Natural killer cells from psoriasis vulgaris patients have reduced levels of cytotoxicity associated degranulation and cytokine production.

Psoriasis vulgaris is a chronic inflammatory disease of the skin with a strong genetic component and immune system involvement. Although some evidence suggests that Natural Killer (NK) cells may play a part in psoriasis, their role is relatively unstudied and results are controversial. In this current study, NK cells from psoriasis patients exhibited reduced degranulation and produced lower levels of the pro-inflammatory cytokines IFN-γ and TNF-α. Further investigation found that NK cells from psoriasis patients and healthy controls expressed similar levels of activation markers, NK cell receptors and apoptosis-inducing molecules. In addition, comparable levels of several cytokines important in NK cell biology were found in the serum of psoriasis patients and healthy controls. Genotyping analysis revealed that HLA-C2, which provides a ligand for killer-cell immunoglobulin-like receptors (KIR) expressed by NK cells, was strongly associated with psoriasis susceptibility. However, no link between the KIR genes themselves and disease was found.

Increased frequencies of the killer immunoglobulin-like receptor genes KIR2DL2 and KIR2DS2 are associated with neuroblastoma.

Neuroblastoma is the most common extra-cranial solid tumour in children. Natural killer (NK) cells are innate lymphocytes that are known to mediate the direct cytotoxicity of neuroblastoma tumour cells. Natural variation in the highly polymorphic killer immunoglobulin-like receptors (KIR) and their cognate human leukocyte antigen (HLA) class I ligands results in considerable diversity in NK cell function. As the early onset of neuroblastoma suggests the contribution of genetic factors, we investigated if individual KIR genes, combined KIR gene haplotypes or compound KIR-HLA ligand genotypes could influence susceptibility to neuroblastoma. Genotype analysis of the KIR genes as well as their three major HLA class I ligand groups, HLA-C1, HLA-C2 and HLA-Bw4, was carried out in a cohort of 201 neuroblastoma patients compared with 240 healthy control subjects using polymerase chain reaction with sequence-specific primers. We found a significant increase in the frequency of KIR2DL2 (P = 0.019) as well as KIR2DS2 (P = 0.008) in patients with neuroblastoma compared with the healthy control group. While the incidence of the least inhibitory compound KIR-HLA-C genotype, KIR2DL3 in the presence of HLA-C1 was slightly reduced in neuroblastoma patients, this did not reach statistical significance (P = 0.069). In summary, while KIR-HLA compound genotypes have previously been implicated in predicting treatment outcomes in neuroblastoma, here we show that the presence of the individual KIR genes, KIR2DL2 and KIR2DS2, irrespective of HLA-C genotype is associated with the onset of this embryonal malignancy.

2DL1, 2DL2 and 2DL3 all contribute to KIR phenotype variability on human NK cells.

Natural killer (NK) cells are lymphocytes that function as part of the innate immune system. Their activity is controlled by a range of inhibitory and activating receptors, including the important killer-cell immunoglobulin-like receptors (KIR). The KIR are a multi-gene family of receptors that interact with the human leukocyte antigen (HLA) class I family of molecules and are characterised by extensive allelic polymorphism. Their expression on the cell surface of NK cells is highly variable, but the factors responsible for this variability are not yet clearly understood. In the current study, we investigated KIR expression in a healthy human cohort that we had previously characterised in depth at a genetic level, with KIR allele typing and HLA class I ligand genotypes available for all donors (n=198). Allelic polymorphism significantly affected the phenotypic expression of all KIR analysed, whereas HLA ligand background influenced the expression levels of 2DL1 and 2DL3. In particular, we found that although 2DL2 may influence 2DL1 expression, this appears to be owing to variation in 2DL1 copy number. Finally, the inhibitory receptor LILRB1 had higher expression levels in individuals with B/B KIR genotypes, suggesting a possible relationship between KIR and non-KIR receptors, which serves to balance NK cell activation potential.

Signatures of natural selection and coevolution between killer cell immunoglobulin-like receptors (KIR) and HLA class I genes.

Natural killer (NK) cells are lymphocytes of the innate immune system. In humans, NK cell activities are partly controlled by the diverse killer immunoglobulin-like receptor (KIR) gene family. The importance of NK cells in both immunity to infection and reproduction makes KIR strong candidates for genes undergoing dynamic evolution in the human genome. Using high-resolution allelic typing, we investigated the potential role of natural selection in the diversification of KIR in the Irish population. Higher diversity than expected is observed at several loci, consistent with a history of balancing selection acting to maintain several allelic variants at high frequency in the population. KIR diversity is enhanced further at the haplotype level with functional polymorphisms at KIR2DL4, KIR3DL1 and KIR2DS4 defining nine 'core' haplotypes. Analysis of these core haplotypes in combination with human leukocyte antigen (HLA) class I ligands revealed several nonrandom associations. In particular, the KIR:HLA association for the core haplotype defined by KIR3DL1(*)01502 was female specific and a likely consequence of negative selection acting against KIR3DL1(*)01502 on an HLA-C1/C1 background. Many of the associations between KIR and HLA in the Irish differ from those previously reported, which argues against universal selective pressures for specific KIR:HLA combinations in diverse human populations.

Receptor systems controlling natural killer cell function are genetically stratified in Europe.

Natural killer (NK) cells are components of the innate immune system that function in identifying and destroying aberrant or pathogen-infected cells. These functions are largely controlled by killer cell immunoglobulin-like receptors (KIRs). KIRs inhibit and activate NK cell functions through interactions with their ligands, epitopes encoded by human leukocyte antigen (HLA) class I genes (HLA-C1, C2 and Bw4). Genes that encode KIR and their HLA ligands vary in frequency across human populations. Here, we characterize two Irish populations for KIR and HLA and determine the spatial distribution of functionally important KIR:HLA systems in Europe, a region known for its considerable underlying genetic stratification. We find that Southern Europe is a region characterized by higher frequencies of activatory KIR and strong inhibitory HLA ligand systems (2DL1:HLA-C2 and 3DL1:Bw4). A lower frequency of activatory KIR and the predominance of a comparatively weaker inhibitory ligand system (2DL3:HLA-C1) are observed northwards. Despite contrasting KIR:HLA systems in Northern and Southern Europe, there is a clear balance between inhibitory and activatory repertoires, and their ligands in both regions. These findings show 'functional stratification' of the epistatic KIR:HLA receptor system in Europe, the presence of which will likely affect NK cell-mediated immunity across different populations.

Different NK cell surface phenotypes defined by the DX9 antibody are due to KIR3DL1 gene polymorphism.

KIR3DL1 and KIR3DL2 are NK cell receptors for polymorphic HLA-B and -A determinants. The proportion of NK cells that bind anti-KIR3DL1-specific Ab DX9 and their level of binding vary between individuals. To determine whether these differences are due to KIR polymorphism, we assessed KIR3D gene diversity in unrelated individuals and families. Both KIR3DL1 and KIR3DL2 are highly polymorphic genes, with KIR3DS1 segregating like an allele of KIR3DL1. A KIR haplotype lacking KIR3DL1 and KIR3DS1 was defined. The two KIR3DL1 alleles of a heterozygous donor were expressed by different, but overlapping, subsets of NK cell clones. Sequence variation in KIR3DL1 and KIR3DL2 appear distinct; recombination is more evident in KIR3DL1, and point mutation is more evident in KIR3DL2. The KIR3DL1 genotype correlates well with levels of DX9 binding by NK cells, but not with the frequency of DX9-binding cells. Different KIR3DL1 alleles determine high, low, and no binding of DX9 Ab. Consequently, heterozygotes for high and low binding KIR3DL1 alleles have distinct subpopulations of NK cells that bind DX9 at high and low levels, giving characteristic bimodal distributions in flow cytometry. The Z27 Ab gave binding patterns similar to those of DX9. Four KIR3DL1 alleles producing high DX9 binding phenotypes were distinguished from four alleles producing low or no binding phenotypes by substitution at one or more of four positions in the encoded protein: 182 and 283 in the extracellular Ig-like domains, 320 in the transmembrane region, and 373 in the cytoplasmic tail.

Identification of seventeen novel KIR variants: fourteen of them from two non-Caucasian donors.

The killer-cell immunoglobulin-like receptors (KIR) expressed by human natural killer (NK) cells are encoded by a family of genes on chromosome 19. The number of KIR genes varies with haplotype and the individual genes exhibit polymorphism. To investigate KIR diversity we studied KIR cDNA and genes of four human donors: two Caucasians, one Black American and one Asian Indian. From analysis of these donors seventeen novel KIR variants were identified and characterized. Fifteen of the new variants appear to have a simple allelic relationship with a known KIR, whereas two of them combine the sequences of two different KIR genes. Fourteen of the seventeen KIR variants were isolated from the two non-Caucasoid blood donors. These data show that much human KIR diversity remains to be characterized, particularly in non-Caucasoid populations.

Gene structure and promoter variation of expressed and nonexpressed variants of the KIR2DL5 gene.

Two variants of the novel KIR2DL5 gene (KIR2DL5.1 and.2) were identified in genomic DNA of a single donor. However, only the KIR2DL5.1 variant was transcribed in PBMC. In this study, analysis of seven additional donors reveals two new variants of the KIR2DL5 gene and indicates that transcription, or its lack, are consistently associated with particular variants of this gene. Comparison of the complete nucleotide sequences of the exons and introns of KIR2DL5.1 and KIR2DL5.2 reveals no structural abnormalities, but similar open reading frames for both variants. In contrast, the promoter region of KIR2DL5 shows a high degree of sequence polymorphism that is likely relevant for expression. Substitution within a putative binding site for the transcription factor acute myeloid leukemia gene 1 could determine the lack of expression for some KIR2DL5 variants.

KIR2DL5, a novel killer-cell receptor with a D0-D2 configuration of Ig-like domains.

Four novel killer-cell Ig-like receptor (KIR) genes were discovered by analysis of genomic DNA from a human donor. One gene, KIR2DL5, is expressed by subpopulations of NK cells and T cells, whereas expression of the other three genes could not be detected. KIR2DL5 has two extracellular Ig-like domains of the D0 and D2 type, a structural configuration that was previously unique to KIR2DL4. Although having a similar structure overall, the KIR2DL4 and KIR2DL5 receptors have distinctive amino acid sequences in the ligand-binding extracellular domains and differ in the transmembrane and cytoplasmic motifs that determine signal transduction. Whereas the KIR2DL4 gene is present on all KIR haplotypes and is expressed by all human NK cells, the KIR2DL5 gene is restricted to the "B" subset of KIR haplotypes and is clonally expressed by NK cells within an individual. Chimpanzee genes for KIR2DL4 and KIR2DL5 have been defined and are very similar in sequence to their human orthologs. The donor in whom KIR2DL5 was first detected bears two variants of it that differ by five nucleotide substitutions in the coding region. Although the substitutions are not predicted to affect gene expression, transcription of only one of the two KIR2DL5 variants could be detected.

Discovery of two novel variants of KIR2DS5 reveals this gene to be a common component of human KIR 'B' haplotypes.

The gene encoding the non-inhibitory receptor KIR2DS5 has so far been represented by a single cDNA sequence, NKAT9. A previous study by polymerase chain reaction using sequence-specific primers (PCR-SSP) failed to detect NKAT9 in genomic DNA of 52 donors, which suggested that KIR2DS5 could be a rare gene. Here, we have characterized two novel variants of KIR2DS5 that differ from NKAT9 by 8 and 10 nucleotide substitutions. The frequency of KIR2DS5 was then re-assessed by PCR-SSP using primers specific for conserved sequences of all three known KIR2DS5 variants. We found KIR2DS5 is not a rare gene, but one present in 26% of 34 donors representing the major ethnic groups. Like other non-inhibitory KIR, the distribution of KIR2DS5 is restricted to the 'B' subset of KIR-gene haplotypes. Transcription of the KIR2DS5 gene was studied by reverse transcriptase (RT)-PCR in natural killer (NK) cells from one donor and shown to follow the clonal distribution seen for most other KIR genes.

Differential cytokine regulation of natural killer cell-mediated necrotic and apoptotic cytotoxicity.

Natural killer (NK) cells can kill target cells by either necrotic or apoptotic mechanisms. Using the 51Cr-release assay to measure necrotic death of target cells, neonatal NK cells had low NK activity (K562 targets) and high lymphokine-activated killer (LAK) activity (Daudi targets) compared with adult cells, as has been previously reported. Using a 125I-deoxyuridine (125I-UdR) release assay, cord cells were shown to also have higher apoptotic LAK activity against YAC-1 target cells. Interleukin-4 (IL-4) inhibited interleukin-2 (IL-2)-induced necrotic killing of target cells by adult effectors but had no such inhibitory effect on cord cells. In contrast, IL-4 inhibited both adult and cord LAK cytotoxicity of YAC-1 target cells by apoptotic mechanisms with higher suppression observed in cord cell preparations. Using a colorimetric substrate conversion assay, IL-2 induced higher, and IL-4 had a more significant suppressive effect on, cord cell granzyme B enzyme activity compared with adult cells, paralleling apoptosis cytotoxicity data. Co-culture of either adult or cord LAK cells with IL-4 had a similar inhibitory effect on granzyme B protein expression, as detected by Western blotting. In contrast, IL-4 did not inhibit perforin expression, thereby defining IL-4 as a cytokine that can differentially regulate the NK cell-mediated cytotoxicity processes of apoptosis and necrosis. The differential sensitivity of cord cells to cytokine regulation of cytotoxicity may also have implications for cord blood transplantations, as NK cells are known to function as an effector cell in both graft-versus-host disease and in the graft-versus-leukaemia phenomena.

Differential cytotoxicity of cord blood and bone marrow-derived natural killer cells.

Allogeneic cord blood is now being widely used as a source of stem cells for hematologic reconstitution after myeloablative therapy, with reported significantly lower levels of graft-versus-host disease (GVHD) compared with the use of allogeneic bone marrow (BM). This study was undertaken to investigate biologic aspects of natural killer (NK) cell activity, as recognized effector cells of the GVHD and graft-versus-leukemia (GVL) response, from cord blood and conventional BM. NK-cell activity levels of freshly isolated cells from cord blood and BM against K562 targets were comparable. Lymphokine activated killer (LAK) cells from both hematopoietic cell sources were compared for their ability to kill target cells by necrotic or apoptotic mechanisms using specific target cell lines. Cord blood cells had significantly higher necrosis-mediated cytotoxic activity against Daudi target cells compared with BM-derived cells. Cord blood LAK cells had relatively high levels of apoptotic-mediated cytotoxicity against YAC-1 target cells, whereas BM-derived LAK cells were unable to induce apoptosis in these cells. Interleukin-2 (IL-2) induced significant granzyme B activity in cord cells in contrast to BM cells, in which very little activity was measured. Western blotting confirmed these findings, with IL-2 inducing granzyme B protein expression in cord cells but not detectable levels in BM cells. BM cells had significantly lower cell surface expression of IL-2R and prolonged culture in IL-2 was only partially able to restore their deficient apoptotic cytotoxic activity. Thus, major differences exist between cord blood-derived and BM-derived mononuclear cells with respect to their NK-cell-associated cytotoxic behavior. This could have important implications for stem cell transplantation phenomena, because it suggests that cord blood may have increased potential for a GVL effect.

Recognition of unusual presentation of natural killer cell leukemia.

Expansion of the natural killer (NK) subset of lymphocytes represents a rare leukemia phenotype with variations in clinical presentation, morphology, surface phenotype, and effector function. This paper reports on a 5-year-old male patient who had an unusual presentation of an NK cell leukemia that was initially diagnosed as neuroblastoma. A bone marrow (BM) aspirate showed clumps of undifferentiated cells with the following phenotype: CD56bright+, CD33dim+, CD45-, CD2-, CD19-, CD16-, and CD57-. Cytochemistry was noncontributory. The patient, having failed to respond to conventional neuroblastoma chemotherapy, was subsequently diagnosed as having NK cell leukemia based on functional in vitro assays. The patient responded to acute lymphoblastic leukemia (ALL) chemotherapy but relapsed 4 weeks into treatment and eventually died 25 weeks after initial presentation. The cell surface phenotype observed is consistent with a rare NK cell subset, the biology of which has not been well defined. Freshly isolated BM cells killed K562 cells in a conventional 51Cr-release assay. Both interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) induced LAK activity against the Daudi cell line. IL-2 induced proliferation of the leukemic cells. TNF-alpha, IFN-gamma, IL-6, IL-1ra, and TGF-beta levels were assessed and found to be concentrated in BM, in contrast to plasma samples. TNF-alpha was present at a high concentration in BM (150.9 pg/ml), probably a reflection of the associated disease pathology of severe bone pain and pyrexia. In summary, this paper details clinical and laboratory investigations of a leukemia of a rare NK cell subset.