Role of interleukin 2 on Foxp3+CD4+ regulatory T (Treg) cell gene expression and chromatin modeling in thymus and lymph node, and on SATB1 DNA binding in single positive CD4 thymocytes

Interleukine 2 (IL-2) is still one of the most interesting cytokines in T cell biology with its ability to control immune homeostasis by maintaining the functional identity of Foxp3+ regulatory T (Treg) cells and the expansion of activated conventional T (Tconv) cells. Yet, how IL-2 exactly enables Treg cells to suppress autoreactive Tconv cells and to maintain their identity is unclear. Using a mouse model in which IL-2 signaling via its high affinity receptor CD25 is selectively impaired, the “Il2ramut/mut” mouse, we report that Treg cells that only receive low IL-2 signals keep Il2ramut/mut but not WT Tconv cells “in check”, suggesting equal IL-2 signals in Treg and Tconv cells is essential to safeguard immune homeostasis. Furthermore, the comparative analysis of gene expression and epigenetic landscape of Il2ramut/mut and WT Treg cells support a model in which IL-2 "locks in” Treg cell identity and functions in vivo by controlling their genome-wide chromatin accessibility. Microarrays:LNs from 6-8 weeks old aged-matched females either Il2ramut/mut (2 or 3 mice) or WT (2 or 3 mice) Foxp3-RFP+ reporter mice were harvested and RFP+ Treg or RFPneg Tconv cells were enriched by negative selection (dynabeads) and flow-sorted (Aria III) before proceeding to extraction of total RNA and cDNA RT transcription for microarray analysis ATAC-seq: LNs from 6-8 weeks old aged-matched females either Il2ramut/mut (2 mice) or WT (2 mice) Foxp3-RFP+ reporter mice were harvested and RFP+ Treg cells were enriched by negative selection (dynabeads) and flow-sorted (Aria III) before proceeding to extraction of nuclei, transposition and library preparation for sequencing (ATAC-seq). Thymuses from ~20 weeks old aged-matched females either Il2ramut/mut (4 mice) or WT (4 mice) Foxp3-RFP+ reporter mice were harvested and RFP+ Treg cells were enriched by negative selection (dynabeads) and flow-sorted (Aria III) before proceeding to extraction of nuclei, transposition and library preparation for sequencing (ATAC-seq). ChIP-seq: Thymus from 6-8 weeks old aged-matched females either Il2ramut/mut (2 mice) or WT (2 mice) mice were harvested and 2x10e6 single positive (SP) CD4+ thymocytes were pre-enriched by negative selection (dynabeads), stained with fluorescent labeled antibodies against CD4 (GK1.5) and CD8α (53-6.7) and flow-sorted (Aria III) before proceeding to fixation (cross-linking), lysis, immunoprecipation against SATB1 and library preparation for sequencing.