Abstract
Dendritic cells (DCs) are a heterogenous population of professional antigen-presenting cells (APCs) that play a major role in the initiation of immune responses. DC subsets differ in their anatomical locations together with their intrinsic abilities to capture, process, and present antigens on their major histocompatibility (MHC) class I and class II molecules. These features enable each DC subset to have distinct roles in immunity to infection and in the maintenance of self-tolerance. The discrete features of DC subpopulations have largely been defined by cell surface phenotype and anatomical location, rather than function. We have developed direct ex vivo methods to efficiently isolate small numbers of DCs from lymph node (LN) draining tissues and infectious sites to allow fine probing of their function using very sensitive antigen-specific LacZ hybridomas and in vitro proliferation of CFSE-labeled T cells. These approaches are particularly sensitive for detecting endogenous antigens derived from pathogens and self-tissues. Understanding these interactions has begun to allow us to understand how integration of different populations in the DC network responds to multiple scenarios of infection.
Key words
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 subscriptionsReferences
Steinman, R.M. (1991). The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9, 271–296.
Reis e Sousa, C. (2006). Dendritic cells in a mature age. Nat. Rev. Immunol. 6, 476–483.
Wilson, N.S., El-Sukkari, D., Belz, G.T., Smith, C.M., Steptoe, R.J., Heath, W.R., Shortman, K., and Villadangos, J.A. (2003). Most lymphoid organ dendritic cell types are phenotypically and functionally immature. Blood 102, 2187–2194.
Allan, R.S., Waithman, J., Bedoui, S., Jones, C.M., Villadangos, J.A., Zhan, Y., Lew, A.M., Shortman, K., Heath, W.R., and Carbone, F.R. (2006). Migratory dendritic cells transfer antigen to a lymph node-resident dendritic cell population for efficient CTL priming.Immunity 25, 153–162.
Belz, G.T., Smith, C.M., Kleinert, L., Reading, P., Brooks, A., Shortman, K., Carbone, F.R., and Heath, W.R. (2004). Distinct migrating and nonmigrating dendritic cell populations are involved in MHC class I-restricted antigen presentation after lung infection with virus. Proc. Natl. Acad. Sci. USA 101, 8670–8675.
Vremec, D., and Shortman, K. (1997). Dendritic cell subtypes in mouse lymphoid organs: cross-correlation of surface markers, changes with incubation, and differences among thymus, spleen, and lymph nodes. J. Immunol. 159, 565–573.
Masson, F., Mount, A.M., Wilson, N.S., and Belz, G.T. (2008). Dendritic cells: driving the differentiation programme of T cells in viral infections. Immunol. Cell Biol. 86, 333–342.
Belz, G.T., Mount, A., and Masson, F. (2008). Dendritic cells in viral infections. Handbook of Experimental Pharmacology: Dendritic cells. 188:51–77, 2009.
Smith, C.M., Belz, G.T., Wilson, N.S., Villadangos, J.A., Shortman, K., Carbone, F.R., and Heath, W.R. (2003). Cutting Edge: Conventional CD8α+ dendritic cells are preferentially involved in CTL priming after footpad infection with herpes simplex virus-1. J. Immunol. 170, 4437–4440.
Belz, G.T., Behrens, G.M., Smith, C.M., Miller, J.F., Jones, C., Lejon, K., Fathman, C.G., Mueller, S.N., Shortman, K., Carbone, F.R., and Heath, W.R. (2002). The CD8α+ dendritic cell is responsible for inducing peripheral self-tolerance to tissue-associated antigens. J. Exp. Med. 196, 1099–1104.
Quah, B.J., Warren, H.S., and Parish, C.R. (2007). Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Nat. Protoc. 2, 2049–2056.
Lyons, A.B., and Parish, C.R. (1994). Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171, 131–137.
Hawkins, E.D., Hommel, M., Turner, M.L., Battye, F.L., Markham, J.F., and Hodgkin, P.D. (2007). Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data. Nat. Protoc. 2, 2057–2067.
Malarkannan, S., Mendoza, L.M., and Shastri, N. (2001). Generation of antigen-specific, lacZ-inducible T-cell hybrids. Methods Mol. Biol. 156, 265–272.
Fiering, S., Northrop, J.P., Nolan, G.P., Mattila, P.S., Crabtree, G.R., and Herzenberg, L.A. (1990). Single cell assay of a transcription factor reveals a threshold in transcription activated by signals emanating from the T-cell antigen receptor. Genes Dev. 4, 1823–1834.
Karttunen, J., Sanderson, S., and Shastri, N. (1992). Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens. Proc. Natl. Acad. Sci. USA 89, 6020–6024.
Sanderson, S., and Shastri, N. (1994). LacZ inducible, antigen/MHC-specific T cell hybrids. Int. Immunol. 6, 369–376.
Belz, G.T., Bedoui, S., Kupresanin, F., Carbone, F.R., and Heath, W.R. (2007). Minimal activation of memory CD8+ T cell by tissue-derived dendritic cells favors the stimulation of naive CD8+ T cells. Nat. Immunol. 8, 1060–1066.
Badovinac, V.P., Haring, J.S., and Harty, J.T. (2007). Initial T cell receptor transgenic cell precursor frequency dictates critical aspects of the CD8+ T cell response to infection. Immunity 26, 827–841.
Acknowledgements
Research in the author’s laboratory is supported by grants from the National Health and Medical Research Council (Australia), the Howard Hughes Medical Institute (USA), and the Sylvia and Charles Viertel Foundation (Australia). We are grateful to current and past members of this laboratory who have made key contributions to the development of these procedures and for their help.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Belz, G.T. (2010). Direct Ex Vivo Activation of T cells for Analysis of Dendritic Cells Antigen Presentation. In: Naik, S. (eds) Dendritic Cell Protocols. Methods in Molecular Biology, vol 595. Humana Press. https://doi.org/10.1007/978-1-60761-421-0_23
Download citation
DOI: https://doi.org/10.1007/978-1-60761-421-0_23
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-420-3
Online ISBN: 978-1-60761-421-0
eBook Packages: Springer Protocols