Transcriptome and translatome of human regulatory T cells differentiated with TGFbeta and mTOR inhibitor everolimus

Regulatory T cells (Tregs) inhibit effector T cells and maintain immune system homeostasis. Treg maturation in peripheral sites (pTregs) is poorly understood but is known to require inhibition of protein kinase mTORC1 (e.g.RAD001) and exposure to TGFb. Paradoxically, Treg maturation requires protein synthesis yet mTORC1 inhibition downregulates it, leaving unanswered how Tregs carry-out essential mRNA translation for development and immune suppression activity. Using human CD4+ T cells differentiated in culture (iTregs) and genome-wide transcription and translation profiling, we show that TGFb transcriptionally reprograms naïve T cells to express iTreg differentiation and immune suppression mRNAs, while mTORC1 inhibition impairs translation of T cell mRNAs but not those induced by TGFb. TGFb-induced Treg mRNAs were found to utilize an alternate mechanism for cap-dependent mRNA translation directed by the DAP5/eIF3d complex rather than canonical mTORC1/eIF4E during Treg development, which is directed by their 5’-untranslated regions. iTreg differentiation is therefore mediated by combined TGFb transcriptional and translational reprogramming and a privileged mechanism of mRNA translation Naïve human T cells were isolated from healthy donor PBMCs. Cells were activated with anti-CD3/anti-CD28 and IL-2, treated with TGFbeta + RAD001 (mTOR inhibitor), TGFbeta alone, RAD001 alone, or left untreated. Cells were maintained in culture fo 13 days with treatments, then harvested. Double and single treated cells were enriched for Tregs by magnetic-beads sorting, and processed for RNA extraction. RNA was sedimented on a sucrose gradient to separate actively translating ribosomes (polysomes). Polysomal RNA (HD - high density) and total RNA (TOT) was loaded on Affymetrix chip for microarray analysis