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Mouse CD8+ T Cell Migration in vitro and CXCR3 Internalization Assays
体外小鼠CD8+ T细胞迁移和CXCR3内化测定   

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Abstract

Chemokines are molecules that regulate the positioning of cells during homeostasis and inflammation. CXCL10 is an interferon-induced chemokine that attracts cells that express the chemokine receptor CXCR3 on their surface. CXCL10 expression is often induced upon inflammation and guides lymphocytes, such as T and NK cells, into the injured tissues. Notably, CXCL10 binding to CXCR3 induces receptor internalization and, therefore, low CXCR3 levels in cells positive for CXCR3 expression can be indicative of chemokine signaling.

Here, we describe an in vitro method to evaluate the ability of murine CD8+ T cells to migrate towards recombinant murine CXCL10; and a flow cytometry assay to measure CXCR3 expression levels at the surface of T cells, after exposure to different doses of chemokine.

Keywords: Chemokines(趋化因子), Lymphocytes(淋巴细胞), Migration(迁移), Transwell(跨室), Receptor-internalization(受体内化)

Background

Chemokine-mediated T cell trafficking is an important process during homeostasis and inflammation. Activated CD8+ T cells express chemokine receptors, such as CXCR3, allowing them to migrate towards the chemokines CXCL9, 10 and 11, often upregulated at the injured tissue. The evaluation of molecular cues that modulate T cell migration is important to understand the biology behind their functions but the complex mechanisms operating in vivo are sometimes hard to deconvolve. Here, we provide detailed information on an in vitro method to evaluate chemokine functions on CD8+ T cells, focusing on CXCL10-mediated chemo-attraction and CXCR3 internalization. We use antigen-specific transgenic CD8+ T cells that can be easily expanded and activated in vitro, therefore providing enough number of phenotypically identical lymphocytes (e.g., high chemokine-receptor expression on their surface), required to perform an assay using enough replicates for biologically significant observations and statistical analysis. The cells used per assay originate from one single animal, therefore accounting for reduction of animal usage. This assay is combined with flow cytometry analysis, permitting simultaneous evaluation of 1) number of migrating CD8+ T cells; and 2) phenotypic characterization of chemokine receptor levels on their surface.

Materials and Reagents

  1. 6 well plate (Fisher Scientific, catalog number: 08-772-33 )
  2. Sterile pipettes  
    5 ml (Corning, catalog number: 4051 )
    10 ml (Corning, catalog number: 4101 )
    25 ml (Corning, catalog number: 4251 )
  3. 70 μm cell strainer (Corning, catalog number: 431751 )
  4. 5 ml syringes, without needle (BD, catalog number: 309646 )
  5. Sterile conical tubes
    15 ml (Corning, catalog number: 352099 )
    50 ml (Corning, catalog number: 352098 )
  6. 24 well plate (Corning, catalog number: 353226 )
  7. Corning® HTS Transwell® 96 well permeable supports, 5.0 μm pore size (Corning, catalog number: 3387 )
  8. 96 well plates, U bottom (Fisher Scientific, catalog number: 12-565-500 )
  9. 1.5 ml Eppendorf tubes (Fisher Scientific, catalog number: 05-408-129 )
  10. Flow cytometry tubes (Round-Bottom Polystyrene Tubes, Corning, catalog number: 352052 )
  11. CD8+ T cell TCR transgenic mouse, such as OT-1 (C57BL/6-Tg[TcraTcrb]1100Mjb/J, THE JACKSON LABORATORIES, catalog number: 003831 ) or Pmel-1 (B6.Cg-Thy1a/Cy Tg[TcraTcrb]8Rest/J, THE JACKSON LABORATORIES, catalog number: 005023 ) mouse (age 8-12 weeks)
  12. TCR-specific peptide
    If using OT-1 cells – SIINFEKL (AnaSpect, catalog number: AS-60193-1 )
    If using Pmel-1 cells – human gp100 (AnaSpec, catalog number: AS-62589 )
  13. 1x PBS (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 )
  14. Recombinant human IL-2 (Miltenyi Biotec, catalog number: 130-097-742 )
  15. Recombinant murine CXCL10 (Peprotech, catalog number: 250-16 )
  16. AccuCheck Counting Beads reagent (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: PCB100 )
  17. RPMI 1640 (Thermo Fisher Scientific, GibcoTM, catalog number: 11875093 )
  18. Fetal bovine serum (Seradigm, catalog number: 1500-100 )
  19. Non-essential amino acids (Thermo Fisher Scientific, GibcoTM, catalog number: 11140-050 )
  20. Sodium pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11360-070 )
  21. HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630-080 )
  22. Beta-mercaptoethanol (Thermo Fisher Scientific, GibcoTM, catalog number: 21985-023 )
  23. Gentamycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15710-064 )
  24. Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14025092 )
  25. Protease-free BSA (GE Healthcare, catalog number: SH30574.02 )
  26. Mouse CD16/CD32 Fc Blocking antibodies (BD, catalog number: 553142 )
  27. LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: L34957 ). Reconstitute each vial with 50 μl of DMSO provided with the kit
  28. Pacific Blue-conjugated anti-mouse CD3 (clone 145-2C11) (BioLegend, catalog number: 100334 )
  29. APC-conjugated CD8 (clone 53-6.7) (BD, catalog number: 553035 )
  30. PE-conjugated CXCR3 (clone CXCR3-173) (BioLegend, catalog number: 126506 )
  31. R10 media (see Recipes)
  32. Migration media (see Recipes)
  33. Blocking buffer (see Recipes)
  34. Staining mix (see Recipes)

Equipment

  1. Sterile dissection tools such as small scissors and tweezers (tools can be purchased from Carolina or Sigma, for example)
  2. Bench-top refrigerated centrifuge (such as Thermo Fisher Scientific, Thermo ScientificTM, model: Thermo ScientificTM SorvallTM LegendTM X1 )
  3. Incubator (37 °C, 5% CO2, 95% humidity, such as Thermo Fisher Scientific, Thermo ScientificTM, model: Forma Steri-Cycle CO2 incubator )
  4. Tissue culture hood (Biosafety cabinet, such as The Baker Company, model: SterilGARD e3 )
  5. Bench-top orbital shaker (such as Thermo Fisher Scientific, model: Thermo Barnstead 4625 Titer Plate Shaker , catalog number: THERMO BARNSTEAD 4625 TITER PLATE SHAKER-1478)
  6. Optical microscope (such as Leica, model: DM IL LED )
  7. Neubauer cell counting chamber (such as InCyto, catalog number: DHC-N01 )
  8. Flow cytometer (such as the BD, model: LSRFortessa )

Software

  1. Flow cytometer software: DIVA
  2. Computer analysis software: FlowJo (Treestar)

Procedure

  1. Obtaining CXCR3-expressing CD8+ T cells
    Note: This step of the protocol explains how to generate enough numbers of CD8+ T cells used in the subsequent steps.
    1. Sacrifice one OT-1 or Pmel-1 transgenic mouse.
    2. Collect spleen in sterile conditions using sharp scissors and tweezers.
    3. Place spleen in a well of a sterile 6 well plate, containing 2 ml of sterile ice cold 1x PBS.
    4. Mash spleen using the back part of a 5 ml syringe plunger.
    5. Collect cells with a 5 ml pipette (pipette up and down at least 3 times in order to homogenize the cell suspension) and pass them through a 70 μm cell strainer, into a 50 ml sterile conical tube.
    6. Wash filter with 7 ml of ice cold sterile 1x PBS and collect. Wash it into the same conical tube.
    7. Centrifuge cells at 350 x g, 5 min, 4 °C.
    8. Discard supernatant and resuspend cells in 10 ml of R10.
    9. Add 1 nM of TCR-specific peptide (SIINFEKL or human gp100, respectively if you use an OT-1 transgenic or Pmel-1 transgenic mouse).
      Note: The TCR-specific peptide will initiate T cell activation.
    10. Incubate at room temperature, in a bench-top orbital shaker with agitation for 1 h.
      Note: Set up shaker speed so the liquid inside the conical tube is moving – speed 3 or 4 in the orbital shaker indicated in the Equipment section.
    11. Add 10 ml of fresh R10 (room temperature) and centrifuge cells at 350 x g, 5 min, 4 °C.
    12. Discard supernatant and resuspend cells in 50 ml of fresh R10 (room temperature), supplemented with 30 U/ml of recombinant human IL-2.
      Note: IL-2 promotes T cell expansion and survival.
    13. Place cells in a 24 well plate (2 ml per well) and place plate in an incubator at 37 °C, 5% CO2, 95% humidity.
      Note: There is no need to isolate CD8+ T cells during this procedure. The percentage of CD8+ T cells found in a spleen of a TCR transgenic mouse is approximately 10-20% of total hematopoietic cells, and they will expand during the next steps.
    14. Split cells every two to three days by collecting 500 μl of cell suspension from each well and placing them in a new 24 well plate, filled with 1.5 ml of fresh R10 (room temperature), supplemented with 30 U/ml of recombinant human IL-2 (dilution of cells is 1 to 4).
    15. After 6-7 days (about 2 cell splits), the cells collected should be over 90% CD8+ T cells and harbor high CXCR3 expression on their surface (please refer to the flow cytometry protocol below for evaluation of CXCR3 expression).
      Note: By the end of the expansion, each plate should have at least 10 million CD8+ T cells.

  2. In vitro migration assay
    Note: This step of the protocol explains how to evaluate the ability of CXCL10 to induce migration of CD8+ T cells generated in Procedure A.
    1. Collect activated CD8+ T cells (around 45 ml total volume per plate) in a 50 ml conical tube and spin at 350 x g, 5 min, 4 °C. Cells are collected by pipetting up and down 3-5 times per well.
    2. Discard supernatant and resuspend cells in 5 ml of migration media.
    3. Count cells.
    4. Adjust volume so you have 5 x 105 cells/100 μl of migration media.
    5. Remove transwell insert from the Transwell 96 well plate, exposing the wells from the receiving plate.
    6. Prepare dilutions of chemokine, in migration media, and add them to the wells of the receiving plate. We advice to do triplicates for each condition.
      Note: Final volume in the receiving plate is 200 μl. Please refer to the notes at the end of this step of the protocol for suggested dilutions of CXCL10.
    7. Carefully place the transwell onto the receiving plate. The liquid from each well will be in contact with the transwell.
    8. Carefully add 100 μl of CD8+ T cell suspension (5 x 105 cells) to each transwell. Close the plate and place in the incubator at 37 °C, 5% CO2, 95% humidity for 1 h and 30 min.
    9. After the incubation time, carefully remove the transwell plate away from the receiving plate.
    10. Collect the cells from the wells of the receiving plate (by pipetting up and down 3-5 times) onto a new 96-well U-bottom plate. We advise that this step is performed with a multichannel pipet.
    11. Spin the 96 U bottom plate at 350 x g, 2 min and discard supernatant.
    12. Resuspend cells with blocking buffer (50 μl per well). Incubate for 20 min, 4 °C in the dark.
    13. Add 150 μl of 1x PBS, spin the plate at 350 x g, 2 min and discard supernatant.
    14. Resuspend cells with staining mix (50 μl per well). Incubate for 30 min, 4 °C in the dark.
    15. Add 150 μl of 1x PBS, spin the plate at 350 x g, 2 min and discard supernatant.
    16. Resuspend cells in 200 μl of 1x PBS.
    17. Transfer cells to flow cytometry tubes.
    18. Add 10 μl of counting beads to each tube and run samples in the flow cytometer. Acquire at least 2,000 events on the fluorescent beads gate and 2,000 events on the CD3+CD8+ T cell gate, as 1,000 events is the minimum number of events required for accurate cell counting using the AccuCheck Counting Beads Kit.
      Note: In cases where T cell migration is residual (e.g., conditions without chemoattractant) it might be difficult to reach the required number of events on the T cell gate, but make sure you get the maximum events possible, by running the entire sample.
    19. Analysis of flow cytometry data is done using the Flow Jo software.
    Note: Concentrations of murine CXCL10 that induced migration of activated CD8+ T cells are in between 10 ng/ml and 1,000 ng/ml. We recommend performing 2 fold dilutions. It is very important to include conditions where no chemokine is added, in order to determine non-chemokine induced T cell migration (background of the assay). The calculation of migrating cells using this method is done accordingly to the instructions provided with the AccuCheck Counting Beads.

  3. CXCR3 internalization assay
    Note: This step of the protocol explains how to evaluate the ability of CXCL10 to induce internalization of the chemokine receptor CXCR3, expressed at the surface of CD8+ T cells generated in the Procedure A.
    1. Collect activated CD8+ T cells in a 50 ml conical tube and spin at 350 x g, 5 min, 4 °C.
    2. Discard supernatant and resuspend cells in 5 ml of migration media.
    3. Count cells.
    4. Adjust volume so you have 5 x 105 cells/100 μl of migration media.
    5. Plate cells in a 96-well U-bottom well plate (100 μl per well).
    6. Add 100 μl of migration media supplemented with different concentrations of murine CXCL10 (200 μl total volume per well). Use the same concentration range as in Procedure B. Alternatively, you can do 10 fold dilutions, including 1,000; 100; 10; and 1 ng/ml.
    7. Place plate in the incubator at 37 °C 5% CO2 95% humidity for 1 h and 30 min.
      Note: The time point for this assay (1 h and 30 min) was chosen to match with the time point used in migration assay, described in Procedure B of this protocol. For new chemotatic stimuli, we recommend a time course study to be done (e.g., evaluation of chemokine-receptor internalization after different time points of incubation with chemokines).
    8. After the incubation time, spin the plate at 350 x g, 2 min and discard supernatant.
    9. Resuspend cells with blocking buffer (50 μl per well). Incubate for 20 min, 4 °C in the dark.
    10. Add 150 μl of cold 1x PBS, spin the plate at 350 x g, 2 min and discard supernatant.
    11. Resuspend cells with staining mix (50 μl per well). Incubate for 30 min, 4 °C in the dark.
    12. Add 150 μl of cold 1x PBS and resuspend them in 200 μl of 1x PBS.
    13. Transfer cells to flow cytometry tubes and acquire at least 10,000 events per tube.
    14. Analysis of flow cytometry data is done using the Flow Jo software.
    Note: Evaluation of CXCR3 internalization can be done independently or as a part of the CD8+ T cell migration assay. Please see Data analysis section for an example on how CXCR3 levels can be determined in migrating CD8+ T cells collected from the lower chamber of the transwell plate.

Data analysis

After exclusion of debris and dead cells (Aqua+ cells) the flow cytometry plots are visualized as represented in Figure 1. For calculation of the number of T cells that migrated into the lower chamber, gate on CD8+ cells (red gate) and on fluorescent beads (blue gate) and follow the instruction for calculation of cell numbers by the provider of the beads. For evaluation of CXCR3 levels on the surface of T cells, plot gated CD8+ T cells as histograms (Figure 2). You can obtain the values of mean fluorescence intensity (MFI) on the FlowJo software and plot them as graphs as well. It is recommended to run at least 3 technical replicates, per experiment.


Figure 1. Example of flow cytometry plots corresponding to the cells collected from the receiving chambers of the transwell plate, after exposure to migration media (left plot) or migration media supplemented with 1,000 ng/ml of recombinant murine CXCL10 (right plot). Cells were stained and fluorescent counting beads (gated in blue) were added to the cells as described in the Procedure B section. Debris and Aqua+ (dead) cells were excluded before this representation. Live CD3+CD8+ T cells are gated in red. APC: Allophycocyanin.


Figure 2. Example of flow cytometry histograms representing CXCR3 staining on CD3+CD8+ T cells collected from the receiving chambers of the transwell plate, after exposure to migration media (black) or migration media supplemented with 1,000 ng/ml of recombinant murine CXCL10 (red). % of max: percentage of maximum staining intensity; PE: R-Phycoerythrin.

Recipes

  1. R10 media – Store at 4 °C for 1 month
    RPMI 1640
    10% fetal bovine serum
    0.1 mM non-essential amino acids
    1 mM sodium pyruvate
    10 mM HEPES
    70 μM beta-mercaptoethanol
    23 μg/ml gentamycin
  2. Migration media – Prepare fresh for each experiment
    1x HBSS
    0.1% protease-free BSA
  3. Blocking buffer – Use 50 μl per condition, prepare fresh for each experiment
    1x PBS
    Mouse CD16/CD32 Fc Blocking antibodies (used at 1 to 50 dilution)
    Fixable Aqua Dead Cell Stain reagent (used at 1 to 500 dilution)
  4. Staining mix – Use 50 μl per condition, prepare fresh for each experiment
    1x PBS
    Pacific Blue-conjugated anti-mouse CD3 (clone145-2C11, used at 1 to 100 dilution)
    APC-conjugated CD8 (clone 53-6.7, used at 1 to 400 dilution)
    PE-conjugated CXCR3 (clone CXCR3-173, used at 1 to 100 dilution)

Acknowledgments

When using this protocol, please refer to Barreira da Silva et al. (2015) Nat Immunol. Funding was provided by the Pasteur-Roux post-doctoral fellowship (RBdS), the Ligue Contre le Cancer and the Fondation ARC pour la recherche sur le cancer (MLA) and the French government’s Invest in the Future Program, managed by the Agence Nationale de la Recherche (LabEx Immuno-Onco [RBdS, MLA]). Animal experimental protocols were approved by the comité d’éthique pour l’expérimentation animale (The ethics committee for animal experimentation) Paris.

References

  1. Barreira da Silva, R., Laird, M. E., Yatim, N., Fiette, L., Ingersoll, M. A. and Albert, M. L. (2015). Dipeptidylpeptidase 4 inhibition enhances lymphocyte trafficking, improving both naturally occurring tumor immunity and immunotherapy. Nat Immunol 16(8): 850-858.

简介

趋化因子是调节体内平衡和炎症期间细胞定位的分子。 CXCL10是干扰素诱导的趋化因子,其吸引在其表面上表达趋化因子受体CXCR3的细胞。 CXCL10表达通常在炎症诱导并引导淋巴细胞如T和NK细胞进入受损组织。值得注意的是,CXCL10与CXCR3结合诱导受体内化,因此CXCR3表达阳性细胞中的CXCR3水平降低可能是趋化因子信号传导的指示。
 这里,我们描述体外方法来评估鼠CD8 + T细胞向重组鼠CXCL10迁移的能力;以及暴露于不同剂量的趋化因子后在T细胞表面测量CXCR3表达水平的流式细胞术测定。

背景 趋化因子介导的T细胞运输是稳态和炎症期间的重要过程。活化的CD8 + T细胞表达趋化因子受体,例如CXCR3,允许它们向趋化因子CXCL9,10和11迁移,通常在损伤组织上上调。调节T细胞迁移的分子线索的评估对于了解其功能背后的生物学非常重要,但是在体内运行的复杂机制有时难以去卷积。在这里,我们提供有关体外方法的详细信息,以评估CD8 + T细胞上的趋化因子功能,重点是CXCL10介导的化学吸引和CXCR3内化。我们使用可以容易地在体外扩增和活化的抗原特异性转基因CD8 +细胞,因此提供足够数量的表型相同的淋巴细胞(例如, >,表面上的高趋化因子受体表达),需要使用足够的重复进行生物学显着性观察和统计分析的测定。每次测定使用的细胞来自一只单一动物,因此减少了动物使用量。将该测定与流式细胞术分析结合,允许同时评估1)迁移的CD8 +细胞的数量;和2)表面上趋化因子受体水平的表型表征。

关键字:趋化因子, 淋巴细胞, 迁移, 跨室, 受体内化

材料和试剂

  1. 6孔板(Fisher Scientific,目录号:08-772-33)
  2. 无菌移液器
    5毫升(康宁,目录号:4051)
    10毫升(康宁,目录号:4101)
    25毫升(康宁,目录号:4251)
  3. 70μm细胞过滤器(Corning,目录号:431751)
  4. 5毫升注射器,无针(BD,目录号:309646)
  5. 无菌锥管
    15毫升(康宁,目录号:352099)
    50毫升(康宁,目录号:352098)
  6. 24孔板(Corning,目录号:353226)
  7. Corning ® HTS Transwell ® 96孔渗透性支撑体,孔径5.0μm(Corning,目录号:3387)
  8. 96孔板,U底(Fisher Scientific,目录号:12-565-500)
  9. 1.5ml Eppendorf管(Fisher Scientific,目录号:05-408-129)
  10. 流式细胞仪(圆底聚苯乙烯管,Corning,目录号:352052)
  11. CD8 + T细胞TCR转基因小鼠,例如OT-1(C57BL/6-Tg [TcraTcrb] 1100Mjb/J,THE JACKSON LABORATORIES,目录号:003831)或Pmel-1(B6.Cg -Thy1a/Cy Tg [TcraTcrb] 8Rest/J,JACKSON LABORATORIES,目录号:005023)小鼠(8-12周龄)
  12. TCR特异性肽
    如果使用OT-1细胞 - SIINFEKL(AnaSpect,目录号:AS-60193-1)
    如果使用Pmel-1细胞 - 人gp100(AnaSpec,目录号:AS-62589)
  13. 1x PBS(Thermo Fisher Scientific,Gibco TM,目录号:14190144)
  14. 重组人IL-2(Miltenyi Biotec,目录号:130-097-742)
  15. 重组鼠CXCL10(Peprotech,目录号:250-16)
  16. AccuCheck Counting Beads试剂(Thermo Fisher Scientific,Molecular Probes TM,目录号:PCB100)
  17. RPMI 1640(Thermo Fisher Scientific,Gibco TM,目录号:11875093)
  18. 胎牛血清(Seradigm,目录号:1500-100)
  19. 非必需氨基酸(Thermo Fisher Scientific,Gibco TM,目录号:11140-050)
  20. 丙酮酸钠(Thermo Fisher Scientific,Gibco TM,目录号:11360-070)
  21. HEPES(Thermo Fisher Scientific,Gibco TM ,目录号:15630-080)
  22. β-巯基乙醇(Thermo Fisher Scientific,Gibco TM,目录号:21985-023)
  23. 庆大霉素(Thermo Fisher Scientific,Gibco TM,目录号:15710-064)
  24. Hank的平衡盐溶液(HBSS)(Thermo Fisher Scientific,Gibco TM,目录号:14025092)
  25. 无蛋白酶BSA(GE Healthcare,目录号:SH30574.02)
  26. 小鼠CD16/CD32 Fc阻断抗体(BD,目录号:553142)
  27. LIVE/DEAD ®可固定Aqua死细胞染色试剂盒,用于405nm激发(Thermo Fisher Scientific,Molecular Probes TM,目录号:L34957)。使用试剂盒
    提供的50μlDMSO重构每个小瓶
  28. 太平洋蓝缀合的抗小鼠CD3(克隆145-2C11)(BioLegend,目录号:100334)
  29. APC缀合的CD8(克隆53-6.7)(BD,目录号:553035)
  30. PE缀合的CXCR3(克隆CXCR3-173)(BioLegend,目录号:126506)
  31. R10媒体(见配方)
  32. 移民媒体(见食谱)
  33. 阻塞缓冲区(见配方)
  34. 染色混合(见配方)

设备

  1. 无菌解剖工具如小型剪刀和镊子(工具可以从卡罗莱纳州或西格玛购买)
  2. 台式冷冻离心机(例如Thermo Fisher Scientific,Thermo Scientific TM ,型号:Thermo Scientific TM Sorvall TM Legend /sup> X1)
  3. 孵育器(37℃,5%CO 2,95%湿度,例如Thermo Fishes Scientific,Thermo Scientific,Thermo Scientific,型号:Forma Steri-Cycle CO 2 孵化器)
  4. 组织文化罩(生物安全柜,如贝克公司,型号:SterilGARD e3)
  5. 台式轨道摇床(如Thermo Fisher Scientific,型号:Thermo Barnstead 4625 Titer Plate Shaker,目录号:THERMO BARNSTEAD 4625 TITER PLATE SHAKER-1478)
  6. 光学显微镜(如徕卡,型号:DM IL LED)
  7. Neubauer细胞计数室(如InCyto,目录号:DHC-N01)
  8. 流式细胞仪(如BD,型号:LSRFortessa)

软件

  1. 流式细胞仪软件:DIVA
  2. 计算机分析软件:FlowJo(Treestar)

程序

  1. 获得CXCR3表达CD8 + T细胞
    注意:本协议的这一步骤解释了如何在随后的步骤中生成足够数量的CD8 + T细胞。
    1. 牺牲一个OT-1或Pmel-1转基因小鼠。
    2. 使用锋利的剪刀和镊子在无菌条件下收集脾脏。
    3. 将脾脏置于无菌6孔板的孔中,含有2ml无菌冰冷的1x PBS
    4. 使用5ml注射器柱塞的背部进行麻疹脾脏。
    5. 用5ml移液管收集细胞(移液管至少3次,以使细胞悬浮液均匀化),并将其通过70μm的细胞过滤器,放到50ml无菌锥形管上。
    6. 用7毫升冰冷的无菌1x PBS清洗过滤器,并收集到同一锥形管上
    7. 以350 x g,5分钟,4℃离心细胞。
    8. 弃去上清并将细胞重悬于10ml的R10中
    9. 加入1nM TCR特异性肽(SIINFEKL或人gp100,如果您使用OT-1转基因或Pmel-1转基因小鼠)。
      注意:TCR特异性肽将启动T细胞活化。
    10. 在室温下孵育,在台式轨道摇床中搅拌1小时。
      注意:设置振动器速度,使锥形管内的液体在设备部分所示的轨道振动筛中移动 - 速度为3或4。
    11. 加入10ml新鲜的R10(室温),并以350 x g,5分钟,4℃离心细胞。
    12. 丢弃上清液并将细胞重新悬浮在50ml新鲜的R10(室温)中,补充有30U/ml的重组人IL-2。
      注意:IL-2促进T细胞扩增和存活。
    13. 将细胞置于24孔板(每孔2 ml)中,并将板放在37℃,5%CO 2,95%湿度的培养箱中。
      注意:在此过程中,不需要隔离CD8 + T细胞。在TCR转基因小鼠的脾脏中发现的CD8 + T细胞的百分比约占总造血细胞的10-20%,并且它们将在下一步骤期间扩大。 >
    14. 通过从每个孔中收集500μl细胞悬浮液并将它们置于新的24孔板中,每孔填充1.5ml新鲜的R10(室温),补充有30U/ml的重组人IL- 2(细胞稀释度为1〜4)。
    15. 6-7天后(约2个细胞分裂),收集的细胞应超过90%CD8 + T细胞,并在其表面含有较高的CXCR3表达(请参考流式细胞术方案进行评估的CXCR3表达)。
      注意:在扩展结束之前,每个平板应该具有至少1000万CD8 + T细胞。

  2. 体外移植分析
    注意:该方案的这一步骤解释了如何评估CXCL10诱导在步骤A中生成的CD8 + T细胞迁移的能力。
    1. 在50ml锥形管中收集活化的CD8 + T细胞(每片约45ml总体积),并以350xg,5分钟,4℃旋转。细胞通过每孔浸泡3-5次收集。
    2. 弃去上清液并将细胞重新悬浮在5ml的迁移培养基中。
    3. 计数单元格。
    4. 调整音量,所以你有5 x 10 5 细胞/100μl迁移介质。
    5. 从Transwell 96孔板中取出transwell插入片,从接受板上暴露孔
    6. 在迁移介质中制备趋化因子的稀释液,并将其加入到接受板的孔中。我们建议每个条件一式三份。
      注意:接收板中的最终体积为200μl。请参阅CXCL10建议稀释方案本步骤结束时的说明。
    7. 小心地将transwell放置在接收板上。来自每个井的液体将与transwell接触。
    8. 小心地向每个transwell添加100μlCD8 + T细胞悬液(5×10 5个细胞)。关闭平板并置于37℃,5%CO 2,95%湿度的培养箱中1小时30分钟。
    9. 培养时间后,小心地将transwell板从接收板上取出。
    10. 从接收板的孔中(通过上下移动3-5次)将细胞收集到新的96孔U形底板上。我们建议使用多通道移液器执行此步骤。
    11. 旋转96 U底板,350 x g,2 min,弃上清
    12. 用封闭缓冲液(50μl/孔)重悬细胞。孵育20分钟,4度在黑暗中。
    13. 加入150μl1x PBS,旋转板350 g,2 min,弃上清。
    14. 用染色混合物(50μl/孔)重悬细胞。在黑暗中孵育30分钟,4°C。
    15. 加入150μl1x PBS,旋转板350 g,2 min,弃上清。
    16. 将细胞重悬于200μl1x PBS中
    17. 转移细胞流式细胞仪管。
    18. 向每个管中加入10μl计数珠,并在流式细胞仪中运行样品。在CD3 + CD8 + T细胞门上获得至少2,000次荧光珠门事件和2,000次事件,因为1,000个事件是准确细胞所需的最少事件数使用AccuCheck Counting Beads Kit进行计数。
      注意:如果T细胞迁移是残留的(例如没有化学引诱物的条件),可能难以在T细胞门上达到所需数量的事件,但确保您可以获得最大的事件,通过运行整个样本。
    19. 流式细胞仪数据的分析使用Flow Jo软件完成。
    注意:诱导活化的CD8 T细胞迁移的鼠CXCL10的浓度在10ng/ml和1,000ng/ml之间。我们建议进行2倍稀释。包括没有添加趋化因子的条件是非常重要的,以便确定非趋化因子诱导的T细胞迁移(测定的背景)。使用该方法计算迁移细胞是按照AccuCheck Counting Beads提供的说明进行的。


  3. CXCR3内化分析
    注意:该方案的这一步骤解释了如何评估CXCL10诱导趋化因子受体CXCR3内源化的能力,在CD8表面表达 在步骤A中生成的T细胞
    1. 将活化的CD8 + T细胞收集在50ml锥形管中,并以350xg,5分钟,4℃旋转。
    2. 弃去上清液并将细胞重新悬浮在5ml的迁移培养基中
    3. 计数单元格。
    4. 调整音量,所以你有5 x 10 5 细胞/100μl迁移介质。
    5. 平板细胞在96孔U底孔板(100μl/孔)中。
    6. 加入100μl补充有不同浓度的鼠CXCL10(200μl总体积/孔)的迁移培养基。使用与过程B相同的浓度范围。或者,您可以进行10倍稀释,包括1,000; 100; 10;和1ng/ml。
    7. 将培养箱放置在37℃5%CO 2/95%湿度的培养箱中1小时30分钟。
      注意:选择该测定的时间点(1小时和30分钟)以与在该方案的方法B中描述的迁移测定中使用的时间点相匹配。对于新的化学刺激,我们建议进行时间过程研究(例如,在与趋化因子孵育的不同时间点后评估趋化因子 - 受体内化)。
    8. 培养时间后,将板旋转至350×g,2分钟,弃去上清液。
    9. 用封闭缓冲液(50μl/孔)重悬细胞。孵育20分钟,4度在黑暗中。
    10. 加入150μl冷的1x PBS,以350 x g的速度旋转板2 min,弃去上清液。
    11. 用染色混合物(50μl/孔)重悬细胞。在黑暗中孵育30分钟,4°C。
    12. 加入150μl冷的1x PBS,并将其重悬于200μl1x PBS中
    13. 转移细胞流式细胞仪,每管获取至少10,000次事件
    14. 流式细胞仪数据的分析使用Flow Jo软件完成。
    注意:CXCR3内在化的评估可以独立完成,也可以作为CD8的一部分 T细胞迁移测定。请参阅数据分析部分,了解如何在迁移CD8中确定CXCR3水平的例子 从transwell下腔室收集的T细胞板。

数据分析

在排除碎片和死细胞(Aqua + 细胞)后,流式细胞术图可视化如图1所示。为了计算迁移到下腔室的T细胞数量, sup> + 细胞(红色门)和荧光珠(蓝色门),并按照珠的提供者计算细胞编号的说明。为了评估T细胞表面的CXCR3水平,将门控CD8 + T细胞作为直方图(图2)。您可以在FlowJo软件上获得平均荧光强度(MFI)的值,并将其绘制为图形。建议每个实验至少运行3次技术重复。


图1.与暴露于迁移培养基(左图)或补充有1,000ng/ml重组鼠CXCL10的迁移培养基(右图)相对应的从transwell板的接收室收集的细胞的流式细胞术图的实例)。细胞被染色,荧光计数珠(门控为蓝色)加入到细胞中,如步骤B部分所述。在此表示之前,排除了碎片和Aqua + (死亡)细胞。活细胞CD3 + CD8 + T细胞以红色门控。 APC:别藻蓝蛋白。


图2.在暴露于迁移培养基之后,从transwell板的接收室收集的CD3 + CD8 + T细胞的CXCR3染色的流式细胞术直方图的实施例(黑色)或补充有1,000ng/ml重组鼠CXCL10(红色)的迁移培养基。最大染色强度百分比最大值%。 PE:R-藻红蛋白

食谱

  1. R10媒体 - 在4°C储存1个月
    RPMI 1640
    10%胎牛血清 0.1mM非必需氨基酸
    1mM丙酮酸钠
    10 mM HEPES
    70μMβ-巯基乙醇
    23μg/ml庆大霉素
  2. 迁移媒体 - 为每个实验准备新鲜
    1x HBSS
    0.1%无蛋白酶BSA
  3. 阻塞缓冲液 - 每个条件使用50μl,为每个实验准备新鲜
    1x PBS
    小鼠CD16/CD32 Fc阻断抗体(以1〜50稀释使用)
    可固定水性死细胞染色剂(1〜500稀释使用)
  4. 染色混合 - 每个条件使用50μl,为每个实验准备新鲜
    1x PBS
    太平洋蓝缀合的抗小鼠CD3(克隆145-2C11,以1至100稀释使用)
    APC缀合的CD8(克隆53-6.7,以1〜400稀释使用)
    PE缀合的CXCR3(克隆CXCR3-173,以1至100稀释使用)

致谢

使用此协议时,请参阅Barreira da Silva et al。。 (2015)Nat Immunol。资助由巴斯德·鲁夫博士后研究金(RBdS),Ligue Contre le癌症以及法国政府的ARC pour la recherche sur le cancer(MLA)和法国政府的"未来计划投资计划"提供,由国民政府管理la Recherche(LabEx Immuno-Onco [RBdS,MLA])。动物实验方案由巴黎的动物实验委员会(动物实验伦理委员会)批准。

参考文献

  1. Barreira da Silva,R.,Laird,ME,Yatim,N.,Fiette,L.,Ingersoll,MA和Albert,ML(2015)。  二肽基肽酶4抑制增强淋巴细胞运输,改善天然存在的肿瘤免疫和免疫治疗。 Nat Immunol 16 (8):850-858。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Barreira da Silva, R. and Albert, M. L. (2017). Mouse CD8+ T Cell Migration in vitro and CXCR3 Internalization Assays. Bio-protocol 7(6): e2185. DOI: 10.21769/BioProtoc.2185.
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