Abstract
CD8 T cells play an important role in the regulation of allergic disease. Human and murine CD8 T cells have been shown to be capable of differentiating into distinct subsets defined by cytokine profiles analogous to the Th1 and Th2 subsets and termed T cytotoxic 1 (Tc1, IFN-γ producing) and 2 (Tc2, IL-4 producing).
Effector cell phenotype can be analyzed in vitro on a single cell basis using intracellular cytokine staining and flow cytometry or analysis of other phenotypic markers. Human PBMC usually contain only very low percentages of effector cells which produce relatively high levels of cytokines required for this kind of analysis. It is therefore necessary to activate the T cells to induce rapid accumulation of cytoplasmic cytokines before analysis. This makes it difficult to analyze the antigen specificity of responding T cells but will indicate the type 1/type 2 bias of the population, reflecting previous exposures to antigen. In this chapter, we provide protocols for the generation of polarized populations of CD8 T effector cells using polyclonal stimulation and for their subsequent analysis by intracellular cytokine staining.
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References
Kelso, A. and Groves, P. (1997) A single peripheral CD8+ T cell can give rise to progeny expressing type 1 and/or type 2 cytokine genes and can retain its multipotentiality through many cell divisions. Proc. Natl Acad. Sci. USA 94, 8070–8075.
Sepulveda, H., Cerwenka, A., Morgan, T., and Dutton, R. W. (1999) CD28, IL-2-independent costimulatory pathways for CD8 T lymphocyte activation. J. Immunol. 163, 1133–1142.
Croft, M., Carter, L., Swain, S. L., and Dutton, R. W. (1994) Generation of polarized antigen-specific CD8 effector populations, reciprocal action of interleukin IL-4 and IL-12 in promoting type 2 versus type 1 cytokine profiles. J. Exp. Med. 180, 1715–1728.
Sad, S., Marcotte, R., and Mosmann, T. R. (1995) Cytokine-induced differentiation of precursor mouse CD8+ T cells into cytotoxic CD8+ T cells secreting Th1 or Th2 cytokines. Immunity 2, 271–279.
Vukmanovic-Stejic, M., Vyas, B., Gorak-Stolinska, P., Noble, A., and Kemeny, D. M. (2000) Human Tc1 and Tc2/Tc0 CD8 T-cell clones display distinct cell surface and functional phenotypes. Blood 95, 231–240.
Salgame, P., Convit, J., and Bloom, B. R. (1991) Immunological suppression by human CD8+ T cells is receptor dependent and HLA-DQ restricted. Proc. Natl Acad. Sci. USA 88, 2598–2602.
Inoue, T., Asano, Y., Matsuoka, S., Furutani-Seiki, M., Aizawa, S., and Nishimura, H. (1993) Distinction of mouse CD8+ suppressor effector T cell clones from cytotoxic T cell clones by cytokine production and CD45 isoforms. J. Immunol. 150, 2121–2128.
Noble, A., Macary, P. A., and Kemeny, D. M. (1995) IFNγ and IL-4 regulate the growth and differentiation of CD8+ T cells into subpopulations with distinct cytokine profiles. J. Immunol. 155, 2928–2937.
Carter, L. L. and Dutton, R. W. (1995) Relative perforin-and Fas-mediated lysis in T1 and T2 CD8 effector populations. J. Immunol. 155, 1028–1031.
Noble, A., Zhao, Z., and Cantor, H. (1998) Suppression of immune responses by CD8 cells, 2. Qa-1 on activated B cells stimulates CD8 cell suppression of T helper 2 responses. J. Immunol. 160, 566–571.
Chen, Y., Inobe, J., and Weiner, H. L. (1995) Induction of oral tolerance to myelin basic protein in CD8-depleted mice, both CD4+ and CD8+ cells mediate active suppression. J. Immunol. 155, 910–916.
Farrar, J. D., Smith, J. D., Murphy, T. L., and Murphy, K. M. (2000) Recruitment of Stat4 to the human interferon-alpha/beta receptor requires activated Stat2. J. Biol. Chem. 275, 2693–2697.
Carter, L. L. and Murphy, L. M. (1999) Lineage-specific requirement for signal transducer and activator of transcription Stat4 in interferon-γ production from CD4+ versus CD8+ T cells. J. Exp. Med. 189, 1355–1360.
Rock, K. L., Gamble, S., and Rothstein, L. (1990) Presentation of exogenous antigen with class I major histocompatibility complex molecules. Science 249, 918–921.
McMenamin, C. and Holt, P. G. (1993) The natural immune response to inhaled soluble protein antigens involves major histocompatibility complex (MHC) class Irestricted CD8+ T cell-mediated but MHC class II-restricted CD4+ T cell-dependent immune deviation resulting in selective suppression of immunoglobulin E production. J. Exp. Med. 178, 889–899.
MacAry, P. A., Holmes, B. J., and Kemeny, D. M. (1998) Ovalbumin-specific, MHC class I-restricted, alpha beta-positive, Tc1 and Tc0 CD8+ T cell clones mediate the in vivo inhibition of rat IgE. J. Immunol. 160, 580–588.
Ying, S., Humbert, M., Barkans, J., Corrigan, C. J., Pfister, R., and Menz, G. (1997) Expression of IL-4 and IL-5 mRNA and protein product by CD4+ and CD8+ T cells, eosinophils, and mast cells in bronchial biopsies obtained from atopic and nonatopic (intrinsic) asthmatics. J. Immunol. 158, 3539–3544.
Stanciu, L. A., Shute, J., Promwong, C., Holgate, S. T., and Djukanovic, R. (1997) Increased levels of IL-4 in CD8+ T cells in atopic asthma. J. Allergy Clin. Immunol. 100, 373–378.
Sawicka, E., Noble, A., Walker, C., and Kemeny, D. M. (2004) Tc2 cells respond to soluble antigen in the respiratory tract and induce lung eosinophilia and bronchial hyperresponsiveness. Eur. J. Immunol. 34, 2599–2608.
Brosterhus, H., Brings, S., Leyendeckers, H., Manz, R. A., Miltenyi, S., and Radbruch, A. (1999) Enrichment and detection of live antigen-specific CD4+ and CD8+ T cells based on cytokine secretion. Eur. J. Immunol. 29, 4053–4059.
Jung, T., Schauer, U., Heusser, C., Neumann, C., and Rieger, C. (1993) Detection of intracellular cytokines by flow cytometry. J. Immunol. Methods 159, 197–207.
Bucy, R. P., Karr, L., Huang, G. Q., Li, J., Carter, D., and Honjo, K. (1995) Single cell analysis of cytokine gene coexpression during CD4+ T-cell phenotype development. Proc. Natl Acad. Sci. USA 92, 7565–7569.
Kelso, A., Groves, P., Troutt, A. B., and Francis, K. (1995) Evidence for the stochastic acquisition of cytokine profile by CD4+ T cells activated in a T helper type 2-like response in vivo. Eur. J. Immunol. 25, 1168–1175.
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Vyas, B., Noble, A. (2008). Short-Term Culture of CD8 Cells and Intracellular Cytokine Staining. In: Jones, M.G., Lympany, P. (eds) Allergy Methods and Protocols. Methods in Molecular Medicine, vol 138. Humana Press. https://doi.org/10.1007/978-1-59745-366-0_7
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DOI: https://doi.org/10.1007/978-1-59745-366-0_7
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Print ISBN: 978-0-89603-896-7
Online ISBN: 978-1-59745-366-0
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