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. This article provides protocols with top ticks and pitfalls for preparation and successful generation of mouse and human DC from different cellular sources, such as murine BM and HoxB8 cells, as well as human CD34⁺ cells from cord blood, BM, and peripheral blood or peripheral blood monocytes. We describe murine cDC1, cDC2, and pDC generation with Flt3L and the generation of BM‐derived DC with GM‐CSF. Protocols for human DC generation focus on CD34⁺ cell culture on OP9 cell layers for cDC1, cDC2, cDC3, and pDC subset generation and DC generation from peripheral blood monocytes (MoDC). Additional protocols include enrichment of murine DC subsets, CRISPR/Cas9 editing, and clinical grade human DC generation. 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.

The major human pathogen Staphylococcus aureus has very efficient strategies to subvert the human immune system. Virulence of the emerging community-associated methicillin-resistant S. aureus depends on phenol-soluble modulin (PSM) peptide toxins, which are known to attract and lyse neutrophils. However, their influences on other immune cells remain elusive. In this study, we analyzed the impact of PSMs on dendritic cells (DCs) playing an essential role in linking innate and adaptive immunity. In human neutrophils, PSMs exert their function by binding to the formyl peptide receptor (FPR) 2. We show that mouse DCs express the FPR2 homolog mFPR2 as well as its paralog mFPR1 and that PSMs are chemoattractants for DCs at noncytotoxic concentrations. PSMs reduced clathrin-mediated endocytosis and inhibited TLR2 ligand-induced secretion of the proinflammatory cytokines TNF, IL-12, and IL-6, while inducing IL-10 secretion by DCs. As a consequence, treatment with PSMs impaired the capacity of DCs to induce activation and proliferation of CD4⁺ T cells, characterized by reduced Th1 but increased frequency of FOXP3⁺ regulatory T cells. These regulatory T cells secreted high amounts of IL-10, and their suppression capacity was dependent on IL-10 and TGF-β. Interestingly, the induction of tolerogenic DCs by PSMs appeared to be independent of mFPRs, as shown by experiments with mice lacking mFPR2 (mFPR2^−/−) and the cognate G protein (p110γ^−/−). Thus, PSMs from highly virulent pathogens affect DC functions, thereby modulating the adaptive immune response and probably increasing the tolerance toward the pathogen.

CD4⁺ T cells are essential for the control of Yersinia enterocolitica (Ye) infection in mice. Ye can inhibit dendritic cell (DC) antigen uptake and degradation, maturation and subsequently T-cell activation in vitro. Here we investigated the effects of Ye infection on splenic DCs and T-cell proliferation in an experimental mouse infection model. We found that OVA-specific CD4⁺ T cells had a reduced potential to proliferate when stimulated with OVA after infection with Ye compared to control mice. Additionally, proliferation of OVA-specific CD4⁺ T cells was markedly reduced when cultured with splenic CD8α⁺ DCs from Ye infected mice in the presence of OVA. In contrast, T-cell proliferation was not impaired in cultures with CD4⁺ or CD4⁻CD8α⁻ DCs isolated from Ye infected mice. However, OVA uptake and degradation as well as cytokine production were impaired in CD8α⁺ DCs, but not in CD4⁺ and CD4⁻CD8α⁻ DCs after Ye infection. Pathogenicity factors (Yops) from Ye were most frequently injected into CD8α⁺ DCs, resulting in less MHC class II and CD86 expression than on non-injected CD8α⁺ DCs. Three days post infection with Ye the number of splenic CD8α⁺ and CD4⁺ DCs was reduced by 50% and 90%, respectively. The decreased number of DC subsets, which was dependent on TLR4 and TRIF signaling, was the result of a faster proliferation and suppressed de novo DC generation. Together, we show that Ye infection negatively regulates the stimulatory capacity of some but not all splenic DC subpopulations in vivo. This leads to differential antigen uptake and degradation, cytokine production, cell loss, and cell death rates in various DC subpopulations. The data suggest that these effects might be caused directly by injection of Yops into DCs and indirectly by affecting the homeostasis of CD4⁺ and CD8α⁺ DCs. These events may contribute to reduced T-cell proliferation and immune evasion of Ye. Dendritic cells (DCs) are crucial in promoting immune responses against pathogens. Mouse DCs consist of different subpopulations but their role in immunity to pathogens and immune evasion is largely unclear. The enteric pathogen Yersinia enterocolitica (Ye) was shown to evade DC functions in bone marrow-derived DCs in vitro inhibiting antigen uptake and degradation, maturation and subsequently T-cell activation. However, it is controversial whether and, if so, which virulence factors of Ye (e.g. Yops) contribute to immune evasion of DCs in vivo. Using an experimental mouse infection model and a β-lactamase reporter system to track Yop injection into host cells we demonstrate here for the first time that distinct DC subpopulations are affected by Ye infection in vivo in terms of antigen uptake and degradation, cytokine production, and T-cell proliferation. Moreover, Ye infection causes the loss of 90% of CD11c^hiCD4⁺ DCs in a TLR4- and TRIF-signaling dependent manner. These data combined with results reported from infection with e.g. Mycobacterium tuberculosis, Salmonella typhimurium, or Escherichia coli suggest that the response of DCs to bacterial infections is manifold, reflecting the diversity of DC subpopulations, pathogenicity factors, and life styles of the pathogens.

Dendritic cells (DCs) as professional antigen-presenting cells play an important role in the initiation and modulation of the adaptive immune response. However, their role in the innate immune response against bacterial infections is not completely defined. Here we have analyzed the role of DCs and their impact on the innate anti-bacterial host defense in an experimental infection model of Yersinia enterocolitica (Ye). We used CD11c-diphtheria toxin (DT) mice to deplete DCs prior to severe infection with Ye. DC depletion significantly increased animal survival after Ye infection. The bacterial load in the spleen of DC-depleted mice was significantly lower than that of control mice throughout the infection. DC depletion was accompanied by an increase in the serum levels of CXCL1, G-CSF, IL-1α, and CCL2 and an increase in the numbers of splenic phagocytes. Functionally, splenocytes from DC-depleted mice exhibited an increased bacterial killing capacity compared to splenocytes from control mice. Cellular studies further showed that this was due to an increased production of reactive oxygen species (ROS) by neutrophils. Adoptive transfer of neutrophils from DC-depleted mice into control mice prior to Ye infection reduced the bacterial load to the level of Ye-infected DC-depleted mice, suggesting that the increased number of phagocytes with additional ROS production account for the decreased bacterial load. Furthermore, after incubation with serum from DC-depleted mice splenocytes from control mice increased their bacterial killing capacity, most likely due to enhanced ROS production by neutrophils, indicating that serum factors from DC-depleted mice account for this effect. In summary, we could show that DC depletion triggers phagocyte accumulation in the spleen and enhances their anti-bacterial killing capacity upon bacterial infection. Dendritic cells (DCs) are professional antigen-presenting cells playing a crucial role in the initiation of T-cell responses to combat infection. DCs adapt their immune response according to the type of pathogen. For example, in response to intracellular bacteria, DCs produce IL-12, thereby initiating Th1 polarization, whereas in response to extracellular parasites or extracellular bacteria, DCs instruct Th2 or Th17 polarization, respectively. Nevertheless, their role in innate immunity is less well understood. To address this, we studied the role of DCs upon infection with the Gram-negative enteropathogenic bacteria Yersinia enterocolitica (Ye) and used a mouse model to deplete DCs. We found that DCs have an unexpected role during severe infection as depletion of these cells resulted in better outcome of infection as well as less bacterial load. We also found that DC depletion increased the number of phagocytes with improved anti-bacterial capacity in the spleen. Our study provides new insights into the role of DCs in innate immune response upon bacterial infection and points towards a complex interaction between DCs and phagocyte homeostasis. DC alteration during infection might also be an interesting target for immunotherapy in the future to guide the outcome of infection.

Summary: Antigen uptake by dendritic cells is essential for the induction of antigen‐specific T‐cell responses. Here, we investigate the ability of dendritic cells from different mouse strains to endocytose antigens. The uptake of different fluorescently labelled soluble antigens by bone marrow‐derived dendritic cells from BALB/c, C57BL/6 and C3H/HeN mice was analysed by flow cytometry. Using transferrin as a specific marker for clathrin‐mediated endocytosis, we observed no significant differences of transferrin uptake by dendritic cells from BALB/c, C57BL/6 and C3H/HeN mice. Similar results were obtained by analysing macropinocytosis with lucifer yellow. In contrast, analysing the uptake of ovalbumin, which is predominantly mediated by clathrin‐mediated endocytosis via the macrophage mannose receptor, we found that dendritic cells from C3H/HeN mice take up three‐ to fivefold more ovalbumin than dendritic cells from BALB/c or C57BL/6 mice. Blocking the uptake of ovalbumin via the macrophage mannose receptor by using mannan led to a comparable uptake of ovalbumin by dendritic cells from all three mouse strains. Consistently, dendritic cells from C3H/HeN mice displayed significantly increased expression of the macrophage mannose receptor compared to dendritic cells from BALB/c or C57BL/6 mice. In conclusion, receptors involved in antigen uptake such as the macrophage mannose receptor may be differentially expressed and may explain variations of T‐cell responses after vaccination in different individuals.