Unknown,Transcriptomics,Genomics,Proteomics

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T cell dependent immune responses: B cell activation and differentiation Group1 Fo, Group2 GC1, Group3 GC2

ABSTRACT: Upon immunization with a T cell dependent antigen naive follicular B cells (Fo) are activated and a germinal center reaction is induced. Within the next 2 weeks large germinal centers develop where the process of affinity maturation takes place. To analyze the gene expression profile of resting and activated B cells, follicular B cells (Fo), B cells from early (GC1) and late germinal centers (GC2) were isolated and their gene expression profile compared. Gene expression profiles of Fo versus GC1 and GC2 B cells, respectively. Naïve Fo B cells were isolated from non-immunized BALB/c mice. Germinal center B cells sorted from spleen cell suspensions of BALB/-c mice immunized with the T cell dependent antigen 2-phenyl Oxazolone. GC1 B cells were isolated 7 days after primary immunization. GC2 cells were isolated 15 days after primary immunization. After total RNA extraction, reverse transcription, cDNA extraction, the biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized in duplicates for each of the three groups to the GeneChip arrays. Group1: Fo, Group2: GC1, Group3: GC2. Lists of differentially regulated genes were created using High Performance Chip Data Analysis (HPCDA) with Bioretis database (http://www.bioretis-analysis.de). Worldwide data sharing is possible via Bioretis, please ask the authors.

ORGANISM(S): Mus musculus

SUBMITTER: Joachim Grün

PROVIDER: E-GEOD-28237 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

In silico subtraction approach reveals a close lineage relationship between follicular dendritic cells and BP3(hi) stromal cells isolated from SCID mice.

Wilke Gordon G Steinhauser Gudrun G Grün Joachim J Berek Claudia C

European journal of immunology 20100801 8

Organization of the stromal compartments in secondary lymphoid tissue is a prerequisite for an efficient immune reaction. In particular, follicular dendritic cells (FDC) are pivotal for the activation and differentiation of B cells. To investigate the development of FDC, FDC together with tightly associated B cells (FDC networks) were micro-dissected from frozen tissue sections and follicular B cells were sorted by FACS. Using an in silico subtraction approach, gene expression of FDC was determi ...[more]

PMID: 20518031

Publication: 1/2

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Project description:Human Natural Killer (NK) cells comprise two main subsets, CD56bright and CD56dim cells, that differ in function, phenotype and tissue localization. To further dissect the heterogeneity of CD56dim cells, we have performed transcriptome analysis and functional ex vivo characterization of human NK cell subsets according to the expression of markers related to differentiation, migration or competence. Here, we show for the first time that the ability to respond to cytokines or to activating receptors is mutually exclusive in almost all NK cells with the exception of CD56dim CD62L+ cells. Indeed, only these cells combine the ability to produce interferon (IFN)-gamma after cytokines and proliferate in vivo during viral infection with the capacity to kill and produce cytokines upon engagement of activating receptors. Therefore, CD56dim CD62L+ cells represent a unique subset of polyfunctional NK cells. Ex vivo analysis of their function, phenotype, telomere length, frequencies during ageing as well as transfer experiments of NK cell subsets into immunodeficient mice suggest that CD56dim CD62L+ cells represent an intermediate stage of NK cell maturation, which after restimulation can accomplish multiple tasks and further develop into terminally differentiated effectors. Gene expression profiles of FACSAria sorted CD3- CD56bright CD62L+, CD3- CD56dim CD62L+ and CD3- CD56dim CD62L- NK cells from human peripheral blood of three donors were compared using Affymetrix GeneChip Human Genome HG-U133_Plus_2. After total RNA extraction, reverse transcription, cDNA extraction, the biotinylated cRNA was transcribed, fragmented, and 15 Âµg cRNA hybridized in triplicates for each of the three groups to the GeneChip arrays. Group1: CD3- CD56bright CD62L+,.Group2: CD3- CD56dim CD62L+, Group3: CD3- CD56dim CD62L-. Lists of differentially regulated genes were created using High Performance Chip Data Analysis (HPCDA) with Bioretis database (http://www.bioretis-analysis.de). Worldwide data sharing is possible via Bioretis, please ask the authors.

Project description:Gene expression profiling of cells isolated ex vivo is a unique tool to assess gene expression in vivo. Exemplified for CD4+CD45RO+ effector/memory T helper (T E/M) lymphocytes of human peripheral blood, we have analyzed different isolation procedures and storage conditions for the introduction of bias. Cells from clinical samples can be sorted to high purity by a flow cytometric sorter and assessed for their gene expression pattern but storage/shipping conditions and often necessary pre-enrichment due to low initial population frequencies bears the danger of introducing artificial changes. To test for changes introduced by different procedures, cells were enriched either by magnetic cell sorting (MACS) with Whole-Blood CD4 beads, density gradient centrifugation, alone or in combination with CD3 MACS, or by lysis of erythrocytes followed by MACS depletion of CD15+ cells prior to fluorescence-activated cell sorting (FACS) as CD3+CD4+CD45RO+ cells. A total of 7 different protocols were compared among each other and to T cells stimulated for 3 hours after isolation with Phorbol-12-myristate-13-acetate and Ionomycin (PMA/Iono). Preparation-induced de novo transcription was blocked in one of the protocols by addition of 2 M-BM-5g/ml Actinomycin D (ActD). To test for changes introduced by delays in sample preparation blood samples were drawn and either processed directly or kept at 4 M-BM-0C or room temperature (RT) for 2 or 6 hours before T E/M cells were isolated. Gene expression was compared among the various cell preparations and to T E/M cells isolated from 1 day old buffy coat fractions stored at RT. Sorted cells were lysed in TRIzol (Invitrogen) and frozen at -80M-BM-0C until used for RNA extraction. Total RNA was extracted using the miRNeasy kit (Qiagen). The integrity and amount of isolated RNA was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Double-stranded complementary RNA was synthesized from 1 M-BM-5g total RNA using Message AmpII Biotin (Ambion, USA). Fifteen micrograms of fragmented cRNA of each sample were hybridized to 26 HG-U133A plus 2.0 GeneChips (Affymetrix). Hybridization was performed in a Hybridization Oven 640, and chips were washed and stained in the Fluidics Station 400 (both Affymetrix). Finally, the arrays were scanned with a GeneChip Scanner 3000 using the GCOS software, version 1.4, both Affymetrix. All relevant GCOS data of quality checked microarrays were analyzed with High Performance Chip Data Analysis (HPCDA, unpublished), using the BioRetis database (www.bioretis-analysis.de), as described and validated previously.

Project description:In order to identify genes that are activated in differentiating WT ESCs, but are missing in Tal-1-/- and Runx1-/- ESCs, and which might be involved in the generation of definitive hematopoietic progenitors and their specification thereafter, we performed microarray analyses on purified Flk-1+ cells, differentiated from these ESCs for 4, 5, and 6 days M-bM-^@M-^\in vitroM-bM-^@M-^]. Gene-expression profiling of three biological replicates was performed at days 4, 5, and 6 during the differentiation process of WT J1 ESCs (9 samples), and at day 6 during the differentiation process of either Runx1-/- J1 or Tal-1-/- J1 ESCs (3 samples each). Total RNA was extracted using the RNeasy Mini kit (Qiagen). The integrity and amount of isolated RNA was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Complementary DNA was synthesized from 3-5 M-NM-<g total RNA, using reagents recommended in the technical manual GeneChip Expression Analysis (Affymetrix, Santa Clara, CA). The in vitro transcription, necessary for the synthesis of biotinylated complementary RNA (cRNA) was performed using the Enzo RNA Transcript Labeling kit (Affymetrix). Fifteen micrograms of fragmented cRNA of each sample were hybridized to nine Mouse Genome 430-2 arrays (Affymetrix). Hybridization was performed in a Hybridization Oven 640, and chips were washed and stained in the Fluidics Station 400 (both Affymetrix), according to procedure 2 as described in the technical manual. Finally, the arrays were scanned with a GeneChip Scanner 3000 using the GCOS software, both Affymetrix. All relevant GCOS data of quality checked microarrays were analyzed with High Performance Chip Data Analysis (HPCDA, unpublished), using the BioRetis database (www.bioretis-analysis.de), as described and validated previously (55). Used query parameters for database filtering process was described earlier several times (56). For hierarchical cluster analysis, we used the program Genes@Work (57) with gene vectors for normalization and Pearson w/mean for similarity measure. As cluster type, we used center of mass.

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Dataset Information

T cell dependent immune responses: B cell activation and differentiation Group1 Fo, Group2 GC1, Group3 GC2

Publications

In silico subtraction approach reveals a close lineage relationship between follicular dendritic cells and BP3(hi) stromal cells isolated from SCID mice.

Similar Datasets

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