Modulation of ongoing human immunoglobulin synthesis by natural killer cells

Freshly separated human NK cells (NKH-1+) inhibited IgE synthesis from IgE myeloma U266/AF-10 as much as 70% whereas they enhanced IgG and IgA synthesis 200 and 500% from the lymphoblastoid cell lines GM-1500 and GM-1056, respectively. The inhibition of IgE synthesis by NK cells was due to a direct cytolytic effect on AF-10. This could be reversed using K562 cells in a cold target competition assay. NK cells also inhibited spontaneous IgE as well as IgG and IgA synthesis from B cells of highly atopic donors. On the other hand the enhancement of Ig secretion by NKH-1+ cells was shown to be mediated by soluble factors released from NK cells. Furthermore when NK cells were preincubated with immune complexes (IgE-IC) constructed of human IgE and mouse IgG1 monoclonal anti-human IgE, inhibition of IgE synthesis was reversed, and in some cases actual enhancement of IgE synthesis was observed, while enhancement of IgG and IgA synthesis was not affected. In contrast to NK cells, T cells depleted of NK cells (T-NK), when activated by IgE-IC, suppressed IgE synthesis in an isotype specific fashion. Thus, NK and T-cell modulation of ongoing Ig synthesis involve distinct mechanisms.     

Suppressive effect of human natural killer cells on pokeweed mitogen-induced B cell differentiation

The suppressive effect of human natural killer (NK) cells on B cell differentiation induced by pokeweed mitogen (PWM) was investigated. By using Percoll discontinuous density gradient centrifugation, peripheral blood nonphagocytic and nonadherent mononuclear cells were divided into low and high density fractions for which NK cells (Large granular lymphocytes, LGL) and T cells were enriched, respectively. These fractionated mononuclear cells were co-cultured with purified autologous B cells in the presence of PWM, and were examined for their helper and suppressor activities on differentiation of B cells to immunoglobulin-(IgM and IgG) producing cells by a highly sensitive reversed hemolytic plaque assay. The T cell-enriched high density fractions provided help for B cell differentiation to levels higher than that of unfractionated mononuclear cells. On the other hand, the NK-enriched low density fractions did not show helper activity, and when added to the culture of B cells plus helper T cells, they markedly suppressed B cell differentiation. This suppressive activity, as well as the NK cytotoxicity of the NK-enriched fractions, was abrogated by treatment of the cells with monoclonal antibody against human NK cells (HNK-1), but not against T cells (OKT3) in the presence of complement. NK cells also suppressed PWM-driven B cell differentiation in the presence of T4+ (helper/inducer T) but not T8+ (cytotoxic/suppressor T) cells; however, they showed no inhibition of soluble factor-induced B cell differentiation assayed in the absence of helper T cells. It is thus concluded that human peripheral blood NK cells exhibit an ability to suppress PWM-driven B cell differentiation, possibly by acting through the effect on helper T cells but not directly on B cells.     

Limiting dilution analysis of Epstein-Barr virus-induced immunoglobulin production

The major goal of this work is to establish a culture system for the growth of human B lymphocytes at the single-cell level so that the immunoglobulin secreted by the clonal progeny of that cell can be analyzed. A method which involves culturing small numbers (1–1000) of lymphocytes, which have been infected with Epstein-Barr virus (EBV) prior to plating, in round-bottom microtiter plates is described. A feeder layer of irradiated (2500 R) umbilical cord blood lymphocytes to which phytohemagglutinin has been added was found to be optimal. Culture supernatants collected from 3 to 6 weeks postinfection are assayed for the production of IgG and IgM by radioimmunoassay in order to determine the overall cloning efficiency of the system. We have shown that up to 33% of surface Ig-positive cells produce detectable clones in this system. Umbilical cord blood cells are superior to T-cell and macrophage cell lines as feeder layers. Furthermore, culture supernatants from phytohemagglutinin-stimulated umbilical cord lymphocytes do not adequately replace these cells. We also observed that while most IgM-secreting clones continued to produce immunoglobulin during the 7-week time period analyzed, the majority of IgG-secreting clones had a relatively short half-life in vitro. This culture system allows us to examine a significant proportion of the human B-cell population and carry out studies on the frequency of specific antibody- and isotype-producing clones.     

Suppressive effect of interferon-beta-activated natural killer cells on lipopolysaccharide-induced B cell differentiation of MRL/Mp-lpr/lpr mice

The suppressive effects of mouse recombinant interferon-beta (IFN-beta) on B cell differentiation of MRL/Mp-lpr/lpr (MRL/1) mouse, a model of autoimmune diseases, and C3H/H2 mice, a normal situation, were investigated. Spleen mononuclear cells were cultured in the presence of lipopolysaccharide (LPS), and the suppressive effect of IFN-beta was examined on differentiation of B cells to plaque-forming cells (PFCs) by highly sensitive reversed hemolytic plaque assay. IFN-beta (5,000-10,000 units/ml) suppressed more than 50% of PFCs of both MRL/1 and C3H/H2 mice. This suppressive activity as well as the cytotoxicity of natural killer (NK) cells enhanced by IFN-beta was abrogated by treatment of the spleen cells with anti-asialo GM1 antibody in the presence of complement. This suppressive activity was also abrogated by intravenous administration of 20 microliter/mouse of anti-asialo GM1 12 hr before cultivation of spleen cells. These results suggest that NK cells activated by IFN might be responsible for the immunoregulation in autoimmune diseases.     

Cytotoxic action of natural killer cells on human tumor cells

Natural killer cell cytotoxicity was studied in a 18-hour 51Cr-release assay in the cultures of human tumor target cells: K562 leukemia and lung adenocarcinoma (LAC) cells. The mean cytotoxic value was similar for K562 and LAC cells: 36.13 +/- 3.23% and 40.78 +/- 3.43%, respectively, although significant individual variability was recorded. The similar cytolytic action of blood mononuclear cells (MNC) on the two tumor lines was observed in 30% of normal donors. MNC from 30% donors produced more pronounced lytic action on K562 cells while MNC from other 30% donors lysed mainly LAC cells. In the competitive inhibition test cold K562 cells more effectively than cold LAC cells suppressed the MNC-induced lysis of both K562 and LAC cells.     

Evidence for early infection of nonneoplastic natural killer cells by Epstein-Barr virus

We examined lymph nodes and tonsils from patients with infectious mononucleosis by combined detection of EBV-encoded RNA and a specific marker of natural killer (NK) cells, PEN5. A small number of Epstein-Barr virus (EBV) latently infected nonneoplastic NK cells were detected. Our data demonstrate that NK cells are natural targets of EBV and that infection of these cells is an early event observed during primary EBV infection.     

Mechanism of Epstein-Barr virus-induced human B-lymphocyte activation

The mechanism of Epstein-Barr virus (EBV) activation of human B lymphocytes toward Ig synthesis was investigated in a direct anti-sheep red blood cell (SRBC) antibody plaque-forming cell (PFC) system. Exposure of human peripheral blood lymphocytes to EBV in vitro resulted in an anti-SRBC PFC response in 12 of 16 normal donors. The EBV-induced anti-SRBC PFC response did not require the presence of autologous helper T lymphocytes, but was inhibited by the presence of autologous concanavalin A-generated suppressor T cells. Live virus was required for B-cell activation since the EBV-induced PFC response was inhibited by exposure of EBV to ultraviolet light. Using fluorescent techniques which detected simultaneous intracytoplasmic (ICP) Ig production and the presence of EB nuclear antigen, we found that most, if not all, EBV-activated ICP Ig-positive cells were virally infected. Thus, these studies suggest that viral infection of Ig-producing B lymphocytes is required for EBV-induced polyclonal B-lymphocyte activation. Although the participation of T lymphocytes is not required for the induction of EBV-triggered B-lymphocyte Ig production, activated T lymphocytes can serve as modulators of this response.     

Recognition of vaccinia virus-infected cells by human natural killer cells depends on natural cytotoxicity receptors

Natural Killer (NK) cells are important in the immune response to a number of viruses; however, the mechanisms used by NK cells to discriminate between healthy and virus-infected cells are only beginning to be understood. Infection with vaccinia virus provokes a marked increase in the susceptibility of target cells to lysis by NK cells, and we show that recognition of the changes in the target cell induced by vaccinia virus infection depends on the natural cytotoxicity receptors NKp30, NKp44, and NKp46. Vaccinia virus infection does not induce expression of ligands for the activating NKG2D receptor, nor does downregulation of major histocompatibility complex class I molecules appear to be of critical importance for altered target cell susceptibility to NK cell lysis. The increased susceptibility to lysis by NK cells triggered upon poxvirus infection depends on a viral gene, or genes, transcribed early in the viral life cycle and present in multiple distinct orthopoxviruses. The more general implications of these data for the processes of innate immune recognition are discussed.     

Radiosensitivity of human natural killer cells: binding and cytotoxic activities of natural killer cell subsets

The sensitivity of human natural killer (NK) cell activities (both binding and killing) after exposure of peripheral blood mononuclear cells to different doses of gamma radiation was studied. A panel of monoclonal antibodies was used to identify the NK and T-lymphocyte subsets and to evaluate their radiosensitivity. Peripheral blood mononuclear cells were irradiated with low (2-6 Gy) and high (10-30 Gy) doses and NK cell binding and cytotoxic activity against K562 target cells were studied after 3 h and 48 h in culture. The primary damage to NK cell activity was identified at the postbinding level and affected mainly the lytic machinery. After 48 h culture postirradiation, an overall depression of cytotoxic activity was observed, but ionizing radiation produced either a selection of the more cytotoxic NK cell subsets, which therefore might be considered more resistant to radiation damage than the less cytotoxic NK cells, or a long-term stimulation of cytotoxic activity in surviving cells.     

Characterization of human natural killer cells for therapeutic use

As a part of the innate immune system, natural killer (NK) cells are cytotoxic lymphocytes that can exert cytotoxic activity against infected or transformed cells. Furthermore, due to their expression of a functional Fc receptor, they have also been eluded as a major effector fraction in antibody-dependent cellular cytotoxicity. These characteristics have led to multiple efforts to use them for adoptive immunotherapy against various malignancies.  There are now at least 70 clinical trials testing the safety and efficacy of NK cell products around the world in early-phase clinical trials. NK cells are also being tested in the context of tumor retargeting via chimeric antigen receptors, other genetic modification strategies, as well as tumor-specific activation strategies such as bispecific engagers with or without cytokine stimulations. One advantage of the use of NK cells for adoptive immunotherapy is their potential to overcome HLA barriers. This has led to a plethora of sources, such as cord blood hematopoietic stem cells and induced pluripotent stem cells, which can generate comparatively high cytotoxic NK cells to peripheral blood counterparts. However, the variety of the sources has led to a heterogeneity in the characterization of the final infusion product. Therefore, in this review, we will discuss a comparative assessment strategy, from characterization of NK cells at collection to final product release by various phenotypic and functional assays, in an effort to predict potency of the cellular product.     

Lysis of human solid tumor cells by lymphokine-activated natural killer cells

The ability of NK cells to lyse noncultured solid tumor cells was investigated, and the results were compared with lysis of K562. Purified NK cell fractions separated by either Percoll centrifugation or a cell sorter exhibited higher level of lysis against noncultured melanoma cells than did NK-depleted cell fractions. However, the level of lysis was low (less than 10% lysis). Adding recombinant interleukin 2 (rIL 2) to the 4-hr assay induced significant lysis (more than 10%) of noncultured melanoma cells in 18 of 23 (78%) Percoll-enriched NK cell fractions and seven of 11 (64%) sorted Leu-11a+ cells at an E:T ratio of 80 and 10, respectively. In contrast, only two of 13 (14%) PBMC, five of 17 (29%) Percoll-decreased NK cell fractions, and one of 12 (8%) sorted Leu-11a- cells lysed noncultured melanomas in the presence of rIL 2. rIL 2 induced NK cells to lyse noncultured lung and breast cancer cells, as well as melanoma tumors. Exposure of NK cells to 2000 rad radiation abrogated the rIL 2-induced cytotoxicity against noncultured melanomas. Preculture of PBMC for 18 hr with recombinant interferon-gamma (rIFN-gamma) resulted in a modest level of lysis of non-cultured melanomas by sorted Leu-11a+ cells. Adding rIL 2 to the assay increased the cytotoxic activity in both rIFN-gamma-activated Leu-11a+ and Leu-7+ NK subsets. The level of noncultured tumor lysis correlated well with that of K562 lysis in all of the experiments. Purified NK cell fractions in rIL 2 cultures increased cytotoxic activity against noncultured tumor cells with incubation time for up to 3 days, and the level of NK cell-mediated lysis was dependent on both doses of rIL 2 and length of incubation. In contrast, both NK-depleted and sorted Leu-11a- cells demonstrated very low levels of solid tumor lysis after 3-day cultures with a high dose of rIL 2. Killer cell precursors induced by 3-day cultures of sorted cell fractions with rIL 2 and rIFN-gamma were found in both Leu-11a+ and Leu-7+ NK subsets, but not Leu-4+ or Leu-3a+ T lymphocytes. These results indicate that NK cells become cytotoxic for noncultured solid tumor cells by a brief contact with rIL 2, and increase cytotoxic activity after culture with rIL 2.     

Lysophosphatidylglycerol stimulates chemotactic migration in human natural killer cells

We observed that lysophosphatidylglycerol (LPG) stimulates chemotactic migration in human natural killer (NK) cells. The LPG-induced chemotactic migration of NK cells was completely inhibited by pertussis toxin (PTX). LPG also stimulated the extracellular signal-regulated kinase (ERK) and Akt activities in NK cells. LPG-stimulated ERK activity was inhibited by PTX, indicating the involvement of PTX-sensitive G-proteins. The preincubation of NK cells with an ERK inhibitor (PD98059) or phosphoinositide-3-kinase (PI3K) inhibitors (wortmannin and LY294002) completely inhibited LPG-induced chemotactic migration, suggesting the essential role of ERK and PI3K in the process. Moreover, LPG-induced chemotactic migration in NK cell was inhibited by Ki16425, an LPA_1/3 receptor-selective antagonist, suggesting the involvement of the Ki16425-sensitive G-protein coupled receptor (GPCR) in the process. Taken together, the results indicate that LPG stimulates chemotactic migration in NK cells through GPCR, suggesting a new function of LPG as a modulator of NK cell functioning.     

Effect of interferon-alpha on immunoglobulin synthesis by human B cells

We have investigated the effect of human recombinant interferon-alpha (IFN-alpha) on mitogen-induced immunoglobulin (Ig) production by peripheral blood mononuclear cells from normal individuals. Low concentrations (1 to 100 IU/ml) of IFN-alpha enhanced pokeweed mitogen-stimulated Ig production. In contrast, high concentrations of IFN-alpha (10(5) IU/ml) suppressed pokeweed mitogen-induced Ig production. Irradiation of T cells did not ablate the high dose suppression, indicating that suppression was not due to a radiation-sensitive T cell. Kinetic experiments revealed that IFN-alpha needed to be added to 10 day cultures within the first 72 hr for either enhancement or suppression to be noted. Preincubation of purified B cells with IFN-alpha suppressed Ig production as completely as when unfractionated mononuclear cells were incubated with IFN-alpha. On the other hand, preincubation of T cells or monocytes with IFN-alpha had no effect on subsequent Ig production in reconstituted mononuclear cell cultures. Mitogen-induced proliferation of purified B cells was not affected by IFN-alpha at any concentration, but Ig production by purified B cells stimulated with Staphylococcus aureus Cowan I or anti-mu and B cell differentiation factors responded to IFN-alpha with low concentration enhancement and high concentration suppression. Studies of Ebstein-Barr virus-transformed B cell lines showed that IFN-alpha caused a similar effect on the CESS line as on peripheral blood B cells, with low dose enhancement and high dose suppression of Ig production. Thus one IFN-alpha effect is to modulate Ig production, and this appears to be a direct effect on B cells. Combined with the data in the accompanying paper, the effects of IFN-alpha on B cell function are similar in vivo and in vitro.     

Evidence for a beta-adrenoceptor-mediated regulation of human natural killer cells

Pretreatment of lymphocytes (16 hr, 37 degrees C) with adrenaline at final concentrations of 10(-7) to 10(-9) M, followed by removal of the drug, increased natural killer (NK) cell activity vs K562 leukemic cells in a 4-hr 51Cr-release assay. The most efficient concentration of adrenaline was 10(-8) M; mean increase of NK activity over base-line activity for all donors examined was 30%. However, the individual response to adrenaline pretreatment was variable; in some donors, the effect was equal to maximal interferon (IFN) stimulation. Effects of adrenaline pretreatment were consistently reduced to base-line activity by co-incubation with the nonselective beta-adrenoceptor antagonist propranolol at 100-fold higher concentrations. The enhancing effect of adrenaline (10(-8) M) pretreatment was also observed after 1-hr pretreatment; this effect was prevented by simultaneous incubation with propranolol but was not affected by dex-propranolol. Direct addition of adrenaline to lymphocyte/target cell mixtures was inhibitory at 10(-6) M adrenaline concentration. The inhibitory effect of adrenaline in this assay was again completely prevented by propranolol and unaffected by dex-propranolol. The observed stimulatory effect of adrenaline pretreatment could not be ascribed to IFN production. Data presented indicate a dual effect of adrenaline on NK cell activity and suggest both a positive and a negative beta-adrenoceptor-mediated regulation of human NK cells.     

Characterization of an antigen expressed by human natural killer cells

A monoclonal antibody, anti-N901, was produced by fusing NS-1 myeloma cells with spleen cells of a mouse immunized with human CML cells. This antibody was reactive with a subpopulation of peripheral blood LGL, including the natural killer cells. Monocytes, granulocytes, B cells, T cells (T3+ cells), erythrocytes, and platelets were nonreactive. The N901-positive cells in the peripheral blood were heterogeneous with respect to expression of other cell surface antigens. The majority of N901+ cells co-expressed T11, Mo1, and HNK-1, whereas a smaller percentage expressed T8. Ia, T3, T4, Mo2, or B1 antigens were very uncommon on N901+ cells. The heterogeneity of the N901+ LGL was further investigated by examining the expression of N901 antigen on a series of cloned normal human NK cell lines. N901 antigen was expressed by each of the NK cell lines tested, and by a minority of cloned T cell lines without NK activity. Anti-N901 does not block NK activity and can be used to rapidly purify functional NK cells for further study.