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In vivo and in vitro techniques for comparative study of antiviral T-cell responses in the amphibian Xenopus

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

  1. Doherty P, Allan W, Eichelberger M. Role of ab and gd T cell subsets in viral immunity. Annu Rev Immunol 1992;10:123–151.

    Article  PubMed  CAS  Google Scholar 

  2. 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.

    Google Scholar 

  3. 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.

  4. 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.

    Google Scholar 

  5. Lyons AB. Analysing cell division in vivo and in vitro using flow cytometric measurements of CFSE dilution. J Immunol Methods 2000; 243:147–154.

    Article  PubMed  CAS  Google Scholar 

  6. Tough DF, Sprent J. Turnover of naïve and memory phenotype T cells. J Exp Med 1994; 179:1127–1135.

    Article  PubMed  CAS  Google Scholar 

  7. 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.

    Article  PubMed  CAS  Google Scholar 

  8. 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.

    Article  PubMed  CAS  Google Scholar 

  9. Vanderlaan M, Thomas CB. Characterization of monoclonal antibodies to bromodeoxyuridine. Cytometry 1985; 6: 501.

    Article  PubMed  CAS  Google Scholar 

  10. Lyons AB, Parish CR. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 1994; 171: 131.

    Article  PubMed  CAS  Google Scholar 

  11. Pantaleo G, Harari A. Functional Signatures in antiviral T cell Immunity for monitoring virus associated diseases. Nature Rev Immunol 2006; 6:417–422.

    Article  CAS  Google Scholar 

  12. Klenerman P, Hill A. T cells and viral persistence: lessons from diverse infections. Nature Immunol. 2005; 6:873–879.

    Article  CAS  Google Scholar 

  13. 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.

    Article  CAS  Google Scholar 

  14. 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.

    Article  PubMed  CAS  Google Scholar 

  15. 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.

    Article  CAS  Google Scholar 

  16. 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.

    Article  PubMed  CAS  Google Scholar 

  17. 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.

    Article  PubMed  CAS  Google Scholar 

  18. 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.

    Article  PubMed  CAS  Google Scholar 

  19. 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.

    CAS  Google Scholar 

  20. Hsu E, Du Pasquier L. Studies in Xenopus immunoglobulins using monoclonal antibodies. Mol Immunol 1984;21:257–270.

    Article  PubMed  CAS  Google Scholar 

  21. Cohen N. Phylogeny of Lymphocyte Structure and Function. Amer Zool 1975; 15(1):119–133.

    Google Scholar 

  22. 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.

    Article  CAS  Google Scholar 

  23. 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.

    Article  PubMed  CAS  Google Scholar 

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Correspondence to Jacques Robert Ph.D..

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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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

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