Single cell sequencing of human primary LCs
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE142298 | GEO | 2022/08/30
REPOSITORIES: GEO
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Project description:Langerhans cells (LC) coordinate immune homeostasis at the human epidermis, proficent in initiating immunogenic and tolerogenic immune responses. To investigate the transcriptomic mediators of human LC immunogenic vs tolerogenic immune responses, we performed single cell RNA-sequencing of migratory LCs extracted from epidermal sheets stimulated with or without TNF.
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Project description:Langerhans cells (LCs) in the epidermis promote immune homeostasis, efficiently activating tolerogenic and immunogenic T cell responses. To understand genomic programming in human Langerhans cells we performed whole transcriptome (bulk RNA-seq and single cell RNA-seq) profiling and analysis of H3K4Me3 and H3K27Ac histone modifications across LC genome in primary human cells from 6 independent donors. Primary LCs were either unstimulated and stimulated with TNF-alpha. Additionally we performed a CRISPR editing experiment for IRF4
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Project description:miRNA profiling of CD34+ derived, in vitro-generated Langerhans cells (LCs) and interstitial-type dendritic cells (intDCs). Epidermal Langerhans cells (LCs) form a network of dendritic cells (DCs) in basal/suprabasal layers of the skin and are characterized by a unique phenotype (CD207+ CD1abright CD324+ CD11b-). Due to their specific location at body surfaces, LCs are constantly exposed to environmental stimuli. Conversely, interstitial-type DCs (intDCs) are located in adjacent tissues and can be discriminated from LCs phenotypically (CD207- CD1adim CD324- CD11b+). These DCs constitute a second line of defense against pathogens that have crossed the epithelial barrier. During development both DC subsets originally arise from myeloid progenitor cells via monocyte-committed intermediates in response to specific microenvironmental signals. Additionally certain pools of DCs can be replenished by tissue resident cells or monocytes in response to specific microenvironmental signals (2, 4, 5). In vitro generated GM-CSF/IL-4-dependent DCs derived either from CD14+ monocytes or via CD14+CD11b+ monocyte intermediates from CD34+ progenitor cells share many characteristics with intDCs in vivo. On the other hand, LCs generated from CD34+ cells under serum-free TGF-beta1-dependent conditions phenotypically resemble epidermal-resident LCs. Since these two DC subsets are of considerable interest for clinical cell therapy studies, an improved understanding of their development and function is of substantial relevance. To identify differentially expressed miRNAs in DC subsets, we performed a miRNA screen. Therefore, we generated LCs or intDCs from human CD34+ cord blood haematopoietic progenitor cells as described. For intDCs, CD34+ cells (1 x 104 to 2 x 104/ml per well) were cultured in 24-well tissue culture plates in serum free CellGro® DC medium (CellGenix, Freiburg, Germany) supplemented with GM-CSF (100 ng/ml), SCF (20 ng/ml), FL (50 ng/ml) and TNFalpha (2.5 ng/ml) for 3-5 days and subsequently with GM-CSF (100 ng/ml) and IL-4 (2.5 ng/ml) for 4-5 days. For LCs, CD34+ cells (1 x 104 to 2 x 104/ml per well) were cultured in 24-well tissue culture plates in serum free CellGro® DC medium (CellGenix, Freiburg, Germany) supplemented with GM-CSF (100 ng/ml), SCF (20 ng/ml), FL (50 ng/ml), TNFalpha (2.5 ng/ml) and TGF-beta1 (0.5 ng/ml). The well-established immunophenotype of LCs (CD1ahi CD11b- CD207+ CD324+) and intDCs (CD1a+ CD11b+ CD207- CD324-) was confirmed by FACS. These two DC subsets were purified and submitted to differential miRNA profiling. Two conditioned experiment LCs vs. intDCs. Biological replicates: 3 LCs, 3 intDCs, independently differentiated, harvested and purified. 3 replicates each were pooled and hybridized on one array. Supplementary files linked below.
2010-06-24 | E-GEOD-20215 | biostudies-arrayexpress
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2015-08-31 | E-GEOD-68789 | biostudies-arrayexpress
Project description:Purpose: to characterise the steady state gene expression and transcriptomic response of resident murine epidermal immune cells (dendritic epidermal T cells, DETC; Langerhans cells, LCs) and epithelial cells (interfollicualr keratinocytes) to non-microbial stress in the form of ultraviolet B (UVB) radiation Methods: 6 C57Bl/6 mice were administered 300mJ/cm^2 UVB radiation to the dorsal side of both ears 24hr prior to tissue harvest, and another 6 administered the same dose 4 hr prior. Ears were harvested and dorsal and ventral ear sheets were separated. Samples were pooled from 2 mice to generate 3 biological replicates per timepoint. Dorsal sheets were used for UV samples, while ventral sheets from 24hr UV mice were used as unchallenged skin. Epidermis was separated and digested, and epidermal populations were FACS sorted. Libraries were prepared and sequenced on an Illumina HiSeq 2500. Conclusions: these data reveal the transcriptomic response of LC, DETC and interfollicular keratinocytes to acute epithelial stress in the form of DNA damage
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Project description:miRNA profiling of CD34+ derived, in vitro-generated Langerhans cells (LCs) and interstitial-type dendritic cells (intDCs). Epidermal Langerhans cells (LCs) form a network of dendritic cells (DCs) in basal/suprabasal layers of the skin and are characterized by a unique phenotype (CD207+ CD1abright CD324+ CD11b-). Due to their specific location at body surfaces, LCs are constantly exposed to environmental stimuli. Conversely, interstitial-type DCs (intDCs) are located in adjacent tissues and can be discriminated from LCs phenotypically (CD207- CD1adim CD324- CD11b+). These DCs constitute a second line of defense against pathogens that have crossed the epithelial barrier. During development both DC subsets originally arise from myeloid progenitor cells via monocyte-committed intermediates in response to specific microenvironmental signals. Additionally certain pools of DCs can be replenished by tissue resident cells or monocytes in response to specific microenvironmental signals (2, 4, 5). In vitro generated GM-CSF/IL-4-dependent DCs derived either from CD14+ monocytes or via CD14+CD11b+ monocyte intermediates from CD34+ progenitor cells share many characteristics with intDCs in vivo. On the other hand, LCs generated from CD34+ cells under serum-free TGF-beta1-dependent conditions phenotypically resemble epidermal-resident LCs. Since these two DC subsets are of considerable interest for clinical cell therapy studies, an improved understanding of their development and function is of substantial relevance. To identify differentially expressed miRNAs in DC subsets, we performed a miRNA screen. Therefore, we generated LCs or intDCs from human CD34+ cord blood haematopoietic progenitor cells as described. For intDCs, CD34+ cells (1 x 104 to 2 x 104/ml per well) were cultured in 24-well tissue culture plates in serum free CellGro® DC medium (CellGenix, Freiburg, Germany) supplemented with GM-CSF (100 ng/ml), SCF (20 ng/ml), FL (50 ng/ml) and TNFalpha (2.5 ng/ml) for 3-5 days and subsequently with GM-CSF (100 ng/ml) and IL-4 (2.5 ng/ml) for 4-5 days. For LCs, CD34+ cells (1 x 104 to 2 x 104/ml per well) were cultured in 24-well tissue culture plates in serum free CellGro® DC medium (CellGenix, Freiburg, Germany) supplemented with GM-CSF (100 ng/ml), SCF (20 ng/ml), FL (50 ng/ml), TNFalpha (2.5 ng/ml) and TGF-beta1 (0.5 ng/ml). The well-established immunophenotype of LCs (CD1ahi CD11b- CD207+ CD324+) and intDCs (CD1a+ CD11b+ CD207- CD324-) was confirmed by FACS. These two DC subsets were purified and submitted to differential miRNA profiling.
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Project description:The ontogeny of human Langerhans cells (LCs) remains poorly characterized, in particular the nature of LC precursors and the factors that may drive LC differentiation. Through a systematic transcriptomic analysis of TSLP-activated dendritic cells (DCs), we unexpectedly identified markers that have been associated with a skin-homing potential as well as with a LC phenotype. We performed transcriptomic analysis of TSLP-activated blood DCs, as compared to freshly purified, Medium-, and TNF-activated DCs. Among TSLP up-regulated genes, we identified molecules associated with skin homing, LC phenotype, and LC function, as determined by a literature-based survey. Conversely, genes not expressed in LCs were not found among TSLP-induced genes. Further experiments showed that TGF-? synergized with TSLP leading to the differentiation of blood BDCA-1+ DCs into bona fide Birbeck granule-positive LCs. The ontogeny of human Langerhans cells (LCs) remains poorly characterized, in particular the nature of LC precursors and the factors that may drive LC differentiation. Through a systematic transcriptomic analysis of TSLP-activated dendritic cells (DCs), we unexpectedly identified markers that have been associated with a skin-homing potential as well as with a LC phenotype. We performed transcriptomic analysis of TSLP-activated blood DCs, as compared to freshly purified, Medium-, and TNF-activated DCs. Among TSLP up-regulated genes, we identified molecules associated with skin homing, LC phenotype, and LC function, as determined by a literature-based survey. Conversely, genes not expressed in LCs were not found among TSLP-induced genes. Further experiments showed that TGF-? synergized with TSLP leading to the differentiation of blood BDCA-1+ DCs into bona fide Birbeck granule-positive LCs. Dendritic cells (DC) were purified from the blood of healthy human donors. Each replicate comes from a different donor. There are 6 freshly purified samples (Control_0h), 4 DC samples cultured during 6h with no additional stimulus than the medium (Control_6h), 5 samples cultured during 6h with recombinant TSLP (Thymic Stromal Lymphopoietin at 20 ng/uL) (TSLP_6h) and 3 samples cultured during 6h with recombinant TNF (Tumor necrosis factor at 20 ng/mL) (TNF_6h).
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Project description:The potential involvement of PI3Kγ in immune-related diseases has been recently investigated leading to efforts in developing specific inhibitors of this isoform as therapeutic strategies. (Takeda et al 2010, Rommel et al 2007). Of particular significance to cutaneous disease, murine studies have indicated the importance of PI3Kγ in Langerhans cell (LC) function. Recently, it has been postulated, PI3Kγ is a molecular switch that controls immune suppression in macrophages, a cell type closely related to LCs. [Nature letter 2016]. In our analysis PIK3CG, encoding PI3Kγ was highly expressed by LC, in contrast to dermal dendritic cells (Polak et al 2014). Here we sought to examine the effect of PI3Kg inhibition on the function of human epidermal LCs.
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Project description:Langerhans dendritic cells represent abundantly occuring and evolutionary highly conserved DCs specifically located in the stratified epithelial tissues. LCs are unique among DC family members in that they express epithelial-type adhesion molecules, allowing them to form a tight three-dimensional network in basal and suprabasal epidermal keratinocyte layers and developmentally dependent on the cytokine TGF-M-NM-21. In the present study, we identified BMP-7 as another key factor inducing LC differnetiation. Here we have performed comparative analysis of highly purified CD207+/CD1a+ in vitro generated Langerhans cells in the presence of BMP-7 and TGF-M-NM-21. We have identified that both BMP-7-LCs and TGF-M-NM-21-LCs are closely related to each other. CD34+ hematopoietic stem cells were isolated from freshly obtained cord blood and cultured in the presence of GM-CSF, SCF, Flt3L, TNFa and either TGF-M-NM-21 or BMP-7 for 7 days to get immature LCs. RNA was isolated from these in vitro genetared LCs.
2013-10-18 | E-GEOD-49085 | biostudies-arrayexpress
Project description:TGF-β family ligands are key regulators of dendritic cell (DC) differentiation and activation. Epidermal Langerhans cells (LCs) require TGF-β family signaling for their differentiation and canonical TGF-β1 signaling secures a non-activated LC state. LCs reportedly control skin inflammation and are replenished from peripheral blood monocytes, which also give rise to pro-inflammatory monocyte-derived DCs (moDCs). By studying mechanisms in inflammation, we previously screened LCs vs moDCs for differentially expressed miRNAs. miR-424/503 was the most strongly inversely regulated (moDCs > LCs). We found that miR-424/503 is induced during moDC differentiation and promotes moDC differentiation in human and mouse. Inversely, forced repression of miR-424 during moDC differentiation facilitated TGF-β1-dependent LC differentiation. Mechanistically, miR-424/503 deficiency in monocyte/DC precursors leads to the induction of TGF-β1-response genes critical for LC differentiation. Therefore, the miR-424/503 gene cluster plays a decisive role in anti-inflammatory LC vs pro-inflammatory moDC differentiation from monocytes.
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