Human leukocyte antigen G (HLA-G), a non-classical MHC class I molecule abundantly expressed by invading trophoblast cells, and CD4+CD25+FOXP3+ regulatory T cells (Tregs) are both upregulated in successful pregnancy and reduced in unexplained recurrent spontaneous abortion, yet it remains unresolved whether soluble HLA-G (sHLA-G) causally induces or stably enriches functional Tregs in humans. A 2020 clinical and mixed-lymphocyte-culture (MLC) study found culture-driven Treg expansion regardless of added recombinant sHLA-G and inconclusive cytokine readouts, concluding only to a 'possible role for HLA-G in the enrichment of Treg cells'. Adjacent mechanistic evidence — mesenchymal-stromal-cell-derived HLA-G5 expanding Tregs in co-culture, HLA-G-positive extravillous trophoblast co-cultures enriching decidual Treg subsets, and HLA-G-expressing tolerogenic dendritic cells inducing IL-10-producing Tr1 cells — does not isolate a direct soluble-HLA-G effect in the pregnancy context, and reviews through 2026 continue to treat the HLA-G–Treg axis as partly putative. The open problem is to establish, by appropriately controlled interventional experiments and/or causal in-vivo evidence, whether soluble HLA-G drives Treg induction or enrichment in maternal-fetal tolerance.

Background

Pregnancy requires the maternal immune system to tolerate the semi-allogeneic fetus. Human leukocyte antigen G (HLA-G) is a non-classical major histocompatibility complex (MHC) class I molecule — meaning an HLA gene product with limited polymorphism and immunoinhibitory function, unlike the highly variable classical HLA-A and HLA-B — that in pregnancy is expressed almost exclusively by extravillous trophoblasts (EVTs), the fetal cells that invade the uterine lining (decidua) during placentation. HLA-G exists as seven isoforms: four membrane-bound (G1–G4) and three soluble ones (G5–G7, lacking the transmembrane region), and membrane HLA-G1 can additionally be shed or carried on extracellular vesicles, so 'soluble HLA-G' (sHLA-G) in plasma is a mixture of secreted and shed forms. HLA-G engages inhibitory receptors — ILT2/LILRB1 found on T cells, B cells and monocytes, and ILT4/LILRB2 found on myeloid antigen-presenting cells (APCs) — which is why HLA-G is generally tolerogenic: it inhibits T-cell proliferation and natural-killer cytotoxicity and can reprogram APCs toward a tolerogenic phenotype.

A second pillar of maternal-fetal tolerance is the regulatory T cell (Treg), a suppressive CD4+ T-cell lineage defined by CD25 (the interleukin-2 receptor alpha chain) and the transcription factor FOXP3. Tregs are enriched at the implantation site, expanded systemically during healthy pregnancy, and comparatively deficient in unexplained recurrent spontaneous abortion (URSA, recurrent miscarriage without an identified cause). Because both HLA-G and Tregs rise in successful pregnancy and fall in abortion, a mechanistic link — HLA-G driving Treg induction or enrichment — has long been proposed, and it matters practically: circulating sHLA-G is used as a tolerance biomarker in reproductive and transplantation medicine.

Several results inform, but do not settle, this link. Mesenchymal stromal cells (MSCs) suppress immune responses partly by secreting the soluble isoform HLA-G5; Selmani and colleagues (2008) showed with neutralizing anti-HLA-G antibodies that MSC-derived HLA-G5 contributes to expanding CD4+CD25highFOXP3+ Tregs in MSC–T-cell co-cultures — but HLA-G5 secretion there was IL-10-dependent and required cell contact, so the effect was not attributable to purified soluble HLA-G acting alone, and the context was transplantation-oriented rather than pregnancy. Salvany-Celades and colleagues (2019) identified three functional decidual CD4+ Treg types in healthy pregnancy (CD25HIFOXP3+, PD1HIIL-10+, and TIGIT+FOXP3dim) and showed that co-culture of HLA-G-positive EVTs or decidual macrophages with blood CD4+ T cells directly increased the proportion of CD25HIFOXP3+ Tregs; however, blockade experiments pointed to HLA-C- and CD3-dependent (antigen-specific) mechanisms for part of this induction rather than to HLA-G itself. Separately, work on tolerogenic 'DC-10' dendritic cells (Amodio and Gregori, 2012) established that HLA-G-expressing, IL-10-producing APCs induce IL-10-secreting type 1 regulatory (Tr1) cells — a regulatory T-cell subset distinct from FOXP3+ Tregs — an indirect, APC-mediated route. A 2011 review (Carosella, Gregori and LeMaoult) concluded that although HLA-G had been directly involved in generating regulatory cells, little was known about how HLA-G, APCs, and regulatory cells actually cooperate.

Against this background, Al-Khunaizi, Tabbara and Farid (2020) measured plasma sHLA-G and Treg percentages in healthy pregnant multigravida women, women with URSA, and non-pregnant controls, and cultured peripheral blood lymphocytes of non-pregnant multigravida women with their partners' lymphocytes in a mixed lymphocyte culture (MLC, an in-vitro co-culture of immune cells from two individuals in which allogeneic recognition drives T-cell activation) with and without added recombinant sHLA-G. sHLA-G and Tregs were lower in URSA, consistent with earlier studies. But in the MLC, Tregs expanded in both arms, with no significant difference between cultures with and without sHLA-G; extracellular IL-10 and IFN-γ were unchanged (IFN-γ slightly higher, not significant), and intracellular TGF-β in CD4+ cells was higher after sHLA-G incubation. The authors therefore concluded only to a 'possible role for HLA-G in the enrichment of Treg cells'. The study's simplified MLC design — partner lymphocytes rather than uterine APCs, without a decidual or trophoblast microenvironment — left the causal question unresolved. The question of whether HLA-G is a mechanistic driver of Treg-mediated maternal tolerance — directly on T cells via ILT2, indirectly via APC reprogramming, or not at all — was thereby sharpened but left open, and reviews as late as 2024–2026 continue to present the HLA-G–Treg interplay as part of the tolerance network while marking parts of the mechanism as putative rather than directly demonstrated.

Problem Statement

Determine whether soluble HLA-G plays a causal role in the induction or stable enrichment of CD4+CD25+FOXP3+ regulatory T cells during healthy human pregnancy — the question posed by Al-Khunaizi, Tabbara and Farid (2020) as whether HLA-G plays a role in the upregulation of regulatory cells, and left unresolved by their own study, in which partner-lymphocyte mixed lymphocyte cultures with and without recombinant sHLA-G showed Treg increases in both arms with no significant difference, inconclusive cytokine readouts, and only a 'possible role for HLA-G in the enrichment of Treg cells' as the authors' conclusion. A complete answer establishes, by appropriately controlled experiments and/or causal in-vivo evidence in humans, whether soluble HLA-G induces or stably enriches functional Tregs in the pregnancy context. Demonstrating instead that no direct soluble-HLA-G-to-Treg pathway operates in the pregnancy setting, with the observed sHLA-G/Treg associations arising from other mechanisms, would equally answer the question.

Scientific considerations relevant to any answer, advisory rather than prescriptive, include: dose-dependence of the effect; whether the mechanism is direct, via T-cell ILT2/LILRB1 engagement, or indirect, via reprogramming of antigen-presenting cells such as decidual macrophages or tolerogenic dendritic cells; whether HLA-G-modified Tregs are functionally suppressive and mechanistically stable (e.g., sustained FOXP3 expression, with contributions of IL-10, TGF-β, and related mediators); whether the experimental system preserves trophoblast/decidual features sufficiently to bear on pregnancy, given that the effect could not be detected in the simplified partner-lymphocyte MLC; and the variables — such as partner HLA compatibility, membrane-bound versus soluble HLA-G forms, other regulatory cell subsets, HLA-G polymorphisms, or assay/serum batch effects — that may govern or confound the relationship between circulating sHLA-G concentration and Treg abundance observed in vivo.

References

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