Transcriptional analysis of human tonsil Tfh cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Peter Wilkinson
PROVIDER: E-GEOD-50391 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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2013-11-12 | E-GEOD-50392 | biostudies-arrayexpress
Project description:Tfh cells are required for T cell help to B cells, and BCL6 is the defining transcription factor of Tfh cells. However, the functions of Bcl6 in Tfh have largely remained unclear. Here we defined the BCL6 cistrome in primary human germinal center Tfh cells to assess mechanisms of BCL6 regulation of CD4 T cells, comparing and contrasting BCL6 function in T and B cells. BCL6 primarily acts as a repressor in Tfh cells, and BCL6 binding was associated with control of Tfh cell migration and repression of alternative cell fates. Interestingly, although some BCL6 bound genes possessed BCL6 DNA binding motifs, many BCL6-bound loci were instead characterized by the presence of DNA motifs for AP1 or STAT. AP1 complexes are key positive downstream mediators of TCR signaling and external stimuli. We show that BCL6 can directly bind AP1, and BCL6 depends on AP1 for recruitment to BCL6 binding sites with AP1 motifs, suggesting that BCL6 subverts AP1 activity. These findings reveal that BCL6 has broad and multifaceted effects on Tfh biology, and provide insight into how this master regulator mediates distinct cell-context dependent phenotypes. Sorted naM-CM-/ve tonsil cells were activated with anti-CD3+CD28 Ab coated beads and transduced with BCL6 or control lentiviral vectors as described. RNA was isolated at day 5 following LV infection and microarrays performed as discussed herein. RNA was isolated with the RNeasy Micro Kit (QIAGEN). The quantity and quality of the RNA were confirmed with a NanoDrop 2000c (Thermo Fisher Scientific) and an Experion Electrophoresis System (Bio-Rad). Samples (20 ng) were amplified with Illumina MessageAmp II aRNA Amplification Kits (Ambion), hybridized to HumanHT-12_V4 BeadChips (Illumina), and quantified with Genome Studio (Illumina). Normalized data (heatmap of blood cells) or raw data (heatmap of tonsil cells and Volcano plot) were analyzed with the GenePattern software suite.
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Project description:Tight control of follicular helper T(Tfh) cells is required for optimal maturation of the germinal center (GC) response. The molecular mechansims controlling Tfh cell differentiation remain incompletely understood. Here we sought to to identfiy miRNAs that might regulate Tfh cell development and/or function. To identfiy miRNAs that are differentially expressed in Tfh cells, we performed Agilent microRNA microarray analysis comparing Tfh cells with the other main T cell subsets (naive T cells, non-Tfh effector or non-Tfh memory T cells, CD57+ Tfh cells and CD57- Tfh cells). RNA was extracted from 4 separate human tonsils (biological replicates) using mirVANA RNA isolation kit (Ambion) according to the manufacturerM-bM-^@M-^Ys protocol. Each biological sample was analysed individually. Live lymphocytes were sorted based on negative 7AAD staining, and thefollowing four subsets were purified: Naive T cells (CD4+ CD45RO-); CD57+ Tfh cells (CD4+ CD45RO+ PD-1_high CXCR5_high CD57+); CD57- Tfh cells (CD4+ CD45RO+ PD-1_high CXCR5_high CD57-) and non-Tfh effector or memory T cells (CD4+ CD45RO+ PD-1_neg CXCR5_neg). RNA quantity and quality was determined using a Nanodrop 1000 and an Agilent 2100 Bioanalyzer respectively. Hybridization on Agilent Human miRNA Microarray (V1) Kit, 8x15K and was performed at The Ramaciotti Center for Genomics (University of New South Wales, Australia). The mean of 3-4 biological replicates from each population of each population were calculated and values were plotted using Prism.
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