Project description:We have observed that follicular B cells from mice with a hypomorphic mutation (IkL/L) in the Ikzf1 gene (which encodes the Ikaros transcription factor) exhibit an increased proliferative response to anti-IgM stimulation (Kirstetter et al, Eur J Immunol, 32:720-30, 2002). We asked if Ikaros controls the transcriptional response that unfolds after activation, or if differences in the transcriptional landscape of resting B cells could explain the altered response. To this end, we have determined the transcriptome of unstimulated WT and IkL/L follicular B cells, as well as that of cells stimulated for 3h and 12h with anti-IgM. Samples from 2 independent experients were analyzed. Follicular splenic B cell were sorted from 6-week old WT or IkL/L mice, and stimulated for 3 or 12h with anti IgM, or cultured without anti-IgM for 3h (unstimulated samples) 2 independant experiments were performed
Project description:We have observed that follicular B cells from mice with a hypomorphic mutation (IkL/L) in the Ikzf1 gene (which encodes the Ikaros transcription factor) exhibit an increased proliferative response to anti-IgM stimulation (Kirstetter et al, Eur J Immunol, 32:720-30, 2002). We asked if Ikaros controls the transcriptional response that unfolds after activation, or if differences in the transcriptional landscape of resting B cells could explain the altered response. To this end, we have determined the transcriptome of unstimulated WT and IkL/L follicular B cells, as well as that of cells stimulated for 3h and 12h with anti-IgM. Samples from 2 independent experients were analyzed.
Project description:CD69 is a transmembrane protein expressed on the surface of activated leukocyte. The ligand for CD69 and the intracellular signaling pathway of this molecule are yet unknown. It is widely used as a marker of activated lymphocyte, but its function in immune system is not known. We used micro-array to define genes whose expression is regulated by activation antigene CD69. CD4 T cells were isolated from the spleen of wt B6 and CD69-deficient B6 mice and in vitro activated with anti-CD3/anti-CD28 coated beads. On one groupe of wt B6 cells, CD69 was activated using a anti-CD69 and secoundary antibody. RNA extraction and hybridization on Affymetrix microarrays was performed for wt B6, CD69-activated wt B6 and CD69-deficient B6 CD4 T cells.
Project description:This RNA-seqexperiment was designed to find transciptional differences between wildtype and Themis2-deficient B cells either directly ex-vivo or stimulated for 6 h with various stimuli in vitro. It is part of a larger study on the function of Themis2 in B cells. Therefore splenic, live, B220+ CD93- IgM+ CD23+ follicular B cells were sorted by flow cytometry from wildtype or Themis2-deficient (Themis2KO/KO) C57BL/6J mice. Unstimulated samples were were lysed directly after the sort. Stimulated samples were stimulated in vitro for 6 h at 37 degree Celsius at a concentration of 3 million cells/mL with either 10 microgram/mL anti-IgM or 10 microgram/mL LPS or 1 microgram/mL CD40L with 0.1 microgram/mL IL-4 and then lysed. RNA was extracted using Trizol reagent (Life Technologies) and cleaned up using the RNEasy Mini Kit (Quiagen). Single end, unstranded, poly-A-enriched libraries were made using the TruSeq RNA sample preparation kit (Illumina). Samples were analysed with an Illumina HiSeq 2000, collecting 13.2 to 76.1 million reads of 75 bases per sample.