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. To grasp the intricacies of the immune system and design innovative treatments, the modeling of human dendritic cell differentiation and function is essential. selleck chemicals llc The infrequent occurrence of dendritic cells in human blood underscores the importance of in vitro systems that effectively generate 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.
The heterogeneous population of antigen-presenting cells, dendritic cells (DCs), significantly contributes to both innate and adaptive immunity. By mediating tolerance to host tissues, DCs also coordinate protective responses against both pathogens and tumors. Successful identification and characterization of dendritic cell types and functions relevant to human health have been enabled by the evolutionary conservation between species, leading to the effective use of murine models. Type 1 classical dendritic cells (cDC1s), exceptional among dendritic cell subtypes, are uniquely adept at eliciting anti-tumor responses, rendering them a noteworthy therapeutic target. Nonetheless, the scarcity of dendritic cells, particularly cDC1, poses a constraint on the number of cells that can be isolated for analysis. In spite of considerable work, advancements in this field have been limited due to the lack of adequate techniques for producing large quantities of fully functional DCs in a laboratory setting. This challenge was overcome by designing a culture system that involved the co-cultivation of mouse primary bone marrow cells with OP9 stromal cells, expressing the Notch ligand Delta-like 1 (OP9-DL1), which produced CD8+ DEC205+ XCR1+ cDC1 (Notch cDC1) cells. Facilitating functional investigations and translational applications, including anti-tumor vaccination and immunotherapy, this novel method provides a valuable tool for generating unlimited cDC1 cells.
The protocol for generating mouse dendritic cells (DCs) frequently involves isolating cells from bone marrow (BM) and cultivating them with growth factors promoting DC development, such as FMS-like tyrosine kinase 3 ligand (FLT3L) and granulocyte-macrophage colony-stimulating factor (GM-CSF), according to the Guo et al. (2016) study in J Immunol Methods 432(24-29). The in vitro culture period, in the presence of these growth factors, facilitates the expansion and maturation of DC progenitors, simultaneously causing the demise of other cell types, thus resulting in a relatively homogeneous DC population. selleck chemicals llc This chapter discusses a different method for in vitro conditional immortalization of progenitor cells with dendritic cell potential, employing an estrogen-regulated version of Hoxb8 (ERHBD-Hoxb8). Retroviral transduction of largely unseparated bone marrow cells, facilitated by a retroviral vector expressing ERHBD-Hoxb8, leads to the creation of these progenitors. The administration of estrogen to ERHBD-Hoxb8-expressing progenitor cells results in the activation of Hoxb8, which obstructs cell differentiation and allows for the increase in homogenous progenitor cell populations in the presence of FLT3L. The ability of Hoxb8-FL cells to create lymphocytes, myeloid cells, and dendritic cells, is a key feature of these cells. With the inactivation of Hoxb8, brought about by estrogen removal, Hoxb8-FL cells differentiate into highly homogenous dendritic cell populations under the influence of GM-CSF or FLT3L, much like their endogenous counterparts. 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. This document outlines the method for creating Hoxb8-FL cells from mouse bone marrow, along with the subsequent steps for dendritic cell production and gene editing using lentiviral delivery of CRISPR/Cas9.
Found in both lymphoid and non-lymphoid tissues are mononuclear phagocytes of hematopoietic origin, commonly known as dendritic cells (DCs). DCs, sentinels of the immune system, are equipped to discern both pathogens and signals indicating danger. Upon activation, dendritic cells proceed to the draining lymph nodes, showcasing antigens to naive T cells to induce the adaptive immune reaction. In the adult bone marrow (BM), hematopoietic progenitors for dendritic cells (DCs) are found. Subsequently, BM cell culture systems were created to produce large quantities of primary dendritic cells in vitro in a convenient manner, facilitating the examination of their developmental and functional characteristics. This review examines diverse protocols for in vitro DC generation from murine bone marrow cells, analyzing the cellular diversity within each culture system.
The immune system's performance is determined by the complex interactions occurring between diverse cell types. In vivo investigation of interactions, traditionally conducted using intravital two-photon microscopy, faces a significant obstacle in the molecular characterization of interacting cells, as retrieval for downstream analysis is typically impossible. We have recently developed an approach to label cells undergoing specific interactions in living organisms, which we have named LIPSTIC (Labeling Immune Partnership by Sortagging Intercellular Contacts). Genetically engineered LIPSTIC mice facilitate the tracking of CD40-CD40L interactions between dendritic cells (DCs) and CD4+ T cells, as detailed in this document. This protocol demands significant proficiency in animal experimentation and multicolor flow cytometry. selleck chemicals llc With mouse crossing having been achieved, the subsequent period required to complete the experiment is typically three days or more, contingent on the researcher's specific interaction focus.
Confocal fluorescence microscopy is a prevalent technique for investigating tissue structure and cellular arrangement (Paddock, Confocal microscopy methods and protocols). Methods used in the study of molecular biology principles. In 2013, Humana Press, based in New York, detailed its findings across pages 1 to 388. By combining multicolor fate mapping of cell precursors, a study of single-color cell clusters is enabled, providing information regarding the clonal origins of cells within tissues (Snippert et al, Cell 143134-144). A significant advancement in our understanding of cellular processes is presented in the research paper published at https//doi.org/101016/j.cell.201009.016. The year 2010 saw the unfolding of this event. Tracing the progeny of conventional dendritic cells (cDCs) using a multicolor fate-mapping mouse model and microscopy, as outlined by Cabeza-Cabrerizo et al. (Annu Rev Immunol 39, 2021), is the focus of this chapter. 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. In this chapter, imaging methods take precedence over image analysis, even though the software for measuring cluster formation is also highlighted.
As sentinels of invasion, dendritic cells (DCs) in peripheral tissues help to maintain tolerance. Antigens are ingested, carried to draining lymph nodes, and presented to antigen-specific T cells, triggering acquired immune responses. Accordingly, an in-depth examination of DC migration from peripheral tissues and its influence on cellular function is imperative for grasping DCs' contribution to immune equilibrium. The KikGR in vivo photolabeling system, a crucial tool for examining precise cellular locomotion and connected processes within a living system under normal and disease-related immune responses, was introduced here. The labeling of dendritic cells (DCs) in peripheral tissues, facilitated by a mouse line expressing photoconvertible fluorescent protein KikGR, can be achieved. This labeling method involves the conversion of KikGR fluorescence from green to red through violet light exposure, enabling precise tracking of DC migration from each tissue to the respective draining lymph node.
At the nexus of innate and adaptive immunity, dendritic cells (DCs) are instrumental in combating tumors. Only through the diverse repertoire of mechanisms that dendritic cells employ to activate other immune cells can this critical task be accomplished. Because dendritic cells (DCs) possess a remarkable ability to prime and activate T cells through antigen presentation, their investigation has been substantial over the previous decades. The substantial research on dendritic cells has revealed a complex system of different cell types, prominently categorized as cDC1, cDC2, pDCs, mature DCs, Langerhans cells, monocyte-derived DCs, Axl-DCs, and other similar cell 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).
Antigen presentation and the initiation of innate and adaptive immune reactions are the specialized functions of dendritic cells, which are hematopoietic in origin. Lymphoid organs and the majority of tissues host a heterogeneous assortment of cells. The three major subsets of dendritic cells are delineated by differences in developmental paths, phenotypic expressions, and 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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