Resident effector memory Tregs of the uterus regulate trophic functions during mouse early pregnancy

Regulatory T cells (Tregs) contribute to successful pregnancy by promoting maternal-fetal tolerance. We identified a novel population of CD25-Foxp3+ resident uterine Tregs in non-pregnant mouse uterus. They have phenotypic and migration properties of tissue-resident effector-memory T cells (Trm), thus identifying regulatory Trm (regTrm). Lack of CD25 expression in regTrm is caused by the IL-2-deprived uterine microenvironment. We performed a complete transcriptome analysis by RNA-seq to determined whether uterine regTrm have a unique molecular signature, as has been described for other tissue Tregs. For this, we purified CD25- regTrm from non-pregnant and pregnant uterus at day 6 to 8 of pregnancy, and we compared them to CD4+Foxp3- (CD4conv) T cells from non-pregnant uterus and to CD25+ Tregs from paraaortic uterine draining lymph nodes. We used criteria of (i) a two-fold or greater change in expression and (ii) a p-value <0.05 (cut-off at 5% FDR) to determine differentially expressed genes (DEGs). DEGs were annotated by their functional relevance in pregnancy using ingenuity pathway analysis (IPA) and using the PubMed Query: (Gene main name OR Gene Synonymous 1 OR Gene Synonymous 2 OR …) AND "pregnancy" OR “regulatory T cells” OR "uterus". We found that uterine regTrm have a unique transcriptional profile, impregnated with a uterine signature comprising up-regulation of genes related to the endometrium and to uterine receptivity. Pregnancy triggers regTrm expression of genes involved in trophic functions such as uterine development, extracellular matrix remodeling, hypoxia and vasculogenesis. Samples from regulatory T cells mRNA profiles from uterus of non-pregnant and pregnant BALB/c foxp3 mice at day 6 to 8 of pregnancy, and from paraaortic lymph nodes of non-pregnant mice. Data were generated by RNA sequencing, in quadruplicate, using Illumina NextSeq.