IL-35 is a recently identified heterodimeric cytokine critical for the regulatory functions of Tregs. As a member of the IL-12 cytokine family, IL-35 comprises an α chain (p19 or p28) and a β chain [p40 or Epstein-Barr virus-induced gene 3 (EBI3)] (21). It is part of a group of five major heterodimeric cytokines: IL-12, IL-23 (p19 and p40), IL-27 (p28 and EBI3) and the recently proposed IL-39 (p19 and EBI3) (22). Specifically, IL-35 consists of IL-12p35 and EBI3, which form the IL-12p35/IL-27EBI3 dimer. EBI3, a glycoprotein containing a signal peptide with a repeated Alu sequence, plays a role in cellular pathways and maternal immune tolerance during embryogenesis. EBI3 is also expressed in Hodgkin lymphoma cells, as well as in acute and chronic myeloid leukemia cells (23). While IL-12p35 is involved in promoting inflammatory responses, IL-35 serves to regulate and suppress inflammation. As shown in Fig. 1, IL-35 regulates the functions of various immune cells (such as Tregs, Bregs, macrophages, T cells, and B cells) to regulate the body's immunity. IL-35 is secreted by various immune cells, including tolerogenic dendritic cells (DCs), myeloid-derived suppressor cells, tumor-associated macrophages, neutrophils and natural killer (NK) cells, and primarily by IL-10/IL-35-secreting subsets of Bregs. As an inhibitory cytokine, IL-35 suppresses T-cell proliferation and induces the generation of iTr35 cells to control inflammatory responses (24).

The expression of IL-35 is linked to forkhead box P3 (Foxp3)+ Tregs, with the EBI3 subunit being highly expressed in murine CD4+ Foxp3+ Tregs but absent in activated effector CD4+ T cells. Upon activation, Tregs generate distinct effector subsets (IL-35-producing and IL-10-producing) to maintain immune tolerance (25). iTr35, a newly identified subset of Tregs that are non-Foxp3 dependent, is induced by IL-35 from murine T cells (26). iTr35 cells release IL-35, promote Treg proliferation, block effector T-cell activation, and convert effector cells into iTr35 cells. The differentiation and function of iTr35 cells are driven primarily by IL-35 and these cells do not rely on Foxp3 or mediate immune suppression via IL-10 or TGF-β (26). Studies have highlighted Bregs as another significant source of IL-35 (27-29). Upon B-cell receptor activation, Toll-like receptor 4 (TLR4) binds to CD40, triggering the transcription of EBI3 and p35, leading to IL-35 secretion (27). Additional research using TLR-deficient B cells demonstrated that TLR4 and CD40L costimulation is necessary for the transformation of B cells into Breg subsets (i35-Bregs) capable of secreting IL-35 (30). In addition to Tregs and Bregs, IL-35 may also be produced by other immune cells, such as immature DCs, CD8+ T cells, and certain tumor cells (31-35). Under inflammatory conditions, additional tissues may have the potential to secrete IL-35. While IL-27 is produced primarily by activated antigen-presenting cells (APCs), IL-35 is secreted predominantly by activated Tregs (36).

IL-35 plays a critical role in modulating immune responses by inhibiting effector T-cell differentiation and IL-17 production, exerting a negative immunoregulatory effect that helps maintain immune homeostasis (37). It promotes the generation of Bregs and the secretion of IL-10 by Breg subsets, further contributing to its immunosuppressive effects (38). Activated B cells can produce both IL-35 and IL-10, mediating negative immune regulation (38). In combination with TGF-β, IL-35 also suppresses immune reactions by stimulating the proliferation of CD4+CD25+ T cells and enhancing IL-10 expression, which inhibits inflammatory responses (38). Studies in mouse models have shown that B cells deficient in IL-35 expression fail to recover from T-cell-induced autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE) (39). These findings highlight the potent immunoregulatory functions of IL-35 in various conditions, including cancer, autoimmunity and infection. As summarized in Table I (40-45), IL-35 plays a pivotal role in inflammatory and autoimmune diseases, positioning it as a potential therapeutic target for these disorders (22,32).

The current research on the source of IL-35 in MG is extremely weak and relies mainly on animal models for speculation, lacking human evidence, cell tracing analysis, and clinical translation studies. In the future, multiomics studies targeting the immune microenvironment of patients with MG are needed to clarify the production of cells, regulatory mechanisms and therapeutic potential of IL-35.

The receptors for IL-35 signaling, although distinct, partially overlap with those of other members of the IL-12 family (46). IL-35 signals through four primary receptor types: IL12Rβ2/IL27Rα and glycoprotein 130 (gp130)/gp130. The functional IL-35 receptor comprises IL12Rβ2, gp130 and possibly IL-27Rα. IL-35 transduces immunosuppressive signals primarily via the IL-12Rβ2:gp130 receptor complex. The p35 subunit binds to IL-12Rβ2, while the EBI3 subunit binds to gp130, leading to receptor dimerization, JAK-STAT activation, and the formation of either heterodimers (STAT1-STAT4) or homodimers (STAT1-STAT1). However, IL-35 can also signal through two types of homodimeric receptors formed by gp130 and IL-12Rβ2, namely, gp130:gp130. The IL-12Rβ2 subunit is typically expressed by activated NK and T cells, with some expression on DCs and B cells, whereas gp130 is expressed on nearly all cell types. The expression level of IL-12Rβ2 determines the functional scope of IL-35 signaling. IL-35 can initiate signaling not only by binding to the IL-12Rβ2:gp130 heterodimeric receptor but also by engaging gp130:gp130 homodimers (47). As shown in Fig. 2, unlike other cytokines in the IL-12 family, IL-35 can signal through homodimeric receptors. Specifically, it can interact with IL-12Rβ2:IL-12Rβ2 homodimers, initiating signal transduction via the STAT1 or STAT4 pathway, respectively. However, since only one pathway is activated in this case, the immunoregulatory function of IL-35 is partially compromised (47). Full immunosuppressive activity, including the induction of iTr35 cells, requires binding to the IL-12Rβ2:gp130 heterodimer and simultaneous activation of both the STAT1 pathway and the STAT4 pathway (48). While homodimers can suppress T-cell proliferation, they are less effective than heterodimers in regulating the activity of IL-35-induced Tregs (iTr35 cells) (47). At present, research on the role of the IL-35 receptor in MG is still in its early stages, and its specific mechanism of action, receptor expression regulation, and clinical translational potential are not yet clear.

A key function of the IL-12 family of cytokines is their mediation of activity through the activation of the JAK-STAT signaling pathway (49). Each member of the IL-12 family induces the activation of specific STAT family members, which results in distinct but sometimes overlapping gene transcription patterns. Upon activation of the IL-35 receptor, JAK family members are activated, initiating signal transduction (49). Phosphorylated JAKs subsequently phosphorylate STAT1 and STAT4, members of the STAT family. These phosphorylated STATs then translocate to the nucleus, where they regulate the expression of the p35 and EBI3 genes, promoting IL-35 expression (50). The activation of JAKs by the IL-12Rβ2 and gp130 receptor subunits primarily involves JAK1 and JAK2, respectively, which act on downstream STAT4 and STAT1 molecules. The binding of IL-35 to a specific homodimeric receptor activates a corresponding signaling branch. Thus, the IL-12Rβ2:IL-12Rβ2 homodimer predominantly mediates the JAK1-STAT4 signaling pathway, whereas the gp130:gp130 homodimer primarily activates the JAK2-STAT1 signaling pathway (Fig. 3).

In Tregs, IL-35 transduces signals via either heterodimeric receptors (IL-12Rβ2/gp130) or homodimeric receptors (gp130/gp130), leading to the activation of STAT4, either concurrently or independently (51). When IL-35 binds to its corresponding receptor, only one signaling branch is activated (either STAT1 with gp130:gp130 or STAT4 with IL-12Rβ2:IL-12Rβ2 homodimers), which inhibits T-cell proliferation (52). This results in partial loss of its immunosuppressive activity. However, signaling through the IL-12Rβ2:gp130 heterodimeric receptor can also induce the production of iTr35 cells, which fully exert their immunosuppressive functions (Fig. 3).

IL-35 can bind to a heterodimeric receptor composed of IL-12Rβ2, activating downstream STAT1 and STAT3 signaling molecules. This activation induces the production of both IL-10 and IL-35 and promotes the proliferation of Bregs (28). These findings demonstrate that IL-35 regulates its biological functions through different receptors and STAT molecules in various cell types (Fig. 3).

The combination of IL-35 with its corresponding receptor activates STAT4 in T cells, which then inhibits the MAPK and NF-κB pathways. This reduces proinflammatory responses and inhibits the maturation of monocyte-derived DCs (53). As shown in Figs. 3-4 and Table II (54-58), IL-35 plays a critical role in maintaining immune homeostasis by modulating different target cells and effector pathways. Failure to achieve effective immunosuppression can lead to the development of immune-related diseases.

Inflammatory signaling pathways are often stimulated under inflammatory conditions, leading to the release of proinflammatory factors. During inflammation, the interaction of TLRs, TNF receptors, or IL-18 receptors with their ligands rapidly activates MyD88. This in turn activates TRAF6 or enzymes such as IRAK-1, promoting the expression of NF-κB and increasing the production of inflammatory factors (59). The transcription factors c-Fos and c-Jun, which are activated by MAPKs, form AP-1 (60), which can increase the levels of inflammatory cytokines (60). Additionally, the IκB inhibitor is affected by IKK through ubiquitination, leading to NF-κB release and its translocation to promote inflammatory responses (59). Together, these two pathways contribute to the production of proinflammatory factors such as IL-1, IL-6, and interferon (IFN).

In addition to stimulating proinflammatory cytokines, NF-κB also plays a pivotal role in increasing the levels of IL-35 (composed of EBI3 and p35 subunits) and IL-37, as shown in Fig. 5 (53,61). IL-35 has been found to inhibit the p38 MAPK pathway in various inflammatory diseases (62). IL-37 can inhibit the MAPK pathway (63), thus suppressing inflammatory responses. In summary, both IL-35 and IL-37 control inflammation by inhibiting the production of inflammatory mediators, with a preference for blocking NF-κB activation over MAPK signaling.

Th1 and Th17 cells play essential roles in preventing cancer development and pathogen invasion, whereas Tregs are critical for inhibiting autoimmune diseases (64). Under noninflammatory conditions, a balance exists between Tregs and Th1 and Th17 cells (65), contributing to the regulation of autoimmune diseases and tumor progression. An imbalance between Th17 cells and Tregs can lead to increased Th1 and Th17 cell production in conditions such as IBD (66), driving uncontrolled inflammation. IL-35 may help maintain T-cell balance by influencing the genetic regulation of T-cell factor 1 (TCF) (47). IL-35 also reduces Th1 differentiation by lowering the level of RORγt (67) and contributes to the maintenance of Tregs by promoting Foxp3 expression (68). Through these mechanisms, IL-35 helps prevent inappropriate and excessive inflammatory responses.

Innate immunity is a complex and adaptive system, and both IL-35 and IL-37 play critical roles in regulating inflammation (Figs. 4 and 5). IL-37 markedly inhibits pathways that produce proinflammatory cytokines (Figs. 4 and 5) (69). Specifically, IL-37 suppresses the MAPK pathway by inhibiting the IL-18 pathway (70), whereas IL-35 prevents the overexpression of proinflammatory cytokines such as IL-1 and TNF-α through inhibition of the p38 MAPK pathway (Fig. 5) (71). Additionally, IL-37 suppresses inflammation by enhancing TGF-β activity (71). Treatment with IL-35 and IL-37 results in an increased ratio of Tregs and a reduction in Th17 cells, key outcomes for managing autoimmune diseases and tumors. An imbalance in the Th17/Treg ratio is a significant factor in the development of autoimmune diseases or tumors (72). An excess of Th17 cells over Tregs increases susceptibility to autoimmune conditions such as IBD (73), whereas a greater number of Tregs than Th17 cells increases the risk of cancer (74). IL-35 and IL-37 also regulate Th1 activity and maintain the Th17/Treg balance by reducing T-bet and RORγ levels while increasing FOXP3 expression. These actions help alleviate the inflammatory reactions associated with IBD (75,76), as illustrated in Fig. 5. Although studies have shown that IL-35 is abnormally expressed in patients with MG and may be involved in the regulation of immune imbalance, research on this pathway is still highly limited to the level of basic science and has not yet entered the stage of drug development or clinical intervention.

MG is an autoimmune disorder characterized by autoantibodies targeting AChRs or other associated proteins, such as muscle-specific kinase (MuSK) and low-density lipoprotein receptor related protein 4 (LRP4), on the postsynaptic membrane (77). The pathogenesis of MG is multifactorial and involves humoral and cellular immunity, thymic abnormalities, and genetic predispositions (77). The primary target organ is skeletal muscle, with patients predominantly presenting with muscle fatigability, which can be alleviated by anticholinergic drugs or rest. While various antibody types are present in patients with MG, the most common immunopathological subtype is the presence of autoantibodies against AChRs, accounting for ~85% of cases (78,79). The AChR is a pentameric transmembrane glycoprotein ion channel, and the autoantibodies targeting it are primarily of the IgG1 subclass, with a smaller proportion of IgG3. The active or passive transfer of human AChR antibodies into animal models induces myasthenic symptoms and disease progression, demonstrating a direct pathogenic role for these autoantibodies in MG. Although the production of these antibodies is well understood in theory, the specific cellular immune mechanisms and characteristics underlying antibody production still require further investigation.

The core pathogenesis of MG centers on autoantibodies that target key proteins at the neuromuscular junction (NMJ), impairing synaptic transmission. Of AChR antibody-positive patients with MG, ~70% exhibit thymic abnormalities, such as thymic hyperplasia or thymoma, with the thymus considered the origin of the autoimmune response (80). Thymomas are typically observed in patients over 50 years of age, whereas younger or female patients are more likely to present with thymic lymphoid hyperplasia accompanied by B-cell infiltration (81). By contrast, MuSK-positive patients with MG do not exhibit thymic abnormalities (81). A small subset of LRP4-positive patients with MG also shows thymic hyperplasia resulting from lymphoid hyperplasia (82).

As a primary lymphoid organ, the thymus plays a critical role in T-cell differentiation. Although the frequency of CD8+ T cells exported to peripheral tissues remains unchanged in the thymuses of patients with MG, naturally differentiated Tregs exhibit impaired function, and partially dysfunctional Tregs are also present in peripheral tissues (83). These alterations in T-cell immunoregulatory function have been linked to functional deficiencies in Tregs in patients with MG (83). Additionally, effector T cells (Teffs) in patients with MG can resist the immunosuppressive effects of Tregs (79), likely due to the inflammatory microenvironment within the thymus.

Beyond humoral immunity, T cells, particularly the imbalance among Th1, Th17 and Tregs, play a critical role in the pathogenesis of MG (84,85). These findings suggest that the inflammatory environment alters the functionality and plasticity of CD4+ T cells, leading to abnormal Treg and effector T-cell functions.

AChR antibodies directly damage the postsynaptic membrane by activating the complement cascade (such as C3 and the MAC complex). AChR-specific CD4+ helper T (Th) cells are essential for the progression of MG (85). Some studies suggest that the imbalance between Th1 and Th2 cells, as well as the cytokines they secrete, is closely associated with the pathogenesis of MG (86-88). In an experimental autoimmune MG model, Th17 cells influence autoantibody release by shifting the Th1/Th2 cytokine balance, reducing Treg numbers and downregulating Foxp3 expression (89). One study reported that serum IL-35 concentrations are markedly lower in patients with MG than in controls and are associated with anti-AChR antibody titers (90), indicating a regulatory role for IL-35 in the onset and progression of MG.

MGs are associated with specific HLA alleles, such as HLA-DR3 and HLA-B8, as well as non-HLA genes, such as PTPN22 and CTLA4 (91). Genetic studies have identified MHC types carrying risk alleles for MG (91). GWAS findings have confirmed that CTLA4 and TNFRSF11A are involved in MG pathogenesis (92-94). CTLA4 transmits signals to T cells, whereas TNFRSF11A regulates interactions between T cells and DCs (93,94). Additionally, factors such as infections (such as EBV), medications (such as D-penicillamine) and vitamin D deficiency may trigger MG (95). A deeper understanding of these mechanisms will help pave the way for precision therapies for MG and provide insights into how novel biologics may target these pathological processes, improving the prediction of the durability of their therapeutic effects.

Various treatment options are available for MG, including symptomatic therapy, thymectomy, immunomodulatory therapy and long-term immunosuppressive therapy (96). For severe, widespread MG, prompt initiation of immunosuppressive therapy is critical (97). Commonly used immunomodulatory treatments for patients with MG include corticosteroids, immunosuppressants such as azathioprine, mycophenolate mofetil and methotrexate and newer therapies such as calcineurin inhibitors. As outlined in Table III (96-100), each treatment method offers distinct advantages and limitations. Recently, treatment strategies have evolved from traditional immunosuppressive approaches to precision-targeted therapies aimed at achieving disease remission or improved symptom control, minimizing treatment side effects. For example, oral corticosteroid doses are often reduced to ≤5 mg/day, with side effects maintained at minimal levels (≤1). As shown in Table IV (101-103), precision-targeted therapeutic drugs such as FcRn inhibitors, complement inhibitors and B-cell depletion agents offer diverse regulatory mechanisms, therapeutic benefits, and target populations for MG treatment. These therapies have led to rapid improvements in MG-Activities of Living (ADL) and Quantitative Myasthenia Gravis (QMG) scores, as well as reductions in corticosteroid use (104). Notably, rozalizumab is the first and only approved biological agent in China for patients with AChR and MuSK antibody positivity. Plasma IL-35 levels markedly increase with improvements in MG following immunomodulatory therapy (105). Therefore, plasma IL-35 levels can serve as a valuable biomarker for evaluating the therapeutic efficacy of MG treatments. Further studies revealed that in an experimental autoimmune MG (EAMG) model, treatment with IL-35 combined with low-dose tacrolimus (30% of the conventional dose) resulted in greater clinical improvement (40% increase in muscle strength scores) than did full-dose tacrolimus alone, with a more significant reduction in serum anti-AChR antibody levels (65% decrease in the combination group vs. 45% decrease in the tacrolimus-alone group) (106-108). In anti-AChR antibody-positive MG mice, IL-35 (10 μg per dose; twice weekly) combined with eculizumab (dose reduced by half) was administered for 4 weeks. Postsynaptic membrane complement deposition was decreased by 80% (vs. 50-60% in the single agent group) and the muscle strength recovery time was shortened by 40% compared with that in the single agent group (109). In the anti-MuSK antibody-positive MG mouse model, comparisons were made between IVIG alone (1 g/kg), IL-35 alone (20 μg/kg) and combination therapy (IVIG 0.5 g/kg + IL-35 10 μg/kg). The combination group showed a 32% improvement in the muscle strength recovery rate (compared with IVIG alone); anti-MuSK IgG4 levels decreased by 58% (only a 35% reduction in the IVIG-alone group); and the postsynaptic membrane AChR cluster density recovered to 85% of normal levels (compared with 60-70% in the single-agent groups) (110). Although current immunomodulatory therapy can effectively control the symptoms of most MG patients, there are still key bottlenecks, such as delayed onset, significant toxic side effects, difficulty in treating some patients, and high treatment costs. The future direction lies in developing more precise, safe, and durable targeted immune intervention strategies combined with personalized assessment to optimize treatment pathways.

IL-35 plays a pivotal role in the onset and progression of MG. Through IL-35, Tregs and Bregs modulate various pathways and proteins, influencing the development and progression of this disease. As shown in Table V (9,111-116), the complex interactions between various cytokines and inflammatory factors markedly influence the pathogenesis of MG. Previous studies have shown that the ratio of Tregs is decreased in patients with MG and is negatively associated with disease activity, suggesting that Treg deficiency is linked to the progression of MG (117,118). Tregs are the primary source of IL-35 (119), and their deficiency may contribute to reduced IL-35 expression in patients with MG. Furthermore, IL-35 levels are markedly inversely associated with neurological function scores (QMGs) and ADL scores in these patients (119), indicating that IL-35 is a reliable marker for assessing the severity of MG (120). IL-35 can inhibit Th cell proliferation and promote the generation of Tregs, which, in turn, release IL-35. A reduction in IL-35 levels may disrupt the balance of T-cell subsets, leading to the release of cytokines that exacerbate the condition of MG (121). The imbalance between T and B-cell subsets plays a critical role in the progression and outcome of MG, particularly within the T-cell compartment (122). IL-35 levels are lower in untreated patients with MG, particularly those with GMG or comorbid thymoma (116), but they increase following treatment and are negatively associated with functional disability scores (123), highlighting the importance of IL-35 in MG disease outcomes. The reduction in IL-35 during the acute phase may be related to a decrease in Treg and Breg proportions. Compared with those in healthy controls (HCs), both the ratio and function of Bregs are diminished in patients with MG (124). Additionally, IL-35 is negatively associated with Th17 cells and their secreted factor, IL-17 (125), suggesting that IL-35 may exert its immunosuppressive effects by regulating Th17 cells in MG. These studies suggest that IL-35 is an important anti-inflammatory factor that regulates MG and has the potential to be used for the treatment of MG. However, some studies suggest that it may promote disease progression in certain chronic inflammatory or tumor environments, exhibiting a 'double-edged sword' characteristic. In MG, a highly heterogeneous autoimmune disease, further research is needed to investigate whether IL-35 accidentally activates other immune pathways or leads to immune escape.

B cells, as precursors to plasma cells, play a key role in promoting humoral immune responses through T-cell activation (126). Studies have highlighted the specific protective functions of B cells in regulating immune responses (126-128). In a study involving 41 patients with MG who did not receive any medication treatment and 30 HCs, the proportions of CD19+ IL-10+ cells and CD19+CD24hiCD38hi cell subsets in patients with MG were markedly lower than those in HCs; in thymus tissues, the percentage of CD19+ IL-10+ cells was highest in healthy children (~8%), followed by healthy adults (~3%) and was lowest in patients with MG (~0.5%) (129). In another study involving 112 patients with MG, Breg infiltration in the TME decreased with MG aggravation, whereas the opposite trend was observed for Tfh cells. The Breg/Tfh ratios in the peripheral blood and TME were broadly consistent and the levels of both types of cells were markedly lower in patients with aggravated MG. Therefore, Breg cells have been confirmed to have an immunosuppressive function and play an important role in MG (130). These B-cell subsets, known as Bregs, downregulate immune reactions by secreting cytokines such as IL-10 and TGF-β (131). As shown in Fig. 6, IL-10 secreted by Bregs inhibits Th17 cell differentiation, enhances Th2 polarization, and suppresses DC activation; IL-35 secreted by Bregs inhibits Th1/Th17 activation and promotes Treg expansion; and TGF-β secretion by Bregs inhibits Th1 activation and further promotes Treg expansion (131).

IL-35 is secreted primarily by Tregs and Bregs (28). Under conditions such as TLR activation, an inflammatory microenvironment (such as in autoimmune diseases or tumors) and Treg-Breg interactions, Bregs secrete IL-35 (28,132,133). Naïve B cells can differentiate into immunosuppressive Breg subsets, termed IL-35-induced Bregs (i35-Bregs) upon IL-35 stimulation. These i35-Bregs have been shown to prevent immune responses by blocking Th1/Th17 cells (134), representing a promising new avenue for future autoimmune disease therapies. IL-35 promotes the differentiation of naïve B cells into Bregs through the STAT1/STAT3 signaling pathway (135) and enhances Breg function. IL-35-stimulated i35-Bregs exhibit stronger immunosuppressive capabilities, producing and releasing anti-inflammatory factors such as TGF-β (15). Additionally, IL-35 suppresses Th1/Th17 responses, reduces proinflammatory cytokines such as IL-17, promotes Treg expansion, inhibits CD8+ T cells, facilitates tumor immune escape, and enhances the suppressive function of Bregs (136). These findings suggest that IL-35 may serve as a therapeutic agent for treating MG.

In models of autoimmune diseases, such as rheumatoid arthritis, IL-35-producing Breg (IL-35+ Breg) cells markedly ameliorate inflammatory damage. In EAE and IBD, IL-35+ Bregs alleviate disease symptoms by reducing effector T-cell proliferation (137,138). Further studies have shown that IL-35 treatment increases the proportion of Bregs (such as CD19+CD24hiCD38hi or IL-10+ Bregs) and suppresses the production of pathogenic antibodies, such as anti-AChR antibodies (15,139,140). IL-35 likely promotes Breg differentiation via the STAT3 pathway while preventing the transformation of B cells into plasma cells (141). IL-35+ Bregs block the proliferation of proinflammatory T cells (such as Th1 and Th17) by releasing IL-10, which in turn reduces the expression of IFN-γ and IL-17 (142). These findings suggest that IL-35 can improve the prognosis of patients with autoimmune diseases by regulating Bregs.

In MG, T follicular helper (Tfh) cells induce germinal center reactions and autoantibody production, whereas IL-35 may indirectly inhibit Tfh differentiation through Bregs (143). In an EAMG mouse model, injection of recombinant IL-35 alleviated muscle weakness symptoms, reduced serum AChR antibody titers, and increased the proportion of Bregs (144). Combination therapy using IL-35 and adoptive transfer of Bregs (such as CD19+CD24hiCD38hi Bregs) has synergistic therapeutic effects (144). Delivery of the IL-35 gene via adenoviral vectors, such as AAV-IL-35, can maintain long-term Breg function and delay EAMG progression (145). Bregs in MG exhibit functional defects (such as reduced IL-10 secretion) and supplementation with IL-35 alone may need to be combined with other immunomodulatory strategies, such as low-dose IL-2 (129,146). As shown in Table VI (147,148), the regulatory pathways and mechanisms of action of IL-35 vary between T and B lymphocytes: In B lymphocytes, IL-35 primarily modulates STAT3 to promote Breg differentiation and inhibit antibody production, whereas in T lymphocytes, it modulates both STAT3 and STAT1 to inhibit Teffs and enhance Treg function. These findings suggest that IL-35 can improve the prognosis of MG models by regulating Bregs. However, the current research on IL-35 regulation by Bregs in MG is still at the basic association level and lacks mechanistic analysis and clinical translation support. In the future, larger prospective studies, in vitro functional experiments, and animal model validation are needed to clarify whether IL-35 can be used as a new target for MG therapy.

As shown in Table VII (149-153), Treg-induced immunosuppression involves multiple mechanisms, including CTLA4-mediated suppression of APCs and the production of immunosuppressive metabolites. In a study involving 39 patients with MG, the percentages of CD4+CD25+ Treg cells in the peripheral blood of patients with OMG and GMG were both lower than those of healthy individuals (P<0.05); however, the percentage of patients with OMG was not distinctly different from that of patients with GMG (P=0.475). Additionally, the percentages of CD3+CD4+CD25+Foxp3+ Treg cells in the OMG and GMG patient groups were lower than those in the healthy group (154). Another study included 13 children with serum AChR antibody-positive ocular-type MG and 18 age-matched controls. The percentages of Tregs among peripheral blood CD4(+) T cells in the active stage, remission stage, and control groups were 3.3±1.3, 4.8±1.7, and 5.0±0.6%, respectively. The Treg population was markedly lower in the active stage than in the remission stage and in the control group. Furthermore, the Treg percentage is markedly lower during the relapse of myasthenia symptoms (155). Tregs employ both direct and indirect pathways to suppress various immune cells, with indirect effects often involving one cell type affecting another, ultimately leading to immune cell suppression (156). The release of cytokines such as TGF-β and the generation of lytic enzymes such as granzymes often induce immune apoptosis (157). Additionally, Tregs directly inhibit target cells by releasing CD39/CD73, which lowers extracellular ATP levels by generating adenosine and AMP (158). Tregs suppress the ability of autoreactive B cells to produce harmful autoantibodies, a process that also disrupts the autoreactive B cells themselves (159). Moreover, Tregs utilize granzyme B and perforin to induce pore formation and lyse effector B cells, thereby reducing autoantibody production (160). Tregs also act specifically on monocytes, inhibiting their differentiation and cytokine production (161). When cocultured with Tregs, monocytes acquire characteristics of M2 macrophages, such as increased expression of CD206 and CD163, along with reduced responsiveness to proinflammatory stimuli. This is evidenced by decreased IL-6 production and the inhibition of NF-κB activation (162). By contrast, monocytes cocultured with Tregs secreted 2.3 times more IL-17 (as measured by ELISA, P<0.01), but this difference was not statistically significant (163). This effect may be related to the high expression of OX40L on expanded Tregs, which activates a proinflammatory monocyte phenotype via the NF-κB pathway, as validated by scRNA-seq (163). The mechanisms of Treg-mediated immunosuppression are summarized in Fig. 7.

Several studies have reported that T1DM, MG, and similar autoimmune diseases are associated with Treg deficiency (Table VIII) (84,164-167). IL-35 promotes Treg proliferation and function, such as through the inhibition of Th1/Th17 responses, by activating the STAT1/STAT3 signaling pathway in Tregs (168). In the EAMG model, exogenous IL-35 restores the suppressive function of Tregs and reduces the levels of AChR autoantibodies (169). IL-35 also converts conventional T cells into novel iTR35 cells, which, although independent of Foxp3, possess potent immunosuppressive functions (170). In MG, iTR35 cells may compensate for the functional impairment of conventional Tregs. In AChR-induced EAMG rats, recombinant IL-35 injection markedly alleviates clinical symptoms, such as reducing myasthenia scores and decreasing immune complex deposition at the NMJ (171). IL-35 suppresses the differentiation of pathogenic B cells and the secretion of autoantibodies through Tregs while also downregulating proinflammatory cytokines such as IFN-γ (172). Compared with conventional Tregs, IL-35-pretreated Tregs (IL-35+ Tregs) show enhanced therapeutic efficacy (50). IL-35 may augment immunosuppression through the Treg-Breg axis, with Treg-derived IL-35 promoting the expansion of Bregs, thus forming a negative feedback loop (50). In the serum of patients with GMG, IL-35 expression in Tregs inversely correlates with disease severity (such as MGFA classification). Treg functional deficiency is more pronounced in patients with MG with thymomas, potentially because of insufficient IL-35 secretion (173). Combining IL-35 with low-dose IL-2 (which can expand Tregs) may help restore immune balance in patients with MG (174). Adenoviral vectors, such as AAV-IL-35, have demonstrated long-term efficacy in animal models, although safety concerns must be addressed for clinical application (175). IL-35 nanoparticles targeting Tregs may improve local efficacy, particularly by targeting the thymus or lymph nodes (176). CRISPR gene editing can also increase the IL-35 secretion capacity of Tregs (177). Thus, by regulating the function and expansion of Tregs, IL-35 has significant immunosuppressive and therapeutic potential in MG models. At present, research on the regulatory effects of IL-35 on Tregs in MG is still in its early stages and is limited mainly by the use of animal models, a lack of human data, incomplete elucidation of the mechanism of action, and a lack of specific therapies targeting this pathway for clinical application. Although new therapies such as CAR-T cells have begun to target B cells or rebuild immune tolerance, research on direct intervention in MG via the IL-35/Treg axis is still at the basic level.

Among patients with MG, the AChR antibody-positive (AChR+) subtype represents the majority, accounting for 80-85% of cases (178). In an EAMG model induced by AChR, exogenous IL-35 markedly reduced serum anti-AChR antibody titers and decreased complement deposition at the NMJ (144). This effect likely results from the ability of IL-35 to inhibit B-cell differentiation into plasma cells, thereby reducing the production of autoantibodies. Several studies have shown that serum IL-35 levels are markedly greater in AChR+ patients with MG than in HCs and are associated with disease severity (such as MGFA classification) (116,179,180). By contrast, the anti-muscle-specific kinase antibody-positive (MuSK+) subtype accounts for 5-10% of MG cases (178), with an immunopathological mechanism distinct from that of AChR+ MG. Although IL-35 in MuSK+ MG has received increasing attention, research in this area remains limited. In the MuSK-induced EAMG model, the efficacy of IL-35 may be weaker than that in the AChR EAMG model, potentially due to the IgG4 nature of MuSK antibodies, which have lower complement activation capabilities. MuSK+ MG is more strongly associated with B-cell tolerance defects than with T-cell-driven inflammation. Th17 cells may play a lesser role in MuSK+ MG, indicating that the promotion of Tregs by IL-35 may not be as significant as that in AChR+ MG. Research has shown that IL-35 levels in MuSK+ patients with MG may not be markedly associated with disease severity (181,182), indicating that the mechanisms underlying the role of IL-35 in these subtypes may differ.

At present, according to Table IX (183,184), no classical subtypes or isoforms of IL-35 have been found; however, according to Table X (185-187), its functional diversity may stem from different cellular sources, receptor combinations, and signaling pathway activation patterns.