Numerous studies have investigated the relationship between PE and peripheral blood Treg populations. Some research investigations utilized flow cytometry to examine the populations of Tregs (77, 106, 115–137), whereas others employed qPCR (62, 114, 138, 139). Pregnant women with PE have decreased numbers of circulating Tregs (14, 77, 117, 120, 123–125, 127, 132, 134, 140–144), including CD4⁺CD25⁺ FoxP3⁺ T lymphocytes (125), and CD8⁺CD25⁺FoxP3⁺ cells (138). However, other studies revealed that there was no significant difference in the peripheral proportion of Treg cells between the normal group and the PE group (136, 145, 146). Treg cells might have a diminished suppressive capacity in PE patients (77, 118, 120, 140, 142).

In patients with PE, the proportions of Treg subtypes are distinct from that of healthy expectant women. In comparison to normal pregnant females, the frequency of fully functional effector Tregs (CD4⁺ FoxP3⁺ CD45RA⁻) was lower in patients with PE. Conversely, naive Tregs (CD4⁺ FoxP3⁺ CD45RA⁺) were unchanged (116). In one study, a distinct subset of Treg cells that express HLA-G was characterized (147). These cells, which are hypo-proliferative and thymic-derived, do not show expression of FoxP3 and CD25 molecules. These entities are identified in instances of multiple sclerosis, HIV-1 infection, and transplantation (148). Compared to normal pregnant women, the proportions of peripheral CD4⁺HLA-G⁺, CD8⁺HLA-G⁺, and CD4⁺CD25⁺CD127^low cells are diminished in PE patients (77). Likewise, an additional investigation demonstrated an evident reduction in the circulating CD4⁺HLA-G⁺ T cells in individuals with PE compared to healthy pregnant women (79). Moreover, healthy pregnant women have a significantly higher percentage of circulating CD4⁺CD127^lowCD25⁺, CD4⁺FoxP3⁺, and CD4⁺CD25^high cells compared to patients with PE (14). Furthermore, an increased Th17/Treg ratio was identified in PE by researchers via an examination of the proportions of CD4⁺CD25⁺CD127⁺ and CD4⁺ IL-17⁺ cells (122). In addition, the quantity of CD45RA⁺CD31⁺ recent thymic emigrant Tregs are diminished in patients with PE (119).

In PE cases, the inhibitory function of Treg cells diminishes in tandem with their diminished quantity ( Table 2 ). It has been discovered that CD4⁺CD25⁺CD127^low/neg Treg cells isolated from PE women via magnetic sorting showed a diminished suppressive activity in comparison to Treg cells isolated from healthy expectant individuals (140). In addition, CD4⁺CD25⁺ Treg cells obtained from PE patients via magnetic activated cell sorting (MACS) failed to impede the growth of CD4⁺CD25⁻ responder cells (120). Nevertheless, contradictory studies have been reported that the inhibitory capacity of the Treg cell subset was comparable between PE patients and healthy pregnant controls (14, 126).

Several studies, however, failed to identify any distinctions between the populations of Treg cells in preeclamptic patients and those of healthy pregnancies. However, these studies used frozen PBMCs with a small sample size (145) and investigated CD4⁺CD25⁺ T lymphocytes without specific Treg markers (146). Minimal variation was observed in the quantity and ratio of Treg cells between women with normal pregnancy and PE (146). Additionally, an independent investigation revealed no distinction in the population of activated and resting Treg cells among women with severe and early-onset PE, pregnant women who did not have PE, or non-pregnant females (149). However, an analysis of functional and migratory Treg markers (CCR4 and CTLA-4) revealed that untreated preeclamptic individuals had higher percentages of CTLA-4⁺ and CCR4⁺ resting and inactivated Treg populations than healthy pregnant controls. It is worth noting that ten of eighteen PE patients in this study (149) were treated with glucocorticoids, which may affect the experimental results. It has been reported that glucocorticoids could affect the number and phenotype of Treg cells (150–152). Therefore, it is necessary to select PE patients who have not received glucocorticoids for further research.

The divergent results can likely be attributed primarily to the various definitions of Treg cells. A notable concern pertains to the expression of the critical phenotypic markers, namely CD25 and FoxP3, upon stimulation of conventional CD4⁺ T cells. Determining the precise quantity of “authentic” Treg cells in conditions marked by systemic T-cell stimulation, such as PE, is thus complicated. Diverse flow cytometric methods and markers have been devised to prevent the inclusion of non-suppressive activated Treg cells, which could potentially generate erroneous outcomes. These encompass the identification of suppressive Treg cells characterized by reduced CD4 expression (CD4^dim) relative to conventional CD4 cells (153), as well as diminished or nonexistent expression of the IL-7 alpha receptor subunit CD127 (154). Additionally, the use of CD45RA helps distinguish between suppressive resting Treg cells (FoxP3^dimCD45RA⁺) and non-suppressive, activated FoxP3 expressing T helper cells (FoxP3^dimCD45RA^-) (155).

The dysregulation linked to PE has been illustrated by modifications in transcription factors of T-cells. RORγt and FoxP3 are transcription factors that contribute to the development of Th17 and Treg cells, respectively (122). Ribeiro et al. (115) revealed that early-onset PE women had a greater proportion of CD4⁺ T cells expressing the RORc transcription factor and a consequential decline in the percentage of Treg cells expressing FoxP3, indicating more severe early-onset PE. Additionally, the mRNA expressions of FoxP3 and RORγt in peripheral T cells of patients with PE were higher and lower, respectively, in comparison to healthy expectant women (114, 122).

Altered concentrations of circulating cytokines may influence the equilibrium between Th17 and Treg cells, thus potentially contributing to the pathogenesis of PE. Pro-inflammatory responses result from the stimulation of chemokines (CXCL1, CXCL2, CCL20), cytokines (IL-6 and TNF-α), and inflammatory factors (the complement system and acute-phase proteins) by IL-17 (156). Consequently, IL-17 could facilitate the proliferation of Th17 cells. Circulating IL-17 concentrations are significantly elevated in PE women relative to normal pregnant females and non-pregnant controls (156). Furthermore, IL-17 concentrations in the plasma of pregnant women with severe PE are higher than those of healthy pregnant women (123). Another group, nevertheless, failed to identify a statistically significant difference in serum IL-17 concentrations between pregnant women with PE and those without complications (157). Furthermore, considering the capacity of IL-6 and IL-1β to stimulate the transformation of Treg cells into Th17 cells (158, 159), it is conceivable that heightened concentrations of these cytokines in PE could trigger the transformation from Treg to Th17 cells (160). It has been found that PE induces a rise in IL-6 and IL-1β production (161). IL-6 expression was found to be elevated in chorionic villus sampling (CVS) tissues of women who subsequently develop PE accompanied by fetal growth restriction (FGR) (162). Increased levels of IL-6 expression promote Th17 generation and undermine the stability of Tregs, while concurrently reducing the quantity of alternatively activated M2 macrophages and T cell markers (163).

Variations in the expression of apoptotic molecules contribute to the premature deletion of Tregs, which may account in part for the diminished quantity of Tregs observed in patients with PE (117). Compared with normal pregnancies, the percentage of Tregs expressing the anti-apoptotic molecule Bcl-2 was considerably diminished in PE patients. Furthermore, there was a significant rise in the expression of pro-apoptotic molecule, Bax, in Tregs of PE patients (117). These findings indicate that the presence of PE may increase the vulnerability of Tregs to apoptotic cell death, which is consistent with the reduced Treg counts observed in this specific clinical scenario. Indeed, the signatures of Tregs and effector T cells were found to be differently regulated in women with PE (164). A consistent reduction in STAT5 signaling in Th1 cells was observed in individuals who subsequently experienced PE (164). IL-2/STAT5 signal contributes to the differentiation of T helper (165) and Treg (166) cells, and it also potentially inhibits Th17 differentiation (167). Furthermore, an increase in the p38 signaling pathway, which is essential for Tregs to exert their suppressive function (168), was observed in Tregs from women who were carrying a healthy pregnancy, but not in those who were diagnosed with PE (164). These results suggest the potential utilization of specific signal pathways in maternal blood for the evaluation of PE.

Various studies have revealed decreased Treg cells and increased Th1 and Th17 immune responses in women with PE using various modalities ( Table 3 ). There have been numerous hypotheses regarding the decrease in Tregs observed in women with PE. An initial factor to consider is the potential reduction in decidual Treg differentiation (169). The evidence that peripheral DCs from pregnant women with PE have a stronger ability to stimulate Th1/Th17-like T-cell responses than those from normotensive controls supports this view (170). In addition, alterations in circulating DCs were related to lower peripheral Treg levels in patients with PE (134) and elevated Th17 cell levels in women with early-onset PE (171). Furthermore, the insufficient maturation of decidual lymphatic vessels in PE might impede the penetration of immune cells into this region (172). In fact, a correlation between the number of Tregs and the density of lymphatic vessels in the decidua was established, suggesting that in cases of PE, the entry of circulating Tregs into the decidua may be obstructed (172). Lastly, a study investigating the T-cell receptor (TCR) repertoires of Tregs in the decidua discovered that the proportion of clonally-expanded Tregs decreased obviously in PE pregnancies (173), indicating that the inability of decidual effector Treg cell populations to undergo clonal expansion may be associated with the onset of PE.

Currently, there are some innovative methodologies attempting to explore the molecular mechanisms underlying the onset of PE, including single-cell RNA sequencing (scRNA-seq). In one study, researchers conducted scRNA-seq on the placenta and decidual tissues of individuals with late-onset PE as well as those in a healthy pregnancy (174). It was discovered that the cells in the decidua that do not express extracellular matrix (ECM) are primarily immune cells, which consist of two subtypes of T cells (T1 and T2) (174). T2 cells show the expression of Treg marker genes, and there is a decrease in the down-regulation of the GO term “regulatory T cell differentiation” in T2 cells within the PE group. The pathway for “positive regulation of cytokine production” and several pathways for interleukin production show decreased activity in T cells, suggesting a significant impairment of their regulatory functions in late-onset PE (174). Another study examined transcriptomic changes specific to different cell types through unbiased scRNA-seq of placental samples, including two individuals with PE and two normotensive pregnant women (175). This comprehensive analysis involved 29,008 cells across 11 distinct cell types, encompassing trophoblasts and immune cells (175). They found that compared with normal pregnancies, there are significant differences in the gene expressions of GZMA, CD3E1, and CD3G in T cells from PE placental tissues, suggesting that these genes may be involved in the pathogenesis of PE (175). These findings provide a novel molecular theoretical foundation for the treatment of PE.

Treg cell dysfunction or a deficiency in quantity within the decidua has been linked to diminished invasion of EVT and ineffectual remodeling of spiral arteries. These factors collectively disrupt placental growth and ultimately result in placentation disturbance (176, 177). Therefore, it is not difficult to hypothesize that a deficiency of Treg cells during pre- and peri-conception could act as an initial catalyst for a cascade of events culminating in impaired placental development and shallow placentation, which ultimately manifest as conspicuous symptoms of PE later in pregnancy. Furthermore, in PE, Tregs undergo apoptosis at the feto-maternal interface, and galectin-2 (Gal-2) appears capable of inhibiting this process (178). Moreover, modifications in the activity of uNK cells may result in a potential dysregulation between Th17 and Treg cells. It has been shown that uNK cells communicate with CD14⁺ monocytic cells to induce the development of Treg cells and that IFN-γ produced by decidual NK cells can impede Th17 cells; these effects result in reduced inflammation and increased maternal-fetal tolerance (58, 179).

Treg cells possess a unique combination of anti-inflammatory and immune-regulatory properties that endow them with formidable capabilities to support placental development, facilitate the adaptation of maternal blood vessels, suppress inflammation, and preserve maternal acceptance of the fetus. As a result, enhancing the functionality of Tregs and augmenting their quantity in order to regulate the immune response may represent a viable therapeutic strategy for these pregnancy-related complications, including PE. However, in the context of human reproduction, any experimental evaluation of a method to stimulate Tregs must be conducted extremely cautiously and in strict adherence to robust principles of clinical trial design. Ensuring the safety of mothers and infants is of the utmost importance, and it is critical to recognize that the risk-benefit ratio of reproductive and obstetric conditions differs significantly from that of immune diseases that pose a life-threatening risk. Consideration should be given to the potential adverse effects of artificially increasing maternal Treg cells, such as a diminished capacity to combat pathogens (180) or immune surveillance for cancer (181). Notwithstanding the necessity for a comprehensive assessment of various approaches and identification of appropriate patient cohorts, the advanced immune therapy must take precedence to control over reducing adverse health outcomes and mortality rates associated with PE.

IL-17 is important to the body’s response to bacterial infections and inflammation. In addition to compromised tolerance in conditions such as PE, it has been associated with contact dermatitis, autoimmune disorders, and organ rejection following transplantation. Secukinumab, a monoclonal antibody that specifically targets IL-17, has been employed in treating contact dermatitis (NCT02778711) and discoid lupus erythematosus (NCT03866317) to correct the Th17 imbalance. Tibulizumab (LY3090106) is a tetravalent antibody currently under development for the treatment of Sjögren’s disease (NCT04563195). It functions as a dual antagonist against B cell activating factor and IL-17. The long-term use of recombinant mouse IL-17 receptor C (IL-17RC), a soluble receptor that inhibits IL-17, reduced uterine perfusion pressure (RUPP) in rats as well as reduced levels of circulating IL-17 and placental Th17 cells (200). Thus, the prospective efficacy of IL-17RC in the treatment of pro-inflammatory effects mediated by IL-17 in PE was demonstrated ( Figure 2 ).

In addition to inhibiting immunosuppressive Tregs and Th2 cells, IL-6 enhances the differentiation of Th17 and cytotoxic T cells, thereby leading to inflammation in PE (201). IL-6 inhibitors, such as anti-IL-6 receptor mAbs (sarilumab and tocilizumab) and anti-IL-6 mAbs (siltuximab), have the potential to drive the development of inexperienced CD4⁺ T cells away from inflammatory Th1 and Th17 subsets (202). Clinically, anti-IL-6 therapy has been used to treat atherosclerosis (203) and rheumatoid arthritis (202). Incorporating strategies to decrease IL-6 for the management of PE may potentially promote the transformation of naive CD4⁺ T cells into Treg and Th2 cells, as opposed to Th1 and Th17 cells.

In the RUPP model, IL-10 administration via infusion through an osmotic mini-pump implanted intraperitoneally results in modest reduction in blood pressure as well as a substantial reduction in the levels of circulating Th1 and Th17 cells, an up-regulation of Treg cell differentiation, and the restoration of TNF-α expressions to normal (204). Culturing naive CD4⁺ T cells with IL-10-producing DCs could enhance the differentiation of Treg cells (204), a process that potentially alleviates PE. This study (204) indicate that IL-10 may function as an innovative treatment for PE.

TNF-α, a potential target for modulating CD4⁺ T cells, is produced by Th1 and Th17 cells. TNF-α expressions in PE could be considerably increased, reaching 2-3 times higher than in pregnancies with normal blood pressure (176, 205). These elevated levels have been linked to complications such as gestational hypertension, compromised endothelial function, and unfavorable obstetric outcomes (206). In addition to its essential function in facilitating successful implantation and placental development, TNF-α also functions as an inhibitor of CD4⁺ T cell proliferation via a mechanism dependent on IL-10 (11). By inhibiting effector T cells and repressing the activity of TNF-α, anti-TNF-α neutralizing mAbs promote the development of Tregs (207). The British Association of Dermatologists suggests that TNF-α inhibitors may be used during pregnancy, when large cohort studies that are well-designed, with a follow-up of over 10 years are performed in the future (208). According to the European Crohn’s and Colitis Organization, it is acceptable to utilize TNF-α inhibitors during the third trimester of pregnancy (208). Considering its perceived safety, anti-TNF-α could potentially function as a treatment option for PE by increasing the quantity of Treg cells and restoring the Th1/Th2 and Th17/Treg ratios.

17-hydroxyprogesterone caproate (17-OHPC), a progesterone derivative, is deemed secure for application in obstetrics. In a PE rat model, 17-OHPC induced an elevation in infant weight and a reduction in uterine artery resistance index (UARI) (209). Implementing an intervention such as 17-OHPC to enhance the current treatment for PE patients may have positive effects on both the mother and child. Furthermore, by activating glucocorticoid receptors, progesterone induces the proliferation of Treg cells and advances apoptosis in conventional T cells (210). Consequently, the use of the orally-administered progestogen or dydrogesterone may have the potential to alleviate the adverse pregnancy outcomes in women with PE (211–213).

IVIg is employed in the treatment of various immune-regulated disorders, organ transplantation, and systemic inflammatory conditions by virtue of its capacity to inhibit the proliferation of DCs, increase concentrations of anti-inflammatory IL-10, and diminish the functionality of pro-inflammatory T cells (214). Moreover, IVIg improves pregnancy outcomes by stimulating Tregs’ responses (215). IVIg therapy may, therefore, be contemplated as a prospective therapeutic intervention for PE. In women with a history of recurrent pregnancy loss (RPL) and unexplained infertility, the prevalence of PE was comparable with normal pregnant women when they were treated with prednisone and IVIg (216).

VD has the ability to affect many cell types, such as immune cells, specifically CD4⁺ T cells (217). The circulating concentrations of VD were considerably diminished in pregnant women with PE in comparison to those without (218–220). Furthermore, in comparison to pregnant women with VD levels above 20 ng/mL, those with VD deficiency (25(OH) D < 20 ng/mL) have a five-fold increased risk of developing PE (220). Patients with PE who received VD supplementation exhibited decreased levels of IFN-γ, TNF-α, IL-17, IL-6, and IL-23, whereas IL-10 and TGF-β levels elevated (218, 219). VD exerts its immunomodulatory effects through multifaceted mechanisms, including inhibition of Th1 and Th17 cells, up-regulation of Th2 cell expression, and facilitation of Treg cell proliferation (221–225). Therefore, VD could potentially be suggested as a strategy for regulating the systemic inflammatory response in PE. Moreover, exposure to sunlight (226) and exercise (227) contribute to the maintenance of Treg homeostasis. Nevertheless, the precise mechanisms of VD in human Treg cells remain obscure.

Receiving low-dose aspirin from 11-14 weeks reduced the occurrence of preterm PE in high-risk women compared to those who received a placebo (228). This discovery indicates that giving aspirin early on may decrease the likelihood of PE, potentially by enhancing placental development. A study done on mice revealed that aspirin greatly increased the percentage of functional CD4⁺CD25⁺FoxP3⁺ Treg cells (229). Patients with RPL associated with antiphospholipid syndrome (APS) showed elevated levels of serum cytokines, T cell phenotypes, and transcription factor gene expressions indicative of Th1 responses, while those indicative of Th2 responses were decreased (230). The imbalance between Th1 and Th2 could be restored in patients who responded well to the combination of low-molecular weight heparin (LMWH) and aspirin treatment (230). Moreover, aspirin functions through the production of aspirin-triggered lipoxin, which inhibits the effects of antiphospholipid antibodies on the migration of human trophoblasts and their interactions with endothelial cells (231). In other diseases, aspirin can exert therapeutic effects by regulating T cell function. One example is the potential for aspirin to mitigate the progression of atherosclerosis through restoring balance to the Th17/Treg axis (232). Additionally, aspirin could potentially suppress the autoimmune reaction in atherosclerosis by promoting the expansion of CD4⁺CD25⁺FOXP3⁺ Treg cells (233). In studies on experimental autoimmune encephalomyelitis (EAE), aspirin could enhance Tregs while reducing Th1 and Th17 responses (234, 235). This includes an increase in the levels of FoxP3 and IL-4 in T cells, as well as inhibiting the differentiation of naive T cells into Th17 and Th1 cells (234, 235). The above findings indicate that aspirin may play a role in the treatment of PE by targeting T cells.

Elevated levels of indicators for oxidative stress and reduced levels of antioxidants (such as vitamin E, vitamin C, and lycopene) in women with PE indicate that oxidative stress markers are significantly involved in the development of PE (236–238). AC-11 (AC-11^®, hot-water extract of U. tomentosa) has a potential antioxidant effect (239). The administration of AC-11 resulted in a significant decrease in blood pressure and a significant down-regulation of CD8⁺ T cells and CD8/CD4 ratio in PE mice models induced by angiotensin II, compared with healthy pregnant animals (239). A survey of existing research suggests that the products of the kynurenine (Kyn) pathway have the ability to promote T cell differentiation into Treg cells and trigger the apoptosis of Th1 cells (240). Therefore, the Kyn pathway may be a potential therapeutic target for PE due to the antioxidant effect of its metabolites (240). Moreover, the well-established powerful antioxidant pentoxifylline can mitigate oxidative stress and enhance placentation (241). Its function is to maintain the balance between Th1 and Th2 immunity, reduce Th1-type immune responses, and promote a shift towards Th2 immune reactions (242). A different type of antioxidant, known as heme oxygenase-1 (HO-1), has the ability to stimulate the release of Th2 cytokines (243). Moreover, one study has shown that severe PE is linked to elevated T-cell-endothelium adhesion, and effective antioxidants can prevent this impact (244). Clinically achievable concentrations of antioxidants in the circulation can be attained through oral intake of vitamins E and C, or by intravenous administration of N-acetylcysteine, as demonstrated by this study (244). Accordingly, supplementing with antioxidants for PE patients may be a simple and effective treatment.

The promotion of alloantigen-specific Treg cells is a potential preventive and therapeutic strategy for PE. This could be achieved by increasing exposure to paternal antigen in high-risk population. In addition, medications that enhance the activity of Treg cells by targeting immune checkpoints, such as CTLA-4Ig (245), may represent viable alternatives for the treatment of PE. To develop therapeutics that target immune checkpoints, it is imperative to investigate costimulatory and inhibitory molecules that are unique to antigen-specific Treg cells. Additional investigation about Treg cells at the single-cell level may yield valuable knowledge regarding the progression of immunotherapeutic approach for PE.

In rodent models of PE, proof-of-concept trials have already demonstrated that PD-L1 Fc (246), CD28 superligand (247), and low dose IL-2 (248, 249) were efficacious biological agents for increasing the percentage and stability of Treg cells. In light of the swift advancements in Treg cell immunology, including the development of numerous intervention therapies and the application of flow cytometry for peripheral blood diagnosis, it is important to investigate Treg cell therapy in high-risk women. Prior to or in the early phases of pregnancy, it is essential to develop diagnostic tools and treatment modailities that can halt the progression of disease and prevent fetal or placental damage. Regarding the “window of opportunity” for timely and effective PE treatment, since Treg cells play an essential role in implantation and placentation during early pregnancy, initiating treatments before conception or as soon as pregnancy is identified is the optimal choice (10).