HIV Protective KIR3DL1/S1-HLA-B Genotypes Influence NK Cell-Mediated Inhibition of HIV Replication in Autologous CD4 Targets

Carriage of the genetic combination encoding a high expression inhibitory Killer Immunoglobulin-like Receptor (KIR)3DL1 with its ligand, HLA-B*57 (*h/*y+B*57) is associated with slower time to AIDS and better HIV viral load control than being a Bw6 homozygote (Bw6hmz). Natural Killer (NK) cells from *h/*y+B*57 carriers receive potent educational signals through HLA-B*57 KIR3DL1 ligation leading to high functional potential. NK cells from Bw6hmz are not educated through KIR3DL1 because Bw6 antigens do not interact with this inhibitory receptor. To better understand the impact of KIR/HLA combinations on NK cell mediated anti-viral activity we measured NK cell mediated inhibition of HIV replication in autologous infected CD4 (iCD4) cells by assessing the frequency of p24 positive CD4 targets and supernatant levels of HIV p24 longitudinally in the presence versus absence of NK cells. Forty-seven HIV uninfected subjects were studied, including carriers of *h/*y+B*57, a low expression KIR3DL1 genotype with HLA-B*57 termed *l/*x+B*57, a genotype designated 3DS1+*80I and Bw6hmz. NK cells from *h/*y+B*57 carriers, like those from 3DS1+*80I subjects, inhibited HIV replication in autologous iCD4 cells better than those from Bw6hmz and *l/*x+B*57 carriers. Cell contact between NK and iCD4 cells activated NK cells to inhibit viral replication in a non-contact dependent fashion through secretion of CC-chemokines. iCD4 stimulated NK cells from *h/*y+B*57 and 3DS1+*80I carriers produced higher levels of CC-chemokines than those from Bw6hmz or *l/*x+B*57 carriers. Higher levels of CC-chemokines were produced by KIR3DL1⁺ than KIR3DL1⁻ NK cells. We conclude that NK-mediated inhibition of viral replication in autologous iCD4 cells is partially due to a block at the level of HIV entry into new targets by secreted CC-chemokines.     

Human immunodeficiency virus type 1 infection is associated with increased NK cell polyfunctionality and higher levels of KIR3DL1+ NK cells in ugandans carrying the HLA-B Bw4 motif

Natural killer (NK) cells are important innate effector cells controlled by an array of activating and inhibitory receptors. Some alleles of the inhibitory killer-cell immunoglobulin-like receptor KIR3DL1 in combination with its HLA class I ligand Bw4 have been genetically associated with slower HIV-1 disease progression. Here, we observed that the presence of HLA-B Bw4 was associated with elevated frequencies of KIR3DL1(+) CD56(dim) NK cells in chronically HIV-1-infected individuals from the rural district of Kayunga, Uganda. In contrast, levels of KIR2DL1(+) CD56(dim) NK cells were decreased, and levels of KIR2DL3(+) CD56(dim) NK cells were unchanged in infected subjects carrying their respective HLA-C ligands. Furthermore, the size of the KIR3DL1(+) NK cell subset correlated directly with viral load, and this effect occurred only in HLA-B Bw4(+) patients, suggesting that these cells expand in response to viral replication but may have relatively poor antiviral capacity. In contrast, no association with viral load was present for KIR2DL1(+) and KIR2DL3(+) NK cells. Interestingly, chronic HIV-1 infection was associated with an increased polyfunctional response in the NK cell compartment, and, upon further investigation, KIR3DL1(+) CD56(dim) NK cells exhibited a significantly increased functional response in the patients carrying HLA-B Bw4. These results indicate that chronic HIV-1 infection is associated with increased NK cell polyfunctionality and elevated levels of KIR3DL1(+) NK cells in Ugandans carrying the HLA-B Bw4 motif.     

Influence of Glycosylation Inhibition on the Binding of KIR3DL1 to HLA-B*57:01

Viral infections can affect the glycosylation pattern of glycoproteins involved in antiviral immunity. Given the importance of protein glycosylation for immune function, we investigated the effect that modulation of the highly conserved HLA class I N-glycan has on KIR:HLA interactions and NK cell function. We focused on HLA-B*57:01 and its interaction with KIR3DL1, which has been shown to play a critical role in determining the progression of a number of human diseases, including human immunodeficiency virus-1 infection. 721.221 cells stably expressing HLA-B*57:01 were treated with a panel of glycosylation enzyme inhibitors, and HLA class I expression and KIR3DL1 binding was quantified. In addition, the functional outcomes of HLA-B*57:01 N-glycan disruption/modulation on KIR3DL1ζ⁺ Jurkat reporter cells and primary human KIR3DL1⁺ NK cells was assessed. Different glycosylation enzyme inhibitors had varying effects on HLA-B*57:01 expression and KIR3DL1-Fc binding. The most remarkable effect was that of tunicamycin, an inhibitor of the first step of N-glycosylation, which resulted in significantly reduced KIR3DL1-Fc binding despite sustained expression of HLA-B*57:01 on 721.221 cells. This effect was paralleled by decreased activation of KIR3DL1ζ⁺ Jurkat reporter cells, as well as increased degranulation of primary human KIR3DL1⁺ NK cell clones when encountering HLA-B*57:01-expressing 721.221 cells that were pre-treated with tunicamycin. Overall, these results demonstrate that N-glycosylation of HLA class I is important for KIR:HLA binding and has an impact on NK cell function.     

An early HIV mutation within an HLA-B*57-restricted T cell epitope abrogates binding to the killer inhibitory receptor 3DL1

Mutations within MHC class I-restricted epitopes have been studied in relation to T cell-mediated immune escape, but their impact on NK cells via interaction with killer Ig-like receptors (KIRs) during early HIV infection is poorly understood. In two patients acutely infected with HIV-1, we observed the appearance of a mutation within the B*57-restricted TW10 epitope (G9E) that did not facilitate strong escape from T cell recognition. The NK cell receptor KIR3DL1, carried by these patients, is known to recognize HLA-B*5703 and is associated with good control of HIV-1. Therefore, we tested whether the G9E mutation influenced the binding of HLA-B*5703 to soluble KIR3DL1 protein by surface plasmon resonance, and while the wild-type sequence and a second (T3N) variant were recognized, the G9E variant abrogated KIR3DL1 binding. We extended the study to determine the peptide sensitivity of KIR3DL1 interaction with epitopes carrying mutations near the C termini of TW10 and a second HLA-B*57-restricted epitope, IW9. Several amino acid changes interfered with KIR3DL1 binding, the most extreme of which included the G9E mutation commonly selected by HLA-B*57. Our results imply that during HIV-1 infection, some early-emerging variants could affect KIR-HLA interaction, with possible implications for immune recognition.     

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.     

Analysis of binding of KIR3DS1*014 to HLA suggests distinct evolutionary history of KIR3DS1

NK cell activity is regulated by the integration of positive and negative signals. One important source of these signals for human NK cells is the killer Ig-like receptor (KIR) family, which includes both members that transduce positive and those that generate negative signals. KIR3DL1 inhibits NK cell activity upon engagement by its ligand HLA-Bw4. The highly homologous KIR3DS1 is an activating receptor, which is implicated in the outcome of a variety of pathological situations. However, unlike KIR3DL1, direct binding of KIR3DS1(+) cells to HLA has not been demonstrated. We analyzed four key amino acid differences between KIR3DL1*01502 and KIR3DS1*013 to determine their role in KIR binding to HLA. Single substitutions of these residues dramatically reduced binding by KIR3DL1. In the reciprocal experiment, we found that the rare KIR3DS1 allotype KIR3DS1*014 binds HLA-Bw4 even though it differs from KIR3DS1*013 at only one of these positions (position 138). This reactivity was unexpectedly dependent on residues at other variable positions, as HLA-Bw4 binding was lost in receptors with KIR3DL1-like residues at both positions 199 and 138. These data provide the first evidence, to our knowledge, for the direct binding of KIR3DS1(+) cells to HLA-Bw4 and highlight the key role for position 138 in determining ligand specificity of KIR3DS1. They also reveal that KIR3DS1 reactivity and specificity is dictated by complex interactions between the residues in this region, suggesting a unique functional evolution of KIR3DS1 within the activating KIR family.     

Cutting Edge: Allele-specific and peptide-dependent interactions between KIR3DL1 and HLA-A and HLA-B

Although it is clear that KIR3DL1 recognizes Bw4(+) HLA-B, the role of Bw4(+) HLA-A allotypes as KIR3DL1 ligands is controversial. We therefore examined the binding of tetrameric HLA-A and -B complexes, including HLA*2402, a common Bw4(+) HLA-A allotype, to KIR3DL1*001, *005, *007, and *1502 allotypes. Only Bw4(+) tetramers bound KIR3DL1. Three of four HLA-A*2402 tetramers bound one or more KIR3DL1 allotypes and all four KIR3DL1 allotypes bound to one or more HLA-A*2402 tetramers, but with different binding specificities. Only KIR3DL1*005 bound both HLA-A*2402 and HLA-B*5703 tetramers. HLA-A*2402-expressing target cells were resistant to lysis by NK cells expressing KIR3DL1*001 or *005. This study shows that HLA-A*2402 is a ligand for KIR3DL1 and demonstrates how the binding of KIR3DL1 to Bw4(+) ligands depends upon the bound peptide as well as HLA and KIR3DL1 polymorphism.     

Time to Seroconversion in HIV-Exposed Subjects Carrying Protective versus Non Protective KIR3DS1/L1 and HLA-B Genotypes

Natural killer (NK) cells play a role in the clearance of viral infections. Combinations of alleles at the polymorphic HLA-B locus and the NK cell surface killer immunoglobulin-like receptor locus KIR3DL1/S1 have been shown to influence time to AIDS in HIV-infected individuals and risk of seroconversion in HIV exposed seronegative (HESN) subjects. Here, we assessed time to seroconversion or duration of seronegative status in a group of 168 HIV exposed individuals, including 74 seroconverters and 94 HESN based on carriage or not of KIR3DL1/S1/HLA-B genotypes previously shown to be associated with protection from infection and/or slow time to AIDS. KIR3DL1/S1 genotyping was performed by sequence-specific primer polymerase chain reaction using two pairs of specific primers for each locus. The MHC class IB locus was typed to four-position resolution to resolve Bw4 and Bw6 alleles and the amino acid present at position 80. KIR3DL1/S1 heterozygotes became HIV infected significantly faster than KIR3DS1 homozygotes. Individuals who carried both KIR3DS1 and Bw4*80I did not remain HIV seronegative longer than those from a control group who were homozygous for HLA-Bw6 and carried no HLA-A locus Bw4 alleles Subjects who were *h/*y+B*57 showed a trend towards slower time to serconversion than those with other KIR3DL1 homozygous and KIR3DL1/S1 heterozygous genotypes. Thus, KIR3DS1 homozygosity is associated with protection from HIV infection while co-carriage of KIR3DS1 and Bw4*80I is not. The requirements for protection from HIV infection can differ from those that influence time to AIDS in HIV infected individuals.     

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.     

Interactions of NK cell receptor KIR3DL1*004 with chaperones and conformation-specific antibody reveal a functional folded state as well as predominant intracellular retention

Variable interaction between the Bw4 epitope of HLA-B and the polymorphic KIR3DL1/S1 system of inhibitory and activating NK cell receptors diversifies the development, repertoire formation, and response of human NK cells. KIR3DL1*004, a common KIR3DL1 allotype, in combination with Bw4(+) HLA-B, slows progression of HIV infection to AIDS. Analysis in this study of KIR3DL1*004 membrane traffic in NK cells shows this allotype is largely misfolded but stably retained in the endoplasmic reticulum, where it binds to the chaperone calreticulin and does not induce the unfolded protein response. A small fraction of KIR3DL1*004 folds correctly and leaves the endoplasmic reticulum to be expressed on the surface of primary NK and transfected NKL cells, in a form that can be triggered to inhibit NK cell activation and secretion of IFN-γ. Consistent with this small proportion of correctly folded molecules, trace amounts of MHC class I coimmunoprecipitated with KIR3DL1*004. There was no indication of any extensive intracellular interaction between unfolded KIR3DL1*004 and cognate Bw4(+) HLA-B. A similarly limited interaction of Bw4 with KIR3DL1*002, when both were expressed by the same cell, was observed despite the efficient folding of KIR3DL1*002 and its abundance on the NK cell surface. Several positions of polymorphism modulate KIR3DL1 abundance at the cell surface, differences that do not necessarily correlate with the potency of allotype function. In this context, our results suggest the possibility that the effect of Bw4(+) HLA-B and KIR3DL1*004 in slowing progression to AIDS is mediated by interaction of Bw4(+) HLA-B with the small fraction of cell surface KIR3DL1*004.     

Quantity of HLA-C surface expression and licensing of KIR2DL+ natural killer cells

Natural killer (NK) cells require interaction of inhibitory surface receptors with human leukocyte antigen (HLA) ligands during development to acquire functional competence in a process termed "licensing." The quantity of HLA required for this process is unknown. Two polymorphisms affecting HLA-C surface expression (rs9264942 and rs67384697) have recently been identified, and shown to influence progression of HIV infection. We typed a cohort of healthy donors for the two HLA-C-related polymorphisms, KIR2DL1 and KIR2DL3, and their respective HLA-C ligands and analyzed how HLA ligands influenced licensing status of killer cell immunoglobulin-like receptor (KIR)+ NK cells in terms of degranulation and cytokine production in response to HLA-deficient target cells. The presence of respective HLA class I ligands increased the function of KIR2DL1+ and KIR2DL3+ NK cells in a dose-dependent manner. In contrast, neither of the HLA-C-related polymorphisms nor the quantity of cell surface HLA-C had any significant effect on NK cell function. Interestingly, HLA-Cw7-an HLA-C allele with low surface expression-licensed KIR2DL3+ NK cells more strongly than any other KIR2DL3 ligand. The quantity of cell surface HLA-C does not appear to influence licensing of NK cells, and the HLA-C-related polymorphisms presumably influence HIV progression through factors unrelated to NK cell education.     

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.     

Cutting edge: susceptibility to psoriatic arthritis: influence of activating killer Ig-like receptor genes in the absence of specific HLA-C alleles

NK cell activity is partially controlled through interactions between killer Ig-like receptors (KIR) on NK cells and their respective HLA class I ligands. 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. Inhibitory receptor genes KIR2DL2/3 and KIR2DL1 were present in nearly all subjects sampled in this study, whereas their respective activating homologs, KIR2DS2 and KIR2DS1, are each present in about half of the subjects. In this work we report that subjects with activating KIR2DS1 and/or KIR2DS2 genes are susceptible to developing psoriatic arthritis, but only when HLA ligands for their homologous inhibitory receptors, KIR2DL1 and KIR2DL2/3, are missing. Absence of ligands for inhibitory KIRs could potentially lower the threshold for NK (and/or T) cell activation mediated through activating receptors, thereby contributing to pathogenesis of psoriatic arthritis.     

Synergistic polymorphism at two positions distal to the ligand-binding site makes KIR2DL2 a stronger receptor for HLA-C than KIR2DL3

Interactions between HLA-C ligands and inhibitory killer cell Ig-like receptors (KIR) control the development and response of human NK cells. This regulatory mechanism is usually described by mutually exclusive interactions of KIR2DL1 with C2 having lysine 80, and KIR2DL2/3 with C1 having asparagine 80. Consistent with this simple rule, we found from functional analysis and binding assays to 93 HLA-A, HLA-B, and HLA-C isoforms that KIR2DL1*003 bound all C2, and only C2, allotypes. The allotypically related KIR2DL2*001 and KIR2DL3*001 interacted with all C1, but they violated the simple rule through interactions with several C2 allotypes, notably Cw*0501 and Cw*0202, and two HLA-B allotypes (B*4601 and B*7301) that share polymorphisms with HLA-C. Although the specificities of the "cross-reactions" were similar for KIR2DL2*001 and KIR2DL3*001, they were stronger for KIR2DL2*001, as were the reactions with C1. Mutagenesis explored the avidity difference between KIR2DL2*001 and KIR2DL3*001. Recombinant mutants mapped the difference to the Ig-like domains, where site-directed mutagenesis showed that the combination, but not the individual substitutions, of arginine for proline 16 in D1 and cysteine for arginine 148 in D2 made KIR2DL2*001 a stronger receptor than KIR2DL3*001. Neither residue 16 or 148 is part of, or near to, the ligand-binding site. Instead, their juxtaposition near the flexible hinge between D1 and D2 suggests that their polymorphisms affect the ligand-binding site by changing the hinge angle and the relative orientation of the two domains. This study demonstrates how allelic polymorphism at sites distal to the ligand-binding site of KIR2DL2/3 has diversified this receptor's interactions with HLA-C.     

Cutting edge: resistance to HIV-1 infection among African female sex workers is associated with inhibitory KIR in the absence of their HLA ligands

NK cells are regulated in part by killer Ig-like receptors (KIR) that interact with HLA molecules on potential target cells. KIR and HLA loci are highly polymorphic and certain KIR/HLA combinations were found to protect against HIV disease progression. We show in this study that KIR/HLA interactions also influence resistance to HIV transmission. HIV-exposed but seronegative female sex workers in Abidjan, C?te d'Ivoire, frequently possessed inhibitory KIR genes in the absence of their cognate HLA genes: KIR2DL2/KIR2DL3 heterozygosity in the absence of HLA-C1 and KIR3DL1 homozygosity in the absence of HLA-Bw4. HIV-seropositive female sex workers were characterized by corresponding inhibitory KIR/HLA pairings: KIR2DL3 homozygosity together with HLA-C1 and a trend toward KIR3DL1/HLA-Bw4 homozygosity. Absence of ligands for inhibitory KIR could lower the threshold for NK cell activation. In addition, exposed seronegatives more frequently possessed AB KIR genotypes, which contain more activating KIR. The data support an important role for NK cells and KIR/HLA interactions in antiviral immunity.     

The first Ig domain of KIR3DL1 contacts MHC class I at a secondary site

KIR3DL1 is a highly polymorphic inhibitory killer cell Ig-like receptor (KIR) implicated in resistance to viral diseases such as AIDS. KIR3DL1 contains three Ig domains and is specific for MHC class I (MHC-I) molecules belonging to the HLA-Bw4 serogroup. The receptor's second and third Ig domains confer the Bw4 specificity, but the role of the first Ig domain (D0) in ligand recognition has remained enigmatic. We found that KIR3DL1 expressed in YTS cells and as a soluble receptor can weakly recognize additional MHC-I molecules including HLA-B*0702 and HLA-G. This interaction is highly sensitive to blocking with Abs to the MHC-I α3-domain and the anti-KIR3DL1 Ab Z27, but not the canonical blocking Ab DX9. Using chimeric receptors between KIR3DL1 and KIR2DL1 expressed on YTS cells and as soluble Fc-fusion proteins, we show that the D0 domain confers the broad functional recognition and binding as well as the reactivity with Z27. These results suggest that the presence of a second and independent site of interaction between D0 and MHC-I and that MHC-I could bridge KIR3DL1 molecules together in a manner that facilitates signaling.     

Selection of an HLA-C*03:04-Restricted HIV-1 p24 Gag Sequence Variant Is Associated with Viral Escape from KIR2DL3+ Natural Killer Cells: Data from an Observational Cohort in South Africa

Background

Viruses can evade immune surveillance, but the underlying mechanisms are insufficiently understood. Here, we sought to understand the mechanisms by which natural killer (NK) cells recognize HIV-1-infected cells and how this virus can evade NK-cell-mediated immune pressure.

Methods and Findings

Two sequence mutations in p24 Gag associated with the presence of specific KIR/HLA combined genotypes were identified in HIV-1 clade C viruses from a large cohort of infected, untreated individuals in South Africa (n = 392), suggesting viral escape from KIR+ NK cells through sequence variations within HLA class I—presented epitopes. One sequence polymorphism at position 303 of p24 Gag (T_Gag303V), selected for in infected individuals with both KIR2DL3 and HLA-C*03:04, enabled significantly better binding of the inhibitory KIR2DL3 receptor to HLA-C*03:04-expressing cells presenting this variant epitope compared to the wild-type epitope (wild-type mean 18.01 ± 10.45 standard deviation [SD] and variant mean 44.67 ± 14.42 SD, p = 0.002). Furthermore, activation of primary KIR2DL3+ NK cells from healthy donors in response to HLA-C*03:04+ target cells presenting the variant epitope was significantly reduced in comparison to cells presenting the wild-type sequence (wild-type mean 0.78 ± 0.07 standard error of the mean [SEM] and variant mean 0.63 ± 0.07 SEM, p = 0.012). Structural modeling and surface plasmon resonance of KIR/peptide/HLA interactions in the context of the different viral sequence variants studied supported these results. Future studies will be needed to assess processing and antigen presentation of the investigated HIV-1 epitope in natural infection, and the consequences for viral control.

Conclusions

These data provide novel insights into how viruses can evade NK cell immunity through the selection of mutations in HLA-presented epitopes that enhance binding to inhibitory NK cell receptors. Better understanding of the mechanisms by which HIV-1 evades NK-cell-mediated immune pressure and the functional validation of a structural modeling approach will facilitate the development of novel targeted immune interventions to harness the antiviral activities of NK cells.     

Exploring the Role of Killer Cell Immunoglobulin-Like Receptors and Their HLA Class I Ligands in Autoimmune Hepatitis

Background

Natural killer cells are involved in the complex mechanisms underlying autoimmune diseases but few studies have investigated their role in autoimmune hepatitis. Killer immunoglobulin-like receptors are key regulators of natural killer cell-mediated immune responses.

Methods and Findings

KIR gene frequencies, KIR haplotypes, KIR ligands and combinations of KIRs and their HLA Class I ligands were investigated in 114 patients diagnosed with type 1 autoimmune hepatitis and compared with a group of 221 healthy controls. HLA Class I and Class II antigen frequencies were compared to those of 551 healthy unrelated families representative of the Sardinian population. In our cohort, type 1 autoimmune hepatitis was strongly associated with the HLA-B18, Cw5, DR3 haplotype. The KIR2DS1 activating KIR gene and the high affinity HLA-C2 ligands were significantly higher in patients compared to controls. Patients also had a reduced frequency of HLA-Bw4 ligands for KIR3DL1 and HLA-C1 ligands for KIR2DL3. Age at onset was significantly associated with the KIR2DS1 activating gene but not with HLA-C1 or HLA-C2 ligand groups.

Conclusions

The activating KIR gene KIR2DS1 resulted to have an important predictive potential for early onset of type 1 autoimmune hepatitis. Additionally, the low frequency of the KIR-ligand combinations KIR3DL1/HLA-Bw4 and KIR2DL3/HLA-C1 coupled to the high frequency of the HLA-C2 high affinity ligands for KIR2DS1 could contribute to unwanted NK cell autoreactivity in AIH-1.     

The protein made from a common allele of KIR3DL1 (3DL1*004) is poorly expressed at cell surfaces due to substitution at positions 86 in Ig domain 0 and 182 in Ig domain 1

KIR3DL1 is an inhibitory HLA-B receptor of human NK and T cells that exhibits genetic and phenotypic polymorphism. KIR3DL1*004, a common allotype, cannot be detected on the surface of PBLs using the KIR3DL1-specific Ab DX9. The nature of this phenotype was investigated through comparison of 3DL1*004 with 3DL1*002, an allele giving high DX9 binding to cell surfaces. Analysis of Jurkat T cell transfectants with 3DL1*004 cDNA showed that 3DL1*004 is poorly expressed at the cell surface, but detectable intracellularly. Analysis of recombinant mutants made between 3DL1*004 and 3DL1*002 showed that polymorphism in Ig domains 0 and 1 (D0 and D1) causes the intracellular retention of 3DL1*004. Reciprocal point mutations were introduced into 3DL1*004 and 3DL1*002 at positions 44 and 86 of the D0 domain, where 3DL1*004 has unique residues, and at position 182 of the D1 domain, where 3DL1*004 resembles 3DL1*005, an allotype giving low DX9-binding phenotype. Leucine 86 in 3DL1*004 is the principal cause of its intracellular retention, with a secondary and additive contribution from serine 182. By contrast, glycine 44, which is naturally present in 3DL1*004, slightly increased cell surface expression when introduced into 3DL1*002. In 3DL1*004, the presence of leucine at position 86 corrupts the WSXPS motif implicated in proper folding of the KIR D0 Ig-like domain. This study demonstrates how a difference between KIR3DL1 allotypes in the D0 domain profoundly affects cell surface expression and function.