Dendritic cells are major initiators of immune responses, but are also responsible for the maintenance of peripheral T cell tolerance. A critical decision for induction of immune activation or immune tolerance is dependent on the activation status of DCs, which could be induced by the presence of diverse pathogens. For the recognition of pathogen associated molecular patterns (PAMPs) DCs express pattern recognition receptors (PRR), such as Toll-like receptors (TLR), or C-type lectin receptors (CLR). Previously, we have demonstrated, that steady-state DC subpopulations are specialized regarding processing and presentation of captured antigens. Therefore, DCs tightly control and direct activation of different T cell responses. Here, we investigated how TLR-mediated activation of murine cDC1 and cDC2 DCs influences the expression of molecules involved in the MHC class I antigen processing machinery. We provide evidence that activation of cDC2 DCs induces upregulation of Calreticulin, Calnexin, Erap1, ERp57, TAP1, TAP2, and Tapasin under certain immunostimulatory conditions in vivo. Importantly, delivery of targeted antigens to cDC2 DCs in combination with specific TLR-ligands allowed for cross-presentation and activation of CD8+ T cells in wildtype but also BATF3-/-(missing cDC1 DCs) mice in vivo. Moreover, the complete knock out of the three immunoproteasome subunits (β1i, β2i, β5i) hindered the cross-presentation of antigens by cDC2, but not cDC1 DCs. Our findings suggest that DCs harbor a strong flexibility in counteracting infections and tumor development if they are appropriately stimulated.

Confocal immunofluorescence microscopy is an advanced imaging technique routinely applied in the laboratory and clinics. Histological analyses are performed from tissue material. In general, a single fluorochrome per laser is employed, limiting simultaneous analysis to four antigens in one staining with a conventional 4-laser line microscope. Here, we describe a protocol for combining fluorochromes with the same excitation but different emission properties that allows for the analysis of six different antigens in confocal immunofluorescence microscopy with a conventional 4-laser line microscope. The proposed multiplexed method permits the identification and characterization of complex cell populations in rare tissue material.

In mice, conventional and plasmacytoid dendritic cells (DCs) derive from separate hematopoietic precursors before they migrate to peripheral tissues. Moreover, two classes of conventional DCs (cDC1 and cDC2 DCs) and one class of plasmacytoid DCs (pDCs) have been shown to be transcriptionally and functionally distinct entities. In humans, these three DC subtypes can be identified using the cell surface markers CD1c (cDC2), CD141 (cDC1), and CD303 (pDCs), albeit it remains elusive whether DC functionality is mainly determined by ontogeny or the tissue microenvironment. By phenotypic and transcriptional profiling of these three DC subtypes in different human tissues derived from a large number of human individuals, we demonstrate that DC subpopulations in organs of the lymphohematopoietic system (spleen, thymus, and blood) are strongly defined by ontogeny rather than by signals from the microenvironment. In contrast, DC subsets derived from human lung or skin differed substantially, strongly arguing that DCs react toward modulatory signals from tissue microenvironments. Collectively, the data obtained in this study may serve as a major resource to guide further studies into human DC biology during homeostasis and inflammation.

Confocal laser scanning microscopy is an advanced technique for imaging tissue samples in vitro and in vivo at high optical resolution. The development of new fluorochrome variants do not only make it possible to perform multicolor flow cytometry of single cells, but in combination with high resolution laser scanning systems also to investigate the distribution of cells in lymphoid tissues by confocal immunofluorescence analyses, thus allowing the distinction of various cell populations directly in the tissue. Here, we provide a protocol for the visualization of at least six differently fluorochrome-labeled antibodies at the same time using a conventional confocal laser scanning microscope with four laser lines (405 nm, 488 nm, 555 nm, and 639 nm laser wavelength) in both murine and human tissue samples. We further demonstrate that compensation correction algorithms are not necessary to reduce spillover of fluorochromes into other channels when the used fluorochromes are combined according to their specific emission bands and the varying Stokes shift for co-excited fluorochromes with the same laser line.

Dendritic cells (DCs) are central modulators of immune responses and, therefore, interesting target cells for the induction of antitumor immune responses. Ag delivery to select DC subpopulations via targeting Abs to DC inhibitory receptor 2 (DCIR2, clone 33D1) or to DEC205 was shown to direct Ags specifically to CD11c+CD8− or CD11c+CD8+ DCs, respectively, in vivo. In contrast to the increasing knowledge about the induction of immune responses by efficiently cross-presenting CD11c+CD8+ DCs, little is known about the functional role of Ag-presenting CD11c+CD8− DCs with regard to the initiation of protective immune responses. In this study, we demonstrate that Ag targeting to the CD11c+CD8− DC subpopulation in the presence of stimulating anti-CD40 Ab and TLR3 ligand polyinosinic-polycytidylic acid induces protective responses against rapidly growing tumor cells in naive animals under preventive and therapeutic treatment regimens in vivo. Of note, this immunization protocol induced a mixed Th1/Th2-driven immune response, irrespective of which DC subpopulation initially presented the Ag. Our results provide important information about the role of CD11c+CD8− DCs, which have been considered to be less efficient at cross-presenting Ags, in the induction of protective antitumor immune responses.

Dendritic Cells (DCs) are important regulators of immune responses. In our previous studies we found differential antigen presentation capacities of murine DC subpopulations using an in vivo antigen targeting system [1]. In contrast to murine DCs, the functional role of human tissue DCs is largely unknown.