Reconstitution of Lymphopaenic Mice with Regulatory and Conventional T cell Subsets
在无淋巴细胞的小鼠模型中用调节性T细胞和普通T细胞重建外周T细胞亚群
发布: 2016年05月20日第6卷第10期 DOI: 10.21769/BioProtoc.1814 浏览次数: 8438
评审: Jia LiMareta RusevaSabine Le Saux
下载 PDF 
Q&A
参见作者原研究论文
The authors used this protocol in:
Sep 2015
Abstract
Transfer of mature T cells into immunodeficient mice results in sub-optimal reconstitution of the peripheral T cell pool. Under lymphopenic conditions, dendritic cells are released from tonic control by regulatory T cells (Tregs), and consequently drive activation and proliferation of low affinity T cells specific for endogenous antigens. This oligoclonal proliferation results in a T cell population dominated by T cells possessing an effector/memory phenotype and a limited TCR repertoire. Oligoclonal expansion can be prevented by selectively reconstituting the Treg compartment prior to T cell transfer (Bolton et al., 2015). Reconstitution of the Treg compartment of lymphopenic mice has been tested in immunodeficient mouse strains such as Rag-1-/-or Rag-2-/- mice, and in immunosufficient mice rendered transiently lymphopaenic by lethal whole body irradiation as conditioning for bone marrow transplantation (BMT). Transfer of purified Tregs into these hosts, combined with treatment with exogenous IL-2 for 7 days, is sufficient to reconstitute the Treg compartment and reduce expression of dendritic cell costimulatory molecules, a critical process in preventing inappropriate expansion of self-reactive T cells. T cells transferred after Treg reconstitution do not undergo rapid spontaneous proliferation, and instead undergo slow homeostatic division to repopulate the T cell pool with naive T cells, thus allowing optimal reconstitution of peripheral T cell pool.
Keywords: Lymphopenia (lymphopenia)
Immune reconstitution (免疫重建)
T cell (T细胞)
Regulatory T cell (调节T细胞)
Animal model (动物模型)
Materials and Reagents
- 50 ml conical tube (Corning, Falcon®, catalog number: 352070 )
- 10 ml conical tube (SARSTEDT AG & Co, catalog number: 62.9924.272 )
- 70 µm nylon cell strainer (Corning, Falcon®, catalog number: 352350 )
- 1 ml syringe (BD, catalog number: 309659 )
- 25 gauge needle (BD, catalog number: 305125 )
- 1 ml insulin syringe with 29 G needle (BD, catalog number: 326719 )
- 225 cm tissue culture flasks (Sigma-Aldrich, catalog number: CLS431082 )
- LD columns (Miltenyi Biotec, catalog number: 130-042-901 )
- LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
- Vitamin K1, 10 mg/ml (International Animal Health Products, Koagulon)
- PBS (10x, pH 7.4, without Ca2+ and Mg2+) (Mediatech Inc., catalog number: 46-013-CM )
- Fetal bovine serum (FBS), heat-inactivated at 65 °C (Scientifix, catalog number: FBS-500S )
- Bovine serum albumin (BSA) Fraction V (Amresco, catalog number: 0332-1 kg )
- Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E5134 )
- RPMI-1640 (Thermo Fisher Scientific, GibcoTM, catalog number: 11875-093 )
- Rat anti-mouse Thy1.2 clone 30H12 hybridoma supernatant (prepared in house)
- Rat anti-mouse B220 clone RA3.6B2 hybridoma supernatant (prepared in house)
- Rat anti-mouse CD8a clone 53-6.7 hybridoma supernatant (prepared in house)
- Rat anti-mouse CD11b clone M1/70 hybridoma supernatant (prepared in house)
- Rat anti-mouse erythroid lineage clone Ter119 hybridoma supernatant (prepared in house)
- Anti-CD25 PE clone PC61 (BD Biosciences, catalog number: 553866 )
- Anti-rat IgG microbeads (Miltenyi Biotec, catalog number: 130-048-501 )
- Anti-PE microbeads (Miltenyi Biotec, catalog number: 130-048-801 )
- Recombinant mouse IL-2 (Peprotech, catalog number: 212-12 )
- Anti-IL-2 mAb clone JES6-1 (WEHI monoclonal antibody facility)
- Sulfamethoxazole (400 mg/5 ml) and Trimethoprim (80 mg/5 ml) (Hospira, DBL, catalog number: 618670BAU )
- MACS wash buffer (see Recipes)
- MACS running buffer (see Recipes)
- RPMI + 10% FBS (see Recipes)
- IL-2/JES6-1 (see Recipes)
- Preparation of hybridoma supernatants (see Recipes)
Equipment
- Surgical scissors and forceps
- 80 gauge stainless steel mesh sieves
- MACS MultiStand (Miltenyi Biotec, catalog number: 130-042-303 )
- QuadroMACS separator (Miltenyi Biotec, catalog number: 130-090-976 ) or MidiMACS separator (Miltenyi Biotec, catalog number: 130-042-302 )
- 37 °C water bath
- Benchtop centrifuge
- Gammacell irradiator (Nordion), or equivalent
- Heat lamp
- Perspex mouse restrainer
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Bolton, H. A. and Groth, B. F. D. S. (2016). Reconstitution of Lymphopaenic Mice with Regulatory and Conventional T cell Subsets. Bio-protocol 6(10): e1814. DOI: 10.21769/BioProtoc.1814.
Download Citation in RIS Format
分类
免疫学 > 动物模型 > 小鼠
您对这篇实验方法有问题吗？
在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。
提问指南
+ 问题描述
写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。
发布问题
