Generation of Human iNKT Cell Lines

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Cancer Research
Aug 2012



Natural killer T (NKT) cells comprise an important immunoregulatory T cell subset and express cell surface proteins characteristic of both natural killer cells and T cells. Invariant NKT (iNKT) cells are activated by lipid antigen presented in the context of CD1d molecules, in contrast to classic T cell subsets which recognize peptide antigens presented by MHC molecules. Following activation, iNKT cells rapidly secrete large amounts of cytokines and can lyse tumor cells and virally infected cells; however, iNKT cells are reduced in patients with autoimmune disease and cancer. The potential to characterize and investigate the prospective use of iNKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human iNKT cells. In this protocol, we describe a method to generate and propagate primary human iNKT cells. Specifically, primary iNKT cells were isolated from human peripheral blood mononuclear cells (PBMC), and then expanded periodically with irradiated α-GalCer loaded autologous immature dendritic cells (DC) in the presence of human IL-2.

Materials and Reagents

  1. Blood sample to collect iNKT cells
  2. Ficoll-Paque Plus (GE Healthcare Biosciences, catalog number: 17-1440 )
  3. PE-anti-Vα24Jα18 antibody (6B11) (Biolegend, catalog number: 342904 )
  4. Anti-CD16/32 antibody (Biolegend, catalog number: 101320 )
  5. Anti-Vα24 antibody (Beckman Coulter, catalog number: IM2283 )
  6. Anti-CD3 antibody (Biolegend, catalog number: 300312 )
  7. Recombinant human GM-CSF (R&D Systems, catalog number: 215-GM )
  8. Recombinant human IL-4 (R&D Systems, catalog number: 204-IL )
  9. Recombinant human IL-2 (Proleukin) (BD Biosciences, catalog number: 354043 )
  10. Human dendritic cell isolation kit (human CD14- magnetic microbeads) (MiltenyiBiotec, catalog number: 130-050-201 )
  11. Mitomycin C (Sigma-Aldrich, catalog number: M4287 )
  12. Human iNKT cell isolation kit (Vα24Jα18- magnetic microbeads) (MiltenyiBiotec, catalog number: 130-094-842 )
  13. α-galactosylceramide (α-GalCer, KRN7000) (Enzo Life Sciences, catalog number: BML-SL232 )
  14. RPMI-1640 medium (Life Technologies, Gibco®, catalog number: 11875 )
  15. Non-essential vitamin solution (Life Technologies, Gibco®, catalog number: 11140-050 )
  16. MEM Vitamin solution (Life Technologies, Gibco®, catalog number: 11120-052 )
  17. Sodium Pyruvate (Life Technologies, Gibco®, catalog number: 11360-070 )
  18. 2-mercaptoethanol (Life Technologies, Gibco®, catalog number: 21985-023 )
  19. Nalgene Freezing Container (Thermo Fisher Scientific, catalog number: 15-350-50 )
  20. Antibiotics: penicillin-streptomycin
  21. Heat inactivated fetal bovine serum
  22. MACS buffer (see Recipes)
  23. Complete medium (see Recipes)


  1. Centrifuge with swing out rotor and capable of 300-700 x g
  2. BD LSR II flow cytometer (BD Biosciences)
  3. Sterilized Pasteur pipettes
  4. 50 ml conical tubes
  5. 0.22 μM filter
  6. T-175 flask
  7. T-25 flask
  8. 37 °C 5% CO2 incubator


  1. Collect peripheral blood mononuclear cells (PBMCs). For Ficoll density gradient centrifugation separation of lymphocytes from a buffy coat or leukopheresis pack, first dilute heparinized blood with an equal volume of 1x PBS at room temperature.
  2. Add 15 ml of Ficoll (warmed to room temperature) to 50 ml conical tubes. Then slowly overlay 25 ml of the diluted blood mixture on top of the Ficoll. Centrifuge at 400 x g for 30 min at room temperature with the brake off.
  3. Carefully remove the 2-3 ml lymphocyte interface (white ring between the media and Ficoll, please see schematic diagram in Figure 1) with a sterilized Pasteur pipette and transfer to a new 50 ml conical tube.

    Figure 1. Schematic of Ficoll-Paque Plus separation of human peripheral blood

  4. Wash the cells by resuspending the cell pellets in 5 ml PBS, then combine all of the pellets into one 50 ml tube, and fill the tube with PBS to 50 ml and spin down cells at 400 x g for 10 min at room temperature with the brake on.
  5. Discard the supernatant and combine the tubes from a single individual donor to a single tube and wash the PBMCs again with 20 ml PBS. Then count the PBMCs and resuspend at a concentration of 5 x 107 cells/ml in ice-cold MACS buffer.
    Note: A leukopak is typically used for these studies and the initial volume of blood is 400-450 ml, but the product has been enriched for leukocytes and the final volume used for Ficoll separation in step 1 is around 40 ml. After the completion of step 5, it is best to continue with the rest of the protocol. If not, the cells should be cultured with complete medium at 107 cells/ml in T-175 flask overnight in 37 °C 5% CO2 incubator, and must be processed into the next step in 24 h.
  6. DAY 1- Isolate human CD14+ cells: Use human CD14- magnetic microbeads according to the manufacturer’s instructions. 
    1. For CD14+ PBMCs, culture 107 cells with 8 ml complete medium and 20 ng/ml rhGM-CSF and 20 ng/ml rhIL-4 for 4-5 days in T-25 flask to generate immature dendritic cells (DCs). Freeze the extra cells for future use at 107/vial.
    2. Keep the CD14neg fraction for iNKT cell isolation. In order to maintain the cells, culture in complete medium and 10 U/ml rhIL-2 at 107/ml in a T-175 flask. It is not necessary to replace medium when expanding DC cells.
  7. DAY 5-To expand the primary iNKT cells: Collect CD14neg PBMCs and wash with 10 ml complete medium. Use the human iNKT cell isolation kit according to the manufacturer’s instructions.
    1. To isolate Vα24+ hNKT cells:
      1. Collect CD14- PBMC and wash with wash medium once.
      2. Resuspend the pellets with 30 ml ice cold MACS buffer and pass through a 70 μm sterile cell strainer, then spin at 400 x g for 5 min.
      3. Resuspend pellets with 1 ml MACS buffer, add anti-Vα24 antibody, 5 μg/106 positive cells (NKT cells comprise 0.01%-0.5% of the lymphocyte population in PBMC). Incubate on ice for 30 min.
      4. Wash cells twice in 30 ml ice cold MACS buffer.
      5. Resuspend pellets with 1 ml MACS buffer, add anti-mouse IgG microbeads 20 μl/106 positive cells. Incubate on ice for 20 min.
      6. Wash cells once in 30 ml ice cold MACS buffer, while centrifuging prepare an MS column by adding 500 μl MACS buffer.
      7. Resuspend the cells in 500 μl ml MACS buffer. Then pipette the cells into the MS separating column. Make sure to avoid generating bubbles by pipetting slowly. Rinse the column by adding 500 μl ml MACS buffer. Repeat twice. Add 1 ml fresh MACS buffer and remove column from magnet. Place column into a 15 ml conical tube. Insert plunger and push out contents to obtain purified iNKT cells. Count NKT cell enriched fraction. You should have 0.02-0.5 million cells.
  8. In order to expand primary human iNKT cells:
    1. First, pretreat the immature DCs with mitomycin C. Resuspend immature DCs at 5 x 106 cells/ml in RPMI containing 0.05 mg/ml mitomycin C, incubate at 37 °C in the dark for 30 min, then wash three times with 10 ml complete media.
    2. Pulse immature DCs by incubating the cells in complete medium (12 well plates/1 ml per well) containing 200 ng/ml α-GalCer and 20 U/ml rhIL-2 at 2-3 X 106 cells/ml at 37 °C for 1 h.
  9. To the 12 well plates- Add 1 ml in complete medium containing 20 U/ml rhIL-2 Vα24+ human iNKT cells to wells containing α-GalCer-loaded DCs (DC:iNKT should be 5:1, for example 2 x 106 DC cells + 0.4 x 106 iNKT cells), mix gently.
  10. After 2 days, collect cells with a sterilized serological pipet and transfer into 15 ml tubes. Bring volume to 10 ml with RPMI medium containing 5% FBS. Centrifuge at 300 x g for 10 min with braking, discard supernatant, and resuspend cells with complete medium plus 20 U/ml rhIL-2 at 0.4 x 106 cells/ml, and then culture cells in 12 well plate at 3 ml/well.
  11. Culture cells for 7-10 days, and replace medium every 2 days as step 9.
  12. Flow cytometric analysis of expanding iNKT cells.
    Gate on lymphocytes and check purity by flow cytometric analysis, using PE-anti-Vα24Jα18 antibody (6B11) - see Figure 2.

    Figure 2. A. iNKT cells among human PBMCs; B. iNKT cell line isolated and expanded from human PBMCs

    Flow cytometry procedure:
    1. Collect 0.1 x 106 cells, and transfer into 1.5 ml tube, and filled with 1 ml FACS buffer (0.2% FBS in PBS).
    2. Centrifuge cell 600 x g for 5 min, and then discard supernatant.
    3. Resuspend cells in 50 μl FACS buffer, and add 0.5 μl anti-CD16/32 antibody  for 15 min to block non-specific binding, and then wash as step a.
    4. Resuspend cells in 50 μl FACS buffer, and add 5 μl PE anti-Vα24 antibody and 5 μl APC 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 an LSRII FACS machine.
  13. To maintain iNKT cells culture: Culture iNKT cells in complete medium plus 20 U/ml rhIL-2, and every 2-3 days replaced with fresh medium. When the density of viable cells is greater than 2 x 106 cells/ml, the cells should be diluted to 0.4 x 106 cells/ml with complete medium plus 20 U/ml rhIL-2. To prepare stocks of iNKT cells, iNKT cells with ice-cold RPMI medium plus 10% DMSO and 20% FBS at 5 x 106 cells/ml medium/frozen vial, using Nalgene Freezing Container.


  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


This work was supported by National Institutes of Health (NIH), National Cancer Institute Grants K01 CA131487, R21 CA162273, and R21 CA162277 to T.J. Webb, 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 Exley et al. (1997), Harada et al. (2005) and Shiratsuchi et al. (2009).


  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. 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.
  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. Molecular identification of GD3 as a suppressor of the innate immune response in ovarian cancer. Cancer Res 72(15): 3744-3752.




  1. 血样收集iNKT细胞
  2. Ficoll-Paque Plus(GE Healthcare Biosciences,目录号:17-1440)
  3. PE-anti-Vα24Jα18抗体(6B11)(Biolegend,目录号:342904)
  4. 抗CD16/32抗体(Biolegend,目录号:101320)
  5. 抗-Vα24抗体(Beckman Coulter,目录号:IM2283)
  6. 抗CD3抗体(Biolegend,目录号:300312)
  7. 重组人GM-CSF(R& D Systems,目录号:215-GM)
  8. 重组人IL-4(R& D Systems,目录号:204-IL)
  9. 重组人IL-2(Proleukin)(BD Biosciences,目录号:354043)
  10. 人树突细胞分离试剂盒(人CD14磁珠)(MiltenyiBiotec,目录号:130-050-201)
  11. 丝裂霉素C(Sigma-Aldrich,目录号:M4287)
  12. 人iNKT细胞分离试剂盒(Vα24Jα18磁性微珠)(MiltenyiBiotec,目录号:130-094-842)
  13. α-半乳糖苷神经酰胺(α-GalCer,KRN7000)(Enzo Life Sciences,目录号:BML-SL232)
  14. RPMI-1640培养基(Life Technologies,< Gibco ,目录号:11875)
  15. 非必需维生素溶液(Life Technologies,Gibco ,目录号:11140-050)
  16. MEM维生素溶液(Life Technologies,< Gibco ,目录号:11120-052)
  17. 丙酮酸钠(Life Technologies,  Gibco ,目录号:11360-070)
  18. 2-巯基乙醇(Life Technologies,Gibco ,目录号:21985-023)
  19. Nalgene冷冻容器(Thermo Fisher Scientific,目录号:15-350-50)
  20. 抗生素:青霉素 - 链霉素
  21. 热灭活的胎牛血清
  22. MACS缓冲区(参见配方)
  23. 完整介质(见配方)


  1. 离心机带有旋转转子,能够提供300-700 x g
  2. BD LSR II流式细胞计数器(BD Biosciences)
  3. 灭菌的巴斯德移液器
  4. 50ml锥形管
  5. 0.22μM滤波器
  6. T-175烧瓶
  7. T-25烧瓶
  8. 37℃5%CO 2培养箱


  1. 收集外周血单核细胞(PBMC)。 对于Ficoll密度梯度离心分离淋巴细胞与血沉棕黄层或白细胞提取包,首先用等体积的1x PBS在室温下稀释肝素化的血液。
  2. 加入15ml Ficoll(温至室温)至50ml锥形管中。 然后在Ficoll顶部缓慢覆盖25ml稀释的血液混合物。 在室温下以400×g离心30分钟,并关闭制动器
  3. 小心地取出2-3毫升淋巴细胞界面(介质和Ficoll之间的白色环,请参见图1中的示意图),使用灭菌的巴斯德吸管,并转移到新的50ml锥形管。

    图1. Ficoll-Paque Plus分离人外周血的示意图

  4. 通过将细胞沉淀重悬在5ml PBS中来洗涤细胞,然后将所有沉淀合并到一个50ml管中,并用PBS填充管至50ml,并在400×g离心10分钟分钟,制动器打开
  5. 弃去上清液并将来自单个供体的管合并到单个管中,并再次用20ml PBS洗涤PBMC。然后计数PBMC,并在冰冷的MACS缓冲液中以5×10 7个细胞/ml的浓度重悬。
    注意:白细胞通常用于这些研究,并且血液的初始体积为400-450ml,但产物已富集白细胞,并且步骤1中用于Ficoll分离的最终体积为约40ml。完成步骤5后,最好继续其余的协议。如果不是,细胞应该在T-175培养瓶中以10×10 7个细胞/ml的完全培养基在37℃5%CO 2下培养过夜, CO
  6. 第1-天 - 分离人CD14 + 细胞:根据制造商的说明使用人类CD14 - 磁性微珠。
    1. 对于CD14阳性的PBMC,用8ml完全培养基和20ng/ml rhGM-CSF和20ng/ml rhIL-4培养10天7天的细胞4-5天, T-25烧瓶中以产生未成熟的树突细胞(DC)。冻结额外的储存格,以便日后在10 7 /瓶使用。
    2. 保持CD14 neg 级分用于iNKT细胞分离。为了维持细胞,在T-175烧瓶中以10μL/ml在完全培养基和10U/ml rhIL-2中培养。当扩展DC电池时,不必更换介质
  7. 第5天 - 扩增原代iNKT细胞:收集CD14阴性PBMC,并用10ml完全培养基洗涤。根据制造商的说明使用人类iNKT细胞分离试剂盒。
    1. 为了分离Vα24 + hNKT细胞:
      1. 收集CD14 - PBMC,并用洗涤培养基洗涤一次
      2. 用30ml冰冷的MACS缓冲液重悬沉淀,通过70μm无菌细胞过滤器,然后以400×g离心5分钟。
      3. 用1ml MACS缓冲液重悬沉淀,加入抗-Vα24抗体,5μg/10 6阳性细胞(NKT细胞占PBMC中淋巴细胞群体的0.01%-0.5%)。 在冰上孵育30分钟。
      4. 在30ml冰冷的MACS缓冲液中洗涤细胞两次
      5. 用1ml MACS缓冲液重悬沉淀,加入抗小鼠IgG微珠20μl/10 6阳性细胞。 在冰上孵育20分钟。
      6. 在30ml冰冷的MACS缓冲液中洗涤细胞一次,同时离心通过加入500μlMACS缓冲液制备MS柱。
      7. 重悬细胞在500微升ml MACS缓冲液。然后吸取细胞进入MS分离柱。确保避免通过慢慢吸移产生气泡。通过加入500μlml MACS缓冲液冲洗色谱柱。重复两次。加入1ml新鲜MACS缓冲液,从磁铁中取出柱。将柱放入15ml锥形管中。插入柱塞和推出内容,以获得纯化的iNKT细胞。计数NKT细胞富集级分。您应该有0.02-0.5百万个单元格。
  8. 为了扩展原代人iNKT细胞:
    1. 首先,用丝裂霉素C预处理未成熟的DC。在含有0.05mg/ml丝裂霉素C的RPMI中以5×10 6个细胞/ml重悬未成熟的DC,在37℃下在黑暗中孵育30分钟,然后用10ml完全培养基洗涤三次
    2. 通过将细胞在含有200ng/mlα-GalCer和20U/ml rhIL-2的完全培养基(12孔板/1ml /孔)中在2-3×10 6 /细胞/ml,37℃孵育1小时
  9. 向12孔板中 - 向含有负载α-GalCer的DC(DC:iNKT)的孔中加入1ml在含有20U/ml rhIL-2Vα24+人类iNKT细胞的完全培养基中的比例应为5:1 ,例如2×10 6个DC细胞+ 0.4×10 6个NKT细胞)轻轻混合。
  10. 2天后,用灭菌的血清移液管收集细胞并转移到15ml管中。用含有5%FBS的RPMI培养基使体积达到10ml。以300×g离心10分钟,制动,弃去上清液,并用0.4×10 6个细胞/ml的完全培养基加20U/ml rhIL-2重悬细胞,然后在12孔板中以3ml /孔培养细胞
  11. 培养细胞7-10天,并且每2天更换培养基作为步骤9.
  12. 扩增iNKT细胞的流式细胞术分析 通过流式细胞术分析,使用PE抗Vα24Jα18抗体(6B11)在淋巴细胞上门并检查纯度 - 参见图2.

    图2.人PBMC中的iNKT细胞; B.从人PBMC分离和扩增的iNKT细胞系

    1. 收集0.1×10 6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. 离心细胞600×g 5分钟,然后丢弃上清液
    3. 重悬细胞在50微升FACS缓冲液,并加入0.5微升抗CD16/32抗体 15分钟以阻断非特异性结合,然后作为步骤a洗涤
    4. 重悬细胞在50μlFACS缓冲液,并加入5μlPE抗-Vα24抗体和5μlAPC抗CD3抗体 在冰上在黑暗中30分钟,然后作为步骤a洗涤
    5. 重悬细胞在200μlPBS,并在LSRII FACS机器上运行样品
  13. 保持iNKT细胞培养:将iNKT细胞培养于完全培养基加20U/ml rhIL-2,每2-3天更换新鲜培养基。 当活细胞的密度大于2×10 6个细胞/ml时,应用完全培养基加20U将细胞稀释至0.4×10 6个细胞/ml/ml rhIL-2。 为了使用Nalgene冷冻容器以5×10 6个细胞/ml培养基/冷冻小瓶制备iNKT细胞,iNKT细胞与冰冷的RPMI培养基加10%DMSO和20%FBS的储液。 />


  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


这项工作得到国立卫生研究院(NIH),国家癌症研究所批准K01 CA131487,R21 CA162273和R21 CA162277到T.J. Webb,NIH AI 70258 to M.Tsuji,NIH AI 44129,CA 108835和P01 AI072677 to J.P.Schneck。 该方法公开在Webb等人(2012)中,并且是对Exley等人(1997),Harada等人 。(2005)和Shiratsuchi (2009)。


  1. Dellabona,P.,Padovan,E.,Casorati,G.,Brockhaus,M。和Lanzavecchia,A。(1994)。 不变V alpha 24-J alpha Q/V beta 11 T细胞受体在所有个体中表达通过克隆扩增的CD4-8- T细胞。实验医学 180(3):1171-1176。
  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。
  3. Fowlkes,B.J.,Kruisbeek,A.M.,Ton-That,H.,Weston,M.A.,Coligan,J.E.,Schwartz,R.H.and Pardoll,D.M。(1987)。 主要表达单个Vβ基因的T细胞受体αβ携带胸腺细胞的新群体 329(6136):251-254。
  4. Harada,Y.,Imataki,O.,Heike,Y.,Kawai,H.,Shimosaka,A.,Mori,S.,Kami,M.,Tanosaki,R.,Ikarashi,Y.,Iizuka, Yoshida,M.,Wakasugi,H.,Saito,S.,Takaue,Y.,Takei,M.and Kakizoe,T。(2005)。 在没有动物材料的情况下培养的α-半乳糖苷神经酰胺刺激的Valpha24 + NKT细胞的扩增
  5. Prigozy,TI,Naidenko,O.,Qasba,P.,Elewaut,D.,Brossay,L.,Khurana,A.,Natori,T.,Koezuka,Y.,Kulkarni,A.and Kronenberg, )。 糖脂抗原加工用于CD1d分子的呈递。 291(5504):664-667
  6. Shiratsuchi,T.,Schneck,J.,Kawamura,A。和Tsuji,M。(2009)。 人CD1二聚体蛋白作为用于CD1结合脂质和CD1限制性T细胞研究的不可或缺的工具。免疫方法 345(1-2):49-59
  7. Webb,T.J.,Bieler,J.G.,Schneck,J.P.and Oelke,M。(2009)。
  8. Webb,TJ,Li,X.,Giuntoli,RL,2nd,Lopez,PH,Heuser,C.,Schnaar,RL,Tsuji,M.,Kurts,C.,Oelke,M.and Schneck,JP GD3作为卵巢癌中先天免疫应答的抑制子的分子鉴定。 Cancer Res 72(15):3744-3752。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Li, X., Tsuji, M., Schneck, J. and Webb, T. J. (2013). Generation of Human iNKT Cell Lines. Bio-protocol 3(6): e418. DOI: 10.21769/BioProtoc.418.



Moriya Tsuji
HIV and Malaria Vaccine Program, Aaron Diamond AIDS Research Center, USA
The source of human IL-2 can be E. coli. In fact, we routinely use a recombinant human IL-2 from E.coli. But it needs to be pure and without any contamination of endotoxin. I previously wrote T-75 but it is typo. T-175 is correct. Thanks.
5/18/2018 7:33:51 AM Reply
Burcu İlçe
Ankara University

Ok, thanks for all your answers. I will share the results when I apply the protocol.

All the best,

5/18/2018 10:01:12 AM

Burcu İlçe
Ankara University
Hi Burcu,

You can purchase the iNKT mAb (clone 6B11) from several companies- Biolegend, BD, or, Miltenyi Biotec. It is used to check the purity of your NKT cell population by flow cytometry.

I hope that this helps!

Dear Tonya,
I didn't ask for the 3. item, I asked for the 5th (anti-Va24 ab). When did you use this ab in the protocol? In which step? In the 4th page, 7aiii., I saw anti-Va24 but is this Beckman coulter IM2283? I thought that was anti-inkt microbeads, if not where did you use human iNKT cell isolation kit in the protocol?

Can I express myself clearly? I hope :) TIA
5/18/2018 4:04:43 AM Reply
Tonya Webb
Microbiology and Immunology Department, University of Maryland School of Medicine, USA

Dear Burcu,

Yes, you are correct. You can use the iNKT microbead kit or you can use the Va24 antibody from Beckman Coulter and then use anti-mouse IgG microbeads to enrich for NKT cells.

Thank you Moriya, for providing clarity.

Best wishes,

5/18/2018 6:55:39 AM

Moriya Tsuji
HIV and Malaria Vaccine Program, Aaron Diamond AIDS Research Center, USA
Thanks, Tonya, for your response. Just to add, the concentration of CD14- cells is approx. 2.5 x 10^6/mL, meaning that 5 x 10^7/20 mL in T-75 flask.

Recombinant human IL-2 conversion is usually 2 x 10^4 IU/ug.

5/17/2018 11:52:20 AM Reply
Burcu İlçe
Ankara University

Thank you very much Moriya and Tonya.
Isn't the source of the cytokines that are used important? I thought it should be from human like HEK cells. As I saw you used E.coli origined IL-2, GMCSF etc. It isn't important at that point, right?

5/18/2018 1:36:41 AM

Burcu İlçe
Ankara University
Thanks a lot in advanced!
All the best,
5/8/2018 12:36:07 AM Reply
Burcu İlçe
Ankara University
Dear authors,

I have some questions with the protocol.
1) In 6b, you say culture cells (CD14neg) in T-175 flask at 10^7/ml. It means app. 50x10^7 cells in a flask right? This is for day-1. Than you go with step 7 (day 5). Will we go on with the cells in T-175 flask?
2) In 8b; in previous step (8a) we plate 5x10^6 imDC/ml (means 5x10^6 imDC/per well of 12 well plate). but you want us to add gal cer and IL-2 for 2-3x10^6/ml cell? We should add for 5x10^6/ml cell, shouldn't we?
3) And finally, you gave ng/ml amount for GMCSF and IL-4 but U/ml amount for IL-2. I have 50 ug/ml IL-2 but I don't know how much to add to reach 20U/ml.
5/8/2018 12:35:13 AM Reply
Burcu İlçe
Ankara University

And one more question :)
4) You listed materials for the procedure. I didn't see the useage of 5th item; Anti-Va24 antibody. Where and when will we use it? Thanks again :)

5/8/2018 3:37:18 AM

Tonya Webb
Microbiology and Immunology Department, University of Maryland School of Medicine, USA

Dear Burcu,

1) Regarding the number of cells (10^7) is 10 million cells; step 5 states 50 million cells.
- Each step is a continuation from the previous step.
2) In 8a, you will treat 5 million cells/ml with mitomycin C in a conical tube.
In 8b, after washing, 2-3 million DCs will be pulsed with antigen at the indicated concentration, and then NKT cells will be added to the plates.
3) The company that you purchased the IL-2 should also have the concentration in international units (IU). I am not sure about the conversion.

5/11/2018 8:44:03 AM

Tonya Webb
Microbiology and Immunology Department, University of Maryland School of Medicine, USA

Hi Burcu,

You can purchase the iNKT mAb (clone 6B11) from several companies- Biolegend, BD, or, Miltenyi Biotec. It is used to check the purity of your NKT cell population by flow cytometry.

I hope that this helps!

Good luck,

5/11/2018 8:49:31 AM

Ching-Lien WU
Dear authors,

I got confused with the steps 9 to 11. During the 7-day iNKT cell expansion, do we need to feed with aGC-loaded DC every 2 days?

I had try once with this protocol, and everything went well till the co-culture step. Then my cells didn't expand as expected. It may because I did not keep feeding them. Please help me to solve this problem. Thank you very much!

Best wishes,

6/15/2015 1:05:17 AM Reply
Tonya Webb
Microbiology and Immunology Department, University of Maryland School of Medicine, USA

Dear Ching-Lien,

During the expansion you do not need to add in aGC-loaded DCs. It is a medium exchange, if your cells are rapidly dividing then you should add fresh complete media +IL-2.

Please let me know if you have any additional questions.

Best wishes,

6/15/2015 6:34:30 AM

Moriya Tsuji
HIV and Malaria Vaccine Program, Aaron Diamond AIDS Research Center, USA

Thank you, Tonya, whose response is absolutely correct. MT

6/15/2015 6:54:20 AM

Ching-Lien WU

Dear Tonya,

Firstly, thank you very much for your replay.

In fact, I put the purified NKT and the aGC-loaded DCs together, then I fresh complete medium+ IL-2 every 2 or 3 days. I saw many clusters on Day 2 after co-culture. However, the cell number till today (Day 9) is not increased. So I am wondering what could be the problem.

I saw very low iNKT cells presented in this donor. The purified fraction was not very convincing. Most of them looked dead from my FACS analysis. It could be a reason.

I'm trying to perform the second time. Let's see what I can get. Do you think that, instead of using CD14 beads, collecting the monocytes using adhesion ways would also work to generate the DCs?

I believe that I am going to have many questions about this expansion procedure.

Thank you again!
All the best,


6/15/2015 9:56:23 AM

Tonya Webb
Microbiology and Immunology Department, University of Maryland School of Medicine, USA

Dear Ching-Lien,

Let me know how your results turn out with this second experiment.

Good luck,

6/15/2015 1:23:41 PM

Ching-Lien WU

Ok, I will keep you posted.
Thank you.


6/15/2015 2:29:34 PM

Xiangming Li
HIV and Malaria Vaccine Program, Aaron Diamond AIDS Research Center, USA


Regarding your previous question, it is not necessary to add a-GalCer-loaded DC every 2 days. To expend iNKTs, DCs were only added one time when starting co-culture.

6/16/2015 4:01:51 PM

Ching-Lien WU


It is very helpful. Thank you.


6/17/2015 1:57:00 AM

Ching-Lien WU

Hi again to all,

After performing this protocol several times, I would like to know how to keep the iNKT cell growing? I got enriched iNKT population within my cellular pool (purity >95%), however, my cell counts of iNKT never reach 1 million or even more. The initial iNKT count I had was around 100K to 500K. Then I might have total cells around 1 million till week 4, and then the cell number started to decrease.

After rechecking with this protocol, I would like to know if the 2-mercaptoethanol is important for NKT growing? And it's concentration should be 5 x 10^5 M as mentioned in your protocol or 5 x 10^-5 M? I am confused.

I ever tried to re-stimulate the iNKT cells using their autologue mDC plus aGC at week 5, however, I did not still have huge amount of iNKT as I expected.

Hope you can help me out. Thank you for reading my message.


10/28/2015 8:50:43 AM

Tonya Webb
Microbiology and Immunology Department, University of Maryland School of Medicine, USA

Hi Ching-Lien,

I have found that proliferation is highly variable and the numbers that I get after expansion is donor-dependent. I get a greater yield when the donor has a high starting population (0.4-1%) of NKT cells. After 4-5 weeks, I usually freeze down aliquots. Then I take the cells out of freeze and restimulate them, and the cells will proliferate at a high level again.

The 2-mercaptoethanol is provided at a concentration of 55 mM and we use 1ml per 1L of medium.

Best wishes,

10/29/2015 7:33:49 PM

Mark Exley
Useful to have the NKT protocols available here for all, thanks for posting !

Minor points:

1. to avoid any confusion, T cell medium contains not "Vitamin" but Amino-Acid solutions for those catalogue numbers (and as in ours and others published variations on these protocols):

MEM Non-Essential / Essential Amino Acids Solution, 100X

2. human NKT (like most human ab T cells) can be kept alive almost indefinitely. Some use repeated frequent stimulations (e.g. Rogers. J Immunol Methods. 2004 Feb 15;285(2):197-214), for example if rapid maximal expansion is desired, but when possible, we prefer periodic stimulations when clearly rested cultures (few if any clusters in culture) no longer proliferating effectively, to avoid changing phenotype too much with each cycle of stimulation in vitro (e.g. Exley. Eur J Immunol. 2008 Jun;38(6):1756-66).

Mark Exley
5/7/2013 1:25:42 PM Reply
Antonia Rotolo
Imperial College London
Dear all,
I would like to thank you for your protocol. I much appreciated it!
I am a PhD student at Imperial College London.
I hope you don't mind if I ask for some questions.
1. Do you collect samples from healthy donors?
2. Before leukapheresis, do donors receive GCSF?
3. Which initial volume of peripheral blood would be desirable without leukapheresis?
4. Do you think the initial isolation of CD14 and iNKT can be omitted with an acceptable expansion efficiency?
5. How long can you keep culturing and expanding iNKT cells in your experience?
6. Can you see any difference in the expansion efficiency among donors?
Many thanks in advance for your help! I am very grateful indeed!
Best regards,
Antonia Rotolo

4/9/2013 1:08:28 AM Reply
Tonya Webb
Microbiology and Immunology Department, University of Maryland School of Medicine, USA

Dear Antonia,

Please see below for a point by point response to your questions:
1. Yes, we collect blood from healthy donors.
2. No, the donors do not need to received G-CSF.
3. Without leukapheresis, the minimum amount of blood that I have used is from 2 CPT Vacutainer tubes (16-18ml).
4. If the initial isolation is skipped then the expansion will be minimal unless the donor has a high percentage of circulating NKT cells (>1% of the total T cell population).
5. I can usually keep culturing and expanding for NKT cells 5-6 weeks, but some lines have been maintained for ~3 months without thawing a fresh aliquot.
6. Yes, there is a high amount of variability between the donors, usually the higher the initial population of NKT cells the higher the expansion efficiency.

Good luck with your studies and please let me know if you have any additional questions.

Kind regards,
Tonya Webb

4/9/2013 3:14:32 PM

Antonia Rotolo
Imperial College London

Again thank you very much!

4/9/2013 3:31:15 PM