Suppression of natural killer activity in human blood and bone marrow cultures by bone marrow-adherent OKM1-positive cells

Maintenance and regulation of natural killer (NK) cell activity in human bone marrow cultures were studied using K562 leukemia cells as targets. Culture of bone marrow cells in medium supporting long-term generation of myeloid cells resulted in a rapid loss of NK activity in 1-3 days. In contrast, antibody-dependent cytotoxicity to an NK-resistant tumor was maintained for more than 7 weeks. Horse serum, a component of the myelopoietic culture medium, was found to diminish NK cytotoxicity of blood and bone marrow cultures whereas hydrocortisone supplement did not. In addition, an adherent cell is present in bone marrow which greatly inhibits NK activity. Nonadherent bone marrow cells exhibited higher cytotoxicity than unfractionated cells at all days of culture; adherent cells were not cytotoxic to K562. Purified adherent marrow cells inhibited the cytotoxic capacity of nonadherent blood or marrow mononuclear cells during coculture. Indomethacin, an inhibitor of protaglandin synthesis, augmented levels of NK activity in cultures of bone marrow cells, indicating that macrophages may be suppressing this effector function via prostaglandins. Further identification of the adherent suppressor cells came from experiments in which suppression was prevented by treatment of the adherent cells with monoclonal OKM1 antibody plus complement. This study shows that bone marrow-adherent OKM1-positive cells, presumably macrophages, negatively regulate NK activity, and it defines conditions for analysis of the generation and/or positive regulation of NK cells in human bone marrow.     

Systemic administration of human leukocyte interferon to melanoma patients. III. Increased helper:suppressor cell ratios in melanoma patients during interferon treatment

Malignant melanoma patients treated with human leukocyte interferon (IFN-alpha) displayed increased natural killer (NK) cytotoxicity to K562 targets within the first 2 weeks of therapy. This study explored the possibility of T-cell regulation of this NK response, as evidenced by a variation in T-cell subpopulations. T-cell subset levels were studied in 9 patients who received daily doses of IFN-alpha over a period of 42 days. Five monoclonal antibodies to T-cell surface markers were used: Leu 1 (pan-T), Leu 2a (T suppressor/cytotoxic), Leu 3a (T helper/inducer), HNK-1 (Leu 7, a marker for NK cells), and B73.1 (an antibody against the Fc receptor on NK/K cells). Percentages of these markers were measured on days 0, 3, 7, and 21 of treatment. Percentages of Leu 1-positive cells and Fc-receptor-positive cells remained relatively constant throughout treatment in all patients. A trend toward a decrease in suppressor cells and an increase in helper cells peaking on day 7 and returning to earlier values by day 21 was found in 5 patients. The increase in NK cytotoxicity was not reflected consistently in proportions of HNK-1-positive cells or Fc receptor-bearing cells within the first week of therapy. The most significant finding was an increase in the helper:suppressor cell ratio peaking on day 7 and returning to pretreatment values by day 21. This increase was seen in every patient studied. The average pretreatment Leu 3a:Leu 2a ratio was 0.67 increasing to an average value of 1.47 on day 7 (p less than 0.005). Leu 3a:Leu 2a ratios returned to pretreatment values, in parallel to NK activity, despite continuation of interferon therapy.     

Adoptive transfer of natural killer cells in combination with chemotherapy improves outcomes of patients with locally advanced colon carcinoma

Background

Despite the availability of multiple treatment strategies, patients with advanced colon carcinoma (CC) have poor prognoses. The aim of this study was to evaluate the efficacy and safety of natural killer (NK) cell therapy in combination with chemotherapy in patients with locally advanced CC.

Inhibition of murine lymphokine-activated killer (LAK) cell activity by adherent cells

The effects of adherent cell depletion, indomethacin, and prostaglandin E2 (PGE2) on murine LAK cell activity were investigated. Removal of plastic adherent cells from splenocyte suspensions either prior to 5-day culture with 1000 U/ml of recombinant human IL-2 (rIL-2) or prior to assay resulted in an enhanced LAK cell cytotoxicity compared to that of whole spleen cell suspensions. Indomethacin enhanced LAK cell cytotoxicity of whole splenocyte suspensions if present during the culture period, but had no effect on whole splenocyte or adherent cell-depleted cell suspensions if added just prior to assay. PGE2 suppressed LAK cell activity of nonadherent splenocyte but not whole splenocyte suspensions when present during the culture period. In vivo treatment of mice with indomethacin enhanced cytotoxicity directed toward both LAK sensitive, natural killer (NK) resistant (P-815) and LAK, NK sensitive (YAC-1) tumor cell targets. Splenocytes from indomethacin-treated mice cultured with additional indomethacin and rIL-2 exhibited highest LAK cell activity. The results from this study indicate that LAK cells are regulated by adherent cells which suppress LAK cell activity. This suppression can be reversed both in vitro and in vivo by indomethacin. This study has important implications for the possible clinical use of indomethacin in the potentiation of in vivo and in vitro LAK cell activity for immunotherapeutic protocols.     

Lack of spontaneous and inducible natural killer cell activity in human bone marrow: presence of adherent suppressor cells

Human bone marrow cells collected from ribs of patients undergoing thoracotomy had low or no natural killer (NK) cell activity against K562 in a 4-hour chromium release assay. In vitro overnight treatment with interferon or interleukin 2 of bone marrow cells resulted in no induction or augmentation of NK cell activity. In the presence of adherent bone marrow cells interferon was unable to enhance NK cell activity of blood lymphocytes, although the baseline level of NK cell activity was not suppressed. These results suggest that adherent bone marrow cells regulate the development of active NK cells and that bone marrow components do not provide a favorable environment for the functional differentiation of NK cells.     

Inert particles inhibit natural killer cell function in vitro

Aqueous suspensions of inert particles were found to inhibit the baseline and interferon-enhanced natural killer (NK) cell activity of peripheral blood mononuclear cells (PBMC) and large granular lymphocytes (LGLs). This inhibition was induced with latex, silica, and Sephadex particles. The suppression of NK activity was not related to effector cell death as determined by trypan blue exclusion. The inhibition of NK cell function was more pronounced with prolonged incubation and could be partially reversed with monocyte depletion or the addition of indomethacin, a prostaglandin synthesis inhibitor, but not with the addition of the lipoxygenase inhibitors nordihydroguaiaretic acid and BW755C. Similarly, particle exposure inhibited the NK cell function of monocyte-depleted large granular lymphocytes with and without the add-back of glass adherent cells, implying that monocyte-independent NK suppressive mechanisms were also present. These data demonstrate that inert particles are immunosuppressive in vitro and can inhibit baseline and interferon-stimulated NK cell function of LGLs and PBMC through monocyte-dependent and independent pathways.     

Inhibition of natural killer cell lysis by natural killer-inhibitory substance: mechanism of suppression

The mechanism of suppression of NK-mediated lysis by a soluble product of peritoneal cells (NK-IS, natural killer-inhibitory substance) was investigated. Pretreatment of effector cells resulted in depressed NK lysis while pretreatment of targets had no effect, indicating suppression is due to alterations in effector cell function rather than changes in target cells. NK-IS had no effect on the formation of conjugates between effectors and NK-susceptible targets. When NK-IS was added to effector-target cell mixtures after the binding step had been successfully completed, ensuing lysis was significantly depressed, confirming that NK-IS inhibited a postbinding lytic event. The degree of suppression caused by NK-IS was directly related to the duration of exposure to the inhibitory molecule. In addition, a preliminary temperature-dependent step of binding to and/or intracellular entry of NK-IS into effectors is required before suppression can occur. NK-IS prevents the activation of NK cell lysis by interferon and Corynebacterium parvum and effectively inhibits lysis mediated by already activated effectors. The potent suppression of NK lysis and prevention of interferon and C. parvum-mediated activation of NK lysis by a soluble product of peritoneal cells may explain the extremely low level of NK effector cell function within the peritoneal cavity.     

Cellular suppression of murine ADCC and NK activities induced by Corynebacterium parvum

Summary Administration of a single dose of C. parvum (CP) induces depression of splenic NK activity in mice after a lag period of 3–5 days and this depression lasts about 2 weeks. The depressed levels of NK activity noted in this study depended on time of CP administration and were associated with the induction of suppressor cell activity. Neonatally thymectomized or sublethally irradiated mice had unimpaired ability to generate suppressor cells following CP treatment. Depletion of adherent/phagocytic cells by carbonyl iron plus magnetism, Sephadex G-10 filtration, or both neither enriched NK activity nor removed suppressor activity from the spleens of CP-treated mice. Antibody-dependent cellular cytotoxicity (ADCC) against lymphoma targets was also depressed in CP-treated mice, accompanied by a concomitant appearance of suppressor cells that interfere with ADCC at the effector level.     

Regulation of human natural cytotoxicity by IgG. III. Interaction between negative regulation by monomeric IgG and positive regulation by interferons of different types

Our prior reported results have demonstrated the dose-dependent inhibition of human natural killer (NK) cell activity upon treatment of peripheral blood mononuclear cells (PBMC) with monomeric IgG (mIgG) prior to the cytotoxic assay. In the present study, the combined effects on NK activity of human interferon (IFN) of each of the three types and mIgG, respectively, were determined. NK cells incubated with IFN alpha or IFN beta had augmented cytotoxicity against K562 target cells but remained responsive to negative regulation by mIgG. PBMC treated with human recombinant IFN gamma had unchanged cytotoxic activity but became partially resistant to suppression by mIgG. This ability of IFN gamma to interfere with the negative regulation of NK activity by cytophilic mIgG was seen when the cytokine was preincubated with effector cells prior to, simultaneously with, or after their exposure to inhibitor protein. These data provide some clues regarding the possible biological significance of the mIgG-induced down-regulation of NK cells which, when required for host protection, might be appreciably reversed or blocked by IFN gamma produced by NK cells or T cells in response to various agents.     

Effects of adriamycin on the activity of mouse natural killer cells

Adriamycin, a widely employed anti-neoplastic agent, was found to have either inhibitory or stimulatory effects on NK activity, depending on the site examined. A single i.p. administration of ADM resulted in a rapid increase of cytolytic activity by PEC of various mouse strains. The effector cells appeared to be NK cells, being nonadherent and nonphagocytic; they expressed low amounts of Thy 1.2 antigen and had the same pattern of specificity as splenic NK cells. In contrast to the stimulatory effects of NK activity of PEC, ADM caused a transient dose-dependent depression of NK activity in the spleen, with a peak reduction at day 3 and recovery within a few days thereafter. The depressed NK activity could be reversed by removal of adherent cells by passage through a nylon column. Moreover, ADM induced cytostatic activity against tumor cells by macrophages, suggesting that activated macrophages may be responsible for suppression of splenic NK activity. The possible modulation of the levels of NK activity by ADM-induced macrophages was supported by mixture experiments, in which plastic adherent spleen cells from ADM-treated mice, but not from normal mice, inhibited the NK activity of normal spleen cells.     

Monocyte and NK cell cytotoxic activity in human adherent cell preparations: discriminating effects of interferon and lactoferrin

The comparative cytotoxic specificities of freshly isolated human adherent and nonadherent blood mononuclear cells were examined against seven established target cell lines in 4 and 18 hr chromium release assays. The relative sensitivity of each target cell line to the cytotoxic effects of both adherent and nonadherent effector cells in cultures was identical. Moreover, the relative enhancing effects of interferon on cytotoxicity by both effector cell types were also identical. These adherent cell preparations were contaminated with up to 6% NK cells, as demonstrated by OKM1 staining and flow microfluorometry. These NK cells were loosely adherent and could be removed by vigorous wash procedures. The remaining tightly adherent monocytes also had the capacity to kill K562 cells and Chang cells, but these cytotoxic effects could not be increased by interferon. Enhancement by lactoferrin, however, was consistently observed. Treatment of mononuclear cells with Leu-lla, a monoclonal antibody that reacts with all NK cells, also abolished the enhancing effects of interferon, but not lactoferrin. These studies suggest that caution must be exercised in attributing all cytotoxic activities in adherent cell preparations to monocytes, and that lactoferrin and interferon can be used as functional probes to detect two distinct blood mononuclear cell subsets with natural cytotoxicity.     

Interleukin 2 enhances the natural killer cell activity of acquired immunodeficiency syndrome patients through a gamma-interferon-independent mechanism

Patients with the acquired immunodeficiency syndrome (AIDS) exhibit a variety of disorders of cellular immunity, including a deficient ability to generate cytotoxic T cells and depressed levels of natural killer (NK) cell activity. Interleukin 2 (IL 2) in vitro can markedly augment these depressed immune functions. Because IL 2 can induce the release of interferon-gamma (IFN-gamma) from normal peripheral blood lymphocytes (PBL), and because IFN-gamma may play a role in the regulation of NK cell activity, this study was performed to determine if the IL 2 enhancement of the NK cell activity of patients with AIDS was an IFN-gamma-dependent effect. PBL from eight healthy heterosexual donors and from nine patients with AIDS were studied for their ability to release IFN-gamma in response to IL 2 at a concentration of 100 U/ml. After 60 hr of culture, the PBL of all eight healthy donors produced IFN-gamma with a mean titer of 113 U/ml (range 40 to 320 U/ml). In contrast, the PBL from only two of nine patients with AIDS released measurable amounts of IFN-gamma (40 U/ml each) in response to IL 2 with a mean titer of 13.5 U/ml for all nine. Although the PBL from patients with AIDS were deficient in their capacity to produce IFN-gamma in response to 100 U/ml of IL 2, significant enhancement of NK cell activity could be obtained after only 1 hr of PBL treatment with 10 U/ml of IL 2, with an optimal NK enhancing effect occurring at doses of 50 to 100 U/ml of IL 2. The use of an anti-IFN-gamma monoclonal antibody resulted in complete neutralization of the IFN released from the normal PBL cultured with IL 2, but failed to inhibit the IL 2 enhancement of NK cell activity. Exogenous IFN-gamma exhibited different kinetics of enhancement of NK cell activity when compared to IL 2, requiring substantially more than 1 hr of pretreatment of PBL. These results indicate that the PBL from patients with AIDS usually do not release IFN-gamma when cultured with IL 2, and that IL 2 enhancement of the depressed NK cell activity of these patients may be an IFN-gamma-independent event. These results may have important implications for the therapy of AIDS.     

Decreased natural killer activity in thalassemia major: a possible consequence of iron overload

We have investigated the natural killer (NK) activity of both fractionated (Percoll density gradient) and unfractionated mononuclear cells from patients with beta-thalassemia major who are iron overloaded as a consequence of chronic transfusion therapy. These patients were found to have significantly decreased NK activity against K562 targets at all effector:target ratios tested (p less than 0.001). Both splenectomized and nonsplenectomized patients had normal proportions of Leu-11b-staining (NK) cells. Since they had normal to elevated absolute white cell and lymphocyte counts, a change in the absolute number of NK cells could not account for the decreased killing. We also found that the decrease in NK activity was transfusion related (r = -0.603, p less than 0.005). To determine whether or not this decrease in NK activity could be related to iron overload, we preincubated patient effector cells with desferrioxamine (DFO) or 2,3-dihydroxybenzoic acid (DHB) for 6 hr before addition of K562 targets. Both of these iron-chelating agents consistently increased the NK activity of cells from thalassemia patients. DHB had the greater effect, being able to increase patient NK activity to virtually normal levels. On the other hand, preincubation of cells from normal controls with DHB caused only a slight increase in NK activity, while similar treatment with DFO had little or no effect. When target cells were preincubated with the chelating agents before addition of either normal or patient effector cells, no change in cytotoxicity was seen, demonstrating that the chelating agents act at the effector cell level. Furthermore, if the chelating agents were saturated with iron prior to preincubation with the effectors, no increase in the cytotoxicity of thalassemic NK cells was observed. These results indicate that thalassemia patients have a reversible, transfusion-related decrease in NK function which may arise as a consequence of iron overload.     

Modulation of natural killer cytotoxicity by muramyl dipeptide and trehalose dimycolate incorporated in squalane droplets

Summary The effect on natural killer (NK) cytotoxicity of splenic cells from BALB/c mice pretreated i. v. with squalane-in-water preparations of muramyl dipeptide (MDP), trehalose dimycolate (TDM), or the combination of MDP-plus-TDM was investigated. MDP or TDM augmented the NK cytotoxicity which peaked 48 h after the pretreatment whereas the combination of MDP and TDM induced an inhibition of the NK activity. Infection with influenza virus, a potent stimulator of NK cells, after the pretreatment with biological response modifiers resulted in a markedly enhanced NK activity on day 2 in MDP and control groups. Mice pretreated with TDM or the combination of MDP and TDM showed only moderate NK activity which peaked on day 3 after influenza infection. The NK activity was susceptible to asialo GM₁ and complement treatment. The cytotoxicity of MDP-plus-TDM cells could be significantly enhanced after treatment with anti-macrophage monoclonal antibody and complement. NK activity induced by MDP or TDM was reduced by mixing MDP-plus-TDM cells. Addition of adherent cell-depleted MDP-plus-TDM suspension to MDP or TDM cells had a NK restorative effect. Splenic cells from mice pretreated 2 days earlier with MDP or TDM, but not MDP-plus-TDM, generated enhanced levels of luminol-dependent chemiluminescence.     

High expression of NKR-P1 is not an absolute requirement for natural killer activity in BDIX rats

 NKR-P1 has been identified as a triggering structure selectively expressed on rat natural killer (NK) cells and adherent lymphokine-activated killer (A-LAK) cells. In vivo treatment with anti-NKR-P1 monoclonal antibody (mAb 3.2.3) was shown to induce complete inhibition of NK cytotoxicity and elimination of LAK cell precursors in Lewis and Fisher rat strains. We investigated the effects of mAb 3.2.3 in a colon tumor model in BDIX rats. Inoculation of animals with mAb 3.2.3 even at very high doses induced a strong but incomplete inhibition of NK cytotoxicity in nylon-wool-non-adherent spleen and peripheral blood cells. Generation of adherent A-LAK cells from their spleen precursors was also strongly but not fully inhibited. We also investigated the effect of treatment with mAb 3.2.3 on the tumorigenicity of the NK-sensitive REGb cell line. When subcutaneously inoculated in syngeneic animals, REGb cells induce tumors that first grow for 2 weeks, then spontaneously regress and disappear. In contrast with previous results using anti-asialoGM1, no significant difference in tumor growth was observed between rats treated with mAb 3.2.3 and control animals, even with a long-term treatment. In vitro, mAb 3.2.3 exhibited the same incomplete efficiency. Nylon-wool-non-adherent spleen cells treated with mAb 3.2.3 plus complement were completely free of 3.2.3^bright cells, but retained a substantial NK activity and generated LAK cells after culture with IL-2. After an overnight incubation in standard medium of 3.2.3-depleted spleen cells, 3.2.3^bright cells were partially recovered and the NK cytotoxic activity, as well as the generation of LAK cells, was significantly enhanced. These results suggest that a strong expression of NKR-P1 is not required for BDIX mononuclear cells to exhibit NK function and generate LAK cells under IL-2 activation. Received: 11 July 1995 / Accepted: 16 November 1995     

Normalization of defective monocyte chemotaxis during chemotherapy in patients with small cell anaplastic carcinoma of the lung

Summary Monocyte chemotactic responsiveness (MCR) in 14 patients with small cell anaplastic bronchogenic carcinoma was depressed before treatment compared with the MCR in 28 normal controls (P=0.00004). MCR was subsequently monitored during combination chemotherapy and after 6 months the MCR had become normalized compared with pretreatment values (P=0.00006).In addition, chemotactic factor inhibitor (CFI) activity in plasma was measured before treatment and after 6 months. When incubated with plasma before treatment casein had 62% of normal activity and when incubated with plasma after chemotherapy, 81% of normal activity (P=0.0009). CFI activity decreased by greater amounts in patients in complete remission than in patients in partial remission or in non-responders (P=0.01). This study supports the concept that cancer patients have depressed monocyte function. Chemotherapy seems to enhance monocyte chemotaxis in vitro and to decrease CFI activity in plasma.     

Monocyte involvement in PHA induced cytotoxicity of chicken erythrocytes in man.

Phytohemagglutinin (PHA) induced cell cytotoxicity was studied in man using chromated chicken red blood cells (CRBC) as target cells. A phagocytic, adherent monocyte was found to be necessary for lysis of target cells. Results using E rosette depletion showed that this procedure markedly increased mitogen-induced cellular cytotoxicity (MICC). Carbonyl iron treatment of peripheral blood cell suspensions to remove phagocytic cells abolished MICC, as did removal of adherent cells by glass wool columns. Complement mediated lysis of B cells did not substantially reduce MICC. However, pretreatment of cells with silica or hydrocortisone did reduce MICC. The mechanism of mitogen-induced lysis appears to require direct cell contact between effector cells and target cells.     

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.     

A short-term dietary supplementation with high doses of vitamin E increases NK cell cytolytic activity in advanced colorectal cancer patients

Cancer patients with advanced disease display signs of immune suppression, which constitute a major obstacle for effective immunotherapy. Both T cells and NK cells are affected by a multitude of mechanisms of which the generation of reactive oxygen species is of major importance. Therefore, we hypothesized that two weeks of high-dose treatment with the anti-oxidant vitamin E may enhance NK cell function in cancer patients by protecting from oxidative stress. Seven patients with colorectal cancer (Dukes stage C and D) received a daily dose of 750 mg of vitamin E during a period of two weeks and the function, phenotype and receptor expression of NK cells were analyzed. The short-term vitamin E treatment significantly improved NK cell cytolytic activity in six out of the seven patients analyzed. The increased NK cell activity in patients’ PBMC was not due to increased numbers of NK cells or an increase in the proportion of the CD56^dim NK cell subpopulation. Furthermore, neither an increased perforin expression nor an enhanced ability of NK cells to produce IFN-γ was observed as a result of vitamin E treatment. Finally, vitamin E treatment was associated with a minor, but consistent, induction of NKG2D expression in all patients analyzed. In conclusion, this pilot study demonstrates that vitamin E may boost NK cell function in patients with colorectal cancer. Further studies are warranted to explore the potential of vitamin E as an adjuvant for immunotherapy against cancer and to determine the underlying mechanism(s) behind vitamin E induced NK cell activation.     

培美曲塞化疗对非小细胞肺癌患者T淋巴细胞亚群的影响

目的:探究培美曲塞化疗对非小细胞肺癌患者T淋巴细胞亚群的影响,评价其临床应用价值。方法:选取2016年3月到2017年3月我院肿瘤内科收治的100例非小细胞肺癌患者进行研究,其中,50例非小细胞肺癌患者采用培美曲塞化疗、50例采用多西他赛进行化疗。比较患者治疗前后T淋巴细胞亚群变化情况。结果:与化疗前比较,非小细胞肺癌患者化疗后CD_3~+、CD_4~+、CD_4~+/CD_8~+比值以及NK细胞显著上升,而CD_8~+显著下降(P0.05)。为了验证培美曲塞对非小细胞肺癌患者的安全性,本研究将部分患者采用多西他赛化疗,非小细胞肺癌患者培美曲塞和多西他赛化疗后CD_3~+、CD_4~+、CD_4~+/CD_8~+比值以及NK细胞相比差异不大(P0.05)。此外,两组不良反应如恶心、呕吐,细胞减少,血小板减少,肾功能障碍差异不大(P0.05)。结论:培美曲塞化疗可以显著改变非小细胞患者的T淋巴细胞亚群构成,提高患者免疫功能和治疗效果。