Generation of Mouse iNKT Cell Lines

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Cancer Research
Aug 2012



Natural killer T (NKT) cells bridge the innate and adaptive arms of the immune system, and manipulating their effector functions can have therapeutic significances in the treatment of autoimmunity, transplant biology, infectious disease and cancer. This important lymphocyte subset regulates the immune system through their potent cytokine production following the recognition of lipid antigen present in the context of the MHC class I-like CD1d molecule, in addition their ability to directly mediate cytotoxicity. Here, we describe a method of expanding mouse invariant NKT (iNKT) cell lines from mononuclear cells isolated from the thymus, spleen, or liver using bone marrow derived dendritic cells. These iNKT cell lines can be used study their co-signaling requirements, cytokine profiles and cytotoxic functions which will greatly enhance our knowledge of iNKT cell biology.

Materials and Reagents

  1. 6-12 weeks old C57BL/6 or BALB/c mice
  2. Erythrocyte lysis buffer- ACK Lysing buffer (Life Technologies, Gibco®, catalog number: A10492-01 )
  3. PBS-no calcium, no magnesium, 1x & 10x (Life Technologies, Gibco®, catalog number: 14190 )
  4. FITC-anti-CD3 (BD Biosciences, catalog number: clone 2C11; 553061 )
  5. APC-conjugated CD1d tetramer loaded with PBS-57 lipid antigen (National Institutes of Health Tetramer Core Facility, Atlanta, GA)
  6. PE-anti-NK1.1 (BD Biosciences, catalog number: clone PK136; 561046 )
  7. Recombinant mouse GM-CSF (R&D systems, catalog number: 415-GM )
  8. Recombinant mouse IL-4 (R&D systems, catalog number: 404-IL )
  9. Recombinant mouse IL-2 (Peprotech, catalog number: 212-12 )
  10. Recombinant mouse IL-7 (Peprotech, catalog number: 217-17 )
  11. Mouse Pan T cell isolation kit (MiltenyiBiotec, catalog number: 130-095-130 )
  12. Anti-APC microbeads (MiltenyiBiotec, catalog number: 130-090-855 )
  13. α-galactosylceramide (α-GalCer, KRN7000) (Enzo Life Sciences, catalog number: BML-SL232 )
  14. Lympholyte-M (Accurate Chemical)
  15. Percoll (Amersham-Pharmacia Biotech, catalog number: 17-0891-01 )
  16. RPMI-1640 medium (Life Technologies, Gibco®, catalog number: 11875 )
  17. Non-essential vitamin solution (Life Technologies, Gibco®, catalog number: 11140-050 )
  18. MEM Vitamin solution (Life Technologies, Gibco®, catalog number: 11120-052 )
  19. Sodium Pyruvate (Life Technologies, Gibco®, catalog number: 11360-070 )
  20. 2-mercaptoethanol (Life Technologies, Gibco®, catalog number: 21985-023 )
  21. Anti-CD16/32 antibody (Biolegend, catalog number: 101320 )
  22. Antibiotics: Penicillin-streptomycin
  23. Heat inactivated fetal bovine serum
  24. FBS
  25. EDTA
  26. Complete medium (see Recipes)
  27. MACS Buffer (see Recipes)
  28. Liver MNC isolation (see Recipes)
  29. Cell buffer solution (see Recipes)


  1. 70 μm nylon mesh cell strainer (BD Bioscience, Falcon®, catalog number: 352350 )
  2. Centrifuge with swing out rotor and capable of 300-700 x g
  3. BD LSR II flow cytometer (BD Biosciences)
  4. Gamma irradiator
  5. 0.22 μm filter


  1. To generate immature dendritic cells (BMDCs):
    1. Collect bone marrow from femurs of mice by passing through a 70 μm filter and centrifuge for 5 min at 300 x g. Discard supernatant and lyse red blood cells by gently resuspending the cell pellet in 5 ml ACK lysing buffer and incubating for 2-3 min at room temperature. Then quickly add 5 ml cell buffer solution and centrifuge for 5 min at 300 x g. Wash 2x with 10 ml cold cell buffer solution.
    2. Resuspend pelleted cells (106/ml) in complete medium containing 10 ng/ml mouse GM-CSF.
    3. Plate 5 x 106 cells/well in a 6 well plate for 7 days to generate immature DC.
  2. To isolate iNKT cells:
    1. Make a single-cell suspension of thymocytes or splenocytes by pressing organs through a 70 μm cell strainer (for liver see detailed protocol below) using cell buffer solution and the plunger from a 3 ml syringe.
      Note: This protocol should be performed using 4-6 mice. The total number of NKT cells per organ is approximately one million, however typical yields are 30-40% per organ.
    2. Centrifuge single cell suspension for 5 min at 300 x g. Discard supernatant and lyse red blood cells by gently resuspending the cell pellet in 10 ml ACK lysing buffer and incubating for 2-3 min at room temperature. Then quickly add 10 ml cell buffer solution and centrifuge for 5 min at 300 x g.
    3. Enrich T cells via negative selection by using the mouse Pan T Isolation Kit II according to the manufacturer’s protocol.
    4. Resuspend the cells (108/ml) in MACS buffer, and then select iNKT cells by incubating in the dark on ice with APC-conjugated CD1d tetramer loaded with PBS-57 lipid antigen (5-10 μl/ml of cells; 50 μg/ml) for 30 min.
    5. Next sort iNKT cells by using anti-APC beads following the manufacturer’s protocol.
  3. For Liver Mononuclear cell (MNC) isolation:
    Isolation of hepatic MNC (use 1 liver per tube):
    1. Euthanize mice, open peritoneal cavity, slide intestines over to the right to expose the underside of the liver. Then using a 10 ml syringe and a 21 ga needle, inject 10 ml room tem PBS into the hepatic portal vein.
    2. Remove gall bladder, excise liver, place in tube containing cell buffer solution (on ice).
    3. Mince with liver tissue into very small pieces with scissors (500-700 cuts; < 3 mm3), and add to nylon mesh cell strainer on top of a 50 ml centrifuge tube. Add 2-3 ml cold cell buffer solution and mash through the mesh with the plunger from a 3 ml syringe. This does not need to be performed on ice, but tissue should be kept cold by the addition of ice-cold cell buffer solution and this step should be done quickly.
    4. Rinse with lots of cold cell buffer solution, continue mashing through and bring volume up to 40 ml with cold cell buffer solution. Centrifuge at 300 x g for 7 min at 4 °C.
    5. Resuspend pellet in 25 ml room temperature Percoll solution.
    6. Spin at 700 x g at room temperature for 12 min with the brake on (the cells of interest will form pellet so brake can be left on).
    7. Aspirate off supernatant (MNC are in the pellet), but be careful initially to remove the top layer of hepatocytes before aspirating down to the pellet. This will reduce the amount of hepatocyte contamination of MNC.
    8. Resuspend the pellet in 5 ml of ACK lysing buffer (or a similar RBC lysing buffer) to lyse the RBC, transfer to a 15 ml conical centrifuge tube, stop reaction by adding 5 ml of cell buffer solution. Spin at 300 x g for 7 min at 4 °C.
    9. Wash cell pellet 2x in 5-10 ml cell buffer solution. Resuspend in 5 ml cell buffer solution, media, or staining buffer and count (expect about 3-5 x 106 MNC)
    10. Continue as described above starting with step 3-e.
  4. In vitro expansion of iNKT cells:
    1. Collect the immature DCs by pipetting vigorously with ice cold complete medium and wash cells with 10 ml complete medium.
    2. Resuspend cells in 10 ml fresh medium and irradiate with 2,000 rads.
    3. Incubate 2 x 106 NKT cells with 2 x 105 irradiated immature DCs in the presence of α-GalCer (100 ng/ml) in 10 ml complete medium, and add 2 ml per well in 24 well plate.
    4. On day 4, add IL-2 (10 U/ml) and IL-7 (10 ng/ml) to the media.
    5. On day 10, harvest the cells and remove dead cells and debris using lympholyte-M according to the manufacturer’s protocol.
    6. Then re-stimulate the cells with immature DCs in the presence of 100 ng/ml α-GalCer and 10 U/ml mouse IL-2 at a 1:1 ratio as described in step c.
    7. Culture cells for 7-10 days, and replace medium every 4 days as step 4-d.
      Note: If the cells are maintained in high levels of a-GalCer and cytokines, some specificity and sensitivity to CD1d is lost, so it is best to supplement with a-GalCer and cytokines when the cell line needs to be maintained, but add fresh complete media only if the cells are to be used in functional assays.
    8. Flow cytometric analysis of expanding iNKT cells.
      Gate on lymphocytes and check purity by flow cytometric analysis, using CD1d tetramer (or NK1.1 in C57BL/6 mice) and anti-CD3, see Figure 1.

      Figure 1.

    Flow cytometry procedure:
    1. Collect 1 x 105 cells, and transfer into 1.5 ml tube, and filled with 1 ml FACS buffer (0.2% FBS in PBS).
    2. Centrifuge cells at 600 x g for 5 min, and then discard supernatant.
    3. Resuspend cells in 100 μl FACS buffer, and add 1 μl anti-CD16/32 antibody for 15 min to block non-specific binding, and then wash as step a.
    4. Resuspend cells in 100 μl FACS buffer, and add 0.5 μl APC-PBS57 loaded CD1d tetramer, or 1 μl PE-anti NK1.1 and 1 μl FITC anti-CD3 antibody (for 30 min on ice in dark, and then wash as step a.
    5. Resuspend cells in 200 μl PBS, and run samples on LSRII FACS machine.


  1. Complete medium
    RPMI medium
    100 mM sodium pyruvate
    10 mM non-essential vitamin solution
    100 mM MEM Vitamin solution
    5 x 105 M 2-mercaptoethanol
    50 U/ml penicillin-streptomycin
    10% heat inactivated fetal bovine serum
  2. MACS buffer
    1 L PBS free of Ca2+ and Mg2+
    5 g BSA
    2 mmol EDTA
    sterilized by passing through 0.22 μm filter
  3. For Liver MNC isolation
    Preparation of isotonic Percoll: Make up a 37.5% stock of PercoIl
    337.5 ml of Percoll
    100 ml of 10x PBS free of Ca2+ and Mg2+
    562.5 ml ddH2O
    Filter sterilize through a 0.2 micron filter unit
    Store at 4 °C (very stable as long as it is kept sterile)
  4. Cell buffer solution
    Prepare 1x PBS supplemented with 2% FBS and 0.02% sodium azide


This work was supported by National Institutes of Health (NIH), National Cancer Institute Grants K01 CA131487, R21 CA162273, and R21 CA162277 and grants from the P30 Tumor Immunology and Immunotherapy Program to T.J.W., NIH AI 70258 to M. Tsuji, the NIH AI 44129, CA 108835, and P01 AI072677 to J.P. Schneck. The method was published in Webb et al. (2012) and it is an adaptation of the methods used by Tupin and Kronenberg (2006).


  1. Dellabona, P., Padovan, E., Casorati, G., Brockhaus, M. and Lanzavecchia, A. (1994). An invariant V alpha 24-J alpha Q/V beta 11 T cell receptor is expressed in all individuals by clonally expanded CD4-8- T cells. J Exp Med 180(3): 1171-1176. 
  2. Exley, M., Garcia, J., Balk, S. P. and Porcelli, S. (1997). Requirements for CD1d recognition by human invariant Valpha24+ CD4-CD8- T cells. J Exp Med 186(1): 109-120.
  3. Fowlkes, B. J., Kruisbeek, A. M., Ton-That, H., Weston, M. A., Coligan, J. E., Schwartz, R. H. and Pardoll, D. M. (1987). A novel population of T-cell receptor alpha beta-bearing thymocytes which predominantly expresses a single V beta gene family. Nature 329(6136): 251-254. 
  4. Harada, Y., Imataki, O., Heike, Y., Kawai, H., Shimosaka, A., Mori, S., Kami, M., Tanosaki, R., Ikarashi, Y., Iizuka, A., Yoshida, M., Wakasugi, H., Saito, S., Takaue, Y., Takei, M. and Kakizoe, T. (2005). Expansion of alpha-galactosylceramide-stimulated Valpha24+ NKT cells cultured in the absence of animal materials. J Immunother 28(4): 314-321.
  5. Prigozy, T. I., Naidenko, O., Qasba, P., Elewaut, D., Brossay, L., Khurana, A., Natori, T., Koezuka, Y., Kulkarni, A. and Kronenberg, M. (2001). Glycolipid antigen processing for presentation by CD1d molecules. Science 291(5504): 664-667.
  6. Shiratsuchi, T., Schneck, J., Kawamura, A. and Tsuji, M. (2009). Human CD1 dimeric proteins as indispensable tools for research on CD1-binding lipids and CD1-restricted T cells. J Immunol Methods 345(1-2): 49-59.
  7. Tupin, E. and Kronenberg, M. (2006). Activation of natural killer T cells by glycolipids. Methods Enzymol 417: 185-201.
  8. Webb, T. J., Li, X., Giuntoli, R. L., 2nd, Lopez, P. H., Heuser, C., Schnaar, R. L., Tsuji, M., Kurts, C., Oelke, M. and Schneck, J. P. (2012). Molecular identification of GD3 as a suppressor of the innate immune response in ovarian cancer. Cancer Res 72(15): 3744-3752.
  9. Webb, T. J., Bieler, J. G., Schneck, J. P. and Oelke, M. (2009). Ex vivo induction and expansion of natural killer T cells by CD1d1-Ig coated artificial antigen presenting cells. J Immunol Methods 346(1-2): 38-44.


自然杀伤T(NKT)细胞桥接免疫系统的先天和适应性臂,并且操纵它们的效应子功能可以在治疗自身免疫,移植生物学,传染病和癌症中具有治疗意义。 这种重要的淋巴细胞亚群通过它们在识别存在于MHC I类CD1d分子的背景中的脂质抗原之后的有效细胞因子产生,以及它们直接介导细胞毒性的能力来调节免疫系统。 在这里,我们描述了扩增小鼠不变NKT(iNKT)细胞系从使用骨髓衍生的树突状细胞从胸腺,脾或肝分离的单核细胞的方法。 这些iNKT细胞系可用于研究其共信号要求,细胞因子谱和细胞毒性功能,这将大大增强我们对iNKT细胞生物学的知识。


  1. 6-12周龄的C57BL/6或BALB/c小鼠
  2. 红细胞裂解缓冲液-ACK裂解缓冲液(Life Technologies,< Gibco ,目录号:A10492-01)
  3. PBS-无钙,无镁,1x& 10x(Life Technologies,  Gibco ®,目录号:14190)
  4. FITC-抗CD3(BD Biosciences,目录号:克隆2C11; 553061)
  5. 装有PBS-57脂质抗原的APC-偶联的CD1d四聚体(National Institutes of Health Tetramer Core Facility,Atlanta,GA)
  6. PE-抗-NK1.1(BD Biosciences,目录号:克隆PK136; 561046)
  7. 重组小鼠GM-CSF(R& D systems,目录号:415-GM)
  8. 重组小鼠IL-4(R& D systems,目录号:404-IL)
  9. 重组小鼠IL-2(Peprotech,目录号:212-12)
  10. 重组小鼠IL-7(Peprotech,目录号:217-17)
  11. 小鼠Pan T细胞分离试剂盒(MiltenyiBiotec,目录号:130-095-130)
  12. 抗APC微珠(MiltenyiBiotec,目录号:130-090-855)
  13. α-半乳糖苷神经酰胺(α-GalCer,KRN7000)(Enzo Life Sciences,目录号:BML-SL232)
  14. 淋巴细胞(精确化学)
  15. Percoll(Amersham-Pharmacia Biotech,目录号:17-0891-01)
  16. RPMI-1640培养基(Life Technologies,< Gibco ,目录号:11875)
  17. 非必需维生素溶液(Life Technologies,  Gibco ®,目录号:11140-050)
  18. MEM维生素溶液(Life Technologies,< Gibco ,目录号:11120-052)
  19. 丙酮酸钠(Life Technologies,  Gibco ,目录号:11360-070)
  20. 2-巯基乙醇(Life Technologies,Gibco ,目录号:21985-023)
  21. 抗CD16/32抗体(Biolegend,目录号:101320)
  22. 抗生素:青霉素 - 链霉素
  23. 热灭活的胎牛血清
  24. FBS
  25. EDTA
  26. 完全培养基(见配方)
  27. MACS缓冲区(参见配方)
  28. 肝脏MNC隔离(参见配方)
  29. 细胞缓冲液(参见配方)


  1. 70μM尼龙网孔细胞过滤器(BD Bioscience,Falcon ,目录号:352350)
  2. 离心机带有旋转转子,能够提供300-700 x g
  3. BD LSR II流式细胞仪(BD Biosciences)
  4. 伽玛辐照器


  1. 为了产生未成熟树突细胞(BMDC):
    1. 通过通过70μM过滤器并在300×g离心5分钟从小鼠股骨收集骨髓。弃去上清液并通过轻轻地将细胞沉淀重悬于5ml ACK裂解缓冲液中并在室温下温育2-3分钟来裂解红细胞。然后快速加入5ml细胞缓冲溶液并在300×g离心5分钟。用10ml冷细胞缓冲液洗涤2次
    2. 在含有10ng/ml小鼠GM-CSF的完全培养基中重悬沉淀的细胞(10 6个/ml)。
    3. 板以5×10 6个细胞/孔接种在6孔板中7天以产生未成熟的DC。
  2. 为了分离iNKT细胞:
    1. 通过按下器官通过70微米的细胞过滤器(肝脏参见下面详细的协议)使用细胞缓冲溶液和柱塞从3毫升的单细胞悬浮的胸腺细胞或脾细胞 注射器。
    2. 在300×g离心单细胞悬液5分钟。弃去上清液并通过轻轻地将细胞沉淀重悬于10ml ACK裂解缓冲液中并在室温下温育2-3分钟来裂解红细胞。然后快速加入10ml细胞缓冲液,并在300×g离心5分钟
    3. 通过使用小鼠Pan T Isolation Kit II根据制造商的方案通过负选择富集T细胞。
    4. 在MACS缓冲液中重悬细胞(10×8/ml),然后通过在冰上在黑暗中与APC-偶联的装载有PBS-57脂质抗原的CD1d四聚体孵育来选择iNKT细胞(5-10μl/ml细胞;50μg/ml)30分钟
    5. 接下来按照制造商的方案使用抗APC珠子分选iNKT细胞。
  3. 对于肝脏单核细胞(MNC)分离:
    1. 安乐死小鼠,打开腹膜腔,滑动肠向右到露出肝脏的下面。然后使用10ml注射器和21ga针,将10ml室温PBS注入肝门静脉
    2. 取出胆囊,切除肝脏,置于含有细胞缓冲液(在冰上)的管中
    3. 用剪刀(500-700次切割; <3mm <3 />)将肝脏组织切成非常小的碎片,并在50ml离心管顶部加入尼龙网孔滤器。加入2-3毫升冷的细胞缓冲溶液和捣碎通过从3毫升注射器与柱塞网。这不需要在冰上进行,但通过加入冰冷的细胞缓冲溶液保持组织冷,这一步应该很快。
    4. 用大量冷的细胞缓冲液冲洗,继续糖化通过和使用冷细胞缓冲溶液使体积达到40毫升。在4℃下以300xg离心7分钟。
    5. 在25ml室温Percoll溶液中重悬沉淀
    6. 在室温下旋转700分钟,在制动器打开的情况下旋转12分钟(感兴趣的细胞会形成颗粒,因此可以保持制动)。
    7. 吸出上清液(MNC在沉淀中),但最初小心去除顶层的肝细胞,然后吸出至沉淀。这将减少MNC的肝细胞污染量。
    8. 将沉淀重悬在5ml的ACK裂解缓冲液(或类似的RBC裂解缓冲液)中以裂解RBC,转移至15ml锥形离心管,通过加入5ml细胞缓冲液终止反应。在4℃下以300×g离心7分钟。
    9. 在5-10 ml细胞缓冲液中洗涤细胞沉淀2x。重悬在5ml细胞缓冲液溶液,培养基或染色缓冲液和计数(期望约3-5×10 6 MNC)中
    10. 继续如上所述从步骤3-e开始。
  4. iNKT细胞的体外扩增:
    1. 通过用冰冷的完全培养基剧烈移液收集未成熟的DC,并用10ml完全培养基洗涤细胞
    2. 重悬细胞在10ml新鲜培养基中,并照射2000 rads
    3. 在α-GalCer(100ng/ml)存在下,在10ml完全培养基中用2×10 5个未照射的未成熟DC孵育2×10 6个NKT细胞, 在24孔板中每孔2ml
    4. 在第4天,向培养基中加入IL-2(10U/ml)和IL-7(10ng/ml)。
    5. 在第10天,收获细胞,并使用淋巴细胞-M根据制造商的方案去除死细胞和碎片
    6. 然后如步骤c所述,在100ng/mlα-GalCer和10U/ml小鼠IL-2存在下,以1:1的比例,用未成熟的DC再刺激细胞。
    7. 培养细胞7-10天,并且每4天更换培养基作为步骤4-d。
    8. 扩增iNKT细胞的流式细胞术分析 通过流式细胞术分析,使用CD1d四聚体(或C57BL/6小鼠中的NK1.1)和抗CD3检测淋巴细胞上的淋巴细胞并检查纯度,参见图1.


  5. 流式细胞术程序:
    1. 收集1×10 5个细胞,并转移到1.5ml管中,并填充1ml FACS缓冲液(PBS中的0.2%FBS)。
    2. 在600×g离心细胞5分钟,然后丢弃上清液
    3. 重悬细胞在100微升FACS缓冲液中,加入1微升抗CD16/32抗体15分钟以阻止非特异性结合,然后按步骤a洗涤。
    4. 重悬细胞在100微升FACS缓冲液,并加入0.5微升APC-PBS57加载CD1d四聚体或1微升PE抗NK1.1和1微升FITC抗CD3抗体(在冰上暗处30分钟,然后洗涤步骤 a。
    5. 重悬细胞在200μlPBS,并在LSRII FACS机器上运行样品


  1. 完成媒介
    100mM丙酮酸钠 10 mM非必需维生素溶液
    100mM MEM维生素溶液 5×10 5 M 2-巯基乙醇 50 U/ml青霉素 - 链霉素 10%热灭活的胎牛血清
  2. MACS缓冲区
    1L不含Ca 2+ 2 +和Mg 2+ 2 +
    的PBS 5 g BSA
    2 mmol EDTA
  3. 对于肝脏MNC隔离
    1. 337.5ml Percoll
    2. 100ml不含Ca 2+和Mg 2+的10×PBS +
    3. 562.5ml ddH 2 O
    4. 通过0.2微米过滤器单元过滤灭菌
    5. 储存在4°C(非常稳定,只要它保持无菌)
  4. 细胞缓冲液
    制备补充有2%FBS和0.02%叠氮化钠的1x PBS


这项工作得到国家卫生研究院(NIH),国家癌症研究所批准K01 CA131487,R21 CA162273和R21 CA162277和来自P30肿瘤免疫学和免疫治疗计划授予TJW,NIH AI 70258授予M. Tsuji,NIH AI 44129,CA 108835和JP01ne072677。 该方法公开在Webb等人(2012)中,并且是Tupin和Kronenberg(2006)使用的方法的改编。


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  2. Exley,M.,Garcia,J.,Balk,S.P.and Porcelli,S。(1997)。 人类不变Valpha24 + CD4-CD8-T细胞识别CD1d的要求 J Exp Med 186(1):109-120。
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引用:Li, X., Tsuji, M., Schneck, J. and Webb, T. J. (2013). Generation of Mouse iNKT Cell Lines. Bio-protocol 3(6): e419. DOI: 10.21769/BioProtoc.419.