Influence of low-dose beta-interferon on natural killer cell activity in breast cancer patients subjected to chemotherapy

Summary The present study was designed to test whether immunomodulating doses of human beta-interferon would affect the natural cell-mediated cytotoxic function in untreated breast cancer patients or in those subjected to antitumor therapy. Analyses were performed on 11 breast cancer patients, 3 at stage 1 and 8 at stage 2, the latter being subjected to cyclophosphamide, methotrexate, 5-Fluorouracil (CMF) adjuvant chemotherapy. Five patients treated with CMF and 3 patients not subjected to adjuvant chemotherapy, received human beta-interferon (IF, 2×10⁶ IU/patient, i.m.), on days 0,7, and 15 for 6 cycles of 31 days each. The natural killer (NK) activity (NKA) of peripheral blood mononuclear cells (MNC) was tested 24 and 48 h after low-dose IF administration. The results of NKA determinations carried out for the 6 cycles of treatment show that (1) chemotherapy alone depressed NKA; (2) IF alone increased NKA in stage 1 patients not treated with CMF; (3) IF antagonized the depressive activity of CMF on NK function and significantly augumented NKA in the case of low basal cytotoxic activity detectable in MNC collected before IF administration.Parallel in vitro studies showed that the inhibitory effect on NKA provoked by CMF is due to cyclophosphamide present in the association and is effectively antagonized by IF. These data provide rational bases for using IF in immunochemotherapy regimens, when tumor cells are supposed to be susceptible to host control by the natural resistance function.     

Modulation of natural killer cell activity in stage I postmenopausal breast cancer patients on low-dose aminoglutethimide

Summary Natural killer (NK) cells are important in surveillance against malignant cells. The activity of NK cells can be modulated by naturally occurring mediators; interferon, interleukin-2, and hormones. Low-dose aminoglutethimide (Ag 250 mg/day) inhibits the peripheral aromatization of androstenedione hence decreasing circulating estrogens. Of ten patients treated, seven were evaluable. There was a statistically significant increase in NK activity (P=0.0025) following the administration of Ag. There was no consistent shift in NK cell number (Leu-11b positive cells). In vitro Ag did not alter NK activity whereas 17--estradiol did. These data are consistent with an indirect effect of Ag on NK activity. Hence in vivo Ag which causes a reduction in serum estrogens in postmenopausal patients, also induces an increase in NK activity.     

Analysis of natural killer activity and natural killer cell subsets in patients with bladder cancer

Summary In order to analyze the state of the natural resistance system of bladder cancer patients in vivo, we measured natural killer (NK) activity and NK cell subsets of peripheral blood lymphocytes (PBL) from 46 patients with bladder cancer and 25 age- and sex-matched healthy volunteers. The mean NK activity in patients with lowstage bladder cancer was similar to that in the controls, while NK activity in patients with high-stage bladder cancer was significantly depressed. The mean proportions of Leu7⁺ cells in patients with both low-stage and highstage bladder cancer were significantly higher than that in the controls. The mean proportion of Leu11a⁺ cells in patients with low-stage bladder cancer was similar to that in the controls, while in patients with high-stage bladder cancer it was significantly higher. This study demonstrates the abnormal immunological state of bladder cancer patients; namely, abnormalities exist not only in NK activity but also in the proportions of circulating NK cell subsets.     

Effect on natural killer and antibody-dependent cellular cytotoxicity of adjuvant cytotoxic chemotherapy including melphalan in breast cancer

Natural killer (NK) cell activity and antibody-dependent (K) cell activity were studied sequentially in 30 patients with early node-positive breast cancer entered into an adjuvant chemotherapy trial. The drugs used were melphalan, and melphalan with methotrexate, given for 12 months. Estimations were made 3-monthly during chemotherapy, and then at 15 and 24 months to assess recovery. Mean values for NK-cell activity during chemotherapy were significantly lower than the mean pre-chemotherapy baseline value at all time-points from 3 to 15 months, but there was recovery by 24 months. Mean values for K-cell activity during chemotherapy did not appear to differ from the mean pre-chemotherapy value, but variability in individual values was high. Over a 4-year follow-up period, a comparison of 16 patients who did not develop recurrent breast cancer with 14 who did showed that NK-cell activity was significantly lower in the latter group 12 months after the start of chemotherapy.     

Mutations in mice that influence natural killer (NK) cell activity

A series of mutations in mice was tested for splenic NK-cell activity against YAC-1 target cells. Mutations at six loci that reduce NK-cell activity in the homozygous state were identified, including beige (bg), hairless (hr), motheaten (me), obese (ob), steel (Sl) and, to a lesser extent, dominant spotting (W). Motheaten mice displayed the most profound NK-cell deficiency, with NK-cell activity virtually absent. Two mutations, nude (nu) and lymphoproliferation (Ipr), produced elevated NK-cell-mediated lysis. The double homozygous recessivenu/nu bg/bg nude-beige mouse was viable and NK-cell-deficient, with activity slightly higher than that of +/?bg/bg beige littermate controls. Pigmentation mutants related to beige, including pale ears (ep), pearl (pe), and ruby eyes (ru ^2J ) did not dramatically influence NK-cell levels. Unlike the obese gene, other mutations leading to obesity, diabetes (db) and yellow (Asuy), did not impair NK-cell function. The possible site of gene action of these mutants in the NK-cell pathway is discussed.     

Evaluation of natural killer cell activity

Natural killer (NK) cells are being appreciated not only for their ability to recognize and lyse tumor cells and virus-infected cells but also for their immunoregulatory properties. NK cells provide a first line of defense against invading pathogens with a two pronged attack, lysis of infected cells and secretion of cytokines and chemokines with potent antipathogen effects. This article describes the standard chromium release assay, which measures the ability of NK cells derived from the peripheral blood to lyse appropriate target cells.     

In vivo modulation of natural killer cell activity by tamoxifen in patients with bilateral primary breast cancer

Natural killer (NK) cell activity, the autologous mixed lymphocyte reaction (AMLR) and proportions of T cell subpopulations (CD3⁺/CD4⁺ and CD3⁺/CD8⁺) and NK cells (CD16⁺) were studied in 21 patients with bilateral primary breast cancer (BBC), 10 patients with single-breast cancer (SBC) and 20 healthy controls. All patients studied had no evidence of disease and had been off radiotherapy and/or chemotherapy for at least 1 year. Ten patients with BBC were also treated with tamoxifen. Patients with SBC had NK cell activity, AMLR responses and T cell subpopulations that were comparable to those of normal controls. In patients with BBC, a significant (P<0.01) increase in NK activity compared to that in normal controls (42±13% versus 21±10%, effector-to-target cell ratio, 251) and a significant (P<0.05) decrease in CD4⁺ T cell proportions (30±15% versus 49±13%) and absolute numbers (472±82/mm³ versus 953±131/mm³) were found. However, the proliferative response of BBC patients' T lymphocytes in AMLR was in the range of the normal controls. Lymphocytes derived from 10 BBC patients treated with tamoxifen exhibited NK cell activity that was comparable to that of normal controls and patients with SBC, and was significantly (P<0.01) reduced compared to the pretreatment period. BBC patients who received tamoxifen also show a reduction in the proportion of CD4⁺ T cells and in AMLR proliferative responses, which decreased compared to levels in normal controls. Taken together, these results indicate that long-term tamoxifen treatment modulates immune responses in BBC patients.     

The influence of natural mineral water on aquaporin water permeability and human natural killer cell activity

Aquaporins are the intrinsic membrane proteins functioning as water channel to transport water and/or mineral nutrients across the biological membrane systems. In this research, we aimed to clarify if the selected mineral water can affect aquaporin functions in vitro and the assumption of the mineral water can modify aquaporin expression and activate natural killer cell activity in human body. First, we expressed six human and eight plant aquaporin genes in oocytes and compared the effect of different kinds of natural mineral water on aquaporin activity. The oocyte assay data show that Hita tenryosui water could promote water permeability of almost all human and plant aquaporins in varying degrees, and freeze-dry and organic solvent extraction could reduce AQP2 activity but pH change and boiling could not. Second, each volunteer in two groups (10 in one group) received an oral Hita tenryosui or tap water load of 1000 ml/day for total four weeks. We found that these two kinds of water did not directly affect the relative expression levels of AQP1 and AQP9 in the blood cells, but intriguingly, the natural killer cell activities of the volunteers drinking Hita tenryosui water were significantly improved, suggesting that Hita tenryosui water has obvious health function, which opens a new and interesting field of investigation related to the link between mineral water consumption and human health and the therapies for some chronic diseases.     

Renewal of natural killer cells in mice having elevated natural killer cell activity

The present study was designed to examined the dynamics of splenic natural killer (NK) cells under two conditions of enhanced NK cell activity: (1) CBA/J mice given polyinosinic-polycytidylic acid (poly-I:C), an NK-cell-enhancing agent, and 62) untreated athymic nude (nu/nu) mice. The 'total NK cell activity' of the spleen (percentage specific lysis corrected for changes in organ cellularity) increased 5-fold and 2.7-fold after poly-I:C treatment for 1 day and 4 days, respectively. An injection of hydroxyurea (HU), a cell-cycle-toxic drug, given together with either poly-I:C or saline to CBA/J mice resulted in both cases in a 25% reduction in total NK cell activity 1 day later. This suggests that the renewal rate of nondividing NK cells is similar in poly-I:C-treated and saline-injected mice, and that the NK-enhancing effect of poly-I:C is not due to a stimulation of proliferation among NK cell precursors. HU administered simultaneously with poly-I:C or saline for 4 days eliminated NK cell activity in both cases, indicating that spleen NK cell activity is mediated almost entirely by newly formed (less than or equal to 4 days) cells. In nude mice, NK cell activity was assayed at various intervals after an HU depletion period of 2 days. NK depletion was initially more rapid in nu/nu mice than in control (nu/+) mice, although equally profound, and the subsequent recovery of NK cell activity after cessation of HU was also more rapid than in control (nu/+) mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro modulation of natural killer cell activity in non-Hodgkin's lymphoma patients after therapy

Summary The depressed natural killer (NK) activity, antibody-dependent cellular cytotoxicity (ADCC) and NK cytotoxic factor cytotoxicity in untreated non-Hodgkin's lymphoma patients were found to be elevated after chemotherapy. In vitro treatment of the effector NK cells with interferon could augment the NK activity in normal subjects and treated patients to a comparable degree. Chemotherapy mainly affected the post-binding events in the NK cytotoxic process by causing an increase in the active killing potential of the NK cells. This study provides a better understanding of changes in the NK cytotoxic mechanism in non-Hodgkin's lymphoma patients and the role of interferon in this process.B. A. Mehta is a recipient of the Lady Tata Memorial Trust, India, Senior Scholarship     

A phase II study of allogeneic natural killer cell therapy to treat patients with recurrent ovarian and breast cancer

BackgroundNatural killer (NK) cells derived from patients with cancer exhibit diminished cytotoxicity compared with NK cells from healthy individuals. We evaluated the tumor response and in vivo expansion of allogeneic NK cells in recurrent ovarian and breast cancerMethodsPatients underwent a lymphodepleting preparative regimen: fludarabine 25 mg/m² × 5 doses, cyclophosphamide 60 mg/kg × 2 doses, and, in seven patients, 200 cGy total body irradiation (TBI) to increase host immune suppression. An NK cell product, from a haplo-identical related donor, was incubated overnight in 1000 U/mL interleukin (IL)-2 prior to infusion. Subcutaneous IL-2 (10 MU) was given three times/week × 6 doses after NK cell infusion to promote expansion, defined as detection of ≥100 donor-derived NK cells/μL blood 14 days after infusion, based on molecular chimerism and flow cytometryResultsTwenty (14 ovarian, 6 breast) patients were enrolled. The median age was 52 (range 30–65) years. Mean NK cell dose was 2.16 × 10⁷cells/kg. Donor DNA was detected 7 days after NK cell infusion in 9/13 (69%) patients without TBI and 6/7 (85%) with TBI. T-regulatory cells (Treg) were elevated at day +14 compared with pre-chemotherapy (P = 0.03). Serum IL-15 levels increased after the preparative regimen (P = < 0.001). Patients receiving TBI had delayed hematologic recovery (P = 0.014). One patient who was not evaluable had successful in vivo NK cell expansionConclusionsAdoptive transfer of haplo-identical NK cells after lymphodepleting chemotherapy is associated with transient donor chimerism and may be limited by reconstituting recipient Treg cells. Strategies to augment in vivo NK cell persistence and expansion are needed.     

Characterization of natural killer cell activity in Macaca nemestrina

Natural killer (NK) cell activity was evaluated in three groups of Macaca nemestrina that varied with respect to SAIDS D retrovirus serotype 2 (SRV-2/W) and viremic status. Target cells used were Raji and K562 cells. No significant differences (ANOVA) in mean NK activity were detected among the three groups of animals studied. Using Raji targets, mean LU₃₀/10⁶ ± SEM was 6.3 ± 1.6 for seronegative (V-Ab−) animals, 7.3 ± 1.5 for seropositive (V-Ab+) animals, and 10.2 ± 3.5 for persistently viremic (V + Ab−) animals. Using K562 targets, mean LU₃₀/10⁶ was 7.6 ± 1.7 for seronegative (V-Ab−) animals, 6.5 ± 2.5 for seropositive (V-Ab+) animals, and 5.1 ± 1.9 for persistently viremic (V+Ab−) animals. Percentage blood CD16⁺ and CD8⁺cells also were not different in the three groups of animals. NK activity did not always correlate with percentage of CD16⁺ or CD8⁺ cells in peripheral blood at the time the assays were done. In persistently viremic animals, there was a strong positive correlation between percent CD16⁺ and CD8⁺ cells and NK activity using K562 cells but not Raji cells. Depletion experiments indicated that lysis was mediated by both CD8⁺ and CD16⁺ cells with both Raji and K562 cells. However, Raji targets were a better indicator of killing mediated by CD16⁺ cells. Our studies indicate that M. nemestrina may be classified as high or low responders with regard to NK activity, and there was no correlation with SRV-2/W viral or antibody status. Additionally, our results suggested that group housing of M. nemestrina was usually associated with increased NK activity. In conclusion, studies of NK activity in M. nemestrina should consider target cells used, phenotype of effectors, endogenous (high or low) levels of NK activity in individual animals, and housing conditions. © 1996 Wiley-Liss, Inc.     

Inhibition of natural killer cell activity by IgA

The in vitro effect of IgA on natural killer (NK) activities of human peripheral blood mononuclear cells was investigated. Purified myeloma polymeric IgA2 (pIgA2) and secretory IgA (S-IgA) from human colostrum inhibited NK activity, while myeloma polymeric IgA1 (pIgA1), monomeric IgA1 (mIgA1), IgG, and IgM were ineffective. Inhibition was proportional to the concentration of pIgA2 (0.125-1 mg/ml) and was observed after as little as 1 hr of incubation at various effector to K562 target cell ratios. pIgA2 and S-IgA also inhibited NK activity of NK cell-enriched lymphoid cells and gamma-interferon-treated effector cells, but did not interfere with effector-target cell binding. The inhibitory effect was slightly diminished after 24 hr culture in pIgA2-free medium. Inhibition of cytotoxicity was not due to direct toxicity on lymphoid cells by IgA because PBL treated with pokeweed mitogen in the presence of pIgA2 or S-IgA differentiated into immunoglobulin-producing cells. Viability after 24 hr of preculture with pIgA2 and S-IgA was comparable to that of untreated control cells. Morphological examination of effector cells cultured with pIgA2 or S-IgA showed a decrease in the number of granules, and the formation of cytoplasmic vacuoles. These morphological changes appeared to coincide with the depressed cytotoxicity of NK cells. The results demonstrate that purified pIgA2 and S-IgA have significant immunomodulatory effects on human NK activity.     

Increased natural killer cell activity in experimental American trypanosomiasis

When purified murine plasminogen was added to cultures of mouse spleen B cells, active plasmin progressively appeared in the supernatants. This reaction, resulting from the specific cleavage of the plasminogen by lymphocyte plasminogen activator (LPA), was measured in a fibrinolysis assay using 125I-fibrinogen. T cells were totally ineffective; under certain conditions, they could even antagonize the B cell action. Of the various B populations studied, i.e., obtained from spleen, lymph nodes, or blood of various mouse strains, all expressed the same property of plasminogen activation, which concerned mainly medium-sized B cells. Since only slight activities were detected in extracellular or intracellular compartments, a membrane-associated proteolytic enzyme may be responsible for plasminogen activation. Submitted to a series of group-specific antiproteases, the lymphocyte plasminogen activator essentially behaved as a serine-protease, with sensitivity to diisopropyl fluorophosphate, phenyl methyl sulfonyl fluoride, and nitro phenyl guanidino benzoate. The fast renewal of the enzyme in the membrane was also demonstrated by different techniques, using modifiers of cell physiology, like cycloheximide and dexamethasone, or following the reexpression of the enzyme by the cell kinetically.     

Mechanism of cell contact-mediated inhibition of natural killer activity

Natural killer cell activity is inhibited by primary cultures of monolayer cells. In this study, we analyzed the mechanism of the inhibition. Inhibited NK cells showed unaltered binding capacity to NK sensitive K562 cells. The orientation of the effector cells' actin-containing microfilaments, an event known to occur during the programming for the lysis stage in lytic conjugates, was unaffected by the inhibition. In single cell cytotoxicity experiments, the number of killer cells among conjugate-forming cells was reduced. The capacity of the inactivated NK cells to secrete cytotoxic factors upon stimulation with Con A was also impaired. Both NK-resistant inactivating target cells and NK-sensitive K562 cells were sensitive to the toxic factors secreted by NK cells. Thus, the results indicate that the target cell-mediated inactivation of NK cell is based on a block in the lethal hit stage, possibly due to reduced release of toxic factor(s) from the effector cells. The capacity of inactivated effector cells to mediate antibody-dependent cellular cytotoxicity was unimpaired, suggesting that the contact-mediated inhibition of cytotoxicity selectively affects NK cells.