ISOLATION AND IDENTIFICATION OF REGULATORY T CELLS IN PERIPHERAL BLOOD OF RHESUS MONKEYS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANG Shuang ¹ , JIN Xi ¹ , CHENG Hanjia ² , HE Sirong ¹ , CHEN Younan ¹ , LONG Dan ¹ , SHAN Juan ¹ , ZENG Li ¹ , WANG Chengshi ¹ , CHENG Jingqiu ¹ , LU Yanrong ¹

1Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P.R.China;;
2School of Life Science, Sichuan University. Corresponding author: LU Yanrong, E-mail: luyanrong@scu.edu.cn;

Corresponding author：

Keywords：

Regulatory T cells; Effective T cells; Magnetic cell sorting method; Lymphocyte isolation; Rhesus monkey

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Objective To establish a method to isolate the CD4+CD25+ regulatory T cells (Tregs) and to identify the purity and function of these cells. Methods The peripheral blood (8 mL) were collected from the great saphenous vein of 10 rhesus monkeys (4 females and 6 males, aged 4-5 years, and weighing 5-8 kg). The mononuclear cells were isolated with density gradient centrifugation. CD4+ T cells were separated by the Magnetic cell sorting (MACS) negative selection and MACS positive selection. The cell yield rate, the cell viability, and the cell purity were compared between MACS negative selection and MACS positive selection. In CD4+ MACS negative selection, the anti-biotin MicroBeads and biotin-antibody cocktai in CD4+CD25+ Tregs isolation kit non-human primate were used, and in MACS positive selection, the anti-APC MicroBeads in CD4+CD25+ Tregs isolation kit non-human primate and CD4-APC were used. The CD4+ T cells separated by positive selection were selected to obtain CD4+CD25 Tregs with CD25 MicroBeads. The purity, activity, the FoxP3 level, and the suppressive function to concanavalin A (ConA) activated autologous CD4+CD24- effective T cells (Teffs) of CD4+CD25+ Tregs were detected by flow cytometry. Results After CD4+ T cells were separated by MACS negative selection and MACS positive selection, the cell viabilities were all up to 95%, showing no significant difference (P gt; 0.05). The cell yield rate and purity of CD4+ T cells by positive selection were significantly higher than those of CD4+ T cells by negative selection (P lt; 0.05). CD4+CD25+ Tregs can be successfully isolated by MACS double positive selection. The classifying purity was 76.2% ± 8.6%; the cell activity was 93.3% ± 4.7%; and the level of FoxP3 was 74.2% ± 6.9%. The CD4+CD25+ Tregs had suppressive effect on ConA activated autologous CD4+CD25- Teffs. Conclusion MACS double positive selection can be used to isolate high-purity CD4+CD25+ Tregs from the peripheral blood of rhesus monkeys and the process does not influence the activity of CD4+CD25+ Tregs.

Citation： ZHANG Shuang,JIN Xi,CHENG Hanjia,HE Sirong,CHEN Younan,LONG Dan,SHAN Juan,ZENG Li,WANG Chengshi,CHENG Jingqiu,LU Yanrong. ISOLATION AND IDENTIFICATION OF REGULATORY T CELLS IN PERIPHERAL BLOOD OF RHESUS MONKEYS. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(10): 1237-1241. doi: Copy

1.	Fan H, Cao P, Game DS, et al. Regulatory T cell therapy for induction of clinical organ transplantation tolerance. Semin Immunol, 2011, 23(6): 453-461.
2.	Sagoo P, Lombardi G, Lechler RI. Relevance of regulatoty T cell promotion of donor-specific tolerance in solid organ transplantation. Front Immunol, 2012, 3: 184.
3.	Dummer CD, Carpio VN, Gonçalves LF, et al. FOXP3+ regulatory T cells: from suppression of rejection to induction of renal allograft tolerance. Transpl Immunol, 2012, 26(1): 1-10.
4.	Sakaguchi S, Yamaguchi T, Numura T, et al. Regulatory T cells and immune tolerance. Cell, 2008, 133(5): 775-787.
5.	Riley JL, June CH, Blazar BR, et al. Human T regulatory cells therapy: take a billion or so and call me in the morning. Immunity, 2009, 30(5): 656-665.
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7.	陈磊, 李楠, 王学廉, 等. 微电极引导偏侧帕金森病猴脑深部电极植入方法的研究. 中国微侵袭神经外科杂志, 2012, 17(1): 32-35.
8.	Anderson A, Martens CL, Hendrix R, et al. Expanded nonhuman primate tregs exhibit a unique gene expression signature and potently downregulate alloimmune responses. Am J Transplant, 2008, 8(11): 2252-2263.
9.	Yan H, Ding CG, Tian PX, et al. Magnetic cell sorting and flow cytometry sorting methods for the isolation and function analysis of mouse CD4+CD25+ Treg cells. J Zhejiang Univ Sci B, 2009, 10(12): 928-932.
10.	Bresatz S, Sadlon T, Millard D, et al. Isolation, propagation and characterization of cord blood derived CD4+CD25+ regulatory T cells. J Immunol Methods, 2007, 327(1-2): 53-62.
11.	Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor FoxP3. Science, 2003, 299(5609): 1057-1061.
12.	陈祚珈, 高雅懿, 李志远, 等. FOXP3+调节性T细胞. 生命科学, 2010, 22(6): 515-528.
13.	胡义平, 李晓娟, 刘叔文. ConA对CD4+CD25+调节性T细胞早期活化和功能的影响. 细胞与分子免疫学杂志, 2010, 26(2): 118-120, 124.
14.	吕梦捷, 曾耀英, 宋兵. 人参皂苷Rb1 对小鼠T淋巴细胞体外活化、增殖及凋亡的影响. 中草药, 2011, 42(4): 743-748.
15.	Shevach EM. Regulatory T cells in autoimmunity. Ann Rev Immunol, 2000, 18: 423-449.
16.	Campbell DJ, Ziegler SF. FOXP3 modifies the phenotypic and functional properties of regulatory T cells. Nat Rev Immunol, 2007, 7(4): 305-310.
17.	Carrigan SO, Yang YJ, Issekutz T, et al. Depletion of natural CD4+CD25+ T regulatory cells with anti-CD25 antibody does not change the course of Pseudomonas aeruginosa-induced acute lung infection in mice. Immunobiology, 2009, 214(3): 211-222.
18.	Dons EM, Raimondi G, Cooper DK, et al. Non-human primate regulatory T cells: current biology and implications for transplantation. Transplantation, 2010, 90(8): 811-816.
19.	Porter CM, Horvath-Arcidiacono JA, Singh AK, et al. Characterization and expansion of baboon CD4+CD25+ Treg cells for potential use in a non-human primate xenotransplantation model. Xenotransplantation, 2007, 14(4): 298-308.
20.	Wang J, Huizinga TW, Toes RE. De novo generation and enhanced suppression of human CD4+CD25+ regulatory T cells by retinoic acid. J Immunol, 2009, 183(6): 4119-4126.
21.	Fantini MC, Becker C, Monteleone G, et al. Cutting edge: TGF-beta induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and down-regulation of Smad7. J Immunol, 2004, 172(9): 5149-5153.
22.	Mucida D, Park YJ, Kim G, et al. Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science, 2007, 317(5835): 256-260.
23.	Horwitz DA, Zheng SG, Gray JD. Natural and TGF-beta-induced Foxp3(+)CD4(+) CD25(+) regulatory T cells are not mirror images of each other. Trends Immunol, 2008, 29(9): 429-435.
24.	Zhou X, Kong N, Zou H, et al. Therapeutic potential of TGF-β-induced CD4(+) Foxp3(+) regulatory T cells in autoimmune diseases. Autoimmunity, 2011, 44(1): 43-50.

1. Fan H, Cao P, Game DS, et al. Regulatory T cell therapy for induction of clinical organ transplantation tolerance. Semin Immunol, 2011, 23(6): 453-461.
2. Sagoo P, Lombardi G, Lechler RI. Relevance of regulatoty T cell promotion of donor-specific tolerance in solid organ transplantation. Front Immunol, 2012, 3: 184.
3. Dummer CD, Carpio VN, Gonçalves LF, et al. FOXP3+ regulatory T cells: from suppression of rejection to induction of renal allograft tolerance. Transpl Immunol, 2012, 26(1): 1-10.
4. Sakaguchi S, Yamaguchi T, Numura T, et al. Regulatory T cells and immune tolerance. Cell, 2008, 133(5): 775-787.
5. Riley JL, June CH, Blazar BR, et al. Human T regulatory cells therapy: take a billion or so and call me in the morning. Immunity, 2009, 30(5): 656-665.
6. 熊竞, 刘克剑, 吴芳新, 等. 健康中国恒河猴各淋巴组织中Ｔ淋巴细胞表型及分布. 中国畜牧兽医, 2012, 39(2): 86-91.
7. 陈磊, 李楠, 王学廉, 等. 微电极引导偏侧帕金森病猴脑深部电极植入方法的研究. 中国微侵袭神经外科杂志, 2012, 17(1): 32-35.
8. Anderson A, Martens CL, Hendrix R, et al. Expanded nonhuman primate tregs exhibit a unique gene expression signature and potently downregulate alloimmune responses. Am J Transplant, 2008, 8(11): 2252-2263.
9. Yan H, Ding CG, Tian PX, et al. Magnetic cell sorting and flow cytometry sorting methods for the isolation and function analysis of mouse CD4+CD25+ Treg cells. J Zhejiang Univ Sci B, 2009, 10(12): 928-932.
10. Bresatz S, Sadlon T, Millard D, et al. Isolation, propagation and characterization of cord blood derived CD4+CD25+ regulatory T cells. J Immunol Methods, 2007, 327(1-2): 53-62.
11. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor FoxP3. Science, 2003, 299(5609): 1057-1061.
12. 陈祚珈, 高雅懿, 李志远, 等. FOXP3+调节性T细胞. 生命科学, 2010, 22(6): 515-528.
13. 胡义平, 李晓娟, 刘叔文. ConA对CD4+CD25+调节性T细胞早期活化和功能的影响. 细胞与分子免疫学杂志, 2010, 26(2): 118-120, 124.
14. 吕梦捷, 曾耀英, 宋兵. 人参皂苷Rb1 对小鼠T淋巴细胞体外活化、增殖及凋亡的影响. 中草药, 2011, 42(4): 743-748.
15. Shevach EM. Regulatory T cells in autoimmunity. Ann Rev Immunol, 2000, 18: 423-449.
16. Campbell DJ, Ziegler SF. FOXP3 modifies the phenotypic and functional properties of regulatory T cells. Nat Rev Immunol, 2007, 7(4): 305-310.
17. Carrigan SO, Yang YJ, Issekutz T, et al. Depletion of natural CD4+CD25+ T regulatory cells with anti-CD25 antibody does not change the course of Pseudomonas aeruginosa-induced acute lung infection in mice. Immunobiology, 2009, 214(3): 211-222.
18. Dons EM, Raimondi G, Cooper DK, et al. Non-human primate regulatory T cells: current biology and implications for transplantation. Transplantation, 2010, 90(8): 811-816.
19. Porter CM, Horvath-Arcidiacono JA, Singh AK, et al. Characterization and expansion of baboon CD4+CD25+ Treg cells for potential use in a non-human primate xenotransplantation model. Xenotransplantation, 2007, 14(4): 298-308.
20. Wang J, Huizinga TW, Toes RE. De novo generation and enhanced suppression of human CD4+CD25+ regulatory T cells by retinoic acid. J Immunol, 2009, 183(6): 4119-4126.
21. Fantini MC, Becker C, Monteleone G, et al. Cutting edge: TGF-beta induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and down-regulation of Smad7. J Immunol, 2004, 172(9): 5149-5153.
22. Mucida D, Park YJ, Kim G, et al. Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science, 2007, 317(5835): 256-260.
23. Horwitz DA, Zheng SG, Gray JD. Natural and TGF-beta-induced Foxp3(+)CD4(+) CD25(+) regulatory T cells are not mirror images of each other. Trends Immunol, 2008, 29(9): 429-435.
24. Zhou X, Kong N, Zou H, et al. Therapeutic potential of TGF-β-induced CD4(+) Foxp3(+) regulatory T cells in autoimmune diseases. Autoimmunity, 2011, 44(1): 43-50.

Chinese Journal of Reparative and Reconstructive Surgery

ISOLATION AND IDENTIFICATION OF REGULATORY T CELLS IN PERIPHERAL BLOOD OF RHESUS MONKEYS

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