An advanced approach for the characterization of dendritic cell-induced T cell proliferation in situ

• Published in: Immunobiology (1979) Vol. 215; no. 9; pp. 855 - 862
• Main Authors: Florian, Christian, Barth, Thomas, Wege, Anja K, Männel, Daniela N, Ritter, Uwe
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier GmbH 01.09.2010
• Subjects: Adaptive Immunity; Advanced Basic Science; Allergy and Immunology; Animals; Antigen presentation; BrdU incorporation; Bromodeoxyuridine - metabolism; Cell Proliferation; Cell Separation; Cells, Cultured; Cryoultramicrotomy; Feasibility Studies; Flow Cytometry - methods; Fluorochromes; Immunophenotyping; In Situ Hybridization, Fluorescence - methods; Leishmania major - immunology; Leishmania major - pathogenicity; Leishmaniasis, Cutaneous - diagnosis; Leishmaniasis, Cutaneous - immunology; Lymph node; Lymph Nodes - pathology; Mice; Mice, Inbred C57BL; Proliferation; T cells; T-Lymphocytes - immunology; T-Lymphocytes - metabolism; T-Lymphocytes - microbiology; T-Lymphocytes - pathology; TissueQuest; Lymph node; DAB; Fluorochromes; 3′3′diaminobenzidine; Proliferation; T cells; EGFP; L; 5-bromo-2-deoxyuridine; TissueQuest; BrdU incorporation; Leishmania; Enhanced green fluorescent protein; BrdU
• ISSN: 0171-2985; 1878-3279
• DOI: 10.1016/j.imbio.2010.05.017

Abstract It is commonly accepted that dendritic cells (DCs) play a pivotal role in the induction of adaptive T cell-mediated immune responses. The clonal expansion of antigen-specific T cells within secondary lymphoid organs reflects the efficiency of antigen processing and presentation by DCs. Consequently, the quantification of proliferating antigen-specific T cells represents an important read-out for analyzing DC–T cell interactions. Standard proliferation assays are usually performed with cell suspensions of antigen-stimulated lymphocytes labelled with carboxyfluorescein diacetate succinimidyl ester, [3 H] thymidine, or 5-bromo-2-deoxyuridine. However, these assays have important limitations, including the complete loss of information about the localization of proliferating cells in situ . Additionally, the ex vivo stimulation with antigen does not necessarily reflect the in vivo situation of cell proliferation. Therefore, an advanced approach for a detailed characterization of proliferating T cells in situ would be helpful for the interpretation of ongoing adaptive immune responses. Here, we describe the development of a fluorescence-based multicolour assay allowing the characterization and quantification of proliferation in different T cell subtypes in cryosections of lymphoid organs. Our approach combines the major benefits of flow cytometry and immunofluorescent-based histology, such as (i) multicolour phenotyping of cell populations and (ii) description of tissue-specific sites of cell proliferation.