Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse D44highCD62L-CD25- CD4+ T cells of murine (C57BL/6 mice) bone marrow were compared to those of the spleen

ABSTRACT: CD4+ T lymphocytes are key to immunological memory, but little is known about the lifestyle of memory CD4+ T lymphocytes. We showed that in the memory phase of specific immune responses to antigens, most of the memory CD4+ T lymphocytes relocated into the bone marrow (BM) within 3-8 weeks after their generation, a process involving integrin a2. Antigen-specific memory CD4+ T lymphocytes expressed Ly-6C to a high degree, unlike most splenic CD44hiCD62L- CD4+ T lymphocytes. In adult mice, more than 80% of Ly-6Chi CD44hiCD62L- memory CD4+ T lymphocytes were in the BM. In the BM, they are located next to IL-7-expressing VCAM-1+ stroma cells, and were in a resting state. Upon challenge with antigen, they rapidly expressed cytokines and CD154 and induced the production of high-affinity antibodies, indicating their functional activity in vivo and marking them as professional memory T helper cells Experiment Overall Design: FACSAria sorted CD44highCD62L-CD25- CD4+ T cells of murine (C57BL/6 mice) bone marrow were compared to those of the spleen using Affymetrix GeneChip Mouse Genome 430A 2.0 Array. 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 two groups to the GeneChip arrays. Group of bone marrow chips: BMCD4T1, BMCD4T2, BMCD4T3, group of spleen chips: SCD4T1, SCD4T2, SCD4T3. Lists of differentially regulated genes were created using High Performance Chip Data Analysis (HPCDA) with Bioretis database (http://www.bioretis-analysis.de).

ORGANISM(S): Mus musculus

SUBMITTER: Joachim GrÃ¼n

PROVIDER: E-GEOD-15733 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:To compare human memory CD4+ T cell subsets in peripheral blood (PB) and bone marrow (BM) of healthy individuals at transcriptional level, we analyzed the global gene expression of ex vivo PB CD69- as well as BM CD69- and CD69+ memory CD4+ T cells from 4 paired PB and BM samples. The gene expression of these subsets was additionally compared to the transcriptional profile of 8 PB samples taken ex vivo or stimulated with phorbol myristate acetate (PMA) and Ionomycin for 3 hours. Three ex vivo memory CD4+ T cell subsets (CD4+CD45RO+CD69+ and CD4+CD45RO+CD69- cells from bone marrow; CD4+CD45RO+CD69- cells from peripheral blood) were isolated from 4 paired bone marrow and blood samples (two males and two females) or from a different cohort of 8 blood samples (3 males and five females). The purity of sorted cells was higher than 95% as assessed by FACS. Subsequently, a fraction of purified cells from the 8 blood samples were stimulated with phorbol-myristic-acid/ionomycin (PMA/iono) for 3 h and used as high controls. Total RNA of each cell subset was extracted using a NucleoSpin RNA XS Kit (Macherey-Nagel) or 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). 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 28 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.

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Transcription profiling of mouse D44highCD62L-CD25- CD4+ T cells of murine (C57BL/6 mice) bone marrow were compared to those of the spleen

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