Human natural killer cell lysis of Salmonella typhi intracellularly-infected U937 cells

Peripheral blood mononuclear cell (PBMC) cytotoxicity against S. typhi (wild type or mutant strain TYT1231)-infected U937 cells was significantly higher than its lytic effect against noninfected cells (control) at the various effector-to-target cell ratio used (30:1, 50:1 and 70:1). Natural killer cell activity [expressed as % specific lysis (mean +/- SEM); 30:1 (25.4 +/- 3.6, 25.1 +/- 4.2 and 16.3 +/- 3.3); 50:1 (27.8 +/- 3.7, 26.7 +/- 4.5 and 20.9 +/- 2.9) and 70:1 ratio (33.2 +/- 5.9, 29.4 +/- 4.2 and 22.8 +/- 2.8), respectively] appeared to be dependent on such ratios and independent of the S strain studied. Most (80%) of individual samples tested showed at least a 20% specific lysis increase over their own control; essentially no changes or smaller increases in NKC activity were observed in all other samples. Similar results were obtained when using highly purified NKC (HPNKC) preparations as effector cells [NKC activity (mean +/- SEM); 5:1 (46.2 +/- 4.7, 43.2 +/- 5.0 and 25.2 +/- 2.3) and 10:1 effector-to-target cell ratio (49.3 +/- 4.9, 44.7 +/- 5.2 and 27.2 +/- 2.6, respectively)]. All individual samples tested showed at least a 20% specific lysis increase over their own control. These results show that S. typhi-infected U937 cells are a significantly better target for NKCs than control cells and indicate that intracellular bacteria survival capacity is not a critical factor for infected cells becoming a NKC target.     

Human adipose-derived stem cells impair natural killer cell function and exhibit low susceptibility to natural killer-mediated lysis

Human adipose-derived stem cells (hASCs) have been successfully used in treating numerous diseases. However, several aspects need to be considered, particularly in the context of allogeneic cell therapy. To better understand hASCs-host interactions, we studied the phenotype of hASCs and their modulatory effect on natural killer (NK) cells by using bone marrow-mesenchymal stem cells (hBM-MSCs) as a reference. The hASCs displayed a lower susceptibility to NK cell-mediated lysis and a lower expression of ligands for DNAM-1 when compared with hBM-MSCs. Moreover, here we demonstrated that hASCs and hBM-MSCs can modulate NK cells through the action of soluble factors such as indoleamine 2,3-dioxygenase. Altogether, these results suggest that for an adoptive cell therapy based on the transfer of allogeneic hASCs, the NK-hASCs crosstalk will not result in an immediate recognition of the transferred cells. Thus, hASCs may remain in the tissue long enough to balance the immune response before being cleared.     

Human natural killer cells: Molecular mechanisms controlling NK cell activation and tumor cell lysis

Natural killer cells represent a highly specialized lymphoid population with a potent cytolytic activity against virus-infected or tumor cells. Their function is regulated by a series of inhibiting or activating signals. The mechanisms by which NK cells kill susceptible target cells was thought to be elucidated after the discovery of inhibitory receptors specific for MHC-class I molecules: NK cells would kill those target cells that lack MHC-class I molecules. However, the actual scenario revealed more complex with the discovery of activating receptors and their ligands. Thus, in certain pathological conditions, corticosteroid treatment or exposure to TGFbeta, NK cells may under-express activating receptors. In addition, target cells may lack ligands for activating receptors and thus fail to activate NK cells upon cell-to-cell contact. This clearly implies that activation of NK cells and of their potent effector mechanism are under the control of different checkpoints.     

Human antibody-dependent cellular cytotoxicity and natural killer cytotoxicity to herpes simplex virus-infected autologous and allogeneic cells.

Using cultured skin shavings, human cellular cytotoxicity to uninfected and herpes simplex virus (HSV)-infected autologous and allogeneic fibroblasts and Chang liver cells was analysed in a 51Cr release assay. The effector cell requirements and characterization, time kinetics and antibody requirements were similar using each HSV-infected target cell in an antibody-dependent cellular cytotoxicity (ADCC) system. There was lower natural killer cytotoxicity (NKC) to uninfected autologous cells than unrelated cells in an 18 hr assay. NKC to infected autologous and unrelated fibroblasts was similar to that mediated against Chang liver cells. Thus NKC to uninfected fibroblasts correlated with the relationship of effector and target cells while NKC to infected cells correlated with the intrinsic lytic potential of the effector cells. The autologous system offers little advantage in the analysis of ADCC or NKC in normal individuals to virus-infected cells, but is probably crucial for the detection of HLA-restricted T-cell cytotoxicity. The demonstration of autologous anti-viral ADCC and NKC lends further credence to the in vivo importance of the mechanisms.     

Expression of the UL16 glycoprotein of Human Cytomegalovirus protects the virus-infected cell from attack by natural killer cells

Background

Human Cytomegalovirus (HCMV) has acquired through evolution a number of genes to try to evade immune recognition of the virus-infected cell. Many of these mechanisms act to inhibit the MHC class I antigen presentation pathway, but any virus-infected cell which has down-regulated cell surface expression of MHC class I proteins, to avoid CTL attack, would be expected to become susceptible to lysis by Natural Killer cells. Surprisingly, however, HCMV infected fibroblasts were found to be resistant to NK cell mediated cytotoxicity. Expression of the UL16 glycoprotein could represent one mechanism to help the virus to escape from NK cell attack, as it has been shown to bind, in vitro, some of the ligands for NKG2D, the NK cell activating receptor. Here, we explored the role of UL16, in the context of a viral infection, by comparing the susceptibility to NK lysis of cells infected with HCMV and cells infected with a UL16 deletion mutant of this virus.

Results

Cells infected with the UL16 knockout virus were killed at substantially higher levels than cells infected with the wild-type virus. This increased killing could be correlated with a UL16-dependent reduction in surface expression of ligands for the NK cell activating receptor NKG2D.

Conclusions

Expression of the UL16 glycoprotein was associated with protection of HCMV-infected cells from NK cell attack. This observation could be correlated with the downregulation of cell surface expression of NKG2D ligands. These data represent a first step towards understanding the mechanism(s) of action of the UL16 protein.

     

Human natural killer cell development

Summary:  Our understanding of human natural killer (NK) cell development lags far behind that of human B- or T-cell development. Much of our recent knowledge of this incomplete picture comes from experimental animal models that have aided in identifying fundamental in vivo processes, including those controlling NK cell homeostasis, self-tolerance, and the generation of a diverse NK cell repertoire. However, it has been difficult to fully understand the mechanistic details of NK cell development in humans, primarily because the in vivo cellular intermediates and microenvironments of this developmental pathway have remained elusive. Although there is general consensus that NK cell development occurs primarily within the bone marrow (BM), recent data implicate secondary lymphoid tissues as principal sites of NK cell development in humans. The strongest evidence stems from the observation that the newly described stages of human NK cell development are naturally and selectively enriched within lymph nodes and tonsils compared with blood and BM. In the current review, we provide an overview of these recent findings and discuss these in the context of existing tenets in the field of lymphocyte development.     

Human natural killer cell deficiencies

PURPOSE OF REVIEW: Human natural killer cell deficiencies are a relevant clinical entity that provides insight into the role of natural killer cells in host defense, as well as the basic biology of natural killer cells. Since previously reviewing these disorders, significant developments warrant their reconsideration. RECENT FINDINGS: Human natural killer cell deficiencies can occur as part of a more pervasive immunodeficiency syndrome or, rarely, in isolation. The most informative examples of the former are in the context of a known genetic defect, because the deficiency of natural killer cell development or activity can be attributed to the specific gene function. Since last reviewed, there are five human gene mutations that are now appreciated to affect natural killer cells, and additional new insights into natural killer cell biology have been obtained through seven others. Six new reports of isolated natural killer cell deficiencies, as well as a suggested classification scheme, are also reviewed. SUMMARY: Appreciation of human genetic syndromes that include natural killer cell deficiencies, as well as new cases of isolated natural killer cell deficiencies, continue to advance the understanding of natural killer cell biology and solidify the role of natural killer cells in defense against human herpesviral infection.     

Human natural killer cell receptor functions and their implication in diseases

Both active and passive anti-β-amyloid (Aβ) immunotherapies for the treatment of Alzheimer's disease (AD) have demonstrated clearance of brain Aβ deposits. Among passive immunotherapeutics, two Phase III clinical trials in mild-to-moderate AD patients with bapineuzumab, a humanized monoclonal antibody directed at the N-terminal sequence of Aβ, were disappointing. Also solanezumab, directed at the mid-region of Aβ, failed in two Phase III trials in mild-to-moderate AD. Another Phase III trial with solanezumab is ongoing in mildly affected AD patients based on encouraging results in this subgroup. Second-generation active Aβ vaccines (CAD106, ACC-001, and Affitope AD02) and new passive anti-Aβ immunotherapies (gantenerumab and crenezumab) have been developed and are under clinical testing. These new anti-Aβ immunotherapies are being tested in prodromal AD, in presymptomatic subjects with AD-related mutations, or in asymptomatic subjects at risk of developing AD. These primary and secondary prevention trials will definitely test the Aβ cascade hypothesis of AD.     

Target structure for natural killer cells: evidence against a unique role for transferrin receptor

The transferrin receptor (TfR) was recently proposed as putative natural killer (NK) cell target structure. Here data are presented against this hypothesis and it is shown that low TfR expression and high NK sensitivity can occur concommitantly . K562 cells were studied at various stages of cell proliferation. No change in NK sensitivity could be observed between exponential growth and the plateau phase, whereas TfR expression completely disappeared during the latter. Protein synthesis inhibitors such as cycloheximide (1 microgram/ml, 48 h) and actinomycin D (50 micrograms/ml, 48 h), that abolished the TfR expression at the K562 cell surface, had no effect on NK sensitivity. Similarly, hemin induction (0.1 mM, 5 days) did not change NK susceptibility of K562 cells but considerably diminished TfR expression. Moreover, attempts to block NK sensitivity with anti-TfR monoclonal antibodies were unsuccessful, even when the 42.6 antibody, which is known to bind to the active site of TfR, was used. Finally, no blocking of NK sensitivity could be achieved when K562 cells were preincubated with saturating concentrations of transferrin or when transferrin was added during the NK assay. It therefore seems doubtful that TfR is the unique target structure for NK cells. It remains possible that TfR and NK target structures are often coexpressed on actively dividing cells.     

Dissociated expression of natural killer 1.1 and T‐cell receptor by invariant natural killer T cells after interleukin‐12 receptor and T‐cell receptor signalling

Invariant (i) natural killer T (NKT) cells become undetectable after stimulation with α-galactosylceramide (α-GalCer) or interleukin (IL)-12. Although down-modulation of surface T-cell receptor (TCR)/NKR-P1C (NK1.1) expression has been shown convincingly after stimulation with α-GalCer, it is unclear whether this also holds true for IL-12 stimulation. To determine whether failure to detect iNKT cells after IL-12 stimulation is caused by dissociation/internalization of TCR and/or NKR-P1C, or by block of de novo synthesis of these molecules, and to examine the role of IL-12 in the disappearance of iNKT cells after stimulation with α-GalCer, surface (s)/cytoplasmic (c) protein expression, as well as messenger RNA (mRNA) expression of TCR/NKR-P1C by iNKT cells after stimulation with α-GalCer or IL-12, and the influence of IL-12 neutralization on the down-modulation of sTCR/sNKR-P1C expression by iNKT cells after stimulation with α-GalCer were examined. The s/cTCR⁺s/cNKR-P1C⁺ iNKT cells became undetectable after in vivo administration of α-GalCer, which was partially prevented by IL-12 neutralization. Whereas s/cNKR-P1C⁺ iNKT cells became undetectable after in vivo administration of IL-12, s/cTCR⁺ iNKT cells were only marginally affected. mRNA expression of TCR/NKR-P1C remained unaffected by α-GalCer or IL-12 treatment, despite the down-modulation of cTCR and/or cNKR-P1C protein expression. By contrast, cTCR⁺cNKR-P1C⁺ sTCR⁻ sNKR-P1C⁻ iNKT cells and cNKR-P1C⁺ sNKR-P1C⁻ iNKT cells were detectable after in vitro stimulation with α-GalCer and IL-12, respectively. Our results indicate that TCR and NKR-P1C expression by iNKT cells is differentially regulated by signalling through TCR and IL-12R. They also suggest that IL-12 participates, in part, in the disappearance of iNKT cells after stimulation with α-GalCer by down-modulating not only sNKR-P1C, but also sTCR.     

Rat natural killer cell and cytotoxic T cell lysis of H-2-negative murine embryonal carcinoma cells

H-2-lacking murine embryonal carcinoma (EC) cells have been proposed as universal targets for natural killer (NK) effectors from different species because their killing appeared to be uncomplicated by potential T cell effector mechanisms (Stern, P. L. et al., Int. J. Cancer 1981. 27:679). While some previous studies had shown that murine cytotoxic T cells were unable to lyse EC cells, rat T killers are shown here to be active against these targets and to be distinguishable from NK cells. Percoll density fractionation of rat peripheral blood lymphocytes enriches in parallel for NK-mediated lysis of both EC or YAC target cells. These NK cells unlike T cells, do not mediate lectin-dependent and cell-mediated cytotoxicity (LDCC) of NK-insensitive target cells. This procedure is thought to reveal the total cytolytic potential of stimulated T cell populations, regardless of specificity. In contrast to previous results with mice, we found that allogeneically primed rat cytotoxic T cells can kill murine EC cells in LDCC and, further, that rat cytotoxic T cells, generated by stimulation with mouse spleen cells in vitro, can lyse murine EC cells directly. This demonstration of T cell lysis of EC cells suggests that either there is a novel mechanism of lysis operating without requirement for major histocompatibility complex (MHC) structures, or EC cells express some hitherto unidentified MHC-like structures on their cell surface.     

Effects of Chlamydia trachomatis infection on the expression of natural killer (NK) cell ligands and susceptibility to NK cell lysis

Natural killer (NK) cells are an important component of the immediate immune response to infections, including infection by intracellular bacteria. We have investigated recognition of Chlamydia trachomatis (CT) by NK cells and show that these cells are activated to produce interferon (IFN)-gamma when peripheral blood mononuclear cells (PBMC) are stimulated with CT organisms. Furthermore, infection of epithelial cell lines with CT renders them susceptible to lysis by human NK cells. Susceptibility was observed 18-24 h following infection and required protein synthesis by the infecting chlamydiae, but not by the host cell; heat or UV inactivated chlamydiae did not induce susceptibility to NK cell lysis. CT infection was also shown to decrease the expression of classical and non-classical major histocompatibility complex (MHC) molecules on infected cells, thus allowing recognition by NK cells when combined with an activating signal. A candidate activating signal is MICA/B, which was shown to be expressed constitutively on epithelial cells.     

Transferrin receptors and natural killer cell lysis. A study using Colo 205 cells exposed to 60 Hz electromagnetic fields

The role of target cell transferrin receptors (TfR) in natural killer (NK) cell-induced cytolysis has been studied using Colo 205 cells in which prior exposure to 60 Hz electromagnetic fields produced constitutive expression of maximum numbers of TfR. It was found that NK cell cytolysis was decreased in those cells expressing the highest levels of TfR, while cells expressing the lowest level of TfR were lysed to the greatest extent. Furthermore, pretreatment of target cells with, and the continued presence in the assay medium of, iron transferrin produced, in general, only a slight decrease in cytolysis. Incubation of cells with apotransferrin also produced only a slight decrease in cytolysis, while incubation with zinc transferrin produced a greater decrease in cytolysis. This latter effect was particularly dramatic for cells that had been exposed previously to either a 60 Hz magnetic field alone (1.0 gauss) or to a combined electric + magnetic field (current density 300 mA/m2, 1.0 gauss). In addition to indicating a discordance between TfR expression on target cells and their sensitivity to NK cell cytolysis, the data presented provide a further indication of cellular membrane changes produced by exposure to 60 Hz electromagnetic fields.     

Prostaglandin E2 receptor activity and susceptibility to natural killer cells.

We have described a high-affinity receptor for prostaglandin E2 (PGE2) present on metastatic murine mammary tumor cells. Pharmacologic antagonism of this receptor increases metastatic potential. In the present study, we have asked whether the binding activity of PGE on tumor target cells plays a role in natural killer (NK)-target cell interactions. We have used three unrelated PGE-receptor antagonists, SC19220, LEO101, and AH6809, to show inhibition of [3H]PGE2 binding to YAC-1 cells and inhibition of PGE2-mediated elevation of intracellular cyclic AMP (cAMP). Addition of any of these three receptor antagonists to standard 4-h 51Cr-release assays inhibits YAC-1 lysis by NK-enriched populations from murine spleen. This is the first report that antagonism of PGE binding affects NK activity. Our studies demonstrate that these effects are mediated through inhibition of target-effector cell conjugate formation. Studies in which effector and target cells were pretreated separately indicate that the PGE-mediated effects are expressed at the target cell level.     

Human natural killer cells suppress the proliferation of B cells

We have investigated the suppressive effect of human natural killer (NK) cells on autologous B-cell proliferation. Removal of NK cells by anti-NK-cell monoclonal antibodies (CD16, Leu 11b; Leu 7) increased by 2-3-fold the proliferative response of purified B cells activated by anti-mu and B-cell growth factor (BCGF). The inhibitory effect of NK cells was observed using recombinant IL-2 or semi-purified BCGF-I as sources of BCGF. Moreover NK cells, highly purified by centrifugation on a Percoll discontinuous density gradient, suppressed the proliferative response of purified autologous B cells activated by anti-mu and BCGF. These results show a suppressive effect of human NK cells on B-cell proliferation in vitro.     

Hybrid antibody-mediated lysis of virus-infected cells

Previously, cytotoxic T lymphocytes (CTL) had only been focused by hybrid antibodies to normal, cell-surface proteins and haptenated surface structures. In this report, we extend the application of this technology to mediate lysing of virus-infected cells by nonspecific CTL. Heteroconjugated antibodies between the anti-T cell antigen receptor antibody, F23.1, and monoclonal antibodies against either the hemagglutinin or nucleoprotein of the influenza virus PR/8 were constructed. We show in the present report that these bispecific constructs can target virus-infected cells for lysis according to the specificity of the virus-protein reactive monoclonal antibodies. Furthermore, even a virus protein that is only expressed in small quantities on the cell surface, such as nucleoprotein, can be exploited as target structure for heteroconjugated antibodies. These studies show that hybrid antibodies can focus a CTL response on virus-infected cells which might in the future be used to mount an immune response in situations without sufficient normal cellular defense.