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In vivo and in vitro techniques for comparative study of antiviral T-cell responses in the amphibian Xenopus
Biological Procedures Online volume 10, pages 1–8 (2008)
Abstract
Activation of lymphocytes in mammals is often quantified by measuring the amount of proliferation during the expansion phase of an immune response. Bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assays are some of the techniques widely used in mammalian studies of pathogeninduced proliferation and provide a convenient way of quantifying the cellular response. We have extended the use of these proliferation assays to the amphibian Xenopus laevis. We have developed this species as a valuable comparative model to study immunity against a wellknown amphibian pathogen, Frog Virus 3 (FV3). Fluorescence activated cell sorting was used to assess the level of BrdU incorporation of lymphocytes in vivo and CFSE dilution in an in vitro activation assay. Both techniques have shown that splenic lymphocytes proliferate specifically upon FV3 challenge. This indicates that common methods for detection of proliferation upon immunologic challenge are easily applied to other vertebrate species, as it highlights the evolutionary conservation of the proliferative nature of immune responses throughout vertebrate phyla.
Abbreviations
- Ag:
-
antigen
- APBS:
-
amphibian phosphate buffered saline
- BrdU:
-
bromodeoxyuridine
- CFSE:
-
carboxyfluorescein diacetate succinimidyl ester
- FACS:
-
fluorescence activated cell sorting
- mAb:
-
monoclonal antibody
- MHC:
-
major histocompatibility complex
References
Doherty P, Allan W, Eichelberger M. Role of ab and gd T cell subsets in viral immunity. Annu Rev Immunol 1992;10:123–151.
Du Pasquier L, Flajnik MF, Guiet C, Hsu E. Methods used to study the immune system of Xenopus (Amphibia, Anura). Immunol Methods 1985; 3:425–465.
Robert J, Maniero G, Cohen N, Gantress J. Xenopus as an model system to study evolution of HSP-immune system interactions. In: Methods: A Companion to Methods in Enzymology (HSPImmune System Interactions) 2004 Ed. P. Srivastava, Academic Press. Vol 32: 4253.
Carayon P, Bord A. Indentification of DNA replicating lymphocyte subsets using a new method to label the bromodeoxyuridine incorporated into the DNA. J Immunol Methods 1992; 142: 225–230.
Lyons AB. Analysing cell division in vivo and in vitro using flow cytometric measurements of CFSE dilution. J Immunol Methods 2000; 243:147–154.
Tough DF, Sprent J. Turnover of naïve and memory phenotype T cells. J Exp Med 1994; 179:1127–1135.
Hasbold J, Gett AV, Rush JS, Deenick E, Avery D, Jun J, Hodgkin PD. Quantitative analysis of lymphocyte differentiation and proliferation in vitro using carboxyfluorescein diacetate succinimidyl ester. Immunol Cell Biol 1999; 77:516–522.
Gonchoroff NJ, Katzmann JA, Surriw RM, Evans EL, Houck DW, Kline BC, Greipp PR, Loken MR. Sphase detection with an antibody to bromodeoxyuridine. Role of DNase pretreatment. J immunol Methods 1986; 93:97.
Vanderlaan M, Thomas CB. Characterization of monoclonal antibodies to bromodeoxyuridine. Cytometry 1985; 6: 501.
Lyons AB, Parish CR. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 1994; 171: 131.
Pantaleo G, Harari A. Functional Signatures in antiviral T cell Immunity for monitoring virus associated diseases. Nature Rev Immunol 2006; 6:417–422.
Klenerman P, Hill A. T cells and viral persistence: lessons from diverse infections. Nature Immunol. 2005; 6:873–879.
Migueles SA, Laborico AC, Shupert WL, Sabbaghian MS, Rabin R, Hallahan CW, Van Baarle D, Kostense S, Miedema F, McLaughlin M, Ehler L, Metcalf J, Liu S, Connors M. HIV specific CD8T cell proliferation is coupled to perforin expression in nonprogressors. Nature Immunol 2002;3:1061–1068.
Weekes M, Wills MR, Mynard K, Hicks R, Sissons JG, Carmichael AJ. Large clonal expansions of human virusspecific memory cytotoxic T lymphocytes within the CD57+CD38CD8+ T cell population. Immunology 1999;98:443–449.
Estepa A, Alvarez F, Ezquerrra A, Coll JM. Viralantigen dependence and T cell receptor expression in leucocytes from rhabdovirus immunized trout. Vet Immunol Immunopath 1999; 68: 73–89.
Gantress J, Maniero GD, Cohen N, Robert J. Development and characterization of a model system to study amphibian immune responses to iridoviruses. Virology 2003; 311(2):254–262.
Robert J, Morales H, Wayne B, Cohen N, Marr S, Gantress J. Adaptive immunity and histopathology in frog virus 3-infected Xenopus. Virology 2005; 332: 667–675.
Morales H, Robert J. In vivo characterization of primary and secondary anti-ranavirus CD8 T cell responses in Xenopus laevis. J Virology 2007; 81: 2240–2248.
Flajnik MF, Taylor E, Canel C, Grossberge, D, Du Pasquier L. Reagents specific for MHC I antigens of Xenopus. Amer Zool 1991; 31: 580–591.
Hsu E, Du Pasquier L. Studies in Xenopus immunoglobulins using monoclonal antibodies. Mol Immunol 1984;21:257–270.
Cohen N. Phylogeny of Lymphocyte Structure and Function. Amer Zool 1975; 15(1):119–133.
Maniero GD, Morales H, Gantress J, Robert J. Generation of a longlasting, protective, and neutralizing antibody response to the ranavirus FV3 by the frog Xenopus. Devel Comp Immunol 2006; 30: 649–657.
Maniero GD, Robert J. Phylogenetic conservation of gp96mediated antigen specific cellular immunity: New evidence from adoptive cell transfer in Xenopus. Transplantation 2004; 78(10): 1415–1421.
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Morales, H., Robert, J. In vivo and in vitro techniques for comparative study of antiviral T-cell responses in the amphibian Xenopus . Biol. Proced. Online 10, 1–8 (2008). https://doi.org/10.1251/bpo137
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DOI: https://doi.org/10.1251/bpo137