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Analysis of T Cell Proliferating and Polarizing Potential of Murine Dendritic Cells in Allogeneic-mixed Leukocyte Reaction
建立可同时研究T淋巴细胞增殖和树突细胞极化的共培养体系   

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Abstract

Dendritic cells (DCs) play a critical role in mounting the T cell response against different infectious agents. Nature and intensity of the induced T cell responses are defined by activation status of DCs. It is generally accepted that IL-12, IL-4/IL-5 and IL-23 producing DCs induce TH1, TH2 and TH17 type of immune responses, respectively (Kumar et al., 2015). Besides cytokines, levels of co-stimulatory molecules on DCs also influence the response of T cells.
The activation status of DCs can be determined by examining DC culture supernatants for different cytokines and by analyzing expression of co-stimulatory molecules on these cells. However, these approaches provide indirect information about T cell activating potential of DCs. Analysis of T cell responses in a co-culture system is a more direct approach to examine T cell proliferating and polarizing efficacy of DCs.
A protocol to analyze the T cell proliferating and polarizing potential of DCs in an allogeneic mixed leukocyte reaction (allo-MLR) is described here.

Materials and Reagents

  1. RPMI-1640 medium (HiMedia Laboratories, catalog number: AT028 )
  2. Dulbecco’s Phosphate Buffered Saline (HiMedia Laboratories, catalog number: TS1006 )
  3. Heat-inactivated fetal bovine serum (Biological industries, catalog number: 04-121-1A )
  4. Antibiotic-antimycotic (penicillin-streptomycin) solution, 100x (HiMedia Laboratories, catalog number: A002A )
  5. Round bottom, 96-well cell culture plates (Corning, catalog number: 3799 )
  6. Dendritic cells (derived by culturing mouse bone marrow cells in the presence of recombinant GM-CSF) (PeproTech, catalog number: 315-03 )
  7. Untouched CD4+ and CD8+ T cells from allogeneic mouse strain (isolated from spleen of Balb/c mice using CD4 T cell enrichment kit and CD8 T cell enrichment kit (BD, catalog number: 558131 and 558471 , respectively)
  8. Fluorochrome-conjugated FITC anti-mouse CD3, PE anti-mouse CD4 and PE anti-mouse CD8 antibodies (BD Pharmingen, catalog number: 555274 , 553730 and 553032 , respectively)
  9. Concanavalin A (Sigma-Aldrich, catalog number: C5275 )
  10. 3H-thymidine (BARC)
  11. Trypan Blue (Sigma-Aldrich, catalog number: T8154 )
  12. RPMI-10 (see Recipes)

Equipment

  1. Haemocytometer
  2. Humidified CO2 incubator
  3. Laminar air flow bio-safety cabinet
  4. Centrifuge
  5. Gamma-irradiator
  6. Microscope
  7. Flow-cytometer

Procedure

  1. Harvest mouse bone marrow-derived dendritic cells (BMDCs) from plates by gentle pipetting, give a wash with PBS and prepare the suspensions of 1.0 x 105, 2.0 x 105 and 4.0 x 105 cells per ml in RPMI-10 medium.
    (BMDCs are derived by culturing mouse bone marrow cells in the presence of GM-CSF. Briefly, add 4 x 106 bone-marrow cells per well of 6-well plate in RPMI-10 medium supplemented with 20 ng/ml GM-CSF. Remove culture medium along with non-adherent cells on day 3 and day 5, and fresh 4.0 ml GM-CSF-supplemented medium to each well. Harvest immature DCs on day 7 by gently pipetting. After giving a wash in RPMI-10 medium, cells can be used for subsequent experiments. Purity of DCs derived following this protocol is ~85%. These cells can be used directly in allo-MLR or can be further purified.)
  2. To analyze the ability of DCs to induce T-cell proliferation, add 50 µl of DC suspensions (equivalent to 0.5 x 104, 1.0 x 104 and 2.0 x 104 DCs) per well in a round bottom plate in triplicates.
    (It is necessary to plate the increasing number of DCs to achieve an increasing ratio of stimulator cells to responder cells. It is advised to not add DCs into outer wells of the plate because culture media tend to evaporate from these wells at higher rates. Instead, these wells can be filled with autoclaved distilled water).
  3. To analyze the ability of DCs to induce T-cell polarization, similarly add 50 µl of 2.0 x 105 cells/ml DC suspension (= 1.0 x 104 cells) per well in a round bottom plate in triplicates.
  4. Add the desired stimuli such as LPS or heat-killed mycobacteria to plated DCs and adjust final volume of total contents per well to 100 µl.
    [Dilute stock solution of LPS or mycobacterial suspension to required concentration in RPMI-10 medium. LPS could be used at a concentration of 0.1 to 1.0 µg/ml, whereas heat-killed bacteria (prepared by autoclaving) can be used preferably at a multiplicity of infection (MOI) of 5 to 10].
  5. Keep plates in a humidified CO2 incubator for 24 h.
  6. Next day, isolate CD4+ and CD8+ T lymphocytes from the spleen of naïve allogeneic mice using a negative selection kit as suggested by manufacturer. Determine purity of lymphocytes using anti-mouse CD3/CD4 and CD3/CD8 antibodies by flow cytometry.
    Note: If DCs are derived from C57BL/6 mice, lymphocytes can be prepared from Balb/c mice.
  7. Irradiate DCs with gamma-rays in a gamma-irradiation chamber (irradiation dose, 25 Gy). Irradiation will prevent the proliferation of DCs, which could otherwise give false results.
  8. Adjust concentration of lymphocytes to 1.0 x 106 cells/ml. Add 100 µl of cell suspension to irradiated DCs.
  9. Set positive controls by stimulating CD4+ T cells and CD8+ T cells with Concanavalin A (final concentration, 5 µg/ml).
  10. Keep plates at 37 °C in a humidified CO2 incubator.
  11. After 72 h, add 1.0 µCi 3H-thymidine per well of the plate set up with T cell proliferation assay. Keep plates back into the CO2 incubator.
  12. After 18 h, transfer plates to -20 °C. Plates can be thawed immediately or next day.
  13. Harvest the cells onto a filter paper and wash them using an automated cell harvester.
  14. Measure the 3H-thymidine levels on filter paper using a beta scintillation counter.
  15. Collect culture supernatants from plate set up with T cell polarization assay, after 96 h. Store supernatants at -80 °C or immediately analyze for TH1, TH2, TH17 signature cytokines by ELISA.

Recipes

  1. RPMI-10
    RPMI-1640 base medium supplemented with 10% heat-inactivated FBS and 1% antibiotic-antimycotic solution

References

  1. Kumar, P., John, V., Marathe, S., Das, G. and Bhaskar, S. (2015). Mycobacterium indicus pranii induces dendritic cell activation, survival, and Th1/Th17 polarization potential in a TLR-dependent manner. J Leukoc Biol 97(3): 511-520.
  2. Muul, L. M., Silvin, C., James, S. P. and Candotti, F. (2008). Measurement of proliferative responses of cultured lymphocytes. Curr Protoc Immunol Chapter 7: Unit 7 10 11-17 10 24.

简介

树突细胞(DC)在安装针对不同感染剂的T细胞应答中起关键作用。诱导的T细胞应答的性质和强度由DC的活化状态定义。通常接受的是,产生IL-12,IL-4/IL-5和IL-23的DC诱导T H 1,T H 2和T H (Kumar等人,2015年)中的免疫应答类型。除了细胞因子,DC上的共刺激分子的水平也影响T细胞的响应。通过检查不同细胞因子的DC培养上清液并通过分析这些细胞因子上的共刺激分子的表达,可以确定DC的活化状态细胞。然而,这些方法提供关于DC的T细胞活化潜能的间接信息。在共培养系统中分析T细胞应答是更直接的方法来检查DC的T细胞增殖和极化功效。
在同种异体混合白细胞反应中分析DC的T细胞增殖和极化潜能的方案(allo-MLR)。

材料和试剂

  1. RPMI-1640培养基(HiMedia Laboratories,目录号:AT028)
  2. Dulbecco's磷酸盐缓冲盐水(HiMedia Laboratories,目录号:TS1006)
  3. 热灭活的胎牛血清(Biological industries,目录号:04-121-1A)
  4. 抗生素 - 抗真菌剂(青霉素 - 链霉素)溶液,100x(HiMedia Laboratories,目录号:A002A)
  5. 圆底96孔细胞培养板(Corning,目录号:3799)
  6. 树突状细胞(通过在重组GM-CSF存在下培养小鼠骨髓细胞得到)(PeproTech,目录号:315-03)
  7. 使用CD4 T细胞富集试剂盒和CD8 T细胞富集试剂盒(BD,USA)从同种异体小鼠品系(从Balb/c小鼠的脾中分离)的未接触的CD4 +和CD8 +目录号:558131和558471)
  8. 荧光染料偶联的FITC抗小鼠CD3,PE抗小鼠CD4和PE抗小鼠CD8抗体(BD Pharmingen,目录号:555274,553730和553032)
  9. 刀豆蛋白A(Sigma-Aldrich,目录号:C5275)
  10. 3 H-胸苷(BARC)
  11. 台盼蓝(Sigma-Aldrich,目录号:T8154)
  12. RPMI-10(见配方)

设备

  1. 血细胞计数器
  2. 加湿CO 2培养箱
  3. 层流气流生物安全柜
  4. 离心机
  5. 伽玛辐照器
  6. 显微镜
  7. 流式细胞仪

程序

  1. 通过温和移液从板中收获小鼠骨髓来源的树突细胞(BMDC),用PBS洗涤,并制备1.0×10 5,2.0×10 5,和在RPMI-10培养基中的4.0×10 5个细胞/ml。
    (BMDCs是通过在GM-CSF存在下培养小鼠骨髓细胞而得到的)简单地说,在6孔板的每个孔中加入4×10 6个骨髓细胞的RPMI-10培养基20ng/ml GM-CSF,在第3天和第5天除去培养基以及非粘附细胞,向每孔中加入新鲜的4.0ml GM-CSF补充培养基,在第7天通过轻轻吸取收集未成熟DC,在RPMI-10培养基中洗涤,细胞可用于随后的实验。根据该方案得到的DC的纯度为?85%,这些细胞可以直接用于allo-MLR或可以进一步纯化)。
  2. 为了分析DC诱导T细胞增殖的能力,将50μl的DC悬浮液(相当于0.5×10 4个,1.0×10 4个和2.0×10 8个)在圆底板中每孔一式三份重复培养 (有必要铺板数量增加的DC以实现刺激细胞与响应细胞的增加比例,建议不要将DC添加到板的外孔中,因为培养基倾向于以更高的速率从这些孔中蒸发,而是,这些孔可以用高压灭菌的蒸馏水填充)
  3. 为了分析DCs诱导T细胞极化的能力,同样地每孔加入50μl2.0×10 5个细胞/ml DC悬浮液(= 1.0×10 4个细胞)在一个圆底板上一式三份
  4. 添加所需的刺激,如LPS或热灭活分枝杆菌到平板DCs,并调整每孔的总内容物的最终体积为100微升。
    [在RPMI-10培养基中稀释LPS或分枝杆菌悬浮液至所需浓度的储备溶液。 LPS可以以0.1至1.0μg/ml的浓度使用,而热灭活细菌(通过高压灭菌制备)可以优选以5至10的感染复数(MOI)使用。
  5. 将板在加湿的CO 2培养箱中保持24小时
  6. 第二天,使用阴性选择试剂盒如制造商所建议的从初始同种异体小鼠的脾中分离CD4 +和CD8 + T淋巴细胞。使用抗小鼠CD3/CD4和CD3/CD8抗体通过流式细胞术确定淋巴细胞的纯度。
    注意:如果DC源自C57BL/6小鼠,则可以从Balb/c小鼠制备淋巴细胞。
  7. 在γ-照射室中用γ-射线照射DC(照射剂量,25Gy)。照射将防止DC的增殖,否则可能会导致错误的结果
  8. 将淋巴细胞的浓度调节至1.0×10 6个细胞/ml。加入100微升的细胞悬浮液照射DCs
  9. 通过用伴刀豆球蛋白A(终浓度为5μg/ml)刺激CD4 + T细胞和CD8 + sup + T细胞来设置阳性对照。
  10. 将板在37℃下在加湿的CO 2培养箱中保持。
  11. 72小时后,在用T细胞增殖测定设置的板的每个孔中加入1.0μCi3 H-胸苷。保持板回到CO 2培养箱。
  12. 18小时后,将板转移至-20°C。平板可以立即或第二天解冻。
  13. 收获细胞在滤纸上,并使用自动细胞收获器清洗。
  14. 使用β闪烁计数器测量滤纸上的 3 H-胸苷水平
  15. 在96小时后,用T细胞极化测定法收集来自培养板的培养物上清液。将上清液存储在-80℃或立即通过ELISA分析T H H 1,T H H 2,T H H 17标志性细胞因子。 />

食谱

  1. RPMI-10
    补充有10%热灭活的FBS和1%抗生素 - 抗真菌溶液的RPMI-1640基础培养基

参考文献

  1. Kumar,P.,John,V.,Marathe,S.,Das,G.and Bhaskar,S。(2015)。 Mycobacterium indicus pranii 诱导树突状细胞活化,存活和Th1/Th17极化电位以TLR依赖性方式。 Leukoc Biol 97(3):511-520。
  2. Muul,L.M.,Silvin,C.,James,S.P.and Candotti,F。(2008)。 测量培养淋巴细胞的增殖反应 Curr Protoc Immunol 第7章:单位7 10 11-17 10 24.
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kumar, P. and Bhaskar, S. (2016). Analysis of T Cell Proliferating and Polarizing Potential of Murine Dendritic Cells in Allogeneic-mixed Leukocyte Reaction. Bio-protocol 6(5): e1750. DOI: 10.21769/BioProtoc.1750.
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