Effects of in vitro hyperthermia on human natural killer cells

The present study demonstrates that human natural killer (NK) cells isolated from peripheral blood of normal individuals are highly sensitive to hyperthermia. The effect was time and dose dependent, and treatment of peripheral blood lymphocytes at 42 degrees for 1 hr almost completely abolished NK activity. The effect was not a consequence of cell death since only a small decrease in cell viability was observed and the viability of density gradient fractions enriched for NK activity was normal. Analysis of NK activity at the single-cell level by application of a conjugation assay in agarose revealed that hyperthermia interfered with target cell binding as well as the lytic cycle. Attempts to rescue NK activity after hyperthermia treatment by incubation overnight with human alpha-interferon or activation in mixed leukocyte culture was unsuccessful, indicating that even pre-NK cells are heat sensitive. In contrast, the proliferative response to alloantigens in mixed leukocyte culture and to the T-cell mitogen concanavalin A was unaffected. Hyperthermia exposure of cytotoxic T-lymphocyte generated in mixed leukocyte culture immediately before assay against allogeneic blast cells strongly inhibited their activity. Some alterations in the kinetics of stimulation with the B-cell mitogen Staphylococcus aureus bacteria were observed after heat exposure although maximal stimulation was at control levels. Thus, NK cells, including their precursors, seem to be preferentially sensitive to hyperthermia among various lymphoid subclasses.     

3-methylcholanthrene: transient inhibition of the lytic step of mouse natural killer cells

Female mice belonging to NMRI stock were inoculated neonatally with daily doses of 5 micrograms diethylstilbestrol (DES) or olive oil for the first 5 days after birth and in adult life with 20 or 100 micrograms 3-methylcholanthrene (MCA) or the vehicle tricaprylin only. The cytotoxic activity of spleen natural killer (NK) cells against YAC-1 target cells was studied in a 51Cr release assay at 2, 5, 10, or 15 days after the MCA injection. A dose of 20 micrograms MCA to neonatally olive oil-injected females did not influence the NK activity, whereas an injection of 100 micrograms MCA significantly depressed the NK activity to about half the value seen in controls. This suppression was transient, and the normal level was reached again 15 days after the injection. The depressed NK activity could not be related to humoral or cellular suppressor mechanisms. A study at the single-cell level revealed that the inhibition was due to interference with the lytic step of the NK cell without affecting target-binding capacity. In vitro exposure of spleen cells from MCA-treated animals to interferon fully restored the NK activity. Neonatal DES treatment resulted in a depressed NK activity in adult females to a level about half of that seen in olive oil-injected controls. The NK activity in DES-treated females was not influenced by either 20 or 100 micrograms MCA. The MCA-induced suppression of NK activity was discussed in relation to the earlier reported difference in incidence of MCA-induced sarcomas between DES-treated and control females after they were given 20 micrograms MCA in adult life, as well as in relation to the same incidence in the 2 groups treated with 100 micrograms MCA. The results are compatible with a significant role for NK cells during MCA carcinogenesis and indicate that a possible part of the tumorigenic effect of MCA is its early suppressive effect on NK cell activity.     

Effects of local hyperthermia on natural killer activity in mice

The effect of local hyperthermia on natural killer (NK) activity in C3H mice was investigated, because there have been reports on both the enhancing and suppressing effects of hyperthermia on NK activity. When right hind legs of mice were treated at 43°C for 45 min, NK activity was first suppressed. It reached its lowest level 2 days after the treatment, then recovered, and was significantly enhanced on the 7th day. When mice were treated at a lower temperature (41°C), NK activity was enhanced even 2 days after the treatment. In addition, the suppression of NK activity, which was observed 2 days after the treatment at 43°C, was diminished to some extent by i.p. injection of liposomal recombinant human superoxide dismutase (L-r-hSOD). From these results it is suggested that local hyperthermia had an enhancing effect on NK activity, which plays an important role in the anti-tumour mechanism of hyperthermia, and that transient suppression of NK activity after hyperthermia at 43°C was partially cancelled by the administration of L-r-hSOD.     

Effects of local hyperthermia on natural killer activity in mice

The effect of local hyperthermia on natural killer (NK) activity in C3H mice was investigated, because there have been reports on both the enhancing and suppressing effects of hyperthermia on NK activity. When right hind legs of mice were treated at 43 degrees C for 45 min, NK activity was first suppressed. It reached its lowest level 2 days after the treatment, then recovered, and was significantly enhanced on the 7th day. When mice were treated at a lower temperature (41 degrees C), NK activity was enhanced even 2 days after the treatment. In addition, the suppression of NK activity, which was observed 2 days after the treatment at 43 degrees C, was diminished to some extent by i.p. injection of liposomal recombinant human superoxide dismutase (L-r-hSOD). From these results it is suggested that local hyperthermia had an enhancing effect on NK activity, which plays an important role in the anti-tumour mechanism of hyperthermia, and that transient suppression of NK activity after hyperthermia at 43 degrees C was partially cancelled by the administration of L-r-hSOD.     

Hyperthermic inactivation, recovery and induced thermotolerance of human natural killer cell lytic function

Natural killer (NK) cytotoxic activity in human peripheral blood mononuclear cells or preparations of large granular lymphocytes was assayed after a hyperthermia treatment. Human NK cells are very sensitive to hyperthermic inactivation; 30 min at 42 degrees C reduced NK lytic activity toward K562 target cells by 40-50%, 43 degrees C by 85-90% and 44 degrees C or 45 degrees C by 100%, but similar treatment of the target cells did not alter their sensitivity to lysis. However, holding at 37 degrees C allowed heated NK cells to recover their lytic activity. The extent of the recovery was inversely correlated with the temperature as well as the recovery time. Heated and subsequently recovered NK cells were more thermotolerant to loss of lytic function by a further hyperthermia exposure. About 0.65 degrees C increase in temperature was required for a 50% loss of lysis ability in the NK cells made thermotolerant by a previous hyperthermic exposure at 43 degrees C for 30 min. The Vmax for NK lytic activity of cells heated at 42 degrees C for 30 min was reduced by 70% compared with that of normal NK cells. When heated cells were incubated the Vmax recovered. There are at least two heat-sensitive processes involved in NK cytotoxic activity. Cells completely inactivated for target cell lysis by a 44 degrees C exposure still showed recognition and binding functions and acted as competitive inhibitors of unheated NK cells. Cells heated at 45 degrees C caused less inhibition and had lower ability to recognize and bind target cells. The lytic function is therefore a more heat-sensitive process than the recognition and binding functions, but both are heat-inhibitable. Individual variations in thermal sensitivity, recovery and induced thermotolerance were evident.     

KIR不相合的NK细胞对乳腺癌细胞的体外杀伤作用

放化疗导致的淋巴细胞减少，改变了免疫细胞亚群的格局，启动了免疫细胞的内源性增殖，重建了一个新的免疫微环境。其机制主要与抑制肿瘤免疫的调节性T细胞比例与功能显著下调；内源性细胞因子IL-7、IL-15等供应增多，淋巴细胞高效扩增；抗原提呈细胞功能增强和以非对称性为特点的淋巴细胞自稳性增生的改变等相关。经历了淋巴细胞减少状态下肿瘤免疫格局的改变后，机体消除了抑制肿瘤免疫的重要因素，打破了肿瘤免疫耐受。肿瘤放化疗后选择合适时间点与生物治疗联合治疗，可以诱导出优于单一生物治疗的抗肿瘤效应，部分修正了以往认为放化疗导致的淋巴细胞减少不利于抗肿瘤免疫生物治疗的观念，为临床攻克肿瘤开辟新的治疗途径。     

Tumor cells regulate the lytic activity of tumor-specific cytotoxic t lymphocytes by modulating the inhibitory natural killer receptor function

Tumor-infiltrating p58+ T cells from a renal tumor were specifically expanded in response to tumor cell stimulation and cloned. These p58+ T cells were found to express a memory phenotype and corresponded to clonal TCRBV3 T-cell expansion. Functionally, p58(+) CTLs displayed a low lytic activity for HLA-A2 tumor and normal cells. However, this lytic activity was significantly increased after blockade of p58 with specific monoclonal antibodies. Interestingly, we demonstrated that stimulation by tumor cells was required to trigger the inhibitory effect of p58 on the lytic activity of antigen-specific CTLs and that stimulation of the inhibitory function of p58 by tumor cells correlated with an inhibition of nuclear factor-kappaB activation in p58+ tumor-specific CTLS.     

Phenotype and function of natural killer cells in patients with bronchogenic carcinoma.

Decreased peripheral blood natural killer (NK) cell lytic activity may be associated with tumor presence. We evaluated peripheral blood NK lytic activity in 38 patients with bronchogenic carcinoma and compared this to ten normal volunteers of comparable age. The patients with carcinoma had significantly (P less than 0.001) less NK activity (20 +/- 17 lytic units at 25% specific lysis)/10(6) peripheral blood lymphocytes) against the K562 tumor target compared to normal (69 +/- 9, SD). NK subpopulations can be defined phenotypically using CD56, CD16, and CD3 monoclonal antibodies and express differing degrees of lytic activity. NK cells from patients with carcinoma had the same absolute number of CD56+ cells and percentage of CD56+CD16+ NK cells (the most lytic subpopulation). However, patients with carcinoma had significantly (P less than 0.001) more CD56+CD16-CD3- cells in their overall NK population. This is of note, since this subpopulation is the least lytic. Patient NK cells bound to tumor cells as effectively as those from normal volunteers; however, the maximum rate of kill of patient NK cells was significantly (P less than 0.001) less. Thus, decreased NK lytic activity in patients with carcinoma was not due to decreased numbers or proportion of NK cells in peripheral blood or to defective tumor cell binding, but rather to the large CD56+CD16-CD3-NK subpopulation which is characterized by minimal lytic activity. The relation of this NK cell population with the presence of carcinoma is currently under investigation.     

Leuprolide vs. diethylstilboestrol: effect on natural killer cells

Recent studies suggest even low dosages of oestrogen currently used for treatment of prostate cancer increase cardiovascular morbidity. In addition, relapse following growth of hormone - insensitive cells, and the present observations of further evidence of immunosuppression demonstrated by the significant (p less than 0.001) effect of DES on the lytic activity of natural killer cells vs. the negligible effect of the luteinizing - hormone - releasing - hormone, leuprolide (Lupron) raises concern that the palliative effects of oestrogen therapy are possibly further compromised by a reduction in immunosurveillance to tumour, or equally important, by a decreased capacity to cope with infectious agents.     

自然杀伤细胞对小鼠HepS肝癌移植瘤的抑制作用研究

目的 研究自然杀伤(NK)细胞对原发性肝癌Heps移植瘤小鼠的治疗作用和机理。方法 建立小鼠移植性肝癌(Heps)实体瘤模型,随机分为生理盐水(NS)组、治疗1组(输注的NK细胞数为1×10⁴/只)、治疗2组(输注的NK细胞数为1×10⁵/只)和治疗3组(输注的NK细胞数为1×10⁶/只),每组15只。接种肝癌细胞当天,分别于尾静脉注射不同数量的NK细胞,对照组注射NS。观测小鼠移植瘤体积及各组小鼠平均生存时间。各组于输注NK细胞后第15天分别处死2只小鼠,对移植瘤组织切片进行HE染色,用流式细胞术(FCM)检测脾细胞NK的含量;乳酸脱氢酶(LDH)释放法检测脾细胞杀伤活性。结果 接种移植瘤后第8天,治疗3组移植瘤的生长速度较其他组小鼠显著减慢(P＜0.05)。第20天时治疗1组移植瘤体积明显小于对照组(P＜0.05),治疗2组和3组移植瘤体积非常显著小于对照组(P＜0.01)。治疗1组小鼠平均生存期明显长于对照组和治疗2、3组(P＜0.01)。治疗1-3组脾细胞杀伤活性以及NK细胞的含量高于对照组(P＜0.01)。结论 适量输注NK细胞可以有效抑制HepS肝癌移植瘤的生长,延长小鼠的生存期,为临床应用NK细胞治疗时数量的选择提供了实验依据。     

Cytolytic function and survival of natural killer cells are severely altered in myelodysplastic syndromes.

Natural Killer (NK) cells are critical in host defense against malignant transformation and are potent antileukemic cytotoxic effectors. In the present study, we investigated the peripheral NK function in patients with myelodysplastic syndromes (MDS). We demonstrated that the peripheral NK cell population was quantitatively normal in MDS patients. Furthermore, NK cells displayed an expression of the activating natural cytotoxicity receptors (NCR) NKp46 and NKp30 as well as NKG2D similar to that observed in donors, but exert a highly decreased constitutive cytolytic activity compared to resting normal NK cells. Although activation with IL-2 resulted in the upregulation of NKp46 expression by MDS-NK cells, their cytolytic function remained deeply altered as compared to activated donor NK cells. In addition, MDS NK cells did not proliferate in vitro, and displayed an increased rate of apoptosis in response to IL-2 stimulation although the spontaneous apoptosis was not significantly increased. Interestingly, a proportion of peripheral MDS-NK cells were derived from the MDS clone as the cytogenetic anomaly found in bone marrow karyotype was also detected in 20-50% of circulating NK cells. In conclusion, NK cells' cytolytic function and proliferative capacities in response to activation by cytokines are profoundly altered in MDS.     

Dissecting the role of hyperthermia in natural killer cell mediated anti-tumor responses.

The effects of hyperthermia on natural killer (NK) cell cytotoxicity against tumor cell targets are not yet fully understood. A more complete understanding of these effects could be important for maximizing the clinical benefits obtained by using hyperthermia for cancer therapy. Here, we summarize results in the literature regarding the effects of elevated temperatures on NK cells and our own recent data on the effects of fever-range temperatures. At treatment temperatures above 40 degrees C, (which is above the physiological body temperatures normally achieved during fever or exercise), both enhancing and inhibitory effects on cytotoxic activity of NK cells against tumor cells have been reported. Our own results have shown that fever-range thermal stress (using a temperature of 39.5 degrees C) enhances human NK cell cytotoxicity against tumor target cells. This effect requires function of the NKG2D receptor of NK cells, and is maximal when both NK and tumor cell targets are heated. Reported heat sensitive cellular targets affected by hyperthermia on tumor cells include heat shock proteins, MICA and MHC Class I. In NK cells, plasma membrane reorganization may occur after mild heat stress. We conclude this review by listing several unresolved questions that should be addressed for a more complete understanding of the molecular mechanisms which underlie the effects of thermal stress on the function of NK cells. Altogether, the available data indicate a strong potential for heat-induced enhancement of NK cell activity in mediating, at least in part, the improved clinical responses seen when hyperthermia is used in combination with other therapies.     

Regulation of natural killer cell function

Individuals lacking natural killer (NK) cells have persistent viral infections and as a consequence die prematurely. In addition, mice with decreased NK cell function are more susceptible to carcinogen-induced cancers. Current evidence strongly suggests that downregulation of MHC by certain tumors and virally-infected cells results in NK cell attack due to the inability to trigger inhibitory Ly49, KIR, and NKG2A/CD94 class Ia and Ib MHC receptors. Extreme haplotype diversity is present in both mouse and human chromosomal segments coding for NK cell class Ia MHC receptors resulting in different numbers and types of receptors being expressed in individuals and different inbred mouse strains. Whether the absence or presence of a particular NK cell receptor gene is advantageous or deleterious for an individual with respect to immunity to pathogens and cancer is a question of paramount importance. Recent advances in our understanding of NK cell function are due to the identification of activating NK cell receptors, such as Ly49H and NKG2D, for specific viral and tumor ligands (m157 and Rae1, respectively). In a clinical setting, such MHC class I receptor diversity is advantageous with respect to preventing leukemic relapse in individuals treated for leukemia and receiving bone marrow transplants. Further delineation of NK cell receptors and tumor ligands will help researchers to exploit the innate immune system to better treat such diseases.     

Adenosine-mediated inhibition of the cytotoxic activity and cytokine production by activated natural killer cells

Adenosine is an important signaling molecule that regulates multiple physiologic processes and exerts major anti-inflammatory actions. Tumors have high concentrations of adenosine, which could inhibit the function of tumor-infiltrating lymphoid cells. We investigated the ability of adenosine and its stable analogue 2-chloroadenosine (CADO) to inhibit cytokine production and cytotoxic activity of lymphokine-activated killer (LAK) cells and determined whether both these effects are initiated via a common pathway. CADO strongly inhibited cytotoxic activity of LAK cells and attenuated the production of IFN-gamma, granulocyte macrophage colony-stimulating factor, tumor necrosis factor alpha, and macrophage inflammatory protein-1alpha by LAK cells stimulated by cross-linking of the Ly49D receptor. These inhibitory effects were associated with the ability of CADO to stimulate cyclic AMP (cAMP) production and activate protein kinase A (PKA). Using cAMP analogues with different affinities for the A and B sites of the regulatory subunits of PKA types I and II, we found that activation of PKA I, but not PKA II, mimicked the inhibitory effects of CADO on LAK cell cytotoxic activity and cytokine production. Inhibitors of the PKA catalytic subunits (H89 and PKI(14-22) peptide) failed to abrogate the inhibitory effects of CADO whereas Rp-8-Br-cAMPS, an antagonist of the RI subunit, blocked the inhibitory effects of CADO. We conclude that the inhibitory effects of adenosine are probably mediated via cAMP-dependent activation of the RI subunits of PKA I but are independent of the catalytic activity of PKA. Tumor-produced adenosine could be a potent tumor microenvironmental factor inhibiting the functional activity of tumor-infiltrating immune cells.     

Studies of human natural killer cells. I. in vivo parameters affecting normal cytotoxic function

The results of natural killer cell (NK) studies on 539 normal healthy donors tested from once to 213 times over a seven-year time span have been presented. NK activity did not vary with donor blood group, Rh type or (in a small sample) HLA type. There was a slight but significant increase in NK activity from birth to adulthood, and between males and females. The male/female difference was present at birth and persisted through adulthood. The relative NK activity of individual donors tested repeatedly over many years was remarkably consistent in spite of variability in the absolute cytotoxidty observed. The expression of NK data in terms of relative NK was found to be superior to other methods, and the values obtained were found to be independent of the NK-sensitive target cell used. Although age and sex differences in NK activity are slight, their existence should be considered when studies of NK activity in patients are analysed.     

Effect of whole-body hyperthermia and cyclophosphamide on natural killer cell activity in murine erythroleukemia

Mice infected with the polycythemia-inducing strain of Friend virus complex (FVC-P) develop a fatal erythroid disease similar in some respects to leukemia. Six- to eight-week-old DBA/2 female mice were injected i.v. with 0.5 ml of a virus suspension containing approximately 5 X 10(4) plaque-forming units and 5 X 10(3) spleen focus-forming units. Four treatment regimens were begun 3 days postinjection: (a) no treatment; (b) whole-body hyperthermia (WBH) alone; (c) cyclophosphamide (CY) alone; (d) WBH combined with CY. WBH treatment utilized a microwave generator operating at 2450 MHz. The i.p. temperature of the mice receiving WBH was maintained at 39.5-40 degrees C for 30 min. The CY was given i.p. at a dosage of 20 mg/kg of body weight. The various treatments, CY, WBH, CY + WBH were given once a week for 2 weeks. Natural killer cell activity was examined in all four groups of mice and was found to be significantly higher in the animals treated with WBH or CY. Our results show that WBH, either alone or in combination with CY, can prolong the lifespan of mice infected with lethal dosages of the FVC-P, possibly via a mechanism involving natural killer cells.     

γ干扰素对人NK细胞识别功能的负调节作用

目的 探讨γ干扰素(IFN-γ)对人NK细胞识别功能的负调节作用。方法 用MTT法测定人NK细胞系(NK92,NKL)的细胞毒活性及细胞增殖能力;用RT-PCR检测NK细胞受体(NKG2D、NKG2A／B、KIR2DLI、KIR2DSI)及NKG2D的识别配体主要组织相容性复合体Ⅰ类链相关分子A(MICA)的表达。结果 NK细胞系(NK92、NKL)对MICA表达阳性的肿瘤细胞杀伤活性明显高于对MICA表达阴性者;IFN-γ 1000U／ml以上可明显抑制NK细胞对MICA表达阳性肿瘤细胞的细胞毒活性,并轻度抑制NK细胞的增殖,而对MICA表达阴性肿瘤细胞的杀伤活性无明显抑制作用;IFN-γ可抑制NK细胞系活化受体NKG2D的表达,增强抑制性受体NKG2A／B和KIR2DLI的表达。结论 IFN-γ可能通过下调NK细胞活化受体的表达,上调抑制性受体的表达,使NK识别的信号平衡向抑制性方向倾斜,从而对NK细胞功能发挥负调节作用,这种作用可能是NK细胞自我调节功能的表现。     

Presence of natural killer cells in specific-pathogen-free chickens.

Spleen cells of normal specific-pathogen-free N, P, and 15 X 7 chickens were cytotoxic in vitro for target cells of Marek's disease lymphoma line MSB-1. The natural killer (NK) cell activity, best expressed in chickens over 7 weeks old, varied among several genetic lines of chickens tested. The NK cells were thermolabile, and incubation of the cells at 37 degrees C for 30--60 minutes resulted in substantial loss of cytotoxicity. The specificity of NK effector cells was directed against common antigen(s) on tumor cell lines of diverse origin but not on normal adult or embryonic cells.     

Human natural killer cells: a comprehensive review

The senior author of this comprehensive review observed and reported in 1969 that his lymphocytes killed allogeneic tumor cells in vitro. Some of his research associates and technicians and other healthy individuals also yielded such killer lymphocytes. The team considered pre-immunization to cancer occurring in individuals after in-family or professional exposure to patients with cancer (in an era when the concept of viral etiology of cancer was receiving major support); or that lymphocytes can acquire through blastic transformation immune reactivity to allogeneic cells anew in vitro. The phenomenon was eventually referred to as 'lymphocytes practicing Burnet's immunosurveillance.' Project site visitors of the USA NCI first viewed these observations as a matter of 'in vitro artifacts' being in opposition to strong tumor- specific cytotoxicity of tumor-bearing patients' lymphocytes recognized in the vast majority of other assays. After NCI funds were released for intramural studies on the phenomenon of non-specific cytotoxicity by lymphocytes, recipients (other than the senior author) of these NCI funds later characterized (1973-1975) the 'indiscriminately' cytotoxic lymphocyte populations as those of 'natural killer (NK) cells.' In this article, the original observations made in 1969-1971 are reviewed based on genuine material preserved by the senior author and are explained in view of recent discoveries that were not available at the time of the original observations. NK cells display a fascinating history arising first in urochordates during the cambrian explosion. At that level, NK cells protected their hosts from incompatible cell colony fusions and against intracellular, especially viral, pathogens. Since then, viruses evolved evasive maneuvers to escape NK cell attack on the infected cells. NK cells persisted after the evolution of adaptive immunity in cartilaginous fish fitting seamlessly into the new system. In mammals, NK cells assumed the role of chief arbitrators between the fetal trophoblast and maternal immune reactions to the semi-allograft fetus. Tumors induce in NK cells the same (inactivating; mediating Th2-type immunity) reactions the fetal trophoblast engenders in utero, but NK cells may overcome the host's tolerance to its tumor and kill tumor cells, especially when converted into lymphokine-activated killer (LAK) cells by molecular mediators of Th1-type immunity. The authors prepare and utilize LAK cells and IL-2 for adoptive immunotherapy of patients with metastatic melanoma and kidney carcinoma. A patient with malignant ascites due to ovarian carcinoma entered remission on LAK cell therapy. Just as dendritic cells, the major antigen presentors, may undergo malignant transformation, NK cells are also subject to transformation into FasL-producer virulent lymphoma-leukemia cells. The senior author reported in 1970 a patient with 'lymphosarcoma cell leukemia' whose circulating lymphoma cells killed indiscriminately human sarcoma and carcinoma cells. The exemplary case history of another patient with NK cell lymphoma-leukemia treated by the authors is presented.