CD4⁺CD25⁺ regulatory T cell therapy in neurological autoimmune diseases

Development of CD4⁺CD25⁺Tregs

A population of thymus-derived suppressor T lymphocytes were discovered more than 50 years ago (Gershon & Kondo, 1971; Nishizuka & Sakakura, 1969). Subsequently, an increasing number of studies have been conducted on this cell population (Chen et al., 1994; Sakaguchi et al., 1985). Sakaguchi et al. (1995) first identified the specific surface expression of CD25 (IL-2 receptor [IL-2R] alpha-chain) molecules on CD4⁺ T cells in the peripheral blood of mice. Upon elimination of CD4⁺CD25⁺ cells, the general immunosuppression is relieved, thereby accelerating immune responses to non-self-antigens, and causing autoimmune responses to certain self-antigens. Decades of research have extensively explored the phenotypes and regulation of Tregs (Fig. 1).

Based on their developmental origin, Tregs can be further classified into thymus-derived Tregs (tTregs) and peripherally derived Tregs (pTregs) (Martin-Moreno, Tripathi & Chandraker, 2018; Robertson et al., 2019). Others divide Tregs into natural Tregs (nTregs) and induced Tregs (iTregs) based on the characteristic of natural occurrence (Edinger, 2009; Schmitt & Williams, 2013). However, others argue that there are three subgroups of Tregs: nTregs, pTregs, and iTregs (Göschl, Scheinecker & Bonelli, 2019; Zhang, Guo & Jia, 2021). nTregs and tTregs are fundamentally the same CD4⁺ T cells subpopulations, undergoing natural development and maturation within the thymus. Mature CD4⁺CD25⁺ Tregs can also be induced from naïve T cells by TGF-β and IL-2 stimulation; the resulting cells are termed iTregs or pTregs when generated in vitro or in vivo. Unlike nTregs, iTregs have been demonstrated to be unstable, and approaches to generate stable iTregs have been developed for clinical utility (Fig. 2) (Kanamori et al., 2016; Pacholczyk, Kraj & Ignatowicz, 2002).

Differentiation and functional expression of Tregs are regulated by a multitude of cytokines. T helper type 17 (Th17) cells originate from the same subset of CD4⁺ T cells. Initially, precursor Th (Th0) cells differentiate into intermediate cells. The subsequent differentiation trajectory primarily depends on the type of cytokines present in the microenvironment of the organism. Th17 cells and Tregs play opposing roles. The former hinders immune tolerance, whereas the latter induces it. A dynamic imbalance between them is pivotal for the onset of autoimmune disorders (Astier & Kofler, 2023; Lee, 2018; Yasuda, Takeuchi & Hirota, 2019).

IL-6 plays a significant role in modulating the equilibrium between Th17 cells and Tregs. It collaboratively induces the differentiation of naïve CD4⁺ T cells into Th17 cells with TGF-β, while suppressing the differentiation of TGF-β-induced Tregs (Kimura & Kishimoto, 2010).

Foxp3, presently the most discerning molecular marker of Tregs, participates in the differentiation and functional regulation of Tregs. Foxp3 is expressed in the thymic development of nTregs, or highly expressed in iTregs induced by TGF-β and retinoic acid (Ilnicka et al., 2019; Schmidt et al., 2016; Sun, Yi & O’Connell, 2010). In the absence of cytokines, Foxp3 can suppress the function of the retinoic acid receptor-related orphan receptor (ROR) γt and promote Treg differentiation. However, when naïve T cells receive signals from cytokines such as IL-6, the function of Foxp3 is inhibited, inducing the differentiation of Th17 cells (Deng et al., 2019; Zeng et al., 2023; Ziegler & Buckner, 2009). Therefore, the expression and regulatory levels of Foxp3 and RORγt determine the differentiation direction of Th17 cells/Tregs, resulting in the development of autoimmune diseases (Lee, 2018; Zeng et al., 2023). Additionally, in orchestrating the differentiation and function of Tregs, Foxp3 is influenced by various protein complexes with which it interacts. Prior to initiating Foxp3 transcription, Tregs have already been identified with homologous antigens and received T cell receptor (TCR) signals, triggered by TCR-induced transcription factors (NFAT, AP-1, and NF-κB), inducing the interaction between Foxp3 and AML1/Runx1 and NFAT, thereby exerting regulatory effects (Grover, Goel & Greene, 2021; Long, Luo & Zhu, 2022; Ono, 2020).

Signal transducer and activator of transcription 5 (STAT5) is another pivotal factor involved in Treg differentiation and activation. Mounting evidence indicates that IL-2 binds to the IL-2R on the surface of CD4⁺CD25⁺ Tregs, thereby activating STAT5. This, in turn, leads to the binding of STAT5 to the Foxp3 promoter, ultimately facilitating Treg cell generation (Jones, Read & Oestreich, 2020; Li & Park, 2020; Mao et al., 2019; Paradowska-Gorycka et al., 2020). Consequently, IL-2 regulates the differentiation of CD4⁺CD25⁺Foxp3⁺ Tregs via the STAT5 pathway, concurrently suppressing Th1 and Th17 cells as well as their secretion of IFN-γ and IL-17. This may be crucial for the onset and progression of autoimmune diseases (Mao et al., 2019; Paradowska-Gorycka et al., 2020; Xie et al., 2023).

Thymic development of nTregs is contingent upon the synergistic stimulation of TCR and CD28. CD28 is indispensable for the peripheral homeostasis, expansion, and survival of nTregs (Kitazawa et al., 2009). The maturation of iTregs necessitates the production of IL-2 and TGF-β, rather than the cooperative stimulation with CD28 (Cassis, Aiello & Noris, 2005). Additionally, IL-2, in conjunction with TGF-β, has the capacity to jointly induce the conversion of initial CD4⁺CD25⁻T cells into CD4⁺CD25⁺ T cells, thereby expressing Foxp3 (Zheng et al., 2007). Figure 2 illustrates the specific Treg differentiation process.

Migration and residency of CD4⁺CD25⁺Tregs

Upon maturation in the thymus, CD4⁺CD25⁺ Tregs enter systemic circulation or lymphatic tissues, with a subset further migrating to various non-lymphoid tissues, where they form transiently resident tissue Tregs in small numbers (Burton et al., 2024). In contrast to lymphoid tissue Tregs, these non-lymphoid tissue Tregs typically exhibit common molecular phenotypes, including CD69, CD103, programmed cell death protein-1 (PD-1), and so on, which facilitate their short-term residence in tissues and enable them to perform specific function (Burton et al., 2024; Shou et al., 2021; Evrard et al., 2023; Pasciuto et al., 2020).

Unlike Tregs in other tissues, brain Tregs undergo a distinct migration and residence process. Under physiological conditions, only a small subset of Tregs can cross the blood-cerebrospinal fluid barrier (not the intact blood–brain barrier) to enter the brain parenchyma (Korin et al., 2017; Hrastelj et al., 2021). The low IL-2 microenvironment within the CNS further limits their long-term residence, typically resulting in their renewal after approximately three weeks (Burton et al., 2024). During inflammation, Tregs are activated in lymphoid organs and are directed by chemokine C-C motif ligands (CCL) such as CCL2 and CCL5. These cells upregulate chemokine C-C motif receptors (CCR) like CCR2 and CCR5, which allow them to recognize self-antigens within the blood–brain barrier (Liston et al., 2024; Ben-Yehuda et al., 2021). Consequently, Tregs migrate through capillary endothelial cells, the basal lamina, and other barriers, entering the perivascular space. Once within this space, Tregs express high levels of specific TCRs, which interact with major histocompatibility complex II (MHC II) molecules on CNS target cells (Liston et al., 2024; Liston, Dooley & Yshii, 2022). This interaction drives their passage through the astrocyte limitans, allowing them to enter the brain parenchyma and target the corresponding inflammatory regions within the CNS.

Moreover, compared to other non-brain tissue Tregs, brain Tregs exhibit distinct gene expression and protein secretion patterns, which are associated with their unique role in neural repair (as discussed in the next section).

Multiple sclerosis

MS is an inflammatory demyelinating disease occurring within the CNS. There are concerns that CD4⁺CD25⁺ Tregs may participate in the pathogenic process of MS through the induction or modulation of immune responses within the CNS (Dendrou, Fugger & Friese, 2015; Bhise & Dhib-Jalbut, 2016). Previous studies have revealed a significant decrease in the effector function of CD4⁺CD25⁺ Tregs in peripheral blood of MS patients compared to normal controls (Tarighi et al., 2023; Haas et al., 2005). Following immunotherapies such as IFN-β or anti-CD52 monoclonal antibodies, there is a marked increase in both the quantity and functionality of these cells (Chiarini et al., 2020; Kiapour et al., 2022), indicating an immunological deficiency of Tregs in MS. The dysfunction and reduced quantity of Tregs in MS attenuate the inhibition against other pro-inflammatory immune cells (such as CD4⁺ effector T cells, CD8⁺ T cells, Th1, Th17 cells, B cells, etc.), and the neural repair function of myelin slacken off, which further leads to the progression of neuroinflammation and demyelination. In addition, Tregs expressed certain chemokines or their receptors (such as CCL17 or C-X-C motif chemokine receptor 3 (CXCR3)) are recruited into neuroinflammatory sites of brain, inhibiting inflammatory response (Yang et al., 2015; Moreno Ayala et al., 2023). However, due to the migratory receptor dysregulation of Tregs in MS patients, migration to the brain is restricted and Tregs cannot serves various purposes in alleviating MS pathology (Danikowski, Jayaraman & Prabhakar, 2017).

Myasthenia gravis

MG is an organ-specific autoimmune disease, primarily elicited by highly specific autoantibodies directed against skeletal muscle acetylcholine receptors (AChR). Currently, the thymus is believed to play a pivotal role in its pathogenesis (Thapa & Farber, 2019). Within the thymus, a substantial population of activated autoreactive CD4⁺ T cells exists, particularly AChR-reactive CD4⁺ Tregs. However, abnormalities in the thymus of MG patients may lead to a deficiency or impairment in Treg function, subsequently resulting in an immunodynamic imbalance (Niu et al., 2020; Dong, 2021). A prior study indicated that the mRNA and protein expression of Foxp3 in peripheral blood CD4⁺CD25⁺CD127^low Tregs of MG patients exhibit a decline compared with the healthy control group, resulting in impairment of their mediated inhibition on AChR-reactive T cells (Thiruppathi et al., 2012). This suggests that the functional deficiency of Tregs in MG patients may be correlated with a diminished expression of critical functional molecules such as Foxp3 (Kohler et al., 2017). Few studies have demonstrated that Tregs express dysfunctional migratory receptors in MG (Danikowski, Jayaraman & Prabhakar, 2017; Wei, Kryczek & Zou, 2006). Most research indicate that CD4⁺ follicular helper T cells expressing chemokine receptors CXCR5 involved in Tregs migration into germinal centers (Saito et al., 2005; Wen et al., 2016). However, the intensive mechanism of Tregs migration dysfunction is not yet fully clarified.

Guillain-Barré syndrome

GBS, characterized by demyelination of peripheral nerves and nerve roots along with vasculitic cellular infiltration, stands as an autoimmune peripheral neuropathy. Studies indicate a significant reduction in both the quantity and proportion of CD4⁺CD25⁺CD127⁻ Tregs in the peripheral blood of GBS patients compared to the normal control group, suggesting immunological dysfunction (Wang et al., 2015; Súkeníková et al., 2024). Similarly, in comparison to the healthy control group, GBS patients (especially during the progressive or relapsing phases) exhibit a marked decrease in both the quantity and suppressive function of Tregs, along with reduced mRNA expression of Foxp3 in Tregs (Chi, Wang & Wang, 2008). In addition, GBS patients who receive intravenous immunoglobulin therapy experience a significant decrease in the frequency of Th1 and Th17 cells, along with an increase in the quantity of Tregs and enhanced suppression of effector T cell function (Maddur et al., 2014). This suggests the involvement of Th17/Treg cells in the pathogenesis of GBS. The functional deficiency of Tregs eventuate in an obvious abatement in the immunosuppressive function of effector T cells or B cells, which attack the self-myelin antigen of PNS, thereby promoting peripheral nerve inflammation. At present, there is no consensus on whether Tregs has the ability to promote peripheral nerve myelin regeneration, and more research is needed to explore.

Neuromyelitis optica spectrum disorders

NMOSD is an autoimmune, inflammatory, demyelinating disorder of the CNS. The pathogenic mechanisms of NMOSD are primarily influenced by B cell-mediated humoral immune regulation, with cellular immunity also playing a role (Murtagh et al., 2022). Only a few studies have shown that the percentage of CD4⁺CD25⁺Foxp3⁺ Tregs in peripheral blood T cells of acute phase of NMOSD patients is significantly lower than healthy controls. At the same time, animal experiments have found that the consumption of Tregs significantly enhances the loss of astrocytes and demyelination in the mice (Ma et al., 2021). The implications of these researches may herald an important role of Tregs abnormality in NMOSD pathogenesis. Paradoxically, Tregs primarily exert their immunomodulatory effects by influencing B cell humoral immunity, rather than directly participating in this process (Brill, Lavon & Vaknin-Dembinsky, 2019; Cao et al., 2023). Further researches will be needed on the specific role of Tregs in NMOSD.

Expanding of CD4⁺CD25⁺Tregsin vivo

Tregs can be selectively expanded in vivo using different methods, allowing for the polyclonal expansion of Tregs to mediate non-specific immune suppression. This in vivo expansion method is generally more straightforward than the adoptive Treg therapy. However, these methodologies often lack durability, specificity, and targeting precision, making it challenging to control the potential toxic side effects (Balcerek et al., 2021).

Several methodologies have been proposed for in vivo induction of Tregs (Fig. 4A). Initially, in the treatment of autoimmune diseases with anti-CD3 antibodies, CD4⁺ Treg expansion is induced, concomitant with the selective depletion of T-effector cells (Notley et al., 2010). Another approach to induce Tregs involves in vivo inhibition of mammalian target of rapamycin (mTOR) function. Administration of rapamycin, an mTOR antagonist, selectively augments the population of endogenous Tregs (Bagherpour et al., 2018; Zhao et al., 2022), concurrently demonstrating heightened immunosuppressive capabilities (Chen et al., 2010). Furthermore, a variety of studies have revealed that those expressing IL-2Rαβγ exhibit a higher affinity and sensitivity to IL-2 compared to conventional T cells expressing IL-2Rβγ (Jones, Read & Oestreich, 2020), thus low doses of IL-2 can stimulate the proliferation of Tregs (Dong et al., 2021; Graßhoff et al., 2021; Harris, Berdugo & Tree, 2023). Consequently, low-dose IL-2 therapy has been utilized as a new practical method to induce Treg expansion in vivo (Harris, Berdugo & Tree, 2023; Qiao et al., 2023; Zhou, 2022). However, implementing this approach is challenging. Given the small size of IL-2, it is quickly excreted in the urine, resulting in a relatively short half-life and diminished therapeutic sustainability. Moreover, as CD25 expression is not exclusive to Tregs and also at lower levels in activated T cells, low-dose IL-2 could stimulate the proliferation and activation of effector T cells (Harris, Berdugo & Tree, 2023; Qiao et al., 2023).

Current approaches for inducing antigen-specific Tregs in vivo rely primarily on the introduction of exogenous antigens into the body to stimulate and induce initial T cell responses. However, the safety of this method requires significant scrutiny. In recent years, microbial vaccines have garnered attention as granulocyte-macrophage colony-stimulating factor (GM-CSF)-neuronal antigen T-cell source vaccines, relying on the recognition of low-affinity T-cell antigen receptors, inducing the expansion and activation of CD4⁺CD25^highFoxp3⁺ Tregs in myelin-specific TCR transgenic mice (Moorman et al., 2018). Furthermore, biomedical engineering for in vivo induction of Tregs has emerged as a prominent area of research. Rhodes et al. (2023) recently developed a novel method for inducing the expansion and activation of Tregs in vivo. They achieved this by designing new types of biodegradable bioengineered particle-biodegradable microparticles (MP) to induce immune tolerance. A schematic of this process is shown in Fig. 4B.

Adoptive transfer therapy of CD4⁺CD25⁺Tregs

To date, ongoing clinical/preclinical trials for Treg therapies have been conducted, with the majority of treatment protocols using intravenous infusion of ex vivo-expanded autologous Tregs.

Methods for the ex vivo expansion of polyclonal Tregs have now reached a relatively mature and stable stage. This protocol began with the isolation of CD4⁺CD25^highCD127^low Tregs from the peripheral blood, followed by stimulation with CD3/CD28 magnetic beads in the presence of IL-2, collectively activating and proliferating Tregs (Fig. 4C) (Balcerek et al., 2021). This approach has successfully yielded a sufficient number of well-functioning Tregs in graft-versus-host and autoimmune disease patients, with some trials showing signs of disease amelioration (Brunstein et al., 2011; Chwojnicki et al., 2021; Marek-Trzonkowska et al., 2016).

Although the adoptive transfer of polyclonal Tregs to induce immune tolerance has been proven safe and effective, the current research emphasis lies in the development of antigen-specific Tregs. Various studies suggest that, in comparison to polyclonal Tregs, antigen-specific Tregs hold distinct advantages over polyclonal Tregs in more efficiently suppressing effector T cells and promoting immune tolerance (Sagoo et al., 2011; Selck & Dominguez-Villar, 2021). In the context of cell therapy through adoptive transfer, antigen-specific Tregs require fewer cells to induce immune tolerance and simultaneously reduce the side effects associated with broad immunosuppression.

Several studies have demonstrated that co-culturing sorted Tregs with APCs or B cells in vitro can induce the expansion of antigen-specific CD4⁺CD25^highCD127^low Tregs (Putnam et al., 2013). Previous studies have shown that naïve CD4⁺ T cells can be induced and expanded from the peripheral blood of patients with MS to yield myelin basic protein 85–99 (MBP₈₅₋₉₉) specific CD4⁺CD25⁺ Tregs (Fig. 4D) (Xiang et al., 2016). Another method for generating antigen-specific Tregs in vitro involves the use of synthetic receptors, including engineered TCRs and chimeric antigen receptors (CARs), to alter the specificity of polyclonal Tregs. A variety of studies have revealed that the adoptive transfer of Tregs engineered with MBP-specific TCRs can effectively alleviate symptoms (Fig. 4D) (Kim et al., 2018). However, the clinical translation of Tregs manufactured using TCR engineering is, to some extent, constrained by MHC limitations. The development of CARs enables Tregs to recognize their antigens directly in a non-MHC-restricted fashion (Arjomandnejad, Kopec & Keeler, 2022; Sadelain, Brentjens & Rivière, 2013). A recent study demonstrated that myelin oligodendrocyte glycoprotein (MOG)-specific CAR-Tregs can, by binding to Foxp3 in effector T cells, enable MOG-specific Tregs to breach the blood–brain barrier and exert immunosuppressive effects within the CNS, thereby ameliorating disease symptoms (Fig. 4D) (Fransson et al., 2012).

Multiple sclerosis

Based on the pathogenesis and therapeutic principles of Tregs in MS, various studies have favored Treg-enhancing therapies. Current therapeutic strategies for MS encompass indirect Treg expansion and functional augmentation. A phase II randomized controlled trial (RCT) revealed that low-dose IL-2 therapy selectively activates Tregs, mitigates symptoms of relapsing-remitting MS (RRMS), and markedly decreases radiologically detectable gadolinium-enhancing lesions (Louapre et al., 2023). Pharmacologic modulation of Treg activity via small-molecule inhibitors or biologic agents represents a promising avenue for MS therapeutics. Nevertheless, such interventions carry substantial adverse effects, rendering the adoptive transfer therapy a critical focus for future MS research.

As is well known, the experimental autoimmune encephalomyelitis (EAE) models produce various phenotypes of human MS, universally accepted as the quintessential animal model for MS (Procaccini et al., 2015). Currently, the literature on adoptive transfer of polyclonal Tregs in the EAE model of MS is extensive. Kohm et al. (2002) reported that mouse CD4⁺CD25⁺ polyclonal Tregs reduced inflammatory cell infiltration in the spinal cord of EAE mice and inhibited the progression of EAE. Subsequent studies have confirmed the efficacy of polyclonal Tregs in improving EAE symptoms (McGeachy, Stephens & Anderton, 2005; Zhang et al., 2004). However, only a few clinical trials have been conducted on polyclonal Tregs in patients with MS. Only one Phase I clinical trial indicated that intravenous or intrathecal injections of ex vivo-expanded polyclonal Tregs led to symptom improvement, lesion reduction, and no significant adverse reactions in relapsing-remitting patients with MS (Chwojnicki et al., 2021).

Given the limitations of polyclonal Tregs, the preparation of antigen-specific Tregs and autologous transplantation for MS treatment have become current research hotspots. Several recent studies have shown that antigen-specific Tregs induced by intraventricular inoculation with microbe-based vaccines or engineered carrier particles can significantly improve EAE symptoms in mice. Moreover, histopathological examination showed a marked reduction in inflammatory infiltration compared with that in the control group (Moorman et al., 2018; Rhodes et al., 2023). To date, limited data are available regarding the cultivation of antigen-specific Tregs through in vitro induction (co-culture with APCs and specific peptide segments, TCR, and CAR engineering) for the treatment of EAE. Previous studies have indicated that, compared to polyclonal Tregs, ex vivo-induced mouse antigen-specific Tregs by MBP demonstrated significant efficacy in preventing symptom recurrence of EAE (Stephens, Malpass & Anderton, 2009). Furthermore, various studies have shown that functional and stable MBP antigen-specific Tregs can be cultured ex vivo from the peripheral blood of mice or patients with MS using TCR and CAR-T engineering techniques. When intravenously administered to EAE mice, these cells suppress the autoimmune pathology of EAE and significantly improve neurological function scores of EAE mice (DePaula Pohl et al., 2020; Fransson et al., 2012; Kim et al., 2018). However, no clinical trials of antigen-specific Tregs have been reported for the treatment of MS. Moreover, these animal experiments lack effective humanized models and produce less compelling experimental results, thus making it challenging to conduct further clinical trials in humans. Animal and clinical trials on adoptive Treg transfer therapy for MS are presented in Table 1.

Myasthenia gravis

Presently, the treatment options for MG primarily include the use of immunomodulators to indirectly enhance the biological activity of Tregs or regulate the dynamic balance between Th17 cells and Tregs. Administering of GM-CSF can augment the inhibitory function of Tregs, elevate Foxp3 expression, thereby exerting a negative regulatory effect (Sheng et al., 2006; Thiruppathi et al., 2012). In a rat model of MG, experimental autoimmune myasthenia gravis (EAMG), crosstalk occurs between the JAK2/STAT3 and AKT/mTOR pathways. JAK2 inhibitors can regulate the Th17 cells/Treg balance by inhibiting both signaling pathways, consequently contributing to the amelioration of EAMG symptoms (Lu et al., 2023). Furthermore, IFN-γ possesses the capacity to promote an increased proportion of CD4⁺CD25⁺ Tregs, enhance Foxp3 expression (Huang, Wang & Chi, 2015). Additionally, MG-related animal experiments have indicated that currently used immunosuppressive drugs, such as fingolimod (Luo et al., 2020) and certain traditional Chinese medicines (i.e., astilbin and artesunate) (Meng et al., 2018; Meng et al., 2016), primarily regulate the balance between Th17 cells and Treg for therapeutic efficacy, providing new potential therapeutic targets for MG immunotherapy.

Regarding the adoptive transfer therapy of Tregs, numerous animal experiments have consistently confirmed their outstanding therapeutic potential (Aricha et al., 2008; Aricha et al., 2016; Souroujon et al., 2012). Intraperitoneal injection of ex vivo-induced and cultured CD4⁺CD25⁺ Tregs from healthy rats into the EAMG significantly ameliorated the clinical symptoms, reduced acetylcholine receptor (AChR) antibody titers, and suppressed the quantity and functionality of Th1 and Th2 cells. Moreover, an 8-week follow-up revealed a markedly higher survival rate than that of controls (Souroujon et al., 2012). Intravenous injection of ex vivo-induced and expanded autologous polyclonal CD4⁺CD25⁺Foxp3⁺ Tregs into EAMG rats showed inhibitory effects on Teff cells proliferation and improved myasthenic symptoms. In parallel with this finding, patients with MG showed decreased AChR-specific antibody levels in the peripheral blood (Aricha et al., 2016). Since numerical animal experiments were initially employed to investigate polyclonal Tregs, the next step should be taken to determine the efficacy and safety of antigen-specific Treg therapies for EAMG. This will shed light on the fundamental basis for conducting clinical trials related to Treg therapy for MG. Preclinical studies on MG are presented in Table 1.

Guillain-Barré syndrome

To date, the modulation of Treg bioactivity in patients with GBS is primarily achieved through immunomodulators or pharmaceutical agents. In vitro, through the activation of anti-CD3 and anti-CD28 antibodies, IFN-γ induced CD4⁺CD25⁻ T cells from patients with GBS to generate CD4⁺CD25⁺ Tregs with heightened functional activity (Huang et al., 2009). Experimental autoimmune neuritis (EAN) is the most effective animal model of GBS. Some studies have indicated that atorvastatin, dexamethasone, and triptolide augment the quantity and function of CD4⁺CD25⁺Foxp3⁺ Tregs, thereby ameliorating EAN symptoms (Fagone et al., 2018; Shao, Fan & Tang, 2023; Xu et al., 2014). In several studies on adoptive transfer therapy using Tregs for animal experiments of GBS, ex vivo-induced and expanded rat polyclonal CD4⁺CD25⁺ Tregs were intravenously infused into the EAN model, leading to a significant reduction in sciatic nerve inflammatory cell infiltration and marked alleviation of myelin sheath and axonal damage (Wang, Cui & Qian, 2018). Building upon this research, Tran et al. (2020) reported that induced and cultured activated rat antigen-specific Tregs from the peripheral nerve myelin sheath and recombinant IL-2 were injected into the EAN, and demonstrated a notable reduction in clinical paralysis symptoms and degree of weight loss, along with accelerated disease recovery. Various animal experiments have provided a robust foundation for subsequent clinical trials (Table 1).

Neuromyelitis optica spectrum disorders

Currently, studies on Tregs therapy for NMOSD are scarce. In an animal experiment, adoptive transfer of Tregs attenuated brain damage in a mouse NMOSD model; reduced macrophage, neutrophil, and T-cell infiltration; and suppressed inflammatory responses by regulating the functional status of macrophages/microglia and reducing chemokines and proinflammatory factors (Table 1) (Ma et al., 2021). The clinical potential of Treg therapy for NMOSD is worth investigating.