B cells from human tonsil and blood were sorted using flow cytometry. The human samples were processed immediately ex-vivo using markers for known B cell subsets.
Analysis of somatic hypermutation in X-linked hyper-IgM syndrome shows specific deficiencies in mutational targeting.
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View SamplesSorted B cells using flow cytometry
Analysis of somatic hypermutation in X-linked hyper-IgM syndrome shows specific deficiencies in mutational targeting.
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View SamplesPioneering studies within the last few years have allowed the in vitro expansion of tissue-specific adult stem cells from a variety of endoderm-derived organs, including the stomach, small intestine and colon. Here we derived organoids from mouse gallbladder tissue (gallbladder organoids), from mouse liver (including the extrahepatic biliary ducts and gallbladder; liver organoids) and from mouse small intestine tissue (intestinal organoids). RNA was prepared from these organoids and used to assay expression of 21,258 genes using Affymetrix gene expression arrays. RNA was also prepared from mouse gallbladder, liver and small intestine tissues and used to assay gene expression in these tissues. Finally, gallbladder organoids were induced to differentiate by removing R-spondin 1 and noggin from the culture media and subjected to gene expression array analysis.
R-spondin 1 and noggin facilitate expansion of resident stem cells from non-damaged gallbladders.
Specimen part
View SamplesProfiling of the transcriptome of FITChigh/FSCdim and FITCdim/FSChigh sub-populations. Three biological replicates were profiled for each cell type. Overall design: Profiling of the transcriptome of FITChigh/FSCdim and FITCdim/FSChigh sub-populations. Three biological replicates were profiled for each cell type.
An autofluorescence-based method for the isolation of highly purified ventricular cardiomyocytes.
Specimen part, Cell line, Subject
View SamplesDiffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma in adults. The disease exhibits a striking heterogeneity in gene expression profiles and clinical outcomes, but its genetic causes remain to be fully defined. Through whole genome and exome sequencing, we characterized the genetic diversity of DLBCL. In all, we sequenced 73 DLBCL primary tumors (34 with matched normal DNA). Separately, we sequenced the exomes of 21 DLBCL cell lines. We identified 322 DLBCL cancer genes that were recurrently mutated in primary DLBCLs. We identified recurrent mutations implicating a number of known and not previously identified genes and pathways in DLBCL including those related to chromatin modification (ARID1A and MEF2B), NF-B (CARD11 and TNFAIP3), PI3 kinase (PIK3CD, PIK3R1, and MTOR), B-cell lineage (IRF8, POU2F2, and GNA13), and WNT signaling (WIF1). We also experimentally validated a mutation in PIK3CD, a gene not previously implicated in lymphomas. The patterns of mutation demonstrated a classic long tail distribution with substantial variation of mutated genes from patient to patient and also between published studies. Thus, our study reveals the tremendous genetic heterogeneity that underlies lymphomas and highlights the need for personalized medicine approaches to treating these patients.
Genetic heterogeneity of diffuse large B-cell lymphoma.
Specimen part, Cell line
View SamplesThis SuperSeries is composed of the SubSeries listed below.
Deep sequencing of the small RNA transcriptome of normal and malignant human B cells identifies hundreds of novel microRNAs.
Specimen part, Cell line
View SamplesBurkitt lymphoma is characterized by deregulation of MYC, but the contribution of other genetic mutations to the disease is largely unknown. Here, we describe the first completely sequenced genome from a Burkitt lymphoma tumor and germline DNA from the same affected individual. We further sequenced the exomes of 59 Burkitt lymphoma tumors and compared them to sequenced exomes from 94 diffuse large B-cell lymphoma (DLBCL) tumors. We identified 70 genes that were recurrently mutated in Burkitt lymphomas, including ID3, GNA13, RET, PIK3R1 and the SWI/SNF genes ARID1A and SMARCA4. Our data implicate a number of genes in cancer for the first time, including CCT6B, SALL3, FTCD and PC. ID3 mutations occurred in 34% of Burkitt lymphomas and not in DLBCLs. We show experimentally that ID3 mutations promote cell cycle progression and proliferation. Our work thus elucidates commonly occurring gene-coding mutations in Burkitt lymphoma and implicates ID3 as a new tumor suppressor gene.
Deep sequencing of the small RNA transcriptome of normal and malignant human B cells identifies hundreds of novel microRNAs.
Specimen part, Cell line
View SamplesNatural Killer (NK) cells are the first lymphocyte population to reconstitute early after non myelo-ablative and T cell-replete haploidentical hematopoietic stem cell transplantations (h-HSCTs) with post-transplant infusion of cyclophosphamide. The present study characterizes the transient and predominant expansion starting from the 2nd week after h-HSCT of a donor-derived unconventional subset of CD56dim/CD16neg (uCD56dim) NK cells expressing remarkable high levels of NKG2A and low levels of NKp46. Both transcription and phenotypic profiles indicated that uCD56dim NK cells are a distinct NK cell subpopulation with features of late differentiation, yet retaining proliferative capability and functional plasticity to generate conventional CD56bright/CD16pos NK cells in response to IL-15 plus IL-18. uCD56dim NK cells represent by far the largest NK cell subset detectable in the following 7 weeks after h-HSCT and they also express high levels of the activating receptors NKGD and NKp30 as well as of the lytic granules Granzyme-B and Perforin. Nonetheless, uCD56dim NK cells displayed a defective cytotoxicity that could be reversed by blocking the inhibitory receptor CD94/NKG2A. These data open new important perspectives to better understand the ontogenesis/homeostasis of human NK cells and to develop a novel immune-therapeutic approach by targeting the inhibitory NKG2A check point, thus enhancing NK cell alloreactivity early after h-HSCT.
The early expansion of anergic NKG2A<sup>pos</sup>/CD56<sup>dim</sup>/CD16<sup>neg</sup> natural killer represents a therapeutic target in haploidentical hematopoietic stem cell transplantation.
Specimen part
View SamplesTranslation and mRNA decay are intimately connected processes, and translational inhibition often precedes and stimulates transcript degradation. Here, we have focused on methods that allow determination of mRNA stability on a transcriptome-wide scale. We describe experimental and computational methods for the two most commonly used approaches (transcriptional inhibition and metabolic labeling), and we discuss associated caveats. Overall design: Metabolic labeling time courses (1, 2, 4, 8, 12, 24 hr) using 4SU were performed in HEK293.
Determining mRNA half-lives on a transcriptome-wide scale.
Treatment, Subject
View SamplesPro-inflammation triggered by microbial lipopolysaccharide (LPS) through Toll-like receptor (TLR) 4 in the presence of interferon (IFN)-g induces cytokine secretion in dendritic cells (DCs) tightly regulated by a defined differentiation program. This DC differentiation is characterized by a dynamic immune activating but also tolerance inducing phenotype associated with irreversible down-modulation of cytokines. CD40L on activated T cells further modifies DC differentiation. Using DNA micro arrays we showed down-regulated mRNA levels of TLR signaling molecules while CD40/CD40L signaling molecules were up-regulated at a time when LPS/IFN-g activated DCs have ceased cytokine expression. Accordingly we demonstrated that CD40/CD40L but not TLR4 or TLR3 signaling mediated by LPS or poly (cytidylic-inosinic) acid (poly I:C) and dsRNA re-established the capacity to secret interleukin (IL)-12 in LPS/IFN-g activated DCs, which have exhausted their potential for cytokine secretion. This resulting TH1 polarizing DC phenotype which lacked accompanying secretion of the crucial immune suppressive IL-10 - enhanced activation of cytotoxic T lymphocytes (CTLs). We therefore conclude that immune modulation is restricted to a secondary T-cell mediated stimulus at an exhausted DC state which prevents an immune tolerant DC phenotype. These findings impacts on the rational design of TLR activated DC-based cancer vaccines for the induction of anti-tumoral CTL responses.
CD40 ligation restores type 1 polarizing capacity in TLR4-activated dendritic cells that have ceased interleukin-12 expression.
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