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Natural killer (NK) cells are CD3-,
sIg-, CD16+ and CD56+ peripheral blood mononuclear cells (large granular
lymphocytes), which are involved in non-specific host defence (CD69 is the
earliest activation marker on T and NK cells). They act as an innate immune
system defence against infected cells (by bacteria, parasites and viruses) and
tumour cells but spare normal cells. They form the first line of defence
especially against viral infections 1-3. They control viral replication during the time required
for activation, proliferation and differentiation of CTL precursors into
functional CTLs at during the first 5 to 7 days of the infection by mediating
direct cytotoxicity and secretion of cytokines such as g-IFN, TNFa and GM-CSF but not IL-2 3-9. The main target for them to attack is a cell that is
missing the self MHC class I molecules 10,11. Unlike cytotoxic T lymphocytes (CTL), NK cells do
not require presentation of a peptide by an MHC molecule. NK cells do not
require activation for cytotoxicity; they always have large granules of
granzymes and perforin in their cytoplasm, which make them constitutively
cytotoxic.
Table
I. Characteristics of NK cell receptor families
|
NK Receptor Family |
Molecular Nature |
Genetic Complex |
Ligands |
Mouse Correspondent |
|
KIR |
Ig-superfamily |
LRC (19q13.4) |
HLA-A, -Bw, -Cw, -G |
gp49 |
|
ILT/LIR |
Ig-superfamily |
LRC |
HLA Class Ia (-G) |
LRC |
|
CD94/NKG2 (KLR) |
C-type lectin-like |
NKC (12p12.3 - 13.2) |
HLA Class Ib (-E) |
NKC / Ly49 |
|
NKG2D (KLRK1) |
C-type lectin-like |
NKC |
MIC and MHC class
I-like |
NKG2D |
|
NCR |
Ig-superfamily |
Various, incl. MHC (NCR3),
LRC |
Viral hemagglutinins
and others |
NCR |
From Refs 12-15
Target cell lysis is controlled by a
number of receptors (Table I). The main ligands for these receptors are MHC
class I and class I-like molecules (more precisely HLA-A, ‑B, ‑Cw, ‑E,
MICA, MICB and others). In general, NK receptors recognise missing self (MHC),
induced self (stress signals) or modified self (stress signals) proteins as
their ligands 16. Crystallographic studies have shown that NK
receptors form an immune synapse directly with the a helices and bound peptide of HLA class I molecules 17. The Ig-like NK receptors interact with the carboxyl
terminus of MHC class I a1 helix 18-20. The peptides within the MHC class I cleft do not
interact with the NK receptors for recognition 21; however, certain side chains at position 7 and 8 of
the nonamer peptide interfere with KIR2DL and KIR3DL binding 22-24. (The amino acid sequence of the peptide in HLA-E is
known to affect the binding by CD94/NKG2A 25 (Sullivan, 2007)).
NK cells recognize conserved
epitopes shared by groups of class I molecules rather than individual alleles.
In the opposing signals model of NK cell activity, the inhibitory and
activating NK receptors may coexist in the same cell but the binding of
inhibitory receptors by MHC class I transmits dominant inhibiting signals 26,27. This way, normal cells expressing MHC molecules are
protected and this is the basis of the missing self hypothesis of NK
cytotoxicity 11. The only exception is the
activating killer
cell lectin-like receptor (KLR)K1 (NKG2D)
which can override inhibitory signals when engaged with its ligands MICA, MICB
and other MHC class I-like molecules such as retinoic acid early inducible-1
(RAE-1), H60 minor histocompatibility molecules and CMV UL-binding proteins
(ULBP) which are identical to NKDL-1, -2, and -3 in mice and humans 28-42. This stimulatory signal
generated by NKG2D, however, is not entirely refractory to inhibitory signals 32. Possible human homologue of
the mouse RAE-1 has been cloned recently on human chromosome 6q24.2 43. In its interaction with the
activating lectin-like NK cell receptor NKG2D, the MICA polymorphism at amino
acid 130 in the a2 domain seems to be functional whereas MICB
polymorphism does not have the same effect 34. NKG2D also provides an activating signal for anti-viral CD8+ CTLs 44 (for general reviews on the NK receptors, see Refs 9,13,14,45-60. Maintenance of the balance between inhibiting and
activating signals is very important in terms of physiological consequences.
Having natural cytotoxic capacity, NK cells are important in defence against
viral infections and malignant transformation. An excess of inhibitory signals
may interfere with this function. In contrast, an unbalanced excess of
triggering signals would be a risk factor for autoimmune disorders. For
examples of disease associations with KIR, see Refs 9,59,61.
Most of the NK receptors belong to
the Ig superfamily and are type I integral membrane proteins (Table I). The
major group is called killer cell Ig-like receptors (KIR) and is encoded in the
leukocyte receptor complex/cluster (LRC) on human chromosome 19q13.42, which
spans approximately 1 Mb 48,62,63. The LRC is polygenic and individual genes exhibit
polymorphism 50,58,64. This region is flanked by Fc alpha receptor (CD89),
Ig-like transcripts (ILT, including CD85 also called Leukocyte
Immunoglobulin-like Receptors ‘LIR’) and monocyte-macrophage inhibitory
receptor (MIR) gene families 48,65-68. The ILTs are also inhibitory receptors using HLA
class I as ligands. They are expressed on monocytic cells, dendritic cells and
some NK and B cells 46. The KIR (or Ly49) genes do not undergo somatic
recombination (unlike TCR or Ig genes) but the number of genes (especially the
non-inhibitory ones) on each haplotype is variable 55,59,64,69,70 (this is similar to variable number of C4, CYP21A2
and DRB loci on MHC haplotypes). Over 100 highly homologous KIR variant
sequences have been deposited in databases and more sequences are reported as
different ethnic groups are examined 71-76. Therefore, the KIR genetic repertoire is
characterized by variable gene content and allelic polymorphism resulting in a
probability of <0.01 for two unrelated individuals to have the same KIR
genotype 58,64. Different clones within an individual may each
express a unique subset of the available KIR repertoire 71,77.
Within the LRC, five different broad
gene families can be identified by phylogenetic analysis, number of
extracellular Ig domains (2D or 3D) and length of cytoplasmic tail (S or L):
p50, p58, KIR103 (with 2 extracellular Ig domains ‘2D’), p70 and p70Dcyt (with three Ig domains ‘3D’). The p58, p70 and
KIR103 have long (L) cytoplasmic tail containing ITIMs , which contribute to
inhibitory signalling. The activating ones, p50 and p70Dcyt, have short (S) cytoplasmic tails lacking ITIMs. A hierarchy of the
strength of inhibition or activation for different KIR-HLA ligand combination
has been recognised 59,78-81. For example, the inhibitory signal generated by
KIR2DL1-HLA-C2 is the strongest. Seemingly paradoxical associations reported
for HCV infection 80 and HPV-induced cervical cancer 81 have been explained by this relationship. A similar
hierarchy for KIR2DL receptors for their inhibitory signal strength in response
to interaction with HLA-C has also been shown (in the order 2DL1 > 2DL2 >
2DL3) 82.
In general, KIR molecules with three
Ig-like domains (KIR3D) are involved in recognition of HLA-A and -B alleles
whereas receptors with two Ig-like domains (KIR2D) bind HLA-C molecules. The
five broad families were originally subdivided into 12 subfamilies based on the
number of Ig domains and cytoplasmic tail length. Sequence homology among
members of the same subfamily is indicated by a number. Each KIR subfamily
consists of one to five members that differ by 1-9 nucleotide substitutions,
while members of different subfamilies differ by at least 20 nucleotides. These
subfamilies are shown in Table II.
Two broad groups A and B have been
proposed for segregating KIR haplotypes in human populations 64,71. The main difference is a 24 kb HindIII
fragment which is exclusive to group B haplotypes. Mutually exclusive members
of the KIR2DL family (p58) are the basis of the haplotypic grouping.
KIR2DL1 and KIR2DL3 are characteristic of group A whereas KIR2DL2 (and KIR2DS2)
are characteristic of group B 71,73,74,83,84. KIR2DS4 is the
only activating receptor on haplotype A but usually occurs as a nonfunctional
deletion variant (KIR1D) 69,85. Haplotype A is
usually the more common one but the frequencies of the haplotypes vary
considerably among the ethnic groups 69,73-76,86. Homozygosity for the
putative haplotype B is highest in Australian Aborigines (26.7%) and around 10% in Caucasians. More recent studies refined this
initial haplotypic division and subgrouped haplotype A into two different
genotype groups: one containing KIR2DS4 and the other containing KIR1D
(KIR2DS4*003) in association with the pseudogene 3DP1 69. In haplotype B, 2DS4 may replace 2DS1 (i.e., they do
not occur on the same haplotype). Because haplotype B is rich in activating
receptors as opposed to the lack of them on haplotype A 69,70,85, individuals who are homozygous for group B
haplotypes will have qualitatively greater potential for providing KIR-mediated
for NK cell activation than group A homozygotes 58. To see the major haplotypic structure of KIR, see IPD KIR Sequence Database.
Table II. Major KIR gene subfamilies
(For a complete list, see KIR Nomenclature Report 2002)
I. Inhibitory subfamilies (haplotypic group) and their ligands:
* KIR2DL1 (NKAT1)/CD158a (A
> B): C2
epitope
* KIR2DL2 (NKAT6)/CD158b (B): C1
epitope (+ C2 epitope? 82)
* KIR2DL3 (NKAT2)/CD158b (A
> B): C1
epitope (+ C2 epitope? 82)
* KIR3DL1 (NKAT3/NKB1) (A > B): HLA-Bw4 (residues 77-83 of the a1 helix of HLA-B molecule 18)
* KIR3DL2 (NKAT4) (AB: framework gene): HLA-A3/A11 87 (but not confirmed in another study 88)
* KIR3DL3 (KIRC1, CD158z)
(AB: framework gene): Ligand
unknown.
II. Non-inhibitory (activating, triggering) subfamilies and their
ligands:
* KIR2DS1 (B): C2
epitope
* KIR2DS2 (NKAT5)/CD158b) (B): C1 epitope (may also occur on haplotype A 74)
* KIR2DS3 (NKAT7) (B): ligand unknown (the least frequent one in
Caucasians 74,84)
* KIR2DS4 (NKAT8) (A > B): ligand unknown but may be a non-MHC molecule 89. The commonest non-inhibitory KIR in Caucasians 74 and the only one that can occur on haplotype A. A
particular allele of this gene (KIR2DS4*003) corresponds to a soluble deletion
variant called KIR1D 69,85
* KIR2DS5 (NKAT9) (B): ligand unknown 90 and very rare 73
* KIR2DL4 (p49, CD158d) (AB: framework gene 91,92): ligand may be HLA-G 50,91,93. Despite having a long cytoplasmic tail (ITIM),
KIR2DL4 exhibits activating function but with inhibitory potential 94-96. Thus, this central framework gene KIR2DL4 is
the sole receptor on haplotype A with activating function. Its expression
varies with allelic forms 96,97.
* KIR3DS1 (NKAT10) (B):
ligand unknown (probably HLA-Bw4) 98
Remarks on Table II
* The framework (anchor) genes 2DL4, 2DP1 (KIRY/Z), 3DP1
(KIRX), 3DL2 and 3DL3 are present on almost
all haplotypes 55,62,69,70. 2DP1 and 3DP1 are pseudogenes.
*
Each gene may show further allelic polymorphism 55,64,69
*
2DL1v is a recombinant also called 2DL1*004 99
*
KIR3DL1 and KIR3DS1 seem to be a pair of inhibitory and non-inhibitory alleles 48,62,77,84,98
*
More recently identified new subfamily KIR2DL5 (2DL5A/B; inhibitory, ligand
unknown) is not shown above 100,101.
Winter et al 82 reported an in vitro binding
assay suggestive of cross-reactivity for the haplotype B locus 2DL2 with
epitope C2. No HLA-Bw6-specific KIR has been shown (but see
Vyas et al. 102).
* Despite the fact that 2DL4 contains an ITIM motif in
its cytoplasmic region, it is an activating receptor as it carries a positively
charged amino acid in the transmembrane region and upon activation induces IFN-g production. The lack of 2DL4 expression in some NK cells 77 suggests that NK cells are heterogeneous with respect
to IFN-g production, and
therefore some NK cells may not produce this cytokine when activated.
* For IHWG Reference Cell Line typing results, see Hsu et al. 2002
and Cook
et al, 2003.
* To order IHWG Reference Cell Line Panel, click here.
* A complete list of KIR
genes and specific features of each gene are given in the NCBI Online Book KIR
Gene Cluster by Carrington &
Norman. See also KIR Nomenclature Committee Report with complete listings 103 (Marsh, 2003) and
complete sequences 104 (Garcia, 2003).
Table III. C1 and C2 epitopes
C1 epitope (HLA-Cw3-related
group): Ser(77S) and Asn(80N) in a1 domain
of HLA-Cw molecule: Cw*01, 03, *07, *08, *12, *13, *14, *1601/4 (original
designation NK2)
C2 epitope (HLA-Cw4-related
group): Asn(77N) and Lys(80K) in a1 domain
of HLA-Cw molecule: Cw*02, *04, *05, *06, *15, *1602, *17, *18 (Refs 19,26,105,106; (original designation NK1)
Click here for a complete list of C1 and C2
epitopes; and here for the listing of Bw4 and Bw6
epitopes.
KIR Expression
The expression of KIR genes has been
shown to be highly diverse and largely independent of one another in NK- or
T-cell clones derived from individuals. Some KIR receptors, such as 2DL4, 3DL2
and 3DL3, reportedly are expressed on all NK cells 71,107, but see Ref 77 for 2DL4 expression (found only in 68% of
the cells examined). A study on single cell expression
of KIR genes concluded that both stochastic and nonstochastic mechanisms of
gene expression may explain the formation of the
complex pattern of NK receptor repertoire in individual NK cells 77. NK cells use DNA
methylation to maintain clonally restricted expression of highly
homologous KIR genes and alleles 92,108.
The expression of KIRs is not regulated by self MHC and is not inherited in an HLA-linked manner 77,109,110. Individuals may have any combination of KIRs
regardless of their HLA type even though the KIRs they have may not have the
correct ligands for them in the HLA type of the individual 111. The impact of HLA is to change the frequencies of
KIR-expressing cells, while they have no effect on the surface levels of KIR
expression 110. The HLA class I genotype dictates the number of KIR
that can serve as inhibitory receptors for autologous HLA class I, and thus the
proportion of NK cells needing CD94:NKG2A expression to be tolerant of self. In
mice, however, the expression of the Ly49 group of (inhibitory) NK receptors
seems to be regulated by the MHC 112.
Random expression of individual KIR receptor genes is
a rule with some exceptions. Significant increases in the frequencies of cells
expressing the combinations 2DL1/2DS1, 2DL2/2DS2 and 2DS1/2DS2 over those
expected from the product of their individual frequencies have been reported 77. This suggests
the presence of a non-stochastic component in the regulation of expression in
addition to the generally stochastic nature. The molecular mechanisms that
regulate the clonally diverse expression of KIR genes on NK and T cells are not
known 53. Methylation, however, plays a role in regulation KIR
expression and may also result in monoallelic expression 92,113-115. Allelic polymorphism of KIR loci may correlate with
expression levels as has been shown for KIR3DL1 116 and KIR2DL4 96,97.
Besides NK cells, a subpopulation of
T lymphocytes (<2% of CD3+ T cells) also express KIR 9,20,107,117,118. KIR+ T cells display a cell surface phenotype
typical of memory CD8+ T cells (CD45RO+CD29+CD28-CD45RA-) 119,120. Likewise in mice, inhibitory Ly49 expressing T cells
are of memory phenotype 121. One subtype of T cells, CD4(+)CD28(null) T cells,
are a highly oligoclonal subset of T cells that is expanded in patients with
rheumatoid arthritis 122. In CD8+ self-reactive T cells, TCR engagement
sustains KIR expression 123. It is believed that KIR expression may mediate
T-cell tolerance to self-antigens by sparing self-reactive T cells.
Transduction of an inhibitory signal requires the presence in the cytoplasmic tail of two immune receptor tyrosine-based inhibitory motifs (ITIMs) The ITIM recruits and activates the tyrosine phosphatase (PTC-1C or SHP-1)