GSE96050
ZFP36 RNA binding proteins restrain T-cell activation kinetics and anti-viral immunity [RNA-seq]
Dynamic post-transcriptional control of RNA expression by RNA-binding proteins (RBPs) is critical during immune response. ZFP36 RBPs are prominent inflammatory regulators linked to autoimmunity and cancer, but functions in adaptive immunity are less clear. We used HITS-CLIP to define ZFP36 targets in T-cells, which confirmed regulation of cytokine expression and revealed unanticipated actions in regulating T-cell activation and proliferation. Transcriptome and ribosome profiling showed that ZFP36 represses mRNA target abundance and translation, most robustly through a novel class of AU-rich sites in coding sequence. Functional studies revealed that ZFP36 regulates early T-cell activation kinetics by attenuating activation marker expression, limiting T-cell expansion, and promoting apoptosis in a cell autonomous manner. Strikingly, loss of ZFP36 in vivo accelerated T-cell responses to acute viral infection, and enhanced anti-viral immunity. These findings uncover a critical role for ZFP36 RBPs in restraining T-cell expansion and effector functions, and suggest ZFP36 inhibition as a novel strategy to enhance immune-based therapies. Overall design: Transcriptome profiling by RNAseq was done for WT and Zfp36 KO CD4+ T-cells activated under Th1 polarizing conditions for 4 hours or 72 hours, to determine differentially expressed transcripts. Naïve CD4+ T-cells were FACS-sorted from pooled spleen and lymph nodes of WT or Zfp36 KO mice, and incubated in co-cultures with formalin-fixed bone-marrow derived dendritic cells (BMDCs), anti-CD3e, 10 U/ml IL-2 and 5 ng/ml IL-12. Total RNA was extracted with Trizol (Life Technologies) four hours post-activation, or 72 hours post-activation after 2 hours? restimulation with 20 ng/ml PMA and 1 ?M ionomycin. RNA was further purified on HiPure columns with DNAse treatment (Roche).  500 ng total RNA was depleted of rRNA with Ribo-Zero (Epicentre), then used to generate libraries with the TruSeq kit (Illumina).  Libraries were analyzed on the Hiseq 2000, pooling 4-5 samples per sequencing lane. RNAseq libraries were aligned against the mouse genome (mm10) with STAR, and read counts per feature were determined with HTSeq.