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嵌合抗原受体T细胞体外肿瘤杀伤的流式检测   

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摘要:嵌合抗原受体T细胞 (CAR-T),是通过基因工程手段进行修饰的T细胞。胞外区含有不受MHC限制的单链可变区 (scFv),可特异识别抗原 (如CD20等);胞内区含有共刺激结构域 (CD28,4-1BB等) 和第一信号激活结构域 (CD3ζ),进行信号传递 (Eshhar等,1993;Sadelain等,2013)。CD20 CAR-T可特异的识别并杀伤表达CD20的人B淋巴瘤细胞系Raji (Kowolik等,2006)。在体外,CD20 CAR-T与靶细胞Raji以不同比例共培养24小时,使用不同抗体分别标记CAR-T细胞和靶细胞,通过流式细胞技术确定CD20 CAR-T杀伤功能。

关键词: CAR-T, 靶细胞, 流式细胞技术, T细胞杀伤

材料与试剂

  1. 96孔板 (U型) (Jet Biofil, catalog number: 011096)
  2. Falcon细胞流式管 (BD, catalog number: 352054)
  3. 离心管 (Jet Biofil, catalog number: CFT011550)
  4. Raji 细胞 (ATCC® CCL-86TM)
  5. 抗体:
    APC-anti-human CD19 (BioLegend, catalog number: 302212)
    FITC-anti-human CD3 (BioLegend, catalog number: 317306)
  6. 胎牛血清 (FBS) (Gibco, catalog number: 10270)
  7. RPMI 1640培养基 (Hyclone, catalog number: SH30809.01)
  8. Penicillin/streptomycin (Hyclone, catalog number: SV30010)
  9. 台盼蓝 (Trypan Blue) (Sigma-Aldrich, catalog number: T8154)
  10. β-巯基乙醇 (2-Mercaptoethanol) (Amresco, catalog number: 0482)
  11. NaCl (Sigma-Aldrich, catalog number: V900058-500g)
  12. KCl (Sigma-Aldrich, catalog number: V900068-500g)
  13. Na2HPO4 (Sigma-Aldrich, catalog number: V900060-500g)
  14. KH2PO4 (Sigma-Aldrich, catalog number: V900041-500g)
  15. Na3N (Amresco, catalog number: 0639-250g) 
  16. IL-2 (北京四环生物制药有限公司,catalog number: 国药准字S10970018)
  17. L-Glutamine (200 mM)
  18. 10× PBS (1 L, pH = 7.3) (见溶液配方)
  19. FACS buffer (1 L) (见溶液配方)
  20. T细胞培养基 (见溶液配方)

仪器设备

  1. 流式细胞仪 (Beckman Coulter, model: Cytoflex S)
  2. 电动吸引器 (斯曼峰,model: YX930D)
  3. 移液器 (Eppendorf)
  4. 正置光学显微镜 (Olympus, model: IX2-SLP)
  5. 离心机 (Eppendorf, model: 5810R)
  6. 医用低温保存箱 (海尔,model: DW-25L262/BD-226W)
  7. 振荡混匀机 (kylin-bell)
  8. 生物安全柜 (LabGard, model: Class II/Labconco)
  9. 天平 (Sartorius, model: BSA124S)
  10. 高压灭菌锅 (上海博迅实业有限公司,model: YXQ-LS-100S11)

软件

  1. FlowJo

实验步骤

  1. 分别收集悬浮细胞Raji、对照T细胞 (未进行基因修饰原代T细胞)、CD20 CAR-T细胞 (能特异识别CD20的嵌合抗原受体T细胞) 到50 ml离心管,取10 μl细胞,加入10 μl台盼蓝 (可根据细胞浓度进行适当稀释),计数;
  2. 500 x g离心5 min,弃去上清,新鲜T细胞培养基重悬细胞,调整细胞浓度 (Raji 2 × 106/ml,对照T细胞或CD20 CAR-T 1 × 106/ml);
  3. 按照不同的比例将Raji (Target cells) 和对照T细胞、CD20 CAR-T (Effector cells)混合于96孔培养板 (平底型),总体积至200 μl,置于37 °C,5% CO2培养箱进行培养:
    E:T = 1:0.5 (1 × 105/100 μl CD20 CAR-T + 5 × 104/25 μl Raji +75 μl T培养基)
    E:T = 1:1 (1 × 105/100 μl CD20 CAR-T + 1 × 105/50 μl Raji + 50 μl T培养基)
    E:T = 1:2 (1 × 105/100 μl CD20 CAR-T + 2 × 105/100 μl Raji);
  4. 24小时,混匀细胞,取100 μl细胞加入96孔板 (U型),进行流式检测;
  5. 加入150 μl FACS buffer,500 x g离心3 min,弃去上清;
  6. 抗体准备 (以下为单个样品):
    50 μl FACS buffer + 0.03 μg FITC-anti-human CD3 + 0.0075 μg APC-anti-human CD19;
  7. 将已配制抗体加入样品中,50 μl/样品,移液枪混匀,4 °C避光孵育25 min;
  8. 加入150 μl FACS buffer洗去未结合抗体,500 x g,离心3 min,弃去上清;
  9. 重复步骤8;
  10. 70 μl FACS buffer重悬样品,将样品置于冰上;
  11. 预热流式细胞仪,进行上样检测;
  12. FlowJo软件分析流式检测结果。

结果与分析

  1. 人淋巴瘤细胞Raji表达CD19,CD20等抗原,为了规避CD20 CAR-T与anti-human CD20竞争性结合CD20位点,使用anti-human CD19抗体进行肿瘤特异性标记。
  2. FSC-A和SSC-A排除死细胞和细胞碎片,确定和收集主要细胞群 (Gate 1,收集10,000个数据点/样品) (图1)。
  3. 通过CD3 (FITC-A) 和CD19 (APC-A) 分别分析T细胞和Raji细胞。
  4. 与未进行基因修饰T细胞相比,随着E:T比例降低,残留靶细胞Raji比例逐渐增加,但是与对照T细胞相比,CD20 CAR-T仍表现出较强的杀伤能力,T cell only和Raji only作为染色的对照。



图1. CAR-T细胞体外杀伤流式检测

注意事项

  1. 对于不同肿瘤细胞的CAR-T杀伤实验,可根据肿瘤自身的抗原表达特点,选取不同的表面分子进行流式分析,以达到区分肿瘤细胞和T细胞的目的,对于选择的抗体也应进行用量滴定,保证最佳的细胞分群效果。
  2. 若肿瘤细胞表达较高FcγR导致抗体非特异结合,应在染色体系中包含FcγR blocker。
  3. 对于某些肿瘤杀伤较慢,可延长杀伤时间至48小时或72小时。

溶液配方

  1. 10× PBS (1 L, pH = 7.3)
    NaCl
    80 g
    KCl
    10 g
    Na2HPO4
    14.4 g
    KH2PO4
    2.4 g
    使用分析天平精准称量上述试剂,加入800 ml ddH2O依次进行溶解,待所有试剂完全溶解后,加入ddH2O定容至1 L,高温湿热灭菌,冷却后室温保存待用
  2. FACS buffer (1 L)
    10× PBS
    100 ml
    FBS
    10 ml
    Na3N (20%)
    2.5 ml
    使用量筒取100 ml 10× PBS,首先加入800 ml ddH2O进行稀释,再加入10 ml FBS和2.5 ml Na3N (20%),混合均匀,加入ddH2O定容至1 L,4 °C备用
  3. T细胞培养基
    RPMI 1640
    500 ml
    2-Me (1 mol/L)
    27 ul
    IL-2
    30 IU/ml
    L-Glutamine (200 mM)
    6 ml
    Penicillin/streptomycin
    5 ml
    FBS
    60 ml

致谢

感谢杨选明实验室全体成员的建议和帮助。该研究受国家自然基金 (81671643)、科技部重点研发计划 (2016YFC1303400) 资助。

参考文献

  1. Eshhar, Z., Waks, T., Gross, G. and Schindler, D. G. (1993). Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors. Proc Natl Acad Sci U S A 90(2): 720-724.
  2. Kowolik, C. M., Topp, M. S., Gonzalez, S., Pfeiffer, T., Olivares, S., Gonzalez, N., Smith, D. D., Forman, S. J., Jensen, M. C. and Cooper, L. J. (2006). CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells. Cancer Res 66(22): 10995-11004.
  3. Sadelain, M., Brentjens, R. and Rivière, I. (2013). The basic principles of chimeric antigen receptor design. Cancer Discov 3(4): 388-398.
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:张晓卿, 杨选明. (2019). 嵌合抗原受体T细胞体外肿瘤杀伤的流式检测. Bio-101: e1010316. DOI: 10.21769/BioProtoc.1010316.
How to cite: Zhang, X. Q. and Yang, X. M. (2019). Analyzing Cytotoxicity Capacity of Chimeric Antigen Receptor T-cell in vitro by Flow Cytometry. Bio-101: e1010316. DOI: 10.21769/BioProtoc.1010316.
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