Guidelines for mouse and human DC functional assays

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

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

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

DC subset-specific induction of T cell responses upon antigen uptake via Fc gamma 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 Fc gamma receptors (Fc gamma Rs) 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 Fc gamma R 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 Fc gamma RIV is able to induce superior CD4(+) and CD8(+) T cell responses. Of note, this effect was independent of Fc gamma R intrinsic activating signaling pathways. Moreover, despite the expression of Fc gamma RIV 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

Efeito da radiação gama em soluções de aflatoxinas

Os fungos filamentosos, seres ubíquos na natureza, são muitas vezes parasitas de produtos alimentares, nomeadamente produtos agrícolas. A sua presença, embora encarada como natural, poderá não ser inócua, uma vez que alguns fungos são capazes de produzir compostos tóxicos, nomeadamente micotoxinas (e.g., aflatoxinas). As aflatoxinas, metabolito secundário produzido por Aspergillus flavus e Aspergillus parasiticus, são altamente tóxicas, mutagénicas e carcinogénicas. Por forma a garantir a segurança alimentar no que se refere à presença destes metabolitos em alimentos várias alternativas têm vindo a ser testadas. A irradiação de alimentos (e.g, radiação gama) é uma dessas alternativas [1]; no entanto, a identificação dos produtos de degradação das micotoxinas pela utilização desta tecnologia, assim como a avaliação da sua toxicidade, está por realizar. Com o trabalho realizado pretendeu-se verificar qual o efeito da radiação gama em soluções de aflatoxinas (B1, B2, G1 e G2). Para tal, soluções de aflatoxinas (B1, B2, G1 e G2) foram submetidas à radiação gama nas seguintes doses: 0, 0,5; 1,0; 3,0; 6,0 kGy. Após irradiação, a quantificação de aflatoxinas e a deteção de produtos de degradação foi efetuada por cromatografia líquida, com deteção por fluorescência e derivatização pós-coluna. Os resultados obtidos mostraram que existe uma diminuição da concentração de aflatoxinas nas soluções irradiadas, embora a sensibilidade de cada toxina à irradiação seja diferente. Para a dose mais elevada de radiação testada (6,0 kGy) a diminuição foi superior a 80 % para G1, G2 e B1. Além disto, em todas as amostras irradiadas, verifica-se a formação de compostos de degradação de aflatoxinas. A concentração destes compostos aumenta até à dose de 1,0 kGy, diminuindo nas restantes doses. O estudo da estrutura e da toxicidade destes compostos tem que ser levados a cabo de modo a ponderar a eficácia desta metodologia no controlo de micotoxinas em alimentos

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