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T Follicular Helper Cell Coculture Assay

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Nature Medicine
Apr 2013


T follicular helper (Tfh) cells constitute a distinct subset of CD4+ T cells specialized in providing help to B cells in germinal centers. Phenotypically, Tfh cells are characterized by their high expression of the chemokine receptor CXCR5 that allows their migration into B cell follicles as well as high expression of PD-1, BTLA, the co-stimulatory molecules ICOS and SLAM and the transcription factors BCL6 and cMaf. Tfh cells are the main producers of IL-21 as well as other cytokines like IL-4 and IL-10 critical for B cell survival and differentiation. Tfh cells drive somatic hypermutation and the generation of long-lived memory B cells and plasma cells having an essential role in the development of protective immunity. Developing a coculture system to measure the effects of Tfh-cell mediated B cell help is of great interest to further our understanding of Tfh-B cell interaction and to allow for the manipulation of culture conditions to investigate the potential effect different microenvironment signals or ligand/receptor interactions could have on Tfh cell function.

Materials and Reagents

  1. Lymph node mononuclear cells (LMNCs)
  2. Benzonse® Nuclease (EMD Millipore, catalog number: 70664 )
  3. LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Life Technologies, catalog number: L34957 )
  4. Staphylococcal enterotoxin B (Toxin Technology Inc, catalog number: BT202 )
  5. RPMI 1640 Medium 1x with L-Glutamine (Fischer Scientific, catalog number: 10-040-CV )
  6. 100x Penicillin-Streptomycin solution (5000 units) (Life Technologies, catalog number: 15070063 )
  7. Fetal Bovine Serum (FBS) (Access Cell Culture)
  8. RPMI 1640 Medium 1x with L-Glutamine and no Phenol red (Gibco®, Life Technologies, catalog number: 11835-030 )
  9. HEPES solution (1 M) (Sigma-Aldrich, catalog number: H0887 )
  10. Antibodies:
    1. anti-CD3 (clone: HIT3a ) (BioLegend)
    2. anti-CD4 (clone: RPA-T4 ) (BioLegend)
    3. anti-CD45RA (clone: IM2711U ) (Beckman Coulter)
    4. anti-CXCR5 (clone: RF8B2 ) (BD Biosciences)
    5. anti-CD19 (clone: HIB19 ) (BioLegend)
    6. anti-IgD (clone: IA6-2 ) (BD Biosciences)
    7. anti-CD38 (clone: HIT2 ) (BioLegend)
  11. Complete media (see Recipes)
  12. Sorting buffer (see Recipes)


  1. Centrifuge (SorvallTM LegendTM XTR, Thermo Fisher Scientific)
  2. 15 ml Falcon tube
  3. BD FACSAria II cell sorter (BD Biosciences)
  4. 37 °C 5% CO2 incubator
  5. 96-well sterile V-bottom plates (Thermo Fisher Scientific, catalog number: 249935 )
  6. 5 ml polystyrene round-bottom tubes with cell-strainer cap (BD FalconTM, catalog number: 352235 )


  1. Thaw lymph node mononuclear cells (LMNCs) and wash once to remove all traces of DMSO carried from the freezing media. For information on lymph node samples and processing protocols see reference 1 (Cubas et al., 2013).
  2. Count the cells and resuspend at 1 x 107 cells/ml in complete media in a 15 ml Falcon tube.
  3. Treat the cells for 30 min in an incubator at 37 °C 5% CO2 by adding 25 U/ml of Benzonase® Nuclease to degrade all DNA/RNA and improve the viability of the cells.
  4. Pellet the cells in ice cold sorting buffer (containing HEPES) at 1,500 rpm for 5 min and resuspend in sorting buffer at a concentration of 5 x 107 cells/ml.
  5. Stain cells with a viability dye such as LIVE/DEAD® Fixable Aqua Dead Cell kit (Vivid) following the manufacturer’s instructions.
  6. Subsequently, proceed to stain the cells with the following antibodies: anti-CD3 (clone: HIT3a), anti-CD4 (clone: RPA-T4), anti-CD45RA (clone: IM2711U), anti-CXCR5 (clone: RF8B2) to gate on Tfh cells. Additionally and based on the population of B cells that you want to include in your coculture assay include B cell markers. For germinal center B cells you could use the following general markers: anti-CD19 (clone: HIB19), anti-IgD (clone: IA6-2), anti-CD38 (clone: HIT2).
  7. Stain the cells for 15 min on ice.
  8. Resuspend the cells in sorting buffer and spin at 1,500 rpm for 5 min.
  9. Remove the supernatant and resuspend at 3 x 107 cells/ml for sorting. Filter the cells by passing them through a 5 ml polystyrene round-bottom tube with cell-strainer cap.
  10. Proceed to cell sorting. Gate Tfh cells and germinal center (GC) B cells after excluding doublets and dead cells as: CD3+CD4+CD45RA-CXCR5++ (Figure 1) and CD3-CD19+CD38+IgD- (Figure 2) respectively.

    Figure 1. Gating strategy for sorting Tfh cells. Tfh cells are gated as CD3+CD4+CD45RA- CXCR5++ cells after excluding doublets and dead cells.

    Figure 2. Gating strategy for sorting GC B cells. GC B cells are gated as CD3-CD19+IgD-CD38+ cells after excluding doublets and dead cells. The different B cell populations in the CD38-IgD dot plot are depicted.

  11. Once the cells are sorted, resuspend in complete media and centrifuge at 1,500 rpm for 5 min. Carefully remove the supernatant and make sure all the media is taken out. Resuspend the cells in either 250 μl or 500 μl of complete media (depending on number of sorted cells) and count the cells. It is highly recommended to have a post-sort check of the different cell populations sorted to confirm the cell purity.
  12. Resuspend the cells (both sorted Tfh cells and GC B cells) in complete media and spin down again at 1,200 rpm for 5 min. Remove the supernatant carefully and thoroughly and resuspend in an appropriate amount of complete media to have 1 x 105 cells in 50 μl (you can decide to resuspend at other concentrations but the coculture will have a final volume of 100 μl so you don’t want to have a volume larger than 50 μl for 1 x 105 cells since you are culturing both Tfh cells and GC B cells).
  13. Transfer 50 μl (1 x 105 cells) from both the sorted Tfh cells and GC B cells into a well of a 96-well V-bottom plate (1:1 ratio).
  14. Add 100 ng/ml of Staphylococcal enterotoxin B (SEB) into each well to mimic the antigen-specific interaction between T cells and B cells. Flick the plate carefully to mix and centrifuge at 900 rpm for 1 min.
  15. Incubate the cells at 37 °C 5% CO2 for 7 days or a different number of days based on the readout being measured. Testing the viability of the cells and readout measurements at later time points is recommended. Measuring the total levels of IgG in the coculture supernatant can be used as a measure of Tfh-mediated B cell help. Negative controls can include the incubation of GC B cells with naïve CD45RA+ cells or CD45RA-CXCR5- cells which do not provide B cell help. Cell characterization by flow cytometry can also be used to measure B cell activation and absolute numbers of live cells at the desired time point.


  1. Complete media
    RPMI 1640 (with L-Glutamine)
    10% FBS
    1x Pen/Strep
  2. Sorting buffer
    RPMI 1640 (with L-Glutamine + no Phenol red)
    2% FBS
    1x Pen/Strep
    25 mM HEPES


This protocol was first used in Cubas et al. (2013).


  1. Cubas, R. A., Mudd, J. C., Savoye, A. L., Perreau, M., van Grevenynghe, J., Metcalf, T., Connick, E., Meditz, A., Freeman, G. J., Abesada-Terk, G., Jr., Jacobson, J. M., Brooks, A. D., Crotty, S., Estes, J. D., Pantaleo, G., Lederman, M. M. and Haddad, E. K. (2013). Inadequate T follicular cell help impairs B cell immunity during HIV infection. Nat Med 19(4): 494-499.


T滤泡辅助(Tfh)细胞构成专门提供对生发中心中的B细胞的帮助的CD4 + T细胞的不同亚群。表型上,Tfh细胞的特征在于其高趋化因子受体CXCR5的表达,其允许它们迁移到B细胞滤泡以及PD-1,BTLA,共刺激分子ICOS和SLAM以及转录因子BCL6和cMaf的高表达。 Tfh细胞是IL-21以及其他细胞因子如IL-4和IL-10对B细胞存活和分化关键的主要生产者。 Tfh细胞驱动体细胞超突变和长寿记忆B细胞和浆细胞的产生在保护性免疫的发展中具有重要作用。开发共培养系统以测量Tfh细胞介导的B细胞帮助的效果是非常感兴趣的进一步我们的Tfh-B细胞相互作用的理解,并允许操纵文化条件调查潜在影响不同的微环境信号或配体/受体相互作用可能对Tfh细胞功能。


  1. 淋巴结单核细胞(LMNC)
  2. Benzonse Nuclease(EMD Millipore,目录号:70664)
  3. LIVE/DEAD ® Fixable Aqua死细胞染色试剂盒(Life Technologies,目录号:L34957)
  4. 葡萄球菌肠毒素B(Toxin Technology Inc,目录号:BT202)
  5. RPMI 1640 Medium 1x用L-谷氨酰胺(Fischer Scientific,目录号:10-040-CV)
  6. 100x青霉素 - 链霉素溶液(5000单位)(Life Technologies,目录号:15070063)
  7. 胎牛血清(FBS)(接入细胞培养)
  8. RPMI 1640 Medium 1x与L-谷氨酰胺且不含酚红(Gibco ,Life Technologies,目录号:11835-030)
  9. HEPES溶液(1M)(Sigma-Aldrich,目录号:H0887)
  10. 抗体:
    1. 抗CD3(克隆:HIT3a)(BioLegend)
    2. 抗CD4(克隆:RPA-T4)(BioLegend)
    3. 抗CD45RA(克隆:IM2711U)(Beckman Coulter)
    4. 抗CXCR5(克隆:RF8B2)(BD Biosciences)
    5. 抗CD19(克隆:HIB19)(BioLegend)
    6. 抗IgD(克隆:IA6-2)(BD Biosciences)
    7. 抗CD38(克隆:HIT2)(BioLegend)
  11. 完成媒体(见配方)
  12. 排序缓冲区(请参阅配方)


  1. 离心机(Thermo Fisher Scientific的Sorvall TM Suppress TM XTR)
  2. 15 ml Falcon管
  3. BD FACSAria II细胞分选仪(BD Biosciences)
  4. 37℃5%CO 2培养箱
  5. 96孔无菌V-底板(Thermo Fisher Scientific,目录号:249935)
  6. 具有细胞 - 滤器盖的5mL聚苯乙烯圆底管(BD Falcon TM,,目录号:352235)


  1. 解冻淋巴结单核细胞(LMNCs),并洗涤一次,以去除从冷冻介质携带的所有痕量的DMSO。 关于淋巴结样品和加工方案的信息,参见参考文献1(Cubas等人,2013)。
  2. 计数细胞,并在15ml Falcon管中的完全培养基中以1×10 7个细胞/ml重悬。
  3. 通过加入25U/ml的Benzonase核酸酶以在所有DNA/RNA中降解所有的DNA/RNA并改进所述DNA/RNA,在37℃,5%CO 2的培养箱中处理细胞30分钟。细胞的活力。
  4. 在冰冷的分选缓冲液(含有HEPES)中以1,500rpm将细胞沉淀5分钟,并以5×10 7个细胞/ml的浓度重悬于分选缓冲液中。
  5. 按照制造商的说明书用活力染料如LIVE/DEAD可固定水族死细胞试剂盒(Vivid)染色细胞。
  6. 随后,用以下抗体染色细胞:抗CD3(克隆:HIT3a),抗CD4(克隆:RPA-T4),抗CD45RA(克隆:IM2711U),抗CXCR5(克隆:RF8B2)至门在Tfh细胞上。此外,基于您想要包括在共培养测定中的B细胞群体包括B细胞标记物。对于生发中心B细胞,可以使用以下通用标记:抗-CD19(克隆:HIB19),抗-IgD(克隆:IA6-2),抗-CD38(克隆:HIT2)。
  7. 在冰上染色细胞15分钟。
  8. 将细胞重悬在分选缓冲液中,并在1,500 rpm下旋转5分钟。
  9. 除去上清液并以3×10 7个细胞/ml重悬以进行分选。通过使细胞通过具有细胞 - 过滤器帽的5ml聚苯乙烯圆底管来过滤细胞。
  10. 继续细胞分选。门Tfh细胞和生发中心(GC)B细胞在排除双联体和死细胞后:CD3 + CD45RA -/- > CXCR5 ++(图1)和CD3 - - - CD19 -/- CD38 -/- > IgD - sup/- (图2 ) 分别。

    图1.用于分选Tfh细胞的门控策略。 Tfh细胞被门控为CD3 + CD4 + CD45RA - CXCR5 ++ 细胞排除双联体和死细胞后

    图2.用于分选GC B细胞的门控策略。GC B细胞被门控为CD3 - CD19 + IgD - CD38 + 细胞。描述了CD38阳性IgD点图中的不同B细胞群体。

  11. 一旦细胞分选,重悬在完全培养基中并在1,500rpm离心5分钟。小心地取出上清液,并确保所有的介质被取出。重悬细胞在250微升或500微升的完全培养基(取决于分选细胞的数量),并计数细胞。强烈建议对分选的不同细胞群进行分选后检查,以确认细胞纯度。
  12. 在完全培养基中重悬细胞(分选的Tfh细胞和GC B细胞),并以1,200rpm再次旋转5分钟。小心地和彻底地除去上清液并且在适当量的完全培养基中重悬以在50μl中具有1×10 5个细胞(可以决定在其他浓度下重悬,但是共培养物将具有最终体积100μl,所以你不想有一个体积大于50微升1×10 5 细胞,因为你正在培养Tfh细胞和GC B细胞)。
  13. 将50μl(1×10 5个细胞)从分选的Tfh细胞和GC B细胞转移到96-孔V-底板(1:1比例)的孔中。
  14. 向每个孔中加入100ng/ml的葡萄球菌肠毒素B(SEB),以模拟T细胞和B细胞之间的抗原特异性相互作用。小心轻拍板以混合并在900rpm离心1分钟。
  15. 基于测量的读数,在37℃,5%CO 2下孵育细胞7天或不同天数。建议在稍后的时间点测试细胞的活力和读数测量。测量共培养上清液中IgG的总水平可以用作Tfh介导的B细胞帮助的量度。阴性对照可以包括将GC B细胞与不提供B细胞帮助的幼稚CD45RA +细胞或CD45RA -/- CXCR5 - 细胞孵育。通过流式细胞术的细胞表征也可以用于在期望的时间点测量B细胞活化和活细胞的绝对数量。


  1. 填写媒体
    RPMI 1640(含L-谷氨酰胺)
    1x Pen/Strep
  2. 排序缓冲区
    RPMI 1640(含L-谷氨酰胺+无酚红) 2%FBS
    1x Pen/Strep
    25 mM HEPES




  1. Cubas,RA,Mudd,JC,Savoye,AL,Perreau,M.,van Grevenynghe,J.,Metcalf,T.,Connick,E.,Meditz,A.,Freeman,GJ,Abesada-Terk, Jacobson,JM,Brooks,AD,Crotty,S.,Estes,JD,Pantaleo,G.,Lederman,MM和Haddad,EK(2013)。 不充分的T滤泡细胞帮助损害HIV感染期间的B细胞免疫力。 Med 19(4):494-499
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Cubas, R. A. and Haddad, E. K. (2014). T Follicular Helper Cell Coculture Assay. Bio-protocol 4(1): e1021. DOI: 10.21769/BioProtoc.1021.