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RNA-seq analysis of regulatory T cells in thymus, placenta, spleen and white adipose tissue of pregnant mice

ABSTRACT: In this study we performed RNA sequencing to determine and compare the transcriptome of regulatory T cells (Tregs) present in the thymus, placenta, spleen and white adipose tissue (FAT) of pregnant mice. We further assessed how the expression of the receptor Rank in the thymic medullary epithelia affects the transcriptional program of these Treg populations. For the isolation of viable and bona fide Tregs, we first generated RankWt (wild type control mice) and RankΔFoxn1 (which lack expression of Rank in the thymic epithelia) and further crossed them to mice that express GFP from the Foxp3 promoter. Neuropilin1 was used to mark the thymic origin of the Tregs. Equal numbers of Tregs (280 cells) were sorted as CD45-CD8-CD4+GFP+Neuropilin1High cells from the thymus and placenta of RankWtFoxp3GFP/GFP and RankΔFoxn1Foxp3GFP/GFP pregnant females at E17.5 (both samples from the same female; n=4 females per genotype). Thymus Tregs from non-pregnant littermate female mice for each genotype cohort were also studied. To increase robustness, the 280 placental Tregs were purified from 5 individual placentas per pregnant female (56 Tregs/placenta). Our study is the first one to determine the transcriptome of thymus and placenta Tregs during pregnancy and reveals that, partially dependent on Rank expression in the thymic epithelia, placental-resident Tregs are molecularly distinct from thymic Tregs. In a separate experiment with different pregnant mice than those used for thymus and placenta analysis, VAT Tregs (20-30 cells) and splenic Tregs (150 cells) were sorted (both samples from the same female) by using the same mouse lines (Rank Wt and RankΔFoxn1), embryological days of analysis (E17.5) and sorting strategies.

ORGANISM(S): Mus musculus

PROVIDER: GSE97666 | GEO | 2020/10/21

REPOSITORIES: GEO

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Project description:The mechanism of egress of mature regulatory T cells (Tregs) from the thymus to the periphery remains enigmatic, as does the nature of those factors expressed in the thymic environment. Here, we examined the fate of thymic Tregs in TNFÎ±/RelA double-knockout (TA-KO) mice, because TA-KO mice retain a Treg population in the thymus but have only a small Treg population at the periphery. Transplantation of whole TA-KO thymus to under the kidney capsule of Rag1 null mice failed to induce the production of donor-derived splenic Tregs expressing neuropilin-1 (Nrp1), which was reported to be a marker of naturally occurring Tregs, indicating that TA-KO thymic Tregs either do not leave the thymus or are lost at the periphery. We next transplanted enriched TA-KO thymic Tregs to the peripheries of TA-KO mice and traced mouse survival. Transplantation of TA-KO thymic Tregs rescued the lethality in TA-KO mice, demonstrating that TA-KO thymic Tregs remain functional at the periphery. The TA-KO thymic Treg population had highly demethylated CpG motifs in the foxp3 locus, indicating that the cells were arrested at a late-mature stage. Also, the population included a large subpopulation of Tregs expressing IL-7RÎ±, which is a possible marker of late-mature Tregs. Finally, TA-KO fetal liver chimeric mice developed an Nrp1+ splenic Treg population from TA-KO cells, suggesting that Treg arrest is caused by a lack of RelA in the thymic environment. Together, these results suggest that egress of mature Tregs from the thymus depends on RelA in the thymic environment. For the isolation of thymic Tregs, CD4+CD8Î±-CD25hi thymocytes were isolated from five 1.5- to 2-week-old TNFÎ±-KO or TA-KO mice by using a FACSAria cell sorter. For the isolation of thymic stromal cells, 10 thymi from 1.5- to 2-week-old TNFÎ±-KO or TA-KO mice were minced with scissors and treated with RPMI 1640 supplemented with 2% FCS, 0.2 mg/ml collagenase (Roche, Basel, Switzerland), 0.2 mg/ml dispase I (Roche), and 100 U/ml DNase I (Life Technologies) for 30 min with stirring. Digested thymi were centrifuged in a Percoll (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) gradient (density, 1.115, 1.065, and PBS) at 1400g for 30 min. Cells in the upper layer were collected, and the CD45-EpCAM+ (thymic epithelial cells) and CD45+EpCAM- populations (enriched thymic stromal cells containing macrophages or dendritic cells) were sorted.

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RNA-seq analysis of regulatory T cells in thymus, placenta, spleen and white adipose tissue of pregnant mice

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