Human Tregs at the materno-fetal interface show site-specific adaptation reminiscent of tumor Tregs

Tregs are crucial for maintaining maternal immunotolerance against the semiallogeneic fetus. We investigated the elusive transcriptional profile and functional adaptation of human uterine Tregs (uTregs) during pregnancy. Uterine biopsies, from placental bed (materno-fetal interface) and incision site (control) and blood were obtained from women with uncomplicated pregnancies undergoing cesarean section. Tregs and CD4+ non-Tregs were isolated for transcriptomic profiling by Cel-Seq2. Results were validated on protein and single cell levels by flow cytometry. Placental bed uTregs showed elevated expression of Treg signature markers, including FOXP3, CTLA-4, and TIGIT. Their transcriptional profile was indicative of late-stage effector Treg differentiation and chronic activation, with increased expression of immune checkpoints GITR, TNFR2, OX-40, and 4-1BB; genes associated with suppressive capacity (HAVCR2, IL10, LAYN, and PDCD1); and transcription factors MAF, PRDM1, BATF, and VDR. uTregs mirrored non-Treg Th1 polarization and tissue residency. The particular transcriptional signature of placental bed uTregs overlapped strongly with that of tumor-infiltrating Tregs and was remarkably pronounced at the placental bed compared with uterine control site. In conclusion, human uTregs acquire a differentiated effector Treg profile similar to tumor-infiltrating Tregs, specifically at the materno-fetal interface. This introduces the concept of site-specific transcriptional adaptation of Tregs within 1 organ.

• Fig. S2. Enrichment of gene signatures of in vitro activated Tregs.
• Fig. S3. Tregs at the maternal-fetal interface share transcriptional adaptation with uTconv. • Fig. S4. uTregs from the maternal-fetal interface share similarities were human Tregs from healthy tissue sites. • Fig. S5. uTregs from the incision site are bona fide Tregs and have a tissue-resident profile.
• Table S1. Clinical characteristics of 20 human subjects undergoing caesarian section.
• Table S2. Antibodies used for sorting and flow cytometric analysis.
• Table S3. Gene signatures used for gene set enrichment analysis and overlap with uTreg signature. • Table S4. Upregulated genes in the uTreg-specific core signature. • Table S5. Downregulated genes in the uTreg-specific core signature.