Controlling the activation of T cells, dendritic cells (DCs) are professional antigen-presenting cells, thereby regulating the adaptive immune response against both pathogens and tumors. A critical aspect of comprehending immune responses and advancing therapeutic strategies lies in modeling the differentiation and function of human dendritic cells. learn more The scarcity of dendritic cells in human blood highlights the critical requirement for in vitro systems accurately producing them. This chapter will detail a DC differentiation method, which relies on the co-culture of CD34+ cord blood progenitor cells with mesenchymal stromal cells (eMSCs) that have been genetically modified to secrete growth factors and chemokines.
DCs, a heterogeneous group of antigen-presenting cells, are instrumental in coordinating both innate and adaptive immune mechanisms. While DCs orchestrate defensive actions against pathogens and tumors, they also mediate 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. Type 1 classical dendritic cells (cDC1s), a distinct subset of dendritic cells (DCs), uniquely facilitate anti-tumor responses, making them a promising area for therapeutic exploration. However, the uncommonness of DCs, particularly cDC1, restricts the number of cells that can be isolated for in-depth examination. Remarkable attempts notwithstanding, the progress in this domain has been hampered by the absence of appropriate techniques for creating substantial numbers of functionally mature DCs 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. This novel method equips researchers with a valuable tool for generating unlimited numbers of cDC1 cells, which is crucial for functional studies and translational applications like anti-tumor vaccination and immunotherapy.
Cells from the bone marrow (BM) are routinely isolated and cultured to produce mouse dendritic cells (DCs) in the presence of growth factors like FMS-like tyrosine kinase 3 ligand (FLT3L) and granulocyte-macrophage colony-stimulating factor (GM-CSF), supporting DC maturation, as detailed in Guo et al. (J Immunol Methods 432:24-29, 2016). DC progenitor cells, in response to these growth factors, augment in number and differentiate, leaving other cell types to decline during the in vitro culture, thus yielding relatively homogenous DC populations. learn more Conditional immortalization of progenitor cells displaying dendritic cell potential in vitro, using an estrogen-regulated form of Hoxb8 (ERHBD-Hoxb8), represents an alternative method, thoroughly investigated in this chapter. Retroviral transduction of largely unseparated bone marrow cells using a retroviral vector carrying the ERHBD-Hoxb8 gene establishes these progenitors. Application of estrogen to ERHBD-Hoxb8-expressing progenitor cells leads to Hoxb8 activation, impeding cellular differentiation and allowing for the augmentation of homogenous progenitor cell populations cultivated with FLT3L. The lineage potential of Hoxb8-FL cells extends to lymphocytes, myeloid cells, and, crucially, dendritic cells. Hoxb8-FL cells, in the presence of GM-CSF or FLT3L, differentiate into highly homogenous dendritic cell populations closely resembling their physiological counterparts, following the inactivation of Hoxb8 due to estrogen removal. Because of their unrestricted ability to multiply and their responsiveness to genetic modification techniques like CRISPR/Cas9, these cells present a diverse range of possibilities for examining dendritic cell (DC) biology. I describe the process for generating Hoxb8-FL cells from mouse bone marrow, including the methods for dendritic cell generation and CRISPR/Cas9 gene deletion via lentiviral vectors.
In lymphoid and non-lymphoid tissues, dendritic cells (DCs), mononuclear phagocytes of hematopoietic origin, reside. The ability to perceive pathogens and signals of danger distinguishes DCs, which are frequently called sentinels of the immune system. 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). Therefore, systems for culturing BM cells in vitro have been developed to generate substantial quantities of primary dendritic cells, providing convenient access to analyzing their developmental and functional attributes. 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 harmonious communication between different cell types is essential for immune system efficacy. Intravital two-photon microscopy, a standard approach for examining interactions in living systems, encounters a bottleneck in the molecular analysis of interacting cells due to the inability to isolate and process them. Our recent work has yielded a method to label cells undergoing precise interactions in living systems; we have named it 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. To execute this protocol, one must possess expert knowledge in animal experimentation and multicolor flow cytometry techniques. learn more Mouse crossing, once established, necessitates an experimental duration spanning three days or more, as dictated by the specific interactions the researcher seeks to investigate.
Tissue architecture and cellular distribution are often examined using the method of confocal fluorescence microscopy (Paddock, Confocal microscopy methods and protocols). Techniques employed in molecular biology research. The publication, Humana Press, New York, released in 2013, explored a wide array of topics from page 1 to 388. 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). This scholarly publication, available at https//doi.org/101016/j.cell.201009.016, presents meticulous research into a pivotal aspect of cell biology. This event took place in the year 2010. Employing a multicolor fate-mapping approach in mice, this chapter outlines a microscopy technique for tracing the progeny of conventional dendritic cells (cDCs), building upon the methodology of Cabeza-Cabrerizo et al. (Annu Rev Immunol 39, 2021). The DOI you've provided, https//doi.org/101146/annurev-immunol-061020-053707, leads to an article. I need the content of that article's sentence to construct 10 different rewrites. To investigate the clonality of cDCs, the 2021 progenitors present in diverse tissues were studied. While the chapter primarily concerns imaging techniques, it also briefly introduces the software employed for quantifying cluster formation.
Tolerance is maintained by dendritic cells (DCs) in peripheral tissue, which act as sentinels for any invasion. By carrying antigens to draining lymph nodes and presenting them to antigen-specific T cells, the system initiates acquired immune responses. It follows that a thorough comprehension of DC migration from peripheral tissues and its impact on their function is critical for understanding DCs' role in maintaining immune homeostasis. The KikGR in vivo photolabeling system, a perfect methodology for monitoring precise cellular movements and related processes inside living organisms under typical conditions and various immune responses during disease, is detailed in this study. 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.
Within the context of antitumor immunity, dendritic cells serve as a key link between innate and adaptive immune responses. The extensive array of activation mechanisms available to DCs is crucial for the successful completion of this significant undertaking. The substantial research into dendritic cells (DCs) during the past decades stems from their exceptional ability to prime and activate T cells through antigen presentation. 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. We present here a review of human DC subset phenotypes, functions, and localization within the tumor microenvironment (TME), facilitated by flow cytometry and immunofluorescence, complemented by high-throughput technologies such as single-cell RNA sequencing and imaging mass cytometry (IMC).
Cells of hematopoietic lineage, dendritic cells excel at antigen presentation, thereby instructing both innate and adaptive immune systems. Lymphoid organs and nearly every tissue are home to a heterogenous assemblage of cells. The three primary dendritic cell subsets are differentiated by their distinct developmental lineages, phenotypic markers, and functional specializations. Given the preponderance of dendritic cell research performed in mice, this chapter will synthesize recent developments and existing knowledge regarding the development, phenotype, and functions of mouse dendritic cell subsets.
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