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.     

Polymorphic sites away from the Bw4 epitope that affect interaction of Bw4+ HLA-B with KIR3DL1

KIR3DL1 is a polymorphic, inhibitory NK cell receptor specific for the Bw4 epitope carried by subsets of HLA-A and HLA-B allotypes. The Bw4 epitope of HLA-B*5101 and HLA-B*1513 is determined by the NIALR sequence motif at positions 77, 80, 81, 82, and 83 in the alpha(1) helix. Mutation of these positions to the residues present in the alternative and nonfunctional Bw6 motif showed that the functional activity of the Bw4 epitopes of B*5101 and B*1513 is retained after substitution at positions 77, 80, and 81, but lost after substitution of position 83. Mutation of leucine to arginine at position 82 led to loss of function for B*5101 but not for B*1513. Further mutagenesis, in which B*1513 residues were replaced by their B*5101 counterparts, showed that polymorphisms in all three extracellular domains contribute to this functional difference. Prominent were positions 67 in the alpha(1) domain, 116 in the alpha(2) domain, and 194 in the alpha(3) domain. Lesser contributions were made by additional positions in the alpha(2) domain. These positions are not part of the Bw4 epitope and include residues shaping the B and F pockets that determine the sequence and conformation of the peptides bound by HLA class I molecules. This analysis shows how polymorphism at sites throughout the HLA class I molecule can influence the interaction of the Bw4 epitope with KIR3DL1. This influence is likely mediated by changes in the peptides bound, which alter the conformation of the Bw4 epitope.     

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.     

Variable NK cell receptors exemplified by human KIR3DL1/S1

Variegated expression of variable NK cell receptors for polymorphic MHC class I broadens the range of an individual's NK cell response and the capacity for populations and species to survive disease epidemics and population bottlenecks. On evolutionary time scales, this component of immunity is exceptionally dynamic, unstable, and short-lived, being dependent on coevolution of ligands and receptors subject to varying, competing selection pressures. Consequently these systems of variable NK cell receptors are largely species specific and have recruited different classes of glycoprotein, even within the primate order of mammals. Such disparity helps to explain substantial differences in NK cell biology between humans and animal models, for which the population genetics is largely ignored. KIR3DL1/S1, which recognizes the Bw4 epitope of HLA-A and -B and is the most extensively studied of the variable NK cell receptors, exemplifies how variation in all possible parameters of function is recruited to diversify the human NK cell response.     

Th17 cells expressing KIR3DL2+ and responsive to HLA-B27 homodimers are increased in ankylosing spondylitis

CD4 Th cells producing the proinflammatory cytokine IL-17 (Th17) have been implicated in a number of inflammatory arthritides including the spondyloarthritides. Th17 development is promoted by IL-23. Ankylosing spondylitis, the most common spondyloarthritis (SpA), is genetically associated with both HLA-B27 (B27) and IL-23R polymorphisms; however, the link remains unexplained. We have previously shown that B27 can form H chain dimers (termed B27(2)), which, unlike classical HLA-B27, bind the killer-cell Ig-like receptor KIR3DL2. In this article, we show that B27(2)-expressing APCs stimulate the survival, proliferation, and IL-17 production of KIR3DL2(+) CD4 T cells. KIR3DL2(+) CD4 T cells are expanded and enriched for IL-17 production in the blood and synovial fluid of patients with SpA. Despite KIR3DL2(+) cells comprising a mean of just 15% of CD4 T in the peripheral blood of SpA patients, this subset accounted for 70% of the observed increase in Th17 numbers in SpA patients compared with control subjects. TCR-stimulated peripheral blood KIR3DL2(+) CD4 T cell lines from SpA patients secreted 4-fold more IL-17 than KIR3DL2(+) lines from controls or KIR3DL2(-) CD4 T cells. Strikingly, KIR3DL2(+) CD4 T cells account for the majority of peripheral blood CD4 T cell IL-23R expression and produce more IL-17 in the presence of IL-23. Our findings link HLA-B27 with IL-17 production and suggest new therapeutic strategies in ankylosing spondylitis/SpA.     

Significant Association of KIR2DL3-HLA-C1 Combination with Cerebral Malaria and Implications for Co-evolution of KIR and HLA

Cerebral malaria is a major, life-threatening complication of Plasmodium falciparum malaria, and has very high mortality rate. In murine malaria models, natural killer (NK) cell responses have been shown to play a crucial role in the pathogenesis of cerebral malaria. To investigate the role of NK cells in the developmental process of human cerebral malaria, we conducted a case-control study examining genotypes for killer immunoglobulin-like receptors (KIR) and their human leukocyte antigen (HLA) class I ligands in 477 malaria patients. We found that the combination of KIR2DL3 and its cognate HLA-C1 ligand was significantly associated with the development of cerebral malaria when compared with non-cerebral malaria (odds ratio 3.14, 95% confidence interval 1.52–6.48, P = 0.00079, corrected P = 0.02). In contrast, no other KIR-HLA pairs showed a significant association with cerebral malaria, suggesting that the NK cell repertoire shaped by the KIR2DL3-HLA-C1 interaction shows certain functional responses that facilitate development of cerebral malaria. Furthermore, the frequency of the KIR2DL3-HLA-C1 combination was found to be significantly lower in malaria high-endemic populations. These results suggest that natural selection has reduced the frequency of the KIR2DL3-HLA-C1 combination in malaria high-endemic populations because of the propensity of interaction between KIR2DL3 and C1 to favor development of cerebral malaria. Our findings provide one possible explanation for KIR-HLA co-evolution driven by a microbial pathogen, and its effect on the global distribution of malaria, KIR and HLA.     

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 polymorphisms that affect NK cell inhibition by HLA-Bw4 ligand

The killer cell Ig-like receptor (KIR) gene family encodes MHC class I receptors expressed by NK cells and several T cell subpopulations. Factors contributing to human KIR haplotype diversity are differences in gene number, gene content, and allelic polymorphism. Whereas functional and clinical consequences of the first two factors are established, knowledge of the effects of KIR gene polymorphism is limited to special cases in which signaling function is reversed or cell surface expression lost. In this study we use retrovirally transduced human cell lines to show that 3DL1*002 is a stronger inhibitory receptor for HLA-Bw4 ligands than 3DL1*007. Analysis of mutant 3DL1*002 and 3DL1*007 molecules demonstrates that residue 238 in the D2 domain and 320 in the transmembrane region contribute to the difference in receptor strength. Neither position 238 nor 320 is predicted to interact directly with HLA-Bw4 ligand. This study also revealed that KIR3DL1 and LILRB1 both contribute to developing an inhibitory response to HLA-Bw4 ligands.     

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.     

Functional polymorphism of the KIR3DL1/S1 receptor on human NK cells

NK cells express both inhibitory and activatory receptors that allow them to recognize target cells through HLA class I Ag expression. KIR3DL1 is a receptor that recognizes the HLA-Bw4 public epitope of HLA-B alleles. We demonstrate that polymorphism within the KIR3DL1 receptor has functional consequences in terms of NK cell recognition of target. Inhibitory alleles of KIR3DL1 differ in their ability to recognize HLA-Bw4 ligand, and a consistent hierarchy of ligand reactivity can be defined. KIR3DS1, which segregates as an allele of KIR3DL1, has a short cytoplasmic tail characteristic of activatory receptors. Because it is very similar to KIR3DL1 in the extracellular domains, it has been assumed that KIR3DS1 will recognize a HLA-Bw4 ligand. In this study, we demonstrate that KIR3DS1 is expressed as a protein at the cell surface of NK cells, where it is recognized by the Z27 Ab. Using this Ab, we found that KIR3DS1 is expressed on a higher percentage of NK cells in KIR3DS1 homozygous compared with heterozygous donors. In contrast to the inhibitory KIR3DL1 allotypes, KIR3DS1 did not recognize HLA-Bw4 on EBV-transformed cell lines.     

Receptor-ligand requirements for increased NK cell polyfunctional potential in slow progressors infected with HIV-1 coexpressing KIR3DL1*h/*y and HLA-B*57

Carriage of the natural killer (NK) receptor genotype KIR3DL1*h/*y with its HLA-B*57 ligand (*h/*y+B*57) is associated with slow time to AIDS and low viral load (VL). To provide a functional basis for these epidemiological observations, we assessed whether HIV-1-infected slow progressors (SP) carrying the *h/*y+B*57 compound genotype would have increased NK cell polyfunctional potential in comparison to SP with other killer immunoglobulin-like receptor (KIR)/HLA compound genotypes and whether this enhanced polyfunctionality was dependent upon the coexpression of both KIR3DL1*h/*y and HLA-B*57. The functional potential of NK cells was investigated by stimulating peripheral blood mononuclear cells with HLA-devoid targets or single HLA transfectants. Multiparametric flow cytometry was used to detect NK cells with seven functional profiles representing all permutations of CD107a expression and gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) secretion. NK cells from individuals carrying KIR3DL1 receptor-HLA-Bw4 ligand pairs had greater trifunctional responses than those from KIR3DL1 homozygotes (hmz), who were Bw6 homozygotes. NK cells from subjects carrying the *h/*y+B*57 genotypes exhibited the highest trifunctional potential, and this was dependent on cocarriage of the NK receptor and its ligand. Trifunctional cells secreted more of each function tested on a per-cell basis than each corresponding monofunctional NK subset. Although VL influenced NK functionality, individuals with defined KIR/HLA genotypes exhibited differences in NK cell polyfunctionality that could not be accounted for by VL alone. The protective effect of HLA-B*57 on slow progression to AIDS and low VL may be mediated through its interaction with KIR3DL1 alleles to educate NK cells for potent activity upon stimulation.     

Common HIV-1 peptide variants mediate differential binding of KIR3DL1 to HLA-Bw4 molecules

Epidemiological studies have shown the protective effect of KIR3DL1/HLA-Bw4 genotypes in human immunodeficiency virus type 1 (HIV-1) infection; however, the functional correlates for the protective effect remain unknown. We investigated whether human leukocyte antigen (HLA)-Bw4-presented HIV-1 peptides could affect the interaction between the inhibitory natural killer (NK) cell receptor KIR3DL1 and its ligand HLA-Bw4. Distinct HIV-1 epitopes differentially modulated the binding of KIR3DL1 to HLA-Bw4. Furthermore, cytotoxic T lymphocyte (CTL) escape mutations within the immunodominant HLA-B57 (Bw4)-restricted Gag epitope TSTLQEQIGW abrogated KIR3DL1 binding to HLA-B57, suggesting that sensing of CTL escape variants by NK cells can contribute to the protective effect of the KIR3DL1/HLA-Bw4 compound genotype.     

Contribution of KIR3DL1/3DS1 to ankylosing spondylitis in human leukocyte antigen-B27 Caucasian populations

Killer cell immunoglobulin-like receptors (KIRs) and human leukocyte antigen (HLA) loci are both highly polymorphic, and some HLA class I molecules bind and trigger cell-surface receptors specified by KIR genes. We examined whether the combination of KIR3DS1/3DL1 genes in concert with HLA-B27 genotypes is associated with susceptibility to ankylosing spondylitis (AS). Two HLA-B27-positive Caucasian populations were selected, one from Spain (71 patients and 105 controls) and another from the Azores (Portugal) (55 patients and 75 controls). All were typed for HLA-B and KIR (3DS1 and 3DL1) genes. Our results show that in addition to B27, the allele 3DS1 is associated with AS compared with B27 controls (p < 0.0001 and p < 0.003 in the Spanish population and Azoreans, respectively). We also observed that the association of KIR3DS1 to AS was found in combination with HLA-B alleles carrying Bw4-I80 in trans position in the Spanish population (30.9% in AS versus 15.2% in B27 controls, p = 0.02, odds ratio (OR) = 2.49) and in Azoreans (27.2% in AS versus 8.7% in B27 controls, p = 0.01, OR = 4.4 in Azoreans). On the other hand, 3DL1 was decreased in patients compared with B27 controls (p < 0.0001 in the Spanish population and p < 0.003 in Azoreans). The presence of this allele in combination with Bw4-I80 had a protective effect against the development of AS in the Spanish population (19.7% in AS, 35.2% in B27 controls; p = 0.03, OR = 0.45). The presence of KIR3DS1 or KIR3DL1 in combination with HLA-B*27s/HLA-B Bw4-I80 genotypes may modulate the development of AS. The susceptibility to AS could be determined by the overall balance of activating and inhibitory composite KIR-HLA genotypes.     

Mapping the locus for hereditary hemochromatosis: localization between HLA-B and HLA-A.

We studied a family with HLA-linked hereditary hemochromatosis in which an informative recombination occurred within the HLA region. The father, an obligate heterozygote for hereditary hemochromatosis, had HLA haplotypes A2,B13 and A11,B27. The mother, also an obligate heterozygote, had HLA haplotypes A29,B44 and A2,B7. Three haplotypes were found among three homozygous affected offspring. Two affected siblings were HLA-identical with haplotypes A2,B13 and A29,B44. The proband had HLA haplotypes A2,B13 and A2,B44, the latter a recombinant haplotype inherited from her mother. Since the maternal hemochromatosis allele was linked to the A29,B44 haplotype, and since the proband has hemochromatosis, the maternal hemochromatosis allele was transmitted to the proband with the B44 antigen. This is the first known example of recombination in an individual with HLA-linked hemochromatosis in whom the hemochromatosis allele appeared to segregate with the HLA-B antigen instead of the -A antigen. The possibility of either a double reciprocal recombination event or a gene conversion event cannot be excluded. Combined with earlier observations of segregation of the hemochromatosis allele with the A locus in HLA recombinants, the findings in this pedigree map the hemochromatosis locus between the HLA-B and HLA-A loci rather than outside the HLA region.     

Allele-specific interactions between the yeast RFC1 and RFC5 genes suggest a basis for RFC subunit-subunit interactions

Replication factor C (RFC) is an essential, multi-subunit ATPase that functions in DNA replication, DNA repair, and DNA metabolism-related checkpoints. In order to investigate how the individual RFC subunits contribute to these functions in vivo, we undertook a genetic analysis of RFC genes from budding yeast. We isolated and characterized mutations in the RFC5 gene that could suppress the cold-sensitive phenotype of rfc1-1 mutants. Analysis of the RFC5 suppressors revealed that they could not suppress the elongated telomere phenotype, the sensitivity to DNA damaging agents, or the mutator phenotype of rfc1-1 mutants. Unlike the checkpoint-defective rfc5-1 mutation, the RFC5 suppressor mutations did not interfere with the methylmethane sulfonate- or hydroxyurea-induced phosphorylation of Rad53p. The Rfc5p suppressor substitutions mapped to amino acid positions in the conserved RFC box motifs IV-VII. Comparisons of the structures of related RFC box-containing proteins suggest that these RFC motifs may function to coordinate interactions between neighboring subunits of multi-subunit ATPases.