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

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, [3H] 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.