Adoptive Transfer of Tumor Expanded Regulatory T Cells (Tregs)
肿瘤扩增调节性T细胞的过继转移
发布: 2016年08月20日第6卷第16期 DOI: 10.21769/BioProtoc.1899 浏览次数: 10784
评审: Lee-Hwa TaiClara Lubeseder-MartellatoToshitsugu Fujita
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参见作者原研究论文
The authors used this protocol in:
Nov 2015
Abstract
Regulatory T cells (Tregs), a subset of CD4+CD25+ T cells, infiltrate tumors and suppress antitumor activity of effector T and NK cells. Depletion of Tregs by anti CD25+ antibodies has been shown to reduce tumor growth and metastasis (Olkhanud et al., 2009). Conversely, adoptive transfer of Tregs induced immune suppression and promoted tumor growth (Smyth et al., 2006; Janakiram et al., 2015). We have adoptively transferred Tregs to evaluate their immunosuppressive function in vivo. Our study (Vences-Catalan et al., 2015) compared the immunosuppressive efficacy of Tregs derived from tumor-bearing wild type to those of CD81KO mice. The following protocol could be adapted to any other source of Tregs.
Lymph node or splenic tumor-induced Tregs are isolated and purified by a two-step procedure using CD4+CD25+ regulatory T cell isolation kit from MACS Miltenyi Biotec. First, CD4+ T cells are enriched by negative selection, followed by positive selection of CD25+ T cells. Tumor-induced purified Tregs (CD3+CD4+CD25+FoxP3+) are then co-injected subcutaneously together with tumor cells into naïve mice (Winn assay) (Winn, 1960). Tregs could also be injected intravenously once or several times, according to the research needs. The effect of the adoptively transferred Tregs on tumor growth is then measured by caliper or by in vivo imaging techniques.
Adoptive transfer (过继转移)
Tumor (肿瘤)
Immune suppression (免疫抑制)
Materials and Reagents
- 70 μm cell strainer (Corning, Falcon®, catalog number: 352350 )
- 1 ml syringe (BD, catalog number: 309659 )
- 30 G x 1/2 needle (BD, catalog number: 305106 )
- LD columns (Miltenyi Biotec, catalog number: 130-042-901 )
- MS columns (Miltenyi Biotec, catalog number: 130-042-201 )
- 15 and 50 ml polypropylene conical tubes (Corning, Falcon®, catalog number: 352096 and 352070 )
- Mice
Note: We use 6-8 weeks old female Balb/c or C57BL6 mice, but the protocol is applicable to any mouse strain as long as both donor and recipient mice are from the same genetic background. - Cells
Note: We use breast cancer cell lines 4T1 or E0771 syngeneic to Balb/c or C57Bl6 respectively. - Fetal calf serum (FCS) (GE life sciences, HyCloneTM, FetalClone®III, catalog numer: SH30109.03 )
- Penicillin-Streptomycin (Pen-Strep) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-22 )
Note: 5 ml of this solution was used in 500 ml of RPMI 1640 media. - PBS (Corning, Cellgro, catalog number: 21-031-CV )
- ACK buffer (Quality Biological, catalog number: 118-156-101 )
- CD4+CD25+ Regulatory T cell Isolation kit (Miltenyi Biotec, catalog number: 130-091-041 )
Components of the kit:- 1 ml CD4+CD25+ Regulatory T cell Biotin-antibody cocktail
- 2 ml Anti-biotin Microbeads
- 1 ml CD25-PE
- 1 ml Anti-PE Microbeads
- RPMI 1640 (Corning, Cellgro, catalog number: 10-040-CV )
- Bovine serum albumin (BSA) (Sigma Aldrich, catalog number: A9647-500G )
Note: A solution of 0.5% in PBS was used. - EDTA (Sigma Aldrich, catalog number: E-5134 )
Note: A solution of 2 mM in PBS was used. - Optional: CD3 FITC (clone:145-2C11) (BD Pharmingen)
- Optional: CD25 PE (clone:PC61.5) (BD Pharmingen)
- Optional: CD4 PerCP (clone:H129.19/RM4-5) (BD Pharmingen)
- Optional: FoxP3 APC (clone:FJK16s) (BD Pharmingen)
- MACS Buffer (see Recipes)
Equipment
- MidiMACS separator (Miltenyi Biotec, catalog number: 130-042-302 )
- MiniMACS separator (Miltenyi Biotec, catalog number: 130-042-102 )
- Centrifuge (Eppendorf, model: 5810 R )
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Vences-Catalán, F. and Levy, S. (2016). Adoptive Transfer of Tumor Expanded Regulatory T Cells (Tregs). Bio-protocol 6(16): e1899. DOI: 10.21769/BioProtoc.1899.
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分类
免疫学 > 免疫细胞分离 > 淋巴细胞
癌症生物学 > 肿瘤免疫学 > 肿瘤微环境
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