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NATURAL KILLER CELL RECEPTORS

Mehmet Tevfik DORAK, M.D., Ph.D.

Comprehensive reviews on KIR and disease:

Williams et al, 2005; Boyton & Altmann, 2007; Zamai et al, 2007; Kulkarni et al, 2008

Review on KIR polymorphisms (alleles & haplotypes) and interactions with HLA:

Middleton & Gonzeles, 2010

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. Although they belong to the innate immune system, there is some evidence that they may have memory against infectious exposures (Sun, 2009). 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 (haplotypes) & Middleton & Gonzeles, 2010.

Table II. Major KIR Gene Subfamilies

(For a complete list, see KIR Nomenclature Report 2002)

I. Inhibitory subfamilies (haplotypic group) and their ligands:

p58 family

* 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)

p70 family

* KIR3DL1 (NKAT3/NKB1) (A > B): HLA-Bw4 (residues 77-83 of the a1 helix of HLA-B molecule ¹⁸; except HLA-B*1301/2) & A23/24/32 (Thananchai, 2007; Stern, 2008; Foley, 2008)

* KIR3DL2 (NKAT4) (AB: framework gene): HLA-A3/A11 ⁸⁷ (but not confirmed in another study ⁸⁸)

p140 family

* KIR3DL3 (KIRC1, CD158z) (AB: framework gene): Ligand unknown.

II. Non-inhibitory (activating, triggering) subfamilies and their ligands:

p50 family

* 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 most common 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

p49 family

* KIR2DL4 (p49, CD158d) (AB: framework gene ^91,92): its ligand is 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.

p70Dcyt family

* 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.

* Although HLA-B*1301/2 are associated with Bw4 epitopes, they may not interact with KIR3DL1 (Foley, 2008)

* For IHWG Reference Cell Line typing results, see Hsu et al. 2002 and Cook et al, 2003.

* To order IHWG - KIR Reference Cell Line Panels, 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). For a graphic display of all KIR haplotypes, see IPD KIR Database & Middleton & Gonzeles, 2010.

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. See Schellekens (2007); Ugolotti (2011); Bari et al (2011) for genotyping protocols of HLA-B and C 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 tomaintain clonally restricted expression of highly homologousKIR 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) ^124,125. The activating KIRs lack ITIMs but associate with DAP12, a signalling subunit similar to CD3 protein ^126,127. Engagement of Ig-like activating receptors with their ligands triggers a signalling cascade similar to that initiated by the T cell receptor ¹²⁶. On the other hand, the major activating lectin-like NK cell receptor NKG2D/KLRK1 is generally associated with DAP10 as its signalling subunit ²⁹ although splice variants may use different signalling adaptor proteins ¹²⁸. The mouse homologue of the human KIR complex is known as gp49 whose ligand is unknown ⁴⁶. It functions in a much smaller magnitude compared to the main NK receptor family of mice (Ly49). (Ly49 is also the primary NK cell receptor family in horse ⁹.

CD94/NKG2:

The major group of NK receptors in mice is the Ly49 group (A-I) and they are type II transmembrane molecules belonging to C-type lectins (C-type-lectin-inhibitory receptors, CLIR). Human KIRs recognize peptides whereas mouse Ly49 recognizes carbohydrates. The direct human correspondent of Ly49 is a pseudogene ¹²⁹. The equivalent functional molecule in humans is CD94 ¹³⁰. This is a promiscuous NK inhibitory receptor found covalently associated with NKG2/KLR members ^131,132 except NKG2D, which is usually associated with a signalling subunit called DAP10 ²⁹ or KARAP/DAP12 ¹²⁸. The family of NKG2 (killer cell lectin-like receptor subfamily C; KLRC) genes and the CD94 (KLRD1) gene make up the NK complex (NKC) on chromosome 12p12.3-p13.2 ^133,134. The LRC and NKC are not evolutionarily related and reside on different chromosomes. Although CD94 is a nonpolymorphic protein, NKG2 occurs as variants (A,C,D,E,F,H) and isoforms (A/B) both in humans and mice ^28,135-140. While NKG2A/B (KLRC1) and NKG2F (KLRC4) are inhibitory in both species, other NKG2 variants (C "KLRC2", D "D12S2489E", E or H "KLRC3") act as activating receptors ^{25,49,136,141,142}. Among these, all but NKG2D show a high degree of identity. NKG2D has a major role in anti-tumour and anti-viral NK cytotoxicity both in mice and humans ^{30-32,42,44,143-145}. The functional relevance of recently shown polymorphism of NKG2D has not been studied yet ¹⁴⁶. Two NKG2D splice variants associate with different signalling adaptor proteins (DAP10 or DAP12), resulting in qualitatively different signals ¹²⁸ and the functional association of this finding has not been studied in disease susceptibility either. It has a variety of ligands: MICA, MICB, ULBPs (RAET) in humans and RAE-1, H-60 and MULT1 in mice ⁴¹. Perhaps in correlation with its dominant importance in NK cell biology and activity, cancers and viruses have plenty of mechanisms to subverse NKG2D function. These vary from releasing soluble MICA to downregulate NKG2D in epithelial tumours ¹⁴⁷ (Kaiser, 2007) an example of ligand-mediated internalisation and downregulation of NKG2D expression by TGF-beta1 ¹⁴⁸ to downregulation/sequestration of its ligands ^149-152.

The CD94/NKG2 receptors and KIRs provide a wide overlap in NK ligand specificity. Due to multiplicity of inhibitory receptors, even a single allelic loss (as opposed to total class I loss) can be recognized by NK cells. Also, a substantial proportion of the population has genes for KIR for which they have no HLA class I ligand ¹⁰⁹. The CD94/NKG2 inhibitory system compensates for possible “holes” in the KIR repertoire because HLA class I of most individuals includes a ligand for CD94/NKG2. The KLRs use HLA-E, MIC-A/B, HSP60 and other MHC class I-related molecules as ligands ^{28-30,32-34,41,137,153-155}. In fact, it is not HLA-E itself but its complex with nonamers (aa 3 to 11: VMAPRTLL) derived from classical HLA class I leader sequences that are the ligand for CD94/NKG2 ¹⁵⁶. Because HLA-E does not have surface expression unless it has bound an HLA class I-derived peptide, the cell surface expression of HLA-E serves as an indicator of the overall level of HLA-A, -B, -C biosynthesis in the cells (similar to what Qa-1(b) does in mice ^132,157,158). HLA-E also presents a peptide derived from the leader sequence of human heat shock protein 60 as a stress signal ¹⁵⁵. This presentation of a stress signal by HLA-E interferes with the normal presentation of MHC leader peptides by HLA-E and leads to recognition of the cellular distress. This route via an altered self molecule provides a mechanism of NK cell activation of cells that still express MHC class I molecules. HLA class I and HLA-E alleles are not equally effective in the inhibition of NK cells ^{25,137,141,156}. The efficiency also depends on the HLA-E allele possessed. Thus, the efficiency of the CD94/NKG back-up system depends on the HLA-E variant as well as the HLA class I haplotype. For example, peptides derived from HLA-B15 and -Cw7 have been shown to bind to both HLA-E variants but with no effect on CD94/NKG receptor ¹⁴¹. Such alleles may be considered to be endogenous competitors for the CD94/NKG2A-mediated inhibitory function of HLA-E.

Another lectin-like NK cell activating receptor is CD69 ^159-162. Its ligand is unknown. Its activity is blocked by anti-CD94 monoclonal antibody. The function of CD69 is believed to sustain the NK cytotoxic activity as it does in T cells ¹⁶⁰.

Other molecules well-known for their unrelated functions can also function as activating receptors for NK cells: LFA-1 ¹⁶³ and the CD2 family members ¹⁶⁴. 2B4 (CD244) is a member of the CD2 family (see below). LFA-1 may be involved in the post-BMT GvL reaction mediated by NK cells.

Natural Cytotoxicity Triggering Receptors (NCR):

CD335	NKp46; NCR1	9437
CD336	NKp44; NCR2	9436
CD337	NKp30; NCR3 (6p21)	259197

Natural cytotoxicity defines the capacity of NK cells to kill targets without any prior activation. This is MHC-independent and also different from ADCC, which is mediated by antigen-bound IgG antibodies recognized by FcgRIII on NK cells. Non-MHC-dependent NK cell cytotoxicity is mediated by NCR both in mice and humans ^{13,49,54,93,165} (as well as CD16). The NCR are NKp46 (CD335) ^166-168, NKp44 (CD336) ¹⁶⁹ and NKp30 (CD337) ¹⁷⁰ all of which are the members of the immunoglobulin superfamily. The genes encoding these molecules are: NCR1 (Gene ID: 9437), NCR2 (Gene ID: 9436) and NCR3 (Gene ID: 259197), respectively. The NCRs are expressed in the early phases of NK cell development (before KIR) and may regulate cell differentiation ¹⁷¹. It appears that anti-viral cytotoxicity may be mediated by NKp44 and NKp46 but not by NKp30 ^168,172,173. The only known ligand for NKp44 and NKp46 is viral hemagglutinin ^168,172. There is a possibility that HSP72 may be a ligand for NCRs ^174,175.

Activation via NKp46 requires the involvement of co-stimulatory receptor 2B4 (CD244), which interacts with CD48 ^164,176-178. The defects in 2B4 signal transduction pathway plays a role in the etiology of EBV-induced X-linked lymphoproliferative disease ^93,179-181.

NKp30 is encoded by NCR3 (also known as D6S2570 or 1C7) ^170,182,183. NCR3 is a polymorphic gene ^184,185 with yet unknown functional relevance. NKp30 is the activation receptor in the interaction between dendritic cells (DC) and NK cells ¹⁸⁶. This interaction between NK cells and DC occurs during DC development to selectively kill immature DCs before they move to secondary lymphoid organs ^186,187. In parallel, DC promote NK cell activation and enhancement of their cytolytic activity ^57,187. NKp44 and NKp46 do not play a role in this interaction. In at least one animal model, NKp30 is also involved in NK cell cytotoxicity in transplant rejection ¹⁸⁸. BAT3/BAG6 has been recognized as a ligand for NCR3 (von Strandmann, 2007) and CMV tegument protein pp65 has been shown to bind and inactivate NKp30 (Arnon, 2005); this is yet another way CMV causes viral immune-evasion (Rajagopalan & Long, 2005).

KIR Typing:

Although the genomic organization of the LRC is still being worked out (Gourraud, 2010; Middleton & Gonzeles, 2010), typing methods exist to study population genetics of the KIR system. Within an individual, each NK cell expresses 2-9 stochastically different KIRs in different combinations along with the CD94/NKG2 heterodimer ⁸⁸. At least one inhibitor KIR specific for a host HLA allele would normally be expressed ^77,88 but if an NK cell lacks an inhibitory KIR, this would be compensated by the expression of the inhibitory receptor CD94:NKG2A ⁸⁸. The framework genes 2DL4, 3DP1, 3DL2 and 3DL3 that are present on all haplotypes ⁶² can be used as internal controls for PCR-based detection methods. By genotyping at the DNA level, an overall impression of the KIR repertoire is obtained. PCR-SSOP ^84,189, PCR-SSP ^{69,71,74,76,189,190} and multiplex PCR-SSP ¹⁹¹ have been established as typing techniques. The minimum requirements are 12 primer pairs for PCR-SSP or 13 probes for PCR-SSOP. These techniques however only address to the variable gene content of KIR haplotypes but do not deal with allelic polymorphism that has recently been recognized ⁶⁴. A medium resolution PCR-SSP typing kit is available from Invitrogene (formerly Pel-Freez/Dynal) (link). This kit detects the presence or absence of major KIR genes and also provides limited information on allelic polymorphism. Sequence-based typing has not been used routinely to type KIR loci or alleles but NCBI dbLRC has an alignment viewer for analysing KIR sequence data ¹⁹². For KIR genotyping protocols, see Martin & Carrington, 2008 & Kulkarni, 2010; Gonzales, 2009 (high-resolution melting analysis); Koehler, 2009 (real-time PCR).

Some Observations in HIV Infection:

* CD3+ HIV-specific CTL population express inhibitory NK receptors (KIR and CD94) and CTL activity is down regulated ^193-196.

* HIV down regulates HLA-A and -B but not HLA-Cw and -E which are the major ligands for NK cell receptors ^197-199.

* HLA-Cw acts as a restriction element for immune response to mostly conserved HIV peptides ^200-203.

* Long-term non progressors may have HLA-Cw-restricted CTL responses to HIV ²⁰⁴.

* Most HLA associations with HIV infection or progression are with class I molecules as opposed to class II.

Rationale for KIR Haplotyping in HIV Infection:

NK cells have been implicated in the defence against infectious diseasesthrough mechanisms involving cytotoxicity and cytokine production ³ presumably mediated in part by activatingKIR molecules. The activity of KIR molecules is regulated by their ligands (HLA class I molecules). Independent segregation of HLA and KIR genes, along with KIR specificity for particular HLA allotypes, raises the possibility that any given individual may express KIR molecules for which no ligand is present. Approximately 10% of the population do not possess a gene for an inhibitory KIR for the C2 epitope in their genetic LRC repertoire ^74,83,84, i.e., they lack the mainly haplotype A-encoded inhibitory receptors. For these individuals, it is important what their HLA-C type is. In the case of homozygosity for the HLA-Cw4 group (the C2 epitope), an individual without the KIR haplotype A will not have an inhibitory signal from HLA-C alleles for any NK clone in circulation. If the same person is lacking HLA-A3/A11 and -Bw4, the only other known inhibitory ligands, the NK cells will rely on only the CD94/NKG system for any inhibitory signal. The efficiency of the CD94/NKG system is also affected by the HLA-E and HLA class I signal peptide polymorphism. This leaves a fraction of normal population with NK cells, which will not receive enough inhibitory signals. These people are expected to be better protected against intracellular pathogens especially against viruses, which have mechanisms to evade the conventional CTL responses. Simultaneously, such individuals may also be more susceptible to autoimmune disorders. An example of this has recently been presented ²⁰⁵. In psoriasis, subjects with activating KIR genes (2DS1 and/or KIR2DS2) are susceptible to developing psoriatic arthritis, but only when HLA ligands for their homologous inhibitory receptors, KIR2DL1 and KIR2DL2/3, are missing. It appears that in the absence of ligands (HLA-C2 group) for theinhibitory KIRs 2DL1 and 2DL2/3 molecules the threshold for NK cell activation islowered, thereby allowing NK cell activation mediated by KIR2DS1and KIR2DS2. In the absence of group HLA-C2 group alleles, the presence of the activating KIR2DS1is significantly associated with psoriasis arthritis. A similar situation would allow better control of viral infections one of which may be HIV infection. HIV down regulates HLA-A and -B but not HLA-Cw and ‑E in an attempt to avoid NK-mediated cytotoxicity since these are the major ligands for NK cell receptors ^197-199. CD3+ HIV-specific CTL population expresses inhibitory NK receptors (KIR and CD94) and CTL activity is down regulated ^193-196. These observations justify the examination of HLA class I (including HLA‑E) and KIR genes in HIV infection. For a review of HLA-KIR and HIV associations, see Carrington et al, 2008.

KIR and HSCT:

Few studies in hemopoietic stem cell transplantation (HSCT) suggested a predominant effect of the HLA-C type in the prediction of NK cell behaviour. One study tested the hypothesis that if donor-recipient pairs were matched at HLA-C, following HSCT there would be no change in the ligands for NK cells and since everybody has at least one inhibitory receptor in each NK clone, no enhancement in anti-leukemic NK cell activity would be expected. Indeed, the results singled out HLA-C "matching" as a significant factor in leukemia relapse ²⁰⁶. Another study went one step further and considered the NK cell repertoire determined by flow cytometry in addition to the HLA type of the recipient and donor ²⁰⁷. The monoclonal antibodies used were specific for KIRs recognizing the two groups of HLA-C epitopes and HLA-Bw4. The analysis of recipient-donor pairs mismatched at these ligand specificities showed that when the inhibition was predicted to be lifted (because of the absence of at least one of the inhibitory ligands for donor NK cell clones in the recipients) there was an enhanced anti-leukemic effect of NK cells in vitro and as an in vivo correlation, these patients had less relapse. This study suggested that KIR-specific epitope mismatching may be a useful strategy for enhancing the graft-versus-leukemia (GVL) effect of allogeneic HSCT ^15,208. In terms of KIR genes themselves, unrelated individuals almost always differ in their KIR repertories ^58,64 (see IMGT KIR Ligand Calculator).

Further work by the Peruggia group reported a beneficial effect of KIR mismatching in haplo-identical HSCT in AML ^207,209,210. A period of studies with inconsistent and unexpected results followed the initial enthusiasm ^211-213. The inconsistencies turned out to be due to the differences between lymphoid and myeloid leukemias, conditioning regimes (whether or not ATG and T-cell depletion are used), dosage of CD34+ stem cells used and whether or not post-transplant immunosuppression has been used. Most recent studies have confirmed and refined the KIR effect on AML relapse ^115,214-216. It appears that incompatibility for KIR ligands, defined by the absenceof a KIR ligand in the recipient that is present in the donor,could lead to a favourable outcome. In the absence of corresponding KIR ligand in an AML patient's HLA repertoire, the presence of inhibitory KIRs on the donor's NK cells and predicts a strong GVL effect with a more favourable clinical outcome ¹⁵. This model is called 'receptor-ligand' or 'missing KIR ligand' model ^215,216. The current model is more compatible with the NK biology and the view that disease can be modified by specific KIR-ligand interactions, rather than by global responsiveness of NK and T cells ⁶¹. See a review by Pegram, 2011.

NK activity in Malignancies:

Although NK cells have MHC-independent activity, melanoma-specific inhibitory KIR+ CTL clones have been described ²¹⁷. In this situation, expression of HLA class I would be deleterious for anti-tumour immunity. In melanoma, NK cells can lyse not only cells which have lost the expression of one or more HLA class I alleles but also cells expressing a decreased amount of class I molecules ²¹⁸. This suggests the involvement of both KIR and CD94/NKG systems in anti-tumour surveillance. In the same model, IFN-g abolished NK cell-mediated lysis by upregulating HLA expression ²¹⁸. The implication for treatment is that a combination of IFN-g treatment and IL2-activated NK cells may provide clinical benefit to cancer patients.

Recent developments lent further support to the cancer immune surveillance theory and particularly the role played by NK cells ^219-224. A Japanese (the Saitama prospective cohort study) has reported the correlation between NKG2D haplotype variants and natural killer cell cytotoxic activity ²²². This activity that appears to be linked to NKG2D genetic variation is inversely correlated with cancer development risk ²²⁵. These recent findings are expected to prompt new studies in cancer immunoepidemiology with renewed interest.

There are also many consistently observed associations between KIR polymorphisms and cancer susceptibility (Middleton, 2009; Al Omar, 2010). Cervical cancer is one of those studied in greatest detail (Carrington, 2005; Martin, 2010).

NK Cells and Reproductive Failure:

Another pathophysiology NK cells are involved with is reproductive failure. Also in this very active field, a lot of genetic associations have been reported (see for example, Hiby, 2008; Hiby, 2010).

Natural Killer Cell Recognition of Target Cells by David Hoskin

POSTER: NK cells: receptors & functions

References

Online Resources

KIR Sequence Database ( IPD ) - see Robinson, 2010

KIR Nomenclature Report 2002: Immunogenetics (PDF); Eur J Immunogenet; Tissue Antigens; Hum Immunol

KIR Gene Cluster (Online NCBI Publication) by Carrington & Norman

KIR Sequence Database (NCBI-dbLRC)

IMGT Tutorial on LRC and KIR