Abstract
Tracking antigen-specific cytotoxic T lymphocyte (CTL) function in vivo can be difficult due to the need to monitor the presence and subsequent destruction of antigen-bearing target cells. In this report, we describe a simple method using the fluorescent dye 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) to evaluate CD8+ T-cell effector function in vivo by flow cytometry. In this assay, peptide-pulsed and control target cells are labeled to different levels with CFSE and coadministered to animals that have been previously immunized or tolerized to the cognate antigen. Because naïve antigen-specific CD8+ T cells cannot acquire effector function within the time frame of this assay, adoptively transferred nonimmunized animals are used as negative controls for in vivo CTL function. Target cells are syngeneic splenocytes pulsed with peptide antigen and control cells are unpulsed syngeneic splenocytes. The loss of antigen-specific target cells is indicative of cytotoxicity and immunity, whereas the lack of killing in the setting of antigen recognition is suggestive of tolerance.
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References
Itano, A. A., McSorley, S. J., Reinhardt, R. L., et al. (2003) Distinct dendritic cell populations sequentially present antigen to CD4 T cells and stimulate different aspects of cell-mediated immunity. Immunity 19, 47–57.
Kearney, E. R., Pape, A., Loh, D. Y., and Jenkins, M. K. (1994) Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. Immunity 1, 327–339.
Curtsinger, J. M., Johnson, M., and Mescher, M. F. (2003) CD8 T cell clonal expansion and development of effector function require prolonged exposure to antigen, costimulation, and signal 3 cytokine. J. Immunol. 171, 5165–5171.
Grabie, N., Delfs, M. W., Westrich, J. R., et al. (2003) IL-12 is required for differentiation of pathogenic CD8+ T cell effectors that cause myocarditis. J. Clin. Invest. 111, 671–680.
Hernandez, J., Aung, S., Marquardt, K., and Sherman, L. A. (2002) Uncoupling of proliferative potential and gain of effector function by CD8(+) T cells responding to self-antigens. J. Exp. Med. 196, 323–333.
Curtsinger, J. M., Lins, D. C., and Mescher, M. F. (2003) Signal 3 determines tolerance versus full activation of naive CD8 T cells: dissociating proliferation and development of effector function. J. Exp. Med. 197, 1141–1151.
Filatenkov, A. A., Jacovetty, E. L., Fischer, U., Curtsinger, J. M., Mescher, M. F., and Ingulli, E. (2005) CD4 T cell-dependent conditioning of dendritic cells to produce IL-12 results in CD8-mediated graft rejection and avoidance of tolerance. J. Immunol. 174, 6909–6917.
Oehen, S., Brduscha-Riem, K., Oxenius, A., and Odermatt, B. (1997) A simple method for evaluating the rejection of grafted spleen cells by flow cytometry and tracing adoptively transferred cells by light microscopy. J. Immunol. Methods 207, 33–42.
Weston, S. A. and Parish, C. R. (1990) New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy. J. Immunol. Methods 133, 87–97.
Lyons, A. B. and Parish, R. (1994) Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171, 131–137.
Aichele, P., Brduscha-Riem, K., Oehen, S., et al. (1997) Peptide antigen treatment of naive and virus-immune mice: antigen-specific tolerance versus immunopathology. Immunity 6, 519–529.
Coles, R. M., Mueller, S. N., Heath, W. R., Carbone, F. R., and Brooks, A. G. (2002) Progression of armed CTL from draining lymph node to spleen shortly after localized infection with herpes simplex virus 1. J. Immunol. 168, 834–838.
Mueller, S. N., Jones, M., Smith, M., Heath, W. R., and Carbone, F. R. (2002) Rapid cytotoxic T lymphocyte activation occurs in the draining lymph nodes after cutaneous herpes simplex virus infection as a result of early antigen presentation and not the presence of virus. J. Exp. Med. 195, 651–656.
Oehen, S. and Brduscha-Riem, K. (1998) Differentiation of naive CTL to effector and memory CTL: correlation of effector function with phenotype and cell division. J. Immunol. 161, 5338–5346.
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© 2007 Humana Press Inc., Totowa, NJ
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Ingulli, E. (2007). Tracing Tolerance and Immunity In Vivo by CFSE-Labeling of Administered Cells. In: Fairchild, P.J. (eds) Immunological Tolerance. Methods in Molecular Biology™, vol 380. Humana Press. https://doi.org/10.1007/978-1-59745-395-0_23
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DOI: https://doi.org/10.1007/978-1-59745-395-0_23
Publisher Name: Humana Press
Print ISBN: 978-1-58829-652-8
Online ISBN: 978-1-59745-395-0
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