Abstract
Spontaneously generated mutant T cells defective in T-cell receptor (TCR) gene expression are detectable at the frequency of 10−4 in vivo, and the mutant fractions are dose-dependently increased by exposure to genotoxic substances such as ionizing radiation. Mutant cells with altered expression of TCR-α or -β among CD4+ T cells can be detected as CD3−/CD4+ cells by two-color flow cytometry using anti-CD3 and anti-CD4 monoclonal antibodies labeled with different fluorescent dyes, because an incomplete TCRαβ/CD3 complex cannot be transported to the cellular membrane. This flow cytometric mutation assay can be applied to CD4+ T cells from human peripheral blood and mouse spleen. Methods for both preparation of target cells and detection of the mutant cells are described.
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References
Cole, J. and Skopek, T. R. (1994) Somatic mutant frequency, mutation rates and mutational spectra in the human population in vivo. Mutat. Res. 304, 33–105.
Kyoizumi, S., Akiyama, M., Hirai, Y., Kusunoki, Y., Tanabe, K., and Umeki, S. (1990) Spontaneous loss and alteration of antigen receptor expression in mature CD4+ T cells. J. Exp. Med. 171, 1981–1999.
Umeki, S., Suzuki, T., Kusunoki, Y., Seyama, T., Fujita, S., and Kyoizumi, S. (1997) Development of a mouse model for studying in vivo T-cell receptor mutations. Mutat. Res. 393, 37–46.
Davodeau, F., Peyrat, M. A., Romagne, F., et al. (1995) Dual T cell receptor beta chain expression on human T lymphocytes. J. Exp. Med. 181, 1391–1398.
Padovan, E., Casorati, G., Dellabona, P., Meyer, S., Brockhaus, M., and Lanzavecchia, A. (1993) Expression of two T cell receptor alpha chains: dual receptor T cells. Science 262, 422–424.
Malissen, M., Trucy, J., Jouvin-Marche, E., Cazenave, P. A., Scollay, R., and Malissen, B. (1992) Regulation of TCR alpha and beta gene allelic exclusion during T-cell development. Immunol. Today 13, 315–322.
Clevers, H., Alarcon, B., Wileman, T., and Terhorst, C. (1988) The T cell receptor/CD3 complex: a dynamic protein ensemble. Annu. Rev. Immunol. 6, 629–662.
Akiyama, M., Kyoizumi, S., Hirai, Y., Kusunoki, Y., Iwamoto, K. S., and Nakamura, N. (1995). Mutation frequency in human blood cells increases with age. Mutat. Res. 338, 141–149.
Kyoizumi, S., Umeki, S., Akiyama, M., et al. (1992) Frequency of mutant T lymphocytes defective in the expression of the T-cell antigen receptor gene among radiation-exposed people. Mutat. Res. 265, 173–180.
Saenko, A. S., Zamulaeva, I. A., Smirnova, S. G, et al. (1998) Determination of somatic mutant frequencies at glycophorin A and T-cell receptor loci for biodosimetry of prolonged irradiation. Int. J. Radiat. Biol. 73, 613–618.
Lantelme, E., Mantovani, S., Palermo, B., et al. (2000) Increased frequency of RAG-expressing, CD4(+)CD3(low) peripheral T lymphocytes in patients with defective responses to DNA damage. Eur. J. Immunol. 30, 1520–1525.
Mei, N., Kunugita, N., Nomoto, S., and Norimura, T. (1996) Comparison of the frequency of T-cell receptor mutants and thioguanine resistance induced by X-rays and ethylnitrosourea in cultured human blood T-lymphocytes. Mutat. Res. 357, 191–197.
Iwamoto, K. S., Mizuno, T., Ito, T., Tsuyama, N., Kyoizumi, S., and Seyama, T. (1996) Gain-of-function p53 mutations enhance alteration of the T-cell receptor following X-irradiation, independently of the cell cycle and cell survival. Cancer Res. 56, 3862–3865.
Ishioka, N., Umeki, S., Hirai, Y., et al. (1997) Stimulated rapid expression in vitro for early detection of in vivo T-cell receptor mutations induced by radiation exposure. Mutat. Res. 390, 269–282.
Iwamoto, K. S., Hirai, Y., Umeki, S., et al. (1994) A positive correlation between T-cell-receptor mutant frequencies and dicentric chromosome frequencies in lymphocytes from radiotherapy patients. J. Radiat. Res. 35, 92–103.
Hirota, H., Kubota, M., Adachi, S., et al. (1994) Somatic mutations at T-cell antigen receptor and glycophorin A loci in pediatric leukemia patients following chemotherapy: comparison with HPRT locus mutation. Mutat. Res. 315, 95–103.
Umeki, S., Kyoizumi, S., Kusunoki, Y., et al. (1991) Flow cytometric measurements of somatic cell mutations in Thorotrast patients. Jpn. J. Cancer Res. 82, 1349–1353.
Akleyev, A. V., Kossenko, M. M., Silkina, L. A., et al. (1995). Health effects of radiation incidents in the southern Urals. Stem Cells 13(suppl. 1), 58–68.
Umeki, S., Kusunoki, Y., Cologne, J. B., et al. (1998). Lifespan of human memory T-cells in the absence of T-cell receptor expression. Immunol. Lett. 62, 99–104.
Kyoizumi, S., Kusunoki, Y., Seyama, T., Hatamochi, A., and Goto, M. (1998) In vivo somatic mutations in Werner’s syndrome. Hum. Genet. 103, 405–410.
Kusunoki, Y., Hayashi, T., Hirai, Y., et al. (1994) Increased rate of spontaneous mitotic recombination in T lymphocytes from a Bloom’s syndrome patient using a flow-cytometric assay at HLA-A locus. Jpn. J. Cancer Res. 85, 610–618.
Suzuki, T., Kusunoki, Y., Tsuyama, N., Ohnishi, H., Seyama, T., and Kyoizumi, S. (2001) Elevated in vivo frequencies of mutant T cells with altered functional expression of the T-cell receptor or hypoxanthine phosphoribosyltransferase genes in p53-deficient mice. Mutat. Res. 483, 13–17.
Shallow, S. O. (1999) Overview of flow cytometry, in Current Protocols in Immunology (Coligan, J. E., Kruisbreek, A. M., Margulies, D. H., Shevach, E. M., and Strober, W., eds.), John Wiley & Sons, New York, Units 5.1 and 5.2.
Otten, G., Yokoyama, W. M., and Holmes, K. L. (1999) Flow cytometry analysis using the Becton Dickinson FACScan, in Current Protocols in Immunology (Coligan, J. E., Kruisbreek, A. M., Margulies, D. H., Shevach, E. M., and Stroer, W., eds.), John Wiley & Sons, New York, Unit 5.4.
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Kyoizumi, S., Kusunoki, Y., Hayashi, T. (2005). Flow Cytometric Measurement of Mutant T Cells With Altered Expression of TCR . In: Keohavong, P., Grant, S.G. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology™, vol 291. Humana Press. https://doi.org/10.1385/1-59259-840-4:197
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DOI: https://doi.org/10.1385/1-59259-840-4:197
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Print ISBN: 978-1-58829-084-7
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