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Generation of Aβ-specific T cell lines and in vivo Transfer
β-淀粉样蛋白(Aβ)特异性T淋巴细胞系的建立和过继转移   

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Abstract

Amyloid-β (Aβ)-containing plaques accumulate in the brains of patients with Alzheimer’s disease (AD). Studies in transgenic mice which over-express amyloid precursor protein and presenilin 1 (APP/PS1 mice) have suggested that T cells that infiltrate the brain may influence the development of Aβ plaques and associated cognitive dysfuncation. Active immunization with Aβ peptides and adjuvants has been evaluated as a therapy for AD, based on the premise that it induces Aβ-specific antibodies that may help to clear the Aβ plaques. However, immunization with Aβ peptides and adjuvants also promotes the development of Aβ-specific T cells (McQuillan et al., 2010) and there is evidence that Aβ-specific T cell may influence the development of Aβ plaques and disease progression in AD patients. In the mouse model, Aβ-specific T cells that secrete IFN-γ (Th1 cells) have been shown to enhance the plaque burden (Browne et al., 2013). Adoptive transfer of Aβ-specific T cells that have been polarized in vitro to Th1, Th2, Th17 or Treg cells can be used to examine the function of these cells in vivo.

Materials and Reagents

  1. C57BL/6 mice (adult, >6 weeks old; typically 4 per experiment)
  2. 1-42 peptide (Life Technologies, InvitrogenTM, catalog number: 03112 )
  3. CpG (CpG-oligodeoxynucleotide 1668; 5′-tccatgacgttccgatgct-3′; Sigma-Genosys)
  4. PBS (Sigma-Aldrich, catalog number: D8537 )
  5. IL-1 (Immunotools, catalog number: 12340013 )
  6. IL-2 (Immunotools, catalog number: 12340024 )
  7. IL-4(Immunotools, catalog number: 12340043 )
  8. IL-12 (Miltenyi, catalog number: 130-096-707 )
  9. IL-23 (Miltenyi, catalog number: 130-096-676 )
  10. Anti–IFNγ (BD, catalog number: 554430 )
  11. Dexamethasone (Sigma-Aldrich, catalog number: D4902 )
  12. High-performance liquid chromatography (HPLC)-grade water (sterile ddH2O)
  13. RPMI medium (Sigma-Aldrich, catalog number: R0883 )
  14. Penicillin-streptomycin (Sigma-Aldrich, catalog number: P4333 )
  15. L-glutamine (Sigma-Aldrich, catalog number: G7513 )
  16. FBS (Sigma-Aldrich, catalog number: F9665 )
  17. Complete RPMI medium (see Recipes)

Equipment

  1. Shaker capable of 200 rpm at 37 °C
  2. 24-well cell culture plates (Greiner Bio-one, catalog number: 662160 )
  3. 1 ml tuberculin syringes (BD, catalog number: 300013 )
  4. 70 μm nylon mesh filter (Corning, catalog number: 352350 )
  5. Tissue culture facilities including class II laminar flow hood
  6. Centrifuge

Procedure

  1. Dissolve Aβ1-42 peptide in HPLC-grade water to provide a 12 mg/ml stock solution, which is diluted to 2 mg/ml using sterile PBS and allowed to aggregate for 48 h at 37 °C and with agitation of 200 rpm. Aβ1-42 is used immediately or stored at -20 °C.
  2. Immunize C57BL/6 mice by subcutaneous injection into the rear footpad with Aβ1-42 (75 μg/mouse) and CpG (25 μg/mouse) in a total volume of 50 μl i.e. 25 μl per foot.
  3. Administer a sec (booster) immunization with the same doses of antigen and adjuvant after 21 days.
  4. After a further 7 days, sacrifice the mice and remove the popliteal lymph nodes (the draining lymph node; see Figure 1) and spleens.


    Figure 1. A schematic of popliteal lymph node

  5. Dissociate lymph node and spleen tissue through a sterile 70 μm nylon mesh filter, wash with complete RPMI and centrifuged at 280 x g for 5 min and perform a cell count.
  6. Stimulate the cells at 2 x 106 cells per ml with Aβ1-42 (25 μg/ml) in the presence of cytokines and antibodies depending on the type of cell line required. To generate Th1 cells, the cells from the lymph nodes and spleens are stimulated with Aβ1-42 (25 μg/ml) and IL-12 (10 ng/ml). Th2 cells are amplified using dexamethasone (1 x 10-8 M), IL-4 (10 ng/ml), and anti-IFNγ (5 mg/ml), and Th17 cells are generated with IL-1 (10 ng/ml), IL-23 (10 ng/ml) and anti-IFNγ (5 mg/ml).
  7. After 4 days, add IL-2 (5 ng/ml) to the Th1 and Th2 cell cultures, or medium only to the Th17 cells and incubation continued for a further 7 days.
  8. Wash cells with complete RPMI, centrifuge at 280 x g for 5 min and count.
  9. Confirm that cells are polarized to Th1, Th2 or Th17 either by performing intracellular cytokine staining for IFN-γ, IL-5 or IL-17 and FACS analysis or by assessing the quantity of these cyokines in supernatants by ELISA.
  10. Inject cells i.p. into recipient mice (typically 1.5 x 107 cells/mouse) in 100 μl serum-free medium or PBS.

Recipes

  1. Complete RPMI medium
    RPMI medium supplemented with 1% penicillin-streptomycin, 1% L-glutamine and 10% FBS

Acknowledgments

This work was funded by a PI grant to Kingston Mills from Science Foundation Ireland.

References

  1. Browne, T. C., McQuillan, K., McManus, R. M., O'Reilly, J. A., Mills, K. H. and Lynch, M. A. (2013). IFN-gamma Production by amyloid beta-specific Th1 cells promotes microglial activation and increases plaque burden in a mouse model of Alzheimer's disease. J Immunol 190(5): 2241-2251.
  2. McQuillan, K., Lynch, M. A. and Mills, K. H. (2010). Activation of mixed glia by Abeta-specific Th1 and Th17 cells and its regulation by Th2 cells. Brain Behav Immun 24(4): 598-607.

简介

含有淀粉样蛋白β(Aβ)的斑块积聚在患有阿尔茨海默病(AD)的患者的脑中。过表达淀粉样蛋白前体蛋白和早老蛋白1(APP/PS1小鼠)的转基因小鼠的研究已经表明浸润大脑的T细胞可能影响Aβ斑块和相关认知功能障碍的发展。使用Aβ肽和佐剂的主动免疫已被评估为AD的治疗,基于其诱导可能有助于清除Aβ斑块的Aβ特异性抗体的前提。然而,用Aβ肽和佐剂免疫也促进Aβ特异性T细胞的发展(McQuillan等人,2010),并且有证据表明Aβ特异性T细胞可能影响Aβ斑块的发展和AD患者的疾病进展。在小鼠模型中,分泌IFN-γ(Th1细胞)的Aβ特异性T细胞已显示增强斑块负荷(Browne等人,2013)。已经在体外极化为Th1,Th2,Th17或Treg细胞的Aβ特异性T细胞的过继转移可用于体内检查这些细胞的功能。

材料和试剂

  1. C57BL/6小鼠(成年,> 6周龄;通常每次实验4次)
  2. Aβ1-42肽(Life Technologies,Invitrogen TM ,目录号:03112)
  3. CpG(CpG-寡脱氧核苷酸1668; 5'-tccatgacgttccgatgct-3'; Sigma-Genosys)
  4. PBS(Sigma-Aldrich,目录号:D8537)
  5. IL-1(Immunotools,目录号:12340013)
  6. 材料和试剂

    1. C57BL/6小鼠(成年,> 6周龄;通常每次实验4次)
    2. Aβ1-42肽(Life Technologies,Invitrogen TM ,目录号:03112)
    3. CpG(CpG-寡脱氧核苷酸1668; 5'-tccatgacgttccgatgct-3'; Sigma-Genosys)
    4. PBS(Sigma-Aldrich,目录号:D8537)
    5. IL-1(Immunotools,目录号:12340013)
    6. ... High-performance liquid chromatography (HPLC)-grade water (sterile ddH2O)
    7. RPMI medium (Sigma-Aldrich, catalog number: R0883)
    8. Penicillin-streptomycin (Sigma-Aldrich, catalog number: P4333)
    9. L-glutamine (Sigma-Aldrich, catalog number: G7513)
    10. FBS (Sigma-Aldrich, catalog number: F9665)
    11. Complete RPMI medium (see Recipes)

    Equipment

    1. Shaker capable of 200 rpm at 37 °C
    2. 24-well cell culture plates (Greiner Bio-one, catalog number: 662160)
    3. 1 ml tuberculin syringes (BD, catalog number: 300013)
    4. 70 μm nylon mesh filter (Corning, catalog number: 352350)
    5. Tissue culture facilities including class II laminar flow hood
    6. 离心机

    程序

    1. 在HPLC级水中溶解Aβ1-42肽以提供12mg/ml储备溶液,使用无菌PBS将其稀释至2mg/ml,并使其在37℃下聚集48小时 和200rpm的搅拌。 Aβ1-42立即使用或储存在-20℃下。
    2. 通过在50μl总体积中用Aβ1-42(75μg/小鼠)和CpG(25μg/小鼠)皮下注射到后足垫中来免疫C57BL/6小鼠,/em> 25μl/英尺
    3. 在21天后用相同剂量的抗原和佐剂进行持续(加强)免疫。
    4. 再过7天后,处死小鼠,取出al淋巴结(引流淋巴结,参见图1)和脾。


      图1。 al淋巴结示意图

    5. 通过无菌的70μm尼龙网过滤器分离淋巴结和脾组织,用完全RPMI洗涤并在280×g离心5分钟并进行细胞计数。
    6. 在细胞因子和抗体的存在下,根据细胞系的类型,用Aβ1-42(25μg/ml)在2×10 6个细胞/ml下刺激细胞需要。为了产生Th1细胞,用Aβ1-42(25μg/ml)和IL-12(10ng/ml)刺激来自淋巴结和脾的细胞。使用地塞米松(1×10 8 -8 M),IL-4(10ng/ml)和抗-IFNγ(5mg/ml)扩增Th2细胞,并用IL产生Th17细胞-1(10ng/ml),IL-23(10ng/ml)和抗-IFNγ(5mg/ml)。
    7. 4天后,向Th1和Th2细胞培养物中加入IL-2(5ng/ml),或仅对Th17细胞加入培养基,继续培养7天。
    8. 用完全RPMI洗涤细胞,在280×g离心5分钟并计数。
    9. 通过对IFN-γ,IL-5或IL-17进行细胞内细胞因子染色和FACS分析或通过ELISA测定上清液中这些细胞因子的量来确认细胞被极化为Th1,Th2或Th17。
    10. 注射细胞i.p。 向100μl无血清培养基或PBS中的受体小鼠(通常为1.5×10 7个细胞/小鼠)中。

    食谱

    1. 完成RPMI培养基
      补充有1%青霉素 - 链霉素,1%L-谷氨酰胺和10%FBS的RPMI培养基中

    致谢

    这项工作是由科学基金会爱尔兰的金斯顿米尔斯的PI拨款资助。

    参考文献

    1. Browne,T.C.,McQuillan,K.,McManus,R.M.,O'Reilly,J.A.,Mills,K.H.and Lynch,M.A。(2013)。 IFN-γ由淀粉状蛋白β特异性Th1细胞的产生促进小胶质细胞活化并增加小鼠的斑块负荷 模型的阿尔茨海默病。 Immunol 190(5):2241-2251。
    2. McQuillan,K.,Lynch,M.A。和Mills,K.H。(2010)。 通过Abeta特异性Th1和Th17细胞激活混合型胶质细胞及其通过Th2细胞的调节。 a> Brain Behav Immun 24(4):598-607。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:McManus, R. M., Lynch, M. A. and Mills, K. H. (2014). Generation of Aβ-specific T cell lines and in vivo Transfer. Bio-protocol 4(18): e1241. DOI: 10.21769/BioProtoc.1241.
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