SRA

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 Rank?Foxn1 (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 Rank?Foxn1Foxp3GFP/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 Rank?Foxn1), embryological days of analysis (E17.5) and sorting strategies. Overall design: RNA-seq analysis of small, FACS-sorted cell pools. Affiliation: Magdalena Paolino, IMBA - Institute of Molecular Biotechnology of the Austrian Academy of Sciences Affiliation: Josef M. Penninger, IMBA - Institute of Molecular Biotechnology of the Austrian Academy of Sciences