ABSTRACT: Gene expressions of murine germinal center and naive B cells on Affymetrix platform The experiment include 3 d14 GC B1-8, 3 d14 GC V23 and 4 NaM-CM-/ve samples
Project description:Since the introduction of new generation pertussis vaccines, resurgence of pertussis is observed in many developed countries. Former whole-cell pertussis vaccines (wP) are able to protect against disease and transmission but have been replaced in several industrialized countries because of their reactogenicity and adverse effects. Current acellular pertussis vaccines (aP), made of purified proteins of Bordetella pertussis, are efficient at preventing disease but fail to induce long-term protection from infection. While the systemic and mucosal T cell immunity induced by the two types of vaccines has been well described, much less is known concerning B cell responses. Taking advantage of an inducible AID-Cre-EYFP fate-mapping mouse model, we sorted and analyzed by scRNAseq the transcriptomic profiles of memory B cells after a combination of prime:boost with the two classes of vaccines. B220+EYFP+GL7-PNA- memory B cells from the draining lymph nodes (dLNs) of tamoxifen-fed mice were FACS sorted 7 weeks after boost, alongside with B220+EYFP+GL7+PNA+ germinal center (GC) B cells and B220+GL7-PNA-EYFP-IgD+ naive B cells as a control population for the unsupervised clustering analysis. Single-cell mRNA sequencing was performed according to an adapted version of the SORT-seq protocol (Muraro et al., 2016, PMID: 27693023, with primers described in van den Brink et al. 2017), with cDNA libraries generation, sequencing and reads alignment performed at Single Cell Discoveries (Utrecht, Netherlands).
Project description:Activation-induced cytidine deaminase (AID) is essential for the generation of antibody memory but also targets oncogenes among others. We investigated the transcriptional regulation of the AID gene, Aicda, in the class switchâinducible CH12F3-2 cells, and found that the Aicda regulation involves derepression by several layers of positive regulatory elements in addition to the 5â promoter region. The 5â upstream region contains functional motifs for the response to signaling by cytokines, CD40-ligand, or stimuli that activate NF-κB. The first intron contains functional binding elements for the ubiquitous silencers c-Myb and E2f and for B cellâspecific activator Pax5 and E-box-binding proteins. To confirm involvement of these transcription factors in Acida regulation, we examined their expression profile of these factors in naïve and germinal center B cells. Experiment Overall Design: Naïve B cells were defined as B220+PNA-Fas- and germinal center B cells were defined as B220+PNA+Fas+ cells. These cells were purified from immunized mouse lymph nodes or mouse Peyer's patches by using FACS Aria. Samples are as follows: pLN GC, pLN naïve, PP GC, PP naïve, (each 1 sample, prepared from pooled 3-5 mice)
Project description:Somatic mutations of the MLL2 methyltransferase gene represent a common genetic lesion in multiple cancer types. In diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) (collectively, over 70% of all lymphoma diagnoses), these mutations are highly recurrent and appear early during transformation, possibly in pre-malignant precursors. Here we show that FL- and DLBCL-associated MLL2 mutations impair its enzymatic activity and lead to diminished global H3K4 methylation in normal germinal-center (GC) B cells and DLBCL, consistent with the enrichment of MLL2 binding at enhancer and promoter regions marked by mono- and tri-methylation. Conditional deletion of Mll2 early during B cell development, but not after initiation of the GC reaction, leads to increased percentages and numbers of GC B cells, which feature a distinct transcriptional profile defined by the enrichment of cell-cycle regulatory and B-cell receptor signaling genes. Consistently, Mll2-deficient B cells exhibit proliferative advantage and accumulation in the S phase of the cell cycle, which is influenced by the number of cell divisions. While GC-specific loss of Mll2 was not sufficient to initiate malignant transformation, compound Mll2-deficient/BCL2-transgenic mice displayed an increased incidence of clonal lymphoproliferations resembling the features of human FL and DLBCL. These findings suggest that early MLL2 loss favors BCL2-induced lymphomagenesis by remodeling the epigenetic landscape of the cancer precursor cells. Eradication of MLL2-deficient cells may represent a rational therapeutic approach targeting early tumorigenic events. Murine germinal center B cells (B220+CD95+PNA+) were sorted from the spleen of conditional Mll2 knock-out, Mll2 heterozygous and Mll2 wild type mice (crossed with CD19-Cre or Cg1-Cre deletor strains) and sacrificed at day 12 after immunization with sheep red blood cells (SRBC)(n=3 mice per genotype). Total RNA was extracted from single cell suspensions and processed according to the Ovation RNA Amplification System and Encore® Biotin Module protocols (both from NuGEN). Samples were analyzed on Affymetrix Mouse Genome 430 2.0 arrays.
Project description:B220+GL7+ (GC) and B220+GL7- (non-GC) B cells were sorted from SRBC-immunized mice deficient for Hdac3 and wild type controls. RNA-sequencing revealed an upregulation of critical regulators of B cell differentiation in Hdac3-deleted animals. 10 days post-immunization with SRBCs, GC and non-GC B cells were sorted and RNA isolated by Trizol extraction for RNA-sequencing. 2 replicates were sequenced for each condition.
Project description:We found that Gm40600 significantly affects plasmablast(PB)-like SP 2/0 cells. To explore the role of Gm40600 in B-cell especially plasma cell (PC) differentiation, we developed Gm40600 Transgenic (Gm40600 Tg) mice. B cells from spleen and lymph nodes (LNs) of WT and Gm40600 Tg mice were sorted by using B220 microbeads. B cells were also stimulated for 3 days by 10 ug/ml LPS. To explore the effect of Gm40600 overexpression on gene expression, we determined mRNA profiles in B cells from WT and Gm40600 Tg mice and LPS-stimulated B cells by RNA-seq. RNA-seq was done with an Illumina HiSeq 2500 instrument at GENEWIZ, Suzhou, China.
Project description:In vitro studies identified TBC1D4 as an regulator of renal ion and water transporting proteins. However, TBC1D4-deficient mice did not show a defective renal salt and water homeostasis. With microarray analysis we aimed to decipher compensatory mechanisms in TBC1D4-deficient mice which might mask the renal phenotype already on transciptomic levels. We used and compared mRNA of COPAS-sorted and -enriched distal convoluted tubule (DCT)-cells, known to express TBC1D4 at high levels, of 8-week old male homogenous C57/Bl6 mice of TBC1D4-deficient and wild-type mice.
Project description:In this study, MLN transcriptome profiling of female BALB/c mice was conducted in order to explore the molecular mechanism(s) of antigen sensitization using three of the most common food allergens; BLG, OVA and PNA. Analysis of cDNA microarray data revealed a complex network of genes that is modulated during BLG, OVA or PNA sensitization. Results from this study implicate the role of differentially expressed genes in the pathogenesis of food allergy. Results from this study will also help better understanding the molecular mechanism of food allergy in animal models and may help in developing new drug target for the therapy of food allergy. This study is likely to add a new dimension in the development of biomarker genes for the determination of potential allergenicity of genetically modified (GM) foods.
Project description:The main goal of our study is to identify the molecular events that determine the gonadal identity in mammals. Although testis and ovary arise from a common embryonic primordium, they represent outcomes of opposing fate determination. This decision to differentiate into a testis or an ovary hinges upon the balance between two antagonizing factors, pro-testis SOX9 and pro-ovary β-catenin. This microarray analysis led to the identification of the genes involved in the fate of XX and XY gonads in absence of SOX9 and beta-catenin We developed mouse genetic models that lack either Sox9, β-catenin, or both specifically in the somatic cells. All embryos used in this study resulted from the crossing between Ctnnb1f/f; Sox9f/f females with Sf1-cre+/Tg ; Ctnnb1+/f; Sox9+/f males. XX and XY fetal gonads were collected at embryonic day E14.5
Project description:Although CD4 T cell senescence plays an important role in immunosenescence, the mechanisms remain unclear. We found that T cell-specific Menin deficiency results in the premature senescence of CD4 T cells, accompanied by the senescence-associated secretory phenotype (SASP) after antigenic stimulation. TH1 and TH2 differentiation was dysregulated in Menin-knockout CD4 T cells. Bach2, which regulates SASP and TH differentiation, was identified as a Menin target. Menin binds to the Bach2 locus, and controls its expression through maintenance of histone acetylation. These findings reveal a critical role of the Menin-Bach2 pathway in regulating CD4 T cell senescence and homeostasis, thus indicating the involvement of this pathway in the inhibition of age-associated development of inflammatory diseases, which are induced by immunosenescence. Examination of transcriptional factor Menin binding and histone modefications in Menin WT and KO CD4 T cells
Project description:The germinal center (GC) is a microanatomical compartment wherein high-affinity antibody-producing B cells are selectively expanded. B cells proliferate and mutate their antibody genes in the dark zone (DZ) of the GC and are then selected by T cells in the light zone (LZ) on the basis of affinity. Here, we show that T cell help regulates the speed of cell cycle phase transitions and DNA replication of GC B cells. Genome sequencing and single-molecule analyses revealed that T cell help shortens S phase by regulating replication fork progression while preserving the relative order of replication origin activation. Thus, high-affinity GC B cells are selected by a mechanism that involves prolonged dwell time in the DZ where selected cells undergo accelerated cell cycles. To determine whether GC B cells receiving high levels of T cell help show a specific change in gene expression, we compared DZ cells in the G1 phase of the cell cycle from αDEC-OVA and control αDEC-CS treated GCs using a fluorescent ubiquitination-based cell cycle indicator (Fucci-tg). RNA sequencing revealed that T cell-mediated selection produced an increase in gene expression programs associated with the cell cycle, metabolism, including the metabolism of nucleotides, and genes downstream of c-Myc and the E2F transcription factors.