Abstract
Specific immune responses are mediated by activated CD4+ T-helper (Th) cells. Two major subsets, denoted Th1 and Th2, have been identified that are characterized by their distinctive cytokine secretion pattern and associated effector functions. The signature cytokines of Th1 and Th2 cells are interferon-γ and interleukin-4, respectively. Because of the dominant role of Th cells in directing specific immunity, the analysis of Th subsets by means of determining their signature cytokines has contributed greatly to the progress that has been made in recent years in the understanding of protective as well as pathogenic immune responses. Several methods, such as reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, ELISpot, and intracellular flow cytometric analysis are used for the analysis of T-cell cytokines and, thus, of Th subsets. Here, we briefly discuss the advantages and disadvantages of these methods and describe in detail a standard protocol for the analysis of human Th subsets by means of detection of cytoplasmic cytokines by flow cytometry.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin, M. A. and Coffman, R. L. (1986) Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 136, 2348–2357.
Abbas, A. K., Murphy, K. M., and Sher, A. (1996) Functional diversity of helper T lymphocytes. Nature 383, 787–793.
Yssel, H., de Waal Malefyt, R., Roncarolo, M. G., et al. (1992) IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells. J. Immunol. 149, 2378–2384.
Sallusto, F., Lanzavecchia, A., and Mackay. C. R. (1998) Chemokines and chemokine receptors in T-cell priming and Th1/Th2-mediated responses. Immunol. Today 19, 568–574.
Nanki, T. and Lipsky, P. E. (2000) Cytokine, activation marker, and chemokine receptor expression by individual CD4(+) memory T cells in rheumatoid arthritis synovium. Arthritis Res. 2, 415–423.
Andersson, U., Andersson, J., Lindfors, A., Wagner, K., Moller, G., and Heusser, C. H. (1990) Simultaneous production of interleukin 2, interleukin 4 and interferongamma by activated human blood lymphocytes. Eur. J. Immunol 20, 1591–1596.
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.
Prussin, C. and Metcalfe, D. D. (1995) Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J. Immunol. Methods 188, 117–128.
Sander, B., Andersson, J., and Andersson, U. (1991) Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunol. Rev. 119, 65–93.
Picker, L. J., Singh, M. K., Zdraveski, Z., et al. (1995) Direct demonstration of cytokine synthesis heterogeneity among human memory/effector T cells by flow cytometry. Blood 86,:1408–1419.
Schulze-Koops, H., Lipsky, P. E., and Davis, L. S. (1998) Human memory T cell differentiation into Th2-like effector cells is dependent on IL-4 and CD28 and inhibited by TCR ligation. Eur. J. Immunol. 28, 2517–2529.
Skapenko, A., Wendler, J., Lipsky, P. E., Kalden, J. R., and Schulze-Koops, H. (1999) Altered memory T cell differentiation in patients with early rheumatoid arthritis. J. Immunol. 163, 491–499.
Maino, V. C. and Picker, L. J. (1998) Identification of functional subsets by flow cytometry: intracellular detection of cytokine expression. Cytometry 34, 207–215.
Skapenko, A., Lipsky, P. E., Kraetsch, H. G., Kalden, J. R., and Schulze-Koops, H. (2001) Antigen-independent Th2 cell differentiation by stimulation of CD28: regulation via IL-4 gene expression and mitogen-activated protein kinase activation. J. Immunol. 166, 4283–4292.
Dimitrova, P., Skapenko, A., Herrmann, M. L., Schleyerbach, R., Kalden, J. R., and Schulze-Koops, H. (2002) Restriction of de novo pyrimidine biosynthesis inhibits Th1 cell activation and promotes Th2 cell differentiation. J. Immunol. 169, 3392–3399.
Skapenko, A., Leipe, J., Niesner, U., et al. (2004) GATA-3 in Human T Cell Helper Type 2 Development. J. Exp. Med. 199, 423–428.
Coffman, R. L., Lebman, D. A., and Rothman, P. (1993) Mechanism and regulation of immunoglobulin isotype switching. Adv. Immunol. 54, 229–270.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Skapenko, A., Schulze-Koops, H. (2007). Analysis of Th1/Th2 T-Cell Subsets. In: Cope, A.P. (eds) Arthritis Research. Methods in Molecular Medicine, vol 136. Humana Press. https://doi.org/10.1007/978-1-59745-402-5_7
Download citation
DOI: https://doi.org/10.1007/978-1-59745-402-5_7
Publisher Name: Humana Press
Print ISBN: 978-1-58829-918-5
Online ISBN: 978-1-59745-402-5
eBook Packages: Springer Protocols