to appreciate that NK cells are far more complex than originally anticipated. NK cells have now been implicated in control and clearance of malignant and virally infected cells, regulation of adaptive immune responses, rejection of bone marrow transplants, autoimmunity and the maintenance of pregnancy . Thus it is clear that NK cells are more than simple killers, but have a multifaceted role in the immune system.
The importance of NK cells in the control of viral infections was first postulated when an adolescent girl with a genetic NK cell deficiency presented with recurrent Herpes virus infections despite normal B cell and T cell counts . Subsequent NK depletion or adoptive transfer experiments in mice demonstrated that NK cells also play a critical role in resistance to murine CMV (MCMV) infection . Furthermore, NK cell cytotoxicity and IFN-γ secretion were both shown to be critical in the control of Herpes simplex (HSV)-1 , influenza  and MCMV infections . NK cell mediated control in diverse viral infections appears to be dependent on different subsets of NK cell receptors. However, the critical role of NK cells in the early containment of viral replication and the induction of strong antiviral adaptive immune responses appears to be common across pathogens [19, 20].
Natural killer cells consist of approximately 5–15% of peripheral blood mononuclear cells, and can be divided into two subsets, based on the level of CD56 and CD16 expressed on their surface . The minor population of circulating NK cells belongs to the CD3negCD56brightCD16neg NK cells, that are poorly cytolytic but are able to secrete large amounts of pro-inflammatory cytokines such as interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α), macrophage inflammatory protein-1β (MIP-1β) and granulocyte macrophage colony-stimulating factor (GM-CSF) upon activation. The bulk of the circulating NK cell population belongs to the CD3negCD56dimCD16pos population that contains large quantities of perforin and granzyme, and secretes moderate levels of the aforementioned cytokines. Interestingly, the distribution of these two subsets is reversed in healthy secondary lymphoid organs such as the lymph nodes, where 90% of the cells belong to the cytokine secreting CD3negCD56brightCD16neg NK cell subset and only 10% of the cells belong to the CD3negCD56dimCD16pos cytolytic compartment of NK cells .
Human immunodeficiency virus-1 infection is associated with significant changes in NK cell subset distributions in the peripheral circulation [22, 23]. Several reports have shown a dramatic reduction in the proportion of CD3negCD56pos NK cells [24–26]. This reduction appears to be partially attributable to the emergence of a novel subset of NK cells that is rare in healthy individuals, the CD3negCD56negCD16pos NK cells [22, 23]. This subset becomes more prominent in individuals with active viral replication at the expense of the two other subsets of cells, resulting in an overall stable number of NK cells over the course of HIV-1 infection. Antiretroviral therapy is associated with a reduction in the CD3negCD56negCD16pos NK cells in the peripheral circulation, but the distribution of subsets does not normalize completely. Unlike the other two subsets of NK cells, these CD3negCD56negCD16pos NK cells lack the majority of NK cell effector functions, including killing, cytokine secretion, antibody-dependent cellular cytotoxicity (ADCC), and exhibit aberrant DC editing activity . It is likely that the redistribution of NK cells towards this anergic subset of cells may contribute to the observed loss of NK cell function over the course of HIV-1 infection. However changes in NK cell subset distribution in different anatomical sites and tissues have been poorly characterized at different stages of HIV-1 infection, and require further investigation.
NK cell receptors, and their role in HIV-1 infection
The complexity of NK cell biology, in part, is attributable to the complicated network of receptors expressed on their cell surface that allows them to recognize target cells [28, 29]. Contrary to the original suggestion that NK cells are ‘null’ cells lacking specific receptors, NK cells express a wealth of receptors in different combinations. These receptors can be either inhibitory or activating in nature, and it is therefore the balance of these opposing signals that dictates the activation state of an individual NK cell clone.
The repertoire of NK cell receptors includes a vast array of molecules that can either be expressed exclusively on NK cells (Fig. 3), whilst others have a more promiscuous distribution on cells of the hematopoietic lineage. The major classes of receptors include the killer immunoglobulin receptors (KIR) that bind to MHC and MHC like molecules, the c-type lectins (NKG2) that bind to stress inducible molecules (MICA/B, and ULBP), the natural cytotoxicity receptors (NCR) that bind viral hemaglutinins, and the FcγRIIIa receptor (CD16) that bind to the Fc-region of IgG antibodies . In addition accumulating work has demonstrated the critical importance of additional receptors such as: 2B4 in activation and co-activation of NK cells , DNAM-1  and CD160 in activation , TRAIL in inducing apoptosis , as well as KLRG1  and SIGLECs  in the inhibition of NK cell functional activation. These receptors are expressed differentially amongst the different subsets of NK cells and confer the ability to respond in a selective manner upon encounter with a target cell. Ultimately, it is the complex integration of signaling cascades from all the activating and inhibitory receptors expressed on a single NK cell clone that determines whether an NK cell will be active or remain quiescent in response to a target cell. For the purpose of this review, we will primarily focus on the role of activating and inhibitory KIRs in determining NK cell function.
The role of KIR+ NK cells in HIV-1 disease
The KIR receptors are particularly interesting due to the fact that this locus is highly polymorphic and has been implicated in differential clinical outcome in various cancers, in transplantation and in infectious diseases . Furthermore, the expression of these receptors is quite complex at the NK cell clonal level, as their expression is determined through a stochastic process that shuts off the expression of some receptors and not others in a single cell . Thus, it is through the combination of KIR that allows different NK cell clones to recognize their targets differentially.
Killer immunoglobulin receptors are preferentially expressed on the cytotoxic CD3negCD56dimCD16pos NK cell subset, and for the most part bind to HLA class I molecules and homologues [12, 39, 40]. Inhibitory KIR are responsible for monitoring the peripheral circulation for cells ‘missing self’, for example in the situation of HLA class I downmodulation as a consequence of viral infection or malignancy, or for recognizing foreign material that lacks autologous HLA class I molecules [12, 39, 40]. In contrast, the activating KIR, which bind similar ligands, have a weaker binding affinity than their inhibitory counterpart for the same ligand, but are likely involved in the recognition of changed self, such as the potential presentation of stress-peptides generated following infection or malignancy and presented in the groove of HLA class I molecules. Thus the KIR network of receptors is tuned to monitor for changes in self antigens that are normally expressed on the surface of cells [12, 39, 40].
The KIR locus has evolved rapidly, independently across primate species, and evolved with HLA [reviewed in 37]. Given the critical role of these receptors in the regulation of NK cell function, their associations with protection/susceptibility in human disease is not surprising. Interestingly, activating and inhibitory signals derived through KIR seem to have opposing roles in different diseases . Activating KIR, or weakly inhibitory KIR, have a beneficial impact in viral infections, whereas they are associated with susceptibility in autoimmune diseases and some cancers . This difference might be related to the fact that NK cells expressing activating receptors have a lower activation threshold than NK cells expressing only inhibitory KIR, allowing them to respond rapidly and more aggressively during infection or in the course of inflammation. However, sustained activation of these ‘trigger happy’ NK cells may aggravate the lesion during an autoimmune inflammation thereby contributing detrimentally to the progression of the disease. On the other hand, inhibitory KIR have also been associated with differential impacts on disease progression. For example, KIR3DL2 has been associated with increased rates of spondylarthritis and some lymphomas but may in contrast play a role in the protection against malaria. Similarly, maternal KIR and fetal HLA haplotypes play a critical impact on the success of the development of fetal/placental engraftment, where too much inhibition results in higher rates of spontaneous abortion, suggesting that KIR are not only important in disease but also in shaping reproductive success [reviewed in 37].
Several groups have described the significant impact of particular HLA class I alleles on HIV-1 disease progression . The data consistently indicate that HLA-B alleles are responsible for the greatest level of control over HIV-1 viral replication . HLA-B alleles can be grouped into two families, HLA-Bw4 and HLA-Bw6, based on a serologically definable epitope located on the outside of the peptide binding groove . It appears that the HLA-Bw4 family of receptors is associated with enhanced protection against HIV-1 disease progression [43, 44]. Interestingly, there are significant differences between these two families of HLA class I receptors with respect to their capacity to engage KIR receptors on NK cells , and co-expression of HLA class I alleles of the HLA-Bw4 family in conjunction with KIR3DL1 allotypes have been shown to be protective in HIV-1 disease . This enhanced protection of HLA-Bw4 alleles when co-expressed with particular KIR alleles further supports a critical role for KIR-expressing NK cells in the protection against disease progression, and are reviewed below.
Potential mechanisms underlying the protective effect of KIR / HLA compound genotypes
A number of studies have started to address the impact of the combined expression of HLA class I alleles and KIR alleles on HIV-1 disease outcome [46–49]. The seminal epidemiologic study by Martin et al. was the first to show that individuals that co-express the KIR3DS1 allele in conjunction with HLA class I alleles from the HLA-Bw4 family that encode an isoleucine at position 80 (referred to as HLA-Bw480I) progressed significantly more slowly towards AIDS than individuals that have only one or neither of these two alleles . While the physical interaction between the KIR3DS1 molecule and HLA-Bw480I molecules has not been shown, KIR3DS1+ NK cells degranulate more potently in response to HIV-infected Bw480I+ CD4+ T cells and suppress viral replication in the presence of Bw480I+ cells potently . Furthermore, studies in HIV-1-infected individuals showed that NK cells derived from individuals that possess a copy of KIR3DS1 responded more potently to HLA class I negative target cells than NK cells from KIR3DS1neg subjects. Interestingly, whilst the expression of KIR3DS1 alone was associated with a significantly elevated NK cell response, the NK cell responses were strongest in individuals that encoded for both KIR3DS1 and HLA-Bw480I . Furthermore, new evidence now indicates that KIR3DS1+ NK cells may expand preferentially during acute HIV-1 infection, and persist at elevated levels, only in subjects that co-express its putative ligand, HLA-Bw480I (Alter and Altfeld, unpublished data). Finally, elevated KIR3DS1 transcripts were identified in persistently HIV-1 negative but highly HIV-1 exposed individuals, suggesting that KIR3DS1 may also be involved in protection from infection . Taken together, these epidemiological and functional data support a cooperative interaction between KIR3DS1 and HLA-Bw480I in the NK cell response to HIV infection.
As mentioned above, the physical interaction between KIR3DS1 and HLA-Bw480I has yet to be shown. Activating KIR interact with HLA class I at a lower affinity than their inhibitory counterparts, and a stress peptide generated during infection or a viral peptide presented by HLA class I might be required to modify the affinity of an activating receptor for its ligand. This concept is supported by recent data from the murine model of Ly49p-mediated protection in MCMV infection, demonstrating that the Ly49p NK cell receptor interacts with its putative ligand, H2Dk, only in the presence of a third undefined protein . Given this observation in the murine model, and the fact that KIR3DS1 in conjunction with HLA-Bw480I is also protective in the setting of HCV , it is more likely that a stress peptide rather than a specific viral peptide may be responsible for this interaction . Overall, these data suggest that the expression of a third molecule induced by viral infection might enhance the affinity of KIR3DS1 for its putative ligand HLA-Bw480I, but the search for this third partner in the interaction has been elusive to date.
In addition to KIR3DS1, which is the sole activating allotype of KIR3DL1, additional inhibitory allotypes of KIR3DL1 have been recently shown to be protective in HIV-1 disease . Amongst the KIR3DL1 alleles, different allotypes exhibit diverse levels of KIR3DL1 protein expression on the surface of NK cells . These varying expression levels of KIR3DL1 correlate with differing NK cell functional potencies. Interestingly, KIR3DL1 alleles encoding for receptors expressed at high levels are associated with slower HIV-1 disease progression if they are expressed in conjunction with HLA-Bw480I . Two possible explanations have been proposed for this epidemiological observation: (i) highly KIR3DL1 expressing NK cells might have a stronger capacity to respond to HLA devoid target cells, suggesting that these NK cells may be more potent in providing a stronger early response to HIV-1 infection prior to the induction of adaptive immune responses, and/or (ii) the level of KIR3DL1 expression may have significant consequences on the acquisition of NK effector functions during development, as discussed in the next section. Overall, it is possible that a combination of the above mechanisms is responsible for the observed protective effect of KIR3DL1 molecules expressed at high levels in HIV-1 infection, and functional studies are needed to understand the mechanisms underlying the protective role of KIR3DL1/HLA-Bw480 in HIV-1 infection observed in epidemiological studies.
NK cell development and its potential consequences on antiviral NK cell function
Natural killer cells develop in the bone marrow, where they undergo a dynamic process of receptor expression before exiting the bone marrow as effector cells to patrol the peripheral circulation [13, 55]. In mice, this process has been well characterized, and signals delivered through direct contact or by the cytokine milieu in the proximity of the bone marrow stroma have been demonstrated to be absolutely critical for NK cell development. During this process, the common hematopoietic progenitor transitions to a common lymphoid progenitor, then to a tri-potential T/NK/NKT progenitor, and to the final NK progenitor (Fig. 4). These transitions, which differentiate an NK cell from a T cell, rely heavily on the presentation of IL-15 by stromal cells. The final step that produces mature NK cells is quite a remarkable step where NK cells in a highly organized sequential manner begin to turn on the transcription of a number of different receptors before developing into a mature NK cell. These changes include the acquisition of CD94/NKG2A/NKG2D, followed by the acquisition of KIR like genes (Ly49), and finally by the acquisition of cytolytic potential (Fig. 4) . It is through the interactions between these sequentially expressed receptors and their ligands on the bone marrow stroma that NK cells gain the right to transition to a mature phenotype.
Killer immunoglobulin receptors and KIR-like molecules are upregulated on NK cells in the bone marrow prior to the acquisition of cytotoxic potential. This led several groups to postulate that KIR may have a crucial additional role in shaping the cytotoxic potential of NK cells during development. A number of models have been proposed to explain this important step in NK cell development. The first two models are referred to as the ‘licensing’ or ‘education’ models [56, 57]. In these models the interaction between inhibitory MHC-specific NK cell receptors and self-MHC during development confers a signal that renders an NK cell functionally competent, whilst NK cells that lack an inhibitory receptor to self-MHC remain inert. The third and fourth model is referred to as the ‘arming’ model or a ‘tuning’ model, which takes into account that some NK cell clones will express activating MHC-binding receptors in the absence of an inhibitory self-binding receptor. In these models, the presence of a dominant inhibitory signal during development results in the ‘arming’ of an NK cell, whereas the presence of no inhibition, and/or too much activation results in the disarming  or tuning  of NK cells, which then circulate in the peripheral circulation as a hyporesponsive subset of cells.
These models have been tested by different groups in the murine model of NK cell development. Recently it was shown that both qualitative and quantitative differences in the interactions between the MHC-engaging receptors and their ligands result in notable differences in the level of NK cell licensing . Thus mice that possessed MHC-molecules with high affinity interactions between MHC and binding KIR-like receptors (Ly49) generated NK cells with far greater cytolytic potential than NK cells derived from a mouse with a weaker Ly49/MHC-receptor interaction. Furthermore, the authors were able to demonstrate that during development, a hierarchy of MHC ligands was established, where some MHC molecules were able to dominate the licensing of NK cells in contrast to others. These data demonstrate the critical nature of the ligand for inhibitory-MHC-binding NK cell receptors in shaping the cytolytic potential of NK cells in the peripheral circulation.
How might NK cell development be important for our understanding of NK cell mediated immune protection in HIV-1 infection? As mentioned above, HLA-Bw480I alleles that have the potential to bind inhibitory KIR (KIR3DL1) appear to be protective in HIV-1 disease . The licensing model of NK cell development would suggest that strong inhibitory interactions between HLA-Bw480I and highly expressed KIR3DL1 during development might generate more cytolytic NK cells able to deal more robustly with HIV-1 infection. It is also possible that different HLA class I molecules may engage KIR with different affinities during development, thereby inducing a similar hierarchical licensing effect, as demonstrated in the murine model where Db dominated licensing over Ld licensed NK cells . This model would also provide a possible mechanism by which HLA-Bw480I alleles, such as HLA-B57, can be protective across different viral infections, independent of the specific viral epitopes presented. Thus it is possible that protective HLA-alleles may have a dual role in presenting antigens to drive effective CD8+ T cell responses but also in generating more functionally competent NK cells during NK cell development.
Genome wide association studies provide further support for NK cells in the control of HIV-1 disease progression
The first genome wide association study in HIV-1 disease was published recently, and revealed three single nucleotide polymorphisms (SNP) associated with lower HIV-1 viral load set points in chronically infected individuals . Remarkably, all three SNPs were located in the HLA region on chromosome 6, including a SNP linked to HLA-B57, a SNP located near HLA-C, and a SNP in an RNA polymerase subunit, ZNRD1. The second SNP was located 35 kb upstream from HLA-C, and was previously associated with higher HLA-C mRNA transcription . It has been speculated that this SNP may also be related to increased HLA-C expression on the surface of cells. Given that the protective effect of this SNP is not attributable to any specific HLA-C allele, which each present very different epitopes to CD8+ T cells, suggests that the protective effect of elevated HLA-C expression may be mediated through a non-CD8 T cell dependent mechanism. As HLA-C serves as the main ligand for KIRs of the KIR2D family , the potential influence of this polymorphism on NK cell functionality has been proposed. HLA-C alleles can be sub-classified into two different groups by a serological marker on the outside of the antigen binding group: Group C1 and C2 [64, 65]. KIR2DL1/2DS1 bind to group C2 alleles, where KIR2DL3/2DS2/2DS3 bind to group C1 . Recently it was shown that KIR2DL2 can engage both group C1 and C2 alleles . The protective effect of higher HLA-C expression in HIV-1- disease is observed for individuals encoding for HLA-C alleles both of the C1 and C2 group, suggesting that this effect is not attributable to any specific HLA-C binding KIR. Two alternative explanations have therefore been proposed for a potential role of NK cells in mediating this protective effect: (i) KIR2DL2+ NK cells, which interact with both subgroups of HLA-C alleles, recognize a stress peptides presented by HLA-C alleles in a C-group independent manner; or (ii) elevated levels of HLA-C expression may provide stronger licensing signals during NK cell development to all HLA-C-binding inhibitory KIRs expressed on NK cells. Studies testing these different models, as well as alternative models exploring a role of HLA-C-restricted HIV-1-specific CD8+ T cells, are currently underway.
Taken together, studies aimed at assessing the role of NK cells in HIV-1 infection have been driven and guided by epidemiological population studies demonstrating that the combined expression of specific KIRs in conjunction with their HLA class I ligands is protective in HIV-1 disease. Understanding the mechanisms underlying the protective effect of KIR-mediated activation of NK cells in HIV-1 infection will be crucial to harness the potency of these cyctotoxic innate effector cells for vaccine development and therapeutic interventions.
HIV evasion from NK cell mediated immune pressure
Viruses have developed multiple elegant ways to evade the host’s immune pressure, and the evolution of these evasion mechanisms are a direct reflection of the immune selection pressure mediated by the immune system . Viral evasion strategies from NK cell mediated immune pressure have been best studied for the Herpes viruses. As an example, CMV has evolved multiple genes specifically designed for the evasion of NK cells, providing strong evidence of the critical pressure this subset of effector cells places on this virus. The immune-evasion genes encoded by CMV include genes that alter antigen presentation, that down-regulate HLA class I alleles, and genes that mimic HLA class I molecules to provide inhibitory signals to NK cells [20, 68]. CMV is a large DNA virus that has the opportunity to accommodate multiple genes for the evasion of both innate and adaptive immune responses. However, HIV is a small RNA virus that only encodes nine genes. Yet, a number of studies have shown that HIV has evolved the capacity to evade both innate and adaptive immunity as well.
The HIV-1 Nef protein has been shown to have many different functions, one of which is immune evasion. When expressed, the Nef protein triggers the accelerated endocytosis or retention of HLA class I molecules in the Golgi, thereby reducing HLA class I expression on the surface of infected cells . Theoretically, this function would allow infected cells to subvert attacks from HIV-specific CD8+ T cells, however reduced HLA class I expression also sends a strong alarm to patrolling NK cells. To overcome this problem, Nef downregulates HLA class I molecules differentially to protect infected cells from NK cell mediated lysis. Nef profoundly down regulates HLA-A, partially down regulates HLA-B, but spares HLA-C [70, 71]. Given that HLA-B and C serve as the primary ligands for inhibitory NK cell receptors, the virus has apparently evolved to find an elegant balance between evasion of T cells and NK cells, reflecting the importance of both arms of the immune response to HIV-1 infection.
In addition to Nef-mediated modulation of KIR ligands, new data suggest that Nef is also able to modulate the expression of the ligands for a second NK cell receptor, NKG2D . NKG2D recognizes HLA class I homologues (MIC A, ULBP1, 2, and 3) expressed on surface of stressed cells. Similar to Nef-mediated retention of HLA class I molecules, HIV Nef partially down-modulates MIC A, ULBP-1 and -2 from the surface of infected cells . Nef-mediated down-modulation of NKG2D-ligands suggests that this receptor may also be involved in the recognition and control of HIV-1 infected cells during infection, but the precise mechanisms are currently not understood.
NK cell based therapeutic intervention
Therapeutic strategies specifically targeting NK cells have been used with remarkable success in the setting of transplantation and cancer therapy [15, 73], but have not yet been applied to viral infections. One of the greatest successes of NK cell based therapies is the use of rituximab, a CD20 specific antibody , for the treatment of non-hodgkin lymphomas. This antibody induces strong ADCC, through NKs and monocytes, and thereby eliminates the tumour [74–76]. Thus antibodies can direct the potent cytolytic activity of CD16+ NK cells directly to the malignant cells for the efficient elimination of these targets. Elevated HIV-specific ADCC function has been correlated with protection against disease progression ; however, the utility of ADCC in the context of HIV vaccine design has not been well studied. Recently it was shown that the protective antiviral function of the neutralizing antibody B12 was in large part mediated by the Fc-portion of the antibody , suggesting that ADCC is possibly involved in both the control of HIV-1 infection and the prevention of the infection. Thus, the incorporation of antibodies that can target the cytolytic effector function of NK cells directly to HIV-infected cells may serve as a new strategy in HIV-1 vaccine design, linking innate and adaptive antiviral immunity.
Therapeutic strategies aimed at manipulating inhibitory HLA class I binding receptors, such as KIR, have also shown some success in the context of cancer and transplantation [15, 73]. Epidemiologic evidence suggests that individuals with low NK cell activity tend to be more susceptible to cancers . Furthermore, the success of allogenic transplantation in some leukememias is enhanced when donor KIR/HLA mismatch results in elevated NK cell outgrowth and function, leading to improved engraftment and post-transplant survival . New approaches including antibodies that block inhibitory KIR have been shown to increase NK cell effector function and clearance of the tumour. In vitro NK cell expansions and re-infusion have been shown to be well tolerated in humans, and had some success enhancing engraftment and clearance of the tumour in vivo . Finally, new strategies are underway to expand specific populations of NK cells ex vivo and in vivo to gain more effective control over the tumour , suggesting that a greater understanding of the role of specific KIRs in the control of HIV-1 infection may provide us with some clues as to how therapeutic targeting of specific KIR+ NK cell populations can add an innate component to both prophylactic and therapeutic vaccines.
Natural killer cell cross talk with DCs plays a critical role in determining the quality of the adaptive immune response following infection and vaccination [15, 73]. NK cells play a central role in performing quality control over of DC populations [80, 81], and are involved in the deletion of aberrant immature DCs. NK cells have now been shown to both limit the potency of DC based vaccinations if the NK cells delete DCs too rapidly following injection, and have also been shown to enhance antigen cross-presentation when NK cells induce apoptosis of antigen-loaded DCs . Novel vaccine approaches aimed at modulating the adjuvant effect of DCs using NK cells have aimed at inducing NKG2D ligands or TRAIL-ligands on reinfused antigen-loaded DCs, thereby providing apoptotic targets to NK cells for more efficient antigen delivery in vivo by cross-presentation . This strategy could provide a new angle to enhance the quality of the adaptive immune response in therapeutic interventional trials by enhancing NK cell cross-talk with ex vivo manipulated DCs.
Over the past two decades, significant advances have been made in the understanding of the role of NK cells in viral infections, and how NK cells can be manipulated in vivo. The failure of recent HIV-1 vaccine trials to induce protective B and T cell immunity in humans has highlighted our lack of understanding of the precise correlates of immune protection in HIV-1 infection, and that we have to explore novel concepts reaching beyond traditionally studied adaptive immunity to gain control over HIV-1. Thus novel strategies aimed at harnessing the power of NK cells to mediate or modulate anti-HIV activity may provide a new approach to enhance the quality of immune control induced by prophylactic and therapeutic vaccinations.