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Murine in vitro Memory T Cell Differentiation

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Aug 2013



Upon pathogen encounter, naïve CD8+ T cells are primed and undergo massive clonal expansion. A fraction of effector CD8+ T cells remains during the contraction phase and differentiate into memory T cells critical for mounting robust recall responses in response to secondary infection. Low frequency of memory T cells in vivo is a major obstacle to investigate their functional aspects including migration capacity and genetic regulation. Here, we describe detailed protocol for memory T cell differentiation developed by von Andrian’s group to generate large number of CD44hiCD62Lhi antigen-specific memory T cells in vitro.

Keywords: Ag-specific memory CD8 T cell (记忆CD8 T细胞抗原特异性), OT-I (催产素- I), IL-15 (白细胞介素15), In vitro (在体外), CD44+CD62L+ central memory T cell (CD44 + CD62L +中央记忆T细胞)

Materials and Reagents

  1. Recombinant mouse IL-15 (rmIL15) (BioLegend, catalog number: 566302 )
  2. RPMI-1640 medium (Life Technologies, Gibco®, catalog number: 11875-119 )
  3. Fetal bovine serum (Atlanta Biologicals, catalog number: S11055H )
  4. Penicillin/streptomycin (Gemini Bio-Products, catalog number: F52M00E )
  5. L-Glutamine (Life Technologies, Gibco®, catalog number: 25030-081 )
  6. 100x 1 M Hepes (Life Technologies, Gibco®, catalog number: 15630-080 )
  7. 100x MEM non-essential amino acids (Life Technologies, Gibco®, catalog number: 11140-050 )
  8. 100x sodium pyruvate (100 mM) (Life Technologies, Gibco®, catalog number: 11360-070 )
  9. 100x 2-mercaptoethanol (Life Technologies, Gibco®, catalog number: 21985-023 )
  10. OVA257-264 synthetic peptide (Sigma-Aldrich, catalog number: S7951 )
  11. Ficoll-PaqueTM Premium 1.084 (GE Healthcare, catalog number: 17-5446-02 )
  12. Antibodies:
    1. Anti-CD44 PerCpCy5.5 (clone: IM7) (eBioscience, catalog number: 45-0441 )
    2. Anti-CD62L APC (clone: MEL-14) (eBioscience, catalog number: 17-0621 )
  13. RBC lysis buffer (eBioscience, catalog number: 00-4333-57 )
  14. Bovine serum albumin (Thermo Fisher Scientific, catalog number: BP1605-100 )
  15. NaN3  (Sigma-Aldrich, catalog number: S8032 )
  16. T cell media (see Recipes)
  17. Staining buffer (in PBS) (see Recipes)


  1. Centrifuge (Thermo Fischer Scientific, SorvallTM Legend RT)
  2. 70 µm cell strainer (BD Biosciences, Falcon®, catalog number: 352350 )
  3. 15 ml and 50ml Falcon tubes
  4. 24 well plates (BD Biosciences, Falcon®, catalog number: 353226 )
  5. T75 culture flask (Corning, catalog number: 430641 )
  6. 37 °C, 5% CO2 cell culture incubator


  1. CD44hiCD62Llo Memory T cell differentiation proceeds under sterile tissue culture conditions
    1. Euthanize a OT-1 CD8 TCR transgenic mouse and take spleen, and (optional) lymph nodes.
    2. Splenocytes are RBC lysed followed by washing with PBS twice.

  2. OT-1 TCR stimulation with cognate peptide antigen
    1. Resuspend cells in 1 ml of T cell media and add OVA257-264 synthetic peptide to 1 µM.
    2. Incubate in the 5% CO2 at 37 °C for 1 h.
    3. Spin down cells at 1,500 rpm for 3 min at 4 °C and wash once with T cell media.
    4. Resuspend cells in 12 ml of T cell media and plate 1ml/well of a 24 well plate.
    5. Incubate in the 5% CO2 at 37 °C for 2 days.
    6. Harvest the cells by pipetting up and down, and pellet cells.
    7. Resuspend cells in 5 ml of T cell media, and load on to 2.5 ml of Ficoll.
    8. Spin down at 400 x g for 15 min at 4 °C.
    9. Transfer live cells on the interphase to a new 15 ml tube and fill up the tube with T cell media.
    10. Spin down cells at 1,500 rpm for 3 min at 4 °C.

  3. Memory T cell culture in the presence of IL-15
    1. Resuspend cells in 24 ml of T cell media containing rmIL15 (20 ng/ml). Culture cells in T75 flask for four days.
    2. Harvest and pellet cells for Ficoll gradient (repeat steps 9-12).
    3. Resuspend cells in 40 ml of T cell media containing rmIL15 (20 ng/ml). Culture in T75 flask for two days.
    4. Staining cells with anti-CD44 and CD62L antibodies in staining buffer for 15 min on ice.
    5. Wash with staining buffer twice, then proceeds flow cytometry analysis.

      Figure 1. CD44 and CD62L expression of differentiated memory T cells


  1. T cell media
    10% fetal bovine serum
    1% penicillin/streptomycin
    1% L-Glutamine
    1x 1 M Hepes
    1x MEM non-essential amino acids
    1x sodium pyruvate 100 mM
    1x 2-mercaptoethanol
  2. Staining buffer (in PBS)
    1% BSA
    0.02% NaN3


The protocol was adapted from a previously described study (Manjunath et al., 2001). This work was supported by the Starr Cancer Consortium (13-A123 to M.O.L. and M.Q.Z.), the Rita Allen Foundation (M.O.L.), the NBRPC (2012CB316503 to M.Q.Z), and the NIH (HG001696 to M.Q.Z.).


  1. Kim, M. V., Ouyang, W., Liao, W., Zhang, M. Q. and Li, M. O. (2013). The transcription factor Foxo1 controls central-memory CD8+ T cell responses to infection. Immunity 39(2): 286-297.
  2. Manjunath, N., Shankar, P., Wan, J., Weninger, W., Crowley, M., Hieshima, K., Springer, T., Fan, X., Shen, H. and Lieberman, J. (2001). Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes. J Clin Invest 108(6): 871-878.


在病原体遭遇时,初始CD8 + T细胞被引发并经历大量克隆扩增。 效应CD8 + sup T细胞的一部分在收缩阶段期间保持并分化为响应于继发感染而对于安装鲁棒回忆反应至关重要的记忆T细胞。 体内低频记忆T细胞是研究其功能方面(包括迁移能力和遗传调节)的主要障碍。 在这里,我们描述由von Andrian的小组开发的记忆性T细胞分化的详细方案,以在体外产生大量的CD44 - 高 - 抗原特异性记忆T细胞 。

关键字:记忆CD8 T细胞抗原特异性, 催产素- I, 白细胞介素15, 在体外, CD44 + CD62L +中央记忆T细胞


  1. 重组小鼠IL-15(rmIL15)(BioLegend,目录号:566302)
  2. RPMI-1640培养基(Life Technologies,Gibco ,目录号:11875-119)
  3. 胎牛血清(Atlanta Biologicals,目录号:S11055H)
  4. 青霉素/链霉素(Gemini Bio-Products,目录号:F52M00E)
  5. L-谷氨酰胺(Life Technologies,Gibco ,目录号:25030-081)
  6. 100x 1M Hepes(Life Technologies,Gibco ,目录号:15630-080)
  7. 100x MEM非必需氨基酸(Life Technologies,Gibco ,目录号:11140-050)
  8. 100x丙酮酸钠(100mM)(Life Technologies,Gibco ,目录号:11360-070)
  9. 100x 2-巯基乙醇(Life Technologies,Gibco ,目录号:21985-023)
  10. OVA 257-264合成肽(Sigma-Aldrich,目录号:S7951)
  11. Ficoll-Paque TM Suppressor 1.084(GE Healthcare,目录号:17-5446-02)
  12. 抗体:
    1. 抗CD44 PerCpCy5.5(克隆:IM7)(eBioscience,目录号:45-0441)
    2. 抗CD62L APC(克隆:MEL-14)(eBioscience,目录号:17-0621)
  13. RBC裂解缓冲液(eBioscience,目录号:00-4333-57)
  14. 牛血清白蛋白(Thermo Fisher Scientific,目录号:BP1605-100)
  15. NaN 3 (Sigma-Aldrich,目录号:S8032)
  16. T细胞培养基(见配方)
  17. 染色缓冲液(在PBS中)(参见配方)


  1. 离心机(Thermo Fischer Scientific,Sorvall Legend RT)
  2. 70μm细胞滤器(BD Biosciences,Falcon ,目录号:352350)
  3. 15 ml和50ml Falcon管
  4. 24孔板(BD Biosciences,Falcon ,目录号:353226)
  5. T75培养瓶(Corning,目录号:430641)
  6. 37℃,5%CO 2细胞培养箱中培养


  1. CD44高级记忆T细胞分化在无菌组织培养条件下进行
    1. 安乐死OT-1 CD8 TCR转基因小鼠,并取脾脏和(可选)淋巴结。
    2. 脾细胞被RBC裂解,然后用PBS洗涤两次。

  2. 用同源肽抗原的OT-1 TCR刺激
    1. 将细胞重悬于1ml T细胞培养基中,并将OVA 257-264合成肽加至1μM。
    2. 在37℃下在5%CO 2中孵育1小时
    3. 在4℃下以1,500rpm旋转细胞3分钟,并用T细胞培养基洗涤一次
    4. 将细胞重悬在12ml T细胞培养基中,并铺平1ml /孔的24孔板
    5. 在37℃下在5%CO 2中孵育2天
    6. 通过上下吹吸收细胞,并使细胞沉淀
    7. 重悬细胞在5ml的T细胞培养基,并加载到2.5ml的Ficoll
    8. 在4℃下以400xg 旋转15分钟。
    9. 转移间期的活细胞到一个新的15毫升管,并填充管的T细胞培养基。
    10. 在4℃下以1,500rpm旋转细胞3分钟。

  3. 在IL-15存在下的记忆T细胞培养
    1. 重悬细胞在24毫升含有rmIL15(20毫微克/毫升)的T细胞培养基。 培养细胞在T75瓶中培养4天
    2. 收获和Ficoll梯度的沉淀细胞(重复步骤9-12)。
    3. 重悬细胞在40毫升含有rmIL15(20毫微克/毫升)的T细胞培养基。 在T75瓶中培养两天。
    4. 在染色缓冲液中用抗CD44和CD62L抗体在冰上染色细胞15分钟。
    5. 用染色缓冲液洗涤两次,然后进行流式细胞术分析



  1. T细胞培养基
    10%胎牛血清 1%青霉素/链霉素 1%L-谷氨酰胺 1x 1 M Hepes
  2. 染色缓冲液(在PBS中)
    0.02%NaN 3


该方案改编自先前描述的研究(Manjunath et al。,2001)。 这项工作由Starr癌症联盟(13-A123到M.O.L.和M.Q.Z.),Rita Allen基金会(M.O.L.),NBRPC(2012CB316503到M.Q.Z)和NIH(HG001696到M.Q.Z.)支持。


  1. Kim,M.V.,Ouyang,W.,Liao,W.,Zhang,M.Q.and Li,M.O。(2013)。 转录因子Foxo1控制中枢记忆CD8 + T细胞应答 感染。 免疫力 39(2):286-297
  2. Manjunath,N.,Shankar,P.,Wan,J.,Weninger,W.,Crowley,M.,Hieshima,K.,Springer,T.,Fan,X.,Shen,H.and Lieberman, 2001)。 效应细胞分化不是产生记忆细胞毒性T淋巴细胞的先决条件。临床投资 108(6):871-878。
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引用:Kim, M. V., Ouyang, W., Liao, W., Zhang, M. Q. and Li, M. O. (2014). Murine in vitro Memory T Cell Differentiation. Bio-protocol 4(13): e1171. DOI: 10.21769/BioProtoc.1171.