K562-4-1BBL-MICA工程细胞构建及其联合IL-21对NK细胞体外扩增的研究
摘要
肿瘤是机体在各种理化诱变剂或生物致癌因素作用下，细胞在基因水平上失去对其生长的正常调控而异常增生形成的病变。肿瘤的发生意味着免疫系统内的免疫器官、免疫细胞或免疫分子部分发生故障，使得机体免疫监视功能受损而不能正常行使清除自发突变细胞的功能。晚期癌症患者的治疗以放射疗法、化学药物疗法为主，但是放、化疗的副作用明显，能引起骨髓造血抑制、免疫细胞功能低下。由于肿瘤的发生发展和预后同免疫细胞功能紧密相关，而激活的免疫细胞具有强大的肿瘤杀伤能力，免疫细胞过继疗法为主的肿瘤生物疗法逐渐成为继外科手术、放射疗法、化学药物疗法之后的第四大肿瘤治疗方法。淋巴因子激活的杀伤细胞（lymphokine-activated killercells，LAK）成分复杂且在转移至患者机体后仍需要高浓度IL-2的刺激来维持肿瘤杀伤活性，而高浓度IL-2会诱发严重的毒副反应如毛细血管渗漏综合征（capillary leaksyndrome，CLS）。肿瘤浸润T细胞（tumor-infiltrating lympbocytes，TIL）过继转移的免疫疗法在部分黑色素瘤患者中取得了显著的成功，但是限制其广泛应用的最大困难是无法分离得到足够数量的免疫细胞。在一些研究中，基因改造T细胞，让它表达嵌合抗原的受体，能够识别多种肿瘤相关抗原，在小鼠模型和患者的过继免疫治疗中，可以有效对抗多种类型肿瘤。至今，过继疗法的效应细胞主要是具有MHC限制性的CD8+T细胞，越来越多的研究报道了过继性转移自然杀伤（Natural Killer，NK）细胞能诱导更有效的抗肿瘤、抗感染免疫反应。
NK细胞是固有免疫系统的主要效应细胞，能无需预先刺激，通过使用穿孔素和颗粒酶以及其它机制，无MHC限制性杀伤肿瘤细胞和病毒感染细胞。NK细胞表达众多的激活性受体，能有效识别肿瘤细胞或感染细胞产生的应激性配体，而抑制性受体在靶细胞MHC表达下调或消失时则失去对NK细胞杀伤作用的抑制作用。同种异体或自体移植NK细胞在临床应用中很有前景，研究显示NK细胞有很强的抗肿瘤活性。但是NK细胞在外周血中比例小，体外扩增难，扩增后细胞毒活性弱，都是限制其应用于临床免疫治疗的瓶颈。
本实验的目的是通过基因工程的方法，构建基因修饰的K562工程细胞，使其双表达激活性配体4-1BBL和MICA，将其同体外分离的外周血单个核细胞（Pefipheral BloodMononuclear Cells，PBMCs）共培养，观察NK细胞体外扩增、检测细胞表面激活性、抑制性受体等表达、测定扩增前后NK细胞毒活性变化、分析扩增前后NK细胞亚群异同，为NK细胞过继疗法从实验室研究向临床应用的转化提供理论依据。
1K562-4-1BBL-MICA工程细胞的制备
1.14-1BBL和MICA表达质粒的构建
从人PBMCs中提取总RNA，根据NCBI参考序列（NCBI Reference Sequence：NM_003811.3），设计引物，进行逆转录PCR获得4-1BBL的全长CDS序列。再次设计引物，将4-1BBL序列前后分别加入酶切位点BmaHI/XbaI，酶切后，连接到pcDNA3.1-Hygro中，构建成重组表达质粒pcDNA3.1-Hygro-4-1BBL。
同样的方法，从人K562细胞系中提取RNA，通过RT-PCR获得MICA全长CDS序列（NCBI Reference Sequence：NM_000247.1）。设计引物，将MICA序列加入酶切位点XhoI/Nhe I，酶切之后连接到pVITRO2-Neo-mcs的MCS2中，构建成重组表达质粒pVITRO2-Neo-MICA。
1.2构建并克隆化K562-4-1BBL-MICA细胞
将pcDNA3.1-Hygro-4-1BBL用脂质体转染法转染K562细胞，潮霉素筛选抗性细胞，流式细胞术测定表达4-1BBL的K562细胞比例，有限稀释法克隆出并通过流式细胞术鉴定高表达4-1BBL的K562细胞。接着将pVITRO2-Neo-MICA转染K562-4-1BBL细胞，G418和潮霉素共同筛选抗性细胞，同样的方法获得双表达4-1BBL和MICA的K562细胞。经过多次有限稀释法进行单克隆以及流式细胞术鉴定，最终获得单克隆化、可以稳定遗传的K562-4-1BBL-MICA细胞株。
2K562-4-1BBL-MICA工程细胞体外扩增PBMCs中NK细胞
2.1体外扩增NK细胞
首先用一定浓度的丝裂霉素C灭活K562-4-1BBL-MICA工程细胞，按照1：1的比例，在IL-21存在下将其与体外分离的PBMCs在24孔板中共培养，分别在第0天、7天、14天、21天和28天用流式细胞术测定NK细胞在PBMCs中的比例并计算NK细胞总量。在5ng/mLIL-21存在时，NK细胞在共培养第21天获得最高的比例以及数量。
2.2扩增后NK细胞表面受体分析
选取第0天、21天的NK细胞，分析其表面ADCC效应受体CD16、激活性受体CD69、CD38和NKG2D、抑制性受体CD94、CD158a和CD158f、自然细胞毒受体NKp46、NKp44和NKp30以及CD8、CD62L、CD57表达情况。NK细胞活化受体CD69、CD38、NKG2D和抑制性受体CD94表达均显著上调，NK细胞终末分化标志CD57几乎不表达。
2.3扩增前后NK细胞毒活性试验
使用流式细胞术和CCK8试验分别检测NK细胞对K562敏感细胞的特异性杀伤和对常见细胞株Hela、HepG2和SKOV3的细胞毒活性。扩增后的NK细胞相对于新分离的NK细胞，在不同的效：靶比下，对NK敏感K562细胞杀伤力显著增加；扩增后的NK细胞对Hela、HepG2和SKOV3细胞株也有增强的细胞毒作用。
2.4刺激前后NK细胞分泌IFN-γ能力检测
经过胞内染色检测，扩增后NK细胞可以分泌IFN-γ，而扩增前NK细胞中仅仅部分CD56brightNK细胞分泌IFN-γ。
2.5扩增后NK细胞亚群变化
体外扩增21天后，NK细胞中主要亚群为CD56brightCD16+NK细胞，而新分离的NK细胞中CD56dimNK细胞约占约90％，激活的D56brightCD16+NK细胞依然高水平表达穿孔素，表现出对K562细胞更强的杀伤活性。
综上所述，我们成功构建了可以稳定遗传的单克隆化的K562-4-1BBL-MICA工程细胞，用其联合5ng/mL的IL-21，可以体外从PBMCs中扩增出活化性受体和抑制性受体同时表达上调的NK细胞，扩增后的NK细胞毒活性相对于新鲜分离的NK细胞显著增强，且分泌免疫调理细胞因子IFN-γ。在扩增后的NK细胞中，CD56brightCD16+NK细胞占主要地位，高水平表达穿孔素，同时相对于CD56dimCD16+NK细胞表现出增强的细胞毒活性。本研究对于NK细胞体外扩增后用于免疫过继疗法治疗肿瘤提供了实验依据，同时也为CD56brightCD16+NK细胞亚群的细胞生物学分析提供了体外扩增的方法。
Tumorgenesis occurrs when normal cells are exposed to various mutagens, and itsuggests an impaired immune function. Besides surgical resection, radiotherapy andchemotherapy are the main means in the treatment of cancer, although there are a number ofserious side effects, including suppression of hematopoiesis and impaired immunology. Inconsideration of the close relation between tumorgenesis and cellular immunity, adoptiveimmunotherapy become more and more important in tumor therapy, following the threecommon methods mentioned above. Lymphokine-activated killer cells (LAK) are potentkiller cells in the lysis of tumor cells, however, high dose IL-2that induces serious capillaryleak syndrome (CLS), is needed after infusion of LAK cells to maintain their killing ability.Adoptive immunotherapy involving transfer of tumor infiltrating T cells (TIL) has shownremarkable success in a proportion of melanoma patients. However the use of TIL therapyfor most malignancies has been hampered due to difficulties in isolating sufficient numbersof these cells. Many of these studies to date have involved in the transfer of predominantlyCD8+effector T cells. However, more recent studies have reported that adoptive transfer ofNK cells can in some cases induce more effective anti-tumor responses.
NK cells are a potent innate immune cell type which are able to respond quickly to adisease threat, and can kill target cells using perforin and granzymes, among othermechanisms. They express a number of different activating and inhibitory receptors whichenable these cells to effectively recognize stress ligands on tumor cells that have otherwisedown-regulated or lost MHC expression. The transfer of allogeneic NK cells has shownpromise in the clinical setting. Studies demonstrate that NK cells possess strong anti-tumoractivity and potential, however the difficulty in generating sufficient numbers of NK cellshas posed difficulties for universal use of these cells. This indicated the feasibility tomanipulate and redirect NK killing for a specific antigen target, and highlights the potentialuse of gene-modified NK cells in clinic.
This study was aimed to construct a genetically modified k562cell line with stablesurface expression of4-1BBL and MICA, and tried to expand NK cells in vitro with isolatedPBMCs. Expression of activating and inhibitory receptors is detected later, as well as specific cytotoxicity against tumor cells. Our research would provide biological evidence forthe clinical application of adoptive NK cells in tumor therapy.
1Preparation of modified K562-4-1BBL-MICA cell line
1.1Construction of expression vectors with4-1BBL or MICA
RNA was obtained from newly isolated PBMCs and Reverse transctiption wasconducted to aquire cDNA.4-1BBL was obtained through PCR from cDNA, subsequentlyinserted into multiple cloning site of pcDNA3.1/Hygro (+)(Invitrogen), namedpcDNA3.1-Hygro-4-1BBL. MICA was obtained from K562cell line (ATCC), using reversetranscriptase PCR, and was inserted into MCS2of pVITRO2-noe-mcs plasmid (Invivogen),called pVITRO2-Neo-MICA.
1.2Construction and cloning of K562cells with surface expression of4-1BBL and MICA
At the beginning, Lipofectamine2000was used to transfect K562cells withpcDNA3.1-Hygro-4-1BBL. Culture medium with hygromycin was used to select K562cellswith stable transfection. K562cells with resistance to hygromycin were cloned with limitingdilution assay and identified the expression of4-1BBL by flow cytometry. And then, therecombinant pVITRO2-Neo-MICA was transduced into K562-4-1BBL cells.K562-4-1BBL-MICA cells were reconfirmed again in the same way mentioned above andmaintained with G418and hygromycin. Limiting dilution assay was conducted four times toclone a stable K562-4-1BBL-MICA cell strain.
2Long-term culture of PBMCs with K562-4-1BBL-MICA cells
2.1Expansion of NK cells in vitro
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll Hypaque densitygradient centrifugation. K562-4-1BBL-MICA stimulator cells were treated with mitomycinC and then washed four times with PBS. PBMCs (1.0×106) were incubated in24-welltissue-culture plates with106K562-4-1BBL-MICA stimulator cells in the presence IL-21.The proportion of CD3CD56+CD16+NK cells in stimulated PBMCs was analyzed by flowcytometry on day0,7,14,21and28. The proportion of NK cell reached70﹪at day21, inthe presence of5ng/mL IL-21.
2.2Phenotype analysis of expanded NK cells
Expanded NK cells with K562-4-1BBL-MICA+IL-21were analyzed for detailedmarkers by flow cytometry on day21, as well as fresh isolated PBMCs. Cells were stainedwith mixed Antibodies CD3, CD56, CD16; CD8; CD94; CD62L; CD57; CD69; CD38;CD314(NKG2D); CD158a; CD158f; CD337(NKp30); CD336(NKp44); CD335. Up-regulation of activating receptors like CD69, CD38and NKG2D was observed inexpanded NK cells, so was inhibitory receptor CD94. Expanded NK cells seldomlyexpressed CD57, a terminal differential marker of NK cells.
2.3Specific lysis of expanded NK cells against sensitive K562cells and cytotoxicityagainst Hela, HepG2and SKOV3cell strains.
For lysis of fresh and expanded NK Cells, the effectors were mixed with log-phaseK562-EGFP target cells at different ratios. Cytotoxicity against Hela, SKOV3and HepG2cell strains was analyzed by Cell Counting Kit (CCK-8/WST-8)(SAB). Expanded NK cellsshowed higher killing ability against K562cells at each ratio, compared with freshly isolatedNK cells. Enhanced cytotoxicity was detected with stimulated NK cells against Hela,SKOV3and HepG2cell strains.
2.4The expression of IFN-γ in expanded NK cells
Expanded NK cells produced the immune-regulatory cytokine IFN-γ, which wouldpositively regulate Th1-type immunity in the cancer patients, compared with expression ina very few proportion of fresh NK cells, mainly in CD56brightNK subset.
2.5Identification of NK subsets in expanded NK cells
After expansion with K562-4-1BBL-MICA feeder cells, CD56brightCD16+NK subsetdominated in expanded NK cells and expressed similar level of perforin, wherever, morethan90﹪of newly isolated NK cells were CD56dimCD16+. CD56brightCD16+NK cellsshowed enhanced cytotoxicity against K562cells than CD56dimCD16+subset.
According to results above, expansion of activated NK cells stimulated withK562-4-1BBL-MICA cells+IL-21for21days was confirmed, in which the CD56brightCD16+NK subset dominated. Our research provided a method to expand CD56brightCD16+NK cellsin vitro for further biological study, meanwhile, the data on expanded NK cells partiallysupports the application of stimulated NK cells in clinical immunotherapy against tumor inthe future. However, further investigations have to be done to generate sufficient clinical NKcells for adoptive immunotherapy.
NK细胞是固有免疫系统的主要效应细胞，能无需预先刺激，通过使用穿孔素和颗粒酶以及其它机制，无MHC限制性杀伤肿瘤细胞和病毒感染细胞。NK细胞表达众多的激活性受体，能有效识别肿瘤细胞或感染细胞产生的应激性配体，而抑制性受体在靶细胞MHC表达下调或消失时则失去对NK细胞杀伤作用的抑制作用。同种异体或自体移植NK细胞在临床应用中很有前景，研究显示NK细胞有很强的抗肿瘤活性。但是NK细胞在外周血中比例小，体外扩增难，扩增后细胞毒活性弱，都是限制其应用于临床免疫治疗的瓶颈。
本实验的目的是通过基因工程的方法，构建基因修饰的K562工程细胞，使其双表达激活性配体4-1BBL和MICA，将其同体外分离的外周血单个核细胞（Pefipheral BloodMononuclear Cells，PBMCs）共培养，观察NK细胞体外扩增、检测细胞表面激活性、抑制性受体等表达、测定扩增前后NK细胞毒活性变化、分析扩增前后NK细胞亚群异同，为NK细胞过继疗法从实验室研究向临床应用的转化提供理论依据。
1K562-4-1BBL-MICA工程细胞的制备
1.14-1BBL和MICA表达质粒的构建
从人PBMCs中提取总RNA，根据NCBI参考序列（NCBI Reference Sequence：NM_003811.3），设计引物，进行逆转录PCR获得4-1BBL的全长CDS序列。再次设计引物，将4-1BBL序列前后分别加入酶切位点BmaHI/XbaI，酶切后，连接到pcDNA3.1-Hygro中，构建成重组表达质粒pcDNA3.1-Hygro-4-1BBL。
同样的方法，从人K562细胞系中提取RNA，通过RT-PCR获得MICA全长CDS序列（NCBI Reference Sequence：NM_000247.1）。设计引物，将MICA序列加入酶切位点XhoI/Nhe I，酶切之后连接到pVITRO2-Neo-mcs的MCS2中，构建成重组表达质粒pVITRO2-Neo-MICA。
1.2构建并克隆化K562-4-1BBL-MICA细胞
将pcDNA3.1-Hygro-4-1BBL用脂质体转染法转染K562细胞，潮霉素筛选抗性细胞，流式细胞术测定表达4-1BBL的K562细胞比例，有限稀释法克隆出并通过流式细胞术鉴定高表达4-1BBL的K562细胞。接着将pVITRO2-Neo-MICA转染K562-4-1BBL细胞，G418和潮霉素共同筛选抗性细胞，同样的方法获得双表达4-1BBL和MICA的K562细胞。经过多次有限稀释法进行单克隆以及流式细胞术鉴定，最终获得单克隆化、可以稳定遗传的K562-4-1BBL-MICA细胞株。
2K562-4-1BBL-MICA工程细胞体外扩增PBMCs中NK细胞
2.1体外扩增NK细胞
首先用一定浓度的丝裂霉素C灭活K562-4-1BBL-MICA工程细胞，按照1：1的比例，在IL-21存在下将其与体外分离的PBMCs在24孔板中共培养，分别在第0天、7天、14天、21天和28天用流式细胞术测定NK细胞在PBMCs中的比例并计算NK细胞总量。在5ng/mLIL-21存在时，NK细胞在共培养第21天获得最高的比例以及数量。
2.2扩增后NK细胞表面受体分析
选取第0天、21天的NK细胞，分析其表面ADCC效应受体CD16、激活性受体CD69、CD38和NKG2D、抑制性受体CD94、CD158a和CD158f、自然细胞毒受体NKp46、NKp44和NKp30以及CD8、CD62L、CD57表达情况。NK细胞活化受体CD69、CD38、NKG2D和抑制性受体CD94表达均显著上调，NK细胞终末分化标志CD57几乎不表达。
2.3扩增前后NK细胞毒活性试验
使用流式细胞术和CCK8试验分别检测NK细胞对K562敏感细胞的特异性杀伤和对常见细胞株Hela、HepG2和SKOV3的细胞毒活性。扩增后的NK细胞相对于新分离的NK细胞，在不同的效：靶比下，对NK敏感K562细胞杀伤力显著增加；扩增后的NK细胞对Hela、HepG2和SKOV3细胞株也有增强的细胞毒作用。
2.4刺激前后NK细胞分泌IFN-γ能力检测
经过胞内染色检测，扩增后NK细胞可以分泌IFN-γ，而扩增前NK细胞中仅仅部分CD56brightNK细胞分泌IFN-γ。
2.5扩增后NK细胞亚群变化
体外扩增21天后，NK细胞中主要亚群为CD56brightCD16+NK细胞，而新分离的NK细胞中CD56dimNK细胞约占约90％，激活的D56brightCD16+NK细胞依然高水平表达穿孔素，表现出对K562细胞更强的杀伤活性。
综上所述，我们成功构建了可以稳定遗传的单克隆化的K562-4-1BBL-MICA工程细胞，用其联合5ng/mL的IL-21，可以体外从PBMCs中扩增出活化性受体和抑制性受体同时表达上调的NK细胞，扩增后的NK细胞毒活性相对于新鲜分离的NK细胞显著增强，且分泌免疫调理细胞因子IFN-γ。在扩增后的NK细胞中，CD56brightCD16+NK细胞占主要地位，高水平表达穿孔素，同时相对于CD56dimCD16+NK细胞表现出增强的细胞毒活性。本研究对于NK细胞体外扩增后用于免疫过继疗法治疗肿瘤提供了实验依据，同时也为CD56brightCD16+NK细胞亚群的细胞生物学分析提供了体外扩增的方法。
Tumorgenesis occurrs when normal cells are exposed to various mutagens, and itsuggests an impaired immune function. Besides surgical resection, radiotherapy andchemotherapy are the main means in the treatment of cancer, although there are a number ofserious side effects, including suppression of hematopoiesis and impaired immunology. Inconsideration of the close relation between tumorgenesis and cellular immunity, adoptiveimmunotherapy become more and more important in tumor therapy, following the threecommon methods mentioned above. Lymphokine-activated killer cells (LAK) are potentkiller cells in the lysis of tumor cells, however, high dose IL-2that induces serious capillaryleak syndrome (CLS), is needed after infusion of LAK cells to maintain their killing ability.Adoptive immunotherapy involving transfer of tumor infiltrating T cells (TIL) has shownremarkable success in a proportion of melanoma patients. However the use of TIL therapyfor most malignancies has been hampered due to difficulties in isolating sufficient numbersof these cells. Many of these studies to date have involved in the transfer of predominantlyCD8+effector T cells. However, more recent studies have reported that adoptive transfer ofNK cells can in some cases induce more effective anti-tumor responses.
NK cells are a potent innate immune cell type which are able to respond quickly to adisease threat, and can kill target cells using perforin and granzymes, among othermechanisms. They express a number of different activating and inhibitory receptors whichenable these cells to effectively recognize stress ligands on tumor cells that have otherwisedown-regulated or lost MHC expression. The transfer of allogeneic NK cells has shownpromise in the clinical setting. Studies demonstrate that NK cells possess strong anti-tumoractivity and potential, however the difficulty in generating sufficient numbers of NK cellshas posed difficulties for universal use of these cells. This indicated the feasibility tomanipulate and redirect NK killing for a specific antigen target, and highlights the potentialuse of gene-modified NK cells in clinic.
This study was aimed to construct a genetically modified k562cell line with stablesurface expression of4-1BBL and MICA, and tried to expand NK cells in vitro with isolatedPBMCs. Expression of activating and inhibitory receptors is detected later, as well as specific cytotoxicity against tumor cells. Our research would provide biological evidence forthe clinical application of adoptive NK cells in tumor therapy.
1Preparation of modified K562-4-1BBL-MICA cell line
1.1Construction of expression vectors with4-1BBL or MICA
RNA was obtained from newly isolated PBMCs and Reverse transctiption wasconducted to aquire cDNA.4-1BBL was obtained through PCR from cDNA, subsequentlyinserted into multiple cloning site of pcDNA3.1/Hygro (+)(Invitrogen), namedpcDNA3.1-Hygro-4-1BBL. MICA was obtained from K562cell line (ATCC), using reversetranscriptase PCR, and was inserted into MCS2of pVITRO2-noe-mcs plasmid (Invivogen),called pVITRO2-Neo-MICA.
1.2Construction and cloning of K562cells with surface expression of4-1BBL and MICA
At the beginning, Lipofectamine2000was used to transfect K562cells withpcDNA3.1-Hygro-4-1BBL. Culture medium with hygromycin was used to select K562cellswith stable transfection. K562cells with resistance to hygromycin were cloned with limitingdilution assay and identified the expression of4-1BBL by flow cytometry. And then, therecombinant pVITRO2-Neo-MICA was transduced into K562-4-1BBL cells.K562-4-1BBL-MICA cells were reconfirmed again in the same way mentioned above andmaintained with G418and hygromycin. Limiting dilution assay was conducted four times toclone a stable K562-4-1BBL-MICA cell strain.
2Long-term culture of PBMCs with K562-4-1BBL-MICA cells
2.1Expansion of NK cells in vitro
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll Hypaque densitygradient centrifugation. K562-4-1BBL-MICA stimulator cells were treated with mitomycinC and then washed four times with PBS. PBMCs (1.0×106) were incubated in24-welltissue-culture plates with106K562-4-1BBL-MICA stimulator cells in the presence IL-21.The proportion of CD3CD56+CD16+NK cells in stimulated PBMCs was analyzed by flowcytometry on day0,7,14,21and28. The proportion of NK cell reached70﹪at day21, inthe presence of5ng/mL IL-21.
2.2Phenotype analysis of expanded NK cells
Expanded NK cells with K562-4-1BBL-MICA+IL-21were analyzed for detailedmarkers by flow cytometry on day21, as well as fresh isolated PBMCs. Cells were stainedwith mixed Antibodies CD3, CD56, CD16; CD8; CD94; CD62L; CD57; CD69; CD38;CD314(NKG2D); CD158a; CD158f; CD337(NKp30); CD336(NKp44); CD335. Up-regulation of activating receptors like CD69, CD38and NKG2D was observed inexpanded NK cells, so was inhibitory receptor CD94. Expanded NK cells seldomlyexpressed CD57, a terminal differential marker of NK cells.
2.3Specific lysis of expanded NK cells against sensitive K562cells and cytotoxicityagainst Hela, HepG2and SKOV3cell strains.
For lysis of fresh and expanded NK Cells, the effectors were mixed with log-phaseK562-EGFP target cells at different ratios. Cytotoxicity against Hela, SKOV3and HepG2cell strains was analyzed by Cell Counting Kit (CCK-8/WST-8)(SAB). Expanded NK cellsshowed higher killing ability against K562cells at each ratio, compared with freshly isolatedNK cells. Enhanced cytotoxicity was detected with stimulated NK cells against Hela,SKOV3and HepG2cell strains.
2.4The expression of IFN-γ in expanded NK cells
Expanded NK cells produced the immune-regulatory cytokine IFN-γ, which wouldpositively regulate Th1-type immunity in the cancer patients, compared with expression ina very few proportion of fresh NK cells, mainly in CD56brightNK subset.
2.5Identification of NK subsets in expanded NK cells
After expansion with K562-4-1BBL-MICA feeder cells, CD56brightCD16+NK subsetdominated in expanded NK cells and expressed similar level of perforin, wherever, morethan90﹪of newly isolated NK cells were CD56dimCD16+. CD56brightCD16+NK cellsshowed enhanced cytotoxicity against K562cells than CD56dimCD16+subset.
According to results above, expansion of activated NK cells stimulated withK562-4-1BBL-MICA cells+IL-21for21days was confirmed, in which the CD56brightCD16+NK subset dominated. Our research provided a method to expand CD56brightCD16+NK cellsin vitro for further biological study, meanwhile, the data on expanded NK cells partiallysupports the application of stimulated NK cells in clinical immunotherapy against tumor inthe future. However, further investigations have to be done to generate sufficient clinical NKcells for adoptive immunotherapy.
引文
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