Effector regulatory T (eTreg) cells — the most differentiated suppressive CD4+^{+} T-cell subset in the pregnancy-transformed uterine lining (decidua) — undergo oligoclonal T-cell-receptor (TCR) expansion in late normal human gestation, and this expansion is absent or altered in preeclampsia (PE). Which antigens drive these clonal expansions, and whether their failure contributes causally to PE, is unknown. Tsuda et al. (2018) identified the expansions by single-cell TCRβ\beta sequencing and left four unresolved issues: the antigen specificity of the expanded clonotypes, the lack of causal evidence linking insufficient expansion to PE, methodological limitations (unpaired TCR chains, shallow repertoire sampling, no antigen-agnostic assay for CD4+^{+} TCRs), and the untested relationship between clonotype and effector phenotype. Later work has largely closed the methodological gap — paired single-cell TCRαβ\alpha\beta sequencing with transcriptomics is now routine and applied to decidua, and pan-HLA TCR-ligand discovery platforms now exist — and has sharpened the specificity question: term decidual Tregs appear largely thymus-derived with a self-reactive TCR repertoire, challenging the default assumption of fetal-alloantigen specificity. Epitope-level identification of the antigens driving human decidual eTreg clonal expansion, the conserved phenotypic identity of the expanded clonotypes, and direct causal evidence in PE remain open.

Background

Regulatory T (Treg) cells are CD4+^{+} T lymphocytes whose defining transcription factor FOXP3 endows them with the ability to suppress other immune cells. The fetus and placenta are semi-allogeneic (they carry paternally inherited antigens foreign to the mother), so maternal immune tolerance must be established locally at the maternal-fetal interface. The decidua is the endometrium transformed by pregnancy; it is invaded by fetal extravillous trophoblast (EVT), the placental cell type that remodels maternal spiral arteries (the uterine vessels that deliver maternal blood to the placenta). Uniquely among fetal tissues, EVT expresses a restricted set of human leukocyte antigen (HLA) molecules — the polymorphic classical HLA-C together with the non-classical HLA-E, HLA-F, and HLA-G of limited polymorphism — while lacking the broadly expressed HLA-A and HLA-B. Paternally inherited fetal HLA-C is therefore the principal polymorphic alloantigen presented at the interface.

T cells recognize peptide antigens bound to major histocompatibility complex (MHC; HLA in humans) molecules through the T-cell receptor (TCR), a heterodimer of α\alpha and β\beta chains. The hypervariable complementarity determining region 3 (CDR3) sequence defines a clonotype — a unique receptor marking a T cell and its clonal descendants, so that an expanded clonotype indicates antigen-driven proliferation. Treg cells recognize antigen through the same TCR machinery, but on MHC class II molecules, which present extracellularly derived peptides and are carried by professional antigen-presenting cells (dendritic cells, macrophages, B cells) rather than by trophoblast, which does not express MHC class II.

Treg cells are required for fetal tolerance in mice, and human decidua accumulates FOXP3+^{+} Tregs whose most differentiated suppressive subset, the effector Tregs (CD45RA−^{-}CD25hi^{hi}FOXP3hi^{hi}), dominates in late gestation. Tsuda et al. (2018) single-cell-sorted decidual eTregs from normal and complicated pregnancies and sequenced TCRβ\beta CDR3. They found oligoclonal expansion of decidual eTregs in normal third-trimester pregnancy that was not seen in preeclampsia, recurrence of some clonotypes between successive pregnancies of the same woman (suggesting antigen-specific memory), failure to amplify TCRα\alpha, and no functional validation of any TCR; they proposed fetal HLA (HLA-C/E/F/G) as candidate antigens and posed the unresolved issues tracked here. Preeclampsia — new-onset hypertension with organ dysfunction after mid-gestation, generally traced to defective placentation and downstream systemic inflammation — is the hypertensive disorder of pregnancy most consistently associated with Treg insufficiency, yet in humans this association remains observational.

Subsequent work reshaped the context. Wienke et al. (2020) showed that placental-bed decidual Tregs acquire a late-stage effector, chronically activated, tumor-Treg-like transcriptional profile. Salvany-Celades et al. (2019) identified three functional decidual Treg types and showed that HLA-G+^{+} EVT can induce Tregs in an HLA-C- and CD3-dependent manner, suggesting antigen-specific induction. Mahajan et al. (2023) demonstrated fetus-specific decidual CD8+^{+} T cells that are not uniformly suppressed, and Koenig et al. (2026) showed that decidual CD4+^{+} memory T cells respond to fetal cord-blood allo-antigens — so fetal-antigen recognition at the interface is real, but at the population level only. Chasman et al. (2023), Tsuda et al. (2024), and Verleng et al. (2026) then brought paired single-cell transcriptomics plus TCR sequencing to decidual T cells: TCRαβ\alpha\beta pairing is now recoverable at scale; decidual FOXP3+^{+} Tregs are clonally expanded with clonotypes largely distinct from other CD4+^{+} subsets; and, most consequentially, term decidual Tregs appear predominantly thymus-derived (tTreg) — a lineage whose TCRs are selected for self-reactivity — leading Verleng et al. (2026) to state that the antigen specificity of Tregs responding in pregnancy is currently unknown and may be self-directed rather than alloantigen-directed. In parallel, antigen-agnostic TCR-ligand discovery platforms (T-FINDER, SABR-II, TCR-MAP) now permit functional identification of MHC class II-restricted ligands for orphan CD4+^{+} TCRs, though none has been applied to decidual Treg clonotypes. In mice, transient Treg depletion during placentation causally impairs spiral-artery remodeling and fetal growth, positioning Tregs upstream of the vascular lesion of PE — but as a lineage, not as specific clonal expansions.

Problem Statement

The problem, posed by Tsuda et al. (2018) and refined by the subsequent literature, is to explain the antigen-driven basis of clonally expanded decidual effector regulatory T cells (eTregs) in normal human pregnancy and whether their failure contributes to preeclampsia. It has three linked parts serving this single objective.

(1) Antigen specificity: identify, at the peptide-MHC class II ligand level, the antigens recognized by the clonally expanded decidual eTreg TCR clonotypes of normal late gestation, using paired TCRαβ\alpha\beta sequences. The candidate antigen classes to be distinguished are fetal/paternal alloantigens (for example, peptides from paternally inherited HLA-C — or from the non-classical HLA-E/-F/-G — taken up from trophoblast debris and presented by maternal decidual antigen-presenting cells on maternal MHC class II), maternal self-antigens (consistent with the thymus-derived Treg lineage), and microbial or other environmental antigens. The identification must be validated functionally (for example by recombinant-TCR reporter assays, in which the cloned receptor reports ligand recognition through an activation or fluorescent readout, or by peptide-MHC class II tetramer staining — multivalent fluorescent peptide-MHC reagents that selectively label T cells whose TCR recognizes the displayed peptide — of the original decidual clones) and must be shown to account for the observed expansion — for example through ligand-reactivity of clonotypes recurring between successive pregnancies of the same woman (the within-subject recurrence pattern reported by Tsuda et al. 2018), through tetramer staining of a fraction of ex vivo decidual eTregs carrying the expanded clonotypes, or through other evidence linking the identified ligand to the expansion.

(2) Causal role in preeclampsia: determine whether the quantitative or qualitative failure of these antigen-responsive decidual eTreg clonal expansions (insufficient expansion, or expansion with an exhausted/dysfunctional state, as the 2018 and 2024 datasets respectively suggest) contributes causally to preeclampsia, or is a secondary consequence of the disease, through direct mechanistic evidence such as antigen-specific reconstitution or perturbation in ex vivo decidual co-culture systems, humanized or adoptive-transfer models, or adequately powered longitudinal human cohorts.

(3) Clonotype-phenotype correspondence: establish whether the expanded eTreg clonotypes map to conserved transcriptional and functional effector identities (shared across subjects and gestational ages), linking TCR specificity to the effector-Treg differentiation program observed at the interface.

Answers that resolve only the antigen specificity, only the causal question, or only the clonotype-phenotype correspondence are admissible contributions toward this single objective; the complete answer establishes which antigens drive decidual eTreg clonal expansion in normal pregnancy, whether defective or dysfunctional expansion of these antigen-specific cells is causal in preeclampsia, and whether the expanded clonotypes map to conserved transcriptional and functional effector identities.

References

  1. Tsuda S, Zhang X, Hamana H, Shima T, Ushijima A, Tsuda K, Muraguchi A, Kishi H, Saito S (2018). Clonally Expanded Decidual Effector Regulatory T Cells Increase in Late Gestation of Normal Pregnancy, but Not in Preeclampsia, in Humans. Frontiers in Immunology 9. DOI: 10.3389/fimmu.2018.01934. https://doi.org/10.3389/fimmu.2018.01934 (source of the open question)
  2. Tsuda S, Shichino S, Tilburgs T, Shima T, Morita K, Yamaki-Ushijima A, Roskin K, Tomura M, Sameshima A, Saito S, Nakashima A (2024). CD4+ T cell heterogeneity in gestational age and preeclampsia using single-cell RNA sequencing. Frontiers in Immunology. DOI: 10.3389/fimmu.2024.1401738. https://doi.org/10.3389/fimmu.2024.1401738
  3. Chasman DA, Welch Schwartz R, Vazquez J, Chavarria M, Jenkins ET, Lopez GE, Tyler CT, Stanic AK, Ong IM (2023). Proteogenomic and V(D)J Analysis of Human Decidual T Cells Highlights Unique Transcriptional Programming and Clonal Distribution. Journal of Immunology. DOI: 10.4049/jimmunol.2200061. https://doi.org/10.4049/jimmunol.2200061
  4. Verleng LJ, Busselaar J, Mensink M, Schrama E, Kapsenberg H, van der Keur C, Lei X, Eikmans M, Xiao Y, de Kivit S, Borst J (2026). Defining nature, responses and drivers of regulatory- and conventional CD4+ T cells in the human term decidua. Frontiers in Immunology 17. DOI: 10.3389/fimmu.2026.1865340. https://doi.org/10.3389/fimmu.2026.1865340
  5. Salvany-Celades M, van der Zwan A, Benner M, Setrajcic-Dragos V, Bougleux Gomes HA, Iyer V, Norwitz ER, Strominger JL, Tilburgs T (2019). Three Types of Functional Regulatory T Cells Control T Cell Responses at the Human Maternal-Fetal Interface. Cell Reports. DOI: 10.1016/j.celrep.2019.04.109. https://doi.org/10.1016/j.celrep.2019.04.109
  6. Wienke J, Brouwers L, van der Burg LM, Mokry M, Scholman RC, Nikkels PG, van Rijn BB, van Wijk F (2020). Human Tregs at the materno-fetal interface show site-specific adaptation reminiscent of tumor Tregs. JCI Insight. DOI: 10.1172/jci.insight.137926. https://doi.org/10.1172/jci.insight.137926
  7. Morita K, Tsuda S, Kobayashi E, Hamana H, Tsuda K, Shima T, Nakashima A, Ushijima A, Kishi H, Saito S (2020). Analysis of TCR Repertoire and PD-1 Expression in Decidual and Peripheral CD8+ T Cells Reveals Distinct Immune Mechanisms in Miscarriage and Preeclampsia. Frontiers in Immunology. DOI: 10.3389/fimmu.2020.01082. https://doi.org/10.3389/fimmu.2020.01082
  8. Mahajan S, Alexander A, Koenig Z, Saba N, Prasanphanich N, Hildeman DA, Chougnet CA, DeFranco E, Andorf S, Tilburgs T (2023). Antigen-specific decidual CD8+ T cells include distinct effector memory and tissue-resident memory cells. JCI Insight. DOI: 10.1172/jci.insight.171806. https://doi.org/10.1172/jci.insight.171806
  9. Koenig ZT, Eswar S, Tsuda S, Schuldt N, Chougnet CA, Forde B, Andorf S, Tilburgs T (2026). Decidual CD4 Effector Memory and Resident Memory T Cells Respond to Cord Blood Allo-Antigens. American Journal of Reproductive Immunology. DOI: 10.1111/aji.70213. https://doi.org/10.1111/aji.70213
  10. Tsuda S, Nakashima A, Morita K, Shima T, Yoneda S, Kishi H, Saito S (2021). The role of decidual regulatory T cells in the induction and maintenance of fetal antigen-specific tolerance: Imbalance between regulatory and cytotoxic T cells in pregnancy complications. Human Immunology. DOI: 10.1016/j.humimm.2021.01.019. https://doi.org/10.1016/j.humimm.2021.01.019
  11. Robertson SA, Care AS (2026). Regulatory T cells and decidual vascular remodelling - A new piece in the preeclampsia puzzle. Journal of Reproductive Immunology. DOI: 10.1016/j.jri.2026.104847. https://doi.org/10.1016/j.jri.2026.104847
  12. Papúchová H, Meissner TB, Li Q, Strominger JL, Tilburgs T (2019). The Dual Role of HLA-C in Tolerance and Immunity at the Maternal-Fetal Interface. Frontiers in Immunology. DOI: 10.3389/fimmu.2019.02730. https://doi.org/10.3389/fimmu.2019.02730
  13. Tian X, Krop J, Ijsselsteijn ME, Kapsenberg JM, Anholts JDH, van der Meeren L, Mei H, Huigen MHJ, van der Keur C, Roelen DL, Lashley LEELO, van Beelen E, Koning F, van der Hoorn MP, Eikmans M (2026). Myeloid-T cell proximity is prominent in healthy pregnancies with extreme fetal-maternal HLA incompatibility. iScience. DOI: 10.1016/j.isci.2025.114179. https://doi.org/10.1016/j.isci.2025.114179
  14. Morgan JC, Hynes GE, Pollard JM, Andrade MS, Jeong JC, Chong AS (2025). Hyporesponsive fetus-specific T cell responses in multiparous human pregnancy. Frontiers in Immunology. DOI: 10.3389/fimmu.2025.1634430. https://doi.org/10.3389/fimmu.2025.1634430
  15. Cetin M, Pinamonti V, Schmid T, Boschert T, Mellado Fuentes A, Kromer K, Lerner T, Zhang J, Herzig Y, Ehlert C, Hernandez-Hernandez M, Samaras G, Maldonado Torres C, Fisch L, Dragan V, Kouwenhoven A, Van Schoubroeck B, Wils H, Van Hove C, Platten M, Green EW, Stevenaert F, Felix NJ, Lindner JM (2024). T-FINDER: A highly sensitive, pan-HLA platform for functional T cell receptor and ligand discovery. Science Advances. DOI: 10.1126/sciadv.adk3060. https://doi.org/10.1126/sciadv.adk3060
  16. Zdinak PM, Trivedi N, Grebinoski S, Torrey J, Martinez EZ, Martinez S, Hicks L, Ranjan R, Makani VKK, Roland MM, Kublo L, Arshad S, Anderson MS, Vignali DAA, Joglekar AV (2024). De novo identification of CD4+ T cell epitopes. Nature Methods. DOI: 10.1038/s41592-024-02255-0. https://doi.org/10.1038/s41592-024-02255-0
  17. Kohlgruber AC, Dezfulian MH, Sie BM, Wang CI, Kula T, Laserson U, Larman HB, Elledge SJ (2025). High-throughput discovery of MHC class I- and II-restricted T cell epitopes using synthetic cellular circuits. Nature Biotechnology. DOI: 10.1038/s41587-024-02248-6. https://doi.org/10.1038/s41587-024-02248-6
  18. Maurice NJ, Erickson JR, DeJong CS, Mair F, Taber AK, Frutoso M, Islas LV, Vigil ALBG, Lawler RL, McElrath J, Newell E, Sullivan LB, Shree R, McCartney SA (2025). Cytokine and metabolite networks shape T cell residency and functionality at the term human maternal-fetal interface. Journal of Immunology. DOI: 10.1093/jimmun/vkaf093. https://doi.org/10.1093/jimmun/vkaf093
  19. Whillock A, Smith P, Burger S, Sridhar A, Lindgren A, Berg J, Tsuda S, Mahajan S, Tilburgs T, Schuldt NJ (2025). Natural microbial exposure populates the maternal fetal interface with diverse T cells. Frontiers in Immunology. DOI: 10.3389/fimmu.2025.1616491. https://doi.org/10.3389/fimmu.2025.1616491
  20. Nguyen NNM, Brady AE, Russi A, Pham G, Li B, Turner LH, Kotliar M, Barski A, Sun X, Way SS (2026). Maternal Krüppel-like factor 2 (KLF2)+ CD4 T cells promote fertility and fetal tolerance. Proceedings of the National Academy of Sciences USA. DOI: 10.1073/pnas.2519393123. https://doi.org/10.1073/pnas.2519393123