Migratory response of human natural killer cells to lymphotactin

Lymphotactin (Lptn) is a new protein belonging to the C or γ subfamily of chemokines with only two of the four cysteine residues. Lptn was reported to act specifically on T lymphocytes and not on monocytes and neutrophils. To understand better the spectrum of action of Lptn we have examined its ability to induce natural killer (NK) cell migration. Freshly isolated human NK cells as well as long-term cultured NK cells propagated in interleukin-2 (IL-2)-containing medium migrated in response to Lptn. Optimal activity was observed at concentrations ranging from 50 to 200 ng/ml, and the efficacy was comparable to that of MCP-1, the prototype of C-C chemokines. Migration in response to Lptn was chemotaxis rather than chemokinesis as determined in a checkerboard analysis. Migration of NK cells was comparable to that observed with T lymphocytes from the same donor, under the same experimental conditions. Finally, in contrast to other cytokines (IL-2 and IL-12) which in addition to chemotaxis augment NK cell adhesion to endothelial cells in vitro, Lptn did not affect the adhesiveness of NK cells to vascular endothelium.     

Cells with natural killer activity in human rabies.

Cytotoxic lymphocyte function in 13 patients with rabies was studied by counting the number of CD56 cells and assessing natural killer (NK) cell activity. There was no significant difference in the number of killer cells between rabies patients and 31 normal controls (P greater than 0.05). Two of six non-fatal encephalitic patients due to causes other than rabies had reduced CD56 numbers. Base-line NK cell responses versus K562 cell targets did not differ among the normal control and rabies groups (P greater than 0.05). Study of the non-rabies encephalitis group showed heterogeneous results with wide variation. Significant enhancement of NK activity was seen in four rabies patients and in 10 normal control subjects tested after interferon-alpha (IFN-alpha) and IL-2. None of the four patients with encephalitis due to causes other than rabies showed such enhancement. Our results suggest that NK cells of rabies patients are not fully stimulated and that this might contribute to the virulence of rabies. The cause of this phenomenon remains unknown.     

Biological response modifier enhances the activity of natural killer cell against human cytomegalovirus-infected cells

Peripheral blood lymphocytes (PBL) from two human cytomegalovirus (CMV)-seronegative donors and eight CMV-seropositive donors were cultured for 3 days with or without the biological response modifier OK-432 and examined for lysis of K562 cells and CMV-infected MRC-5 cells. OK-432-stimulated PBL exhibited significantly greater natural killer (NK) activity than did unstimulated PBL. There was no difference in activity of NK cells in PBL prepared from CMV-seronegative and -seropositive donors. Antibody-complement depletion studies suggested that OK-432-stimulated NK activity was associated with Leu-7-positive cells. The ability of OK-432 to sustain the NK activity in PBL was decreased when the CD4-positive population of lymphocytes was eliminated by antibody-complement depletion prior to OK-432 stimulation. The ability of OK-432 to sustain the NK activity of PBL was also significantly decreased in the presence of monoclonal antibody against recombinant human interleukin-2. The results suggest that the activity of human NK cells against K562 and CMV-infected MRC-5 target cells can be sustained in vitro by OK-432-stimulated T-helper cells and that the effect of the T-helper cells is mediated, at least in part, by interleukin-2.     

In vitro expanded human invariant natural killer T-cells promote functional activity of natural killer cells

Invariant natural killer T (iNKT) cells play a pivotal role in cancer immunity through trans-activation of effector cells via swift cytokine secretion. In mice, iNKT cell activation by alpha-galactosylceramide (alpha-GC) induces potent NK cell-mediated anti-tumour effects. Here we investigated whether human iNKT cells could enhance NK cell functional activity in vitro. iNKT cell activation by alpha-GC treatment of peripheral blood mononuclear cells (PBMC) was not sufficient to enhance NK cell effector functions. However, addition of in vitro expanded iNKT cells to PBMC enhanced NK cell-mediated cytotoxicity in an alpha-GC-dependent manner. NK cell activation by iNKT cells was primarily mediated by soluble factors, and could be enhanced by the NK cell activating cytokine IL-21. These results suggest that adoptive transfer of ex vivo expanded iNKT cells will enhance NK cell function and is expected to enhance the efficacy of cancer immunotherapy, particularly in combination with IL-21 and alpha-GC.     

Phospholipase C activation in the cytotoxic response of human natural killer cells requires protein-tyrosine kinase activity.

Treatment of highly purified natural killer (NK) cells with the protein-tyrosine kinase (PTK) inhibitors, genistein and herbimycin A, diminished their ability to lyse K562 target cells by as much as 100%. The ability of NK cells to bind to K562 cells was not affected by PTK inhibition. However, activation of phospholipase C (PLC) in response to K562 cell binding (as measured by inositol phosphate turnover) was decreased by as much as 75% when PTK activity was inhibited. Furthermore, there was an increase in tyrosine phosphorylation of NK cell PLC gamma 2 after exposure to K562 target cells. These data indicate that a PTK is involved in the activation of NK PLC by tumour target cells in the cytotoxic response.     

Lectin-binding characteristics of human natural killer cells.

Human natural killer (NK) cells separated initially by density centrifugation of lymphocytes (E+) forming rosettes with sheep red blood cells (SRBC), were further fractionated on gradients of bovine serum albumin (BSA). Low density fractions contained effector cells which displayed high cytotoxicity against the NK-sensitive erythroleukaemic cell line, K562. These low density cells, which expressed receptors for Fc and the monoclonal antibody OKMI, showed enhanced cytotoxicity when treated with lymphoblastoid interferon (IFN-alpha). They also showed an increased response to phytomitogen in comparison with unseparated cells or those recovered from high density fractions. Two lymphocyte subsets one of high and one of low lectin binding capacity were identified in the E+ populations by their reactivity with Lens culinaris agglutinin (LCA). High LCA binding was observed only in low density fractions and was associated with a marked enrichment of NK activity. This property was used to separate the NK active population in E+ cells by fluorescence-activated cell sorting (FACS). These data add a new dimension to the cell surface properties of human NK cells and suggest the presence of LCA-reactive glycoproteins which are either enriched in, or uniquely associated with, cells of the NK subset. The experiments indicate that lectins can serve as useful probes of lymphocyte function and provide the basis for effective cell sorting.     

Compartmentalization of human natural killer cells

Human natural killer (NK) cells are bone marrow-derived cells that are found in the bloodstream, but can extravasate into various tissue sites upon inflammation. NK cells that migrate toward inflamed sites must be activated prior to their extravasation. However, the factors responsible for NK cell compartmentalization are not clearly defined. Resting human NK cells (CD16(-) and CD16(+)) express constitutive chemokine receptors, as well as receptors that have both constitutive and inflammatory functions. Upon activation, NK cells up-regulate the expression of the inflammatory chemokine receptors which facilitate their distribution into inflammatory sites. However, chemokines are not expected to play any role in maintaining resting NK cells in the blood circulation. In contrast, members of the lysolipids which are abundant in the bloodstream may be the major factors responsible for maintaining resting NK cells in the bloodstream, and also for facilitating their extravasation into inflamed tissues. Both resting and activated NK cells express receptors for various lysolipids. Hence, chemoattractants which include chemokines and lysolipids have important roles in determining the compartmentalization of NK cells where resting NK cells are found in the blood circulation, and activated NK cells extravasate into inflamed sites.     

Two monoclonal antibodies identifying a subset of human peripheral mononuclear cells with natural killer cell activity

Two monoclonal antibodies (H25 and H366) raised against the cultured T lymphoid cell line HSB-2 were shown to define a subset of peripheral mononuclear cells with natural killer and killer cell activity. The two antibodies show similar tissue distributions. Radioimmune trace binding assay shows that H25 and H366 react with all T cell lines tested and with the monocyte line U937, but weakly or not at all with cell lines of B, myeloid or erythroid origin. Both antibodies react with about 10% of E rosette-forming cells and a proportion of nonrosetting lymphocytes and monocytes. They do not react with B lymphocytes, granulocytes, red cells or platelets. A proportion of thymocytes and low numbers of tonsil and spleen mononuclear cells are positive. H25+ and H366+ lymphocytes are medium-sized cells with abundant granular cytoplasm, and most carry Fc receptors for IgG. H25 and H366 immunoprecipitate two polypeptide chains of 96 and 53kDa from surface-labeled HSB-2 cells.     

Regulation of human natural killer activity with autologous interferon

A study is performed of the effects of α-interferon and γ-interferon induced in 8 healthy donors and 9 patients with multiple sclerosis on thein vitro cytotoxic activity of natural killers in an autologous and allogeneic systems. The general characteristics of regulation are estimated on the basis of the results. There is found to be an inhibitor regulating the effect of interferon on natural killer activity, which is produced in parallel with interferon in response to interferon induction, the efficacy of this inhibitor being dependent on the initial natural killer activity; the inhibitor is absent in commercial interferon preparations. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 9, pp. 281–284, September, 1994     

Modulation of natural killer cells by human cytomegalovirus.

Human cytomegalovirus (HCMV) causes lifelong, persistent infections and its survival is under intense, continuous selective pressure from the immune system. A key aspect of HCMV's capacity for survival lies in immune avoidance. In this context, cells undergoing productive infection exhibit remarkable resistance to natural killer (NK) cell-mediated cytolysis in vitro. To date, six genes encoding proteins (UL16, UL18, UL40, UL83, UL141 and UL142) and one encoding a microRNA (miR-UL112) have been identified as capable of suppressing NK cell recognition. Even though HCMV infection efficiently activates expression of ligands for the NK cell activating receptor NKG2D, at least three functions (UL16, UL142 and miR-UL112) act in concert to suppress presentation of these ligands on the cell surface. Although HCMV downregulates expression of endogenous MHC-I, it encodes an MHC-I homologue (UL18) and also upregulates the expression of cellular HLA-E through the action of UL40. The disruption of normal intercellular connections exposes ligands for NK cell activating receptors on the cell surface, notably CD155. HCMV overcomes this vulnerability by encoding a function (UL141) that acts post-translationally to suppress cell surface expression of CD155. The mechanisms by which HCMV systematically evades (or, more properly, modulates) NK cell recognition constitutes an area of growing understanding that is enhancing our appreciation of the basic mechanisms of NK cell function in humans.     

Direct activity of human T lymphocytes and natural killer cells against Cryptococcus neoformans.

Lymphocytes constitute a critical component of host defenses against cryptococcosis. Previously, we demonstrated that human lymphocytes cultured with interleukin-2 formed conjugates with, and directly inhibited the growth of, Cryptococcus neoformans. Here, we explore the anticryptococcal activity of freshly isolated, highly purified populations of human peripheral blood lymphocytes. Lymphocytes were incubated with encapsulated C. neoformans for 24 h, after which the lymphocytes were lysed, dilutions and spread plates were made, and CFU were counted. Fungistasis was determined by comparing growth in wells with and without lymphocytes. Nylon wool-nonadherent peripheral blood mononuclear cells (NWNA PBMC) were highly fungistatic, even if either T cells or natural killer (NK) cells were depleted by panning. A mixed population of T cells and NK cells, obtained by rosetting NWNA PBMC with sheep erythrocytes, completely inhibited cryptococcal growth, whereas the nonrosetting cells had little fungistatic activity. CD4+, CD8+, and CD16/56+ lymphocytes, isolated by positive immunoselection, had potent growth-inhibitory activity. In contrast, purified B cells had no activity. Fungistasis was seen even in the absence of opsonins. Antifungal activity was markedly diminished when surface receptors on NWNA PBMC were cleaved by treatment with trypsin or bromelain. Supernatants from stimulated lymphocytes or concentrated lymphocyte sonicates were not active. Lymphocyte-mediated fungistasis was seen with two different strains of C. neoformans. CD4+, CD8+, and CD16/56+ lymphocytes formed conjugates with C. neoformans, as observed under Nomarski differential interference contrast microscopy and videomicroscopy. These data demonstrate that freshly isolated peripheral blood T cells and NK cells have the capacity to bind and directly inhibit the growth of C. neoformans.     

Proliferative and cytotoxic response of human natural killer cells exposed to transporter associated with antigen-processing-deficient cells

In transporter associated with antigen-processing (TAP)-deficient patients affected by a severe downmodulation of human leucocyte antigen class I (HLA-I) molecules, natural killer (NK) cells have an increased expression of the inhibitory receptor CD94/NKG2A. Focusing our attention on NK cells, we have investigated the phenotype, function and proliferative response of peripheral blood lymphocytes (PBLs) derived from healthy donors after coculturing with TAP (T2)- or HLA-I-deficient (721.221) cell lines and their related HLA-I-expressing transfectants (T3 and DT360, respectively). After 4 days, NK cells cocultured with T2 cells had a threefold increased CD94 expression compared to NK cells cocultured with T3. This increase was due to proliferation of the CD56brightCD94bright subset. In contrast, expression of other inhibitory receptors [killer cell immunoglobulin (Ig)-like receptors] was variable during time and was not related to HLA-I molecules expressed by stimulating cells. Similar results were obtained using HLA-I-deficient cells (721.221). The PBLs cocultured for 4 days with T2 cells displayed enhanced cytotoxic responses. The results suggest that CD56brightCD94bright NK cells are induced to proliferate and kill in response to a TAP-deficient environment. The changes seen in the NK-cell compartment were partially contributed by T lymphocytes present in the coculture. These data could explain the increased CD94 expression and autoimmune manifestations observed in TAP-deficient patients.     

Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells. IX. The quantitation of natural killer cell activity

On analysis ofin vitro assays of human natural killer (NK) cell function the inadequacy of commonly used methods of expressing lytic activity was apparent. A comparison was made of the data obtained using modifications of two equations—the simple exponential fit and the von Krogh equations. Both of these equations were found to satisfy the following essential criteria for use in these assays. First, the majority of the results obtained in the chromium-release assay could be used in data reduction; second, the resultant dose-response curve was reduced to linearity; and third, a single numerical expression was obtained which was directly proportional to the cytotoxic activity. Of the two methods the more conventional exponential fit was found to be the simpler to use. The closeness of fit of the experimentally derived data to the ideal curves did not support the possibility that normal lymphocyte preparations contain suppressor cells capable of inhibiting NK activity. Data have also been presented showing that NK-sensitive targets could be categorized with respect to their susceptibility by comparing the slopes of the target cell survival curves obtained using the exponential fit equation. These observations are relevant to the accurate assessment of NK activity in patient populations and to the determination of the effects of disease and its treatment on this activity.     

The dialogue between human natural killer cells and dendritic cells

The interaction of NK cells with dendritic cells (DCs) appears to play an important role in both innate and adaptive immune responses to pathogens. In peripheral inflamed tissues the simultaneous engagement of receptors for danger (e.g. Toll-like receptors), which are expressed by both NK cells and DCs, results in cell activation and the acquisition of functional properties necessary for controlling, and possibly rapidly eliminating, pathogens by innate effector mechanisms. Moreover, NK cells are needed to select the most appropriate DCs that display the functional properties suitable for subsequent T-cell priming. This NK-cell-mediated programming of DC maturation is modulated by cytokines released during the early stages of inflammatory responses (i.e. IL-12, IFN-gamma, IL-4). NK cells and DCs continue their interactions in secondary lymphoid organs where both cell types play a role in the control of T-cell priming.     

Effects of biological response modifiers on lung natural killer activity.

Natural killer (NK) activity plays an important role in host defense against tumors, especially once augmented by immunomodulators. It is likely that the modulation of NK cells is a reflection of the environment in which they reside. The current study was undertaken to characterize the response profile of lung interstitial lymphocyte natural killer (LLNK) activity to various biological response modifiers (BRM) in vitro after short term incubation (18h). The presented data show that treatment of lung lymphocytes with human recombinant interleukin 2 (rIL-2), purified rat interferon alpha/beta (IFN-alpha/beta), or murine recombinant tumor necrosis factor alpha (rTNF-alpha) resulted in a dose-dependent increase in LLNK activity. The maximum stimulation was similar for rIL-2 and IFN-alpha/beta, although a much higher concentration of IFN-alpha/beta was required to reach this level of stimulation. The maximum response to rTNF-alpha treatment was about half that seen with rIL-2 or IFN-alpha/beta and it, too, required a high concentration. By contrast, rat recombinant interferon gamma (rIFN-gamma) or murine recombinant interleukin 1 (rIL-1) failed to alter LLNK activity significantly when used alone. Furthermore, doses of IFN-alpha/beta and rTNF-alpha that had little enhancing effect were able to synergize with a suboptimal dose of rIL-2, whereas rIL-1 and rIFN-gamma failed to do so. These data demonstrate the response of lung NK activity to BRM treatment, which is important for the responsible and effective use of BRM. However the spectrum of lung NK cell response to BRM is smaller than that previously reported for NK cells from other anatomic compartments.     

Studies of human natural killer cells. III. Neutropenia associated with unusual characteristics of antibody-dependent and natural killer cell-mediated cytotoxicity

A 52-year-old Caucasian man with chronic neutropenia and recurrent infections was found to have an increased proportion of peripheral T lymphocytes having Fc receptors for IgG (T(). Although levels of antibody-dependent cell-mediated cytotoxicity (ADCC) and natural killing (NK) by unfractionated lymphocytes were similar to those of a control donor, the frequency of NK cells was markedly increased. Removal of E rosette-forming cells eliminated both NK and ADCC by the patient's peripheral blood, in marked contrast to theenhanced cytotoxicity seen with control lymphocytes. Both normal and patient ADCC and NK functions were removed by depletion of Fc receptor-bearing cells. These depletion experiments proved that all of the patient's killer cells were E rosetteforming T cells, in contrast to the heterogeneous pattern of Null and T killer cells seen in the blood of normal donors. The homogeneity of the T proliferation suggested that ADCC and NK were mediated by the same cell type, albeit acting by different mechanisms. The addition of the patient's serum and lymphocytes to chromiumlabeled normal granulocytes caused a low but significant level of cytotoxicity, indicating that the patient's neutropenia may have been caused by a similar mechanismin vivo. There was no evidence of complement-dependent serum antibody-mediated neutrophil lysis, but one serum sample taken over the course of the patient's disease agglutinated granulocytes from four of five donors tested.     

Reduced IL-12 level correlates with decreased IFN-gamma secreting T cells but not natural killer cell activity in asthmatic children.

BACKGROUND: Accessory cells such as macrophages and natural killer cells, and their cytokines such as IL-10, IL-12, and IFN-gamma have been suggested to play a critical role in the development of T helper cells. OBJECTIVE: Both natural killer cells and peripheral blood mononuclear cells were isolated and stimulated for their ability in producing cytokines. In addition, the percentage of IFN-gamma-secreting cells was analyzed with the method of intracellular staining. RESULTS: The data suggested (1) no significant difference between asthmatic children and normal controls in number, cytotoxicity, and IFN-gamma production of purified NK cells; (2) decreased secretion of IL-12 by stimulated peripheral blood mononuclear cells in asthmatic children compared with normals (P < .05); (3) decreased production of IFN-gamma by PBMC from asthmatic children compared with normals (P < .05); and (4) intracellular expressed IFN-gamma level was lower in CD4+ T cells of asthmatic children (P < .05). CONCLUSION: The results suggested that IL-12 produced predominantly by macrophages and associated decreased IFN-gamma-secreting CD4+ T cells play a critical role in the pathogenesis of asthma.     

Association of alpha interferon production with natural killer cell lysis of U937 cells infected with human immunodeficiency virus.

Mononuclear leukocytes from human immunodeficiency virus (HIV)-seronegative and -seropositive homosexual men lysed HIV-infected U937 cells to a significantly greater degree than uninfected U937 cells. Depletion of cell subsets with monoclonal antibodies and complement indicated that the effector cells were primarily of the CD16+ phenotype. Acid-stable alpha interferon (IFN-alpha) production induced by the HIV-infected cells correlated with, although was not an absolute requisite for, preferential lysis of the infected targets. The activity of these CD16+, natural killer (NK) cells decreased in relation to the duration of HIV infection and the presence of acquired immunodeficiency syndrome. Pretreatment of peripheral blood mononuclear cells from HIV-seronegative subjects, but not HIV-seropositive men, with IFN-alpha or recombinant interleukin-2 enhanced lysis of both uninfected and HIV-infected U937 cells. These results suggest that IFN-alpha-associated, NK-like mechanisms are active in the cytotoxic response against HIV-infected cells and that HIV infection results in an early and progressive depression of such responses. Prospective investigations may be useful in determining the role of this NK cell response in the natural history and pathogenesis of HIV infection and the efficacy of therapeutic modalities.     

Heterogeneity of human natural killer cells in the spleen.

Natural killer cells have an important role in tumour and viral defence and immunoregulation. In the present study the pan-NK cell monoclonal antibody NKH1 was utilized to study the heterogeneity of NK cells in the human spleen. Using one and two colour analysis it could be demonstrated that the majority of NKH1+ NK cells are located in the red pulp and marginal zone whereas only a minor subset is found in the lymph follicles. In the red pulp, NK cells resemble those of peripheral blood in terms of function and phenotype. In contrast, the few NK cells in the lymph follicles are mostly NKH1+, CD2- and CD16- and thus express a unique phenotype. The analysis of tissue distribution provides a basis for further studies on NK cell kinetics and differentiation.