Gene expression profiling of thymically-derived regulatory T cells from miR-142-deficient versus wild-type mice

Regulatory T cells (Tregs) play a fundamental role in immune tolerance via control of self-reactive effector T cells (Teffs). The development and function of the Treg lineage is critically dependent on the transcription factor Forkhead box P3 (Foxp3). Mice with a Treg-specific deletion of miRNAs, also develop a spontaneous, lethal autoimmune disease virtually indistinguishable from that seen in Foxp3-decicient mice demonstrating that miRNAs are critical for establishment of Treg-mediated peripheral tolerance. However, the set of miRNAs responsible for this functional deficiency has yet to be fully defined. Using Foxp3 ChIP-seq data we identified Mir142 as the only miRNA gene associated with a super-enhancer bound by FOXP3 suggesting that miR-142 is important for Treg function. To identify genes directly regulated by miR-142 in Tregs we profiled the changes in gene expression upon conditional deletion of miR-142 in thymically-derived Treg cells. Tregs were obtained by magnetic positive selection of CD4+ cells from the lymph nodes and spleens of FoxP3YFP-Cre x Mir142fl/fl and FoxP3YFP-Cre x Mir142+/+ mice followed by flow-cytometric sorting of CD4+ CD25+ YFP+ cells to >95% purity. Complementary DNA (cDNA) from total RNA was prepared using the Nugen WT-Ovation Pico kit (Nugen Technologies) and hybridized to Affymetrix Mouse Gene ST 2.0 microarrays. All cell populations used for the microarray analysis were generated in duplicates and individually processed. Raw data were processed with the robust multi-array average (RMA) algorithm for probe-level normalization and differentially expression was estimated using the limma package in Bioconductor.