Crucial for the regulation of adaptive immune responses to pathogens or tumors, dendritic cells (DCs) are specialized antigen-presenting cells that effectively control T cell activation. For our comprehension of immune responses and the development of novel therapies, a critical focus is placed on modeling human dendritic cell differentiation and function. Selitrectinib Because of the low concentration of dendritic cells in human blood, the demand for in vitro systems capable of producing them accurately is substantial. The DC differentiation method, described in this chapter, leverages co-culture of CD34+ cord blood progenitors with mesenchymal stromal cells (eMSCs) genetically modified to release growth factors and chemokines.
Dendritic cells (DCs), a heterogeneous population of antigen-presenting cells, are vital components in both innate and adaptive immune systems. DCs act in a dual role, mediating both protective responses against pathogens and tumors and tolerance toward host tissues. The successful application of murine models in the determination and description of human health-related DC types and functions is a testament to evolutionary conservation between species. In the realm of dendritic cells (DCs), type 1 classical DCs (cDC1s) are uniquely equipped to initiate anti-tumor responses, presenting them as a valuable therapeutic target. However, the limited abundance of dendritic cells, especially cDC1, constrains the achievable number of cells that can be isolated for study. Though considerable work was performed, the development of this field has been impeded by inadequate methods for creating large amounts of functionally mature dendritic cells in vitro. A novel culture method was constructed by co-culturing mouse primary bone marrow cells with OP9 stromal cells expressing Delta-like 1 (OP9-DL1) Notch ligand, which yielded CD8+ DEC205+ XCR1+ cDC1 cells (Notch cDC1), addressing the challenge. A novel approach offers an invaluable resource, facilitating the creation of an unlimited supply of cDC1 cells for functional investigations and translational applications, including anti-tumor vaccination and immunotherapy.
Bone marrow (BM) cells, cultured with growth factors essential for dendritic cell (DC) maturation, such as FMS-like tyrosine kinase 3 ligand (FLT3L) and granulocyte-macrophage colony-stimulating factor (GM-CSF), are commonly used to generate mouse dendritic cells (DCs), as reported by Guo et al. in J Immunol Methods 432(24-29), 2016. In response to the provided growth factors, DC progenitor cells multiply and mature, while other cell types undergo demise during the in vitro culture period, ultimately resulting in relatively homogeneous DC populations. Selitrectinib An alternative methodology, comprehensively explained within these pages, depends on in vitro conditional immortalization of progenitor cells that could mature into dendritic cells, using an estrogen-regulated Hoxb8 protein (ERHBD-Hoxb8). The establishment of these progenitors involves the retroviral transduction of largely unseparated bone marrow cells with a retroviral vector that expresses ERHBD-Hoxb8. Progenitors expressing ERHBD-Hoxb8, when exposed to estrogen, experience Hoxb8 activation, thus inhibiting cell differentiation and facilitating the growth of uniform progenitor cell populations in the presence of FLT3L. The capacity of Hoxb8-FL cells to differentiate into lymphocytes, myeloid cells, and dendritic cells remains intact. The inactivation of Hoxb8, achieved by removing estrogen, results in the differentiation of Hoxb8-FL cells into highly uniform dendritic cell populations closely mirroring their natural counterparts, when cultured in the presence of GM-CSF or FLT3L. The cells' unrestricted proliferative potential and susceptibility to genetic manipulation, exemplified by CRISPR/Cas9, afford a considerable number of opportunities to delve into the intricacies of dendritic cell biology. The following describes the technique for deriving Hoxb8-FL cells from murine bone marrow, detailing the methodology for dendritic cell creation and the application of lentivirally-delivered CRISPR/Cas9 for gene modification.
Mononuclear phagocytes of hematopoietic origin, dendritic cells (DCs), are situated within lymphoid and non-lymphoid tissues. Sentinels of the immune system, DCs are frequently recognized for their ability to detect pathogens and danger signals. Dendritic cells, stimulated, migrate towards the draining lymph nodes, displaying antigens to naïve T cells, thus inducing adaptive immunity. The adult bone marrow (BM) serves as the dwelling place for hematopoietic progenitors that are the source of dendritic cells (DCs). Hence, BM cell culture systems were established to allow for the convenient generation of substantial quantities of primary dendritic cells in vitro, thereby enabling the examination of their developmental and functional properties. This study reviews the diverse protocols used for producing dendritic cells (DCs) in vitro from murine bone marrow cells and assesses the cellular variability within each culture environment.
The immune system's performance is determined by the complex interactions occurring between diverse cell types. In the traditional study of interactions in vivo using intravital two-photon microscopy, a key obstacle is the difficulty in retrieving the cells for further downstream molecular characterization. We have pioneered a technique for labeling cells participating in specific in vivo interactions, which we have termed LIPSTIC (Labeling Immune Partnership by Sortagging Intercellular Contacts). To track CD40-CD40L interactions between dendritic cells (DCs) and CD4+ T cells, we leverage genetically engineered LIPSTIC mice and provide detailed instructions. Animal experimentation and multicolor flow cytometry expertise are essential for this protocol. Selitrectinib The researcher's investigation of the interactions, initiated after the mouse crossing procedure, requires at least three days, potentially longer.
Tissue architecture and cellular distribution are often examined using the method of confocal fluorescence microscopy (Paddock, Confocal microscopy methods and protocols). Molecular biology: exploring biological processes through methods. Humana Press's 2013 publication in New York, encompassing pages 1 to 388, offered a wealth of information. To ascertain the clonal relationship of cells within tissues, multicolor fate mapping of cell precursors is combined with analysis of single-color cell clusters, as demonstrated in (Snippert et al, Cell 143134-144). An in-depth analysis of a key cellular process is detailed in the research article accessible at https//doi.org/101016/j.cell.201009.016. In the calendar year 2010, this phenomenon was observed. A microscopy technique and multicolor fate-mapping mouse model are described in this chapter to track the progeny of conventional dendritic cells (cDCs), inspired by the work of Cabeza-Cabrerizo et al. (Annu Rev Immunol 39, 2021). Regarding the provided DOI, https//doi.org/101146/annurev-immunol-061020-053707, I am unable to access and process the linked article, so I cannot rewrite the sentence 10 times. Scrutinizing the clonality of cDCs, the progenitors from 2021 in various tissues were examined. The chapter is primarily structured around imaging techniques, steering clear of image analysis procedures, though the software utilized for determining cluster formation is presented.
In peripheral tissues, dendritic cells (DCs) function as vigilant sentinels against invasion, upholding immune tolerance. Ingested antigens are transported to draining lymph nodes, where they are presented to antigen-specific T cells, thereby initiating acquired immunity. In order to fully grasp the roles of dendritic cells in immune stability, it is critical to study the migration of these cells from peripheral tissues and evaluate its impact on their functional attributes. This report introduces the KikGR in vivo photolabeling system, an ideal approach for tracking precise cellular movements and related functions in living organisms under physiological conditions, as well as during various immune responses in disease states. Photoconvertible fluorescent protein KikGR, expressed in mouse lines, allows for the labeling of dendritic cells (DCs) in peripheral tissues. The color shift of KikGR from green to red, following violet light exposure, facilitates the precise tracking of DC migration from these peripheral tissues to their corresponding draining lymph nodes.
The antitumor immune response relies heavily on dendritic cells, acting as a vital connection point between innate and adaptive immunity. The diverse and expansive collection of activation mechanisms within dendritic cells is essential for the successful execution of this important task. Dendritic cells (DCs), recognized for their remarkable proficiency in priming and activating T cells through antigen presentation, have been under thorough investigation throughout the past decades. A multitude of studies have pinpointed novel dendritic cell (DC) subtypes, resulting in a considerable array of subsets, frequently categorized as cDC1, cDC2, pDCs, mature DCs, Langerhans cells, monocyte-derived DCs, Axl-DCs, and numerous other types. Within this review, the specific phenotypes, functions, and localization of human dendritic cell subsets within the tumor microenvironment (TME) are analyzed, capitalizing on flow cytometry and immunofluorescence, as well as advanced technologies such as single-cell RNA sequencing and imaging mass cytometry (IMC).
Hematopoietic cells called dendritic cells are proficient at presenting antigens, and in turn, instruct both innate and adaptive immune responses. Lymphoid organs and the majority of tissues host a heterogeneous assortment of cells. Three distinct dendritic cell subsets are commonly identified, which are characterized by divergent developmental lineages, phenotypic distinctions, and specific functional roles. Research on dendritic cells has largely been conducted in mice; therefore, this chapter will compile and discuss recent progress and current understanding of mouse dendritic cell subsets' development, phenotype, and functions.
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