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.

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.

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。

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。

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.

The Skin Whitening Effect of Co-Cultured Conditioned Medium: Involvement of Synergy between Stem Cells and Immune Cells

Many researchers have described that mesenchymal stem cells conditioned medium and immune cells conditioned medium have a clear whitening effect when they are used as cosmetic ingredients. In this study, we confirmed the whitening efficacy of various concentrations of immune cells and stem cell conditioned media. The author tried to study a conditioned medium that has a strong whitening effect even with a composition of less than 20% (the most used concentration in cosmetics). Because of the fact that the conditioned medium contains various cytokines and growth factors secreted by stem cells or immune cells, it is known to have effects such as wound healing, antioxidant, and whitening effect. Recently, stem cells have been used not only in the development of cosmetic raw materials but also in skincare procedures, and there are reports being released of cosmetics using immune cells conditioned medium. The concentration-dependent whitening effect equivalently increased as the concentration of the mono-cultured conditioned medium was obtained through the stem cells or immune cells culture. In the case of co-culture, whitening results are like the effect of positive control such as arbutin in the medium carrying only 10% of the co-cultured conditioned medium. It is possible that enhanced whitening efficiency in co-cultured conditioned medium leads to a major innovation in the global cosmetic market.

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。

CAR-NK细胞治疗技术专利分析

目的 从专利视角揭示CAR-NK细胞治疗技术的研究进展,以期为我国创新主体的研发提供有益参考。方法 通过中国专利检索与服务系统数据库检索CAR-NK相关专利,对其专利申请趋势、地区分布情况和主要申请人的研发路线进行分析。结果 CAR-NK专利申请呈稳步增长态势,国内外多家企业正在该领域加速布局,国内公司研究主要集中于对不同靶点的CAR-NK的设计,对联合用药及上下游技术涉及较少,专利申请分布的技术方向相对单一,处于较为初始的研发阶段,而国外大型医药公司的专利申请注重多角度布局,涉及产业链各个环节。结论 我国CAR-NK领域高价值专利数量相对较少,今后还需在专利布局和成果转化上给予更高的重视。

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.

肿瘤浸润性自然杀伤细胞在免疫治疗中的研究进展

自然杀伤(NK)细胞是一种具有强大细胞毒性和活性的先天淋巴细胞。基于NK细胞的免疫疗法在血液系统肿瘤的治疗中取得显著疗效,但在实体恶性肿瘤的治疗中疗效较低。该文就NK细胞的功能、表型及其肿瘤免疫治疗的研究进展作一综述。

糖萜素对小鼠NK细胞活性影响的研究

目的 :探讨糖萜素对机体 NK细胞活性的影响。方法 :1体外试验 ,不同浓度糖萜素体外直接作用小鼠脾细胞和 YAC-1细胞 ,用 MTT法测定细胞生长代谢和 NK细胞活性 ;2体内试验 ,以糖萜素饲料和普通饲料分别喂养正常和免疫抑制 (环磷酰胺抑制 )小鼠 ,在不同时间段检测 NK细胞活性。结果 :1体外 <5μg/ ml的糖萜素浓度 ,对小鼠脾细胞生长和 NK细胞活性无明显影响 ( P>0 .0 5) ;2以糖萜素饲料喂养的正常小鼠和免疫抑制小鼠 ,各时间段 NK细胞活性明显高于喂养普通饲料的对照小鼠 ( P<0 .0 5)。结论 :口服糖萜素能明显增强小鼠 NK细胞活性 ,且使其稳定维持在高水平。

不同肿瘤细胞表面MICA的表达及NK细胞杀伤活性的研究

目的观察不同肿瘤细胞表面MICA的表达及其对NK细胞杀伤活性的影响。方法以体外培养的K562(人慢性髓原白血病细胞株)、MCF-7(乳腺癌细胞株)、HR-8348(直肠腺癌细胞株)、CNE-2(人鼻咽癌细胞株)、Hela(人宫颈癌细胞株)为靶细胞,流式细胞仪检测细胞表面MICA的表达,以K562细胞作为对照,应用LDH释放法检测不同效靶比情况下体外培养的NK细胞对靶细胞的杀伤活性。结果K562、MCF-7、HR-8348、CNE-2、Hela细胞表面均表达MICA,体外杀伤试验表明NK细胞对上述肿瘤细胞均有杀伤活性,anti-MICA单抗可部分封闭NK细胞对靶细胞的杀伤活性。结论NK细胞对K562、MCF-7、HR-8348、CNE-2、Hela细胞具有较高的杀伤活性,可作为一种生物治疗手段。

MICA分子在NK细胞杀伤乳腺癌中的作用

目的观察乳腺肿瘤细胞膜MHCⅠ链相关分子A(MHC class I chain-related gene A,MICA)及血清中可溶性MICA表达,探讨MICA在NK细胞杀伤乳腺癌中的作用。方法流式细胞术检测膜型MICA(mMICA)及NK细胞表面NKG2D表达,观察膜型MICA、可溶性MICA(sMICA)对NKG2D表达的影响;免疫组织化学检测膜型MICA及可溶性MICA的表达及分布;抗体封闭法观察膜型MICA与NKG2D相互作用。结果乳腺细胞膜型MICA在正常组织不表达,在良性肿瘤表达量为(38.5±7.5)%,恶性肿瘤表达量为(53.2±5.6)%。可溶性MICA含量在健康成年人为阴性,在乳腺良性肿瘤患者血清中含量(76.8±22.3)pg/mL,在恶性肿瘤患者中含量(205.36±71.27)pg/mL。经NKG2D或MICA抗体封闭后,NK细胞的杀瘤活性显著减弱。含可溶性MICA的血清可明显下调NKG2D的表达。结论大部分乳腺肿瘤细胞膜都有MICA的表达,可作为乳腺肿瘤相关性抗原。膜型MICA与NKG2D的相互识别在介导NK细胞抗乳腺癌中起重要作用,而可溶性MICA通过下调NKG2D表达介导肿瘤免疫逃逸。

结直肠癌中自然杀伤细胞表型及功能初探

目的·初步探究结直肠癌(colorectal cancer,CRC)患者肿瘤组织中免疫微环境的组成,分析自然杀伤细胞(natural killer cell,NK细胞)在CRC中的比例、表型及功能特征。方法·收集CRC患者肿瘤组织及配对的癌旁组织和匹配的CRC患者外周血样本,消化组织制备成单细胞悬液,使用流式细胞术检测各类免疫细胞比例,通过t-SNE(t-distributed stochastic neighbor embedding)降维与统计学分析,描述CRC肿瘤免疫微环境的特征。运用流式细胞术检测肿瘤组织和癌旁组织中NK细胞表面活化分子的表达情况,包括CD16、CD27、CD69、晚期活化标志人类白细胞抗原-DR(human leukocyte antigen-DR,HLA-DR)、细胞耗竭标志T细胞免疫球蛋白与黏蛋白结构域3(T cell immunoglobulin domain and mucin domain-3,TIM-3)。运用流式细胞术探究CD38分子在NK细胞上的表达水平,描述CD38highNK细胞的表型特征。利用细胞刺激试剂盒激活NK细胞,通过流式细胞术胞内染色检测NK细胞的功能,包括细胞因子γ干扰素(interferon-γ,IFN-γ)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)的表达水平。结果·收集25例CRC患者肿瘤组织及配对的癌旁组织和15例匹配的CRC患者外周血样本。CRC肿瘤组织中包含T淋巴细胞、B淋巴细胞、NK细胞及髓系细胞等免疫细胞。与癌旁组织相比,肿瘤组织中T淋巴细胞(P=0.000)和髓系细胞(P=0.026)的比例显著升高,NK细胞(P=0.007)的比例显著降低,B淋巴细胞比例差异无统计学意义。与癌旁组织相比,肿瘤组织中NK细胞表面CD27(P=0.000)、CD69(P=0.001)表达水平显著降低,同时CD16(P=0.008)、HLA-DR(P=0.000)和TIM-3(P=0.024)的表达水平显著升高,呈现晚期活化和耗竭表型。NK细胞按CD38的表达水平可分为CD38highNK和CD38lowNK 2个亚群。与癌旁组织相比,肿瘤组织CD38highNK(P=0.003)比例显著降低,CD38lowNK比例差异无统计学意义。相比CD38lowNK,CD38highNK的CD27高表达,CD16、NKp46、CD57、CD94、HLA-DR和CD158a低表达(均P=0.000),处于早期分化和未活化的状态。肿瘤浸润的NK细胞与癌旁组织相比,分泌细胞因子IFN-γ(P=0.032)、TNF-α(P=0.042)和GM-CSF(P=0.019)的水平显著降低,表明NK细胞杀伤功能受损。结论·CRC患者肿瘤组织中NK细胞浸润减少,早期分化状态的CD38highNK细胞亚群下降,分泌细胞因子的能力下降并呈现耗竭表型。这些结果提示NK细胞在CRC中功能受损,介导的抗肿瘤免疫应答下降。

NK细胞肿瘤免疫治疗技术的挑战

自然杀伤(NK)细胞具有以MHC I非依赖识别机制和快速杀伤病变细胞能力、低移植物抗宿主反应(GVHD)风险、可采用异体细胞回输、体内存活周期短和无细胞因子风暴等长期和不可预期风险较低等特点和优势,使其在肿瘤免疫治疗中展现出巨大的应用潜力。虽然外周血单个核细胞(PBMC)来源NK细胞相比干细胞来源NK和NK细胞系在安全性和肿瘤杀伤能力上相对更好,但细胞制备技术的效率、稳定性和安全性仍有待完善;NK细胞被认为是较理想的嵌合抗原受体(chimeric antigen receptor,CAR)载体,但外周血来源NK细胞转染效率较低,影响了CAR-NK的研发进程。由于NK细胞来源和培养技术的多样性,使细胞制品的活性不一,虽然NK细胞在抗血液肿瘤治疗中表现相对突出,但对实体瘤的治疗效果仍有待验证。总之,NK细胞应用开发近年已取得显著进步,但仍面临生产技术和临床疗效等诸多挑战。

同种异体NK细胞对不同肿瘤细胞的体外杀伤活性及其机制的初步探讨

为了观察同种异体NK细胞对不同肿瘤细胞的体外杀伤活性,并初步探讨其分子机制。以K562细胞为对照,应用LDH释放法检测不同效靶比时同种异体NK细胞杀伤CNE2、KG1a和U251细胞的活性。应用RT-PCR和流式细胞仪分别检测4种细胞MHCI类链相关分子(MICA/B)和人巨细胞病毒糖蛋白UL16结合蛋白(ULBP1~3)基因和分子的表达情况。效靶比20∶1时用AMO-1、BMO-1、M295、M310和M551单抗分别阻断肿瘤细胞表面MICA、MICB、ULBP1、ULBP2和ULBP3分子,观察NK细胞对其杀伤活性的变化。结果:NK细胞对K562、CNE2和KG1a细胞均有杀伤活性,对U251细胞无杀伤活性。在mRNA水平4种细胞均表达MICA/B和ULBP1~3基因。K562细胞表达MICA/B和ULBP1~3全部分子;KG1a和U251细胞均不表达5种分子;CNE2细胞表达MICA/B和ULBP2,不表达ULBP1和ULBP3。CNE2、KG1a和U251细胞均高表达HLAI分子,而K562细胞不表达。用单抗分别阻断靶细胞表面相应的NKG2D配体分子,NK细胞对KG1a和U251细胞的杀伤活性无变化。...

Harnessing natural killer cells to develop next-generation cellular immunotherapy

Cellular immunotherapy harnesses the body’’s own immune system to fight cancer by using engineered T cells,macrophages,or natural killer(NK)cells.Compared to chimeric antigen receptor T(CAR-T)cells that are commonly used to treat hematological malignancies,CAR-NK cells have shown remarkable therapeutic effectiveness while exhibiting enhanced safety,reduced risk of graft-versus-host disease,fewer side effects,and amplified antitumor efficacy.Preclinical trials have unveiled the high potential of adoptive CAR-NK cell therapy to curtail or even eliminate both hematological malignancies and solid tumors in animal models.We brought forth herein the design principle of CAR-NK cells,highlighted the latest progress in the preclinical testing and clinical trials of CAR-NK cells,briefly delved into discussed major roadblocks in CAR-NK therapy,and discussed potential solutions to surmount these challenges.Given the accelerated progress in both basic and translational studies on immune cell engineering,CAR-NK cell therapy promises to become a serious contender and important addition to the next-generation cell-based immunotherapy.

IL2、IL12、IL15的不同组合对人外周血源NK细胞功能的影响

本研究旨在探讨细胞因子IL2,IL12,IL15的不同组合对体外扩增的人外周血来源的NK细胞功能的影响。IL2、IL12和IL15等细胞因子的不同组合培养的人外周血NK细胞分为IL2组、IL2+IL12组、IL2+IL15组、IL2+IL15+IL12组;无细胞因子培养的NK细胞组为对照组。用细胞计数盒分别测定不同效靶比下NK细胞对靶细胞(K562)的杀伤作用;用ELISA方法检测培养液上清中IFN-γ的分泌水平;用建立竞争性RT-PCR方法定量检测穿孔蛋白、颗粒酶BmRNA的表达水平。结果显示,各组培养的NK细胞对K562细胞的杀伤率增强,在不同效靶比下IL2+IL15和IL2+IL15+IL12组NK细胞对K562细胞的杀伤率显著高于其他组,但此两组间无显著性差异(p>0.05);各细胞因子组培养液中分泌IFN-γ水平均显著高于未加细胞因子的空白对照组(p<0.01),在不同细胞因子条件下,IFN-γ分泌水平依次为IL2+IL12+IL15组>IL2+IL12组>IL2+IL15组>IL2组(p<0.01);各培养组的NK细胞穿孔蛋白及颗粒酶B基因的表达量均较对照组明显增加(p<0.01),且IL2+IL15组和IL2+IL12+IL15组上述两种基因的表达量均显著高于其他组(p<0.01),但组间差别较小,与NK细胞杀伤实验的结果相一致。结论:不同细胞因子组合诱导的外周血NK细胞功能有差异。IL2和IL15在增强NK细胞杀伤功能及促进其扩增作用方面有协同作用,IL12主要刺激NK细胞分泌细胞因子(如IFN-γ),增强其免疫调节功能。