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Natural Killer Cells Kill Virally Infected Cells

Natural Killer Cells Kill Virally Infected Cells
Thus far, we have dealt with situations that deal primarily with infectious agents that reside in the extracellular space. But what if an infectious agent manages to enter cells of the host, where they are protected from the attentions of the soluble PRRs (e.g., complement) and are also shielded from phagocytosis by macrophages and neutrophils? To deal with this situation, another type of immune cell has evolved – the natural killer (NK) cell, which is endowed with the ability to inspect host cells for signs of abnormal patterns of protein expression that may indicate that such cells might be harboring a virus. NK cells are also capable of killing cells that have suffered mutations and are on the way to malignant transformation into tumors. Note that although NK cells constitute a component of the innate response, under certain circumstances they exhibit immunological memory, a feature usually confined to adaptive responses.

Natural killer (NK) cells kill host cells that appear abnormal
NK cells are large granular leukocytes with a characteristic morphology. They choose their victims on the basis of two major criteria. The first, termed “missing self,” relates to the fact that practically all nucleated cells in the body express molecules on their surface called major histocompatibility complex (MHC) proteins. The latter molecules have a very important role in activating cells of the adaptive immune system, which we will deal with later in this chapter, but for now, it is sufficient to know that a cell lacking MHC molecules is not a good proposition from the perspective of the immune system. NK cells exist as a countermeasure to such an eventuality and cells lacking the normal pattern of expression of MHC molecules are swiftly recognized and killed by NK cells. As we saw in the previous section dealing with interferons, one way in which the expression of MHC molecules can be reduced is as a consequence of interferon‐responsive gene products that can interfere with protein translation within cells infected by viruses, or in the vicinity of such cells.
In addition to reduced or absent MHC expression, NK cells are also capable of inspecting cells for the expression of MHC‐related molecules (called nonclassical MHC molecules) and other proteins that are not normally expressed on cells, but become so in response to certain stresses such as DNA damage. This scenario represents “altered self” and also results in such cells being singled out for the attentions of NK cells, culminating in swift execution. NK receptors have also been found to be capable of detecting certain viral proteins directly, such as hemagglutinin from the influenza virus, that qualifies such receptors as another class of PRRs. There are additional receptors on the surfaces of NK cells that enable these cells to recognize infected or transformed cells that we will discuss in Chapter 4. Clearly an NK is not a cell to get on the wrong side of.

NK cells kill target cells via two different pathways
Upon recognition of a target cell, through either of the mechanisms mentioned in the preceding section, the NK cell has two main weapons at its disposal, either of which is sufficient to kill a target cell within a matter of 30–60 minutes (see Video clip 3). In both cases the target cell dies through switching on its own cell death machinery as a result of encounter with the NK cell; thus, NK killing represents a type of assisted cellular suicide. During NK‐mediated killing, killer and target are brought into close apposition (Figure 1.39) as a result of detection of either missing self or altered self on the target cell. This can engage either the death receptor pathway or the granule‐dependent pathway to apoptosis (Figure 1.40). We shall consider these in turn, although the outcomes are very similar.

Death receptor‐dependent cell killing
Death receptors are a subset of the TNF receptor superfamily, which includes the receptors for Fas, TNF, and TRAIL, and these molecules derive their name from the observation that ligation of such receptors with the appropriate ligand can result in death of the cell bearing the receptor (Figure 1.40). When this observation was first made, it was a fairly astonishing proposition as it suggested that a cell could be killed through the simple expedient of tickling a membrane receptor in the correct way. Clearly, this is a very different type of killing compared with that seen upon exposure of a cell to a toxic chemical or physical stress that can kill through disruption of normal cellular processes. Here we have a physiological receptor/ligand system that exists for the purpose of killing cells on demand– something it has to be said that the immune system does a lot of. Naturally, this sparked a lot of investigation directed towards understanding how ligation of Fas, TNF, and related receptors culminates in cell death and this is now understood in fine detail as a consequence. Engagement of Fas or TNF receptors with their trimeric ligands results in the recruitment of a protease, called caspase‐8, to the receptor complex that becomes activated as a result of receptor‐induced aggregation of this protease that now undergoes auto activation (Figure 1.41). Activation of caspase‐8 at the receptor then results in propagation of the signaling cascade in two possible ways, either via proteolysis of Bid, which routes the signal through mitochondria, or by direct processing of other effector caspases (caspases‐3 and ‐7) downstream. In each case, activation of the effector caspases culminates in death of the cell via apoptosis, which, as we mentioned earlier in this chapter, represents a programmed mode of cell death. NK cells can kill target cells in a Fas ligand dependent manner, but ca also kill through the related TNF ligand to some extent.
Cytotoxic lymphocyte killing. In this time‐lapse series, an NK cell (red arrows) is observed to come into close contact with a target cell (green arrows), which is rapidly followed by rounding up and vigorous membrane blebbing within the target cell as it undergoes apoptosis. The interval between each frame is 80 minutes. (Source: Dr. Sean Cullen, Martin Laboratory, Trinity College Dublin, Ireland. Reproduced with permission.)

Granule‐dependent cell killing
NK cells also possess cytotoxic granules that contain a battery of serine proteases, called granzymes, as well as a poreforming protein called perforin. Activation of the NK cell leads to polarization of granules between nucleus and target within minutes, and extracellular release of their contents into the space between the two cells followed by target cell death. Polarization of the granules towards the target cell takes place as a result of the formation of a synapse between the killer and target that is composed of an adhesion molecule called LFA‐1 and its cognate receptor ICAM‐1.
Perforin bears some structural homology to C9; it is like that protein, but without any help other than from Ca2+ it can insert itself into the membrane of the target, apparently by binding to phosphorylcholine through its central amphipathic domain. It then polymerizes to form a transmembrane pore with an annular structure, comparable to the complement membrane attack complex (Figure 1.41). This pore then facilitates entry of the additional cytotoxic granule constituents, the granzymes, which do the actual killing. Perforin deficient animals are severely compromised in terms of their ability to kill target cells, as the granule‐dependent pathway no longer functions in the absence of a mechanism to deliver the granzymes into the target.
Natural killer (NK) cells can kill target cells by two major mechanisms: the death receptor and granule‐dependent pathways. In both cases, the target cell dies as a result of the activation of a battery of cytotoxic proteases within the target cell, called caspases. See Figure 1.41 for further details of the molecular mechanisms of killing in either case.

Granzymes kill through proteolysis of a variety of proteins within the target cell. Most of the killing potential resides in granzymes A and B, with the function of several additional granzymes (H, K, and M in humans) still unclear. The mode of action of granzyme B is particularly well understood and it has been found that this protease in essence mimicks the action of caspase‐8 in the death receptor pathway to apoptosis, as described above. Thus, upon entry into the target cell, granzyme B can initiate apoptosis by cleaving Bid or through directly processing and activating the downstream effector caspases (Figure 1.41). Both routes result in the activation of the effector caspases that coordinate the dismantling of the cell through restricted proteolysis of hundreds of key cellular proteins.

NK cell activity can be enhanced by PAMPs as well as type I interferons
NK cells also express a subset of the TLRs that are focused towards detecting PAMPs, such as double‐stranded RNA, that are typically associated with viruses. TLR3, TLR7, and TLR8 all appear to be functional in NK cells and upon engagement of these receptors, NK cells become activated and their killing potential is enhanced. Interferon‐α and interferon‐β are also important activators of NK cells, the effects of which can increase the killing activity of such cells by up to 100‐fold (Figure 1.42). Recall from our earlier discussion of PRRs, especially those that detect intracellular infections such as the cytoplasmic DNA sensor, STING, and the viral RNA sensors within RIG‐I‐like receptor family (Figure 1.22 and Figure 1.23), that activation of these PRRs induces the expression of Type I interferons, such as IFN‐α and IFN‐β. This is an excellent example of cooperation between cells of the innate immune system, where cytokines produced by macrophages or other cells upon detection of a pathogen results in the activation of other cells, NK cells in the present context, that may be better adapted to dealing with the infectious threat.

Signal transduction events involved in natural killer (NK) cell‐mediated apoptosis. NK cells can kill target cells by two major pathways (I) or (II) as shown. In the cytotoxic granule‐dependent pathway (I), binding of the NK receptors to the surface of the virally infected cell triggers the extracellular release of perforin (a pore‐forming protein) and granzymes (which are a diverse collection of proteases) from the NK cell cytotoxic granules; perforin polymerizes within the target cell membrane to form transmembrane channels that permit entry of granzymes into the target cell. Granzymes induce apoptotic cell death through activation of the caspase protease cascade, either by directly processing and activating caspases, or through release of cytochrome c from mitochondria that activates the “apoptosome” pathway to caspase activation. In the second pathway (II) to cell death (called the death receptor pathway), membrane‐bound Fas ligand (FasL) on the NK cell engages and trimerizes surface Fas receptors on the target cell. Engagement of Fas receptors recruits the adaptor protein FADD, followed by caspase‐8, which then becomes activated at the receptor. Caspase‐8 can then promote further caspase activation through directly processing other caspases, or via the mitochondrial apoptosome pathway similar to granzymes. In both pathways, the final common pathway to apoptosis occurs as a result of the activation of several “executioner caspases” that coordinate cell death through restricted proteolysis of hundreds of cellular proteins.

Activated NK cells can amplify immune responses through production of IFNγ
Another consequence of the activation of NK cells is the production of another type of interferon, IFNγ, a very important cytokine that has a set of activities distinct from that of IFNα and IFNβ. Macrophages respond to IFNγ by greatly enhancing their microbicidal activities and also by producing other cytokines (such as IL‐12) that shape the nature of the ensuing immune response by T‐cells within the adaptive immune system (Figure 1.42). Another effect of IFNγ is to enhance the antigen presentation function of dendritic cells, which is also important for activation of the adaptive immune system. This cytokine can also influence the type of adaptive immune response that is mounted by helping to polarize T‐cells towards a particular response pattern; we shall discuss this issue at length in Chapter 8.

Type I interferons, or direct PAMP‐mediated stimulation, activates NK cells leading to IFNγ secretion. Activated macrophages can produce type I interferons, as shown, leading to 100‐fold enhancement of NK killing activity. NK cells can also be activated through direct stimulation with PAMPs. In turn, activated NK cells are an important source of IFNγ, which greatly enhances killing of intracellular microbes by macrophages and also leads to production of IL‐12 by the latter. As we shall see in Chapter 8, IL‐12 is an important T‐cell polarizing cytokine. Production of IFNγ by NK cells also enhances antigen presentation by dendritic cells.