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Generation and Expansion of T Helper 17 Lymphocytes Ex Vivo

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Suppression and Regulation of Immune Responses

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1371))

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Abstract

CD4+ T helper (Th) lymphocytes are essential elements of the complex cellular networks regulating the initiation, development, and termination of adaptive immune responses. Different independent and specialized subsets of Th cells can be distinguished based on their dedicated transcription factor and cytokine expression profiles. Th17 lymphocytes have been described about a decade ago as CD4+ Th cells producing high quantity of IL-17A as a signature cytokine. Since their initial discovery, Th17 have drawn intense scrutiny for their dominant role in the pathogenesis of multiple autoimmune, infectious diseases and allergy. The influence of Th17 lymphocytes in cancer remains however ambiguous. The plethoric functions of Th17 may rely on the remarkable plasticity of these cells, endowed with the ability to trans-differentiate into other Th subpopulations depending on the environmental cytokine context. The possibility to generate Th17 ex vivo has facilitated the elucidation of the signals and transcription factors required for their differentiation and functions and has allowed for the evaluation of their functions following adoptive transfer in vivo. Several protocols have been developed to produce Th17 in vitro. The intent of this chapter is to provide examples of procedures for generating and expanding Th17 ex vivo.

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References

  1. Sun JC, Bevan MJ (2003) Defective CD8 T cell memory following acute infection without CD4 T cell help. Science 300:339–342

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. Sun JC, Williams MA, Bevan MJ (2004) CD4+ T cells are required for the maintenance, not programming, of memory CD8+ T cells after acute infection. Nat Immunol 5:927–933

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Mitsdoerffer M, Lee Y, Jager A, Kim HJ, Korn T, Kolls JK, Cantor H, Bettelli E, Kuchroo VK (2010) Proinflammatory T helper type 17 cells are effective B-cell helpers. Proc Natl Acad Sci U S A 107:14292–14297

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Kelly MN, Zheng M, Ruan S, Kolls J, D'Souza A, Shellito JE (2013) Memory CD4+ T cells are required for optimal NK cell effector functions against the opportunistic fungal pathogen Pneumocystis murina. J Immunol 190:285–295

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. DeNardo DG, Barreto JB, Andreu P, Vasquez L, Tawfik D, Kolhatkar N, Coussens LM (2009) CD4(+) T cells regulate pulmonary metastasis of mammary carcinomas by enhancing protumor properties of macrophages. Cancer Cell 16:91–102

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Veldhoen M, Moncrieffe H, Hocking RJ, Atkins CJ, Stockinger B (2006) Modulation of dendritic cell function by naive and regulatory CD4+ T cells. J Immunol 176:6202–6210

    Article  CAS  PubMed  Google Scholar 

  7. Shevach EM, DiPaolo RA, Andersson J, Zhao DM, Stephens GL, Thornton AM (2006) The lifestyle of naturally occurring CD4+ CD25+ Foxp3+ regulatory T cells. Immunol Rev 212:60–73

    Article  CAS  PubMed  Google Scholar 

  8. Romagnani S (1997) The Th1/Th2 paradigm. Immunol Today 18:263–266

    Article  CAS  PubMed  Google Scholar 

  9. Mosmann TR, Coffman RL (1989) TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 7:145–173

    Article  CAS  PubMed  Google Scholar 

  10. Zheng W, Flavell RA (1997) The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89:587–596

    Article  CAS  PubMed  Google Scholar 

  11. Baeten DL, Kuchroo VK (2013) How Cytokine networks fuel inflammation: interleukin-17 and a tale of two autoimmune diseases. Nat Med 19:824–825

    Article  CAS  PubMed  Google Scholar 

  12. Muranski P, Restifo NP (2013) Essentials of Th17 cell commitment and plasticity. Blood 121(13):2402–2414

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Knutson KL, Disis ML (2005) Tumor antigen-specific T helper cells in cancer immunity and immunotherapy. Cancer Immunol Immunother 54:721–728

    Article  CAS  PubMed  Google Scholar 

  14. Tangye SG, Ma CS, Brink R, Deenick EK (2013) The good, the bad and the ugly – TFH cells in human health and disease. Nat Rev Immunol 13:412–426

    Article  CAS  PubMed  Google Scholar 

  15. Jabeen R, Kaplan MH (2012) The symphony of the ninth: the development and function of Th9 cells. Curr Opin Immunol 24:303–307

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Yang XO, Pappu BP, Nurieva R, Akimzhanov A, Kang HS, Chung Y, Ma L, Shah B, Panopoulos AD, Schluns KS, Watowich SS, Tian Q, Jetten AM, Dong C (2008) T helper 17 lineage differentiation is programmed by orphan nuclear receptors ROR alpha and ROR gamma. Immunity 28:29–39

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Ivanov BS II, McKenzie L, Zhou CE, Tadokoro A, Lepelley JJ, Lafaille DJC, Littman DR (2006) The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126:1121–1133

    Article  CAS  PubMed  Google Scholar 

  18. Kryczek I, Banerjee M, Cheng P, Vatan L, Szeliga W, Wei S, Huang E, Finlayson E, Simeone D, Welling TH, Chang A, Coukos G, Liu R, Zou W (2009) Phenotype, distribution, generation, and functional and clinical relevance of Th17 cells in the human tumor environments. Blood 114:1141–1149

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Kebir H, Ifergan I, Alvarez JI, Bernard M, Poirier J, Arbour N, Duquette P, Prat A (2009) Preferential recruitment of interferon-gamma-expressing TH17 cells in multiple sclerosis. Ann Neurol 66:390–402

    Article  CAS  PubMed  Google Scholar 

  20. Korn T, Bettelli E, Gao W, Awasthi A, Jager A, Strom TB, Oukka M, Kuchroo VK (2007) IL-21 initiates an alternative pathway to induce proinflammatory T(H)17 cells. Nature 448:484–487

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Korn T, Bettelli E, Oukka M, Kuchroo VK (2009) IL-17 and Th17 cells. Annu Rev Immunol 27:485–517

    Article  CAS  PubMed  Google Scholar 

  22. Kryczek I, Zhao E, Liu Y, Wang Y, Vatan L, Szeliga W, Moyer J, Klimczak A, Lange A, Zou W (2011) Human TH17 cells are long-lived effector memory cells. Sci Transl Med 3:104ra100

    Article  PubMed Central  PubMed  Google Scholar 

  23. Martin-Orozco N, Muranski P, Chung Y, Yang XO, Yamazaki T, Lu S, Hwu P, Restifo NP, Overwijk WW, Dong C (2009) T helper 17 cells promote cytotoxic T cell activation in tumor immunity. Immunity 31:787–798

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Wilke CM, Bishop K, Fox D, Zou W (2011) Deciphering the role of Th17 cells in human disease. Trends Immunol 32:603–611

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Alizadeh D, Katsanis E, Larmonier N (2013) The multifaceted role of Th17 lymphocytes and their associated cytokines in cancer. Clin Dev Immunol 2013:957878

    Article  PubMed Central  PubMed  Google Scholar 

  26. Alizadeh D, Trad M, Hanke NT, Larmonier CB, Janikashvili N, Bonnotte B, Katsanis E, Larmonier N (2014) Doxorubicin eliminates myeloid-derived suppressor cells and enhances the efficacy of adoptive T-cell transfer in breast cancer. Cancer Res 74:104–118

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Muranski P, Boni A, Antony PA, Cassard L, Irvine KR, Kaiser A, Paulos CM, Palmer DC, Touloukian CE, Ptak K, Gattinoni L, Wrzesinski C, Hinrichs CS, Kerstann KW, Feigenbaum L, Chan CC, Restifo NP (2008) Tumor-specific Th17-polarized cells eradicate large established melanoma. Blood 112:362–373

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Muranski P, Borman ZA, Kerkar SP, Klebanoff CA, Ji Y, Sanchez-Perez L, Sukumar M, Reger RN, Yu Z, Kern SJ, Roychoudhuri R, Ferreyra GA, Shen W, Durum SK, Feigenbaum L, Palmer DC, Antony PA, Chan CC, Laurence A, Danner RL, Gattinoni L, Restifo NP (2011) Th17 cells are long lived and retain a stem cell-like molecular signature. Immunity 35:972–985

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Alizadeh D, Larmonier N (2014) Chemotherapeutic targeting of cancer-induced immunosuppressive cells. Cancer Res 74:2663–2668

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Iida T, Iwahashi M, Katsuda M, Ishida K, Nakamori M, Nakamura M, Naka T, Ojima T, Ueda K, Hayata K, Nakamura Y, Yamaue H (2011) Tumor-infiltrating CD4+ Th17 cells produce IL-17 in tumor microenvironment and promote tumor progression in human gastric cancer. Oncol Rep 25:1271–1277

    Article  CAS  PubMed  Google Scholar 

  31. He S, Fei M, Wu Y, Zheng D, Wan D, Wang L, Li D (2011) Distribution and clinical significance of th17 cells in the tumor microenvironment and peripheral blood of pancreatic cancer patients. Int J Mol Sci 12:7424–7437

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Kryczek I, Wei S, Gong W, Shu X, Szeliga W, Vatan L, Chen L, Wang G, Zou W (2008) Cutting edge: IFN-gamma enables APC to promote memory Th17 and abate Th1 cell development. J Immunol 181:5842–5846

    Article  CAS  PubMed  Google Scholar 

  33. Koenen HJ, Smeets RL, Vink PM, van Rijssen E, Boots AM, Joosten I (2008) Human CD25highFoxp3pos regulatory T cells differentiate into IL-17-producing cells. Blood 112:2340–2352

    Article  CAS  PubMed  Google Scholar 

  34. Dong C (2009) Mouse Th17 cells: current understanding of their generation and regulation. Eur J Immunol 39:640–644

    Article  CAS  PubMed  Google Scholar 

  35. Korn T, Mitsdoerffer M, Croxford AL, Awasthi A, Dardalhon VA, Galileos G, Vollmar P, Stritesky GL, Kaplan MH, Waisman A, Kuchroo VK, Oukka M (2008) IL-6 controls Th17 immunity in vivo by inhibiting the conversion of conventional T cells into Foxp3+ regulatory T cells. Proc Natl Acad Sci U S A 105:18460–18465

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Yang L, Anderson DE, Baecher-Allan C, Hastings WD, Bettelli E, Oukka M, Kuchroo VK, Hafler DA (2008) IL-21 and TGF-beta are required for differentiation of human T(H)17 cells. Nature 454:350–352

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  37. Nurieva R, Yang XO, Martinez G, Zhang Y, Panopoulos AD, Ma L, Schluns K, Tian Q, Watowich SS, Jetten AM, Dong C (2007) Essential autocrine regulation by IL-21 in the generation of inflammatory T cells. Nature 448:480–483

    Article  CAS  PubMed  Google Scholar 

  38. Parham C, Chirica M, Timans J, Vaisberg E, Travis M, Cheung J, Pflanz S, Zhang R, Singh KP, Vega F, To W, Wagner J, O'Farrell AM, McClanahan T, Zurawski S, Hannum C, Gorman D, Rennick DM, Kastelein RA, de Waal Malefyt R, Moore KW (2002) A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rbeta1 and a novel cytokine receptor subunit, IL-23R. J Immunol 168:5699–5708

    Article  CAS  PubMed  Google Scholar 

  39. Stritesky GL, Yeh N, Kaplan MH (2008) IL-23 promotes maintenance but not commitment to the Th17 lineage. J Immunol 181:5948–5955

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  40. Cua DJ, Sherlock J, Chen Y, Murphy CA, Joyce B, Seymour B, Lucian L, To W, Kwan S, Churakova T, Zurawski S, Wiekowski M, Lira SA, Gorman D, Kastelein RA, Sedgwick JD (2003) Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421:744–748

    Article  CAS  PubMed  Google Scholar 

  41. Langrish CL, Chen Y, Blumenschein WM, Mattson J, Basham B, Sedgwick JD, McClanahan T, Kastelein RA, Cua DJ (2005) IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J Exp Med 201:233–240

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  42. Murphy CA, Langrish CL, Chen Y, Blumenschein W, McClanahan T, Kastelein RA, Sedgwick JD, Cua DJ (2003) Divergent pro- and antiinflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation. J Exp Med 198:1951–1957

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  43. Chung Y, Chang SH, Martinez GJ, Yang XO, Nurieva R, Kang HS, Ma L, Watowich SS, Jetten AM, Tian Q, Dong C (2009) Critical regulation of early Th17 cell differentiation by interleukin-1 signaling. Immunity 30:576–587

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  44. Chen Q, Yang W, Gupta S, Biswas P, Smith P, Bhagat G, Pernis AB (2008) IRF-4-binding protein inhibits interleukin-17 and interleukin-21 production by controlling the activity of IRF-4 transcription factor. Immunity 29:899–911

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  45. Acosta-Rodriguez EV, Rivino L, Geginat J, Jarrossay D, Gattorno M, Lanzavecchia A, Sallusto F, Napolitani G (2007) Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells. Nat Immunol 8:639–646

    Article  CAS  PubMed  Google Scholar 

  46. Wilson NJ, Boniface K, Chan JR, McKenzie BS, Blumenschein WM, Mattson JD, Basham B, Smith K, Chen T, Morel F, Lecron JC, Kastelein RA, Cua DJ, McClanahan TK, Bowman EP, de Waal Malefyt R (2007) Development, cytokine profile and function of human interleukin 17-producing helper T cells. Nat Immunol 8:950–957

    Article  CAS  PubMed  Google Scholar 

  47. Ghoreschi K, Laurence A, Yang XP, Tato CM, McGeachy MJ, Konkel JE, Ramos HL, Wei L, Davidson TS, Bouladoux N, Grainger JR, Chen Q, Kanno Y, Watford WT, Sun HW, Eberl G, Shevach EM, Belkaid Y, Cua DJ, Chen W, O'Shea JJ (2010) Generation of pathogenic T(H)17 cells in the absence of TGF-beta signalling. Nature 467:967–971

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  48. Manel N, Unutmaz D, Littman DR (2008) The differentiation of human T(H)-17 cells requires transforming growth factor-beta and induction of the nuclear receptor RORgammat. Nat Immunol 9:641–649

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  49. Hebel K, Rudolph M, Kosak B, Chang HD, Butzmann J, Brunner-Weinzierl MC (2011) IL-1beta and TGF-beta act antagonistically in induction and differentially in propagation of human proinflammatory precursor CD4+ T cells. J Immunol 187:5627–5635

    Article  CAS  PubMed  Google Scholar 

  50. Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK (2006) Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441:235–238

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The authors thank Martin Asimis for technical assistance with the characterization of human Th17 cells and Claire Larmonier for technical assistance with real-time PCR.

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Correspondence to Darya Alizadeh Ph.D. or Nicolas Larmonier Ph.D. .

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Alizadeh, D., Larmonier, N. (2016). Generation and Expansion of T Helper 17 Lymphocytes Ex Vivo. In: Cuturi, M., Anegon, I. (eds) Suppression and Regulation of Immune Responses. Methods in Molecular Biology, vol 1371. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3139-2_7

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  • DOI: https://doi.org/10.1007/978-1-4939-3139-2_7

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3138-5

  • Online ISBN: 978-1-4939-3139-2

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