Influence of interleukin IL-2 and IL-12 + IL-18 on surface expression of immunoglobulin-like receptors KIR2DL1, KIR2DL2, and KIR3DL2 in natural killer cells

Natural killer (NK) cells express killer cell inhibitory receptors (KIRs) that recognize polymorphic class I MHC molecules. In the present study, we analyze the modulatory effect of IL-2 alone or a combination of IL-12 with IL-18 on surface expression of killer cell immunoglobulin-like receptors KIR2DL1, KIR2DL2, and KIR3DL2 in NK cells. Thus, it was found that IL-2 causes a significant increase in the proportion of cells with given studied receptors. Stimulation by a mixture of IL-12 and IL-18 caused significant increase in the fraction of cells with the KIR2DL1 and KIR2DL2, however no significant change in the percentage of cells with KIR3DL2 receptor on their surface was observed. The results of the study show the presence of KIRs on both resting and activated NK cells, this may suggest that KIRs have also an important role in the regulatory processes after activation of this subpopulation of cells.     

Conjunctival Scarring in Trachoma Is Associated with the HLA-C Ligand of KIR and Is Exacerbated by Heterozygosity at KIR2DL2/KIR2DL3

Background

Chlamydia trachomatis is globally the predominant infectious cause of blindness and one of the most common bacterial causes of sexually transmitted infection. Infections of the conjunctiva cause the blinding disease trachoma, an immuno-pathological disease that is characterised by chronic conjunctival inflammation and fibrosis. The polymorphic Killer-cell Immunoglobulin-like Receptors (KIR) are found on Natural Killer cells and have co-evolved with the Human Leucocyte Antigen (HLA) class I system. Certain genetic constellations of KIR and HLA class I polymorphisms are associated with a number of diseases in which modulation of the innate responses to viral and intracellular bacterial pathogens is central.

Methodology

A sample of 134 Gambian pedigrees selected to contain at least one individual with conjunctival scarring in the F₁ generation was used. Individuals (n = 830) were genotyped for HLA class I and KIR gene families. Family Based Association Tests and Case Pseudo-control tests were used to extend tests for transmission disequilibrium to take full advantage of the family design, genetic model and phenotype.

Principle findings

We found that the odds of trachomatous scarring increased with the number of genome copies of HLA-C2 (C1/C2 OR = 2.29 _BHP-value = 0.006; C2/C2 OR = 3.97 _BHP-value = 0.0004) and further increased when both KIR2DL2 and KIR2DL3 (C2/C2 OR = 5.95 _BHP-value = 0.006) were present.

Conclusions

To explain the observations in the context of chlamydial infection and trachoma we propose a two-stage model of response and disease that balances the cytolytic response of KIR expressing NK cells with the ability to secrete interferon gamma, a combination that may cause pathology. The data presented indicate that HLA-C genotypes are important determinants of conjunctival scarring in trachoma and that KIR2DL2/KIR2DL3 heterozygosity further increases risk of conjunctival scarring in individuals carrying HLA-C2.     

KIR3DL1/S1 genotypes and KIR2DS4 allelic variants in the AB KIR genotypes are associated with Plasmodium-positive individuals in malaria infection

The importance of innate immunity in malaria has been suggested for early protection from maturation and multiplication of Plasmodium parasites injected via infected mosquitoes. In this study, the killer cell immunoglobulin-like receptor (KIR) genes in innate immunity were investigated for an association with malaria in the comparison between Plasmodium-positive and Plasmodium-negative Melanesian individuals in the Solomon Islands, one of the most hyperendemic malaria regions in the world. The higher frequency of a pair of KIR3DL1 and KIR2DS4 was observed in the Plasmodium-positive individuals, which led to the investigation of KIR3DL1/S1 genotypes in concert with KIR2DS4 allelic variants. The positive individuals showed the highest frequency of KIR3DL1/KIR3DS1 heterozygosity, which might suggest the masking of activating KIR3DS1 by inhibitory KIR3DL1 at allelic levels to maintain the KIR3DS1-driven activation of natural killer cells diminished in controlling Plasmodium proliferation. The extended analysis with A/B genotypes further revealed the trend of parasitic positive individuals to be KIR3DL1/KIR3DS1 heterozygous in pair with KIR2DS4 nondeleted variants in a set of KIR genes inheritable as the AB genotypes. To the best of our knowledge, this study is the first KIR investigation of the malaria-infected population, which strengthened the potential associations of KIR with malaria pathogenesis. The balance of inhibitory and activating KIR3D genes (KIR3DL1/S1) and membrane-bound or secreted status of KIR2DS4 alleles in the interaction with the other KIR genes in the AB genotypes might constitute a part of KIR characteristics to determine resistance or susceptibility to Plasmodium parasitic infection.     

The Structure of the Atypical Killer Cell Immunoglobulin-like Receptor,KIR2DL4

The engagement of natural killer cell immunoglobulin-like receptors (KIRs) with their target ligands, human leukocyte antigen (HLA) molecules, is a critical component of innate immunity. Structurally, KIRs typically have either two (D1-D2) or three (D0-D1-D2) extracellular immunoglobulin domains, with the D1 and D2 domain recognizing the α1 and α2 helices of HLA, respectively, whereas the D0 domain of the KIR3DLs binds a loop region flanking the α1 helix of the HLA molecule. KIR2DL4 is distinct from other KIRs (except KIR2DL5) in that it does not contain a D1 domain and instead has a D0-D2 arrangement. Functionally, KIR2DL4 is also atypical in that, unlike all other KIRs, KIR2DL4 has both activating and inhibitory signaling domains. Here, we determined the 2.8 Å crystal structure of the extracellular domains of KIR2DL4. Structurally, KIR2DL4 is reminiscent of other KIR2DL receptors, with the D0 and D2 adopting the C2-type immunoglobulin fold arranged with an acute elbow angle. However, KIR2DL4 self-associated via the D0 domain in a concentration-dependent manner and was observed as a tetramer in the crystal lattice by size exclusion chromatography, dynamic light scattering, analytical ultracentrifugation, and small angle x-ray scattering experiments. The assignment of residues in the D0 domain to forming the KIR2DL4 tetramer precludes an interaction with HLA akin to that observed for KIR3DL1. Accordingly, no interaction was observed to HLA by direct binding studies. Our data suggest that the unique functional properties of KIR2DL4 may be mediated by self-association of the receptor.     

Enrichment of variations in KIR3DL1/S1 and KIR2DL2/L3 among H1N1/09 ICU patients: an exploratory study

Background

Infection by the pandemic influenza A (H1N1/09) virus resulted in significant pathology among specific ethnic groups worldwide. Natural Killer (NK) cells are important in early innate immune responses to viral infections. Activation of NK cells, in part, depend on killer-cell immunoglobulin-like receptors (KIR) and HLA class I ligand interactions. To study factors involved in NK cell dysfunction in overactive immune responses to H1N1 infection, KIR3DL1/S1 and KIR2DL2/L3 allotypes and cognate HLA ligands of H1N1/09 intensive-care unit (ICU) patients were determined.

Methodology and Findings

KIR3DL1/S1, KIR2DL2/L3, and HLA -B and -C of 51 H1N1/09 ICU patients and 105 H1N1-negative subjects (St. Theresa Point, Manitoba) were characterized. We detected an increase of 3DL1 ligand-negative pairs (3DL1/S1⁺ Bw6⁺ Bw4⁻), and a lack of 2DL1 HLA-C2 ligands, among ICU patients. They were also significantly enriched for 2DL2/L3 ligand-positive pairs (P<0.001, Pc<0.001; Odds Ratio:6.3158, CI95%:2.481–16.078). Relative to St. Theresa aboriginals (STh) and Venezuelan Amerindians (VA), allotypes enriched among aboriginal ICU patients (Ab) were: 2DL3 (Ab>VA, P = 0.024, Pc = 0.047; Odds Ratio:2.563, CI95%:1.109–5.923), 3DL1*00101 (Ab>VA, P<0.001, Pc<0.001), 3DL1*01502 (Ab>STh, P = 0.034, Pc = 0.268), and 3DL1*029 (Ab>STh, P  = 0.039, Pc = 0.301). Aboriginal patients ligand-positive for 3DL1/S1 and 2DL1 had the lowest probabilities of death (R_d) (R_d = 28%), compared to patients that were 3DL1/S1 ligand-negative (R_d = 52%) or carried 3DL1*029 (R_d = 52%). Relative to Caucasoids (CA), two allotypes were enriched among non-aboriginal ICU patients (NAb): 3DL1*00401 (NAb>CA, P<0.001, Pc<0.001) and 3DL1*01502 (CAP = 0.012, Pc = 0.156). Non-aboriginal patients with ligands for all three KIRs (3DL1/S1, 2DL2/L3, and 2DL1) had the lowest probabilities of death (R_d = 36%), compared to subjects with 3DL1*01502 (R_d = 48%) and/or 3DL1*00401 (R_d = 58%).

Conclusions

Specific KIR3DL1/S1 allotypes, 3DL1/S1 and 2DL1 ligand-negative pairs, and 2DL2/L3 ligand-positive pairs were enriched among ICU patients. This suggests a possible association with NK cell dysfunction in patients with overactive immune responses to H1N1/09, leading to severe disease.     

KIR2DL4 is an IL-2-regulated NK cell receptor that exhibits limited expression in humans but triggers strong IFN-gamma production

Killer cell Ig-like receptor (KIR)2DL4 (2DL4, CD158d) was previously described as the only KIR expressed by every human NK cell. It is also structurally atypical among KIRs because it possesses a basic transmembrane residue, which is characteristic of many activating receptors, but also contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM). We expressed epitope-tagged 2DL4 in an NK-like cell line to study receptor function. Three distinct 2DL4 cDNA clones were analyzed: one encoding the "conventional" 2DL4 with the cytoplasmic ITIM (2DL4.1) and two encoding different cytoplasmic truncated forms lacking the ITIM (2DL4.2 and 2DL4(*)). Surprisingly, one truncated receptor (2DL4.2), which is the product of a prevalent human 2DL4 allele, was not expressed on the cell surface, indicating that some individuals may lack functional 2DL4 protein expression. Conversely, both 2DL4.1 and 2DL4(*) were expressed on the cell surface and up-regulated by IL-2. Analysis of primary NK cells with anti-2DL4 mAb confirmed the lack of surface expression in a donor with the 2DL4.2 genotype. Donors with the 2DL4.1 genotype occasionally expressed receptor only on CD56(high) NK cells, although their expression was up-regulated by IL-2. Interestingly, Ab engagement of epitope-tagged 2DL4 triggered rapid and robust IFN-gamma production, but weak redirected cytotoxicity in an NK-like cell line, which was the opposite pattern to that observed upon engagement of another NK cell activating receptor, NKp44. Importantly, both 2DL4.1 and 2DL4(*) exhibited similar activation potential, indicating that the ITIM does not influence 2DL4.1 activating function. The unique activation properties of 2DL4 suggest linkage to a distinct signaling pathway.     

Endosomal Signaling and a Novel Pathway Defined by the Natural Killer Receptor KIR2DL4 (CD158d)

In addition to ligand‐induced activation of receptors at the cell surface, certain internalized receptor–ligand complexes are activated in endosomes which are, now recognized as important intracellular platforms of signal transduction. The major receptor families that signal from endosomes and illustrate the diversity and complexity of endosomal signaling include receptor tyrosine kinases (RTKs), G‐protein‐coupled receptors (GPCRs) and toll‐like receptors (TLRs). Natural killer (NK) cells, an important component of the innate immune system, not only provide a rapid defense against foreign invaders, such as bacteria and viruses, but also positively shape local responses by cytokine and chemokine secretion. The NK cell receptor KIR2DL4 (CD158d) utilizes a new mode of endosomal signaling after binding its ligand, soluble HLA‐G, in the extracellular milieu. Internalization of the receptor and its ligand into endosomes and initiation of signaling at this site result in a proinflammatory and proangiogenic response with important functions at sites of ligand expression, such as at the maternal–fetal interface during early pregnancy. After a brief overview of the modes of endosomal signaling and its value in generating distinct physiological responses, this review will highlight the mechanism and physiological significance of a novel intracellular signaling pathway used by the endosome‐resident immune receptor KIR2DL4.     

Characterization of constitutive and inducible transcription factors binding to the P2 NF-AT site in the human interleukin-4 promoter

Interleukin-4 (IL-4) is a pleiotropic immunomodulatory cytokine secreted by T helper 2 cells. The IL-4 promoter contains multiple sites with DNA sequences homologous to the IL-2 NF-AT binding site. One of these sites—the P2 site—located between –173 and –150 was previously found to be flanked by two octamer-like motifs. NF-ATp/c and octamer proteins were suggested to bind to this region and to cooperatively activate the promoter activity (Chuvpilo et al., 1993). To precisely analyze the P2-binding factors we used antibodies against NF-ATp, NF-ATc, Fos, Jun, Oct-1 and Oct-2 in EMSA. We show here that nuclear extracts from T-cells form two P2-binding complexes—a PMA/ionomycin-inducible and a constitutive one. The PMA/ionomycin-inducible complex contains NF-ATp/c, Fos and Jun. No octamer binding factors could be detected in either of the two complexes. Analysis of the precise DNA contact points of the two complexes showed that both complexes are formed in the center of the NF-AT consensus site. No DNA contact points could be detected in the octamer-like motif site. Furthermore, purified recombinant POU domains of Oct-1 and Oct-2 failed to bind to the P2 site, suggesting that this site is not an independent octamer-binding site. Therefore, the DNA sequence at –173 to –150 of the IL-4 promoter is a binding site for NF-ATp/c and AP-1. Octamer proteins are unlikely to cooperate with NF-ATp/c at this site.© 1997 Elsevier Science B.V. All rights reserved.     

KIR2DL4 (CD158d) genotype influences expression and function in NK cells

The expression and function of the NK cell receptor KIR2DL4 are controversial. Two common alleles of the transmembrane domain of KIR2DL4 exist. The 10A allele with 10 adenines at the end of the transmembrane exon encodes a full length receptor, whereas the 9A allele has only 9 adenines resulting in a frame shift which in turn generates a stop codon early in the first cytoplasmic exon. The possibility that the 10A and 9A alleles might result in differences in expression and function of KIR2DL4 was explored using mAbs to KIR2DL4. Transfection experiments with cDNA from the 10A and 9A alleles revealed significant membrane expression only with the protein encoded by the 10A allele. Analysis of peripheral blood NK cells demonstrated that only in subjects with at least one 10A allele was cell surface expression of KIR2DL4 detectable, and then only on the minor CD56(bright) NK cell subset. The major CD56(dim) NK cell subset did not cell surface express KIR2DL4 but, interestingly, did so after in vitro culture. Functional analysis using cultured NK cells in redirected lysis assays demonstrated that KIR2DL4 is an activating receptor for NK cells with at least one 10A allele. No significant activity was detected for NK cells generated from subjects homozygous for the 9A allele. These data show that genotype influences cell surface expression and function of KIR2DL4 which may account for reported differences in KIR2DL4 expression and function.     

Rapid production of human KIR2DL4 extracellular domain and verification of its interaction with HLA-G

Killer cell immunoglobulin-like receptor (KIR) 2DL4 is the only KIR member reported to be expressed by all human natural killer (NK) cells. It differs from other KIR members in both structure and function. Its specific interaction with HLA-G, a non-classical MHC class I molecule, has been suggested to play an important role in regulating NK cell-mediated cytotoxicity. However, this interaction is still in doubt. In addition, the soluble KIR2DL4 extracellular domain used in many studies was produced by eukaryotic expression, which is less efficient than prokaryotic expression. In this study, we describe a method of rapid production of a large amount of soluble KIR2DL4 extracellular domain based on a prokaryotic expression system. With this soluble KIR2DL4, we verified the interaction between KIR2DL4 and HLA-G1.     

Rapid production of human KIR2DL4 extracellular domain and verification of its interaction with HLA-G

Killer cell immunoglobulin-like receptor (KIR) 2DL4 is the only KIR member reported to be expressed by all human natural killer (NK) cells. It differs from other KIR members in both structure and function. Its specific interaction with HLA-G, a non-classical MHC class I molecule, has been suggested to play an important role in regulating NK cell-mediated cytotoxicity. However, this interaction is still in doubt. In addition, the soluble KIR2DL4 extracellular domain used in many studies was produced by eukaryotic expression, which is less efficient than prokaryotic expression. In this study, we describe a method of rapid production a large amount of soluble KIR2DL4 extracellular domain based on a prokaryotic expression system. With this soluble KIR2DL4, we verified the interaction between KIR2DL4 and HLA-G1.