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Immune cell imaging Immune cell isolation Immune cell staining Immune mechanisms Immunotherapy Inflammatory disorder Mucosal immunologyIn Vitro Bone Marrow–Derived Dendritic Cells (BMDC) Generation for Antigen Presentation Assay
Dendritic cells (DC) are sentinel cells of the immune system that process and present antigens to activate T cells, thus serving to bridge the innate and adaptive immune systems. DCs are particularly efficient at cross-presentation whereby exogenously acquired antigens are processed and presented in context with MHCI molecules to activate CD8+ T cells. Assaying antigen presentation by DCs is a critical parameter in assessing immune functionality. However, the low abundance of bona fide DCs within the lymphoid compartments limits the utility of such assays. An alternative approach employing the culturing of bone marrow cells in the presence of factors needed for DC lineage commitment can result in the differentiation of bone marrow dendritic cells (BMDCs). This protocol details the process of in vitro generation of BMDCs and demonstrates their subsequent utility in antigen presentation assays. The protocol described can be adapted to various conditions and antigens.
Measurement of CD74 N-terminal Fragment Accumulation in Cells Treated with SPPL2a Inhibitor
The recent discovery of human signal peptide peptidase-like 2a (SPPL2a) deficiency in humans revealed the toxicity associated with the accumulation of one of its substrates, CD74 N-terminal fragment (CD74-NTF), for certain type of dendritic cells (cDC2). We developed a two-step protocol for monitoring the accumulation of this molecule in different subsets of PBMCs and immortalized B cells, in which SPPL2a is chemically inhibited and CD74-NTF levels are then assessed by flow cytometry or western blotting. The chemical inhibition of SPPL2a has been described elsewhere, but this is the first time that this inhibition has been reported as a protocol.
Evaluation of Cross-presentation in Bone Marrow-derived Dendritic Cells in vitro and Splenic Dendritic Cells ex vivo Using Antigen-coated Beads
Antigen presentation by MHC class I molecules, also referred to as cross-presentation, elicits cytotoxic immune responses. In particular, dendritic cells (DC) are the most proficient cross-presenting cells, since they have developed unique means to control phagocytic and degradative pathways.
This protocol allows the evaluation of antigen cross-presentation both in vitro (by using bone marrow-derived DC) and ex vivo (by purifying CD8+ DC from spleen after incorporation of particulate antigen) using ovalbumin (OVA)-coupled particles. Cross-presentation efficiency is measured by three different readouts: the B3Z hybridoma T cell line (Karttunen et al., 1992) and stimulation of antigen-specific CD8+ T cells (OT-I) (Kurts et al., 1996), either analyzing OT-I activation by CD69 expression or OT-I proliferation after labeling them with carboxyfluorescein succinimidyl ester (CFSE). By using this approach, we could show recently that DCs are able to increase cross-presentation efficiency transiently upon engagement of TLR4 (Alloatti et al., 2015).
This protocol allows the evaluation of antigen cross-presentation both in vitro (by using bone marrow-derived DC) and ex vivo (by purifying CD8+ DC from spleen after incorporation of particulate antigen) using ovalbumin (OVA)-coupled particles. Cross-presentation efficiency is measured by three different readouts: the B3Z hybridoma T cell line (Karttunen et al., 1992) and stimulation of antigen-specific CD8+ T cells (OT-I) (Kurts et al., 1996), either analyzing OT-I activation by CD69 expression or OT-I proliferation after labeling them with carboxyfluorescein succinimidyl ester (CFSE). By using this approach, we could show recently that DCs are able to increase cross-presentation efficiency transiently upon engagement of TLR4 (Alloatti et al., 2015).
Analysis of Phagosomal Antigen Degradation by Flow Organellocytometry
Professional phagocytes internalize self and non-self particles by phagocytosis to initiate innate immune responses. After internalization, the formed phagosome matures through fusion and fission events with endosomes and lysosomes to obtain a more acidic, oxidative and hydrolytic environment for the degradation of its cargo. Interestingly, phagosome maturation kinetics differ between cell types and cell activation states. This protocol allows to quantify phagosome maturation kinetics on a single organelle level in different types of phagocytes using flow cytometry. Here, ovalbumin (OVA)-coupled particles are used as phagocytosis model system in dendritic cells (DC), which are internalized by phagocytosis. After different time points, phagosome maturation parameters, such as phagosomal degradation of OVA and acquisition of lysosomal proteins (like LAMP-1), can be measured simultaneously in a highly quantitative manner by flow organellocytometry. These read-outs can be correlated to other phagosomal functions, for example antigen degradation, processing and loading in DC.
Phagocytosis Assay to Measure Uptake of Necroptotic Cancer Cells by BMDCs
This protocol is a flow cytometry-based method to measure the phagocytosis efficiency of necroptotic target cells by bone marrow-derived dendritic cells (BMDCs) in vitro (Aaes et al., 2016). The method is a slightly modified and updated version of the protocols used in previously published papers (Krysko et al., 2006; Brouckaert et al., 2004). In brief, the target cells are labeled with a CellTrackerTM dye before they are induced to undergo cell death. After a co-culture period of 2 h with BMDCs, the cells are immunostained with a dendritic cell marker and dead cell marker, and the phagocytic efficiency is quantified using a flow cytometer. This protocol can readily be used for target cells undergoing cell death modalities other than necroptosis.
Skin TRITC Painting to Track Dendritic Cells Migrating to the Lymph Nodes
Our protocol describes a simple method that allows tracking of dendritic cells (DC) migration from the flank skin to draining lymph nodes (LN) using a red fluorescent dye tetramethylrhodamine-5-isothiocyanate (TRITC). TRITC is a photostable dye that readily labels cells including DC in the skin and can survive repeated exposure of two-photon laser excitation for prolonged imaging duration. This method can be combined with various fluorescent antibody labels or transgenic mouse strains (such as CD11c-EYFP) to visualize distinct DC populations simultaneously.
In vivo DCs Depletion with Diphtheria Toxin and MARCO+/MOMA1+ Cells Depletion with Clodronate Liposomes in B6.CD11c-DTR Mice
To evaluate precisely the relative roles of different splenic phagocytic cells during an immune response, efficient methods for the depletion of specific populations are needed. Here, we describe the protocols for the depletion of splenic dendritic cells (DCs) by human diphtheria toxin (DTx) treatment in target mice (which express the human DTx receptor in all CD11c+ DCs) and for the specific depletion of MARCO+/MOMA-1+ marginal zone macrophages (MZMΦs) with clodronate liposomes (ClLip) treatment (when a small dose of ClLip is ministered, MZMΦs preferentially uptake ClLip, and clodronate is released inside those cells causing apoptosis-mediated cell death). These protocols are adaptations from previous works (Jung et al., 2002; McGaha et al., 2011), and were used to evaluate the respective roles of DCs and of MZMΦs during the acute phase of experimental blood-stage malaria infection (Borges da Silva et al., 2015).
Cell Tracer Violet and CellTracker Red CMTPX Staining of Purified Mature Plasmodium-infected Red Blood Cells
Efficient staining methods to identify Plasmodium-infected red blood cells (iRBCs) are crucial to discriminate precisely the immune cells responsible for their elimination from circulation. Here, we describe the protocol for the purification of iRBCs and their subsequent staining with the vital dyes Cell Tracer Violet (CTV) or CellTracker Red CMTPX (CMTPX), both of which readily diffuse into cells and bind covalently to intracellular amines. The iRBCs stained by using this protocol were used in ex vivo phagocytosis assays, to determine the ability of splenic dendritic cells of phagocytizing these parasites (Borges da Silva et al., 2015).
Analysis of T Cell Proliferating and Polarizing Potential of Murine Dendritic Cells in Allogeneic-mixed Leukocyte Reaction
Dendritic cells (DCs) play a critical role in mounting the T cell response against different infectious agents. Nature and intensity of the induced T cell responses are defined by activation status of DCs. It is generally accepted that IL-12, IL-4/IL-5 and IL-23 producing DCs induce TH1, TH2 and TH17 type of immune responses, respectively (Kumar et al., 2015). Besides cytokines, levels of co-stimulatory molecules on DCs also influence the response of T cells.
The activation status of DCs can be determined by examining DC culture supernatants for different cytokines and by analyzing expression of co-stimulatory molecules on these cells. However, these approaches provide indirect information about T cell activating potential of DCs. Analysis of T cell responses in a co-culture system is a more direct approach to examine T cell proliferating and polarizing efficacy of DCs.
A protocol to analyze the T cell proliferating and polarizing potential of DCs in an allogeneic mixed leukocyte reaction (allo-MLR) is described here.
The activation status of DCs can be determined by examining DC culture supernatants for different cytokines and by analyzing expression of co-stimulatory molecules on these cells. However, these approaches provide indirect information about T cell activating potential of DCs. Analysis of T cell responses in a co-culture system is a more direct approach to examine T cell proliferating and polarizing efficacy of DCs.
A protocol to analyze the T cell proliferating and polarizing potential of DCs in an allogeneic mixed leukocyte reaction (allo-MLR) is described here.
Generation and Maturation of Human Monocyte-derived DCs
Dendritic cells (DC) are antigen-presenting cells, which play a critical role in the regulation of the adaptive immune response. They act as a bridge between the innate and the adaptive immune systems. An approach to study their function and potentiality is to generate DC-like cells by culturing CD14+ monocyte-enriched peripheral blood mononuclear cells (PBMC). In the presence of GM-CSF and IL-4, these cultures give rise to large numbers of DC-like cells. Generating human-DC from PBMC is a useful tool to study biological functions of human DC.
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