Qualitative changes in cortical thymic epithelial cells drive postpartum thymic regeneration.

During gestation, sex hormones cause a significant thymic involution which enhances fertility. This thymic involution is rapidly corrected following parturition. As thymic epithelial cells (TECs) are responsible for the regulation of thymopoiesis, we analyzed the sequential phenotypic and transcriptomic changes in TECs during the postpartum period in order to identify mechanisms triggering postpartum thymic regeneration. In particular, we performed flow cytometry analyses and deep RNA-sequencing on purified TEC subsets at several time points before and after parturition. We report that pregnancy-induced involution is not caused by loss of TECs since their number does not change during or after pregnancy. However, during pregnancy, we observed a significant depletion of all thymocyte subsets downstream of the double-negative 1 (DN1) differentiation stage. Variations in thymocyte numbers correlated with conspicuous changes in the transcriptome of cortical TECs (cTECs). The transcriptomic changes affected predominantly cTEC expression of Foxn1, its targets and several genes that are essential for thymopoiesis. By contrast, medullary TECs (mTECs) showed very little transcriptomic changes in the early postpartum regenerative phase, but seemed to respond to the expansion of single-positive (SP) thymocytes in the late phase of regeneration. Together, these results show that postpartum thymic regeneration is orchestrated by variations in expression of a well-defined subset of cTEC genes, that occur very early after parturition. As the swift postpartum thymic regrowth does not require thymic epithelial cell (TEC) expansion, we hypothesized that it might rather depend on qualitative changes in TECs. We therefore performed RNA-sequencing of cortical and medullary TECs (cTECs and mTECs) extracted from controls, and at 15 time points spanning between D-2 (i.e. 2 days before parturition) to D28 postpartum. Thymi were expracted and TEC were isolated through enzymatic digestion of the stroma. Poly-A enriched mRNA sequencing was performed on two populations of TECs (EpCAM+CD45-): cTECs (UEA1-) and mTECs (UEA1+). Purified cell populations from non-pregnant females (NP), pregnant females at 18 days of gestation (D-2) and following parturition (D0 to D28) were extracted using fluorescence activated cell sorter (FACS) after enzymatic digestion of the stroma. Each replicate was extracted from one mouse (each sample containing between 13,800 and 96,700 cells). RNA extraction was performed using TrizolTM as recommended by the manufacturer (Invitrogen), and purified using RNeasy Micro columns (Qiagen). Samples quality was confirmed using Bioanalyzer RNA Pico (Agilent). Transcriptome librairies were synthesized with KAPA RNA HyperPrep PolyA (Roche), and validated with Bioanalyzer (Agilent). Sequencing was performed with the Nextseq500 (NextSeq High Output, 75 cycles) at the genomic core facility of the Institute for Research in Immunology and Cancer (Université de Montréal).