Maternal immune adaptation in the decidua is essential to support fetal development, maintain tolerance to fetal antigens and preserve the capacity to respond to infection. Decidual CD4+ regulatory T cells (Tregs) have been implicated in pregnancy success by restraining potentially harmful conventional T-cell (Tconv) responses. This study set out to define the nature, activation, clonal behavior and drivers of both regulatory and conventional CD4+ T cells in the human term decidua by combining single-cell transcriptomics with paired TCR sequencing and complementary flow cytometry.
Samples were obtained from donors who underwent elective Caesarean section at term before labor. Paired tissues included decidua basalis (DB), decidua parietalis (DP), maternal peripheral blood (mPB) collected approximately 1 hour prior to C-section, and umbilical cord blood (UCB) collected immediately after delivery. Decidual tissues were macroscopically dissected, enzymatically digested with collagenase IV and DNAse I, mechanically dissociated and processed through sequential filtration and Percoll density gradients to isolate mononuclear cells. Blood mononuclear cells were isolated by Ficoll-Paque density centrifugation.
CD4+ fractions were enriched by magnetic separation for flow cytometry and panels included markers to delineate Tregs and Tconvs as well as activation and checkpoint molecules. Opt-SNE dimension reduction of concatenated flow data revealed tissue-specific clustering and higher frequencies of CD45RO+ effector phenotypes in decidual samples compared with blood. Decidual CD4+ cells showed increased expression of FOXP3, CD25 and CTLA-4 consistent with an effector Treg phenotype, and higher PD-1 expression consistent with ongoing activation or regulation.
For scRNAseq and scTCRseq analyses, mononuclear cells from each tissue were cryopreserved, thawed, pooled per tissue across donors and sorted by flow cytometry into Treg and Tconv fractions based on CD25, CD26 and CD127 gating patterns adapted for decidua and blood. Sorted populations were mixed to ensure representation of both lineages and were labeled with hashtag oligonucleotide–conjugated antibodies to distinguish tissues prior to library generation. Single-cell transcriptomes and paired TCR sequences were obtained to resolve cellular states and clonal relationships.
Combined transcriptomic and TCR analyses provided evidence that both decidual Tregs and Tconvs undergo antigen recognition and activation in the decidua, followed by local clonal expansion and differentiation toward effector states. Clonal expansion occurred locally within decidual tissue, and expanded clones showed transcriptional profiles indicating effector differentiation and tissue adaptation.
Decidual Tregs at term were largely classified as thymus-derived (tTregs). This classification was supported by enrichment of the tTreg core transcriptional signature — including FOXP3 and other stable lineage markers — and by largely non-overlapping TCR repertoires between Tregs and Tconvs, indicating divergent origins rather than broad conversion of Tconvs into Tregs in situ.
Transcriptomic signatures implicated multiple drivers of local effector differentiation. Costimulation via TNFR2 emerged as a principal driver promoting differentiation of tTregs into non-lymphoid tissue (NLT)–resident effector phenotypes with suppressive functions. Additional shared drivers for both Tregs and Tconvs included interferons, various interleukins and prolactin, suggesting common cytokine-mediated cues shaping decidual effector programs.
Tregs in the decidua adopted NLT-resident effector programs and expressed multiple suppressive molecules consistent with tissue-resident regulatory function. Effector Tconvs acquired proinflammatory and cytotoxic lineage-defining features. Despite these opposing roles, both cell types shared metabolic (glycolytic) and antigen-presenting transcriptional modules and displayed features consistent with local antigen recognition and reactivation. The data support a model in which effector Tconvs are restrained by a combination of intrinsic inhibitory receptor expression and extrinsic suppression by Tregs.
Subpopulations of both decidual Tregs and Tconvs expressed markers associated with exhaustion, interpreted as indicative of chronic antigenic stimulation in the decidua. Together with observed clonal expansion and tissue-resident differentiation, these findings argue for ongoing de novo priming in lymphoid organs, periodic reactivation in the decidua and dynamic turnover of maternal CD4+ T-cell populations throughout pregnancy.
At term pregnancy, the decidua contains a dynamic population of CD4+ T cells characterized by local activation, clonal expansion and effector differentiation. The majority of decidual Tregs are classifiable as tTregs that adapt to the tissue microenvironment under TNFR2-associated signals and exert suppressive functions that help maintain maternal–fetal tolerance. Effector Tconvs display proinflammatory and cytotoxic potential but are kept in check by intrinsic and extrinsic regulatory mechanisms. The molecular definitions and markers identified at mRNA and protein levels can inform diagnostic approaches and enhance mechanistic understanding of maternal–fetal immune regulation. Details on specific quantitative results, statistical measures and full gene lists are reported in the original article figures, tables and supplementary materials.