Gene expression differences between activated B cells expressing GL7 and not in Peyer´s patches
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ABSTRACT: We report two populations of activated germinal centre B cells in the Peyer´s patches that are distinguished by their expression of GL7 or not. Using an antigen-specific system, we sort GL7+ and GL7- activated cells and performe RNASeq. We also included GL7 expressing B cells activated against the gut microbiota, and resting B cells from Peyer´s patches and spleen.
Project description:Peyer`s patches (PP) are underpinned by a dense network of FRCs. To elaborate the subset composition and topological organization of PP FRCs, we employed well-characterized Ccl19-Cre and Col6a1-Cre mouse models that permit FRC targeting in secondary lymphoid organs. To define PP FRC subsets and their relation to the observed FRC lineages, we isolated fibroblasts from Col6a1Eyfp and Ccl19Eyfp PPs and performed droplet-based single cell transcriptomics analysis. Taken together, the molecular definition of the PP FRC landscape reveals two FRC lineages furnishing distinct microenvironmental niches.
Project description:The production of IL-21 by T follicular helper (Tfh) cells is vital in driving the germinal centre reaction and high affinity antibody formation. However, the degree of Tfh cell heterogeneity and function is not fully understood. Using a novel IL-21eGFP reporter mouse strain, we identified a subpopulation of GFP+ highly differentiated Tfh cells in Peyer’s Patches. In vitro, GFP+ cells that did not co-express IL-17 or Foxp3 were induced by CD3 and CD28 stimulation of naïve CD4+ cells from IL-21eGFP mice in cultures containing IL-6, TGFβ and retinoic acid. In vivo, the differentiation of GFP+Tfh cells in Peyer’s Patches was induced by intestinal bacteria, and required CD4+ T cells and B cells with diverse repertoires. Analysis of the TCRβ repertoire of Peyer’s Patches CD4+ T cell subpopulations demonstrated that GFP+Tfh cells are polyclonal and closely related to GFP-Tfh cells. Importantly, ablation of GFP+ cells resulted in a reduced frequency of Peyer’s Patches germinal center B cells and small but significant shifts in gut microbiome composition. Thus, using a new IL-21-reporter mouse we have identified a subpopulation of Tfh cells induced by the gut microbiota and necessary for optimal Peyer’s Patches germinal center responses.
Project description:Germinal center (CD19+Fas+GL7+) and naive (CD19+Fas-GL7-) B cells were sorted from Peyer's patches of littermate 12 weeks old WT C57BL/6 mice. Three biological replicates were analyzed, each composed of a pool of 5 female mice. RNA was purified from pellets of 2-2.5x10^⁴ cells and sequencing libraries were prepared from 100ng of total RNA per replicate.
Project description:Many studies have shown that the mucous membranes and skin are at the interface with different external environments and face the disparity of pathogenic effects, such as biological agents, chemical or physical environment. This difference may demand distinct immune responses. However, the mechanism to induce the distinct immune responses in mucous and skin is largely unknown. Dendritic cells of mucosa and skin are crucial in the initiation of immune responses, maintenance of self-tolerance and antigens presentation T cells. The different functions between mucosal and epidermal dendritic cells may play an important role in different immune responses. To compare the different gene expression of the mucosal DC and Langerhans cells (LC), we utilized microarrays to investigate different gene expression profiles in mucosal DC isolated from PPs (PDC) and epidermal LC from skin (ELC). 3548 genes were shown to be differentially expressed between PDC and ELC. According to genes annotations, 105 genes may be involved in immunity process. The genes involved in immune process were categorized to five groups related to DC function, including antigen presentation, antigen uptake, cytokines chemokines, and receptors, cell surface molecules and signal transduction. 11 of the highest expressed genes were selected as the candidate genes and reformed by real-time PCR. These 11 selected genes might be suitable candidates to further study the difference of gene expression between mucosal DC and epidermal LC and would be used for design for new vaccine. Beads and FACS sort were used to isolate dendritic cells from Peyer's patches and epidermal of skin from 80 four-week-old female BALB/c mice. Dendritic cells from Peyer's patches were pooled as one sample, and dendritic cells from epidermal of skin were pooled as another sample. RNA isolation, amplification, cDNA labelling, microarray hybridization and analyses were performed according to the Affymetrix manual book.
Project description:In a time course study, we characterized global gene expression profile of B. melitensis-infected bovine Peyer patches in the first 4 h p.i. Microarray analysis revealed that 2,916 bovine genes were detected as differentially expressed (z-score p < 0.025) in loops inoculated with virulent B. melitensis 16M compared with controls between 15 min and 4 h post-infection. From these genes, 2,286 (78%) were up- and 630 (22%) were down-regulated. Specific genes and biological processes identified in this study will further help elucidate how both host and Brucella interact during the early infectious process to the eventual benefit of the pathogen and to the detriment of the naM-CM-/ve host. Microarrays were used to examine the transcriptional profiles of bovine intestinal Peyer patches infected with wild type Brucella melitensis 16M across five time points (15 min, 30 min, 1, 2 and 4 hours). Intestinal loops inoculated with cell culture medium were used as a control. Experiments were performed in quadruplicate (bovine ligated ileal loops surgeries were performed with four calves), generating a total of 40 arrays.
Project description:To gain a comprehensive view of the host response to pathogens within these tissues, we determined the transcriptional profiles of intestinal lymphatic tissue infected with Y. enterocolitica. Expression analysis using Affymetrix GeneChips revealed a complex host response in the Peyers patches (PP) and mesenteric lymph nodes (MLN) following oral infection with Y. enterocolitica. Peyer's patches and mesenteric lymph nodes were surgically removed from uninfected (control) and infected (experimental) mice at 12 hours, day 2, 4 and 7 post oral infection. Tissue samples from 10 mice per time-point were combined. Total RNA was extracted using the Trizol method prior to total cDNA syntesis, labeling and hybridization to Affymetrix MGU74Av2 GeneChips. Data was processed and analyzed using MAS 5.0 and custom analysis protocols. Samples from Peyer's patches and mesenteric lymph nodes are included in this series.