Killer cell immunoglobulin-like receptors (KIR or KIR-receptors) are receptors which are mainly present at the plasma membrane of natural killer cells (NK cells). They play a big role in the mammal immune system. NK cells use KIR to detect pathological cells, like tumor or infected cells [2].
Killer immunoglobulin-like receptors (KIR) regulate the function of natural killer cells through interactions with various ligands on the surface of cells, thereby determining whether natural killer (NK) cells are to be activated or inhibited from killing the cell being interrogated. The genes encoding these proteins display extensive variation through variable gene content, copy number and allele polymorphism. The combination of KIR genes and their ligands is implicated in various clinical settings including haematopoietic stem cell and solid organ transplant and infectious disease progression. The determination of KIR genes has been used as a factor in the selection of optimal stem cell donors with haplotype variations in recipient and donor giving differential clinical outcomes [6].
The KIR gene family currently consists of 15 gene loci (KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3 and two pseudogenes, KIR2DP1 and KIR3DP1) encoded within a 100-200 Kb region of the Leukocyte Receptor Complex (LRC) located on chromosome 19 (19q13.4) [7].
The LRC constitutes a large, 1 Mb, and dense cluster of rapidly evolving immune genes which contains genes encoding other cell surface molecules with distinctive Ig-like extra-cellular domains. These genes include, from centromere to telomere, Leukocyte Immunoglobulin-like Receptors (LILR) and Leukocyte-Associated Immunoglobulin-like Receptors (LAIR), FcGammaR as well as the Natural cytotoxicity-triggering Receptor 1 (NCR1). In addition the extended LRC contains genes encoding the Sialic acid binding Immunoglobulin-like Lectins (SIGLEC) and the CD66 family members such as the carcino-embryonic antigen (CEA) genes as well as the genes encoding the transmembrane adaptor molecules DAP10 and DAP12.
KIR Gene Organisation
KIR genes vary in length from 4 to 16 Kb (full genomic sequence) and can contain four to nine exons. KIR genes are classified as belonging to one of three groups according to their structural features: 1) Type I KIR2D genes, which encode two extra-cellular domain proteins with a D1 and D2 conformation; 2) The structurally divergent Type II KIR2D genes which encode two extra-cellular domain proteins with a D0 and D2 conformation and finally; 3) KIR3D genes encoding proteins with three extra-cellular Ig-like domains (D0, D1 and D2).
(Source link — https://www.ebi.ac.uk/ipd/kir/about/#lrc)
KIR Haplotypes
Based on their gene content, KIR genotypes can be divided into two broad haplotypes termed A and B. These haplotype groups were originally distinguished using restriction fragment length polymorphism (RFLP), with the presence of a ~24 Kb HindIII fragment indicating a group B haplotype. However, these haplotype groups are currently distinguished by the number and combination of KIR genes present. According to this new KIR haplotype group definition, group A haplotypes are generally non-variable in its gene organisation with all four framework genes present plus KIR2DL1, KIR2DL3, KIR3DL1, KIR2DS4 and KIR2DP1.
Group B haplotypes show a lot more variation in the number and combination of KIR genes present and are characterized by the presence of one or more of KIR2DL2, KIR2DL5A/B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5 and KIR3DS1 genes. Group B haplotypes possess a greater variability in the number of genes present. They possess from one to five activating KIR (i.e. KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5 and KIR3DS1) and can incorporate inhibitory KIR genes which are known to be absent in group A haplotypes (i.e. KIR2DL2 and KIR2DL5). KIR genotyping techniques used in family segregation analysis have defined over 40 distinct group B haplotypes.
KIR Proteins
KIR proteins possess characteristic Ig-like domains on their extracellular regions, which in some KIR proteins are involved in HLA class I ligand binding. They also possess transmembrane and cytoplasmic regions which are functionally relevant as they define the type of signal which is transduced to the NK cell. KIR proteins can have two or three Ig-like domains (hence KIR2D or KIR3D) as well as short or long cytoplasmic tails (represented as KIR2DS or KIR2DL). Two domain KIR proteins are subdivided into two groups depending on the origin of the membrane distal Ig-like domains present. Type I KIR2D proteins (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4 and KIR2DS5) possess a membrane-distal Ig-like domain similar in origin to the KIR3D D1 Ig-like domain but lack a D0 domain. This D1 Ig-like domain is encoded mainly by the fourth exon of the corresponding KIR genes. The Type II KIR2D proteins, KIR2DL4 and KIR2DL5, possess a membrane-distal Ig-like domain of similar sequence to the D0 domain present in KIR3D proteins, however, Type II KIR2D lack a D1 domain. Long cytoplasmic tails usually contain two Immune Tyrosine-based Inhibitory Motifs (ITIM) which transduce inhibitory signals to the NK cell. Short cytoplasmic tails possess a positively charged amino acid residue in their transmembrane region which allows them to associate with a DAP12 signalling molecule capable of generating an activation signal [1].
(Source link — https://www.ebi.ac.uk/ipd/kir/about/#lrc)
KIR in solid organ transplantation
The role of NK cells has been studied in the solid organ transplant setting. NK cells are known to infiltrate kidney allografts and occur at a higher frequency in the peripheral blood of patients with acute graft rejection. A role for inhibitory KIR-KIR ligand has been shown in the increased risk of chronic rejection and reduction long-term graft survival. KIR2DS4 and KIR2DS5 have been implicated in the outcome of renal transplantation in patients with glomerular nephritis. KIR2DS4 was more frequent in patients with acute graft rejection, whereas KIR2DS5 was associated with decreased rejection. Interindividual differences in KIR and HLA class I ligand genotypes associated with differences in NK cell reactivity impact donor-specific antibody-mediated NK cell antibody dependant cell-mediated cytotoxicity in organ allografts. In addition, differences have been shown in the number of KIR2D expressing CD8+ T cells between liver transplant recipients with and without acute rejection, suggesting a role for KIR receptors in liver allograft outcomes [6].
In the context of infectious disease, KIR molecules have been shown to exhibit an effect in a variety of settings. The protection from HIV disease progression was shown to be associated with the presence of KIR3DL1 and its ligand HLA-B Bw4, particularly where the 80I is present. More recently, in individuals possessing HLA-B*57, a variant of the KIR3DL1 molecule has been shown to be associated with elite control of viral load and delay in disease progression.
KIR in HSCT
KIR typing in haematopoietic stem cell transplantation is used in high-resolution KIR genotyping and is increasingly used in researching allelic variations present in the KIR gene family. KIR gene allelic variation has been linked with the outcome of HSCT and solid organ transplant illustrating the need for further study of these genes at this level. Allelic variation in KIR genes allows for the characterisation of individuals by differing levels of expression, with high and low expression alleles providing stronger and weaker inhibitory signals [6].
KIR and HLA-C genotyping for assessing the risk of pregnancy complications
During the first trimester of gestation, trophoblastic cells from the blastocyst invade the wall of the maternal uterus, contributing to the formation of the placenta. A defect in the placental origin process can lead to various disorders, such as premature births, preeclampsia, fetal growth restriction, or gestational losses. Studies have shown that part of the regulation of the placental formation process occurs under the influence of local immune recognition. Trophoblastic cells express high levels of HLA-C, which are recognized by KIR (killer immunoglobulin-like receptor) and uNK (uterine Natural Killer cells). KIRs are expressed from a highly polymorphic gene family. These genes are grouped and classified into haplotype A (predominantly inhibitory KIRs) and haplotype B (predominantly activating KIRs). The presence of activating KIRs (haplotype B) provides protection against pregnancy complications, while their absence (haplotype A) increases the risk of complications. The balance between activation and inhibition allows the release of cytokines that favor trophoblastic placental invasion.
The Genotyping KIR and HLA-C test is a genetic test that determines the individual’s haplotype based on the described genes and performs molecular typing of human leukocyte antigen alleles (HLA-C class I) at the allelic group level. The result allows the assessment of the risk of pregnancy complications based on the present haplotype, enabling treatment direction for a better reproductive response. This research is conducted using a combination of PCR SSP and PCR SSO methods and is primarily recommended in cases of reproductive disorders [4].
Another a fascinating study by Wilczyńska et al., 2020, published in the esteemed Journal of Assisted Reproduction and Genetics [8]. This pioneering research marks the first exploration of the role of KIR genes in male infertility. Using inno-train’s KIR-Ready Gene kit, Wilczyńska et al. conducted genotype analyses on the participants in this study. Their findings are truly remarkable: "In this work, we managed to show significant differences in the profile of KIR genes between men who have children from natural conception and men who participated with their female partners in in vitro fertilization."
The KIR-Ready Gene KIR typing kit from the aforementioned company inno-train allows for the detection of all 15 KIR genes (incl. pseudogenes) and the detection of all known allelic variants with important phenotypes, e.g.:
- 2DL4: Differentiation of primary alleles 2DL4*001–*006 and deletion alleles 2DL4*007–*009;
- 2DL5: Differentiation of expressed alleles 2DL5A*001, A*005, A*012, or 2DL5*003 and not expressed alleles 2DL5B*002, B*004, B*006–013;
- 2DS4: Differentiation of primary alleles 2DS4*001, *002 and deletion alleles 2DS4*003, *004, *006;
- 3DP1 pseudogen: Discrimination between two polymorphisms mostly associated with presence or absence of 2DL1; differentiation of 3DP1*003 (2DL1 present) and 3DP1*001, *002 (2DL1 absence).
The test kit also includes an integrated negative control, pre-dosed and dried primer mixtures, a ready-to-use PCR buffer (ReadyPCR), and the method itself is based on the principle of sequence-specific priming (SSP-PCR).
inno-train’s molecular biological SSP detection systems are based on the Polymerase Chain Reaction (PCR), which enables amplification of defined DNA sequences. After successful amplification, the genomic DNA target sequence is present in a detectable concentration. Evaluation of the result is performed by agarose gel electrophoresis. In the electric field the amplificates separate according to their size. Under UV light, the bands are visualized by intercalating ethidium bromide and can be photographed for documentation [2].
CONCLUSIONS
Consequently, the number and type of KIR genes vary significantly between individuals. Genotyping based on the SSP-PCR principle is the most common method for assessing KIR gene content due to the simplicity of the procedure, the availability of the kit, and the understandable interpretation of the results, unlike other typing methods that require significant time for comprehensive processing of PCR data and complex interpretation, which significantly limits their usefulness [5]. KIR typing is also increasingly used in the diagnosis of infertility, as it helps to diagnose immunological infertility, the causes of failed embryo implantation and miscarriages, and allows treatment with a high probability of success.
REFERENCES
1. https://www.ebi.ac.uk/ipd/kir/ — IPD-KIR Database.
2. https://www.inno-train.de/en/home-ssp-pcr/kir-typing/.
4. https://www.synlab-sd.com/en/exame/genotipagem-kir-hla-c-2/.
5. Andrea A. Zachary and Mary S. Leffell (eds.), Transplantation Immunology: Methods and Protocols, Second Edition, Methods in Molecular Biology, vol. 1034, DOI 10.1007/978-1-62703-493-7_12.
6. Downing, J., D’Orsogna, L. High-resolution human KIR genotyping. Immunogenetics 74, 369–379 (2022). https://doi.org/10.1007/s00251-021-01247-0.
7. Trowsdale J. Genetic and functional relationships between MHC and NK receptor genes. Immunity. 2001 Sep;15(3):363-374. DOI: 10.1016/s1074-7613(01)00197-2.
8. Wilczyńska, K., Radwan, P., Krasiński, R. et al. KIR and HLA-C genes in male infertility. J Assist Reprod Genet 37, 2007–2017 (2020). https://doi.org/10.1007/s10815-020-01814-6.
(Джерело посилання — inno-train Diagnostik GmbH)
