Research on stress-induced apoptosis of natural killer cells and the alteration of their killing activity in mouse liver

AIM:To investigate the stress-induced apoptosis of natural killer(NK)cells and the changes in their killing activity in mouse livers.METHODS:A restraint stress model was established in mice.Flow cytometry was employed to measure the percentage of NK cells and the changes in their absolute number in mouse liver.The cytotoxicity of hepatic and splenic NK cells was assessed against YAC-1 target cells via a 4 h 51Cr-release assay.RESULTS:The restraint stress stimulation induced the apoptosis of NK cells in the liver and the spleen,which decreased the cell number.The number and percentage of NK cells in the spleen decreased.However,the number of NK cells in the liver decreased,whereas the percentage of NK cells was significantly increased.The apoptosis of NK cells increased gradually with prolonged stress time,and the macrophage-1(Mac-1)+NK cells were more susceptible to apoptosis than Mac-1-NK cells.Large numbers of Mac-1-NK cells in the liver,which are more resistant to stress-induced apoptosis,were observed than the Mac-1-NK cells in the spleen.The stress stimulation diminished the killing activity of NK cells in the spleen was significantly decreased,but the retention of numerous Mac-1-NK cells in the liver maintained the killing ability.CONCLUSION:Significant stress-induced apoptosis was observed among Mac-1+NK cells,but not Mac-1-NK cells in the mouse liver.Stress stimulation markedly decreased the killing activity of NK cells in the spleen but remained unchanged in the liver.

Inositol hexaphosphate-induced enhancement of natural killer cell activity correlates with suppression of colon carcinogenesis in rats

AIM： To investigate the anti-neoplastic effect of inositol hexaphosphate （InsP6 or phytic acid） on dimethylhydrazine （DMH）-induced colon tumor in rats and its effect on blood natural killer （NK） cell activity. METHODS： Healthy Wistar rats, 4 wk old, were divided into control group （fed with common food） and InsP6 group （fed with common food＋2% sodium inositol hexaphosphate in the drinking water）, 15 rats in each group. Both groups were injected with 1,2-dimethylhydrazine subcutaneously （20 mg/kg body weight） once a week for 20 wk. Rats were killed after 21 wk. The whole large intestine was isolated to determine the general condition of tumors and to test blood NK cell activity by lactate-dehydrogenase-release assay. RESULTS： Administration of InsP6 significantly increased blood NK cell activity in DMH-induced colorectal tumor in rats. InsP6 group had a smaller tumor size on average and a smaller number of tumors than the control group. Its mortality was also higher than that in control. However, the variables of body weight and tumor incidence were not significantly different between the two groups. CONCLUSION： InsP6 can increase blood NK cell activity in DMH-induced colon tumor in rats and inhibit tumor growth and metastasis in rats.

Clinical Detection of Peripheral Blood Natural Killer Cell Activity

Natural Killer (NK) cells are specific immune cells in human immune system. They have a quick effect and can exert a cytotoxic function without prior sensitization, and they show great application potential in cell-based immunotherapy, anti-infection in vivo. NK cell activity in peripheral blood can be used as one of the biomarkers of immune function response. It has a great positive guiding significance for the clinical prognosis of tumor patients, the prevention of cancer and anti-aging. The clinical detection strategies of NK cell activity in circulation mainly grouped into five types: methyl thiazolyl tetrazolium colorimetric, lactate dehydrogenase release, radionuclide labeling, flow cytometry and NK Vue cytokine release method. It has played an important role in different stages of clinical application development. This paper will make a comparative review of the above-mentioned detection strategies for the NK cell activity.

REGULATORY EFFECTS OF HUMAN RECOMBINANT IL-6 ON NATURAL KILLER CELL ACTIVITY OF HUMAN FETAL SPLEENS

In the present study it was proved first that human recombinant interleukin-6(HrIL-6) significantly augmented natural killer(NK) cell activity derived from human fetal spleens against K562 target cells in a 4 hours 51Cr release assay. The enhancement of NK activity with 24 hours preincubation in HrlL-6 was dose-dependent, and significantly higher than that of fresh NK cells and controls cultured with RPMI-1640 medium alone (P<0.001). We also found that IL-6 was able to augment NK activity from different fetal spleens at 20 to 40 weeks of gestation (up to 2.24 to 2.78 times), and no difference of NK activity of fetal splenocytes treated by HrIL-6 was observed between different fetal age (32.3% to 45.4%, P>0.05). Furthermore, IL-6-augmented NK activity of fetal splenocytes was very similar to adult levels (P>0.05). These finding strongly indicated that IL-6 plays an important role in the development of NK cell function during the gestational period, suggesting that IL-6 may be of importance in the regulation of host defense mechanisms against malignancies and viral diseases.

Autologous Natural Killer Cell/Natural Killer T Cell Immunotherapy of Malignant Diseases

Adoptive immunotherapy, the therapeutic infusion of ex vivo activated cancer-fighting white blood cells that was pioneered by Dr. Steven Rosenberg over 30 years ago, has become more widespread due to outstanding published research documenting the clinical efficacy of this strategy. Based on the well-established in vivo functions of NK and NKT cells, their integral role in the efficacy of certain chemotherapeutic and immunomodulatory agents, and their direct therapeutic action as displayed in clinical trials, the use of autologous natural killer cell infusions is an appropriate and warranted therapeutic option for the treatment of malignant diseases, especially in patients whose disease is refractory to standard treatments such as chemotherapy and radiation.

Feeder-free differentiation of human iPSCs into natural killer cells with cytotoxic potential against malignant brain rhabdoid tumor cells

Natural killer(NK)cells are cytotoxic immune cells that can eliminate target cells without prior stimulation.Human induced pluripotent stem cells(iPSCs)provide a robust source of NK cells for safe and effective cell-based immunotherapy against aggressive cancers.In this in vitro study,a feeder-free iPSC differentiation was performed to obtain iPSC-NK cells,and distinct maturational stages of iPSC-NK were characterized.Mature cells of CD56^(bright)CD16^(bright)phenotype showed upregulation of CD56,CD16,and NK cell activation markers NKG2D and NKp46 upon IL-15 exposure,while exposure to aggressive atypical teratoid/rhabdoid tumor(ATRT)cell lines enhanced NKG2D and NKp46 expression.Malignant cell exposure also increased CD107a degranulation markers and stimulated IFN-γsecretion in activated NK cells.CD56^(bright)CD16^(bright)iPSC-NK cells showed a ratio-dependent killing of ATRT cells,and the percentage lysis of CHLA-05-ATRT was higher than that of CHLA-02-ATRT.The iPSC-NK cells were also cytotoxic against other brain,kidney,and lung cancer cell lines.Further NK maturation yielded CD56^(-ve) CD16^(bright)cells,which lacked activation markers even after exposure to interleukins or ATRT cells-indicating diminished cytotoxicity.Generation and characterization of different NK phenotypes from iPSCs,coupled with their promising anti-tumor activity against ATRT in vitro,offer valuable insights into potential immunotherapeutic strategies for brain tumors.

自然杀伤(NK)细胞与2019冠状病毒病(COVID-19)发生发展的研究进展

自然杀伤(NK)细胞在清除病毒,执行免疫调节方面发挥着重要的作用,它通过分泌趋化因子以及与其他天然免疫细胞协同作用,被赋予天然免疫调节功能和获得性免疫反应。随着对严重急性呼吸综合征冠状病毒2(SARS-CoV-2)感染的2019冠状病毒病(COVID-19)的研究深入,发现NK细胞在其中发挥着重要作用。SARS-CoV-2感染会影响NK细胞的分布和效应功能,NK细胞的免疫应答或可影响新冠肺炎患者的预后。本文总结了近年来NK细胞在COVID-19感染中作用的研究现状,并且讨论了以NK细胞作为治疗剂的可能性,以期为防治SARS-CoV-2的免疫损害提供潜在思路。

基于自然杀伤(NK)细胞的肝癌免疫疗法研究进展

肝细胞肝癌(HCC)异质性高,晚期诊断和化疗耐药使其成为我国难治性肿瘤之一。自然杀伤(NK)细胞在机体的免疫监视过程中充当重要角色。但在肝癌的进展过程中,NK细胞的功能受损致肿瘤免疫逃逸。该文总结了HCC中基于NK细胞的不同免疫治疗策略,包括直接输注过继性NK细胞免疫疗法、基因工程NK细胞免疫疗法、靶向NK细胞受体免疫疗法、改变免疫抑制微环境免疫疗法、细胞因子增强NK细胞肿瘤毒性免疫疗法以及中医药增强NK细胞肿瘤毒性免疫疗法,这些基于NK细胞的免疫治疗策略已显示出巨大的治疗潜力。

NKG2A-HLA-E轴介导NK细胞应答在病毒感染性疾病中的作用及机制研究进展

自然杀伤(NK)细胞主要通过迅速释放细胞毒性介质以及细胞因子直接杀伤病毒感染的靶细胞。NK细胞表面抑制性和活化性受体的平衡以及靶细胞上相应配体的表达均参与调控NK细胞的杀伤功能。其中NK细胞2组成员A(NKG2A)是NK细胞上备受关注的抑制性受体之一,通过与其配体人类白细胞抗原E(HLA-E)的相互作用,可抑制NK细胞对自身正常组织细胞的杀伤活性。研究发现病毒感染细胞表面HLA-E表达上调,并且可与病毒蛋白来源的多肽结合形成复合物,通过与NKG2A的相互作用调控NK细胞功能,从而参与病毒感染性疾病发病过程。本文总结了NKG2A与HLA-E分子互作特点,以及NKG2A-HLA-E轴参与调控NK细胞的功能机制,有助于了解NK细胞在病毒感染性疾病中的重要作用。

利妥昔单抗对多发性硬化症复发期患者外周血NK细胞 功能

目的观察利妥昔单抗对多发性硬化症(MS)复发期患者外周血NK细胞活化及ADCC功能的影响及机制。方法选取MS复发期病例(n=28),并将无自身免疫疾病及重大感染疾病的受试者作为健康对照组(n=25),提取两组被检者外周血单个核细胞(PBMCs),检测利妥昔单抗处理前后B细胞、自然杀伤细胞(NK)细胞比例及NK细胞活化水平;建立PBMCs与人慢性髓源白血病细胞系K562细胞直接接触共培养体系,检测利妥昔单抗处理后NK细胞抗体依赖细胞介导细胞毒性作用(ADCC)的效应因子释放。结果利妥昔单抗能诱导MS复发期组和健康对照组外周血中B细胞和NK细胞比例显著下调,且能诱导健康对照组的NK细胞活化及ADCC功能的显著上调,但对MS复发期患者NK细胞的活化及ADCC功无明显影响。结论利妥昔单抗可诱导MS复发期患者B细胞及NK细胞比例下调,但对NK细胞的活化及其ADCC功能无明显影响。

桑黄多糖调控MICB/NKG2D信号通路增强机体对脑胶质瘤细胞的免疫监视作用

目的:探讨桑黄多糖对脑胶质瘤细胞免疫监视的增强作用及其对主要组织相容性复合物Ⅰ链相关基因B(MICB)/自然杀伤细胞活化性受体2D(NKG2D)信号通路的调控机制。方法:取6~8周龄C57BL/6小鼠60只和GL261细胞株培养传代,随机取其中的50只小鼠建立脑胶质瘤模型,其余10只小鼠记为对照组,另将建模成功小鼠采用随机数字表分组,榄香烯组予以100 mg/kg榄香烯与1 ml/100 g生理盐水混匀灌胃,桑黄多糖各剂量组分别予以50、100、200 mg/kg桑黄多糖溶于1 ml/100 g生理盐水中灌胃,对照组和模型组均予以等量生理盐水灌胃,均1次/d,共4周。干预后处死小鼠,比较各组瘤重和抑瘤率;苏木素-伊红(HE)染色观察瘤组织病理变化;以CD57抗体标记检测瘤组织中NK细胞浸润;实时-逆转录聚合酶链反应(RT-qPCR)检测瘤组织主要组织相容性复合物Ⅰ链相关基因A(MICA)、MICB、UL16结合蛋白1(ULBP1)、UL16结合蛋白2(ULBP2)、UL16结合蛋白3(ULBP3)mRNA表达,Western blot检测瘤组织MICA、MICB、ULBP1、ULBP2、ULBP3蛋白表达;采用磁珠亲和细胞分选法分离各组组织中NK细胞,采用流式细胞仪检测其表面NKG2D活性。结果:与模型组比较,榄香烯组和桑黄多糖各剂量组瘤重下降,抑瘤率升高,瘤组织细胞变性、坏死增多,NK细胞浸润增多,MICA、ULBP2、ULBP3 mRNA和蛋白表达下降,MICB、ULBP1 mRNA和蛋白表达升高,且NK细胞表面NKG2D活性增强,上述差异均有统计学意义(P<0.05);桑黄多糖的作用呈剂量依赖性,桑黄多糖低剂量组和榄香烯组所有指标比较差异均无统计学意义(P>0.05)。结论:桑黄多糖可抑制脑胶质瘤增长,促进瘤细胞变性、坏死,增加NK细胞浸润,推测d与调控MICB/NKG2D信号通路,增加MICB、ULBP1表达和NK细胞表面NKG2D活性,下调MICA、ULBP2、ULBP3表达,增强对脑胶质瘤细胞的免疫监视有关。

Breast cancer stem-like cells can promote metastasis by activating platelets and down-regulating antitumor activity of natural killer cells

OBJECTIVE:To investigate whether cancer stem cells(CSCs) more efficiently activating platelets and evading immune surveillance than non-CSCs thus promoting metastasis.METHODS:We enriched and identified sphereforming cells(SFCs) and coincubated washed platelets with several platelet activators including collagen,4T1 and SFCs.Platelet-coating tumor cells,platelet activation and TGF-β1 release were analyzed.Then natural kell cells(NK) were incubated with supernatants of different activated platelet samples what we called sample release(SR).The degranulation assay and NKG2 D expression on NK cells were conducted by flow cytometry.Finally tissue factor(TF) expression of SFCs or 4T1 were evaluated by western blot.RESULTS:Breast cancer cell line 4T1 could form spheres in serum-free medium at low adherence.Sphere-forming cells expressed high levels of the CD24-/lowCD44 + stem cell phenotype.Both sphere-forming cells or 4T1 were coated with abundant platelets while sphere-forming cells induced significantly higher expression of platelet activating receptor CD62 p than 4T1 did(P < 0.01).And sphere-forming cells induced platelets to produce more TGF-β1 than 4T1 did(P < 0.01).Furthermore,sample releases induced by sphere-forming cells caused more vigorous inhibition of NK cells antitumor reactivity(P < 0.05) and reduced NKG2 D expression(P < 0.01).The final results showed that sphere-forming cells expressed higher levels of TF than 4T1(P < 0.05).CONCLUSION:Our findings indicate that CSCs could efficiently activate platelets,induce platelets to secrete more TGF-β1,decrease NKG2 D expression and inhibit antitumor activity of NK cell,compared with 4T1.And higher levels of TF expression of CSCs may account for this correlation of CSCs and platelets.

Avian influenza virus directly infects human natural killer cells and inhibits cell activity

Natural killer （NK） cell is a key component of innate immunity and plays an important role in host defense against virus infection by directly destroying infected cells. Influenza is a respiratory disease transmitted in the early phase of virus infection. Evasion of host innate immunity including NK cells is critical for the virus to expand and establish a successful acute infection. Previously, we showed that human influenza HIN1 virus infects NK cells and induces cell apoptosis, as well as inhibits NK cell activity. In this study, we further demonstrated that avian influenza virus also directly targeted NK cells as an immunoevasion strategy. The avian virus infected human NK cells and induced cell apoptosis. In addition, avian influenza virion and HA protein inhibited NK cell cytotoxicity. This novel strategy has obvious advantages for avian influenza virus, allowing the virus sufficient time to expand and subsequent spread before the onset of the specific immune response. Our findings provide an important clue for the immunopathogenesis of avian influenza, and also suggest that direct targeting NK cells may be a common strategy used by both human and avian influenza viruses to evade NK cell immunity.

AUGMENTATION OF IMMUNE FUNCTIONS AND AUTOLOGOUS TUMOR-KILLING ACTIVITY BY KAPPA-SELENOCARRAGEENAN IN MICE BEARING SARCOMA 180

Objective: To study the enhancement of the immune functions and autologous tumor killing (ATK) activity by kappa selenocarrageenan (KSC) in mice bearing sarcoma 180. Methods: To measure the effects of KSC and/or Cyclophosphamide (Cy) on natural killer (NK) activity, lymphokine activated killer (LAK) activity, the produc tion of interleukin 2 (IL 2), ATK activity and the growth of sarcoma 180 (S 180 ). Results: KSC promoted NK activity, LAK activity and ATK activity in vivo , increased IL 2 production at 40 mg/kg/d×9d. It also enhanced the antitumor action of Cy (20 mg/kg/d×9d) and offset the inhibition of Cy on immunocopetent cells. The ATK activity in splenocytes of S 180 bearing mice could be induced and increased by recombinant interleukin 2 (rIL 2) in vitro . Conclusion: KSC has an up regulating effect on the immune functions and ATK activity in tumor bearing mice. It can be used as a biological response modifier (BRM) in cancer biotherapy.

CD226、 TIGIT和CD96调节NK细胞功能参与机体抗肿瘤免疫

CD226是自然杀伤(NK)细胞表面的活化性受体,与具有免疫球蛋白和免疫受体酪氨酸抑制基序结构域的T细胞免疫受体(TIGIT)和CD96等分子通过竞争结合肿瘤细胞表面的CD155等配体,介导NK细胞的杀伤功能。虽然TIGIT和CD96在肿瘤微环境中有其他结合配体,但它们以更高的亲和力竞争结合CD115配体,并抑制NK细胞的活性,使肿瘤细胞逃避杀伤。因此,研究这三种NK细胞表面受体在不同肿瘤中的表达模式,并监测分析它们与配体的结合能力,有助于探索新的肿瘤治疗策略。本文总结了CD226、 TIGIT和CD96等NK细胞受体分子调节NK细胞功能在机体抗肿瘤免疫应答中的作用及其机制。

Phenotypic Characteristics and Function of NK Cell Subsets in cART-Treated HIV-1-Infected Individuals

Natural killer (NK) cell plays an important role in an innate immune response against viral infection. The kinetics regulation and functional consequences of NK cells in the pathogeneses of diseases are uncertain. We analyzed NK cell distribution and function of successfully combination antiretroviral therapy (cART)-treated HIV-1 infected individuals in Khon Kaen Regional Hospital, Thailand. The results demonstrated that increased percentage and the total number of NK cell in cART-treated HIV-1 infected patients with preferential high levels of CD56dimCD16+ and CD56-CD16+ subsets when compared with a control group even in undetectable viral load (<40 copies per milliliter). Concomitantly, decreased cytotoxic activity measured by CD107asurface expression with maintained IFN-γ production implied the impairment of cytolytic activity was not recovered after cART treatment. Thus, altered NK cell frequency and function by HIV-1 infection are not completely recovered with cART, which may contribute to impaired cellular immune response and persistence of HIV-1.

Double potentialities of tumoricide and hematopoiesis of human bone marrow cells activated by IL-2 and IL-3

The biomodulative and hematopoietic potentialities of IL-2 and IL-3 activatedbone marrow(ABM)cells from patients with lung adenocarcinoma were studied in vitro.Human bone marrow(BM)cells could be activated by IL-2 in culture for 7d.TheseIL-2 ABM cells had higher cytolytic activities against cells of H 7402 cell line and freshautologous adenocarcinoma cells and maintained the cytotoxicities longer than IL-2 acti-vated peripheral blood lymphocytes(APBLs),a point of possible importance in adoptiveimmunotherapy for cancer patients.The IL-2 ABM cells also had similar number ofBFU-E and CFU-GM to that had fresh BM cells if 1L-3 was added 48h alter IL-2 inculture.The IL-2 and IL-3 ABM cells might be used to eliminate tumor cells and tosupply reconstitutive elements of BM for autologous bone marrow transplantation.

玉屏风散通过STAT3改善肝细胞肝癌免疫抑制微环境中NK细胞活性的机制研究

目的:阐明玉屏风散在发挥抗肝细胞肝癌(hepatocellular carcinoma,HCC)效应作用中对自然杀伤细胞(natural killer cell,NK细胞)活性的影响及其机制。方法:采用Hepa1-6 HCC小鼠模型,造模后5 d,用玉屏风散药液(20、30、40 g生药/kg)进行灌胃给药,2周后处死小鼠。瘤体质量称重用于抑瘤率的计算;Western Blot检测肿瘤组织中信号转导和转录激活因子3(signal transducer and activator of transcription 3,STAT3)/p-STAT3、程序性死亡因子1的配体(ligands for programmed death factor 1,PD-L1)的蛋白表达情况;ELISA法检测肿瘤组织中吲哚胺2,3-二氧化酶(indoleamine 2,3-dioxygenase,IDO)、TGF-β的表达情况;从癌旁组织中分选出NK细胞,流式细胞术和ELISA法检测NK细胞活性相关因子颗粒酶、穿孔素(Granzyme,Perforin)、IFN-γ的表达情况。结果:与模型组相比,不同剂量的玉屏风散(20、30、40 g生药/kg)均显著抑制HCC小鼠肿瘤的生长(均P<0.05),且呈剂量依赖型,其抑瘤率分别为21.48%、36.12%、49.56%;与模型组比,玉屏风散(20、30、40 g生药/kg)可抑制肿瘤组织中STAT3/p-STAT3、PD-L1的蛋白表达,以及IDO、TGF-β的分泌;且对癌旁组织中NK细胞活性相关因子Granzyme、Perforin、IFN-γ的表达有明显促进作用。结论:玉屏风散是通过抑制STAT3的活化来改善HCC免疫微环境中NK细胞的免疫活性,从而抑制HCC的发生发展。

Case Report: Feasibility and Safety of Autologous NK Cell Therapy in Patients with Cancer

This study reported two cases of Thai cancer patients, including a 36-year-old female with thyroid cancer of more than 5 years and a 64-year-old male with lung and colon cancers of more than 10 years. The written informed consent was provided for autologous natural killer (NK) cell infusion at the anti-ageing and regenerative medicines clinic. Briefly, the blood was taken from the patient for NK cell count and their cytotoxic activity. Then, the patient’s NK cells were expanded in vitro, characterized and then counted before being delivered to the same patient by a single intravenous infusion. The vital signs and general physical examinations were observed for 2 - 6 hours after the infusion. The patients were discharged if there were no adverse effects. The data showed the increasing number of NK cells and level of cytotoxic activity after the NK cell treatment, compared to the pre-treatment. In addition, the increasing total live cell concentration, as identified by the high percentage of CD56dim/CD16bright cytotoxic NK cells, at day 21 of the NK cell expansion was consistent with the increasing cytotoxic activity of the patients after the treatment. Here, we demonstrated that this autologous NK cell therapy might be feasible;however, the study did not aim to evaluate the anti-cancer effect.

用于癌症免疫治疗的CAR-NK细胞递送技术

嵌合抗原受体(chimeric antigen receptor, CAR)工程化T (CAR-T)细胞在治疗血液系统恶性肿瘤方面取得显著成功,成为肿瘤免疫治疗的临床热点。然而,CAR-T细胞在对抗实体恶性肿瘤方面并未表现出太大的优势,因此,需要寻找新的效应细胞作为CAR修饰的候选细胞进行研究。最近,自然杀伤(natural killer, NK)细胞已成为CAR工程化的安全有效平台。CAR-NK细胞可被设计成能够靶向多种抗原的工程化细胞,以增强体内增殖力和持久性,增加对实体瘤的浸润,克服耐药性肿瘤微环境,最终实现有效的抗肿瘤反应。尽管NK细胞具有巨大的潜力,但已被证明难以改造,对细胞凋亡具有高敏感性且基因表达水平低。因此,对基因导入NK细胞的方法进行优化,会促进CAR-NK在研究和临床中的广泛应用。本文综述近年来NK细胞基因工程递送技术的改进,以及为增强NK细胞效应功能而实施的策略,包括NK细胞生物学特性、高效和安全的CAR构建体的设计、递送含CAR转基因载体的种类分析及提高转导效率方法的优化,以更好地靶向恶性肿瘤或抑制肿瘤的微环境,并进一步探讨CAR-NK相关研究中存在的问题和挑战,为恶性肿瘤相关疾病的治疗提供新的思路和方向。