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A protocol to develop T helper and Treg cells in vivo

Cellular & Molecular Immunology volume 14, pages 1013–1016 (2017)Cite this article

An understanding of the development and function of T helper (Th) 1, Th17 and regulatory T (Treg) cells is critical toward revealing pro-inflammatory immune responses, especially in autoimmune diseases. However, there is no standard protocol to monitor the development of these cells over time in vivo. This protocol details a method to generate Th1, Th17, and Treg cells in a T cell-induced colitis model in vivo and monitor their dynamic changes, which can be used for further investigations in their development from naive CD4+ T cells in specific gene knockouts and background strains. This protocol starts with the isolation of naive CD4+CD45RBhigh T cells from C57BL/6 IL-17Agfp mice followed by their i.p. injection into recombinase, activating gene-1-deficient (Rag1−/−) mice. At the indicated time points, mice are killed, and mesenteric lymphocytes and murine lamina propria mononuclear cells are isolated and subjected to flow cytometric analysis to assess the development of Th1, Th17, and Treg cells and Th2, Th9, Th22, and follicular T helper (Tfh) cells. This protocol can be completed within 8 weeks.

CD4+ T cells play a central role in the function of the immune system, particularly in adaptive immunity. They help the activity of other immune cells by releasing T cell cytokines. CD4+ T cells can be subdivided into lineages based on immunological functions, specific transcription factors, and cytokines: Th1, Th2, Th9, Th17, Th22, Tfh and Treg cells. Aberrant activation of Th1, Th17, Th22 and Tfh cells has been implicated in many autoimmune and inflammatory diseases, whereas excessive Th2 activity causes allergic diseases. Conversely, impaired function of Treg cells causes fatal inflammatory disorders both in human and mouse.1, 2

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References

  1. Hu G, Liu Z, Zheng C, Zheng SG . Antigen-non-specific regulation centered on CD25+foxp3+ treg cells. Cell Mol Immunol 2010; 7: 414–418.

    Article  CAS  Google Scholar 

  2. Gu J, Ni X, Pan X, Lu H, Lu Y, Zhao J et al. Human cd39hi regulatory T cells present stronger stability and function under inflammatory conditions. Cell Mol Immunol 2017; 14: 521–528.

    Article  CAS  Google Scholar 

  3. Chen M, Guo Z, Ju W, Ryffel B, He X, Zheng SG . The development and function of follicular helper t cells in immune responses. Cell Mol Immunol 2012; 9: 375–379.

    Article  CAS  Google Scholar 

  4. Liu ZM, Wang KP, Ma J, Guo Zheng S . The role of all-trans retinoic acid in the biology of foxp3+ regulatory t cells. Cell Mol Immunol 2015; 12: 553–557.

    Article  Google Scholar 

  5. Powrie F, Leach MW, Mauze S, Menon S, Caddle LB, Coffman RL . Inhibition of th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45rbhi CD4+ T cells. Immunity 1994; 1: 553–562.

    Article  CAS  Google Scholar 

  6. Coccia M, Harrison OJ, Schiering C, Asquith MJ, Becher B, Powrie F et al. Il-1beta mediates chronic intestinal inflammation by promoting the accumulation of il-17a secreting innate lymphoid cells and CD4(+) th17 cells. J Exp Med 2012; 209: 1595–1609.

    Article  CAS  Google Scholar 

  7. Wu C, Thalhamer T, Franca RF, Xiao S, Wang C, Hotta C et al. Galectin-9-cd44 interaction enhances stability and function of adaptive regulatory T cells. Immunity 2014; 41: 270–282.

    Article  CAS  Google Scholar 

  8. Izcue A, Hue S, Buonocore S, Arancibia-Carcamo CV, Ahern PP, Iwakura Y et al. Interleukin-23 restrains regulatory T cell activity to drive T cell-dependent colitis. Immunity 2008; 28: 559–570.

    Article  CAS  Google Scholar 

  9. Gao Y, Tang J, Chen W, Li Q, Nie J, Lin F et al. Inflammation negatively regulates foxp3 and regulatory T-cell function via dbc1. Proc Natl Acad Sci USA 2015; 112: E3246–E3254.

    Article  CAS  Google Scholar 

  10. Weiss JM, Bilate AM, Gobert M, Ding Y, Curotto de Lafaille MA, Parkhurst CN et al. Neuropilin 1 is expressed on thymus-derived natural regulatory T cells, but not mucosa-generated induced foxp3+ T reg cells. J Exp Med 2012; 209: 1723–1742, S1721.

    Article  CAS  Google Scholar 

  11. Thornton AM, Korty PE, Tran DQ, Wohlfert EA, Murray PE, Belkaid Y et al. Expression of helios, an ikaros transcription factor family member, differentiates thymic-derived from peripherally induced foxp3+ T regulatory cells. J Immunol 2010; 184: 3433–3441.

    Article  CAS  Google Scholar 

  12. Szurek E, Cebula A, Wojciech L, Pietrzak M, Rempala G, Kisielow P et al. Differences in expression level of helios and neuropilin-1 do not distinguish thymus-derived from extrathymically-induced CD4+foxp3+ regulatory T cells. PLoS One 2015; 10: e0141161.

    Article  Google Scholar 

  13. Chen M, Su W, Lin X, Guo Z, Wang J, Zhang Q et al. Adoptive transfer of human gingiva-derived mesenchymal stem cells ameliorates collagen-induced arthritis via suppression of th1 and th17 cells and enhancement of regulatory T cell differentiation. Arthritis Rheum 2013; 65: 1181–1193.

    Article  CAS  Google Scholar 

  14. Nurieva RI, Chung Y, Martinez GJ, Yang XO, Tanaka S, Matskevitch TD et al. Bcl6 mediates the development of t follicular helper cells. Science 2009; 325: 1001–1005.

    Article  CAS  Google Scholar 

  15. Leppkes M, Becker C, Ivanov II, Hirth S, Wirtz S, Neufert C et al. Rorgamma-expressing th17 cells induce murine chronic intestinal inflammation via redundant effects of il-17a and il-17f. Gastroenterology 2009; 136: 257–267.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work is supported in part by grants from the National Key R&D Program of China (2017YFA0105800), NIH R01 AR059103, NIH STAR award, NIH R61 AR073049, National Natural Science Foundation of China (81671611, 81701600), Guangdong Province Innovative Research Program Project (2011Y035), and Zhejiang Provincial Natural Science Foundation of China (LY14H100002).

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Authors and Affiliations

  1. Division of Rheumatology, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, 310003, Zhejiang Province, China

    Weiqian Chen & Jin Lin

  2. Division of Rheumatology, Department of Medicine, Penn State University Hershey College of Medicine Hershey, Hershey, 17033, PA, USA

    Weiqian Chen, Zhenjian Xu, Yongjiang Zheng, Julie Wang, Nancy Olsen & Song Guo Zheng

  3. Department of Clinical Immunology, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510120, China

    Zhenjian Xu, Yongjiang Zheng & Song Guo Zheng

  4. Division of Hematology, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, 310003, Zhejiang Province, China

    Wenbin Qian

  5. Research Service, Memphis VA Medical Center, Memphis, TN, USA

    David Brand

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  1. Weiqian Chen
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Chen, W., Xu, Z., Zheng, Y. et al. A protocol to develop T helper and Treg cells in vivo. Cell Mol Immunol 14, 1013–1016 (2017). https://doi.org/10.1038/cmi.2017.116

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