Essential and non-overlapping IL-2Ra-dependent signaling for thymic development and peripheral homeostasis of regulatory T cells

IL-2R signaling is essential for regulatory T cell (Treg) function. However, the precise contribution for IL-2 during Treg thymic development, peripheral homeostasis, and lineage stability remains unclear. Here we show that IL-2R signaling is essential for thymic Tregs at an early step for expansion/survival and a later step for functional maturation. Using selective deletion of CD25 in peripheral Tregs, we also find that IL-2R signaling was absolutely essential for their persistence whereas Treg lineage stability was IL-2-independent. CD25 knockout peripheral Tregs showed increased apoptosis, oxidative stress, signs of mitochondrial dysfunction, and reduced transcription of key enzymes of lipid and cholesterol biosynthetic pathways. A divergent IL-2 transcriptional signature was noted for thymic Tregs versus peripheral Tregs. These data indicate that IL-2R signaling in the thymus and the periphery leads to distinctive effects on Treg function, where peripheral Treg survival depends on a non-conventional mechanism of metabolic regulation. Overall design: To evaluate IL-2Ra-dependent transcriptional activity in thymic Tregs, CD25 KO Tregs were isolated from thymuses of Treg-targeted CD25 conditional KO animals, as well as CD25 WT controls. Groups of 5 biological replicates (mice) were compared. To evaluate IL-2Ra-dependent transcriptional activity in splenic Tregs, CD25 KO Tregs were isolated from tamoxifen-inducible, Treg-targeted CD25 conditional KO mice as well as CD25 WT reporter controls following tamoxifen induction. Groups of 4 biological replicates (mice) were compared. Libraries were prepared using KAPA's RNA Hyperprep protocol and sequenced on a 75 bp paired-end run using the Illumina NextSeq 500 High Output Kit (150-cycle; 400 M flow cell). Reads from RNA-seq were mapped to the Mus musculus genome GRCm38 using STAR (ver.2.5.0) aligner. Raw counts were generated based on Ensembl genes (GENCODE M13) with featureCounts (ver.1.5.0). Differentially expressed genes between CD25 KO and WT Tregs were identified using DESeq2, and determined by a threshold of false discovery rate (FDR) <0.01.

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Toomer KH, Lui JB, Altman NH, Ban Y, Chen X, Malek TR