Abstract
Regulatory T cells (Tregs) are amongst the most widely studied cells in a variety of immune-mediated conditions, including transplantation and Graft Versus Host Disease (GVHD), cancer and autoimmunity; indeed, there is great interest in the tolerogenic potential of Treg-based therapy. Consequently, the need to establish the mechanisms that determine Treg survival and longevity, in addition to developing new tools to monitor these parameters, is paramount. Using both a mouse model of GVHD and a mouse model of Type 1 Diabetes (T1D), we describe herein a dual reporter system based on Gluc and multiplexed with SEAP and non-secreted Firefly luciferase (Fluc), which permits simultaneous imaging and noninvasive tracking of two different T-cell populations (CD4+CD25+ Tregs and CD4+CD25− Tcon cells) in vivo by transducing the cells with different lentiviruses bearing distinct color signatures. This new technology promises to overcome the limitations of the conventional methods currently available to study lymphocyte survival in vivo. Furthermore, this novel technique has applications not only in autoimmunity and alloimmunity, but also in the wider field of immunology.
Grant K. Lewandrowski and Ciara N. Magee contributed equally to this work.
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References
Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M (1995) Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 155(3): 1151–1164
Itoh M et al (1999) Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J Immunol 162(9):5317–5326
Chen W et al (2003) Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med 198(12): 1875–1886
Curotto de Lafaille MA et al (2008) Adaptive Foxp3+ regulatory T cell-dependent and -independent control of allergic inflammation. Immunity 29(1):114–126
Nishimura E, Sakihama T, Setoguchi R, Tanaka K, Sakaguchi S (2004) Induction of antigen-specific immunologic tolerance by in vivo and in vitro antigen-specific expansion of naturally arising Foxp3+CD25+CD4+ regulatory T cells. Int Immunol 16(8):1189–1201
Sakaguchi S (2005) Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol 6(4):345–352
Collison LW et al (2007) The inhibitory cytokine IL-35 contributes to regulatory T-cell function. Nature 450(7169):566–569
Pandiyan P, Zheng L, Ishihara S, Reed J, Lenardo MJ (2007) CD4+CD25+Foxp3+ regulatory T cells induce cytokine deprivation-mediated apoptosis of effector CD4+ T cells. Nat Immunol 8(12):1353–1362
Campbell DJ, Ziegler SF (2007) FOXP3 modifies the phenotypic and functional properties of regulatory T cells. Nat Rev Immunol 7(4):305–310
Riley JL, June CH, Blazar BR (2009) Human T regulatory cell therapy: take a billion or so and call me in the morning. Immunity 30(5): 656–665
Badr CE, Hewett JW, Breakefield XO, Tannous BA (2007) A highly sensitive assay for monitoring the secretory pathway and ER stress. PLoS One 2(6):e571
Bao R et al (2002) Activation of cancer-specific gene expression by the survivin promoter. J Natl Cancer Inst 94(7):522–528
Bao R, Selvakumaran M, Hamilton TC (2000) Use of a surrogate marker (human secreted alkaline phosphatase) to monitor in vivo tumor growth and anticancer drug efficacy in ovarian cancer xenografts. Gynecol Oncol 78(3 Pt 1): 373–379
Hewett JW et al (2007) Mutant torsinA interferes with protein processing through the secretory pathway in DYT1 dystonia cells. Proc Natl Acad Sci USA 104(17):7271–7276
Hiramatsu N et al (2006) Secreted protein-based reporter systems for monitoring inflammatory events: critical interference by endoplasmic reticulum stress. J Immunol Methods 315(1–2): 202–207
Meng Y et al (2005) Real-time monitoring of mesangial cell-macrophage cross-talk using SEAP in vitro and ex vivo. Kidney Int 68(2): 886–893
Meng Y et al (2005) Continuous, noninvasive monitoring of local microscopic inflammation using a genetically engineered cell-based biosensor. Lab Invest 85(11):1429–1439
Wurdinger T et al (2008) A secreted luciferase for ex vivo monitoring of in vivo processes. Nat Methods 5(2):171–173
Contag CH, Ross BD (2002) It’s not just about anatomy: in vivo bioluminescence imaging as an eyepiece into biology. J Magn Reson Imaging 16(4):378–387
Weissleder R, Ntziachristos V (2003) Shedding light onto live molecular targets. Nat Med 9(1): 123–128
Berger J, Hauber J, Hauber R, Geiger R, Cullen BR (1988) Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene 66(1):1–10
Cullen BR, Malim MH (1992) Secreted placental alkaline phosphatase as a eukaryotic reporter gene. Methods Enzymol 216: 362–368
Hiramatsu N, Kasai A, Hayakawa K, Yao J, Kitamura M (2006) Real-time detection and continuous monitoring of ER stress in vitro and in vivo by ES-TRAP: evidence for systemic, transient ER stress during endotoxemia. Nucleic Acids Res 34(13):e93
Hiramatsu N et al (2005) Alkaline phosphatase vs luciferase as secreted reporter molecules in vivo. Anal Biochem 339(2):249–256
Shiraiwa T et al (2007) Establishment of a non-invasive mouse reporter model for monitoring in vivo pdx-1 promoter activity. Biochem Biophys Res Commun 361(3): 739–744
Abruzzese RV et al (1999) Ligand-dependent regulation of plasmid-based transgene expression in vivo. Hum Gene Ther 10(9): 1499–1507
Chastain M et al (2001) Antigen levels and antibody titers after DNA vaccination. J Pharm Sci 90(4):474–484
Muller L, Saydam O, Saeki Y, Heid I, Fraefel C (2005) Gene transfer into hepatocytes mediated by herpes simplex virus-Epstein-Barr virus hybrid amplicons. J Virol Methods 123(1): 65–72
Phuong LK et al (2003) Use of a vaccine strain of measles virus genetically engineered to produce carcinoembryonic antigen as a novel therapeutic agent against glioblastoma multiforme. Cancer Res 63(10):2462–2469
Hewett JW et al (2008) siRNA knock-down of mutant torsinA restores processing through secretory pathway in DYT1 dystonia cells. Hum Mol Genet 17(10):1436–1445
Tannous BA, Kim DE, Fernandez JL, Weissleder R, Breakefield XO (2005) Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. Mol Ther 11(3):435–443
Venisnik KM, Olafsen T, Gambhir SS, Wu AM (2007) Fusion of Gaussia luciferase to an engineered anti-carcinoembryonic antigen (CEA) antibody for in vivo optical imaging. Mol Imaging Biol 9(5):267–277
Zhou L, Chong MM, Littman DR (2009) Plasticity of CD4+ T cell lineage differentiation. Immunity 30(5):646–655
Tannous BA (2009) Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo. Nat Protoc 4(4):582–591
Ablamunits V, Elias D, Cohen IR (1999) The pathogenicity of islet-infiltrating lymphocytes in the non-obese diabetic (NOD) mouse. Clin Exp Immunol 115(2):260–267
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Lewandrowski, G.K., Magee, C.N., Mounayar, M., Tannous, B.A., Azzi, J. (2014). Simultaneous In Vivo Monitoring of Regulatory and Effector T Lymphocytes Using Secreted Gaussia Luciferase, Firefly Luciferase, and Secreted Alkaline Phosphatase. In: Badr, C. (eds) Bioluminescent Imaging. Methods in Molecular Biology, vol 1098. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-718-1_17
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DOI: https://doi.org/10.1007/978-1-62703-718-1_17
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