免疫学

T follicular helper (Tfh) cells regulate B cell selection for entry into the germinal center (GC) reaction or for differentiation into antibody forming cells. This process takes place at the border between the T and B zones in lymphoid organs and involves physical contacts between T and B cells. During these interactions, T cells endow the B cells with selection signals that promote GC seeding or plasmablast differentiation based on their B cell receptor affinity. In Peyer’s patches (PPs), T cells promote B cell colonization of the subepithelial dome (SED) without effective affinity-based clonal selection. To specifically characterize the T cell population that resides within the SED niche, we performed ex vivo photoactivation of the SED compartment followed by flow cytometry analysis of the labeled cells, as described in this protocol. This technique integrates both spatial and cellular information in studies of immunological niches and can be adapted to various experimental systems.

The main purpose of this sophisticated and highly versatile method is to visualize and quantify structural vessel wall properties, cellular recruitment, and lipid/dextran extravasation under physiological conditions in living arteries. This will be of interest for a broad range of researchers within the field of inflammation, hypertension, atherosclerosis, and even the pharmaceutical industry. Currently, many researchers are using in vitro techniques to evaluate cellular recruitment, like transwell or flow chamber systems with cultured cells, with unclear physiological comparability. The here introduced method describes in detail the use of a sophisticated and flexible method to study arterial wall properties and leukocyte recruitment in fresh and viable murine carotid arteries ex vivo under arterial flow conditions. This model mimics the in vivo situation and allows the use of cells and arteries isolated from two different donors (for example, wildtype vs. specific knockouts) to be combined into one experiment,thereby providing information on both leukocyte and/or endothelial cell properties of both donors. As such, this model can be considered an alternative for the complicated and invasive in vivo studies, such as parabiotic experiments.