Higher expression of inhibitory CD158b and CD158e NK cell receptor and age predicts treatment response in children with chronic hepatitis C.

Treatment with pegylated interferon-α and ribavirin (PEG-IFN/RBV) is the only choice for chronic hepatitis C (CHC) in children. Natural killer (NK) cells were described to play a vital role in CHC. The aim of this study was to analyze the expression of peripheral blood NK cell receptors in their relation to PEG-IFN/RBV treatment response. Study included 26 children with CHC-13 boys, age range 13.42 ± 3.28 years. Blood for biochemical, virological and cytometric testing was taken for evaluation prior to the antiviral treatment. NK cell receptors were detected by flow cytometry and the results were presented as proportion of cells and mean fluorescence intensity (MFI). Therapy consisted of PEG-IFNα-2b (60 µg/m2 s.c 1×/week) and RBV (15 mg/kg p.o. daily). Treatment duration was response-related and varied from 12 to 72 weeks. Rapid virological response (RVR) was evaluated in the 4th week and sustained virological response (SVR) 6 months after completion of the therapy. RVR children were younger (11.67 ± 3.74 vs 15.35 ± 2.42; p = 0.001) and displayed higher CD158b (3.58 ± 0.16 vs 3.45 ± 0.13; p = 0.038) and CD158e expression (4.33 ± 0.21 vs 4.03 ± 0.16; p = 0.039). Density of CD158b (logMFI = 3.68 ± 0.22 vs 3.36 ± 0.16; p = 0.036) and CD158e expression was significantly higher (4.37 ± 0.14 vs 4.12 ± 0.21; p = 0.046) and NKG2D expression significantly lower (97.50 ± 3.46 vs 94.92 ± 5.93; p = 0.049) in SVR children. SVR children were also significantly younger (12.40 ± 3.66 vs 15.13 ± 2.83; p = 0.003). Significance of the age of patients, and expression of CD158b and CD158e were confirmed in univariate and multivariate analysis. Age of patients is negatively related to RVR and SVR. NK cell phenotype with higher expression density of CD158b and CD158e receptor was a positive predictor of SVR.

Enhanced natural killer cell activity is found in exposed uninfected recipients of hepatitis C-contaminated blood.

A minority of injecting drug users, termed exposed uninfected, are resistant to hepatitis C (HCV) infection despite repeated low-dose exposures. We identify for the first time a cohort of blood recipients who remained uninfected despite large-dose exposure to HCV-contaminated blood and characterize immune factors that may confer protection. Of 1340 blood recipients from the English Look Back database who were transfused HCV-contaminated blood, we identified 8 who remained uninfected. In these 8 exposed uninfecteds, we characterized their natural killer (NK) cell populations and HCV-specific T-cell responses. Findings were compared with 10 spontaneous resolvers of HCV infection, 10 patients with chronic HCV infection and 10 healthy controls. Exposed uninfecteds had significantly greater numbers of NK cells with the activating receptor NKp30+ on CD56bright and CD56dim subsets compared with other groups (P < .05). Following interleukin-2 activation, NK cells of exposed uninfecteds had enhanced cytotoxicity that positively correlated with NKp30 expression (P = .02). Differences in NKp80 and KIR2DL3 expression were also observed. HCV-specific T-cell responses were observed in some exposed uninfecteds but of low amplitude. Exposure without infection following transfusion of HCV-contaminated blood is a very rare phenomenon and suggests a high level of resistance to infection. Enhanced NK cell activation and killing, with weak HCV-specific T-cell responses, were observed many years after exposure in uninfected recipients and may contribute to protection from HCV acquisition, although additional protective factors are being sought in this important cohort.

KIR 2D (L1, L3, L4, S4) and KIR 3DL1 protein expression in non-small cell lung cancer.

BACKGROUND: Nature killer (NK) cells are the immune system's first line of defense against both viral infections and tumors. Killer cell immunoglobulin-like receptors (KIRs) are associated with susceptibility to different types of cancers. We investigated KIR 2D (L1, L3, L4, S4) and KIR 3DL1 protein expression and their association with survival in non-small cell lung cancer (NSCLC). METHODS: The expression of KIR 2D (L1, L3, L4, S4) (BC032422/ ADQ31987/ NP_002246/ NP_036446, ABCAM) and KIR 3DL1 (AA 1-444, ABCAM) protein was assessed by immunohistochemistry (IHC) in 62 NSCLC patients. RESULTS: KIR 2D (L1, L3, L4, S4) and KIR 3DL1 were expressed both on NSCLC tumor cells and tumor infiltrating lymphocytes (TILs). Fourteen samples (22.6%) stained positive for KIR 2D (L1, L3, L4, S4) on the tumor cells, and 10 (16.1%) had positive expression on the TILs. Thirty-three samples (53.2%) stained positive for KIR 3DL1 on the tumor cells, and 31 (50.0%) had positive expression on the TILs. Patients with negative KIR 2D (L1, L3, L4, S4) expression on tumor cells or TILs had longer overall survival (OS) than patients who are KIR 2D (L1, L3, L4, S4) positive on tumor cells (40.70 weeks, 95% CI 24.76-56.65 vs. 7.10 weeks, 95% CI 0.00-19.38, P = 0.014) or TILs (40.70 weeks, 95% CI 24.05-57.35 vs. 3.90 weeks, 95% CI 0.00-9.17, P < 0.001). Likewise, longer OS was significantly correlated with negative expression of KIR 3DL1 on tumor cells (62.30 weeks, 95% CI 0.00-177.37 vs. 13.10 weeks, 95% CI 3.42-22.78, P < 0.001) or TILs (62.30 weeks, 95% CI 0.00-152.05 vs. 12.10 weeks, 95% CI 2.61-21.59, P < 0.001). Cox regression analysis showed that KIR 2D (L1, L3, L4, S4) on TILs was correlated with OS (P = 0.032, Odds Ratio 2.628 95%CI 1.089-6.340). CONCLUSIONS: KIR 2D (L1, L3, L4, S4) and KIR 3DL1 expression was correlated with poor prognosis in NSCLC patients.

NK Cells Expressing the Inhibitory Killer Immunoglobulin-Like Receptors (iKIR) KIR2DL1, KIR2DL3 and KIR3DL1 Are Less Likely to Be CD16+ than Their iKIR Negative Counterparts.

Natural Killer (NK) cell education, which requires the engagement of inhibitory NK cell receptors (iNKRs) by their ligands, is important for generating self-tolerant functional NK cells. While the potency of NK cell education is directly related to their functional potential upon stimulation with HLA null cells, the influence of NK cell education on the potency of the antibody dependent cellular cytotoxicity (ADCC) function of NK cells is unclear. ADCC occurs when the Fc portion of an immunoglobulin G antibody bridges the CD16 Fc receptor on NK cells and antigen on target cells, resulting in NK cell activation, cytotoxic granule release, and target cell lysis. We previously reported that education via the KIR3DL1/HLA-Bw4 iNKR/HLA ligand combination supported higher KIR3DL1+ than KIR3DL1- NK cell activation levels but had no impact on ADCC potency measured as the frequency of granzyme B positive (%GrB+) targets generated in an ADCC GranToxiLux assay. A lower frequency of KIR3DL1+ compared to KIR3DL1- NK cells were CD16+, which may in part explain the discrepancy between NK cell activation and target cell effects. Here, we investigated the frequency of CD16+ cells among NK cells expressing other iNKRs. We found that CD16+ cells were significantly more frequent among NK cells negative for the inhibitory KIR (iKIR) KIR2DL1, KIR2DL3, and KIR3DL1 than those positive for any one of these iKIR to the exclusion of the others, making iKIR+ NK cells poorer ADCC effectors than iKIR- NK cells. The education status of these iKIR+ populations had no effect on the frequency of CD16+ cells.

Multiplex and genome-wide analyses reveal distinctive properties of KIR+ and CD56+ T cells in human blood.

Killer cell Ig-like receptors (KIRs) on NK cells have been linked to a wide spectrum of health conditions such as chronic infections, autoimmune diseases, pregnancy complications, cancers, and transplant failures. A small subset of effector memory T cells also expresses KIRs. In this study, we use modern analytic tools including genome-wide and multiplex molecular, phenotypic, and functional assays to characterize the KIR(+) T cells in human blood. We find that KIR(+) T cells primarily reside in the CD56(+) T population that is distinctively DNAM-1(high) with a genome-wide quiescent transcriptome, short telomere, and limited TCR excision circles. During CMV reactivation in bone marrow transplant recipients, KIR(+)CD56(+) T cells rapidly expanded in real-time but not KIR(+)CD56(-) T cells or KIR(+) NK cells. In CMV(+) asymptomatic donors, as much as 50% of CD56(+) T cells are KIR(+), and most are distinguishably KIR2DL2/3(+)NKG2C(+)CD57(+). Functionally, the KIR(+)CD56(+) T cell subset lyses cancer cells and CMVpp65-pulsed target cells in a dual KIR-dependent and TCR-dependent manner. Analysis of metabolic transcriptome confirms the immunological memory status of KIR(+)CD56(+) T cells in contrast to KIR(-)CD56(+) T cells that are more active in energy metabolism and effector differentiation. KIR(-)CD56(+) T cells have >25-fold higher level of expression of RORC than the KIR(+) counterpart and are a previously unknown producer of IL-13 rather than IL-17 in multiplex cytokine arrays. Our data provide fundamental insights into KIR(+) T cells biologically and clinically.

[Expression and significance of the NK cell receptors in primary hepatocellular carcinoma and paracancerous tissues].

AIM: To investigate the expression of the activating and inhibitory receptors on the surface of NK cells of primary hepatocellular carcinoma and its adjacent tissues, and the relationship between these two receptors and occurrence and development of primary liver cancer was analyzed. METHODS: The number and activity of the NK cells, the expression of the activating and the inhibitory receptors on the surface of those cells were detected flow cytometry and immunohistochemistry, which were obtained from 52 cases of primary hepatocellular carcinoma and its adjacent tissues. The relative analysis was done between those results and clinical relative factors. RESULTS: In the tissues of primary hepacellular carcinoma, the number of NK cells is lower than that in the adjacent tissues obviously (P<0.01); the expression of activating receptors, NKG2D and NKP44, is also lower than that in the adjacent tissues obviously (P<0.05); the expression of inhibitory receptors, CD158b and CD159a, is significantly higher than that in the adjacent tissues (P<0.05). A negative correlation was found between the expression of NKG2D, NKP30 and NKP44 and the clinical stage of the liver cancer. The expression of NKG2D, NKP30 and NKP44 was higher in patients with early and middle stages (P<0.05). The content of the inhibitory receptors of NK cells, CD158b and CD159a, is higher in tissues from patients with advanced cancer stage (P<0.05). That's also correlated with the level of AFP and the HBsAg. There is no significant statistical difference between the expression of NK receptors and the distant metastasis, tumor differentiation as well as the tumor size (P>0.05). CONCLUSION: The decrease of NK cell numbers and the activating NK cell receptors and the increase of the inhibitory receptors would be relevant to the incidence of primary hepacellular carcinoma.

Antiviral treatment alters the frequency of activating and inhibitory receptor-expressing natural killer cells in chronic hepatitis B virus infected patients.

Natural killer (NK) cells play a critical role in innate antiviral immunity, but little is known about the impact of antiviral therapy on the frequency of NK cell subsets. To this aim, we performed this longitudinal study to examine the dynamic changes of the frequency of different subsets of NK cells in CHB patients after initiation of tenofovir or adefovir therapy. We found that NK cell numbers and subset distribution differ between CHB patients and normal subjects; furthermore, the association was found between ALT level and CD158b(+) NK cell in HBV patients. In tenofovir group, the frequency of NK cells increased during the treatment accompanied by downregulated expression of NKG2A and KIR2DL3. In adefovir group, NK cell numbers did not differ during the treatment, but also accompanied by downregulated expression of NKG2A and KIR2DL3. Our results demonstrate that treatment with tenofovir leads to viral load reduction, and correlated with NK cell frequencies in peripheral blood of chronic hepatitis B virus infection. In addition, treatments with both tenofovir and adefovir in chronic HBV infected patients induce a decrease of the frequency of inhibitory receptor(+) NK cells, which may account for the partial restoration of the function of NK cells in peripheral blood following treatment.

Sequential recovery of NK cell receptor repertoire after allogeneic hematopoietic SCT.

Alloreactivity of natural killer (NK) cells contributes to the GVL reaction after allogeneic hematopoietic SCT (allo-HSCT). However, various procedure-related factors may affect NK cell maturation and their ability to recognize and kill leukemic cells. In this study, we prospectively evaluated expression of NK cell inhibitory receptors in 83 adults treated with myeloablative, killer cell Ig-like receptor (KIR)-ligand-matched allo-HSCT. NK cell maturation was evaluated by comparing the phenotypic patterns after allo-HSCT with the donor ones. The frequencies of KIR3DL1 were comparable to the donor ones on day +28, while they decreased significantly starting from day +100. The expression of KIR2DL2/3 was significantly lower in patients compared with donors up to day +100. The expression of KIR2DL1, despite continues growth, remained significantly decreased for 1 year after allo-HSCT. NKG2A was over-expressed up to day +180. Within 1 year after allo-HSCT, the NK cell phenotypic pattern tended to recapitulate the donor type. The process was disturbed by the use of steroids with significant differences observed on days +56 (P=0.01) and +100 (P=0.04). Up to day +100, reconstitution of NK cell receptor repertoire correlated with the absolute numbers of circulating CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+) cells. Our observations should be taken into account when trying to predict potential benefit from NK cell alloreactivity.

Expression of complement regulatory proteins on human natural killer cell subsets.

The cell surface complement regulatory (CReg) proteins CD46, CD55 and CD59 are widely distributed on human leucocytes and protect against complement-mediated damage. To investigate heterogeneity in CReg protein expression by human natural killer (NK) cells, levels were assessed on resting and activated NK cell subsets identified phenotypically on the basis of expression of CD56 and CD158 markers. Levels of all three CReg proteins on CD56+ cells were lower than on T cells (p<0.05). Freshly isolated CD56(bright) cells expressed higher levels of CD55 than CD56dim cells (p<0.05). CD158a+ cells expressed significantly lower levels of both CD46 and CD59, and CD158e+ cells expressed significantly lower levels of CD46, than CD158a(-) CD158e(-) cells, respectively (both p<0.05). Stimulation with PHA did not significantly alter NK cell surface CReg protein levels whereas, following culture with IL-2, CD46 and CD59 were decreased on both CD56bright and CD56dim subsets (p<0.05). In the case of CD59, this was independent of T cells. Only CD46 was significantly downregulated on CD158b+ (GL183+) and CD158e (NKB1+) subsets (p<0.05). However, culture in IL-15 significantly increased levels of all three CReg proteins. These observations that CReg proteins are downregulated on certain NK cell subsets following activation with IL-2 are opposite to previous findings for other leucocyte subpopulations. Activated NK cells may instead use other strategies for protection against complement-mediated damage in a local inflammatory response.

Natural killer cells in perinatally HIV-1-infected children exhibit less degranulation compared to HIV-1-exposed uninfected children and their expression of KIR2DL3, NKG2C, and NKp46 correlates with disease severity.

NK cells play an integral role in the innate immune response by targeting virally infected and transformed cells with direct killing and providing help to adaptive responses through cytokine secretion. Whereas recent studies have focused on NK cells in HIV-1-infected adults, the role of NK cells in perinatally HIV-1-infected children is less studied. Using multiparametric flow cytometric analysis, we assessed the number, phenotype, and function of NK cell subsets in the peripheral blood of perinatally HIV-1-infected children on highly active antiretroviral therapy and compared them to perinatally exposed but uninfected children. We observed an increased frequency of NK cells expressing inhibitory killer Ig-like receptors in infected children. This difference existed despite comparable levels of total NK cells and NK cell subpopulations between the two groups. Additionally, NK cell subsets from infected children expressed, with and without stimulation, significantly lower levels of the degranulation marker CD107, which correlates with NK cell cytotoxicity. Lastly, increased expression of KIR2DL3, NKG2C, and NKp46 on NK cells correlated with decreased CD4+ T-lymphocyte percentage, an indicator of disease severity in HIV-1- infected children. Taken together, these results show that HIV-1-infected children retain a large population of cytotoxically dysfunctional NK cells relative to perinatally exposed uninfected children. This reduced function appears concurrently with distinct NK cell surface receptor expression and is associated with a loss of CD4+ T cells. This finding suggests that NK cells may have an important role in HIV-1 disease pathogenesis in HIV-1-infected children.