Clinical Impact of KIR2DS3 and KIR2DL3 Genes in Neuroblastoma Patients.

OBJECTIVE: Neuroblastoma is a common fatal tumor of childhood. Natural killer (NK) cells can exert direct cytotoxicity on tumor cells. The killer immunoglobulin-like receptor (KIR) family of NK cell receptors is involved in activation/inhibition of NK cells. In the KIR gene cluster, six of them (3DS1, 2DS1-5) encode receptors triggering activation, while seven of them (3DL1-3, 2DL1-3, 2DL5) encode receptors triggering inhibition. We aimed to assess the distribution of genetic polymorphisms of KIRs on the clinical course of neuroblastoma and provide guidance on potential therapeutic options. METHODS: Our study group included 50 neuroblastoma patients and 100 healthy children as controls. Twenty-eight patients were boys, and twenty-two were girls; median age was 36 months. Fourteen patients had stage 1, 2, 3, or 4S disease, and 36 patients had stage 4 disease. Isolated DNA from the peripheral blood was amplified for sequence-specific oligonucleotide probe analysis of 16 KIR genes. The Fisher's exact test was used to evaluate the variation of KIR gene distribution. RESULTS: All patients had a lower frequency of KIR2DS3 compared to the control group (p = 0.005). Evaluation of individual KIR genes/genotypes in patients with early stages (stage 1, 2, 3, and 4S) versus stage 4 disease revealed that the frequency of KIR2DS3 was increased in early stages (p = 0.023). Inhibitory KIR2DL3 was increased in the patient group compared to controls (p = 0.038). Furthermore, the frequency of KIR2DL3 was higher in stage 4 neuroblastoma patients compared to the patients with early stages (p = 0.023). CONCLUSION: Our data suggest a role for KIR2DS3 and KIR2DL3 in development of neuroblastoma. Thus, modulation of KIR2SD3 and/or KIR2DL3 expression or function might present a novel therapeutic strategy for neuroblastoma.

Significance of inhibitory maternal killer-cell immunoglobulin-like receptor (KIR) and fetal KIR ligand genotype combinations in placenta related obstetric complications.

Some maternal killer-cell immunoglobulin-like receptor (KIR) and fetal KIR ligand genotypes are associated with obstetric complications, such as recurrent miscarriage, fetal growth restriction, preeclampsia, and preterm birth. However, how KIR/KIR ligand genotypes affect these placenta-related obstetric complications has not been fully understood. We aimed to demonstrate the association of maternal KIR-fetal KIR ligand genotype combinations with immunological/metabolic risk factor associated placenta-related obstetric complications. This study consisted of three groups of pregnant women: 1) Miscarriage group (n = 30), 2) Complicated Pregnancy (CP) group (n = 30), and 3) Control group (n = 30). The observed maternal genotype frequencies of all inhibitory and activating KIRs were similar in all groups (p > 0.05). However, inhibitory 2DL3 was quite frequent in the miscarriage group (p = 0.052). There was no difference between groups in terms of centromeric and telomeric maternal haplotypes (p > 0.05). The fetal group 1 HLA-C genotype was frequently detected in the miscarriage and CP groups with rates of 83.3 % and 93.3 % respectively, while the observed frequency was 70 % in the control group. The fetal group 2 HLA-C genotype was the same in all groups. The results demonstrated significantly less fetal group 2 HLA-C homozygosity in the CP groups when compared to the control group (p = 0.020). The fetal HLA-Bw4 genotype was detected more frequently in the miscarriage and CP groups (p = 0.028 and p = 0.001, respectively). The inhibitory KIR/KIR ligand genotype combinations of 2DL3-C1 and 3DL1-Bw4 were more frequent in the miscarriage and CP groups (p = 0.045 and p = 0.002, respectively). Enhanced NK cell inhibition may be one of the mechanisms underlying placenta-related obstetric complications.

Structural plasticity of KIR2DL2 and KIR2DL3 enables altered docking geometries atop HLA-C.

The closely related inhibitory killer-cell immunoglobulin-like receptors (KIR), KIR2DL2 and KIR2DL3, regulate the activation of natural killer cells (NK) by interacting with the human leukocyte antigen-C1 (HLA-C1) group of molecules. KIR2DL2, KIR2DL3 and HLA-C1 are highly polymorphic, with this variation being associated with differences in the onset and progression of some human diseases. However, the molecular bases underlying these associations remain unresolved. Here, we determined the crystal structures of KIR2DL2 and KIR2DL3 in complex with HLA-C*07:02 presenting a self-epitope. KIR2DL2 differed from KIR2DL3 in docking modality over HLA-C*07:02 that correlates with variabilty of recognition of HLA-C1 allotypes. Mutagenesis assays indicated differences in the mechanism of HLA-C1 allotype recognition by KIR2DL2 and KIR2DL3. Similarly, HLA-C1 allotypes differed markedly in their capacity to inhibit activation of primary NK cells. These functional differences derive, in part, from KIR2DS2 suggesting KIR2DL2 and KIR2DL3 binding geometries combine with other factors to distinguish HLA-C1 functional recognition.

Arsenite suppresses IL-2-dependent tumoricidal activities of natural killer cells.

Chronic exposure to arsenic causes cancers in various organs including the skin, liver, lung, and bladder in humans, but the mechanisms of the multi-organ carcinogenicity of arsenic remain unknown. Natural killer (NK) cells play important roles in the immune surveillance and elimination of tumor cells. Although accumulating evidence has indicated that arsenic has immunosuppressive properties, little is known about the effects of arsenic on the tumoricidal functions of NK cells. We examined the effects of arsenite on the cytotoxic activities of human and mouse NK cells toward target tumor cells. Exposure of human NK-92 cells and primary mouse NK cells to sublethal doses of arsenite reduced the IL-2-activated cytotoxic activities toward human K562 cells and murine YAC-1 cells, respectively. NK cells recognize target cells via integrated signals from both activating and inhibitory receptors and induce apoptosis of target cells via a granzyme/perforin system. We found that exposure of NK-92 cells to arsenite diminished the IL-2-activated down-regulation of the inhibitory receptors, KIR2DL2 and KIR2DL3, and the up-regulation of granzyme B and lymphotoxin-α. The IL-2-activated increases in secretion of interferon-γ and IL-10 were also slightly reduced by arsenite. Thus, arsenite suppressed the IL-2-activated cytotoxic activity of NK cells by disrupting multiple pathways required for the recognition and killing of target tumor cells. Our findings provide new insights into the roles of NK cell-mediated tumor immunity in cancer development by arsenic.

Phenotypic and functional characterization of natural killer cells in rheumatoid arthritis-regulation with interleukin-15.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial inflammation and joint destruction. Previous studies have shown that natural killer (NK) cells may play an important role in the pathogenesis of RA. Interleukin (IL)-15, a pro-inflammatory cytokine which induces proliferation and differentiation of NK cells, is overexpressed in RA. In this present study, we examine various NKRs and adhesion molecule expression on NK cells from RA patients and their response to IL-15 stimulation. We also sought to study cytokine-induced memory-like (CIML) NK cells in RA patients. We established that 1. RA patients had higher NK cell percentages in peripheral blood and their serum IL-15 levels were higher compared to healthy volunteers; 2. NK cells from RA patients showed lower NKp46 expression and an impaired CD69 response to IL-15; 3. NK cells from RA patients showed higher CD158b and CD158e expression but lower CD62L expression; 4. exogenous IL-15 up-regulated CD69, CD158b, CD158e but down-regulated NKp46 and CD62L expression in RA; 5. As to CIML NK cells, restimulation - induced NK cytotoxicity and IFN-γ production was impaired in RA patients, 6. Reduced NKp46, perforin, and granzyme B expression on NK cells was found in RA patients with bone deformity and erosion, 7. RA disease activity (DAS28) showed inverse correlation with the percentages of CD56+CD3- NK cells, and NKp46 and perforin expression on NK cells, respectively. Taken together, our study demonstrated differential expression of various NK receptors in RA patients. NKp46, CD158e, and perforin expression on NK cells may serve as markers of RA severity.

Differential contribution of education through KIR2DL1, KIR2DL3, and KIR3DL1 to antibody-dependent (AD) NK cell activation and ADCC.

The engagement of activating NK receptors (aNKR) stimulates NK cell activity, provided that interactions between inhibitory NK receptors (iNKR) with their HLA ligands do not override them. Abs bound to target cells can also activate NK cells by engaging the CD16 aNKR. NK cell education status is an important factor for Ab-dependent NK cell activation (ADNKA) of some NK cell subsets. However, whether NK cell education also influences Ab-dependent cellular cytotoxicity (ADCC) levels is not fully known. ADCC-GranToxiLux (GTL) assays measured ADCC activity as the frequency of granzyme B positive (%GzB+ ) target cells. Target cells were anti-HIV Immunoglobulin G (HIVIG)-opsonized CEM-NKr.CCR5 (CEM) cells. Lymphocytes and sorted single positive (SP) NKG2A+ , KIR2DL1+ , KIR2DL3+ , and KIR3DL1+ NK cells, to self- and nonself HLA, were used as effectors in ADCC-GTL assays to examine how education status influenced ADCC activity. ADNKA activity was assessed by stimulating lymphocytes with HIVIG-opsonized CEMs and measuring the frequency of NK cell populations defined by their expression of iNKRs, along with IFN-γ, CCL4, and CD107a functions. ADCC: the %GzB+ CEM cells generated by self- versus nonself HLA-specific SPiNKR did not differ. ADNKA: More NK cells educated through KIR2DL1 and KIR3DL1, but not KIR2DL3, responded to ADNKA than their uneducated counterparts. CD16 engagement induced ADCC and ADNKA activity. With the proviso that groups' sizes were small, our results support the notion that NK cell education does not influence ADCC levels but does contribute to ADNKA activity.

A novel antibody combination to identify KIR2DS2high natural killer cells in KIR2DL3/L2/S2 heterozygous donors.

The killer cell immunoglobulin-like receptor (KIR) KIR2DS2 induces natural killer (NK) cell activation upon ligation and in genetic studies is associated with protection against certain cancers and viral infections. One of the difficulties in understanding KIR2DS2 has been that ligands have been hard to define. In part, this is because the high sequence homology between KIR2DS2 and KIR2DL3/KIR2DL2 has made it difficult to make antibodies that specifically detect NK cells expressing KIR2DS2. Using transfected NK cell line (NKL) cells and primary human samples, we report the identification of a novel antibody combination which allows identification of NK cells with relatively high expression of KIR2DS2. This separation is sufficient to examine primary human NK cell activation in response to KIR2DS2 specific ligands.

Stable Frequencies of HLA-C*03:04/Peptide-Binding KIR2DL2/3+ Natural Killer Cells Following Vaccination.

Inhibitory KIRs play a central role in regulating NK cell activity. KIR2DL2/3 bind to HLA-C molecules, but the modulation of these interactions by viral infections and presentation of viral epitopes is not well-understood. We investigated whether the frequencies of KIR2DL2/3+ NK cells recognizing HLA-C*03:04/viral peptide complexes were impacted by YFV vaccination or HIV-1 and HCV infection. Ex vivo HLA class I tetramer staining of primary human NK cells derived from YFV-vaccinated individuals, or HIV-1- or HCV-infected individuals revealed that the YFV/HLA-C*03:04-NS2A4-13-tetramer bound to a larger proportion of KIR2DL2/3+ NK cells compared to HIV-1/HLA-C*03:04-Gag296-304- or HCV/HLA-C*03:04-Core136-144-tetramers. The YFV/HLA-C*03:04-NS2A4-13-tetramer also exhibited a stronger avidity to KIR2DL2/3 compared to the other tested tetramers. The proportional frequencies of KIR2DL2/3+ NK cells binding to the three tested HLA-C*03:04 tetramers were identical between YFV-vaccinated individuals or HIV-1- or HCV-infected individuals, and remained stable following YFV vaccination. These data demonstrate consistent hierarchies in the frequency of primary KIR2DL2/3+ NK cells binding HLA-C*03:04/peptide complexes that were determined by the HLA-C-presented peptide and not modulated by the underlying viral infection or vaccination.

KIR downregulation by IL-12/15/18 unleashes human NK cells from KIR/HLA-I inhibition and enhances killing of tumor cells.

To exploit autologous NK cells for cancer immunotherapy, it is highly relevant to circumvent killer cell immunoglobulin-like receptor (KIR)-mediated self-inhibition of human NK cells by HLA-I-expressing tumor cells. Here, we show that stimulation of NK cells with IL-12/15/18 for two days led to downregulation of surface expression of the inhibitory KIR2DL2/L3, KIR2DL1 and KIR3DL1 receptors on peripheral blood NK cells. Downregulation of KIR expression was attributed to decreased KIR mRNA levels which could be re-induced already 3 days after re-culture in IL-2. Reduced KIR2DL2/L3 expression on IL-12/15/18-activated NK cells resulted in less inhibition upon antibody-mediated KIR engagement and increased CD16-dependent cytotoxicity in redirected lysis assays. Most importantly, downregulated KIR2DL2/L3 expression enabled enhanced cytotoxicity of IL-12/15/18-stimulated NK cells against tumor cells expressing cognate HLA-I molecules. NK cells pre-activated with IL-12/15/18 were previously shown to exert potent anti-tumor activity and memory-like long-lived functionality, mediating remission in a subset of acute myeloid leukemia (AML) patients in a clinical trial. Our study reveals a novel mechanism of IL-12/15/18 in improving the cytotoxicity of NK cells by reducing their sensitivity to inhibition by self-HLA-I due to decreased KIR expression, highlighting the potency of IL-12/15/18-activated NK cells for anti-tumor immunotherapy protocols.

HIV-1-Mediated Downmodulation of HLA-C Impacts Target Cell Recognition and Antiviral Activity of NK Cells.

It was widely accepted that HIV-1 downregulates HLA-A/B to avoid CTL recognition while leaving HLA-C unaltered in order to prevent NK cell activation by engaging inhibitory NK cell receptors, but it was recently observed that most primary isolates of HIV-1 can mediate HLA-C downmodulation. Now we report that HIV-1-mediated downmodulation of HLA-C was associated with reduced binding to its respective inhibitory receptors. Despite this, HLA-C-licensed NK cells displayed reduced antiviral activity compared to their unlicensed counterparts, potentially due to residual binding to the respective inhibitory receptors. Nevertheless, NK cells were able to sense alterations of HLA-C expression demonstrated by increased antiviral activity when exposed to viral strains with differential abilities to downmodulate HLA-C. These results suggest that the capability of HLA-C-licensed NK cells to control HIV-1 replication is determined by the strength of KIR/HLA-C interactions and is thus dependent on both host genetics and the extent of virus-mediated HLA-C downregulation.

The Intergenic Recombinant HLA-B∗46:01 Has a Distinctive Peptidome that Includes KIR2DL3 Ligands.

HLA-B∗46:01 was formed by an intergenic mini-conversion, between HLA-B∗15:01 and HLA-C∗01:02, in Southeast Asia during the last 50,000 years, and it has since become the most common HLA-B allele in the region. A functional effect of the mini-conversion was introduction of the C1 epitope into HLA-B∗46:01, making it an exceptional HLA-B allotype that is recognized by the C1-specific natural killer (NK) cell receptor KIR2DL3. High-resolution mass spectrometry showed that HLA-B∗46:01 has a low-diversity peptidome that is distinct from those of its parents. A minority (21%) of HLA-B∗46:01 peptides, with common C-terminal characteristics, form ligands for KIR2DL3. The HLA-B∗46:01 peptidome is predicted to be enriched for peptide antigens derived from Mycobacterium leprae. Overall, the results indicate that the distinctive peptidome and functions of HLA-B∗46:01 provide carriers with resistance to leprosy, which drove its rapid rise in frequency in Southeast Asia.

The differential impact of natural killer (NK) cell education via KIR2DL3 and KIR3DL1 on CCL4 secretion in the context of in-vitro HIV infection.

Carriage of certain inhibitory natural killer (NK) cell receptor (iNKR)/HLA ligand pairs is associated with protection from infection and slow time to AIDS implicating NK cells in HIV control. NK cells acquire functional potential through education, which requires the engagement of iNKRs by their human leucocyte antigen (HLA) ligands. HIV infection down-regulates cell surface HLA-A/B, but not HLA-C/E. We investigated how NK cell populations expressing combinations of the iNKRs NKG2A, KIR2DL3 (2DL3) and KIR3DL1 (3DL1) responded to autologous HIV infected CD4 (iCD4) cells. Purified NK cells from HIV-uninfected individuals were stimulated with autologous HIV iCD4 or uninfected CD4 T cells. Using flow cytometry we gated on each of the 8 NKG2A+/- 2DL3+/- 3DL1+/- populations and analysed all possible combinations of interferon (IFN)-γ, CCL4 and CD107a functional subsets responding to iCD4 cells. Infected CD4 cells induced differential frequencies of NKG2A+/- 2DL3+/- 3DL1+/- populations with total IFN-γ+ , CCL4+ and CD107a+ functional profiles. 2DL3+ NKG2A+ NK cells had a higher frequency of responses to iCD4 than other populations studied. A higher frequency of 2DL3+ NK cells responded to iCD4 from individuals that were not HLA-C1 homozygotes. These results show that 2DL3+ NK cells are mediators of HIV-specific responses. Furthermore, responses of NK cell populations to iCD4 are influenced not only by NK cell education through specific KIR/HLA pairs, but also by differential HIV-mediated changes in HLA expression.

Killer immunoglobulin receptor genes and their HLA-C ligand are associated with Type 1 diabetes in an Eastern Indian population.

AIM: Killer immunoglobulin-like receptors (KIRs) and their interaction with HLA class I ligands have been shown to be associated with Type 1 diabetes mellitus. The aim of our study was to investigate the influence of KIR genes and their HLA-C ligands for susceptibility to Type 1 diabetes in patients from Eastern India. METHODS: A total of 135 patients with Type 1 diabetes and 98 healthy subjects from Eastern India were typed for KIR genes and HLA-C ligands using PCR-based genotyping. The frequencies of these genes were compared between patients and controls. RESULTS: Comparison of KIR genes between Type 1 diabetes patients and healthy subjects revealed significantly different frequencies of KIRs 2DL2 and 2DS4. The presence of HLA-C1 was negatively associated with disease. The presence of both HLA-C1 and -C2 showed a negative association with Type 1 diabetes, whereas the absence of C1 and presence of C2 was positively associated with disease. Stratification analysis of HLA-C ligands and KIRs showed significant associations between Type 1 diabetes and 2DL2+/C1-, 2DL2-/C1+, 2DL3+/C1+, 2DL3+/C1- and 2DS2+/C1-. CONCLUSIONS: Our results suggest that the interaction of KIRs with HLA-C ligands are significant and certain combinations contribute to susceptibility to and protection against Type 1 diabetes.

[Study of NK cells dysfunction in multiple myeloma patients].

OBJECTIVE: To explore the mechanism of NK cell dysfunction in patients with multiple myeloma (MM). METHODS: The expression of inhibitory receptors (CD158a and CD158b) and activating receptors NKG2D and NCRs (NKp30, NKp44 and NKp46) on CD3-CD56+NK cell of 13 MM patients and 30 healthy controls were analyzed by flow cytometry. The concentration of soluble NKG2D ligands (MICA, MICB, ULBP1, ULBP2 and ULBP3) in serum was detected by enzyme- linked immunosorbent assay (ELISA), and the cytotoxicity of NK cell against MM cell line by flow cytometry. RESULTS: There are no significant differences of percentage and absolute number of NK cells, and the expression level of CD158a and CD158b between MM patients and healthy individuals (P>0.05). No NKp44 expression was detected on fresh isolated NK cells from both groups. There is no difference in inhibitor receptors expression between MM patients and healthy individuals but the expression of NKG2D, NKp30 and NKp46 on NK cells were higher in MM patients as compared with that in healthy individuals. The concentration of soluble NKG2D ligands in serum was higher in MM patients as compared with that in healthy individuals (P<0.05). Cultured healthy individual's NK cells with MM patient's serum could significantly decrease its cytotoxicity against MM cell line U266 cells [(38.5 ± 6.5) % vs (25.4 ± 5.9)%, P=0.044ï¼½. CONCLUSION: The higher level of soluble NKG2D ligands in serum may be the mechanism of NK cell dysfunction in MM patient.

The effect of KIR2D-HLA-C receptor-ligand interactions on clinical outcome in a HIV-1 CRF01_AE-infected Thai population.

OBJECTIVE: Class I human leukocyte antigen (HLA) alleles interact with both cytotoxic T lymphocytes through their T-cell receptors, and natural killer cells through their killer immunoglobulin-like receptors (KIRs). Compared with the reported protective effect of KIR3DL1/S1-HLA-Bw4 interactions in HIV-infected patients, the effect of KIR2D-HLA-C combinations on HIV control remains unclear. Here, we investigate the effect of KIR2D-HLA-C combinations on HIV disease progression. DESIGN: We performed a cross-sectional and longitudinal analysis of a Thai HIV cohort. METHODS: Two hundred and nine HIV-1 CRF01_AE-infected, treatment-naive Thai patients (CD4 T-cell counts of >200/µl) and 104 exposed seronegatives were studied. The effect of KIR-HLA receptor-ligand combinations on viral transmission and survival rate was statistically analyzed. RESULTS: We found the following results: higher frequency of patients expressing both KIR2DL3 and HLA-C1 among infected patients compared with exposed seronegative (odds ratio 4.8, P = 0.004), higher viral load in patients expressing HLA-C1 with KIR2DL3 compared with those without this receptor-ligand combination (median 4.8 vs. 4.2 log copies/ml, P = 0.033), higher numbers of KIR2DL3-HLA-C1 interactions was associated with a higher viral load (ß = 0.13, P = 0.039 by linear regression model), and higher mortality rate in carriers of the KIR2DL3-HLA-C1 combination (adjusted hazard ratio 1.9, P = 0.012 by Cox hazard model). CONCLUSION: We have identified a deleterious effect of the KIR2DL3-HLA-C1 receptor-ligand combination on HIV clinical outcomes in a Thai cohort. Further investigation into mechanisms underlying this susceptibility may aid the understanding of the role of natural killer cells in HIV disease control and pathogenesis.

Enrichment of CD56(dim)KIR + CD57 + highly cytotoxic NK cells in tumour-infiltrated lymph nodes of melanoma patients.

An important checkpoint in the progression of melanoma is the metastasis to lymph nodes. Here, to investigate the role of lymph node NK cells in disease progression, we analyze frequency, phenotype and functions of NK cells from tumour-infiltrated (TILN) and tumour-free ipsilateral lymph nodes (TFLN) of the same patients. We show an expansion of CD56(dim)CD57(dim)CD69 + CCR7 +KIR+ NK cells in TILN. TILN NK cells display robust cytotoxic activity against autologous melanoma cells. In the blood of metastatic melanoma patients, the frequency of NK cells expressing the receptors for CXCL8 receptor is increased compared with healthy subjects, and blood NK cells also express the receptors for CCL2 and IL-6. These factors are produced in high amount in TILN and in vitro switch the phenotype of blood NK cells from healthy donors to the phenotype associated with TILN. Our data suggest that the microenvironment of TILN generates and/or recruits a particularly effective NK cell subset.

Human NK cells licensed by killer Ig receptor genes have an altered cytokine program that modifies CD4+ T cell function.

NK cells are innate immune cells known for their cytolytic activities toward tumors and infections. They are capable of expressing diverse killer Ig-like receptors (KIRs), and KIRs are implicated in susceptibility to Crohn's disease (CD), a chronic intestinal inflammatory disease. However, the cellular mechanism of this genetic contribution is unknown. In this study, we show that the "licensing" of NK cells, determined by the presence of KIR2DL3 and homozygous HLA-C1 in host genome, results in their cytokine reprogramming, which permits them to promote CD4(+) T cell activation and Th17 differentiation ex vivo. Microfluidic analysis of thousands of NK single cells and bulk secretions established that licensed NK cells are more polarized to proinflammatory cytokine production than unlicensed NK cells, including production of IFN-γ, TNF-α, CCL-5, and MIP-1ß. Cytokines produced by licensed NK augmented CD4(+) T cell proliferation and IL-17A/IL-22 production. Ab blocking indicated a primary role for IFN-γ, TNF-α, and IL-6 in the augmented T cell-proliferative response. In conclusion, NK licensing mediated by KIR2DL2/3 and HLA-C1 elicits a novel NK cytokine program that activates and induces proinflammatory CD4(+) T cells, thereby providing a potential biologic mechanism for KIR-associated susceptibility to CD and other chronic inflammatory diseases.

[Changes of natural kill cell in peripheral blood of patients with myelodysplastic syndrome].

OBJECTIVE: To analyze the percentage and functional changes of natural kill (NK) cell in peripheral blood of myelodysplastic syndrome (MDS) patients so as to evaluate the relationships between these changes and hematopoietic functions and explore the role of NK cell in the pathogenesis of MDS. METHODS: By flow cytometry, the percentage of NK cell (CD3⁻CD56⁺CD16⁺) in peripheral blood lymphocytes was detected in 20 MDS patients, 15 acute myelogenous leukemic (AML) and 15 normal controls from March 2013 to November 2013 at our hospital.NK cell activation receptors (NKG2D, NKp44), inhibitory receptors (CD158a, CD158b), perforin and granzyme-ß of patients and normal controls were also detected. The correlation between these changes and hematopoietic functions, including the percentages of neutrophil granulocyte (ANC%), lymphocyte (Lym%), reticulocyte (RET%) and hemoglobin, thrombocyte in peripheral blood and the hematopoietic function in bone marrow (CD34⁺%) were evaluated. RESULTS: (1) The percentage of NK cell (3.87% ± 0.97%) in MDS patients was significantly lower than that of normal controls (6.08% ± 1.37%, P < 0.05) and higher than that of AML patients (2.58% ± 0.78%, P < 0.05).(2) The expression of NKG2D in MDS patients (52.83%) was significantly lower than that of normal controls (86.36%, P < 0.05) and higher than that of AML patients (42.00%, P < 0.05). The expression of NKp44 in MDS patients (2.41%) was significantly higher than that of normal controls (0.62%) and AML patients (0.92%) (both P < 0.05). (3) The expression of CD158a in MDS and AML patients (5.46% ± 2.40%, 4.05% ± 1.89%) were significantly both lower than those of normal controls (7.97% ± 2.85%, both P < 0.05). The expression of CD158b had no significant difference among the 3 groups. (4) The expression of perforin in MDS patients (17.83%) was significantly lower than that of normal controls (59.79%, P < 0.05) and higher than that of AML patients (13.06%, P < 0.05). The expressions of granzyme-ß in MDS and AML patients (21.54%, 30.65%) were significantly both lower than those of normal controls (83.42%, both P < 0.05). (5) The percentage of NK cell and the expression of NKG2D and NKp44 in MDS patients with low-risk group were in turn higher than those of high-risk group (4.06% ± 0.56% vs 3.59% ± 1.37%, 53.42% ± 6.85% vs 47.29% ± 8.08%, 2.46% vs 2.07%, all P < 0.05) , the expression of CD158a and CD158b were in turn lower than those of high-risk group, but both P > 0.05.(6) The percentage of NK was positively correlated with ANC% (r = 0.780, P < 0.05), but negatively with Lym% and the CD34⁺% in bone marrow (r = -0.543, -0.610, both P < 0.05). The expression of CD158a was positively correlated with CD34⁺% in bone marrow (r = 0.612, P < 0.05). The expressions of NKp44, CD158a, perforin and granzyme-ß of NK cells had no correlation with hematopoiesis (all P > 0.05). CONCLUSION: The lowered percentage and function of NK may cause the immunological dysfunction and lead to underkill of pathological hematopoietic cells in MDS.

KIR2DL3⁺NKG2A⁻ natural killer cells are associated with protection from productive hepatitis C virus infection in people who inject drugs.

BACKGROUND & AIMS: Despite continuous high-risk behavior, a subgroup among people who inject drugs (PWID) remains seronegative for hepatitis C virus (HCV) suggesting that a state of "natural resistance" to HCV Infection may exist. Homozygosity for KIR2DL3 and its ligand HLA-C1 group alleles has been associated with control of HCV infection, however, the mechanism mediating this protective effect remained unclear. METHODS: Peripheral NK cells from PWID (n=104) were phenotypically and functionally characterized by multicolor flow cytometry. Expression levels of the NK cell receptor ligands were analysed in liver biopsies and primary human hepatocytes. RESULTS: HCV seronegative PWID (n=34) had increased levels of KIR2DL3(+)NKG2A(-) NK cells compared to healthy controls (n=10; p<0.001) and PWID with chronic (n=38; p<0.001) or resolved infection (n=37; p<0.001). There was an inverse correlation between the frequency of KIR2DL3(+) and NKG2A(+) NK cells (r=-0.53; p<0.0001). Importantly, expression of HLA-E, the ligand for NKG2A, was significantly upregulated in liver biopsies of HCV infected patients (n=51) compared to HBV infected patients (n=22; p<0.01) and correlated with HCV viral load (r=0.32; p<0.0029). In functional analyses KIR2DL3(-)NKG2A(+) NK cells but not KIR2DL3(+)NKG2A(-) NK cells were significantly inhibited by HLA-E ligation. Accordingly, interferon gamma secretion of NK cells from PWID with chronic infection but not from HCV seronegative PWID was significantly suppressed in the presence of HLA-E. CONCLUSIONS: KIR2DL3(+)NKG2A(-) NK cells are not sensitive to HLA-E-mediated inhibition and may thereby control early HCV infection prior to seroconversion and result in an apparent state of "natural resistance" to HCV in PWID.