Project description:Long-lived plasma cells (LLPCs) develop under the help of follicular helper T (Tfh) cells and reside mainly in the bone marrow. However, these cells are unusually abundant in the spleen of several autoimmune models including K/BxNsf mice, yet their pathogenic impact remains unknown. To investigate a previously unappreciated role of splenic LLPCs, we sorted splenic plasma cells (PCs) from K/BxNsf and K/BxN mice, corresponding to LLPCs and conventional short-lived PCs, respectively, and compared their transcriptomes. The B220+CD138+ fraction from K/BxNsf and their control K/BxN mice were sorted by FACSariaIII.
Project description:CD138-high plasma cell subsets were analyzed by single cell RNA-sequencing to identify gene expression differences between long- and short-lived plasma cells. Five different CD138-high subsets were analyzed based upon differential B220 expression and uptake of the fluorescent glucose analog 2NBDG: splenic B220+2NBDG-, B220+2NBDG+, B220-2NBDG-, B220-2NBDG+, and bone marrow B220-2NBDG+ cells.
Project description:CD138-high plasma cell subsets were analyzed by RNA-sequencing to identify gene expression differences between long- and short-lived plasma cells. Five different CD138-high subsets were analyzed based upon differential B220 expression and uptake of the fluorescent glucose analog 2NBDG: splenic B220+2NBDG-, B220+2NBDG+, B220-2NBDG-, B220-2NBDG+, and bone marrow B220-2NBDG+ cells.
Project description:CD138+ B220- plasma cells were sorted from bone marrow and B220+ CD23+ mature follicular B cells were sorted from the spleens. Plasma cells were sorted from C57BL/6 mice 7 days after boosting with antigen, with mice first primed with an i.p. injection of KLH/IFA followed by boost at day 21 with KLH/PBS i.p. Mature B cells were sorted from antigen-naïve C57BL/6 mice. We compared expression profiles of plasma cells and mature B cells to identify differentially expressed transcripts.
Project description:CD138+ B220- plasma cells were sorted from bone marrow and B220+ CD23+ mature follicular B cells were sorted from the spleens. Plasma cells were sorted from C57BL/6 mice 7 days after boosting with antigen, with mice first primed with an i.p. injection of KLH/IFA followed by boost at day 21 with KLH/PBS i.p. Mature B cells were sorted from antigen-naïve C57BL/6 mice. We compared expression profiles of plasma cells and mature B cells to identify differentially expressed transcripts. Seven days after boosting with KLH, plasma cells were enriched by CD138-PE/anti-PE magnetic bead enrichment and then sorted to purity. Affymetrix microarrays were performed on 50,000-70,000 plasma cells and 2,000,000-3,000,000 naïve B cells.
Project description:B cells provide humoral immunity by differentiating into antibody secreting plasma cells. Differentiation is dependent upon division and transcriptional changes, with commitment to B cell lineages associated with epigenetic changes. Analysis of early transcriptional and epigenetic events in B cell differentiation revealed that plasmablasts and plasma cells undergo dynamic changes in gene expression and a progressive DNA hypomethylation targeted to at least 10% of genes/loci. Of the differentially methylated loci, more than 99.7% were demethylated during differentiation and these clustered in cis-regulatory features such as enhancers and transcription factor binding sites. Changes in gene expression and DNA methylation coincided with each other at specific divisions during differentiation and inhibition of DNA methylation resulted in augmented plasma cell commitment in a division-dependent manner. These data identify a major epigenetic reprogramming event during early B cell differentiation coupled division and provide an approach to modulating humoral immune responses. Splenic B cells (B220+ CD43-) from naïve C57/BL6J mice, and Day 3 LPS-induced Plasmablasts (B220mid CD138+) and Plasma Cells (B220low CD138+) were analyzed by Illumina Gene Expression Microarray and Reduced Representation Bisulfite Sequencing
Project description:Long-lived plasma cells (LLPCs) develop under the help of follicular helper T (Tfh) cells and reside mainly in the bone marrow. However, these cells are unusually abundant in the spleen of several autoimmune models including K/BxNsf mice, yet their pathogenic impact remains unknown. To investigate a previously unappreciated role of splenic LLPCs, we sorted splenic plasma cells (PCs) from K/BxNsf and K/BxN mice, corresponding to LLPCs and conventional short-lived PCs, respectively, and compared their transcriptomes.
Project description:in order to document molllecular signature of iMycCα B-cell lymphomas, RNA sequecing of CD138+ and CD138-B-cell lymphomas was performed and normalized expression data was obtained for all protein-coding genes Methods: B220+IgM+IgD+CD138+ and B220+IgM+IgD+CD138- B-cell lymphomas were investigated in the iMycCα mice. Total RNA from lymphoma cells (purified with B220-coupled beads from Miltenyi Biotech) was extracted and analysed by microarray (Génome et Transcriptome, GenoToul, Toulouse, France; get.genotoul.fr). RNA-seq paired-end libraries were prepared according to the Illumina protocol with some adjustments, using the TruSeq Stranded Total RNA Gold library prep Kit (Illumina, San Diego, USA). Libraries were quantified by qPCR using the KAPA Library Quantification Kit (Roche, Basel, Switzerland). Libraries quality was assessed by the HS NGS kit on the Fragment Analyzer (Agilent Technologies, Santa Clara, USA). Libraries were equimolarly pooled and RNA sequencing was then performed on one S4 lane of the Illumina NovaSeq 6000 instrument (Illumina, San Diego, USA), using the NovaSeq 6000 S4 v1.5 Reagent Kit (300 cycles), and a paired-end 2 x 150 pb strategy Results: Bioinformatic analysis defined CD138+ and CD138- B-cell lymphomas as two groups with a different transcriptome signature ( 2522 genes differentially expressed) driving various arrays of the immune responses and expressing different signaling/metabolic pathways. Enrichment of previously established human Burkitt lymphoma (BL) and multiple myeloma signatures was tested in our CD138+ B-cell lymphomas. In contrast to BL, myeloma up and down signatures were significantly enriched, emphasizing the molecular similarity of mouse CD138+ B-cell lymphomas to human multiple myeloma cells and the relevance of our mice as pertinent model of plasma B-cell lymphomas. Conclusions: Our mouse model carrying a homozygous insertion of c-myc into the IgH locus develops B-cell lymphoma with a large part of CD138+ plasma cell neoplasms. Its interesting and large transcriptome similarities with human myelomas make this mouse model an accurate, reliable experimental myeloma model.
Project description:The zinc finger transcription factor growth-factor-independent-1 (Gfi1) has been involved in various cellular differentiation processes. Gfi1 acts as a transcriptional repressor and splicing control factor upon binding to cognate binding sites in regulatory elements of its target genes. Here, we report that Gfi1-deficient mice develop autoimmunity. Gfi1-deficient peripheral B-cells show a hyperproliferative phenotype, leading to expansion of plasma cells, increased levels of nuclear autoantibodies, and immunoglobulin deposition in brain and kidneys. Dysregulation of multiple transcription factors and cell-cycle control elements may contribute to B-cell dependent autoimmunity. Gfi1 thus emerges as a novel master-regulator restricting autoimmunity. Experiment Overall Design: Splenic B220+CD19+ CD138- B cells of 4 week old Gfi1+/+ and Gfi1-/- mice were isolated and RNA was extracted from one sample per group and microarray analysis was performed.