KIR Receptors

KIR receptors are a family of inhibitory and activating receptors, which are expressed by NK cells and CD8⁺ T-cells. Each NK cell from an individual expresses stochastically a specific pattern of KIR receptors, and this pattern differs between

individuals. The killer Immunoglobulin-like receptors (KIR) gene family consists of 14 genes (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3 and KIR3DS1) and 2 pseudogenes (KIR2DP1 and KIR3DP1). These genes are encoded on chromosome 19 in the leukocyte receptor complex (LRC) region. The LRC gene consists of a 1Mb region, which contains several gene coding for several cells surface proteins, such as SIGLEC, LAIR or the Fcg receptor (Fig. 9). There are also genes coding for trans-membrane molecules such as DAP-10 or DAP-12. Among these genes, the KIR genes encode a region of 150bp (26, 27).

Fig. 9. Picture of the LCR region of the human chromosome 19.

In blue, the KIR gene region (28).

4.1.1.1. KIR Genes

The length of KIR genes varies between 4 to 16 Kb and comprises, 4 to 9 exons.

They have been classified in 3 groups, in relation to their extra-cellular structure.

In the first group, the genes coding for KIR2D type I express two extra-cellular domains with a D1 and D2 confirmation. The second group, are the genes for KIR2D type II and express two extra-cellular domains D0 and D2. Finally, the third group represented by the KIR3D, with its 3 extra-cellular domains D0, D1 and D2.

The genes coding for KIR2D type I, which are represented by KIR2DL1, KIR2DL2, KIR2DL3 and KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5 include 8 exons and one pseudoexon, which is inactive in KIR2D type I. KIR2DL1 and KIR2DL2 have a deletion in exon 7, which distinguishes them from the other KIR. The KIR types I, KIR2DL1, KIR2DL2 and KIR2DL3 are different from the KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, and KIR2DS5 in their length in the cytoplasmic region, coding by the exon 9. KIR2D type II, which code for KIR2DL4 and KIR2DL5 do not have an exon 4 and the exon 1 of KIR2DL4 is longer than the one of KIR2DL5. The gene codings for KIR3D, which are represented by KIR3DL1, KIR3DL2, KIR3DL3 and KIR3DS1, include 9 exons. The

ninth exon is coding for the cytoplasmic part of the protein, and notably KIR3DL2 is the longest of all KIR (16'256bp). KIR3DS1 has a shorter exon 8, which coded for the cytoplasmic part, compared to KIR3DL1 and KIR3DL2. KIR genes polymorphisms seem to be the biggest contributor of the KIR diversity. This allows for a tremendous variety of responses against different pathogens. Population studies of KIR genotypes showed variations in KIR gene from individual to individual. KIR are organized in a head-to-tail fashion and each KIR has a length of 10 to 16kb and 2kb separate each pair of genes (29, 30, 31, 32).

4.1.1.2. KIR Haplotype

All KIR haplotypes have the same organization; the centromeric end is delimited by KIR3DL3, and the telomeric end by KIR3DL2. In the centric part, KIR3DP1 and KIR2DL4 can be found. These 4 KIR genes constitute the framework genes, they are present on all KIR haplotypes, and between these genes, the remaining KIR gene can be located. Based on the studies of the KIR genes content within this framework, two different halpotypes appear; haplotype A and haplotype B. The main difference between these two haplotypes is the presence or not of one or more activating KIR (31, 32).

KIR haplotype A express always the same pattern, seven loci, which are KIR3DL3 (at the centromeric end), KIR2DL3, KIR2DL1, KIR2DL4, KIR3DL1, KIR2DS4 and finally KIR3DL2 at the telomeric end. This haplotype has a frequency of expression of 47 to 59% among the European population and include, only one activating KIR, KIR2DS4. By traditional typing, Hsu et al., (33) could not distinguish between the total KIR2DS4 and a variant, which has a 22 bp deletion in the D2 Ig domain. By comparing the sequence of this KIR2DS4 with a 22 bp deletion with other species, a 72% homology of amino acid appears with the Mm-KIR1D receptor, which is found in rhesus monkeys (34). The function of human KIR1D is still unknown. But the frequency of KIR1D in the European population is 78%, in comparison to KIR2DS4, which is 35%. For this reason, KIR haplotype A can be divided into 2 sub-group, the haplotype A-1D and the haplotype A-2DS4, with a frequency of 39% for A-1D and 12% for A-2DS4 (33). As the most frequent KIR haplotype A have KIR1D, which function is unknown, and we can therefore conclude that this haplotype has no

activating KIR. But next to that, it was observed that the framework gene KIR2DL4 retains an activating function (35, 36). In contrast, haplotype B is much more variable and has one to five activating KIR. Its frequency among the European population is the same as haplotype A, however, haplotype B shows a much greater variety of subtypes (26, 32) (Fig. 10).

KIR haplotype consists of 2 parts within the central middle KIR2DL4. The centromeric half is limited by KIR3DL3 upstream and KIR3DP1, and on the other part, the telomeric half is limited by KIR3DL2 downstream and KIR2DL4. KIR2DL2 or KIR2DL3 is found in the centromeric half, although never both combined. Yet, when KIR2DL2 is present, KIR2DS2 will always be present, next to KIR3DL3. The presence of KIR2DL3 is always associated with KIR2DP1, KIR2DL1 and KIR3DP1, which defines a partial haplotype. Nevertheless, the telomeric half is characterized by the presence of KIR3DL1 or KIR3DS1, but not both. The presence of KIR3DL1 indicates a "short" telomeric end with the presence of either KIR2DS4 or KIR1D and finally KIR3LD2. In the case of KIR3DS1, a "long" telomeric end is present with KIR2DL5 paired with KIR2DS3 or KIR2DS5, then by KIR2DS1 and either KIR2DS4 or KIR1D. KIR3LD2 closes the loci (33) (Fig. 10).

Fig. 10. Schematic view of KIR haplotype A (top) and haplotype B (bottom). In pink, the framework genes, in blue inhibitory KIR, in red activating KIR and in grey pseudogenes. Below the KIR haplotype B and shown in parentheses,

the different possibilities of KIR genes, which can form KIR haplotype B (37).

Next to these 2 KIR haplotype, the KIR genomic region possesses a high level of allelic polymorphism. It is generated by homologous recombination or by point mutation (29). It is the most variable region of the human genome, after the MHC polymorphism (38). The great advantage of this KIR allelic polymorphism is to diversify the immune response against pathogens (39). These different KIR alleles can give rise to protein variant with differential binding affinity for the MHC-I

ligand (40). However, frameshift deletion creates premature stop codons that might generate truncated KIR proteins that are not expressed on the cell surface (41).

4.1.1.3. KIR Proteins

KIR receptors are monomeric and include 14 receptors, which can be divided into 8 inhibitory receptors (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2 and KIR3DL3) and 6 activating receptors (KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5 and KIR3DS1). There is a very high sequence homology between these receptors and their nomenclature is based on their structural differences.

They all possess, an extra-cellular, a trans-membrane and a cytoplasmic part. A first difference appears in the extra-cellular domain of the protein. KIR receptors have 2 or 3 Ig-like domains, and this structure is used in the KIR nomenclature. A KIR2D expresses 2 Ig-like domains, whereas a KIR3D possess 3 Ig-like domains. As said before, the extra-cellular domain is divided into D0, D1 and D2 regions. KIR2D type I, which includes KIR2DL1, KIR2DL2, KIR2DL3, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5 expresses only the D1 and D2 Ig-like domains. KIR2D type II with KIR2DL4 and KIR2DL5 possess a D0 and D2; D1 Ig-like domain is absent. Then, KIR3D expresses the three Ig-like domains D0, D1 and D2. A second difference in the KIR structure is situated in the intra-cellular part of the protein. This cytoplasmic region can be either long or short. A long cytoplasmic tail contains two immune tyrosine-based inhibitory motifs (ITIM), which transduces an inhibitory signal to the cells. With a short cytoplasmic tail, a positive charged amino acid arginine (Arg) or lysine (Lys) is expressed in the trans-membrane domain, allowing the binding of a DNAX-activating protein of 12 KDa (DAP-12) molecule. DAP-12 has 2 immune tyrosine-based activating motifs (ITAM), and will generate an activating signal to the cells. This difference in the length in the cytoplasmic domain is also a characteristic in the KIR names. "L" represents a long cytoplasmic tail, and "S"

means a short region. The final digit indicates the number of the gene encoding a protein with this structure. As KIR exists in different allele, they were named similarly as the HLA system. After the last number, 3 digits separated by an

asterisk indicates alleles that differ in the sequences of their encoded proteins (42, 43).

Thus KIR2DL1 for example, is a KIR with 2 Ig-like domains and a long cytoplasmic tail (Fig. 11) (26). KIR2DS1 will be its activating counterpart.

Among all of these KIR receptors, KIR2DL4 has several features, which make it special. First, it has an unusual extra-cellular D0-D2 Ig domain. Secondly, there is a positively charged amino acid (Arg) in its trans-membrane region. Thirdly, KIR2DL4 has a long cytoplasmic tail with only one ITIM motif, instead of two, and finally, KIR2DL4 mRNA is expressed in all cells and its ligand is HLA-G. With all these characteristics, KIR2DL4 shows both inhibitory and activating motifs, but remains an activating KIR (26, 35, 44, 36).

Fig. 11. Schematic view of the different KIR proteins. On the extra-cellular region, the different IgG-like domains; in red, D0; in blue D1 and in green D2. Inside the cell membrane, the red loop represents the DAP-12 molecules with the 2 ITAM motif in grey square. The 2 white squares on the long tail represent the ITIM motif.

Note the KIR2DL4 with a red loop and a white square. Adapted from (45).

KIR are expressed in a stochastic way, which means that each NK cells express one or more KIR at their surface. It should be noted that significant fraction of NK cells do not express any KIR (46).

4.1.1.4. KIR and HLA Interaction

The role of the KIR receptors is to control NK cell response with its environment.

NK cell recognizes cells expressing self-MHC-I, and kill cells, which lack or down-regulate expression of self-MHC-I expression. NK cell reactivity will depend on which type of KIR is bound to its ligand. An inhibitory KIR will give an inhibitory signal to the NK cell, whereas an activating KIR will stimulate the NK cell, and

perforins and granzyms will be secreted in order to kill the target cell. Some studies have suggested that activating KIR have a very weak specificity for MHC-I, while other conducted studies did not show this interaction (47). One of the main ligand for inhibitory KIR is HLA-C, but a difference in the amino acid at position 80, results in two groups, HLA-C1 and HLA-C2. HLA-C1 has an asparagines at position 80 (Asn80), and recognize KIR2DL2 and KIR2DL3, whereas, KIR2DL1 recognized by HLA-C2, has a lysine at position 80 (Lys80) (48).

Behind these 2 ligands, there is an evolutionary process (32). An HLA-C locus was first observed in orangutans (49). This HLA-C, called Popy-C, shows only an Asn80, and no HLA-C with Lys80 were present in orangutans. Then, when orangutan and chimpanzee/human predecessors separated, a mutation of Asn80 to Lys80 in HLA-C1 emerged and produced HLA-C2 (50). Binding measurements show that KIR2DL1 and HLA-C2 are stronger and more specific compared to KIR2DL2/DL3 and HLA-C1, which binding is weaker and less specific. Therefore, most probably, in the evolutionary process, the weak KIR2DL2/2DL3-HLA-C1 interaction appeared first, and then the stronger KIR2DL1-HLA-C2 evolved. In humans, both interactions are present. HLA-C2 did not eliminate HLA-C1, in all probability for the reason that, both interactions are complementary.

For HLA-B, which is the ligand for KIR3DL1, there is also a sequence dimorphism at the C-terminal, which gives 2 groups HLA-Bw4 and HLA-Bw6 (51). The binding strength depends on the amino acid sequence at position 80. HLA-Bw4 with an isoleucine residue at position 80 (Ile80) is the strongest inhibitor (52), whereas, HLA-Bw6 with threonine residue at position 80 (Thr80) is weak. Next to the knowing ligand, it has been shown that KIR3DL2 bind to HLA-A3/-A11. Surprisingly, HLA-A3/-A11 was reported to be expressed by Epstein-Barr virus (EBV) transformed B-cells and direct binding of HLA-A3 to KIR3DL2 is peptide specific. Hansasuta et al.

(53), used tetramer HLA-A3 with several antigenic peptides, and demonstrated that only tetramers, which refold with a peptide from EBV, bind to KIR3DL2. The last knowing KIR ligand involved KIR2DL4, which is mainly expressed by trophoblasts cells, and bind to HLA-G (54).

4.1.1.5. KIR and Diseases

This binding strength between KIR and MHC-I can influence susceptibility to diseases. KIR2DL1, KIR2DL2, KIR2DL3 and KIR3DL1 bind to their ligand HLA-C and HLA-B with varying strength. As KIR2DL3 and HLA-C1 have a weak binding compared to KIR2DL1 and HLA-C2, KIR2DL1 and HLA-C2/C2 homozygote has a stronger inhibition effect compared to KIR2DL3 and HLA-C1/C1 homozygote. On the other hand, KIR haplotype can also play a role; KIR haplotype A/A homozygote has less activation in comparison to KIR haplotype B/B homozygote, which will have between 1 and 5 activating KIR. Autologous KIR-HLA interaction appears in infectious or autoimmune diseases, whereas in pregnancy, maternal KIR will interact with fetal MHC-I, and in transplantation, donor KIR will interact with recipient MHC-I molecules (55, 37).

In pregnancy, which is in a way, a successful allograft transplantation, KIR-MHC-I interaction plays an important role in preeclampsia. Preeclampsia is a serious complication in pregnancy, where the fetus receives too little blood, which as a consequence leads to maternal and fetal mortality. Hiby et al. (56), show that when maternal KIR receptors, with a KIR haplotype A homozygote (no activating KIR), are in the presence of a fetus, which possess a HLA-C2/C2 homozygote ligand, there is an increasing risk of preeclampsia. The authors explain that, as KIR2DL1/HLA-C2 is a strong association, there is too much inhibition and trophoblasts are not able to move into the uterine arteries. They also show, no association with preeclampsia in individual, which have KIR haplotype A homozygote and HLA-C1 ligand. Finally, with maternal activating KIR (haplotype A/B or B/B) and HLA-C2, the strong inhibition of KIR2DL1/HLA-C2 is balanced by the activating receptors.

Human immunodeficiency virus (HIV) is a viral infection causing immunosuppression and progression of acquired immunodeficiency syndrome (AIDS) over a variable period of time. Several publications (57, 58) show an interaction between KIR3DL1/3DS1 and HLA-Bw4. They demonstrated that patients NK cells, which contain KIR3DS1 and its ligand HLA-Bw4-80Ile, show a slower progression of the disease, compared to patients with no KIR3DS1 or no HLA-Bw4. This is interesting;

beside the role of the T-cells that were largely investigated in HIV this KIR-HLA interaction demonstrates an interestingly anti-viral role of the NK cells.

In autoimmune diseases, KIR receptors can also be important. In patients with rheumatoid arthritis (RA), expression of KIR2DS2 is much higher compared to healthy individuals (83% vs. 47%). Moreover, this KIR2DS2 is expressed by a CD4⁺/CD28^- T-cells sub-population. In absence of the corresponding inhibitory receptors, this sub-population can be directly activated through KIR2DS2 receptors, instead of the T-cell receptor (TCR), and become cytotoxic (55, 59).

Still, one of the most interesting roles of the KIR-MHC-I interaction appears in allogeneic BM transplantation (see Chapter 7. NK cells and Transplantation).