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.     

Allele imputation for the killer cell immunoglobulin-like receptor KIR3DL1/S1

Highly polymorphic interaction of KIR3DL1 and KIR3DS1 with HLA class I ligands modulates the effector functions of natural killer (NK) cells and some T cells. This genetically determined diversity affects severity of infections, immune-mediated diseases, and some cancers, and impacts the course of immunotherapies, including transplantation. KIR3DL1 is an inhibitory receptor, and KIR3DS1 is an activating receptor encoded by the KIR3DL1/S1 gene that has more than 200 diverse and divergent alleles. Determination of KIR3DL1/S1 genotypes for medical application is hampered by complex sequence and structural variation, requiring targeted approaches to generate and analyze high-resolution allele data. To overcome these obstacles, we developed and optimized a model for imputing KIR3DL1/S1 alleles at high-resolution from whole-genome SNP data. We designed the model to represent a substantial component of human genetic diversity. Our Global imputation model is effective at genotyping KIR3DL1/S1 alleles with an accuracy ranging from 88% in Africans to 97% in East Asians, with mean specificity of 99% and sensitivity of 95% for alleles >1% frequency. We used the established algorithm of the HIBAG program, in a modification named Pulling Out Natural killer cell Genomics (PONG). Because HIBAG was designed to impute HLA alleles also from whole-genome SNP data, PONG allows combinatorial diversity of KIR3DL1/S1 with HLA-A and -B to be analyzed using complementary techniques on a single data source. The use of PONG thus negates the need for targeted sequencing data in very large-scale association studies where such methods might not be tractable.     

Association of supervillin with KIR2DL1 regulates the inhibitory signaling of natural killer cells

Inhibitory signaling is crucial in the regulation of the cytotoxicity of natural killer (NK) cells. Here, we show that KIR2DL1, an inhibitory receptor of NK cells, associates with supervillin, an F-actin binding protein. Interaction of supervillin with KIR2DL1 is dependent on the KIR2DL1 receptor stimulation and requires the phosphorylation of tyrosines in both ITIM motifs. “Knockdown” of expression of supervillin by RNA interference (RNAi) restores the KIR2DL1-suppressed cytotoxicity of NK cells. Inhibition of supervillin by RNAi also enhances the polarization of cytolytic granules (both granzyme B and perforin) to the synapse formed between YTS-GFP-KIR2DL1 NK cells and 721.221-HLA-Cw4 target cells. Further study reveals that supervillin is required for KIR2DL1-mediated inhibition of Vav1 and ERK phoshorylation. Moreover, we have found that binding of supervillin with KIR2DL1 facilitates the recruitment of SHPs especially SHP-2 to KIR2DL1 receptor. Thus, our findings demonstrate that supervillin is a novel molecule that associates with KIR2DL1 receptor and regulates the inhibitory signaling in NK cells.     

Synergistic proliferation and activation of natural killer cells by interleukin 12 and interleukin 18.

We investigated the effects of IL-12 and IL-18 on unstimulated murine splenocytes and observed that the two cytokines strongly synergized for their proliferation, whereas IL-12 and IL-18 alone were essentially inactive in this respect. Phenotypical and functional analyses of cells proliferating in response to IL-12 and IL-18 revealed that large granular Ly-49C(+)DX5(+)CD3(-)NK blasts were expanded in these cultures and that they displayed cytotoxic activity against Yac-1 cells, a murine NK cell target. Further analyses indicated three major differences between NK cells appearing in response to IL-12 and IL-18 and those derived in the presence of other NK cell growth factors, such as IL-2 or IL-15. First, a population of T-NK cells, i.e. expressing T cell (TCRalphabeta, CD3) and NK cell (Ly-49) markers, was detected amongst cells growing in IL-2 or IL-15 but not in cultures supplemented with IL-12 and IL-18. Second, most NK cells derived with IL-2 or IL-15 expressed the NK1.1 antigen, while those derived with IL-12 and IL-18 did not. Finally, striking differences were observed regarding cytokine production. Cells stimulated with IL-12 and IL-18 in combination, but not with IL-2 or IL-15, produced IFN-gamma, IL-3, IL-6 and TNF. IFN-gamma was not involved in the response of NK cells to IL-12 and IL-18, as indicated by experiments demonstrating that the combination of the two cytokines displayed similar effects on spleen cells from IFN-gammaR-knock-out mice. Receptor (IL-12Rbeta1, IL-12Rbeta2 and IL-18R) gene expression studies did not indicate that the mechanism underlying the synergy between IL-12 and IL-18 involved reciprocal induction of their receptors. Taken together, our results demonstrate that IL-12 and IL-18 exert striking synergistic activities for NK cell proliferation and activation, distinct from those induced by IL-2 or IL-15.     

Characterisation of mouse monoclonal antibodies against rhesus macaque killer immunoglobulin-like receptors KIR3D

Killer immunoglobulin-like receptors (KIRs) represent a highly polymorphic and diverse gene family in rhesus macaques. Analyses of the respective gene products have been hampered until now due to non-availability of specific monoclonal antibodies and failure of cross-reactivity of anti-human KIR antibodies. We utilised one activating (KIR3DSW08) and two inhibitory (KIR3DLW03 and KIR3DL05) rhesus macaque KIR-Fc fusion proteins for generation of monoclonal antibodies in mice. Besides broadly reacting ones, we obtained anti-rhesus macaque KIR antibodies with intermediate and with single specificity. These monoclonal antibodies were tested for binding to a panel of rhesus macaque KIR proteins after heterologous expression on transiently transfected cells. Epitope mapping identified two polymorphic regions that are located next to each other in the mature KIR proteins. The availability of monoclonal antibodies against rhesus macaque KIR proteins will enable future studies on KIR at the protein level in rhesus macaques as important animal models of human infectious diseases.     

Silkworm expression and sugar profiling of human immune cell surface receptor, KIR2DL1

Immune cell surface receptors are directly involved in human diseases, and thus represent major drug targets. However, it is generally difficult to obtain sufficient amounts of these receptors for biochemical and structural studies because they often require posttranslational modifications, especially sugar modification. Recently, we have established a bacmid expression system for the baculovirus BmNPV, which directly infects silkworms, an attractive host for the large-scale production of recombinant sugar-modified proteins. Here we produced the human immune cell surface receptor, killer cell Ig-like receptor 2DL1 (KIR2DL1), by using the BmNPV bacmid expression system, in silkworms. By the direct injection of the bacmid DNA, the recombinant KIR2DL1 protein was efficiently expressed, secreted into body fluids, and purified by Ni²⁺ affinity column chromatography. We further optimized the expression conditions, and the final yield was 0.2 mg/larva. The sugar profiling revealed that the N-linked sugars of the purified protein comprised very few components, two paucimannose-type oligosaccharides, Manα1-6Manβ1-4GlcNAcβ1-4GlcNAc and Manα1-6Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc. This revealed that the protein product was much more homogeneous than the complex-sugar type product obtained by mammalian cell expression. The surface plasmon resonance analysis demonstrated that the purified KIR2DL1 protein exhibited specific binding to the HLA-Cw4 ligand. Moreover, the CD spectrum showed the proper secondary structure. These results clearly suggested that the silkworm expression system is quite useful for the expression of cell surface receptors that require posttranslational modifications, as well as for their structural and binding studies, due to the relatively homogeneous N-linked sugar modifications.     

The effect of interleukin-15 on the expression of killer-cell immunoglobulin-like receptors on peripheral natural killer cells in human

Interleukin (IL)-15 has emerged as a key regulator of both natural killer (NK) cell differentiation and activation. The aim of the present study was to investigate the expansion of the population of cells expressing killer-cell immunoglobulin-like receptors (CD158a and CD158b) in human peripheral lymphocytes by treatment with IL-15. One million peripheral lymphocytes were cultured in RPMI1640 medium alone or in medium containing IL-2 at 100 U/ml or IL-15 at 0.1, 1.0, or 10.0 ng/ml for 48 h. After each incubation, we assessed the natural killing activity and the population of CD16(+)CD158a(+)/b(+) cells and CD8(+)CD158a(+)/b(+) cells. IL-15 increased the NK activity and expanded the populations of CD16(+)CD158a(+)/b(+) cells and CD8(+)CD158a(+)/b(+) cells. These actions were dose dependent, and the effects of IL-15 at 1.0 ng/ml were close to those of IL-2 at 100 U/ml. These findings suggest that IL-15 induces the effector functions of resting NK cells throughout the body, and thereby plays a critical role in the activation of tissue-associated immune responses.     

Self-association of an activating natural killer cell receptor, KIR2DS1

As a major component of the innate immune system, natural killer cells are responsible for activating the cytolytic killing of certain pathogen-infected or tumor cells. The self-recognition of natural killer cells is achieved in part by the killer cell immunoglobulin-like receptors (KIRs) protein family. In the current study, using a suite of biophysical methods, we investigate the self-association of an activating KIR, KIR2DS1. This KIR is of particular interest because when in the presence of the HLA-Cw6 protein, KIR2DS1 becomes a major risk factor for psoriasis, an autoimmune chronic skin disease. Using circular dichroism spectroscopy, dynamic light scattering, and atomic force microscopy, we reveal that KIR2DS1 self-associates in a well-defined fashion. Our novel results on an activating KIR allow us to suggest a working model for the KIR2DS1- HLA class I molecular mechanism.     

Genetic background influences natural killer cell activation during bacterial peritonitis in mice, and is interleukin 12 and interleukin 18 independent

Some mouse strains produce strong pro-inflammatory, T-helper (Th)1 responses (e.g. C57BL/6), or strong anti-inflammatory, Th2 responses (e.g. BALB/c). The exact mechanisms for development of distinct immune responses to infection are not completely understood, although cytokines such as interleukin (IL)-12, IL-18 and IL-4 are known to play roles. Natural killer T (NKT)/natural killer (NK) cells are important regulators of immune responses in infection and non-infection models, and NKT/NK activation is also regulated by IL-12 and IL-18 in many models. We investigated the role of IL-12/IL-18 in NKT/NK activation in murine bacterial peritonitis, as well as differential NKT and NK cell activation in C57BL/6 and BALB/c mice. No differences in NKT or NK cell activation or intracellular interferon (IFN)-gamma were determined between mice given control, anti-IL-12 or anti-IL-18 antibodies or in NKT/NK cell activation in STAT4-/- mice (deficient in IL-12 signaling) or wild type controls. However, there were significant differences in the activation of NKT and NK cells between C57BL/6 mice and BALB/c mice, with NKT/NK cytokine production following Th1 or Th2 lines dependent on strain. This suggests a role for NKT and NK cell activation in the development of Th1 and Th2 responses during bacterial infection independently of IL-12 or IL-18.     

Effect of interleukin 1 on the expression of interleukin 2 receptor (Tac antigen) on human natural killer cells and natural killer-like cell line (YT cells)

The effect of interleukin 1 (IL 1) on the expression of interleukin 2 receptor (IL 2R/Tac antigen) on human natural killer (NK) cells and the NK-like cell line, YT was studied with the use of a fluoresceinated anti-IL 2R monoclonal antibody and a Spectrum III flow cytofluorometer. IL 2R was expressed on approximately 10% of NK cells. The expression of IL 2R on NK cells was increased to approximately 25% by the in vitro culture with monocytes or IL 1 and to a less extent by the culture with IL 2 or interferon-gamma (IFN-gamma). IL 2R was expressed on approximately 50% of YT cells without any stimulations. The expression of IL 2R on YT cells was increased up to almost 100% by the culture with IL 1 or monocytes, but not with IL 2, IFN-alpha, IFN-beta, IFN-gamma, or lectins such as concanavalin A and phytohemagglutinin-P. IL 1 absorbed with YT cells or murine thymocytes lost both IL 1 activity detected by the stimulation of murine thymocyte proliferative response and enhancing activity of IL 2R expression on YT cells, suggesting that IL 1 has both activities. However, the assay system of the expression of IL 2R on YT cells is much more sensitive than the stimulation of murine thymocyte proliferative response. By the kinetic study, the enhancement of IL 2R expression was induced by only a 2-hr incubation of YT cells with IL 1. This enhancement did not proceed at 4 degrees C or by the treatment of YT cells with actinomycin D or cycloheximide, suggesting that this enhancement is energy dependent and requires the synthesis of RNA and protein but not DNA. Thus IL 1 plays an important role for the regulation of the expression of IL 2R on NK cells, and IL 1-dependent IL 2R expression on YT cells may give us a good model for the study of the molecular mechanism of the regulation of IL 2R expression.     

Synergistic effect of IL-2, IL-12, and IL-18 on thymocyte apoptosis and Th1/Th2 cytokine expression

In the periphery, IL-18 synergistically induces the expression of the Th1 cytokine IFN-gamma in the presence of IL-12 and the Th2 cytokines IL-5 and IL-13 in the presence of IL-2. Although the expression of these cytokines has been described in the thymus, their role in thymic development and function remains uncertain. We report here that freshly isolated thymocytes from C57BL/6 and BALB/c mice stimulated in vitro with IL-2-plus-IL-18 or IL-12-plus-IL-18 produce large amounts of IFN-gamma and IL-13. Analysis of the thymic subsets, CD4(-)CD8(-) (DN), CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) revealed that IL-18 in combination with IL-2 or IL-12 induces IFN-gamma and IL-13 preferentially from DN cells. Moreover, DN2 and DN3 thymocytes contained more IFN-gamma(+) cells than cells in the later stage of maturation. Additionally, IL-18 in combination with IL-2 induces CCR4 (Th2-associated) and CCR5 (Th1-associated) gene expression. In contrast, IL-18-plus-IL-12 specifically induced CCR5 expression. The IL-2-plus-IL-18 or IL-12-plus-IL-18 effect on IFN-gamma and IL-13 expression is dependent on Stat4 and NF-kappaB but independent of Stat6, T-bet, or NFAT. Furthermore, IL-12-plus-IL-18 induces significant thymocyte apoptosis when expressed in vivo or in vitro, and this effect is exacerbated in the absence of IFN-gamma. IL-12-plus-IL-18-stimulated thymocytes can also induce IA-IE expression on cortical and medullary thymic epithelial cells in an IFN-gamma-dependent manner. Thus, the combination of IL-2, IL-12, and IL-18 can induce phenotypic and functional changes in thymocytes that may alter migration, differentiation, and cell death of immature T cells inside the thymus and potentially affect the Th1/Th2 bias in peripheral immune compartments.     

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.     

IL-2 induces expression of serine protease enzymes and genes in natural killer and nonspecific T killer cells

The expression of serine protease genes was examined in murine NK cells that were purified by panning spleen cells with PMA. Although unstimulated NK cells were cytolytic, they were found not to express the C11 (chymotrypsin-like) mRNA. Culturing these cells in IL-2 (500 to 800 U/ml) for 5 to 7 days induced both the lytic activities and the protease enzymes by 20- to 30-fold. Concomitant to these activation events, the total steady state mRNA of both C11 and HF (trypsin-like) genes were also elevated. The activation of lysis, serine protease enzymes, and C11 and HF mRNA all peaked around day 5 in culture and was dose dependent. In order to exclude the possibility that PMA synergizes with IL-2 in this system, spleen cells from SCID mice, which contained mainly NK cells, were cultured under the same conditions (800 U/ml IL-2, with or without PMA) and PMA did not appear to enhance the expression of these mRNA. Similarly, IL-2 also induced the lytic activities, enzyme levels, and mRNA in the non-Ag-specific T killer cells isolated from spleens of normal mice. Lytic activity of T killer cells was not as high as the NK cells, however, the addition of PHA into the lytic assay resulted in enhanced lysis comparable to that of NK cells. These results showed that lytic activity increased along with protease enzyme levels and mRNA expression in both NK and resting T cells. Therefore, elevated levels of the protease enzymes could be one mechanism involved in optimal lytic activity of IL-2-induced lymphokine activated killer cells.     

Expression of SCM-1alpha/lymphotactin and SCM-1beta in natural killer cells is upregulated by IL-2 and IL-12.

Recruitment of lymphocytes is an important feature of the host immune response against pathogens. However, the mechanisms by which lymphocytes are attracted are not yet fully understood. Recently, the cDNA of a lymphocyte-specific chemokine, lymphotactin (Lptn), was isolated from murine and human T cells and was also found to be expressed in murine NK cells and human NK cell clones. This study investigated the influence of interleukin (IL)-2 and IL-12 on the expression of Lptn, also known as SCM (single cysteine motif)-1alpha, and SCM-1beta, a 97% homolog of Lptn, in freshly isolated human NK cells and the human NK cell line NK-92. Northern blot analysis and RT-PCR confirmed that nonactivated human NK cells expressed both genes at low level. After activation with IL-2 or IL-12, the expression of both Lptn and SCM-1beta was upregulated within hours. NK-92 cells maintained in medium supplemented with IL-2 constitutively expressed SCM-1 mRNA. However, after 24 h of IL-2 starvation and subsequent culturing at various IL-2 concentrations, the expression of Lptn/SCM-1alpha was upregulated in a dose-dependent manner, whereas the expression of SCM-1beta remained consistently high. These observations indicate that NK cells, in addition to T lymphocytes, express Lptn/SCM-1alpha and SCM-1beta after cytokine activation. The upregulation of these chemokines in NK cells on activation likely acts to increase the number of effector cells reaching the site of an immune response such as inflammation.     

Distribution of killer cell immunoglobulin-like receptors (KIR) and their HLA-C ligands in two Iranian populations

Killer cell immunoglobulin-like receptors (KIR) gene frequencies vary between populations and contribute to functional variation in immune responses to viruses, autoimmunity and reproductive success. This study describes the frequency distribution of 12 variable KIR genes and their HLA-C ligands in two Iranian populations who have lived for many generations in different environments: the Azerbaijanis at high altitude and the Jonobi people at sea level. The results are compared with those published for other human populations and a large group of English Caucasians. Differences were seen in KIR and HLA-C group frequencies, in linkage disequilibrium and inhibitory/activating KIR ratios between the groups. Similarities with geographically close populations in the frequencies of the KIR A and B haplotypes and KIR AA genotype reflected their common ancestry. The extreme variability of the KIR gene family and their HLA-C ligands is highlighted and their importance in defining differences between geographically and culturally isolated communities subject to different environmental pressures who come from the same ethnic grouping.