It is our hope that this method will prove instrumental to both wet-lab and bioinformatics researchers seeking to leverage scRNA-seq data in elucidating the biology of DCs or other cell types, and that it will contribute toward establishing a high standard of practice in the field.
Dendritic cells (DCs), through their dual roles in innate and adaptive immunity, are characterized by their ability to produce cytokines and present antigens. A dendritic cell subtype, plasmacytoid dendritic cells (pDCs), are uniquely adept at synthesizing type I and type III interferons (IFNs). Their fundamental role in the host's antiviral response is demonstrated during the initial, acute phase of infection by viruses from genetically distant groups. Pathogen nucleic acids are detected by endolysosomal sensors, the Toll-like receptors, which primarily initiate the pDC response. Host nucleic acids can induce pDC responses in some disease states, thus playing a role in the etiology of autoimmune diseases like, specifically, systemic lupus erythematosus. It is essential to note that recent in vitro research from our lab and others has demonstrated that infected cell-pDC physical contact activates recognition of viral infections. At the site of infection, this specialized synapse-like structure enables a powerful discharge of type I and type III interferon. Therefore, the targeted and confined response likely minimizes the detrimental consequences of excessive cytokine release within the host, primarily due to the consequential tissue damage. We outline a pipeline of methods for examining pDC antiviral activity in an ex vivo setting. This pipeline investigates pDC activation in response to cell-cell contact with virally infected cells, and the current methodologies for determining the underlying molecular mechanisms leading to an effective antiviral response.
The process of phagocytosis enables immune cells, particularly macrophages and dendritic cells, to engulf large particles. Removal of a broad range of pathogens and apoptotic cells is accomplished by this essential innate immune defense mechanism. Phagocytosis produces nascent phagosomes which, when they fuse with lysosomes, become phagolysosomes. Containing acidic proteases, these phagolysosomes thus enable the degradation of the ingested substance. Streptavidin-Alexa 488 labeled amine beads are utilized in in vitro and in vivo assays for measuring phagocytosis in murine dendritic cells, as detailed in this chapter. Applying this protocol enables monitoring of phagocytosis in human dendritic cells.
Dendritic cells orchestrate T cell responses through antigen presentation and the delivery of polarizing signals. Mixed lymphocyte reactions provide a means of evaluating the capacity of human dendritic cells to polarize effector T cells. This described protocol, usable with any human dendritic cell, aims to assess its capacity to induce the polarization of CD4+ T helper cells or CD8+ cytotoxic T cells.
Crucial to the activation of cytotoxic T-lymphocytes in cellular immunity is the presentation of peptides from foreign antigens on major histocompatibility complex class I molecules of antigen-presenting cells, a process termed cross-presentation. Antigen-presenting cells (APCs) typically obtain exogenous antigens by (i) internalizing soluble antigens present in their surroundings, (ii) ingesting and processing dead/infected cells using phagocytosis, culminating in MHC I presentation, or (iii) absorbing heat shock protein-peptide complexes generated by the cells presenting the antigen (3). Peptide-MHC complexes, preformed on the surfaces of antigen donor cells (such as cancer or infected cells), can be directly transferred to antigen-presenting cells (APCs) without additional processing, a phenomenon termed cross-dressing in a fourth novel mechanism. Z57346765 The impact of cross-dressing on the dendritic cell-mediated responses to both cancerous and viral threats has been recently observed. Z57346765 To examine the cross-dressing of dendritic cells with tumor antigens, the following methodology is described.
Dendritic cells' antigen cross-presentation is a crucial pathway in initiating CD8+ T-cell responses, vital in combating infections, cancers, and other immune-related diseases. The cross-presentation of tumor-associated antigens is vital for an effective antitumor cytotoxic T lymphocyte (CTL) response, particularly in the setting of cancer. The prevailing cross-presentation assay methodology employs chicken ovalbumin (OVA) as a model antigen, subsequently measuring cross-presenting capacity through the use of OVA-specific TCR transgenic CD8+ T (OT-I) cells. We present in vivo and in vitro procedures for evaluating antigen cross-presentation function with cell-associated OVA.
To fulfill their function, dendritic cells (DCs) adjust their metabolism in response to varying stimuli. Employing fluorescent dyes and antibody-based approaches, we provide a description of how diverse metabolic parameters of dendritic cells (DCs), such as glycolysis, lipid metabolism, mitochondrial function, and the function of key metabolic regulators like mTOR and AMPK, can be analyzed. Standard flow cytometry enables these assays, allowing single-cell analysis of DC metabolic properties and the characterization of metabolic diversity within DC populations.
The widespread applications of genetically engineered myeloid cells, including monocytes, macrophages, and dendritic cells, are evident in both basic and translational research projects. Their vital roles within innate and adaptive immune systems render them alluring prospects for therapeutic cellular products. Gene editing in primary myeloid cells presents a unique challenge, arising from their sensitivity to foreign nucleic acids and the relatively low success rates of current editing methods (Hornung et al., Science 314994-997, 2006; Coch et al., PLoS One 8e71057, 2013; Bartok and Hartmann, Immunity 5354-77, 2020; Hartmann, Adv Immunol 133121-169, 2017; Bobadilla et al., Gene Ther 20514-520, 2013; Schlee and Hartmann, Nat Rev Immunol 16566-580, 2016; Leyva et al., BMC Biotechnol 1113, 2011). Gene knockout in primary human and murine monocytes, as well as monocyte-derived and bone marrow-derived macrophages and dendritic cells, is elucidated in this chapter through nonviral CRISPR-mediated approaches. Recombinant Cas9, bound to synthetic guide RNAs, can be delivered via electroporation to achieve population-wide disruption of single or multiple gene targets.
Antigen phagocytosis and T-cell activation, pivotal mechanisms employed by dendritic cells (DCs), professional antigen-presenting cells (APCs), for coordinating adaptive and innate immune responses, are implicated in inflammatory scenarios like tumor development. Despite a lack of comprehensive understanding regarding the precise nature of dendritic cells (DCs) and their interactions with neighboring cells, deciphering DC heterogeneity, particularly in human cancers, continues to pose a significant hurdle. We outline, in this chapter, a procedure for isolating and characterizing dendritic cells that reside within tumors.
With the role of antigen-presenting cells (APCs), dendritic cells (DCs) are integral to the development of both innate and adaptive immune systems. Different functional specializations and phenotypic characteristics define distinct DC subgroups. DCs are ubiquitous, residing in lymphoid organs and throughout multiple tissues. Their presence, though infrequent and scarce at these locations, presents considerable obstacles to their functional exploration. In an effort to create DCs in the laboratory from bone marrow stem cells, several protocols have been devised, however, these methods do not perfectly mirror the multifaceted nature of DCs present within the body. In light of this, the in-vivo increase in endogenous dendritic cells is put forth as a possible solution for this specific issue. We present in this chapter a protocol to amplify murine dendritic cells in vivo by injecting a B16 melanoma cell line that is engineered to express FMS-like tyrosine kinase 3 ligand (Flt3L), a trophic factor. We have examined two magnetic sorting techniques for amplified dendritic cells (DCs), each achieving high total murine DC recoveries, but displaying different representations of the principal DC subtypes encountered in vivo.
In the intricate dance of immunity, dendritic cells, a diverse population of professional antigen-presenting cells, play the role of an educator. Z57346765 Multiple subsets of dendritic cells collectively trigger and coordinate both innate and adaptive immune responses. Advances in single-cell approaches to investigate cellular transcription, signaling, and function have yielded the opportunity to study heterogeneous populations with exceptional detail. From single bone marrow hematopoietic progenitor cells, the isolation and cultivation of mouse dendritic cell subsets, a process called clonal analysis, has uncovered diverse progenitors with different developmental potentials, enriching our comprehension of mouse DC development. Still, efforts to understand human dendritic cell development have been constrained by the absence of a complementary approach for producing multiple types of human dendritic cells. This protocol details a method for assessing the differentiation capacity of individual human hematopoietic stem and progenitor cells (HSPCs) into multiple DC subsets, alongside myeloid and lymphoid cells. The study of human dendritic cell lineage commitment and its associated molecular basis is facilitated.
Monocytes, circulating in the bloodstream, eventually infiltrate tissues where they differentiate into macrophages or dendritic cells, particularly during instances of inflammation. Various signals encountered in the in vivo environment influence monocyte maturation, determining their eventual fate as either macrophages or dendritic cells. Classical methods for human monocyte differentiation lead to the development of either macrophages or dendritic cells, but not both simultaneously in a single culture. The monocyte-derived dendritic cells, additionally, produced with such methodologies do not closely resemble the dendritic cells that appear in clinical specimens. A protocol for differentiating human monocytes into both macrophages and dendritic cells is described, aiming to produce cell populations that closely resemble their in vivo forms observed in inflammatory fluids.