NK Receptors
Natural
killer (NK) cells are a population of leukocytes that, like T‐ and B‐cells,
employ receptors that can provoke their activation, the consequences of which
are the secretion of cytokines, most notably IFNγ, and the delivery of signals
to their target cells via Fas ligand or cytotoxic granules that are capable of
kill ing the cell that provided the activation signal (Figure 1.40 and Figure
1.41; see also Videoclip 3).
However, in addition to activating NK
receptors, NK cells also possess receptors that can inhibit their
function. As we shall see, inhibitory NK cell receptors are
critical to the correct functioning of these cells as these receptors are what
prevent NK cells from indiscriminately attacking healthy host tissue. Let us
dwell on this for a moment because this is quite a different set‐up to the one
that prevails with T‐ and B‐cells. A T‐ or B‐lymphocyte has a single type of
receptor that either recognizes antigen or it doesn’t. NK cells have two types
of receptor: activating receptors that trigger cytotoxic activity upon
recognition of ligands that should not be present on the target cell, and
inhibitory receptors that restrain NK killing by recognizing ligands that ought
to be present. Thus, NK cell killing can be triggered by two different
situations: either the appearance of ligands for the activating receptors or
the disappearance of ligands for the inhibitory receptors. Of course, both
things can happen at once, but one is sufficient.
We have
already discussed NK cell‐mediated killing in some detail in Chapter 1, here we
will focus on how these cells select their targets as a consequence of
alterations to the normal pattern of expression of cell surface molecules, such
as classical MHC class I molecules, that can occur during viral
infection. NK cells can also attack cells that have normal expression levels of
classical MHC class I but have upregulated nonclassical MHC class I‐related
molecules because of cell stress or DNA damage.
NK cells express diverse “hard‐wired”
receptors
Unlike the
antigen receptors of T‐ and B‐lymphocytes, NK receptors are “hard‐wired” and do
not undergo V(D)J recombination to generate diversity. As a consequence, NK
cell receptor diversity is achieved through gene duplication and divergence
and, in this respect, resembles the pattern recognition receptors we discussed
in Chapter 1. Thus, NK receptors are a somewhat confusing ragbag of
structurally disparate molecules that share the common functional property of
being able to survey cells for normal patterns of expression of MHC and
MHC‐related molecules. NK cells, unlike αβ T‐cells, are not MHC‐restricted
in the sense that they do not see antigen only when presented within
the groove of MHC class I or MHC class II molecules. On the contrary, one of
the main functions of NK cells is to patrol the body looking for cells that
have lost expression of the normally ubiquitous classical MHC class I
molecules; a situation that is known as “missing‐self ” recognition (Figure
4.17). Such abnormal cells are usually either malignant or infected with a
microorganism that interferes with class I expression.
We saw in
Chapter 1 that many pathogens activate PRRs such as Toll‐like receptors that
induce transcription of interferon‐regulated factors, which subsequently
direct the transcription of type I interferons (IFNα and IFNβ). PRRs, such as
TLR3, TLR7–9 and the RIG‐like helicases, that reside within intracellular
compartments are particularly attuned to inducing the expression of type I
interferons (see Figure 1.16). Such PRRs typically detect long single or
double‐stranded RNA molecules that are characteristically produced by many
viruses. One of the downstream consequences of interferon secretion is the
cessation of protein synthesis and consequent downregulation of, among other
things, MHC class I molecules. Thus, detection of PAMPs from intracellular
viruses or other intracellular pathogens can render such cells vulnerable to NK
cell‐mediated attack. Which is exactly the point? Many intracellular pathogens
also directly interfere with the expression or surface exposure of MHC class I
molecules as a strategy to evade detection by CD8+ T‐cells that
survey such molecules for the presence of nonself peptides.
 |
Figure 4.17 Natural
killer (NK) cell‐mediated killing and the “missing‐self” hypothesis. (a) Upon
encounter with a normal autologous MHC class I‐expressing cell, NK inhibitory
receptors are engaged and activating NK receptors remain unoccupied because no
activating ligands are expressed on the target cell. The NK cell does not
become activated in this situation. (b) Loss of MHC class I expression
(“missing‐self”), as well as expression of one or more ligands for activating
NK receptors, provokes NK‐mediated attack of the cell via NK cytotoxic granules.
(c) Upon encountering a target cell expressing MHC class I, but also expressing
one or more ligands for activating NK receptors (“induced‐self”), the outcome
will be determined by the relative strength of the inhibitory and activating
signals received by the NK cell. (d) In some cases, cells may not express MHC
class I molecules or activating ligands and may be ignored by NK cells,
possibly owing to expression of alternative ligands for inhibitory NK
receptors.
Because of
the central role that MHC class I molecules play in presenting peptides derived
from intracellular pathogens to the immune system, it is relatively easy to
understand why these molecules may attract the unwelcome attentions of viruses
or other uninvited guests planning to gatecrash their cellular hosts. It is
probably for this reason that NK cells coevolved alongside MHC‐restricted
T‐cells to ensure that pathogens, or other conditions that may interfere with
MHC class I expression and hence antigen presentation to αβ T‐cells, are given
short shrift. Cells that end up in this unfortunate position are likely to soon
find themselves looking down the barrel of an activated NK cell. Such an
encounter typically results in death of the errant cell as a result of attack
by cytotoxic granules containing a battery of proteases and other destructive
enzymes released by the activated NK cell.
NK receptors can be activating or
inhibitory
NK cells
play an important role in the ongoing battle against viral infection and tumor
development and carry out their task using two sets of receptors: activating
receptors, which recognize molecules that are upregulated on stressed or
infected cells, and inhibitory receptors that recognize MHC class I molecules
or MHC‐related molecules that monitor the correct expression of classical MHC
class I molecules. It is the balance between inhibitory and activating stimuli
that will dictate whether NK‐mediated killing will occur (Figure 4.17).
Several
structurally distinct families of NK receptors have been identified: including
the C‐type lectin receptors (CTLRs) and the Ig‐like
receptors. Both receptor types include inhibitory and activating
receptors (Table 4.3). Those that are inhibitory contain ITIMs
(immunoreceptor tyrosine‐based inhibitory motifs) within their
cytoplasmic tails that exert an inhibitory function within the cell by
recruiting phosphatases, such as SHP‐1, that can antagonize
signal transduction events that would otherwise lead to release of NK cytotoxic
granules or cytokines (Figure 4.17). Activating receptors, on the other hand,
are associated with accessory proteins, such as DAP‐12, that
contain positively acting ITAMs within their cytoplasmic tails that can promote
events leading to NK‐mediated attack. Upon engagement with their cognate
ligands (MHC class I molecules), inhibitory receptors suppress signals that
would otherwise lead to NK cell activation. Cells that lack MHC class I
molecules are therefore unable to engage the inhibitory receptors and are
likely to suffer the consequences (Figure 4.18).
NK receptors
are highly diverse and, as this is an area of active investigation, we will
make some necessary generalizations.
Ly49 receptors
The main
class of MHC class I‐monitoring receptors in the mouse is represented by the
Ly49 multigene family of receptors, which contains approximately 23 distinct
genes: Ly49A to
W. These
receptors are expressed as disulfide‐linked homodimers, with each monomer
composed of a C‐type lectin domain connected to the cell membrane via an
α‐helical stalk of ∼ 40
amino acids (Figure 4.18a). Each NK cell expresses from one to four different
Ly49 genes. Individual Ly49 receptors recognize MHC class I molecules in a
manner that is, in most cases, independent of bound peptide. Ly49 dimers make
contact with MHC class I molecules at two distinct sites that do not
significantly overlap with the TCR‐binding area on the MHC (Figure 4.18e).
Killer immunoglobulin‐like
receptors
Rather
remarkably, humans do not use Ly49‐based receptors to carry out the same task,
but instead employ a functionally equivalent, but structurally distinct, set of
receptors for this purpose, the killer immunoglobulin‐like receptors (KIRs)
(Figure 4.18c,d). This is a good example of convergent evolution,
where unrelated genes have evolved to fulfill the same functional role. By
contrast with the mode of binding to MHC displayed by the Ly49 receptors, the
KIRs make contact with MHC class I molecules in an orientation that resembles
the docking mode of the TCR, where contact with bound peptide is part of the
interaction. However, it is worth emphasizing that although KIRs do make
contact with peptide within the MHC class I groove, these receptors do not
distinguish between self and nonself peptides as TCRs do.
CD94/NKG2 receptors
NK cells
also use members of the CD94/NKG2 family, which belong to the
CTLR class of receptor, that are present in human, rat, and mouse genomes.
CD94/NKG2A heterodimers, which are inhibitory receptors, can indirectly monitor
the expression of MHC class I proteins by interacting with an invariant
MHC‐related molecule called HLA‐E (human) and Qa‐1 (mouse), the surface
expression of which is dependent on the proper synthesis of the main MHC class
I A, B, and C proteins as will be discussed in more detail below. If normal
levels of HLA‐E are detected, the inhibitory receptors will suppress NK attack.
CD94/NKG2 heterodimers are expressed on most NK cells as well as γδ T‐cells.
This
receptor system indirectly monitors the expression of MHC class I molecules in
a rather ingenious way. The MHC class I‐related molecules HLA‐E/Qa‐1 are
notable for the fact that they mainly bind invariant peptides that are found in
the leader sequences (amino acids 3–11) of the classical MHC class I A, B, and
C molecules. In the absence of the leader sequences from these peptides, HLA‐E
and Qa‐1 are not expressed on the cell surface, thereby triggering NK attack.
Because many microbial agents, particularly viruses, antagonize the expression
of MHC class I molecules, monitoring the expression level of such molecules is
a neat way of indirectly detecting that all is not well.
Another
member of this receptor family, NKG2D, does not associate with CD94 and instead
forms NKG2D/NKG2D homodimers, which are activating receptors. NKG2D homodimers
recognize the MHC‐related proteins, MHC class I chain‐related A chain (MICA)
and the related MICB, as well as UL16‐binding proteins in human
and the homologous H60/ RAE‐1/MULT‐1 proteins in mice. These ligands become
upregulated in damaged or stressed cells as will be elaborated upon later.
Natural cytotoxicity receptors
Additional
NK receptors that belong to the Ig‐like class are the natural
cytotoxicity receptors, which include NKp30, NKp44, and NKp46, all of
which are activating receptors. The ligands for these receptors remain unclear
but there is some evidence that they can detect certain viral products, such as
hemagglutinin of influenza virus or Sendai virus and may also be sensitive to
altered patterns of heparan sulfate on the surfaces of tumors. BAT‐3 (HLA‐B
associated transcript‐3), a protein that has been implicated in DNA damage
response pathways, has also recently been suggested to be a ligand for NKp30.
 |
Figure 4.18 NK receptors. (a) Schematic
representation of an inhibitory Ly49 receptor dimer composed of two C‐type
lectin domains (CTLDs). The cytoplasmic tails of inhibitory Ly49 receptors
contain immunoreceptor tyrosine‐based inhibitory motifs (ITIMs) that can
recruit phosphatases, such as SHP‐1, capable of antagonizing NK activation.
Activating Ly49 receptors lack ITIMs and can associate with ITAM‐containing
accessory proteins such as DAP‐12 that can promote NK cell activation. (b)
C‐type lectin‐like domain of the Ly49 NK cell receptors. The three‐dimensional
structure shown is the dimeric Ly49A (Protein Data Bank entry code 1Q03), the
monomer A is colored blue and the monomer B is colored green. For clarity,
secondary structural elements α‐helices, β‐strands, disulfide bonds and N and C
termini are labeled only on one monomer. (Source: Dr. Nazzareno Dimasi.
Reproduced with permission.) (c) The human KIRs (killer immunoglobulin‐like
receptors) are functionally equivalent to the murine Ly49 receptors but remain
structurally distinct. These receptors contain two or three Ig‐like
extracellular domains and can also be inhibitory or activating depending on the
presence of an ITIM motif in their cytoplasmic domains, as shown. Activating
receptors can associate with the ITAM‐bearing DAP‐12 accessory complex to
propagate activating signals into the NK cell that result in NK‐mediated
attack. (d) Structure of the extracellular Ig‐like domains (D1 and D2) of a KIR
receptor. (Source: Dr Peter Sun. Reproduced with permission.) (e) Ribbon
diagram of the crystal structure of the Ly49C/H‐2Kb complex. Ly49C, the H‐2Kb heavy
chain, and β ‐microglobulin (β M) are shown in red, gold, and green,
respectively. The MHC‐bound peptide (gray) is drawn in ball‐and‐stick
representation. (Source: Dr. Lu Deng and Professor Roy A. Mariuzza. Reproduced
with permission.)
CD16 Fc receptors
Another
example of an activating NK receptor is CD16, the low‐affinity Fc receptor for
IgG that is responsible for anti body‐dependent cellular cytotoxicity (ADCC).
In this case, the receptor ligand is IgG bound to antigen present on a target
cell, which is clearly an abnormal situation.
Cell stress and DNA damage
responses can activate NK cells
Cellular
stress, such as heat shock, is also a matter for concern for cells of the
immune system as this can also be caused by infection, or alternatively, such
cells may be undergoing malig nant transformation. The HLA‐E/Qa‐1 system,
which as we discussed earlier is involved in monitoring the ongoing expres
sion of MHC class I proteins, is also involved in attracting the attentions of
NK cells in the context of cell stress. In response to diverse forms of
cellular stress, heat‐shock proteins such as HSP‐60 are induced and peptides
derived from the HSP‐60 leader peptide can displace MHC class I‐derived
peptides from the HLA‐E peptide‐binding cleft. Although HLA‐E/HSP‐60 peptide
complexes are trafficked to the cell surface, they are no longer recognized by
CD94/NKG2 heterodimers, which results in NK activation due to “missing self.”
In addition
to recognizing “missing‐self,” NK cells also use their receptors to directly
recognize pathogen components or nonclassical MHC class I‐like proteins, such
as MICA and MICB, which are normally poorly expressed on normal healthy cells.
MICA, and related ligands, have a complex pattern of expression but are often
upregulated on transformed or infected cells and this may be sufficient to
activate NK receptors that are capable of delivering activating signals, a
phenomenon that has been termed “induced‐self” recognition (Figure
4.17). Upon ligation, the activating receptors signal the NK cell to kill the
target cell and/or to secrete cytokines. The potentially anarchic situation in
which the NK cells would attack all cells in the body is normally prevented
because of the recognition of MHC class I by the inhibitory receptors. Thus,
normal pat terns of MHC class I expression suppress NK killing, whereas the
presence of abnormal patterns of self molecules induce NK activation. It is the
relative intensity of these signals that determines whether an attack will
occur.
Recent
studies also suggest that checkpoint kinases, such as Chk1, that are involved
in the DNA damage response can induce expression of a variety of
activating ligands for NKG2 receptors, when a cell is damaged by γ‐irradiation,
or after treatment with DNA‐damaging drugs. This suggests that cells that have
suffered DNA damage may, in addition to activating their DNA repair machinery,
also upregulate NK receptor ligands to alert the immune system. This makes
perfect sense, as such cells are dangerous as they have the potential to escape
normal growth controls and form a tumor owing to faulty or incomplete DNA
repair. Indeed, tumor surveillance is thought to be one of the major roles of
NK cells, a topic we will revisit again in Chapter 16.
