XCR1 expression distinguishes human conventional dendritic cell type 1 with full effector functions from their immediate precursors.

Dendritic cells (DCs) are major regulators of innate and adaptive immune responses. DCs can be classified into plasmacytoid DCs and conventional DCs (cDCs) type 1 and 2. Murine and human cDC1 share the mRNA expression of XCR1. Murine studies indicated a specific role of the XCR1-XCL1 axis in the induction of immune responses. Here, we describe that human cDC1 can be distinguished into XCR1- and XCR1+ cDC1 in lymphoid as well as nonlymphoid tissues. Steady-state XCR1+ cDC1 display a preactivated phenotype compared to XCR1- cDC1. Upon stimulation, XCR1+ cDC1, but not XCR1- cDC1, secreted high levels of inflammatory cytokines as well as chemokines. This was associated with enhanced activation of NK cells mediated by XCR1+ cDC1. Moreover, XCR1+ cDC1 excelled in inhibiting replication of Influenza A virus. Further, under DC differentiation conditions, XCR1- cDC1 developed into XCR1+ cDC1. After acquisition of XCR1 expression, XCR1- cDC1 secreted comparable level of inflammatory cytokines. Thus, XCR1 is a marker of terminally differentiated cDC1 that licenses the antiviral effector functions of human cDC1, while XCR1- cDC1 seem to represent a late immediate precursor of cDC1.

Six-Color Confocal Immunofluorescence Microscopy with 4-Laser Lines.

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.

Maturation of Monocyte-Derived DCs Leads to Increased Cellular Stiffness, Higher Membrane Fluidity, and Changed Lipid Composition.

Dendritic cells (DCs) are professional antigen-presenting cells of the immune system. Upon sensing pathogenic material in their environment, DCs start to mature, which includes cellular processes, such as antigen uptake, processing and presentation, as well as upregulation of costimulatory molecules and cytokine secretion. During maturation, DCs detach from peripheral tissues, migrate to the nearest lymph node, and find their way into the correct position in the net of the lymph node microenvironment to meet and interact with the respective T cells. We hypothesize that the maturation of DCs is well prepared and optimized leading to processes that alter various cellular characteristics from mechanics and metabolism to membrane properties. Here, we investigated the mechanical properties of monocyte-derived dendritic cells (moDCs) using real-time deformability cytometry to measure cytoskeletal changes and found that mature moDCs were stiffer compared to immature moDCs. These cellular changes likely play an important role in the processes of cell migration and T cell activation. As lipids constitute the building blocks of the plasma membrane, which, during maturation, need to adapt to the environment for migration and DC-T cell interaction, we performed an unbiased high-throughput lipidomics screening to identify the lipidome of moDCs. These analyses revealed that the overall lipid composition was significantly changed during moDC maturation, even implying an increase of storage lipids and differences of the relative abundance of membrane lipids upon maturation. Further, metadata analyses demonstrated that lipid changes were associated with the serum low-density lipoprotein (LDL) and cholesterol levels in the blood of the donors. Finally, using lipid packing imaging we found that the membrane of mature moDCs revealed a higher fluidity compared to immature moDCs. This comprehensive and quantitative characterization of maturation associated changes in moDCs sets the stage for improving their use in clinical application.

Impact of Plasma Membrane Domains on IgG Fc Receptor Function.

Lipid cell membranes not only represent the physical boundaries of cells. They also actively participate in many cellular processes. This contribution is facilitated by highly complex mixtures of different lipids and incorporation of various membrane proteins. One group of membrane-associated receptors are Fc receptors (FcRs). These cell-surface receptors are crucial for the activity of most immune cells as they bind immunoglobulins such as immunoglobulin G (IgG). Based on distinct mechanisms of IgG binding, two classes of Fc receptors are now recognized: the canonical type I FcγRs and select C-type lectin receptors newly referred to as type II FcRs. Upon IgG immune complex induced cross-linking, these receptors are known to induce a multitude of cellular effector responses in a cell-type dependent manner, including internalization, antigen processing, and presentation as well as production of cytokines. The response is also determined by specific intracellular signaling domains, allowing FcRs to either positively or negatively modulate immune cell activity. Expression of cell-type specific combinations and numbers of receptors therefore ultimately sets a threshold for induction of effector responses. Mechanistically, receptor cross-linking and localization to lipid rafts, i.e., organized membrane microdomains enriched in intracellular signaling proteins, were proposed as major determinants of initial FcR activation. Given that immune cell membranes might also vary in their lipid compositions, it is reasonable to speculate, that the cell membrane and especially lipid rafts serve as an additional regulator of FcR activity. In this article, we aim to summarize the current knowledge on the interplay of lipid rafts and IgG binding FcRs with a focus on the plasma membrane composition and receptor localization in immune cells, the proposed mechanisms underlying this localization and consequences for FcR function with respect to their immunoregulatory capacity.

CLEC10A Is a Specific Marker for Human CD1c+ Dendritic Cells and Enhances Their Toll-Like Receptor 7/8-Induced Cytokine Secretion.

Dendritic cells (DCs) are major players for the induction of immune responses. Apart from plasmacytoid DCs (pDCs), human DCs can be categorized into two types of conventional DCs: CD141+ DCs (cDC1) and CD1c+ DCs (cDC2). Defining uniquely expressed surface markers on human immune cells is not only important for the identification of DC subpopulations but also a prerequisite for harnessing the DC subset-specific potential in immunomodulatory approaches, such as antibody-mediated antigen targeting. Although others identified CLEC9A as a specific endocytic receptor for CD141+ DCs, such a receptor for CD1c+ DCs has not been discovered, yet. By performing transcriptomic and flow cytometric analyses on human DC subpopulations from different lymphohematopoietic tissues, we identified CLEC10A (CD301, macrophage galactose-type C-type lectin) as a specific marker for human CD1c+ DCs. We further demonstrate that CLEC10A rapidly internalizes into human CD1c+ DCs upon binding of a monoclonal antibody directed against CLEC10A. The binding of a CLEC10A-specific bivalent ligand (the MUC-1 peptide glycosylated with N-acetylgalactosamine) is limited to CD1c+ DCs and enhances the cytokine secretion (namely TNFα, IL-8, and IL-10) induced by TLR 7/8 stimulation. Thus, CLEC10A represents not only a candidate to better define CD1c+ DCs-due to its high endocytic potential-CLEC10A also exhibits an interesting candidate receptor for future antigen-targeting approaches.

DC subset-specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo.

Dendritic cells (DCs) are efficient antigen-presenting cells equipped with various cell surface receptors for the direct or indirect recognition of pathogenic microorganisms. Interestingly, not much is known about the specific expression pattern and function of the individual activating and inhibitory Fcγ receptors (FcγRs) on splenic DC subsets in vivo and how they contribute to the initiation of T cell responses. By targeting antigens to select activating and the inhibitory FcγR in vivo, we show that antigen uptake under steady-state conditions results in a short-term expansion of antigen-specific T cells, whereas under inflammatory conditions especially, the activating FcγRIV is able to induce superior CD4+ and CD8+ T cell responses. Of note, this effect was independent of FcγR intrinsic activating signaling pathways. Moreover, despite the expression of FcγRIV on both conventional splenic DC subsets, the induction of CD8+ T cell responses was largely dependent on CD11c+CD8+ DCs, whereas CD11c+CD8- DCs were critical for priming CD4+ T cell responses.

Direct Delivery of Antigens to Dendritic Cells via Antibodies Specific for Endocytic Receptors as a Promising Strategy for Future Therapies.

Dendritic cells (DCs) are the most potent professional antigen presenting cells and are therefore indispensable for the control of immunity. The technique of antibody mediated antigen targeting to DC subsets has been the basis of intense research for more than a decade. Many murine studies have utilized this approach of antigen delivery to various kinds of endocytic receptors of DCs both in vitro and in vivo. Today, it is widely accepted that different DC subsets are important for the induction of select immune responses. Nevertheless, many questions still remain to be answered, such as the actual influence of the targeted receptor on the initiation of the immune response to the delivered antigen. Further efforts to better understand the induction of antigen-specific immune responses will support the transfer of this knowledge into novel treatment strategies for human diseases. In this review, we will discuss the state-of-the-art aspects of the basic principles of antibody mediated antigen targeting approaches. A table will also provide a broad overview of the latest studies using antigen targeting including addressed DC subset, targeted receptors, outcome, and applied coupling techniques.

Human lymphoid organ dendritic cell identity is predominantly dictated by ontogeny, not tissue microenvironment.

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.