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Murine in vivo CD8+ T Cell Killing Assay
鼠体内 CD8+ T淋巴细胞杀菌分析   

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本实验方案简略版
Immunity
Aug 2013

Abstract

Antigen-specific killing ability of effector CD8+ T cells is critical for protective immunity against infection. Here, we describe in vivo cytotoxic T cell assay to examine effector function of antigen-specific CD8+ T cells. Mice infected with Listeria monocytogenes (L. monocytogenes) expressing chicken ovalbumin as a model antigen mount ovalbumin-specific CD8+ T cell responses. Effector CD8+ T cell function in vivo is determined by mixed transfer of OVA peptide-pulsed target cells with control target cells into the previously immunized mice. Difference in CFSE expression levels clearly marks two distinct populations: Antigen-pulsed target cells-CFSElow vs. unpulsed target cells-CFSEhi. The frequencies between antigen-pulsed target cells and control target cells are used as readouts of antigen-specific killing.

Materials and Reagents

  1. Splenocytes from a wild type mouse
  2. PBS (Thermo Fisher Scientific, catalog number: BP399-20 )
    Note: 10x solution, diluted to 1x in house in distilled water and sterilized by autoclave.
  3. RBC lysis buffer (eBioscience, catalog number: 00-4333-57 )
  4. HBSS without Ca2+ and Mg2+ (Life Technologies, Gibco®, catalog number: 14175-095 )
  5. RPMI-1640 medium (Life Technologies, Gibco®, catalog number: 11875-119 )
  6. Fetal bovine Serum (Atlanta Biologicals, catalog number: S11055H )
  7. Penicillin/streptomycin (Gemini Bio-Products, catalog number: F52M00E )
  8. L-Glutamine (Life Technologies, Gibco®, catalog number: 25030-081 )
  9. Trypan blue solution (Life Technologies, Gibco®, catalog number: 15250-061 )
  10. OVA257-264 synthetic peptide (Sigma-Aldrich, catalog number: S7951 )
  11. 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE) (Sigma-Aldrich, catalog number: 21888 )
  12. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D-8418 )
  13. Collagenase D (Sigma-Aldrich, catalog number: C-5138 )
  14. Percoll (Sigma-Aldrich, catalog number: P-1644 )
  15. Complete RPMI-1640 media (see Recipes)
  16. 100% percoll solution (see Recipes)

Equipment

  1. Centrifuge (Thermo Fischer Scientific, SorvallTM Legend RT )
  2. 37 °C water bath
  3. Hemocytometer
  4. 15 ml and 50 ml Falcon tubes
  5. 6 well plates (USA Scientific, CytoOne®, catalog number: CC7682-7506 )
  6. BD LSRII Flow Cytometer (BD)
  7. 70 µm cell strainer (BD Biosciences, Falcon®, catalog number: 352350 )
  8. 5 ml polystyrene round-bottom tubes with cell-strainer cap (BD Biosciences, Falcon®, catalog number: 352235 )
  9. 3 ml syringe (BD, catalog number: 14-823-435 )

Procedure

  1. Target cell preparation under sterile tissue culture conditions
    This step is for preparing peptide-pulsed target cells and stain cells with CFSE to distinguish peptide-pulsed target cells from control target cells.
    1.  OVA257-264 peptide-loading for the target cells.
      1. Splenocytes are RBC lysed followed by washing with PBS twice.
      2. Resuspend cells in RPMI-1640 complete medium.
      3. Count the mononuclear cells by trypan blue exclusion using a hemocytometer.
      4. Resuspend cells at 5 x 106/ml of RPMI-1640 complete medium.
      5. Divide the cells equally into two separate 50 ml Falcon tubes- one for peptide-pulsed target cells, and the other for unpulsed target cells.
      6. Add OVA257-264 peptide at 1 µl/ml from a 200 µM stock to peptide-pulsed target cells.
      7. Add an equivalent amount of PBS to the unpulsed target cells.
      8. Incubate the cells in a 37 °C water bath for 1 h.
      9. Wash cells twice with RPMI-1640 complete medium.
      10. Centrifuge the cells at 1,500 rpm for 3 min at 4 °C.
      11. Resuspend the cell pellet in HBSS.
    2. CFSE cell labeling under sterile tissue culture conditions.
      1. Count all live cells by trypan blue exclusion using hemocytometer.
      2. Resuspend the cells in HBSS at 5 x 107/ml.
      3. Thaw an aliquot of 5 mM stock CFSE solution.
      4. Make a fresh CFSElow stock solution by diluting 5 mM stock 1:10 in DMSO (a final concentration of 0.5 mM).
      5. Incubate the unpulsed target splenocytes with the higher concentration of CFSE (CFSEhigh): Add 1 µl of the 5 mM stock CFSE for each milliliter of unpulsed target cells (final concentration of 5 µM).
      6. Incubate the pulsed target splenocytes with the lower concentration of CFSE (CFSElow): Add 1 µl of the 0.5 mM stock CFSE for each milliliter of peptide-pulsed cells (final concentration of 0.5 µM).
      7. Pipette cells up and down to mix well and incubate in water bath for 10 min at 37 °C. Gently agitate the cells periodically.
      8. Add 10x the volume of pre-warmed RPMI-1640 complete medium to the CFSE-labeled cells to stop the reaction.
      9. Pellet cells at 1,500 rpm for 3 min at 4 °C.
      10. Remove the supernatant and resuspend the pellet in cold RPMI-1640 complete medium.
      11. Wash the cells two more times with cold RPMI-1640 complete medium.
      12. Count the mononuclear cells by trypan blue exclusion using a hemacytometer.
      13. Wash the cells with cold PBS.
      14. Resuspend each cell populations in PBS at 6.7 x 106/ml.
      15. Combine an equal volume (~equal numbers) of peptide-pulsed CFSElow cells with unpulsed CFSEhi cells and proceed with flow cytometry analysis (Figure 1a).

  2. Intravenous injection of target splenocytes
    To investigate OVA257-264-specific CD8+ T cell killing ability, peptide-pulsed and unpulsed target cells were mixed at a 1:1 ratio and transferred to the previously immunized mice.
    1. Recipient mice were infected with 5,000 colony-forming units (CFU) of Listeria monocytogenes expressing chicken ovalubmin (LM-OVA) 7 days intravenously before the CFSE-labeled cell injection.
    2. Inject intravenously 300 µl of the combined cell populations into the tail vein of each recipient. Each recipient should receive approximately 1 x 107 peptide-pulsed target cells combined with 1 x 107 unpulsed target cells.
    3. Wait for 4 h.

  3. Preparation of splenocytes and lymphocytes in the liver for flow cytometry analysis
    This step is analyzing antigen-specific killing ability in the liver and the spleen by flow cytometric analysis of CFSElow and CFSEhi cell populations.
    1. Prepare splenocytes for flow cytometry analysis.
      1.  Wash once with PBS, and 2-300 µl into a 5 ml round bottom tubes through the cell strainer.
    2. Isolate lymphocytes from the liver.
      1. Harvest livers from the recipients and place them on ice.
      2. Make a fresh collagenase D solution by diluting 20 mg/ml stock 1:20 in PBS (a final concentration of 1 mg/ml).
      3. Chop liver with a blade on a slide glass and transfer them into a 50 ml falcon tube.    
      4. Add 7 ml of collagenase D (1 mg/ml) and vortex well.
      5. Incubate in water bath for 30 min at 37 °C. Vortex every 15 min.
      6. Put tubes on ice and add supernatant on 70 µm filter on a well of a 6 well plate. Grind chunks of the chopped liver with flat portion of 3 ml syringe. Wash the tube with 5 ml of PBS and repeat grinding.
      7. Transfer them to a 50 ml Falcon tube and spin down at 2,000 rpm for 5 min at 4 °C.
      8. Make fresh 44% and 66% percoll solution: Make 44% final concentration by diluting 100% percoll in PBS, and make 66% final concentration by diluting 100% percoll in RPMI-1640 medium.
      9. Resuspend pellet in 7 ml of 44% percoll, and load them on 3 ml of 66% percoll in 15 ml tube.
        Note: The gradient separation is sensitive to agitation. Try not to shake the tube.
      10. Centrifuge at 3,000 rpm for 30 min at 4 °C without brake.
      11. Transfer the interphase lymphocytes to a new 15 ml tube.
      12. Pellet cells at 2,000 rpm for 5 min at 4 °C.
      13. Wash once with PBS, and transfer 2-300 µl into a 5 ml round bottom tubes through the cell strainer.
    3. Proceed to flow cytometry analysis with spleen and liver samples (Figure 1b).


      Figure 1. CFSE expression in antigen-pulsed target cells and unpulsed target cells. a. Peptide-pulsed CFSElow and unpulsed CFSEhi splenocytes were mixed at a 1:1 ratio before transferring to the recipients. b. The mixture of peptide-pulsed and unpulsed splenocytes was transferred into the mice predisposed with LM-OVA at day 7 post infection. The spleen and the liver of the recipients were harvested after 4 h to determine percentages of CFSElow and CFSEhi cells among CFSE+ cells.

Recipes

  1. Complete RPMI-1640 medium
    10% FBS
    1% Penicillin/streptomycin with L-Glutamine
  2. 100% percoll solution
    90% of percoll
    10% of 10x PBS

Notes

  1. The gradient separation is sensitive to agitation. Try not to shake the tube.

Acknowledgments

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.).

References

  1. Ingulli, E. (2007). Tracing tolerance and immunity in vivo by CFSE-labeling of administered cells. Methods Mol Biol 380: 365-376.
  2. 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.

简介

效应CD8 + T细胞的抗原特异性杀伤能力对于感染的保护性免疫是关键的。 在这里,我们描述了在体内细胞毒性T细胞测定来检查抗原特异性CD8 + T细胞的效应子功能。 用表达鸡卵白蛋白作为模型抗原的单核细胞增生性李斯特菌(单核细胞增生李斯特氏菌)感染的小鼠装载卵清蛋白特异性CD8 + T细胞应答。 通过将OVA肽脉冲的靶细胞与对照靶细胞混合转移到先前免疫的小鼠中来测定体内效应物CD8 + T细胞功能。 CFSE表达水平的差异清楚地标记两种不同的群体:抗原脉冲的靶细胞-CFSE 与未突发的靶细胞-FSE hi 。 抗原脉冲的靶细胞和对照靶细胞之间的频率用作抗原特异性杀伤的读数。

材料和试剂

  1. 来自野生型小鼠的脾细胞
  2. PBS(Thermo Fisher Scientific,目录号:BP399-20)
    注意:10×溶液,在蒸馏水中稀释至1×,并通过高压灭菌器灭菌。
  3. RBC裂解缓冲液(eBioscience,目录号:00-4333-57)
  4. 不含Ca 2+和Mg 2+的HBSS(Life Technologies,Gibco ,目录号:14175-095)
  5. RPMI-1640培养基(Life Technologies,Gibco ,目录号:11875-119)
  6. 胎牛血清(Atlanta Biologicals,目录号:S11055H)
  7. 青霉素/链霉素(Gemini Bio-Products,目录号:F52M00E)
  8. L-谷氨酰胺(Life Technologies,Gibco ,目录号:25030-081)
  9. 台盼蓝溶液(Life Technologies,Gibco ,目录号:15250-061)
  10. OVA 257-264合成肽(Sigma-Aldrich,目录号:S7951)
  11. 5(6) - 羧基荧光素二乙酸酯N-琥珀酰亚胺酯(CFSE)(Sigma-Aldrich,目录号:21888)
  12. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D-8418)
  13. 胶原酶D(Sigma-Aldrich,目录号:C-5138)
  14. Percoll(Sigma-Aldrich,目录号:P-1644)
  15. 完成RPMI-1640介质(参见配方)
  16. 100%percoll溶液(见配方)

设备

  1. 离心机(Thermo Fischer Scientific,Sorvall Legend RT)
  2. 37°C水浴
  3. 血细胞计数器
  4. 15 ml和50 ml Falcon管
  5. 6孔板(USA Scientific,CytoOne ,目录号:CC7682-7506)
  6. BD LSRII流式细胞仪(BD)
  7. 70μm细胞滤器(BD Biosciences,Falcon ,目录号:352350)
  8. 具有细胞过滤帽的5mL聚苯乙烯圆底管(BD Biosciences,Falcon ,目录号:352235)
  9. 3ml注射器(BD,目录号:14-823-435)

程序

  1. 无菌组织培养条件下的靶细胞制备
    该步骤用于使用CFSE制备肽脉冲的靶细胞和染色细胞,以区分肽脉冲的靶细胞与对照靶细胞。
    1.   OVA <257> 257> 靶向细胞的肽负载。
      1. 脾细胞被RBC裂解,然后用PBS洗涤两次。
      2. 将细胞重悬在RPMI-1640完全培养基中
      3. 使用血细胞计数器通过台盼蓝排除计数单核细胞
      4. 以5×10 6个/ml的RPMI-1640完全培养基重悬细胞。
      5. 将细胞平均分成两个单独的50毫升Falcon管 - 一个   肽脉冲的靶细胞,另一种用于未脉冲的靶细胞。
      6. 将来自200μM储液的OVA 257-264肽以1μl/ml加入肽脉冲的靶细胞。
      7. 添加等量的PBS到未突发的靶细胞
      8. 孵育细胞在37℃水浴1小时。
      9. 用RPMI-1640完全培养基洗涤细胞两次。
      10. 在4℃下以1,500rpm离心细胞3分钟。
      11. 在HBSS中重悬细胞沉淀。
    2. 在无菌组织培养条件下的CFSE细胞标记。
      1. 使用血细胞计数器通过台盼蓝排除计数所有活细胞
      2. 以5×10 7 /μL/ml重悬细胞于HBSS中
      3. 解冻5mM储备CFSE溶液的等分试样
      4. 通过在DMSO中稀释5mM储备液(1:10)(最终浓度为0.5mM)制备新鲜的CFSE 低储液。
      5. 孵育具有较高浓度的未刺激的靶脾细胞   的CFSE(CFSE ):每毫升添加1μl的5mM储备CFSE   的未刺激靶细胞(最终浓度为5μM)
      6. 孵育具有较低浓度的脉冲目标脾细胞 CFSE(CFSE ):将1μl的0.5mM储备CFSE加入每毫升   肽脉冲的细胞(最终浓度为0.5μM)
      7. 吸管 细胞上下混合,在水浴中孵育10分钟 37℃。 定期轻轻搅拌细胞。
      8. 加入10倍体积的预热RPMI-1640完全培养基到CFSE标记的细胞停止反应。
      9. 颗粒细胞在4℃下以1,500rpm离心3分钟
      10. 取出上清液,并将沉淀重悬在冷RPMI-1640完全培养基中
      11. 用冷RPMI-1640完全培养基洗涤细胞两次以上。
      12. 使用血细胞计数器通过台盼蓝排除计数单核细胞
      13. 用冷PBS洗涤细胞。
      14. 以6.7×10 6 /μL/ml重悬在PBS中的每个细胞群体
      15. 将等体积(〜等数量)的肽脉冲的CFSE低级细胞与未刺激的CFSE sup细胞组合,并进行流式细胞术 分析(图1a)。

  2. 静脉注射靶脾细胞
    为了研究OVA 257-264特异性CD8 + T细胞杀伤能力,将肽脉冲的和未脉冲的靶细胞以1:1的比例混合并转移至先前免疫的 老鼠。
    1. 用表达鸡卵清蛋白(LM-OVA)的5,000个集落形成单位(CFU)的单核细胞增生李斯特氏菌感染受体小鼠7天 在CFSE标记的细胞注射前静脉内
    2. 注入 静脉内300微升的组合细胞群体进入尾静脉   的每个收件人。 每个受体应该接受约1×10 7个肽脉冲的靶细胞与1×10 7个未脉冲靶细胞的组合。
    3. 等待4小时。

  3. 用于流式细胞术分析的肝脏中脾细胞和淋巴细胞的制备
    该步骤通过对CFSE low 和CFSE hi细胞群体的流式细胞术分析来分析肝脏和脾脏中的抗原特异性杀伤能力。
    1. 准备脾细胞进行流式细胞术分析。
      1.  用PBS洗涤一次,并通过细胞过滤器将2-300μl加入5ml圆底管中。
    2. 从肝脏分离淋巴细胞。
      1. 从收件人收获肝脏,并将它们放在冰上
      2. 通过在PBS中稀释20mg/ml储备液1:20(最终浓度为1mg/ml)制备新鲜的胶原酶D溶液。
      3. 用玻片上的刀片切碎肝脏,并将其转移到50ml的falcon管中。  
      4. 加入7毫升胶原酶D(1毫克/毫升),并充分涡旋
      5. 在37℃下在水浴中孵育30分钟。 每15分钟涡旋一次。
      6. 将管置于冰上,并在6孔的孔上加入70μm滤器上的上清液 孔板。 研磨大块的切碎的肝脏与平部分3毫升 注射器。 用5ml PBS洗涤管,并重复磨削。
      7. 将其转移到50ml Falcon管中,在4℃下以2,000rpm离心5分钟
      8. 制备新鲜的44%和66%的percoll溶液:使最终浓度为44% 通过稀释在PBS中的100%percoll,并使66%的最终浓度 在RPMI-1640培养基中稀释100%percoll。
      9. 重悬在7毫升的44%percoll丸,并加载到3毫升的66%percoll在15毫升管中。
        注意:梯度分离对搅拌敏感。 尽量不要摇动管。
      10. 在4℃下以3,000rpm离心30分钟,无制动
      11. 将间期淋巴细胞转移到一个新的15毫升管
      12. 将细胞在4℃以2,000rpm离心5分钟
      13. 用PBS洗涤一次,并通过细胞过滤器转移2-300μl到5ml圆底管。
    3. 进行脾和肝样品的流式细胞术分析(图1b)。


      图   1.在抗原脉冲的靶细胞和未刺激的靶中的CFSE表达 。 肽脉冲的CFSE 和未突变的CFSE hi 脾细胞 在转移到接受者之前以1:1的比例混合。 b。 的 将肽脉冲和未脉冲的脾细胞的混合物转移入 在感染后第7天,LM-OVA易感染小鼠。 脾和   在4小时后收获受体的肝以确定 在CFSE + 细胞中CFSE 和CFSE hi 细胞的百分比。

食谱

  1. 完成RPMI-1640培养基
    10%FBS
    1%青霉素/链霉素和L-谷氨酰胺
  2. 100%percoll溶液
    90%的percoll
    10%的10x PBS

笔记

  1. 梯度分离对搅拌敏感。 尽量不要摇晃管子。

致谢

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。

参考文献

  1. Ingulli,E。(2007)。 通过对所施用细胞的CFSE标记,跟踪体内耐受性和免疫力 。 Methods Mol Biol 380:365-376。
  2. 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 39(2):286-297。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kim, M. V., Ouyang, W., Liao, W., Zhang, M. Q. and Li, M. O. (2014). Murine in vivo CD8+ T Cell Killing Assay. Bio-protocol 4(13): e1172. DOI: 10.21769/BioProtoc.1172.
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