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Single cell RNA-seq (scRNA-seq) of 48h GCBC cultures with either of these two stimuli or with the combination of IL-21R/CD40 and BCR stimulation.

ABSTRACT: Purpose:Flow cytometric analysis showed that after 48h of culture with T cell- and Ag-derived stimuli, GCBC alter their TF expression and begin to differentiate. However, only a subset of cells undergoes this process, for reasons that are unclear. To better define the nature of this heterogeneity, and to further define the differences between IL-21R/CD40 vs BCR/CD40 signals, we performed single cell RNA-seq (scRNA-seq) of 48h GCBC cultures with either of these two stimuli or with the combination of IL-21R/CD40 and BCR stimulation. Methods: we performed scRNA-seq using 10X Genomics Chromium system . Results: Single cell analysis demonstrated surprisingly little overlap between the clusters derived from IL-21/⍺-CD40 stimulation and those derived from BCR/⍺-CD40 ligation, thus establishing at molecular and cellular detail how distinct these two types of selection signals are for GCBC

ORGANISM(S): Mus musculus

PROVIDER: GSE197661 | GEO | 2023/02/16

REPOSITORIES: GEO

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RNA-seq analysis of GCBC and NBC that had been stimulated for either 2h or 4h with IL-21, CD40 ligation, BCR ligation, or combinations of these.

Project description:Purpose: To gain insight into both similarities and differences in the gene expression programs induced by IL-21R, BCR and CD40 signals, we performed RNA-seq on both GCBC and NBC that had been stimulated for either 2h or 4h with IL-21, CD40 ligation, BCR ligation, or combinations of these. Methods : Purified GCBC from d14 NP-CGG immunized MEG mice and NBC from naïve MEG mice were warmed at 37C for 30 minutes and then stimulated with 10ng/mL IL-21, 20μg/ml ⍺-IgM or 2.5μg/mL ⍺-CD40 (prepared as tetramer) alone or combined IL21/⍺-CD40 or ⍺-IgM/⍺-CD40 for 2 and 4 hours. After stimulation, cells were processed for RNA isolation using QIAshredder columns (Qiagen) and the RNeasy Plus Mini Kit (Qiagen) following the manufacturer’s instructions. Samples were sequenced using Illumina NextSeq 500 with 75 bp paired-end reads and aligned to the mm10 genome using the STAR aligner (Dobin et al., 2013). Gene-level counts were determined using featureCounts (Liao et al., 2014), and raw counts were quantile normalized to each other for differential expression using the voom method (Law et al., 2014) in the Limma R package (Ritchie et al., 2015). For normalization of the datasets, the Quantile method was used. Results: Like BCR and CD40 signals, IL-21R plus CD40 signals also synergize to induce c-Myc in GCBC. However, IL-21R plus CD40 stimulation differentially affects GCBC fate compared to BCR plus CD40 ligation—engaging unique molecular mechanisms

2023-02-16 | GSE197662 | GEO

Single cell RNA-seq (scRNA-seq) of 48h GCBC cultures with either of these two stimuli or with the combination of IL-21R/CD40 and BCR stimulation.

Project description:Single cell RNA-seq (scRNA-seq) of 48h GCBC cultures with either of these two stimuli or with the combination of IL-21R/CD40 and BCR stimulation.

| PRJNA811523 | ENA

Gene signature of CLL cells cultured with activated T cells or CD40L-expressing cells

Project description:Chronic Lymphocytic Leukemia (CLL) cells multiply in secondary lymphoid tissue but the mechanisms leading to their proliferation are still uncertain. In addition to BCR-triggered signals, other microenvironmental factors might well be involved. In proliferation centres, leukemic B cells are in close contact with CD4+CD40L+Â T cells. Therefore, we here dissected the signals provided by autologous activated T cells (Tact) to CLL cells. Although the gene expression profile induced by Tact was highly similar to that induced by sole CD40 signaling, an obvious difference was that Tact induced proliferation of CLL cells. We determined that stimulation with only CD40L+IL-21 was sufficient to induce robust proliferation in CLL cells. We then defined an IL-21-induced gene signature in CLL, containing components of JAK-STAT and apoptosis pathways, and this signature could be detected in lymph node (LN) samples from patients. Finally, we could detect IL-21 RNA and protein in LN, and IL-21 productionÂ ex vivoÂ by LN CD4+CXCR5+ follicular helper T cells. These results indicate that, in addition to BCR signaling, activated T cells might contribute to CLL cell proliferation via CD40 and IL-21. Targeting these signaling pathways might offer new venues for treatment of CLL.Â CLL cells were cultured under different conditions for 16 hours and then sorted to purity as CD20+ CD5+ cells.

2013-10-07 | E-GEOD-50572 | biostudies-arrayexpress

Gene signature of CLL cells cultured with activated T cells or CD40L-expressing cells

Project description:Chronic Lymphocytic Leukemia (CLL) cells multiply in secondary lymphoid tissue but the mechanisms leading to their proliferation are still uncertain. In addition to BCR-triggered signals, other microenvironmental factors might well be involved. In proliferation centres, leukemic B cells are in close contact with CD4+CD40L+ T cells. Therefore, we here dissected the signals provided by autologous activated T cells (Tact) to CLL cells. Although the gene expression profile induced by Tact was highly similar to that induced by sole CD40 signaling, an obvious difference was that Tact induced proliferation of CLL cells. We determined that stimulation with only CD40L+IL-21 was sufficient to induce robust proliferation in CLL cells. We then defined an IL-21-induced gene signature in CLL, containing components of JAK-STAT and apoptosis pathways, and this signature could be detected in lymph node (LN) samples from patients. Finally, we could detect IL-21 RNA and protein in LN, and IL-21 production ex vivo by LN CD4+CXCR5+ follicular helper T cells. These results indicate that, in addition to BCR signaling, activated T cells might contribute to CLL cell proliferation via CD40 and IL-21. Targeting these signaling pathways might offer new venues for treatment of CLL.

2013-10-07 | GSE50572 | GEO

IL-21 regulates the expression of chemokine genes in B-chronic lymphocytic leukemia

2013-09-01 | GSE42158 | GEO

In-vitro B cell stimulation RNA-seq

Project description:RNA-seq profiles of sorted B cells undergoing stimulation of BCR, CD40, both, or none.

2021-08-17 | GSE182266 | GEO

IL-21 regulates the expression of chemokine genes in B-chronic lymphocytic leukemia

Project description:B-cell chronic lymphocytic leukemia (CLL) is a common type of leukemia, characterized by the progressive accumulation of CD5+ “mature” monoclonal B lymphocytes in peripheral blood, bone marrow and lymphoid tissues. Although circulating CLL cells are non-dividing cells, prone to spontaneous apoptosis, their progressive accumulation is the result of a dynamic balance between cell death and proliferation and a high turn-over rate has been related to a poor prognosis. Indeed, CLL cells are protected from apoptosis and proliferate in specific niches within the lymphoid tissues and the bone marrow. CLL cells show variable expression of IL-21R that can be up-regulated by cell activation via CD40. CD40-activated CLL cells phosphorylate STAT-1 and STAT-3 and undergo apoptosis in response to IL-21 stimulation. By gene-expression profiling we found out that IL-21 modulates the expression of several genes including cytokine and chemokine genes and genes involved in cell survival and apoptosis in CD40-activated CLL cells. PBMCs were isolated from heparinized blood obtained from 13 untreated patients diagnosed with CLL on the basis of clinical and immunophenotypic criteria. PBMCs were isolated by Ficoll density gradient centrifugation and characterized by immunofluorescence and FACS analysis. When residual non B-cells exceeded 10%, B cells were enriched by negative selection with antibody-coated magnetic beads (CD2-beads, Dynal, Oslo, Norway) to obtain a >95% pure CD19+/CD5+ B cell population. B-CLL cells were pre-activated on adherent CD40L-transduced L cells for 48-36h and then stimulated with IL-21 or medium only for additional 18h. Total RNA was isolated from samples using TriZol (Invitrogen) reagent.

2013-09-01 | E-GEOD-42158 | biostudies-arrayexpress

Expression data from Daudi cells stimulated with anti-CD40 and/or IL-4 for 6 hr

Project description:We used microarrays to detail the transcriptome response to CD40-ligation and IL-4 stimulation in Daudi cells. Keywords: gene expression array-based, response to anti-CD40 and/or IL-4

2006-07-31 | GSE4846 | GEO

Expression data from Daudi cells stimulated with anti-CD40 and/or IL-4 for 6 hr with or without AICAR-pretreatment

Project description:We used microarrays to detail the action of AICAR on the transcriptome response to CD40-ligation and IL-4 stimulation in Daudi cells. Keywords: gene expression array-based, response to anti-CD40 and/or IL-4 with or without AICAR

2006-07-31 | GSE4812 | GEO

RNA-seq of in vitro derived B cells

Project description:IFN-γ has been reported to be the ABC-promoting cytokine combined with signaling from IL-21, CD40L, Ag-BCR and TLR7 agonist. Hence, we adopted the in vitro differentiation cocktails (anti-IgM, R848, anti-CD40, IL-21 plus IFN-γ or IL-4) to determine the role of IFN-γ in ABC differentiation. Mouse splenic B cells were purified by negative selection and cultured in the presence of the above cocktails. Compared with IL-4, IFN-γ-polarized cells preferentially differentiated to ABC-like cells. And RNA-seq were performed on IFN-γ-polarized cells and IL-4-polarized cells.

2023-02-17 | E-MTAB-12569 | biostudies-arrayexpress

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