Analysis of thymic dendritic cells (DCs) subtypes from Foxn1cre x ROSA26TdTOMATO mice by single cell transcription profiling.
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ABSTRACT: Thymi from Foxn1cre x ROSA26TdTOMATO were enzymaticaly digested, cells were MACS enriched for CD11c+ fraction. Dendritic cells were then FACS sorted (BD influx sorter) as Gr-1-CD11c+TdTOMATO+. TdTOMATO DCs represent fraction of thymic DCs which acquire antigen (TdTOMATO) from thymic epithelial cells, through the process of antigen transfer.
Project description:The presentation of self-antigens in the thymus by medullary thymic epithelial cells (mTECs) and dendritic cells (DCs) is crucial for the establishment of central tolerance. While mTECs produce and present self-antigens in an autonomous manner, DCs acquire them from mTECs by Cooperative Antigen Transfer (CAT). Although CAT represents a key process of central tolerance, molecular determinants which drive CAT are largely unknown. To reveal these determinants, we compared the transcriptomes of CAT-experienced and CAT-inexperienced DCs via scRNAseq. Specifically, we sequenced TdTOMATO+ and TdTOMATO– CD11c+ and CD11b+ cells from the thymus of Foxn1CreR26TdTOMATO mice. In this model, fluorescent TdTOMATO protein is exclusively produced by thymic epithelium, thus TdTOMATO expression in DCs (CD11c+ and CD11b+ cells) mark their previous experience of CAT. Using this approach we unraveled several candidate determinants of CAT. We focused on the gene determinant, Cldn1, encoding the tight junction protein Claudin 1, that was found to be highly expressed among the CAT-experienced type 1 lineage DCs. We performed further experiments that confirmed its role in driving CAT. In addition, this scRNAseq approach allowed us to redefine the heterogeneity and maturation states of thymic DCs.
Project description:This SuperSeries is composed of the following subset Series: GSE22127: Expression profiling of small intestine lamina propria dendritic cells GSE22128: Expression profiling of splenic dendritic cells Dendritic cells play a vital role in initiating robust immunity against pathogens as well as maintaining immunological tolerance to self antigens, food antigens and intestinal commensals. However, the intracellular signaling networks that program DCs to become tolerogenic are largely unknown. To address this, we analyzed gene expression profiles using microarray analysis of purified intestinal lamina propria DCs (CD11c+ CD11b+ DCs and CD11c+ CD11b- DCs) and compared it to splenic DCs (CD11c+ DC), from mice. We sought to determine the unique genetic profile of small intestine lamina propria CD11c+ cells compared to splenic CD11c+ cells. We performed a meta-analysis using the expression profiles of Intestinal lamina propria CD11c+ CD11b+ DCs (GSM550122), Intestinal lamina propria CD11c+ CD11b- DCs (GSM550121) and Splenic CD11c+ DCs (GSM550126). This study combined and re-normalized the microarray data from GSE22127 and GSE22128 studies. Refer to individual Series for additional details
Project description:Dendritic cells play a vital role in initiating robust immunity against pathogens as well as maintaining immunological tolerance to self antigens, food antigens and intestinal commensals. However, the intracellular signaling networks that program DCs to become tolerogenic are largely unknown. To address this, we analyzed gene expression profiles using microarray analysis of purified intestinal lamina propria DCs (CD11c+ CD11b+ DCs and CD11c+ CD11b- DCs) from mice. Keywords: Lamina propria, DCs, cell type comparison We sought to determine the expression profile of small intestine lamina propria CD11c+ cells. RNA was extracted from DCs sorted from mouse small intestine (CD11c+CD11b- and CD11c+CD11b+ cells) and hybridized on Affymetrix microarrays.
Project description:Age-associated B cells (ABCs) accumulate in systemic autoimmunity, and contributes to pathogenesis. ABCs are characterized by high expression of CD11c and T-bet. As a transcription factor, T-bet cannot be labeled in live cells with antibodies, so we constructed a reporter mouse strain in which fluorescent protein tdTomato fused to T-bet. Then we sorted ABCs (CD11c+T-bet+CD21- B cell) and non-ABCs (CD11c-T- bet- B cell) from the Bm12-induced lupus model for RNA sequencing.
Project description:Abstract Two major dendritic cell (DC) subsets have been described in the islets of mice: The immunogenic CD8α-CD11b+ DCs and the tolerogenic CD8α+CD103+ DCs. We have recently reported on reduced numbers of the minor population of tolerogenic CD8α+CD103+ DCs in the pancreas of 5 week old pre-diabetic non-obese diabetic (NOD) mice. Aim: To analyze also the larger subset of CD11c+CD8α- DCs isolated from the pancreas of pre-diabetic NOD mice 1) for maturation and tolerance inducing molecules found abnormally expressed on CD8α+CD103+ DCs, and 2) for genome-wide gene expression to further elucidate abnormalities in underlying gene expression networks. Methods: CD11c+CD8α- DCs were isolated from 5 week old C57BL/6 and NOD pancreas. Expression of cell surface markers including CD86, CCR5, CD11b, CD103, Clec9a, CD24 and CD200R3 were measured by FACS. Genome-wide gene expression by microarray was assessed during the steady state and after in vitro LPS stimulation. Results: The steady state pancreatic CD11c+ CD8α- DCs during the pre-diabetic stage showed: 1) A reduced expression of several gene networks important for the prime functions of the cell, such as for cell renewal, immune stimulation and immune tolerance induction, for migration and for the provision of growth factors for beta cell regeneration. This general deficiency state was corroborated by a reduced in vivo proliferation (BrdU incorporation) of the cells and the reduced expression in FACS analysis of CD86, CCR5, CD103, Clec9a, CD24 and CD200R3 on the cells. 2) A hyper reactivity of these cells to LPS correlated with an enhanced pro-inflammatory state characterized by altered expression of a number of classical pro-inflammatory factors and cytokines. Conclusion: The NOD CD11c+CD8α- DCs seem to be Janus-faced depending on the conditions: Deficient in steady state with reduced immune stimulation capabilities also for tolerance induction; over-inflammatory with a molecular profile suggesting a preferential stimulatory capacity for Th1 cells when encountering a Pathogen-Associated Molecular Pattern (PAMP) in the form of LPS. We used microarray gene expression analysis to explain the abnormal expression of several cell surface markers involved in tolerace, migration and maturation in the steady-state and to measure the effect of a PAMP such as LPS We isolated RNA from FACS sorted CD11c+CD8α- DCs in 10 pooled pancreases from pre-diabetic NOD and non-diabetic C57BL/6 mice at 5 weeks. In addition, we treated in another experiment the isolated pancreas DCs with LPS (and PBS), incubated for 18h and measured gene expression. We compared gene expression between strains NOD vs C57BL/6 under steady-state and after in-vitro LPS/PBS stimulation.
Project description:Dendritic cells (DCs) play a vital role in innate immunity. Transcriptome of DCs isolated from mouse spleen was obtained and deposited here. Keywords: Spleen, DCs We sought to determine the expression profile of splenic CD11c+ cells. RNA was extracted from DCs sorted from mouse spleen (CD11c+ cells) and hybridized on Affymetrix microarrays.
Project description:T helper type 2 (Th2) responses are induced by protease allergens and helminthes. However the molecular mechanisms that initiate Th2 responses are poorly understood. To obtain insight into this mechanism, we performed a microarray analysis of lymph node DCs stimulated in vitro with the protease allergen papain, or with LPS, a Th1 inducing stimulus. Key words: Th2 response, LPS, dendritic cells, Papain CD11c+ DCs were isolated from the lymph nodes of C57BL/6 mice, and cultured in vitro (1x106 DCs per ml) with 500 3T3-CD40L fibroblasts, either alone, or in the presence of papain (25 µg/ml) or LPS (1 µg/ml). 4h and 17h later, the cells were harvested and RNA isolated and processed for microarray analyses. RNA was extracted and processed from freshly isolated LN DCs. For a given time point, the expression profile of DCs treated with papain or LPS, were compared to that of untreated DCs
Project description:Analysis of stage-specific gene expression in Zbtb46GFP/+ pre-CD8 DCs, pre-CD4 DCs, CD24 cDCs and CD172a cDCs Bone Marrow and Splenocytes were harvested from 8-10 littermate Zbtb46GFP/+ mice and sorted to >95% purity on the FACS AriaFusion.
Project description:The goal of our study is to determine whether Atg16L1 deficiency leads to differences in the transcriptional profile of CD11c+ Dendritic Cells, ultimately leading to an increased inflammatory phenotype. CD11c+ cell sorted splenic DCs were isolated from 8 week old WT and Atg16L1 hypomorphic mice from spleens of untreated mice and were placed directly into TRIzol LS (Invitrogen). mRNA was isolated, amplified, and hybridized to an Affymetrix GeneChip (MOE430A).