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Ex vivo Human Antigen-specific T Cell Proliferation and Degranulation
人抗原特异性T淋巴细胞的体外纯化和脱粒   

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Abstract

Proliferative capacity and degranulation are important features of antigen-specific CD8+ T cells. By combining tetramer staining with a CFSE staining, we were able to enumerate the total number of antigen-specific T cells, as well as their number of divisions upon antigen-specific stimulation during a week. In addition, we performed restimulation of these cells, to analyze their ability to secrete cytolytic granules, visualized by CD107a staining.

Materials and Reagents

  1. Mouse-anti-human CD107a PE (clone H4A3) (BD Biosciences, catalog number: 555801 )
  2. Mouse-anti-human CD3 PE-Cy7 (clone UCHT1) (Biolegend, catalog number: 300420 )
  3. Mouse-anti-human CD8 Alexa-Fluor-700 (clone 3B5) (Life Technologies, Invitrogen™, catalog number: MCHD0824 )
    Note: The above antibodies have been tested by the author and may be substituted with the antibodies desired by users.
  4. CFSE (Molecular Probes Europe BV)
  5. Iscove’s Modified Dulbecco’s Medium (IMDM) (Life Technologies, Invitrogen™)
  6. Heat-inactivated fetal calf serum (FCS) (Integro)
  7. Human serum (HS) (Sanquin Blood Bank)
  8. Recombinant human IL-2 (Proleukin, Chiron)
  9. Recombinant human IL-15 (Immunotools, catalog number: 11340155 )
  10. Antigen-specific peptide (For example MiHA or CMV-peptide, Thinkpeptides)
  11. APC-conjugated antigen-specific tetramer (Kindly provided by Prof. dr. J. H. F. Falkenburg, Leiden University Medical Centre, the Netherlands)
  12. Sytox blue (Life Technologies, Invitrogen™, catalog number: S34857 )
  13. Mature monocyte-derived DC [cultured following the protocol described in Hobo et al. (2010)]
  14. FACS buffer (see Recipes)

Equipment

  1. Beckman Coulter Navios flow cytometer
  2. 24-wells plate
  3. 37 °C incubator

Procedure

  1. Antigen-specific T cell proliferation and degranulation
    1. Wash PBMC containing low percentages of antigen-specific T cells with 14 ml PBS.
    2. Centrifuge the cells for 5 min at 500 x g at room temperature (RT).
    3. Resuspend the cells in PBS to a concentration of 20 x 106 cells/ml, and add 2.5 μM CFSE at a 1:1 ratio (so in case of 500 μl cell suspension, add 500 μl 2.5 μM CFSE). The end concentration of CFSE will be 1.25 μM.
    4. Incubate the cells for 10 min at RT.
    5. Then, add pure FCS at a 1:1 ratio (so in case of 1 ml CFSE-cell suspension, add 1 ml FCS).
    6. After 2 min, wash the PBMC twice with 14 ml of IMDM/10% HS (centrifuge the cells for 5 min at 500 x g at RT), and count the cell number.
    7. Resuspend the PBMC to a concentration of 2 x 106 cells/ml in IMDM/ 10% HS.
    8. Plate 500 μl PBMC suspension per well of a 24-wells plate.
    9. Use mature monocyte-derived DC expressing the correct HLA-molecule for stimulation.
    10. Wash the DC with 14 ml PBS.
    11. Centrifuge the cells for 5 min at 500 x g at RT.
    12. Resuspend the DC subsequently in 500 μl IMDM containing 10 μM antigen-specific peptide.
    13. Incubate the DC for 30 min at 37 °C in the incubator.
    14. Add 14 ml PBS to the DC.
    15. Centrifuge the cells for 5 min at 500 x g at RT.
    16. Resuspend the DC in IMDM/10% HS to a concentration of 0.2 x 106 cells/ml.
    17. Add 500 μl DC suspension to each well containing CFSE-labeled PBMC.
    18. Culture the cells in the incubator at 37 °C, 5% CO2 and 95% humidity.
    19. After 5 days, remove from each well 500 μl supernatant and add 500 μl IMDM/10% HS containing 100 U/ml IL-2 and 10 ng/ml IL-15, so the end concentration in the wells will be 50 U/ml IL-2 and 5 ng/ml IL-15. Supernatants can be used for cytokine analysis.

  2. Overnight restimulation
    1. At day 7 of culture, 500 μl supernatant was removed.
    2. Add to each well 500 μl IMDM/10% HS containing 10 μM antigen-specific peptide, so the end concentration is 5 μM Antigen-specific peptide.
    3. In addition, add 6.5 μl CD107a-PE antibody to each well.
    4. Culture the cells overnight in the incubator at 37 °C, 5% CO2 and 95% humidity.
    5. The following day, harvest the cultured cells and count the cell numbers.
    6. Centrifuge the cells for 5 min at 500 x g at room temperature.
    7. Resuspend the cells in 100 μl IMDM/10% HS and add 1 μl APC-conjugated antigen-specific tetramer.
    8. Incubate the cells for 20 min in the dark, at RT.
    9. Then (without prior washing!), add 2.5 μl CD3 PE-Cy7 and 1 μl CD8 Alexa-Fluor-700.
    10. Incubate the cells for 30 min at ice, in the dark.
    11. Subsequently, wash cells with 1 ml FACS buffer.
    12. Centrifuge the cells for 5 min at 500 x g at RT.
    13. Resuspend the cells in 300 μl FACS buffer containing 0.1% Sytox blue solution.
    14. Analyze the samples on the Navios flow cytometer.
    15. Flow cytometrical analysis:
      1. Gate on the leukocytes (Forward/ Side scatter)


      2. Gate on viable cells (Sytox blue negative)


      3. Gate on cytotoxic T cells (CD3 positive, CD8 positive)


      4. Gate on tetramer-positive CD8+ T cells


      5. Analyze CFSE dilution pattern of tetramer-positive CD8+ T cells (NB. Use tetramer-negative cells for setting the gate of non-divided cells)


      6. Analyze CD107a-positivity within the tetramer-positive CD8+ T cells (NB. Use tetramer-negative cells for setting the gate of CD107a- cells)


Recipes

  1. FACS buffer (0.5 L)
    2.5 g BSA in 500 ml PBS

Acknowledgments

This protocol has been adapted from the publication by Hobo et al. (2012). We thank Rob Woestenenk for assistance in flow cytometry and Michel Kester for providing us with tetramers. This work was supported by grants from the Dutch Cancer Society (KWF 2008-4018) and Radboud University Nijmegen (UMCN 2007-34).

References

  1. Hobo, W., Maas, F., Adisty, N., de Witte, T., Schaap, N., van der Voort, R. and Dolstra, H. (2010). siRNA silencing of PD-L1 and PD-L2 on dendritic cells augments expansion and function of minor histocompatibility antigen-specific CD8+ T cells. Blood 116(22): 4501-4511.
  2. Hobo, W., Norde, W. J., Schaap, N., Fredrix, H., Maas, F., Schellens, K., Falkenburg, J. H., Korman, A. J., Olive, D., van der Voort, R. and Dolstra, H. (2012). B and T lymphocyte attenuator mediates inhibition of tumor-reactive CD8+ T cells in patients after allogeneic stem cell transplantation. J Immunol 189(1): 39-49.

简介

增殖能力和脱颗粒是抗原特异性CD8 + T细胞的重要特征。 通过将四聚体染色与CFSE染色组合,我们能够列举抗原特异性T细胞的总数,以及它们在一周内抗原特异性刺激时的分裂数。 此外,我们进行这些细胞的再刺激,以分析其分泌细胞溶解颗粒的能力,通过CD107a染色可视化。

材料和试剂

  1. 小鼠抗人CD107a PE(克隆H4A3)(BD Biosciences,目录号:555801)
  2. 小鼠抗人CD3 PE-Cy7(克隆UCHT1)(Biolegend,目录号:300420)
  3. 小鼠抗人CD8 Alexa-Fluor-700(克隆3B5)(Life Technologies,Invitrogen TM,目录号:MCHD0824)
    注意:上述抗体已经被作者测试,可能被用户需要的抗体替代。
  4. CFSE(Molecular Probes Europe BV)
  5. Iscove's Modified Dulbecco's Medium(IMDM)(Life Technologies,Invitrogen TM)
  6. 热灭活的胎牛血清(FCS)(Integro)
  7. 人血清(HS)(Sanquin Blood Bank)
  8. 重组人IL-2(Proleukin,Chiron)
  9. 重组人IL-15(Immunotools,目录号:11340155)
  10. 抗原特异性肽(例如 MiHA或CMV肽,Thinkpeptides)
  11. APC偶联的抗原特异性四聚体(由荷兰莱顿大学医学中心Dr.Fr.F.Falkenburg教授提供)
  12. Sytox blue(Life Technologies,Invitrogen TM,目录号:S34857)
  13. 成熟单核细胞衍生的DC [按照Hobo等人(2010)所述的方案培养]
  14. FACS缓冲区(参见配方)

设备

  1. Beckman Coulter Navios流式细胞仪
  2. 24孔板
  3. 37℃孵育器

程序

  1. 抗原特异性T细胞增殖和脱颗粒
    1. 用14ml PBS洗涤含有低百分比的抗原特异性T细胞的PBMC
    2. 在室温(RT)下以500×g离心细胞5分钟
    3. 将细胞在PBS中重悬浮至浓度为20×10 6个细胞/ml,并以1:1的比例加入2.5μMCFSE(因此在500μl细胞悬浮液的情况下,加入500μl2.5μM CFSE)。 CFSE的最终浓度为1.25μM
    4. 在室温下孵育细胞10分钟。
    5. 然后,以1:1的比例加入纯FCS(因此在1ml CFSE细胞悬浮液的情况下,加入1ml FCS)。
    6. 2分钟后,用14ml IMDM/10%HS洗涤PBMC两次(在室温下以500×g离心细胞5分钟),并计数细胞数。
    7. 在IMDM/10%HS中将PBMC重悬浮至浓度为2×10 6个细胞/ml。
    8. 在24孔板的每孔中加入500μlPBMC悬浮液
    9. 使用表达正确HLA分子的成熟单核细胞衍生的DC进行刺激
    10. 用14ml PBS洗涤DC。
    11. 在室温下以500×g离心细胞5分钟。
    12. 然后将DC重悬在500μl含有10μM抗原特异性肽的IMDM中
    13. 在孵育箱中37℃孵育DC 30分钟
    14. 向DC中加入14 ml PBS
    15. 在室温下以500×g离心细胞5分钟。
    16. 将DC在IMDM/10%HS中重悬浮至浓度为0.2×10 6个细胞/ml。
    17. 向每个含有CFSE标记的PBMC的孔中加入500μlDC悬浮液
    18. 在37℃,5%CO 2和95%湿度的培养箱中培养细胞。
    19. 5天后,从每个孔中取出500μl上清液,并加入500μl含有100U/ml IL-2和10ng/ml IL-15的IMDM/10%HS,因此孔中的终浓度为50U/ml IL-2和5ng/ml IL-15。 上清液可用于细胞因子分析

  2. 过夜再刺激
    1. 在培养的第7天,除去500μl上清液
    2. 向每个孔中加入500μl含有10μM抗原特异性肽的IMDM/10%HS,因此最终浓度为5μM抗原特异性肽。
    3. 另外,向每个孔中加入6.5μlCD107a-PE抗体
    4. 在37℃,5%CO 2和95%湿度的培养箱中培养细胞过夜。
    5. 第二天,收获培养的细胞并计数细胞数
    6. 在室温下以500×g离心细胞5分钟。
    7. 重悬细胞在100微升IMDM/10%HS和添加1微升APC结合的抗原特异性四聚体。
    8. 在黑暗中,室温下孵育细胞20分钟。
    9. 然后(无需预先洗涤!),加入2.5μlCD3 PE-Cy7和1μlCD8 Alexa-Fluor-700。
    10. 孵育细胞在冰上,在黑暗中30分钟。
    11. 随后,用1ml FACS缓冲液洗涤细胞
    12. 在室温下以500×g离心细胞5分钟。
    13. 将细胞重悬在300μl含有0.1%Sytox蓝色溶液的FACS缓冲液中
    14. 在Navios流式细胞仪上分析样品
    15. 流式细胞分析:
      1. 门对白细胞(向前/侧向散射)


      2. 门在活细胞(Sytox蓝色阴性)


      3. 门对细胞毒性T细胞(CD3阳性,CD8阳性)


      4. 在四聚体阳性CD8 + T细胞上的门


      5. 分析四聚体阳性CD8 + T细胞的CFSE稀释模式(NB。使用四聚体阴性细胞设置未分裂细胞的门)


      6. 分析四聚体阳性CD8 + sup + T细胞内的CD107a阳性(NB。使用四聚体阴性细胞设置CD107a细胞的门)


食谱

  1. FACS缓冲液(0.5L)
    2.5g BSA在500ml PBS中的溶液

致谢

该协议已经由Hobo等人的出版物(2012)修改。 我们感谢Rob Woestenenk在流式细胞术中的帮助和Michel Kester为我们提供四聚体。 这项工作是由赠款支持 来自荷兰癌症协会(KWF 2008-4018)和Radboud大学Nijmegen(UMCN 2007-34)。

参考文献

  1. Hobo,W.,Maas,F.,Adisty,N.,de Witte,T.,Schaap,N.,van der Voort,R。和Dolstra,H。 树突状细胞上的PD-L1和PD-L2的siRNA沉默增强了小的组织相容性抗原的扩增和功能 特异性CD8 + T细胞。血液 116(22):4501-4511。
  2. Hobo,W.,Norde,WJ,Schaap,N.,Fredrix,H.,Maas,F.,Schellens,K.,Falkenburg,JH,Korman,AJ,Olive,D.,van der Voort, ,H。(2012)。 B和T淋巴细胞衰减子介导同种异体干细胞移植后患者肿瘤反应性CD8 + T细胞的抑制 。 J Immunol 189(1):39-49
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hobo, W., Norde, W. and Dolstra, H. (2012). Ex vivo Human Antigen-specific T Cell Proliferation and Degranulation. Bio-protocol 2(23): e304. DOI: 10.21769/BioProtoc.304.
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