This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Recent studies have provided evidence for an increasing number of phenotypically distinct conventional DC (cDC) subsets that on one hand exhibit a certain functional plasticity, but on the other hand are characterized by their tissue- and context-dependent functional specialization. Here, we describe a selection of assays for the functional characterization of mouse and human cDC. The first two protocols illustrate analysis of cDC endocytosis and metabolism, followed by guidelines for transcriptomic and proteomic characterization of cDC populations. Then, a larger group of assays describes the characterization of cDC migration in vitro, ex vivo, and in vivo. The final guidelines measure cDC inflammasome and antigen (cross)-presentation activity. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

Dengue viruses (DENVs) are re-emerging pathogens transmitted by mosquitoes mainly in tropical and subtropical regions. Each year, they are estimated to infect 390 million people globally. The major challenge confronting dengue vaccine development is the need to induce balanced, long lasting tetravalent immune responses against four co-circulating virus serotypes (DENV-I, -II, -III, -IV), because primary infection by any one of which may predispose infected individuals to more severe diseases during a heterotypic secondary infection. Another difficulty is to select representative strains in vaccine design to provide cross-protection against most circulating virus strains. In this study, aimed at developing a tetravalent subunit vaccine with a representative single protein, we designed two vaccines (named cE80(D4) and cE80(max)) based on the consensus sequences of the ectodomain of envelope protein of 3127 DENV strains, and then expressed them in the baculovirus expression system. Both vaccines were capable of eliciting specific antibodies against all four DENV serotypes, and the predominant IgG subtype elicited by the two vaccines was IgG1. Moreover, these vaccines activated both type I and type II antigen-specific helper T cells that secreted IFN-γ and IL-4, respectively. This proof-of-concept study has set foundation for further optimization of a single protein-based tetravalent DENV vaccine.