By controlling the activation of T cells, dendritic cells (DCs), as professional antigen-presenting cells, direct the adaptive immune response against pathogens or tumors. 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. selleck inhibitor The scarcity of dendritic cells in human blood highlights the critical requirement for in vitro systems accurately producing them. Employing engineered mesenchymal stromal cells (eMSCs), secreting growth factors and chemokines, in conjunction with CD34+ cord blood progenitors co-culture, this chapter will outline a DC differentiation method.
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. Murine models' successful application in identifying and characterizing DC types and functions relevant to human health stems from evolutionary conservation between species. Type 1 classical DCs (cDC1s) demonstrate a singular capability to induce anti-tumor responses among all dendritic cell types, positioning them as a compelling therapeutic prospect. Despite this, the low prevalence of dendritic cells, specifically cDC1, hinders the isolation of a sufficient number of cells for research. Despite the significant efforts invested, the field's progress has been hindered by the inadequacy of methods for generating large quantities of mature DCs in a laboratory environment. To overcome this impediment, a coculture system was implemented, featuring mouse primary bone marrow cells co-cultured with OP9 stromal cells that expressed Delta-like 1 (OP9-DL1) Notch ligand, leading to the creation of CD8+ DEC205+ XCR1+ cDC1 cells (Notch cDC1). This innovative technique yields a crucial instrument, enabling the production of limitless cDC1 cells for functional analyses and clinical applications such as anti-tumor vaccines and immunotherapeutic strategies.
To routinely generate mouse dendritic cells (DCs), cells are extracted from bone marrow (BM) and nurtured in a culture medium containing growth factors vital for DC differentiation, including FMS-like tyrosine kinase 3 ligand (FLT3L) and granulocyte-macrophage colony-stimulating factor (GM-CSF), as described by Guo et al. (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. selleck inhibitor This chapter details an alternative strategy for immortalizing progenitor cells with dendritic cell potential in vitro. This method utilizes an estrogen-regulated form of Hoxb8 (ERHBD-Hoxb8). Retroviral transduction, using a retroviral vector expressing ERHBD-Hoxb8, is employed to establish these progenitors from largely unseparated bone marrow cells. Estrogen treatment of ERHBD-Hoxb8-expressing progenitor cells triggers Hoxb8 activation, hindering cell differentiation and enabling the expansion of homogeneous progenitor cell populations in the presence of FLT3L. Lymphocyte, myeloid, and dendritic cell lineages retain the developmental potential of Hoxb8-FL cells. Upon the inactivation of Hoxb8, due to estrogen removal, Hoxb8-FL cells, in the presence of GM-CSF or FLT3L, differentiate into highly uniform dendritic cell populations analogous to their naturally occurring counterparts. These cells, boasting an unlimited proliferative capacity and readily amenable to genetic manipulation, for example, via CRISPR/Cas9, provide a substantial number of research avenues for investigating dendritic cell biology. To establish Hoxb8-FL cells from mouse bone marrow (BM), I detail the methodology, including the procedures for dendritic cell (DC) generation and gene deletion mediated by lentivirally delivered CRISPR/Cas9.
Dendritic cells (DCs), mononuclear phagocytes of hematopoietic origin, are positioned in both lymphoid and non-lymphoid tissues. Sentinels of the immune system, DCs are frequently recognized for their ability to detect pathogens and danger signals. Activated dendritic cells, coursing through the lymphatic system, reach the draining lymph nodes, presenting antigens to naïve T cells, initiating adaptive immunity. Adult bone marrow (BM) harbors hematopoietic precursors that ultimately develop into 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. We analyze multiple protocols used for the in vitro production of dendritic cells (DCs) from murine bone marrow cells, and discuss the different cell types identified in each cultivation approach.
For effective immune responses, the collaboration between various cell types is paramount. While intravital two-photon microscopy is a common technique for studying interactions in vivo, a major limitation is the inability to isolate and subsequently characterize at a molecular level the cells participating in the interaction. A novel approach for labeling cells undergoing targeted interactions within living tissue has recently been developed; we named it LIPSTIC (Labeling Immune Partnership by Sortagging Intercellular Contacts). Detailed instructions are offered for the use of genetically engineered LIPSTIC mice to trace CD40-CD40L interactions between dendritic cells (DCs) and CD4+ T cells. The utilization of this protocol mandates a deep understanding of animal experimentation and multicolor flow cytometry. selleck inhibitor Mouse crossing, once established, necessitates an experimental duration spanning three days or more, as dictated by the specific interactions the researcher seeks to investigate.
The analysis of tissue architecture and cell distribution relies heavily upon the use of confocal fluorescence microscopy (Paddock, Confocal microscopy methods and protocols). Techniques employed in molecular biology research. Humana Press, New York, pages 1 to 388, published in 2013. Analysis of single-color cell clusters, when coupled with multicolor fate mapping of cell precursors, aids in understanding the clonal relationships of cells in tissues, a process highlighted in (Snippert et al, Cell 143134-144). The study located at https//doi.org/101016/j.cell.201009.016 investigates a critical aspect of cell biology with exceptional precision. This event took place on a date within the year 2010. To trace the progeny of conventional dendritic cells (cDCs), this chapter showcases a multicolor fate-mapping mouse model and microscopy technique, drawing heavily from the methodology developed by Cabeza-Cabrerizo et al. (Annu Rev Immunol 39, 2021). The referenced article, associated with https//doi.org/101146/annurev-immunol-061020-053707, is unavailable to me; therefore, I cannot furnish 10 different and distinct sentence structures. In diverse tissues, assess 2021 progenitors and scrutinize cDC clonality. Although this chapter mainly centers on imaging approaches instead of image analysis, the software instrumental in assessing cluster formation is nonetheless detailed.
Peripheral tissue dendritic cells (DCs), as sentinels, maintain tolerance to invasion. The process of ingesting and transporting antigens to the draining lymph nodes culminates in the presentation of those antigens to antigen-specific T cells, initiating 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. This study introduces the KikGR in vivo photolabeling system, an ideal instrument for tracking precise cellular movements and corresponding functions within living organisms under typical physiological circumstances and diverse immune responses in pathological contexts. The use of a mouse line expressing photoconvertible fluorescent protein KikGR enables the labeling of dendritic cells (DCs) in peripheral tissues. After exposure to violet light, the color change of KikGR from green to red permits the accurate tracking of DC migration from each peripheral tissue to its respective draining lymph node.
Within the context of antitumor immunity, dendritic cells serve as a key link between innate and adaptive immune responses. Only through the diverse repertoire of mechanisms that dendritic cells employ to activate other immune cells can this critical task be accomplished. 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. Research efforts have highlighted an expanding range of dendritic cell subsets, including the well-known cDC1, cDC2, pDCs, mature DCs, Langerhans cells, monocyte-derived DCs, Axl-DCs, and various other specialized cell types. Employing flow cytometry, immunofluorescence, single-cell RNA sequencing, and imaging mass cytometry (IMC), we analyze the specific phenotypes, functions, and localization of human DC subsets inside the tumor microenvironment (TME).
Dendritic cells, cells of hematopoietic origin, are skilled at antigen presentation and guiding the instruction of both innate and adaptive immune reactions. A collection of heterogeneous cells populate both lymphoid organs and the majority of tissues. Dendritic cells are frequently divided into three principal subtypes, each marked by unique developmental routes, phenotypic markers, and functional activities. The majority of dendritic cell research has been performed using murine models; consequently, this chapter will comprehensively review the recent findings and current understanding regarding mouse dendritic cell subsets' development, phenotype, and functions.
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