CTLs and NK cells mediate cytotoxicity

T cells and NK cells belong to the lymphoid lineage, and are more closely related to each other than they are to B cells. CTLs and NK cells both induce apoptosis in their targets by production of TNF family molecules, cytokines and cytotoxic granules, but they recognize their targets in different ways. CTLs recognize foreign antigens being presented on MHC class I, whereas NK cells respond to cells that fail to express class I. NK cells are also able to recognize stressed or antibody-coated targets directly (Fig. 10.1).

Fig. 10.1 CTLs and NK cells mediate cytotoxicity

CTLs recognize processed antigen presented on the target cell by MHC molecules, using their T cell receptor (TCR). Most CTLs are CD8⁺ and recognize antigen presented by MHC class I molecules, but a minority are CD4⁺ and recognize antigen presented on MHC class II molecules. In contrast, NK cells have receptors that recognize MHC class I on the target and inhibit cytotoxicity. NK cells also express a number of activating receptors to identify their targets positively, including NKG2D and their FC receptor (CD16).

Effector CTLs home to peripheral organs and sites of inflammation

Naive CTLs circulate in the blood and lymphatic system, but in order to kill specific target cells, they must be activated and become effector cells. This process is mediated by APCs in the lymph nodes. Naive T cells in the lymph nodes use their T cell receptor (TCR) to recognize specific antigens being presented by MHC molecules on APCs. Most CTLs are CD8⁺ and therefore recognize antigen presented on MHC class I molecules, although some CD4⁺ cells are cytotoxic and so recognize antigen presented on MHC class II molecules.

If, in addition to signaling through the TCR, the APC also delivers a costimulatory signal through CD28, the CTL becomes activated and proliferates. Activated CTLs downregulate sphingosine phosphate receptor, allowing them to exit the lymph node. They also downregulate molecules associated with homing to the lymph node, such as L-selectin and CCR7, and upregulate molecules that allow them to home to sites of inflammation, such as CD44 and LFA-1. Effector T cells may also express adhesion molecules that allow them to home to specific tissues. CD8⁺ memory T cells appear to preferentially migrate to the tissue in which they first encountered their antigen.

CTLs recognize antigen presented on MHC class I molecules

The most important role of CTLs is the elimination of virally infected cells. CTLs recognize specific antigens (e.g. viral peptides on infected cells) presented by MHC class I molecules, which are expressed by nearly all nucleated cells. Cellular molecules that have been partly degraded by the proteasome are transported to the endoplasmic reticulum, where they become associated with MHC class I molecules and are transported to the cell surface. Normal cells therefore present a sample of all the antigens they produce to CD8⁺ T cells.

Additional interactions may be required to stabilize the bond between a CTL and its target. Like CD4⁺ T cells, CD8⁺ CTLs form an immunological synapse with their target. Signaling molecules including the TCR and CD3 are found in the central zone of the supra-molecular activation cluster (cSMAC), while adhesion molecules segregate in the peripheral zone (pSMAC). In contrast to CD4⁺ T cells, the cSMAC of CTLs and NK cells is divided into signaling and secretory domains (Fig. 10.2). After signaling has occurred, the microtubule organizing center polarizes towards the synapse, directing cytotoxic granules to the secretory domain of the cSMAC. Early CTL signaling occurs within ten seconds of cell–cell contact and granule release follows some two minutes later.

Fig. 10.2 Some interactions involved in the CTL synapse

The TCR and CD8 localize to the signaling domain in the center of the synapse (cSMAC); degranulation occurs in the secretory domain. Adhesion molecules that stabilize the cell–cell interactions are located at the periphery of the supramolecular activation complex (pSMAC).

CTLs and NK cells are complementary in the defense against virally infected and cancerous cells

Natural killer (NK) cells were first identified as a subset of immune cells that were able to kill tumor cells in vitro without prior immunization of the host, even in T cell deficient mice. In 1981 Klas Kärre made the seminal observation that targets killed by NK cells tend to be spared by T cells and vice versa. From this, he reasoned that NK cells must recognize targets that are not expressing a full complement of ‘self’ molecules. Kärre’s ‘missing self’ hypothesis postulates that unlike T cells, which recognize ‘non-self’ (foreign peptide or foreign MHC), NK cells respond to the absence of MHC class I.

Several viruses (particularly herpes viruses) have evolved mechanisms to avoid recognition by CTLs. They reduce the expression of MHC class I molecules on the cell surface to reduce the likelihood that processed viral peptides can be presented to CTLs. Cancerous cells may evade the CTL response in a similar way. NK cells specifically recognize cells that have lost their MHC class I molecules. Therefore, NK cells and CTLs act in a complementary way. In effect:

Not all NK cells mediate cytotoxicity

In humans, most NK cells are CD56⁺CD3⁻ cells. However, not all cells with this phenotype are effective killers:

NK cell development

Like T and B cells, NK cells belong to the lymphoid lineage. However, the pathways and locations of NK cell development are still less well-defined than those of T and B cells. There may also be some differences between humans and mice.

In mice, the bone marrow is essential for the production of NK cells, and a complete pathway of NK development has been described at this location. Human bone marrow does contain CD34⁺ hematopoietic progenitor cells that have the potential to differentiate into NK cells, but NK-committed progenitor cells have not been identified here. On the other hand, a complete scheme of NK cell development has been described in human, but not mouse, secondary lymphoid tissue. Circulating CD34⁺ cells are thought to be recruited from the blood to the lymph nodes, where they progress through an NK-committed immature stage to give rise to CD94⁺CD56^hi mature NK cells, capable of effector functions (Fig. 10.w1). CD56^hi cells can further develop into CD56^low cells, which express KIRs and are more cytotoxic than CD56^hi cells. A thymic pathway of NK cell development has also been described in both humans and mice. However, this pathway is clearly not required for NK cell production as athymic individuals have normal NK cell numbers and function.

Fig. 10.w1 NK cell development

CD34 is a marker of hematopoietic stem cells, which is expressed by cells in early stages of NK development, but not by cells that are committed to the NK lineage. CD117 (also called SCF receptor or c-kit) is expressed by cells before they are committed to becoming NK cells, and in immature NK committed cells, but its expression is lost in more mature cells. CD122 is a marker of NK commitment in mice, but in humans it is not detectable until the mature CD56^hi stage, which is also characterized by expression of CD94. In the final stage of NK cell development, CD56^hi NK cells become CD56^low, and also start to express KIRs.

The factors required for NK cell development have been identified using in vitro cultures of human hematopoietic stem cells and by examining knockout mice. Initially, NK cell development in culture was thought to be absolutely dependent on contact with a stromal cell feeder layer, but it was then discovered that at later stages of development, exogenously added cytokines can substitute for the presence of stroma and that IL-2 or IL-15 alone can mediate the differentiation of NK-committed cells to CD56^hi mature NK cells. Although NK cells will develop in the presence of either IL-2 or IL-15, only IL-15 is made by the stroma. The phenotype of knockout mice and humans with genetic deficiencies also suggests that it is IL-15, and not IL-2, which is the critical cytokine for NK cell development. Flt3L and stem cell factor (SCF) synergistically promote NK cell development, but are not absolutely required.

For the acquisition of KIRs, which occurs at the final stage of development, as CD56^hi NK cells become CD56^low, contact with stromal cells is once again required. Defective Ly49 expression by NK cells in the Tyro/Axl/Mer triple knockout mouse suggests that this may be partially because there is a requirement for an interaction between Protein S or Gas6 on stroma and one of their receptors (Tyro, Axl or Mer) on developing NK cells. Stromal MHC class I molecules are also likely to be important for acquisition of KIR. Several of the genes that are required for immune recognition by NK cells are grouped together on the leukocyte receptor complex on the long arm of chromosome 19 (Fig. 10.w2).

Fig. 10.w2 The leukocyte receptor complex

The leukocyte receptor complex (LRC) is a 1 MB region, including the KIR, LAIR, LILR and SIGLEC, and CD66 (CEACAM) families, as well as other genes relating to NK cell function, such as the DAP adaptor proteins. The 150 kB KIR locus is shown expanded. A number of different haplotypes have been found in this region, but they can broadly be divided into ‘A’ and ‘B’ haplotypes. A haplotypes contain few KIR genes, only one of which (KIR2DS4) is likely to be activating. B haplotypes have many more KIR genes, including several activating KIR. Recombination seems to be possible around the central framework genes, as recombinant haplotypes (A at the telomeric end and B at the centromeric end, or vice versa) do exist.

Most of what is known about the transcriptional control of NK cell development comes from knockout mice, although where attempts have been made to transfect human hematopoietic stem cells with genes of interest, this has confirmed that similar transcription factors are likely to be important in human NK cell development. The master transcriptional regulator of NK cell development has recently been identified as E4bp4, which is also known as Nfil3. E4bp4 knockout mice do not have any NK cells, but are normal in all other aspects of their immune system. Downstream of E4bp4 are other factors involved in commitment to the NK lineage, most notably Id2, and factors required for their migration from the bone marrow to the periphery. T-bet, IRF2 and GATA3 are all thought to be required at this stage, as knockouts of these genes accumulate NK cells in the bone marrow but have low numbers in the periphery. Finally, there are transcription factors that are required for the acquisition of NK cell function. For example, CEBPγ knockout mice have normal numbers of phenotypically mature NK cells, but the cells are unable to kill MHC class I negative target cells, or produce IFNγ.

NK cells recognize cells that fail to express MHC class I

NK cells express inhibitory receptors that bind to MHC class I molecules. When they encounter a target cell that is not expressing MHC class I, this inhibitory signal is lost and tonic activating signals cause the NK cell to degranulate or produce cytokines in response to the target cell. Interestingly, many of the inhibitory receptors expressed by NK cells also have activating counterparts, many of which recognize the same ligands but with lower affinity. The function of these activating receptors is not known (Fig. 10.3).

Fig. 10.3 NK cell receptors with well-defined ligands

NK cell receptors with well-defined ligands.

Killer immunoglobulin-like receptors recognize MHC class I

The killer immunoglobulin-like receptors, or KIRs, are members of the immunoglobulin superfamily. They are present on the majority of CD56^low NK cells, with each individual NK cell expressing a random selection of KIRs. Almost none of the CD56^hi NK cells express KIRs.

KIRs fall into two main subsets:

The KIRs are then further classified by whether they have a long (L) or short (S) cytoplasmic tail. KIRs with long tails are inhibitory and those with short tails are activating (Fig. 10.4). For example, the inhibitory receptor KIR2DL1 has two immunoglobulin domains and a long cytoplasmic tail. It binds alleles of HLA-C that have a lysine residue at position 80 (HLA-C2 alleles). Its activating counterpart, KIR2DS1 also binds HLA-C2 alleles, but with lower affinity. The ligands of most other activating KIRs are not yet known.

Fig. 10.4 Killer immunologlobulin-like receptors

KIRs consist of either two or three extracellular immunoglobulin superfamily domains. The inhibitory forms have long cytoplasmic tails that contain ITIMs (immunoreceptor tyrosine inhibitory motifs). The activating forms have short cytoplasmic tails and a charged lysine residue (K) in their transmembrane domains, which allows them to associate with an ITAM–containing adaptor molecule.

Inhibitory KIRs therefore allow NK cells to recognize and respond to cells that have downregulated specific HLA molecules. This is likely to explain genetic associations whereby those individuals who have both a particular KIR and its cognate HLA molecule experience better outcomes in some viral diseases such as hepatitis B and C. The normal functions of the activating KIRs are unknown, although the presence of activating KIR in the donor is associated with better outcomes when hematopoietic stem cell transplantation is used as a treatment for some leukemias.

NK cells in mice do not express KIRs. Instead, they use Ly49 receptors, which are members of the lectin-like receptor family. Like KIRs, Ly49 receptors come in inhibitory and activating forms, bind to specific MHC class I molecules, and are expressed stochastically. Unlike KIRs, which recognize the top of the peptide binding groove of MHC class I, Ly49 receptors recognize the underside of the molecule. Ly49 and KIRs are unrelated molecules that perform the same function in different species, providing an interesting illustration of convergent evolution.

The lectin-like receptor CD94 recognizes HLA-E

The lectin-like receptor CD94 is present on the majority of CD56^hi NK cells, a large subset of CD56^low NK cells and is also found on a small subset of CTLs. It covalently associates with different members of another group of lectin-like receptors called NKG2, and the dimers are expressed at the cell membrane.

There are at least six members of the NKG2 family (NKG2A-F), of which all except NKG2D associate with CD94. NKG2A-CD94 is an inhibitory receptor that blocks NK cell activation. By contrast, CD94-NKG2C is an activating receptor (Fig. 10.5). The ligand for both CD94-NKG2A and CD94-NKG2C is HLA-E, although the inhibitory CD94-NKG2A has greater affinity for HLA-E than its activating counterpart. The function of CD94-NKG2A is to allow NK cells to recognize and respond to cells, such as those that are virally infected or cancerous, that are expressing low levels of MHC class I molecules

Fig. 10.5 Lectin-like receptors of NK cells

CD94 associates with members of the NKG2 family via a disulphide bond. NKG2A contains intracellular ITIMs (immunoreceptor tyrosine inhibitory motifs) and so forms an inhibitory receptor. NKG2C lacks ITIMs but has a charged lysine residue (K) in its transmembrane segment, which allows it to interact with ITAM (immunoreceptor tyrosine activatory motif) –containing adaptor molecules.

The HLA-E gene locus encodes an MHC class I-like molecule. These are sometimes called non-classical class I molecules, or class Ib molecules, to distinguish them from the classical MHC molecules that present antigen to CTLs. The function of HLA-E is to present peptides from other MHC class I molecules. The leader peptides from other MHC molecules are transported to the endoplasmic reticulum and are loaded into the peptide binding groove of HLA-E molecules, stabilizing them and allowing them to be transported to the plasma membrane (Fig. 10.6). Cells lacking classical MHC class I molecules do not express HLA-E at the cell surface. Thus, surface HLA-E levels provide a sensitive mechanism for monitoring global MHC class I expression by the cell.

Fig. 10.6 HLA-E presents peptides of other MHC class I molecules

Leader peptides from MHC class I molecules are loaded onto HLA-E molecules in the endoplasmic reticulum, a process that requires TAP transporters and tapasin to assemble functional HLA-E molecules. These are presented at the cell surface for review by CD94 receptors on NK cells. The MHC class I molecules meanwhile present antigenic peptides from cytoplasmic proteins that have been transported into the endoplasmic reticulum. These complexes are presented to the TCR on CD8⁺ CTLs.

LILRB1 recognizes all MHC class I molecules including HLA-G

LILRB1 belongs to the LILR family of type I transmembrane proteins with multiple extracellular immunoglobulin domains (previously called the ILT family, or CD85). Of this family, only two inhibitory receptors, LILRB1 and LILRB2 have well-defined ligands, and these interact with a broad spectrum of MHC class I molecules. LILRB1 is expressed by a proportion of NK cells, as well as some T cells, B cells and all monocytes, whereas LILRB2 is only expressed by monocytes and dendritic cells. Thus LILRB1, like CD94-NKG2A, allows NK cells to detect target cells that are expressing low levels of any MHC class I molecule.

LILRB1 recognizes both classical and non-classical MHC class I molecules, but it has a particularly high affinity for the non-classical molecule HLA-G, whose expression is restricted to extravillous trophoblast cells in the placenta. Interaction of LILRB1 with HLA-G inhibits NK cell cytotoxicity more strongly than that with other class I molecules. The significance of this observation is not yet clear.

NK cells are self-tolerant

The MHC and NK receptor loci are polygenic, polymorphic and unlinked. Furthermore, NK cells are highly heterogenous with respect to their receptor repertoire, with some NK cells failing to express any inhibitory receptors that recognize self MHC class I molecules. Since they cannot be inhibited by self MHC class I molecules, these NK cells might be expected to be activated in response to autologous cells and so are potentially autoreactive. Therefore, similar to the cells of the adaptive immune system, there must be some mechanism by which NK cell tolerance to self is established and maintained. This process has been called ‘education’ or ‘licensing’.

The mechanisms that maintain NK tolerance to self have not yet been defined, but some simple rules are clear:

Cancerous and virally-infected cells are recognized by NKG2D

Like other NKG2 receptors, NKG2D is a member of the C-type lectin receptor family, but unlike other NKG2 molecules, NKG2D does not associate with CD94, instead forming a disulphide-linked homodimer (Fig. 10.7). It is an activating receptor expressed by all circulating NK cells.

Fig. 10.7 NKG2D forms disulphide-linked homodimers

NKG2D forms disulphide-linked homodimers. Its transmembrane domain contains an arginine residue (R), which allows it to recruit the adaptor protein DAP10.

In humans, the ligands for NKG2D are the MHC class I-like molecules ULBP1–3, MICA and MICB. Expression of these molecules is upregulated by a variety of cellular stresses including heat shock, oxidative stress, proliferation and viral infection. Thus, NKG2D allows NK cells to recognize cells that are stressed, including virally infected and cancerous cells. For this reason, some viruses encode immune evasion proteins that interfere with NKG2D ligand expression, and some cancers produce soluble NKG2D ligands that block NKG2D recognition of its ligands at the cell surface.

The activating NK cell receptor NKp46 is present on all NK cells, and indeed is currently considered to be the best pan-species marker of NK cells. This receptor has been reported to recognize some viral hemagglutinins, and thus may provide another way for NK cells to recognize virally infected cells directly. Another activating receptor, NKp44, may also be able to recognize viral hemagglutinins.

NK cells can also recognize antibody on target cells using Fc receptors

The Fc receptor CD16 (FcγRIII) is expressed by all CD56^low NK cells, but not by CD56^hi cells. CD16 binds antibody bound to target cells, activating the NK cell so that it degranulates, mediating antibody dependent cell-mediated cytotoxicity (ADCC) (Fig. 10.8). Historically, this was referred to as K cell activity, but this function may also be performed by other cell types with Fc receptors, including T cells. NK cell-mediated ADCC requires both an adaptive immune stimulus (cells coated with antibody) and an innate immune effector mechanism (NK cells) and is thus an example of cross-talk between the innate and adaptive immune systems.

Fig. 10.8 Fluorescence micrograph of two NK cells attacking a tumor cell

Fluorescence micrograph of two NK cells attacking a tumor cell. F-actin is stained in red and perforin in green.

(Courtesy of Dr Pedro Roda Navarro and Dr Hugh Reyburn.)

The balance of inhibitory and activating signals determines whether an NK is activated

During an interaction with a target cell, an NK cell must decide between cytotoxic action and inaction. This decision is thought to depend on the coordination of intracellular signaling pathways, and may involve the balance between activating and inhibitory signals.

Both the KIRs and CD94-associated lectin-like receptors occur as inhibitory or activating receptors.

As well as inhibitory receptors that recognize MHC class I, NK cells express inhibitory receptors for collagens (LAIR1, CD305) and sialic acid (siglecs). These may affect the balance of activation and inhibition at locations where these are present in large amounts, such as in tissues.

It is not yet known precisely how the balance of activation and inhibition is resolved. To degranulate, NK cells require a longer contact period with their targets than CTLs do. This is thought to reflect more complex processing that must occur to integrate activating and inhibitory signals at the NK cell immunological synapse (Fig. 10.9).

Fig. 10.9 Signaling through activating and inhibitory receptors

Signaling through activating receptors leads to the recruitment of adaptor molecules that contain either an ITAM or ITAM-like motif. These recruit and phosphorylate intracellular signaling molecules, leading to NK cell activation. Signaling through inhibitory receptors recruits inhibitory phosphatases, which inhibit the phosphorylation of activating signaling molecules.

Cytotoxicity is effected by direct cellular interactions, granule exocytosis, and cytokines

CTLs and NK cells use a variety of different mechanisms to kill their targets. These include:

All of these mechanisms culminate in target cell death by apoptosis. Apoptosis is a form of programmed cell death in which the nucleus fragments and the cytoplasm, plasma membranes and organelles condense into apoptotic bodies and are digested. Any remnants are phagocytosed by tissue macrophages.

Apoptosis is usually mediated by a family of proteases called the caspases. There are two caspase-dependent pathways of apoptosis:

There are also caspase-independent pathways of apoptosis. All of these may be triggered by CTLs and NK cells. The target cell remains in control of its internal processes throughout apoptosis. Thus CTLs and NK cells effectively instruct their targets to commit suicide (Fig. 10.10).

Fig. 10.10 CTLs and NK cells can trigger apoptosis

CTLs and NK cells can trigger apoptosis in their targets by signaling through TNF receptor family of molecules. These activate caspases 8 and 10, initiating apoptosis via the extrinsic pathway. CTLs and NK cells also trigger apoptosis in their targets by releasing cytotoxic granules. Perforin forms a pore in the target cell membrane, allowing granzymes access to the cytoplasm. Granzyme B activates caspases 3, 7, and 8, triggering apoptosis via the intrinsic pathway. Granzyme A initiates a caspase-independent pathway of apoptosis.

Cytotoxicity may be signaled via TNF receptor family molecules on the target cell

CTLs and NK cells can initiate apoptosis in their targets via the extrinsic pathway using members of the tumor necrosis factor (TNF) receptor group of molecules. These include:

CD4⁺ and CD8⁺ T cells and NK cells can initiate cell death via expression of Fas ligand, a member of the TNF family. Fas ligand recognizes the widely expressed cell surface protein Fas. Cross-linking leads to trimerization of Fas and recruitment of FADD to the death domain in the cytoplasmic tail of Fas. FADD recruits caspase 8 or 10, leading to apoptosis.

Other members of the TNF receptor family can also trigger caspase-dependent cell death upon engagement with their ligand by a similar mechanism. TNFα is primarily produced by macrophages, but is also produced by CTLs and NK cells. TNFR1, one of the receptors for TNFα, recruits caspases 8 and 10 via TRADD. The ability to induce cell death is dependent on the presence of the death domain in the cytoplasmic tail. TNF receptor family members that lack the death domain do not mediate caspase-dependent cell death. Apoptotic signals delivered by members of the TNF family are Ca²⁺ independent.

CTL and NK cell granules contain perforin and granzymes

Activated CTLs and resting CD56^low NK cells contain numerous cytoplasmic granules called lytic granules. Upon recognition of a target cell, these granules polarize to the site of contact, the immunological synapse, releasing their contents into a small cleft between the two cells (Fig. 10.11). The lytic granules contain the pore-forming protein perforin, as well as a series of granule-associated enzymes, called granzymes.

Fig. 10.11 Intracellular reorganizations during effector-target cell interaction

Early events in the interaction of CTLs with specific targets were studied with high-resolution cinematographic techniques. The figure shows four frames (together with interpretative drawings), taken at different times, of a CTL interacting with its target. The location of the granules within the effector cell is indicated in each case. Before contact with the target (1), the effector has granules located in a uropod at the rear, and is seen to move randomly by extending pseudopods from the organelle-free, broad, leading edge of the cell. Within 2 minutes of contacting the target (2), the CTL has begun to round up and initiate granule reorientation (3). After 10 minutes (4) the granules occupy a position in the zone of contact with the target, where they appear to be in the process of emptying their contents into the intercellular space between the two cells.

(Courtesy of Dr VH Engelhard.)

Perforin is a monomeric pore-forming protein that is related both structurally and functionally to the complement component C9.

Perforin is inactive when located within the granules, but undergoes a conformational activation, which is Ca²⁺ dependent. Like C9, perforin is able to form homopolymers, inserting into the membrane to form a circular pore of approximately 16 nm diameter. Unlike C9, perforin is able to bind membrane phospholipids directly in the presence of Ca²⁺ (Fig. 10.12).

Fig. 10.12 Perforin undergoes a conformational activation

Perforin is synthesized with a tailpiece of 20 amino acid residues with a large glycan residue attached. In this form, it is inactive. Cleavage of the tailpiece within the granules allows Ca²⁺ ions to access a site associated with the C2 domain of the molecule, but the molecule is thought to be maintained in an inactive state by the low Ca²⁺ concentration in the granules. Following secretion, the increased Ca²⁺ concentration permits a conformational change, which exposes a phospholipid-binding site, allowing the perforin to bind to the target membrane as a precursor to polymerization.

Perforin-deficient mice show greatly reduced cytotoxicity. The fact that some cytotoxicity remains demonstrates that other mechanisms contribute to CTL- and NK cell-mediated death. Some of the residual killing is likely to come from the Fas ligand and TNF pathways.

Granzymes are serine proteases that are released from the lytic granules alongside perforin. Once perforin has formed a pore in the cell membrane, granzymes may enter the target cell cytoplasm and cleave a number of substrates, leading to apoptosis via the intrinsic pathway:

Granzymes other than A and B have been identified, although mice with these genes knocked out are less severely affected than granzyme A and B knockout mice, suggesting that A and B are the most important death-inducing granzymes. Some of these minor granzymes, such as granzyme C, contribute to apoptosis via a caspase dependent pathway whereas others, such as granzyme K, seem to act in a manner similar to that of granzyme A. The large number of granzymes is likely to provide multiple pathways to trigger apoptosis, ensuring that cell death ensues.

Some cell types are resistant to cell-mediated cytotoxicity

A number of cell types display some resistance to cell-mediated cytotoxicity, including: the CTLs and NK cells themselves. CTLs and NK cells can be killed by other cytotoxic effector cells, but do not destroy themselves when they kill a target cell. A number of mechanisms contribute to this protection:

Neurons, hepatocytes and some placental cell populations are resistant to CTL and NK cell attack under normal circumstances. These cells express little or no MHC class I and for this reason, they are largely resistant to CTL-mediated cytotoxicity. They would, however, be expected to be susceptible to killing by NK cells. They evade NK cell killing in a number of ways:

Cytokine stimulated CTL and NK cells are able to kill these cell types in vitro, and under inflammatory conditions, CTL and NK cells can kill neurons and hepatocytes in vivo. Therefore, these cell types are only resistant to cell-mediated cytotoxicity in the absence of inflammation or infection.

Macrophages and neutrophils primarily kill target cells by phagocytosis

In general, macrophages and neutrophils destroy pathogens by internalizing them and barraging them with toxic molecules and enzymes within the phagolysosome. These include:

Fig. 10.13 Mechanisms that may contribute to the cytotoxicity of myeloid cells

Reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs), cationic proteins, hydrolytic enzymes, and complement proteins released from myeloid cells may damage the target cell, in addition to cytokine-mediated attack.

If the target is engaged by surface receptors but is too large to phagocytose, the phagosome may fail to internalize its target. In this case, molecules from the phagolysosome may be released into the extracellular environment and contribute to localized cell damage. This is called ‘frustrated phagocytosis’. Frustrated phagocytosis can be considered a type of ADCC, but unlike ADCC mediated by NK cells, the mediators produced by the phagocyte damage the target cell, inducing necrosis rather than apoptosis.

Activated macrophages also secrete TNFα, which can induce apoptosis in a similar way to CTLs and NK cells. Macrophages can therefore induce necrosis, apoptosis or a combination of both, depending on the state of activation of the macrophages and the target cell involved.

Eosinophils kill target cells by ADCC

Mature eosinophils contain two types of granules: ‘specific’ granules are unique to eosinophils and have a crystalloid core that binds the dye eosin, whereas ‘primary’ granules are similar to those found in other cells of the granulocyte lineage. Eosinophils are only weakly phagocytic. They are capable of ingesting some bacteria following activation, but are less efficient than neutrophils at intracellular killing.

The major function of eosinophils appears to be the secretion of various toxic granule constituents following activation. They are therefore effective at extracellular killing of microorganisms, particularly of large parasites such as schistosomes.

Eosinophil degranulation can be triggered in a number of ways:

Different modes of activation can alter the balance of toxic proteins released during degranulation, although the details of this are not yet understood.

The components of the eosinophil specific granule include:

Eosinophils are prominent in the inflammatory lesions of a number of diseases, particularly atopic disorders of the gut, skin and respiratory tract, where they are often closely associated with fibrotic reactions. Examples are atopic eczema, asthma, and inflammatory bowel disease. Although eosinophils may play some regulatory role in these conditions, such as inactivating histamine, their toxic products and cytotoxic mechanisms are a major cause of the tissue damage. For example, in asthma, MBP can kill some pneumocytes and tracheal epithelial cells while EPO kills type II pneumocytes. MBP can also induce mast cells to secrete histamine, so exacerbating allergic inflammation.