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.     

Augmentation of natural and antibody-dependent cell-mediated cytotoxicity by pure human leukocyte interferon

Augmentation of the cytolytic activity of human natural killer cells and of antibody-dependent cell-mediated cytotoxicity has been attributed to human interferons. With the purification to homogeneity of human leukocyte interferon, it became possible to test directly whether pure interferon could increase the activity of these effector cells. Treatment of purified blood mononuclear cells with pure interferon resulted in substantial increases in natural killer cell activity and in antibody-dependent cell-mediated cytotoxicity. Concentrations of 10–100 units/ml of antiviral activity were sufficient to augment appreciably natural killer cell activity.     

Modulation of antitumoral antibody-dependent cellular cytotoxicity and natural killer activity by Adriamycin and daunorubicin

Mouse effector cells isolated from various anatomical sources failed to mediate antibody-dependent cellular cytotoxicity (ADCC) against alloantiserum-coated L1210 murine leukemia cell targets, whereas rat spleen cells appeared to be potent mediators of this activity. Following suppression of effector cell function 3 days after a single drug injection, the nylon wool non-adherent population of rat spleen cells from daunorubicin (DM)-treated rats demonstrated an increased ability to mediate ADCC compared to controls. Alternatively, although suppression occurred at day 7, no rebound enhancement was demonstrated by the same cell population isolated from Adriamycin (AM)-treated animals for as long as 12 days post-injection. Natural killer (NK) activity, measured as the ability of the nylon wool non-adherent rat spleen cell population to lyse uncoated L1210 cells, was modulated by drug treatment in a similar manner at each time point although the changes were not significant. In contrast to NK cells for which a substantial amount of activity remained adherent to nylon wool, all K cell activity was found in the non-adherent spleen cell population. The effector cell, in both cases, was not susceptible to antithymocyte serum plus complement treatment; however, NK activity appeared trypsin-sensitive whereas K cell activity did not. Therefore, AM and DM demonstrated different activities with regard toin vivo modulation of antitumoral ADCC.Supported in part by PHS Grant 5T32 GM07230 and a grant from the National Cancer Institute.     

Relevance of soluble cytotoxic factors generated by mycoplasma-contaminated targets to natural killer cell-mediated killing

The possible involvement of soluble cytotoxic factors (SCF) in the lytic mechanism of natural killer (NK) cell-mediated cytotoxicity (CMC) was investigated. Tumor target cells (TC) were examined for their ability to stimulate the release of SCF from human peripheral blood lymphocytes (PBL). SCF were detected when effector cells (EC) were stimulated with mycoplasma-contaminated (MC)-TC and with concanavalin A (Con A), but mycoplasma-free (MF)-TC were unable to induce SCF. Pretreatment of EC with beta-interferon augmented the production of SCF with MC-K562 and Con A, but not with MF-K562. However, MF-K562 once infected with the culture supernatant from MC-K562, as well as mycoplasmas concentrated from culture supernatant, were able to induce SCF. Only Con A was capable of generating cytotoxic supernatants from EC that had been inactivated with MF-K562 and from EC depleted of Leu 11b positive cells. Moreover, when EC were evaluated for NK activity after coculture with MF-K562, their ability to kill fresh MF-K562 in the standard NK-CMC assay was greatly reduced. This loss of lytic potential was not accompanied by SCF release. Collectively, these data suggest that mycoplasma organisms play a crucial role in the induction of human SCF, and the implication that SCF, including NK cytotoxic factors (NKCF), are the lytic mediators in NK-CMC needs to be re-evaluated.     

Azathioprine suppression of natural killer activity and antibody-dependent cellular cytotoxicity in renal transplant recipients

The relative effects of azathioprine (AZA) and prednisone (PRED) on natural killer (NK) activity and the antibody-dependent cellular cytotoxicity (ADCC) of K (killer) cells and the number of FcR and other lymphoid cells were examined in renal transplant recipients. In addition to both long-term (>6 months) and short-term (<6 months) transplant recipients receiving conventional AZA-PRED therapy, an important group of long-term recipients receiving PRED but not AZA was studied for the first time. Both NK activity and ADCC are profoundly reduced in the long-term AZA-PRED group but are normal in the long-term PRED-alone (no-AZA) group. The short-term AZA-PRED group exhibits NK and ADCC levels significantly lower than normal but not as low as those of the long-term AZA-PRED group. Patient groups with low NK and ADCC also have low circulating Fc receptor-bearing (FcR) cells. A single patient in the long-term AZA-PRED group was removed from AZA therapy, and approximately 3 months was required for the patient's suppressed NK and ADCC to return to normal. These findings indicate that AZA rather than PRED is the major drug important in suppressing ADCC and NK activity in renal transplant recipients. Several months are required for combination AZA-PRED therapy to reduce these cytotoxic activities. Similarly, several months are required for suppressed ADCC and NK activity to return to normal upon discontinuation of AZA.     

Natural killer cell activity in the rat. VI. Characterization of rat large granular lymphocytes as effector cells in natural killer and antibody-dependent cellular cytotoxic activities

The present study examined rat natural killer (NK) cells, which mediate not only NK activity but also antibody-dependent cellular cytotoxicity (ADCC). NK and ADCC activities were compared with regard to organ distribution, strain distribution, Percoll fractionation of the effector cells, effects of aging, and potential to be augmented by biological response modifiers (BRM). Like NK activity, appreciable ADCC activity was observed in peripheral blood leukocytes (PBL), splenic leukocytes (SPL), and peritoneal exudate cells (PEC), but not in cell preparations from the peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN), bone marrow (BM), and thymus (THY). ADCC activity, when compared with NK activity, was significantly higher in PBL but the same or lower in SPL and PEC. In terms of strain distribution, a high NK/ADCC strain (rnu/rnu), four intermediate NK and high ADCC strains (PVG/RTLRL, Lewis, PVG/OLA, and F344), an intermediate NK/ADCC strain (WF/N), and a low NK/ADCC strain (Buffalo) were observed. Fractionation of effector cells on discontinuous Percoll gradients revealed that both NK and ADCC activities were associated with relatively high-density large granular lymphocytes (LGL). In contrast, ADCC but little or no NK activity was associated with lower density LGL. However, the NK activity of this lower-density LGL population could be elicited following the in vitro incubation with a number of BRM, including rat interferon (IFN) and OK-432, but not rat interleukin-2 (IL-2). In general, the ADCC activity of both higher and lower density LGL-enriched cell populations correlated with both the frequency of FC gamma R+ LGL and the percentage of LGL binders to antibody-coated P815 target cells. The present study also has shown that in contrast to NK activity, which remained relatively stable with age, ADCC activity from F344 but not WF/N rats increased until 30-50 wk of age. This increase of ADCC activity in older F344 rats was accompanied by an increase in the percentage and absolute number of lower density FC gamma R+ LGL. This study demonstrates a number of similarities and differences between NK and ADCC activities in the rat. These findings should be useful for further examining and comparing the in vivo development and biological role of these two effector arms of the immune system.     

Expression of different lipoprotein receptors in natural killer cells and their effect on natural killer proliferative and cytotoxic activity.

Natural killer (NK) cells take up chylomicrons (CM), very low density (VLDL), low density (LDL), high density (HDL) and acetyl-modified low density (AcLDL) lipoproteins through different receptors, VLDL being the lipoprotein with the highest uptake and HDL the lowest. The uptake of LDL can be selectively blocked by the anti-LDL receptor, which does not affect the uptake of CM, VLDL, HDL and AcLDL. Although the uptake of lipoproteins assessed by flow cytometry using DiI is not very high, the lipoproteins are able to induce an increase in proliferative responses, VLDL, AcLDL and HDL being the most important ones with 12- and 17-fold increments, respectively. CM, VLDL and LDL at low concentrations increase NK cytotoxic activity, while HDL and AcLDL inhibit, in a dose-dependent fashion, the killing of NK cells against K562. These results suggest the presence of four different receptors that are responsible for the cytotoxic and proliferative responses observed.     

Age-dependent decline of natural killer and antibody-dependent cell mediated cytotoxicity activity of human lymphocytes is connected with decrease of their acid phosphatase activity

Decline of cytotoxic potential of lymphocytes of aged persons in ADCC and NK cell mediated cytotoxic reaction was paralleled by a drop in the activity of acid phosphatase. Intensity of ADCC reaction and the level of acid phosphatase activity showed statistically significant correlation when these two parameters were measured in peripheral blood of the same persons. The role of acid phosphatase in cytotoxic function of lymphocytes is discussed.     

Interferon production and natural killer activity induced by staphylococcal enterotoxin in mouse spleen cells

After a single intraperitoneal inoculation of C57BL/6 mice with enterotoxin A of Staphylococcus aureus (SEA) the activity of natural killers (NK) increases and phase change of interferon production by spleen cells upon reinduction in vitro occurs. Multiple daily inoculations of mice with SEA maintain the activity of splenic NK at a similar high level. With an adequate control (multiple administration of the medium) NK activity was maintained at the same high level. Interferon production by spleen cells of mice which were multiply inoculated with the medium upon reinduction in vitro was the same as in the control animals, whereas after multiple inoculations of mice with SEA, spleen cells in vitro produced lower amounts of interferon.     

Dissociation of natural killer activity and antibody-dependent cellular cytotoxicity in spleen cells of tumor bearing mice

Antibody-dependent cellular cytotoxicity (ADCC) increased with the development of tumors in C3H/He mice bearing spontaneous breast cancer or the syngeneic hepatoma MH-134 and in C57BL/6 mice bearing the syngeneic Lewis lung carcinoma 3LL. This cytotoxicity decreased after treatment with guinea pig, monoclonal IgM anti-Thy 1.2 serum and complement to the non-cancer level thus indicating that the increased ADCC in mice with cancer seems mainly attributable to cells with the Thy 1 antigen. On the other hand, NK activity decreased greatly when mice had tumors. Treatment with monoclonal IgM anti-Thy 1.2 serum and complement showed no significant influence on the natural killer (NK) activity of spleen cells of mice bearing MH-134 cancer, but in the 3LL-bearing mice the activity decreased significantly.     

Interferon induces natural killer cell blastogenesis in vivo

Interferon (IFN), types beta and gamma, and IFN inducers polyinosinic-polycytidylic acid and lymphocytic choriomeningitis virus all stimulated the generation of blast-NK cells in mouse spleens. Blast-NK cells were characterized on the basis of size, 3H-thymidine uptake, and NK cell markers. These data indicate that in addition to augmenting NK cell-mediated lysis, IFN may regulate NK cell proliferation in vivo.     

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.     

A microcytotoxicity assay for thyroid-specific cytotoxic antibody, antibody-dependent cell-mediated cytotoxicity and direct lymphocyte cytotoxicity using human thyroid cells

A solid-phase enzyme immunoassay for the detection of antibodies, specific for hemoglobin (Hb) is described. The application of glutaraldehyde resulted in a sensitive assay and allowed the use of urea, which is an important advantage if polypeptides not soluble in aqueous buffers are to be used. Mutation-carrying Hb chains can be purified, solubilized in urea and used in the immunoassay to monitor the purification and selection of antibodies specific for these variants. Specific antibodies are the main tools for the development of a hemoglobin-locus mutation system for detection of potentially mutagenic environmental agents. With erythrocytes as target cells, this system permits in vivo monitoring of subjects under exposure. Conventional antibody production, however, frequently turns out to be unsuccessful. The production of monoclonal antibodies has several advantages over conventional antibody production, but a sensitive antibody screening system is essential. Because of the sensitivity and the ease with which a large panel of antibody fractions against a vast panel of Hb antigens can be examined, the described immunoassay has potential value for the screening of hybridoma cultures.     

Decreased natural and antibody-dependent cellular cytotoxic activities in intravenous drug abusers

Peripheral blood lymphocytes from 14 adult male patients admitted to the hospital with complications of intravenous drug abuse (IDA) were examined for natural killer (NK) and antibody-dependent cellular cytotoxic (ADCC) activities, lectin-dependent cellular cytotoxicity, and interferon (IFN)- and interleukin 2 (IL-2)-induced NK activity. Serum was also assayed for circulating interferon levels and soluble factor(s) capable of suppressing the cytotoxic potential of allogeneic lymphocytes from healthy donors. IDA patients demonstrated significantly decreased levels of NK and ADCC activities compared to age- and sex-matched healthy controls. The lectin, phytohemagglutinin, could significantly enhance the cytotoxicity of IDA lymphocytes; however, activity was not completely restored to normal levels. IDA sera demonstrated a significant inhibitory effect on the NK and ADCC activities of normal allogeneic lymphocytes, and these sera contained negligible levels of circulating IFN. Although the NK activity of IDA lymphocytes could not be restored completely to normal levels by either IFN-alpha or IL-2, the percentage enhancement of cytotoxicity was remarkably higher in IDA patients with significantly reduced NK activity than that observed using PBL from patients with near normal NK activity. The ability of IFN or IL-2 to enhance the decreased cytotoxicity of PBL from drug abusers suggests a novel therapeutic approach to the management of the complications of IDA.     

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.     

Selective expansion of human natural killer cells leads to enhanced alloreactivity

In allogeneic stem cell transplantation （SCT）, natural killer （NK） cells lacking their cognate inhibitory ligand can induce graft-versus-leukemia responses, without the induction of severe graft-versus-host disease （GVHD）. This feature can be exploited for cellular immunotherapy. In this study, we examined selective expansion of NK cell subsets expressing distinct killer immunoglobulin-like receptors （KIRs） within the whole human peripheral blood NK cell population, in the presence of HLA-Cw3 （C1） or Cw4 （C2） transfected K562 stimulator cells. Coculture of KIR＋ NK cells with C1 or C2 positive K562 cells, in the presence of IL-2＋ IL-15, triggered the outgrowth of NK cells that missed their cognate ligand. This resulted in an increased frequency of alloreactive KIR＋ NK cells within the whole NK cell population. Also, after preculture with K562 cells lacking their cognate ligand, we observed that this alloreactive NK population revealed higher numbers of CD107~ cells when cocultured with the relevant K562 HLA-C transfected target cells, as compared to coculture with untransfected K562 cells. This enhanced reactivity was confirmed using primary leukemic cells as target. This study demonstrates that HLA class I expression can mediate the skewing of the NK cell repertoire and enrich the population for cells with enhanced alloreactivity towards leukemic target cells. This feature may support future clinical applications of NK cell-based immunotherapy.     

Mutual interference of HIV and natural killer cell-mediated immune response

Natural killer (NK) cells represent important early effector cells in innate immune defense as they exert their functions without prior sensitization. They participate in regulation of innate and adaptive immune responses and hematopoiesis by producing various cytokines and chemokines. In addition, NK cells lyse virally infected and malignant cells raising them to multifunctional members of the first line of defense. Unlike other lymphocytes they lack specific antigen receptors. They rather bind cells using ubiquitous molecules and communicate via a pattern of receptors specific for MHC-I molecules with their counterparts. In general, successful binding of the receptors delivers an inhibitory signal to NK cells thus sparing the target cell from lysis. In contrast, down-regulated or altered MHC-I expression as frequently observed during virus infection or on malignant cells prevents ligation of inhibitory receptors and MHC-I paralyzing inhibition and thus inducing lysis of the target cell. In human immunodeficiency virus (HIV) infection NK cells are of central importance since they can combat viral infection itself and opportunistic pathogens like fungi and protozoa that usually spread during the course of HIV infection. However, various studies have reported alterations in HIV patients affecting NK cell numbers and functions that might negatively influence course and severity of the disease. This review will focus on the mutual interference of NK cells and the HI virus.     

A simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells

A new, simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells is described. The assay is based on the use of two fluorochromes. The target cell population is stained with one fluorochrome (octadecylamine-fluorescein isothiocyanate, F-18) prior to incubation with the effector cells. F-18 remains in the membrane of the target cells even when they are killed thereby permitting a clear separation between effector and target cells. Dead cells are determined by staining with a second fluorochrome (propidium iodide) after incubation of effector and target cells. staining with a second fluorochrome (propidium iodide) after incubation of effector and target cells. F-18 is not toxic and does not decrease the cytotoxic activity of human natural killer cells. It is also stable (exchange between labeled and non-labeled cells is negligible in a period of at least 4 h at 37 degrees C) and it remains in the membrane of the killed cells. A clear distinction between unlabeled effector and labeled target cells is obtained, even after incubation of target and effector cells for 4 h at 37 degrees C and using a high effector cell-target cell ratio (75:1). A good correlation with the 51Cr release assay was obtained. A potential application of the flow cytometric cytotoxicity assay using whole blood instead of isolated lymphocytes is presented.     

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