From pathogenesis to therapy: the emerging role of regulatory T cells in amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons in the brain and spinal cord, with a pathogenesis that remains incompletely understood. Increasing evidence in recent years has highlighted the pivotal role of neuroinflammation in ALS, in which regulatory T cells (Tregs) emerge as key modulators of the neuroimmune response. This review systematically summarizes recent advances in understanding Treg biology in ALS, including their dynamic alterations across different disease stages and their potential immunoregulatory mechanisms, while also highlighting ongoing clinical trials and emerging cellular therapeutic strategies targeting Tregs. Current evidence suggests that Tregs not only participate in the immunopathology of ALS but also represent a promising target for therapeutic intervention. Nevertheless, there are still significant challenges, including incomplete mechanistic insights, limited clinical validation and obstacles to the implementation of Treg-based therapies. Overall, Treg research in ALS provides valuable directions for elucidating disease mechanisms and developing novel immune-based interventions.

Graphical Abstract

Introduction

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease characterized by the degeneration of motor neurons in the spinal cord, brainstem, and motor cortex, ultimately leading to muscle atrophy and paralysis [1]. ALS is broadly classified into two forms: sporadic and familial. Sporadic ALS accounts for most cases, typically presenting with insidious onset and an unclear etiology. In contrast, familial ALS (fALS), usually have family history, caused by mutations in a series of genes. Several mutations have been identified in genes associated with ALS, with mutations in four causative genes, C9orf72, SOD1, TARDBP, and FUS, accounting for approximately 70% of fALS cases [2, 3]. Drugs currently approved by the U.S. Food and Drug Administration (FDA) for the treatment of ALS include Riluzole, Edaravone, and Tofersen [4]. The primary mechanism of action of Riluzole is that it affects neuronal survival by increasing glutamate reuptake, inhibiting glutamatergic receptors, and blocking sodium channels, which collectively reduce excitotoxicity and thus reduce neuronal death [5]. But Edaravone is to protect motor neurons from damage induced by oxidative stress by scavenging free radicals, thereby delaying disease progression [6]. Tofersen, a SOD1 antisense oligodeoxynucleotide therapy for mutant SOD fALS, reduces the synthesis of SOD1 proteins by degrading SOD1 mRNA [7]. However, these drugs only delay disease progression rather than providing a cure, a limitation that may stem from our incomplete understanding of the pathogenic mechanisms underlying ALS. Till now, multiple hypotheses have been proposed to explain the pathogenesis of ALS, encompassing mitochondrial dysfunction, oxidative stress, defects in DNA repair, dysregulation of RNA metabolism, disturbances in protein homeostasis, neuroimmune inflammation, and impairment of oligodendrocyte function [8]. Among these, neuroimmune inflammation has remained as a key driver of ALS onset and progression [9, 10]. The immune system can be broadly divided into humoral and cellular arms, with the former primarily mediated by B cells and their secreted antibodies, and the latter centered on T cells.Based on surface marker classification, T cells can be divided into two major subsets: CD4⁺ and CD8⁺. CD8⁺ T cells function primarily as cytotoxic lymphocytes that directly mediate target cell lysis and clearance. By contrast, CD4⁺ T cells represent a highly heterogeneous population, in which naïve CD4⁺ T cells differentiate into distinct functional subsets under the influence of antigen-presenting cell–derived signals and specific cytokine milieus. Among them, effector T cells (Teffs), such as Th1 and Th17 subsets, exert pro-inflammatory and neurotoxic effects that drive inflammatory responses, whereas regulatory T cells (Tregs) provide anti-inflammatory, immunosuppressive, and neuroprotective functions [11]. In recent years, an increasing number of studies have demonstrated the importance of Tregs in the regulation of neuroimmune inflammation associated with ALS, and alterations in their number and functional status may profoundly affect ALS progression [12, 13]. Hence, this review provides a systematic overview of recent advances in Treg-based approaches for ALS, with a focus on their potential mechanisms in modulating neuroinflammation, relevant clinical trials, and cell therapy strategies. We aim to offer new theoretical foundations and therapeutic perspectives for immune interventions in ALS, and to explore the potential for clinical translation through Treg-targeted modulation.

Overview of the basic biology of Tregs

Depending on their origins, Tregs can basically be divided into three categories: the first is natural regulatory T cells (nTregs), which are generated during negative selection in the thymus and subsequently migrate to peripheral tissues to exert their suppressive effects, with a phenotype of CD4⁺ CD25⁺, where CD25 is the interleukin-2 receptor alpha chain (IL-2Rα), indicating its interleukin-2 (IL-2) highly dependent [14]. The second subset comprises peripheral regulatory T cells (pTregs), which differentiate from naïve CD4⁺ T cells in the periphery upon antigen stimulation. This process typically depends on a specific cytokine milieu, including transforming growth factor-β (TGF-β), IL-2, and retinoic acid, which act synergistically on the T cell receptor (TCR) signaling pathway; The third category is induced regulatory T cells (iTregs), which are usually induced from conventional T cells under controlled experimental conditions and are widely used in basic and cell therapy research (Fig. 1A) [15]. Tregs can maintain their immunosuppressive phenotype and function only if the lineage-defining transcription factor Forkhead box protein P3 (FOXP3) is continuously expressed [16]. Stable expression of FOXP3 is subject to epigenetic regulation, and in particular relies on a highly conserved noncoding element within its locus, known as the Treg-specific demethylated region (TSDR). Located within the first intron of the FOXP3 gene and enriched in CpG sites, TSDR exhibits a distinct epigenetic profile between cell types. In functionally stable Tregs, this region is fully demethylated, enabling sustained FOXP3 expression and maintenance of the suppressive phenotype. By contrast, in conventional CD4⁺ T cells the TSDR remains completely methylated, precluding stable FOXP3 expression. Thus, the methylation status of the TSDR is regarded as a key molecular hallmark of Treg lineage stability and functional integrity [17, 18]. pTregs are also demethylated at this site, but to a slightly lesser extent than nTregs, and their lineage phenotype and FOXP3 expression remain relatively stable. In contrast, the level of demethylation of this locus is significantly lower in iTreg cells, resulting in a high degree of instability of FOXP3 expression in this cell subpopulation [19, 20]. Additionally, IL-2 plays a central role in the development and maintenance of Tregs. Tregs constitutively express high levels of IL-2Rα (CD25), which combines with IL-2Rβ (CD122) and the common γ chain (CD132) to form a high-affinity trimeric IL-2 receptor complex [21]. Upon IL-2 binding, Janus kinases JAK1 and JAK3 are activated, leading to the phosphorylation of signal transducer and activator of transcription 5 (STAT5). Phosphorylated STAT5 dimerizes and translocates into the nucleus, where it binds to the promoter and enhancer regions of the FOXP3 gene, thereby sustaining FOXP3 transcription and preserving the phenotypic stability and immunosuppressive function of Tregs [22].

Fig. 1.

Origins and Basic Biology of Treg cell. A Origin of Treg cell. nTregs develop in the thymus and express FOXP3. pTregs are induced in the periphery by IL-2 and TGF-β, while iTregs are induced in vitro under the same conditions, promoting the differentiation of conventional CD4 + T cells into Tregs; B Basic Biology of Treg cell. This figure illustrates the key surface markers and molecules associated with Tregs, including CD4, CD25, CTLA-4, TIGIT, LAG3, GITR, CD39, CD73, and TCR. These molecules play critical roles in the function and regulation of Tregs. The transcription factor FOXP3 is essential for the development and function of Tregs. CTLA-4: Cytotoxic T-lymphocyte antigen 4; FOXP3: Transcription factor Forkhead box protein P3; GITR: glucocorticoid-induced tumor necrosis factor receptor; IL-2: Interleukin-2; iTregs: Induced regulatory T cells; LAG-3: Lymphocyte activation gene 3; nTregs: Natural regulatory T cells; pTregs: Peripherally derived regulatory T cells; TCR: T cell receptor; TGF-β: Transforming growth factor-β; TIGIT: T cell immunoglobulin and ITIM domains; Tregs: Regulatory T cells. The figure was created by Biorender.com

Tregs express a variety of key molecules on their surface, including cytotoxic T-lymphocyte antigen 4 (CTLA-4), lymphocyte activation gene 3 (LAG-3), T cell Immunoglobulin and ITIM Domains (TIGIT), CD25, CD39 and CD73. These molecules play important roles in the interactions of Tregs with other immune cells and are key components of their function in mediating immunosuppression (Fig. 1B) [23]. The mechanisms underlying Tregs-mediated immunosuppression include the following: A) Suppressive Cytokine Release: Tregs can secrete anti-inflammatory cytokines, including interleukin − 4 (IL-4), interleukin-10 (IL-10), interleukin-35 (IL-35), and TGF-β, thus inhibiting the function of Teffs (Fig. 2A) [24]; B) Cytolysis Induction: Tregs can produce and release granzyme B and perforin, which undergoes a conformational change in the presence of Ca2 + and inserts into the membrane of target cells, which mediates the entry of granzyme B into the target cells and initiates the intracellular apoptotic pathway, thus inducing cytolysis and apoptosis of effector T cells, B cells and natural killer cells (NK cells) (Fig. 2B) [25]; C) Cell–Cell Contact Suppression: Tregs engage in direct physical interactions with dendritic cells (DCs) through the binding of CTLA-4 on their surface to the costimulatory molecules CD80/CD86 expressed by DCs. This interaction induces DCs to produce indoleamine 2,3-dioxygenase (IDO), a potent immunoregulatory enzyme that suppresses the function of T cells and NK cells. In addition, Tregs can bind to major histocompatibility complex class II (MHC-II) molecules on DCs, thereby inhibiting DC maturation and reducing the activation of Teffs (Fig. 2C) [26]; D) Target Cell Metabolism Blockade: Tregs highly express IL-2 receptors and consume a large amount of IL-2. Since IL-2 is also required for the proliferation and activation of Teffs, competitive binding of Tregs to IL-2 leads to the deprivation of IL-2, thus blocking Teffs from being stimulated to proliferate. In addition, CD39 and CD73, which hydrolyze ATP and adenosine diphosphate into adenosine, are also present on the surface of Treg, inducing inhibitory and antiproliferative effects on Teffs, and Tregs also transfer a large amount of cAMP to Teffs through gap junctions, interfering with their metabolism (Fig. 2D) [11, 27].

Fig. 2.

Suppressive mechanisms of Treg cell. Tregs suppress immune responses through: (A) suppressive cytokine release; (B) cytolysis induction ; (C) cell–cell contact suppression; (D) target cell metabolism blockade. The figure was created by Biorender.com

The role of Tregs in the pathogenesis of ALS

Tregs are essential immunosuppressive cells that play a pivotal role in maintaining immune homeostasis, preventing autoimmunity, and controlling excessive inflammatory responses [28]. In recent years, an increasing number of studies have revealed that Tregs play an important role in the pathogenesis of ALS. ALS is characterized by widespread neuroinflammation, notably involving the activation of astrocytes, microglia, and T cells, all of which actively contribute to neuronal degeneration and disease progression [29, 30]. Under normal physiological conditions, the central nervous system (CNS) is in a state of relative immunosuppression, and microglia maintain a resting state, mainly performing functions such as immune surveillance and removal of metabolic waste products. Upon inflammatory stimulation, microglia can polarize into distinct functional phenotypes: classically activated M1 microglia, which produce high levels of proinflammatory cytokines (e.g., IL-1β, IL-6, TNF-α), reactive oxygen species (ROS), and nitric oxide (NO) and exert neurotoxic effects; or alternatively activated M2 microglia, which secrete anti-inflammatory cytokines (e.g., IL-10, TGF-β) and promote tissue repair and neuroprotection [31]. However, in ALS, damaged motor neurons release pro-inflammatory signals that induce microglia to convert to the M1 phenotype, producing ROS, NO, and a variety of pro-inflammatory factors (e.g., IL-1β, IL-6, and TNF-α), thereby promoting motor neuron apoptosis [32]. Astrocytes also shift from a homeostatic state to an A1-type reactive phenotype, releasing neurotoxic molecules such as glutamate, prostaglandin E2 (PGE2), and leukotriene B4 (LTB4), which further exacerbate neuronal damage [33]. Tregs exert anti-inflammatory and neuroprotective effects in ALS through multiple mechanisms. In the early stages of the disease, based on animal models, Tregs numbers increase and secrete cytokines such as IL-4, IL-10, and TGF-β, promoting the polarization of microglia toward an anti-inflammatory M2-like phenotype [34], and inhibit inflammatory factor expression through signaling pathways such as Peroxisome proliferator-activated receptor gamma (PPAR-γ) and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [35]. In addition, Tregs indirectly inhibit the formation of type A1 astrocytes and directly reduce their pro-inflammatory activity via IL-10 and TGF-β [33]. Tregs were also found to interact with astrocytes and regulate their proliferation and activity by secreting the neurotrophic factor amphiregulin, a low-affinity epidermal growth factor receptor (EGFR) ligand [36]. Notably, the neuroprotective effects of Tregs on motor neurons in ALS are thought to occur primarily through the indirect suppression of toxic inflammatory mediators. Although direct interactions between Tregs and motor neurons have not been conclusively demonstrated, other neurodegenerative disease models (e.g., Parkinson’s disease) suggest that Tregs may directly bind to dopaminergic neurons through CD47-SIRPα signaling axis interactions, thereby attenuating neuroinflammation and neuronal loss [37].

However, research has found that as ALS progresses, the number and function of Tregs gradually decline, the ratio of peripheral Th1 to Th17 cells significantly increases, and the levels of pro-inflammatory factors such as IL-1β, IL-6, and IFN-γ rise, while anti-inflammatory factors such as IL-10 and TGF-β significantly decrease [38]. The “SWITCH model” hypothesis states that temporal factors play a key role in the dynamic evolution of the immune response in ALS. The hypothesis suggests that Tregs play an effective neuroprotective role in the early stages of ALS; however, as the disease progresses, Tregs are gradually replaced by pro-inflammatory CD4⁺ T cells that infiltrate the CNS in large numbers, resulting in a gradual shift from a protective immune state dominated by Tregs to a neurotoxic state dominated by inflammatory cells [39]. In other words, Tregs play a key regulatory role throughout the disease course and exhibit a biphasic trend: in the early stages, the number of Tregs rises, probably reflecting an anti-inflammatory response of the CNS, while in the later stages of the disease, the number of Tregs decreases markedly and the immunosuppressive function is impaired, leading to a further exacerbation of neuroinflammation [40]. Animal experiments also support this time-dependent dynamic evolution. The immunomodulatory effects of Tregs on Teffs exhibit stage-specific differences during ALS progression. Specifically, in mSOD1^G93A mice, in the slow-progression phase (100 days of age), Treg numbers increase significantly, accompanied by elevated expression of IL-4, IL-10, and TGF-β, which synergistically suppress Teff proliferation and IFN-γ production. In the rapid-progression phase (160 days of age), Teffs display enhanced proliferative and cytotoxic capacities, whereas Treg numbers decline and their inhibitory activity is diminished, indicating a shift from an early protective immune state to a pro-inflammatory, dysregulated state [41]. Similarly, it has been reported that in ALS patients with rapid disease progression, the number of Tregs is reduced, and the quantity of Tregs negatively correlates with the rate of disease progression [42]. Furthermore, previous studiesy haves shown that in mSOD1 mice, during the early stages of the disease, the number of Tregs (CD4⁺CD25⁺FOXP3⁺) and FOXP3 expression are elevated, accompanied by an increase in IL-4, IL-10, and M2 microglial markers (Ym1, CD206), forming a neuroprotective immune state. However, during the accelerated phase of the disease, the number of Tregs and FOXP3 expression decrease, IL-10 levels drop, and pro-inflammatory factors such as IL-1β, TNF-α, IL-6, and NOX2 increase, with microglia transitioning to the M1 phenotype. Since 100-day-old mSOD1 mice are in a stable disease phase with an increased number of functional Tregs, researchers isolated CD4 + T cells from these mice. These Tregs were passively transferred into mSOD1/RAG2-/- mice, which lack lymphocyte function, via tail vein injection, with repeated injections every 28 days. The study demonstrated that Treg transplantation significantly prolonged the disease stability phase and survival of recipient mice. Compared to the recipient mice that received CD4 + T cells from wild-type mice, the disease stability phase in mSOD1/RAG2-/- mice was extended by approximately two weeks. However, when CD4 + T cells isolated from 20-week-old mSOD1 mice in the accelerated phase were passively transferred into recipient mice, no extension of the disease stability phase was observed. This suggests that restoring Treg function during the stable phase is more effective, while Treg treatment in the accelerated phase yields poor results. Therefore, expanding Tregs in stable-phase patients may effectively delay disease progression, as the immune system still retains some regulatory capacity. In contrast, for patients in the rapid progression phase, due to significant immune system decline, restoring Treg function becomes more challenging. Thus, Treg therapy may need to be combined with other treatments or initiated during the early stages of the disease for optimal effect.

Beers et al. found that in patients with ALS, the number of Tregs is reduced, and this reduction is associated with an increase in disease progression rate and severity [30]. Additionally, the study revealed that Tregs’ ability to suppress Teff proliferation is impaired, particularly in ALS patients with rapid disease progression, where Tregs’ immune suppressive function is most significantly diminished. Moreover, Beers et al. performed epigenetic analysis and discovered that Tregs in ALS patients have higher methylation levels in the TSDR, which may contribute to Tregs dysfunction. In in vitro experiments, Tregs from ALS patients, after stimulation and expansion with IL-2 and rapamycin, regained the ability to suppress their corresponding Teffs. At a 1:1 ratio of Teffs to Tregs, the suppression level was comparable to that of control Tregs. This finding suggests that, although Tregs in ALS patients are dysfunctional in their natural state, their immune suppressive function can be significantly restored through in vitro expansion, offering a promising direction for Treg cell-based therapy in ALS treatment. Sheean et al. also reached similar conclusions, demonstrating that the total number of Tregs in the peripheral blood of ALS patients is significantly negatively correlated with the rate of disease progression [43]. Additionally, in the SOD1^G93A transgenic mouse, researchers began treatment at 60 days of age, expanding endogenous Tregs using a combination of IL-2/IL-2 monoclonal antibody complexes (IL-2c), which were administered intraperitoneally at a dose of 100µL daily to promote Treg expansion. The results showed that IL-2c treatment significantly delayed disease progression and notably extended the survival of the mice (IL-2c group: 160.6 ± 10.8 days, control group: 144.9 ± 10.6 days, p = 0.003). Furthermore, IL-2c treatment led to significant T cell infiltration in the spinal cord ventral horn of SOD1^G93A mice, indicating that the increase in circulating Tregs induced by IL-2c treatment is closely associated with T cell recruitment to the spinal cord. Further analysis revealed that Treg expansion was closely correlated with the maintenance of motor neuron soma size, inhibition of astrocyte and microglial activation, and upregulation of neurotrophic factor gene expression in the spinal cord and peripheral nerves, providing strong evidence for the neuroprotective role of Tregs in ALS patients. Yazdani et al. conducted a prognostic study in 89 newly diagnosed patients with ALS, analyzing peripheral blood and cerebrospinal fluid samples. They found that a high frequency of FOXP3⁻ effector T cells in both compartments was associated with poorer survival, whereas a high frequency of activated Tregs in blood, as well as a high ratio of activated to resting Tregs, correlated with improved survival [44]. Furthermore, recent single-cell RNA sequencing integrated with proteomics revealed that, in rapidly progressive ALS, Treg stability was reduced, accompanied by marked increases in Th17/Treg, Th1/Treg, and memory CD4/Treg ratios, along with Treg-to-Th17 conversion and a shift of CD8⁺ T cells from a naïve to a cytotoxic phenotype. The Th17/Treg ratio positively correlated with the rate of ALS Functional Rating Scale–Revised (ALSFRS-R) decline, a standardized measure of disease stage and functional deterioration, further supporting a close link between Treg dysfunction and rapid disease progression [45]. In summary, Tregs play a key immunoregulatory role in the pathogenesis and progression of ALS, and their reduced number and dysfunction are closely associated with disease progression and are somewhat reversible. This provides a theoretical basis and potential direction for Treg-targeted immunotherapy.

Clinical trials

Accumulating evidence indicates that Tregs play a pivotal immunomodulatory role in the pathogenesis of ALS and have emerged as a promising therapeutic target. In recent years, multiple clinical trials aimed at enhancing Treg number and function have been initiated, as researchers actively explore Treg-centered cell therapy strategies to assess their potential efficacy and feasibility in ALS intervention (Table 1).

Table 1.

Clinical trials targeting regulatory T cells in amyotrophic lateral sclerosis (ALS)

Trial Name / Registration Number	Drug Name	Sample Size	Phase	Status	Intervention Method	Primary Endpoint	Reference
NCT05695521	CK0803 (Treg cells)	6(1a)/60(1b)	Ia/Ib	Completed (Ia) / Active, not recruiting (1b)	IV injection	Dose-limiting toxicity	Shneider et al. 2025 [13]
NCT06671236	NP001 (Treg cells)	Recruiting	I	Recruiting	IT injection	Safety and tolerability	None
NCT03241784	Tregs + IL-2	3	I	Completed	IV Tregs + SC IL-2	Safety and tolerability	Thonhoff et al. 2018 [46]
NCT04055623	Tregs + IL-2	7	II	Completed	IV Tregs + SC IL-2	Change in Treg suppressive function from screening (baseline value) to week 24	Thonhoff et al. 2022 [47]
NCT06307301	IL-2 + Abatacept (CTLA4-Ig)	6	I	Completed	SC IL-2 + SC CTLA4-Ig	Safety and tolerability	Thonhoff et al. 2024 [48]
IMODALS/NCT02059759	IL-2 + Riluzole	36	IIa	Completed	Oral Riluzole + SC IL-2	Change from baseline in Treg percentage of CD4 + T cells (%Tregs) following a first cycle	Camu et al. 2020 [49]
MIROCALS/NCT03039673	IL-2 + Riluzole	220	II b	Completed	Oral Riluzole + SC IL-2	Survival at 640 days (21 months)	Bensimon et al. 2025 [50]
RAP-ALS/NCT03359538	Ropinirole + Riluzole	63	II	Completed	Oral Riluzole + Oral Ropinirole	Whether Rapamycin administration increases Tregs number in treated patients compared with control arm	Mandrioli et al. 2018 [51]
NCT02525471	RNS60	16	1	Completed	IV + inhalation	Safety and tolerability	Paganoni et al. 2019 [52]
NCT03456882	RNS60 + Riluzole	147	II	Completed	IV + inhalation RNS60, Oral Riluzole	Effects of RNS60 treatment on selected biomarkers of inflammation and neurodegeneration in peripheral blood	Beghi et al. 2023 [53]
FETR-ALS/NCT03766321	FMT + Riluzole	42	Not reported	Completed	FMT infusion + Oral Riluzol	Change in Treg number between FMT-treated patients and control arm from baseline to month 6	Mandrioli et al. 2019 [54]
TEALS /ACTRN12618000534280	Dimethyl fumarate	107	II	Completed	Oral	Change in Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS‐R) at week	Vucic et al. 2021 [55]
VITALS/ NCT02756104	Vitamin D	97	Not reported	Completed	Oral	T cells phenotypes	None

Treg cell transplantation

Treg cell therapy is based on the principle that an effective dose of Treg cells is injected into the patient by isolating, expanding, and infusing back Treg cells in vitro [56]. Thonhoff et al. conducted the first open-label, uncontrolled Phase I clinical trial in 2018, enrolling three ALS patients. The aim was to assess the safety of autologous amplified Tregs in combination with low-dose IL-2 therapy. In the study, Tregs were isolated from peripheral blood and expanded in vitro, then infused intravenously with low-dose IL-2 injected subcutaneously. The results showed that Treg infusions were safe and well tolerated, regardless of the stage of the disease. In addition, increased immunosuppression of Tregs was strongly associated with slower disease progression in ALS, and Spearman’s correlation analysis showed that increased Treg suppression was positively correlated with a slower rate of decline in Appel ALS scores in each patient. However, the generalizability of the findings is limited by the very small sample size [46], https://clinicaltrials.gov/ct2/show/NCT03241784. Building on this, Beers et al. conducted an in-depth analysis of longitudinal serum samples from patients in the trial in 2022 to explore the effects of Treg treatment on peripheral oxidative stress (e.g., ox-LDL, LOX-1) and acute phase protein (sCD14, LBP, CRP) levels. The results showed that Treg infusion significantly suppressed the elevation of the above indexes, and its dynamic changes were highly consistent with the changes in the patients’ clinical status, suggesting that Treg may delay the progression of ALS by mitigating systemic inflammation and oxidative stress [57]. Thonhoff et al. further conducted a randomized, double-blind, placebo-controlled phase 2a clinical trial with an open-label extension in 2022 to systematically assess the safety, biological activity, and its potential impact on disease progression of Treg/IL-2 combination therapy. The study enrolled nine patients with ALS, seven of whom were randomly assigned to the treatment or placebo group, six of whom entered the open-label extension period, and two others who participated directly in the open-label phase. The primary outcome metric was the change in Treg inhibitory function from screening (baseline value) to week 24 during the Randomized controlled trial (RCT). Secondary outcome indicators were change in Treg count from screening to week 24, safety, and tolerability. The results showed that the regimen was well tolerated overall, with no serious adverse events; immunosuppressive function of Tregs was significantly enhanced in patients treated with Treg/IL-2, suggesting that the treatment has a favorable safety profile, tolerability, and potential biological activity in patients with ALS of more than one year’s duration of disease [47], https://clinicaltrials.gov/ct2/show/NCT04055623. Recently, Shneider et al. reported a study in which six patients with ALS received a fixed dose (100 × 10^6 cells) of CK0803, an off-the-shelf, cryopreserved, umbilical cord blood–derived, non–HLA-matched allogeneic Treg product. The treatment regimen consisted of one intravenous infusion per week for 4 consecutive weeks, followed by one infusion per month for a total of 6 infusions. No lymphocyte clearance, immunosuppressants, or IL-2 were administered throughout the course of treatment, and the results of the study showed a significant slowing of disease progression in patients receiving this cell therapy. Before treatment, the patients’ ALSFRS-R scores declined by an average of 1.66 points per month; during the treatment period, the rate of decline slowed significantly to 0.41 points per month; and at the end of the treatment period, the rate of decline increased slightly to 0.60 points per month, suggesting that the effects of the treatment were somewhat sustained. It is worth noting that the clinical improvement was accompanied by a decrease in plasma neurofilament protein levels, which is an important biomarker of neuronal injury. Also no acute safety issues were observed throughout the treatment [13]. In addition, autologous polyclonal Tregs injection (NP001) has received clearance from FDA for an investigational new drug application (IND 031401.0) and has been granted orphan drug designation for the treatment of ALS. NP001 is the world’s first Treg cell therapy delivered via intrathecal injection, with cells derived from highly purified Treg enriched in the patient’s own peripheral blood, expanded to a sufficient volume in vitro, and then infused back into the patient. By injecting regulatory T cells directly into the central nervous system, the therapy is expected to more effectively remodel the local immune microenvironment and promote tissue repair, thereby slowing disease progression. An open-label, multicenter phase I clinical study is underway to systematically evaluate the safety and preliminary efficacy of NP001 in patients with the neurodegenerative disease ALS. In China, the first clinical administration of the product has been completed at the First Affiliated Hospital of Zhengzhou University, with a total of three ALS patients entering the clinical phase. Preliminary results show that the first patient who completed the full course of treatment stabilized after the infusion, with a slower rate of decline in ALSFRS-R score and improved grip strength, suggesting that the therapy has good potential for initial clinical application (https://clinicaltrials.gov/ct2/show/NCT06671236). In summary, Treg-based therapy shows considerable promise for the treatment of ALS. Research strategies have evolved from the initial use of autologous polyclonal Tregs combined with IL-2 to the development of allogeneic “off-the-shelf” cell products, diverse delivery routes such as intravenous and intrathecal administration, and multiple cell sources including peripheral and umbilical cord blood. Together, these advances are driving the continued development of Treg cell therapy in ALS.

IL-2

IL-2 plays a crucial role in the generation, survival, stability, and maintenance of function of Tregs [58]. Tregs consistently express the high-affinity IL-2 receptor complex (consisting of three subunits, IL-2Rα, IL-2Rβ, and IL-2Rγc), whereas Teffs express this receptor only upon TCR activation. In the resting state, Teffs express only the medium-affinity IL-2 receptor (composed of IL-2RB and IL-2RG) [59]. As a result, Tregs are more sensitive to IL-2 and can be preferentially activated at very low doses, whereas Teffs require significantly higher concentrations of IL-2 to be activated. Specifically, the dose of IL-2 required to activate Teffs is approximately 5000 times higher than that required to activate Tregs [60]. Given the differences in IL-2 receptor affinity between the two cell types and their downstream signaling pathways, a low-dose IL-2 (ld-IL-2) strategy has been widely used in animal models and clinical studies to selectively amplify Tregs and avoid excessive activation of Teffs [61]. Recent studies have further revealed the potential metabolic effects of ld-IL-2 beyond its immunomodulatory role. Using a targeted metabolomics approach, researchers analyzed the metabolite changes in plasma of ALS patients before and after ld-IL-2 treatment and found that IL-2 significantly modulates multiple metabolic pathways, some of which were closely associated with increased Treg ratios and decreased levels of the inflammatory factor IL-18 [62], further supporting its potential role in maintaining immune homeostasis.

Camu et al. conducted a phase 2a randomized, double-blind, placebo-controlled trial called IMODALS, which enrolled 36 patients, to evaluate the safety and pharmacodynamics of ld-IL-2 in ALS. The study aimed to verify whether ld-IL-2 improves immune and inflammatory markers in ALS patients and is well tolerated. In the trial, patients received two different doses of IL-2 (1 MIU and 2 MIU) in combination with Riluzole for three consecutive cycles. The results showed that both doses of IL-2 were well tolerated in patients with ALS, and no further safety concerns were observed at the end of treatment. In addition, ld-IL-2 was able to selectively expand functional Treg cells, improve the immune microenvironment, and inhibit inflammation in ALS patients, demonstrating a favorable safety profile and biological activity [49], https://clinicaltrials.gov/ct2/show/NCT02059759. The MIROCALS trial is the first large-scale, long-term, randomized, double-blind, placebo-controlled clinical study in patients with ALS, enrolling 220 participants, to evaluate whether ld-IL-2 (2 million international units) improves survival and functional status. The study was designed as a 1:1 randomized group, with all subjects receiving ld-IL-2 or placebo after 12–18 weeks of riluzole introduction therapy. IL-2 was administered by subcutaneous injection for 5 consecutive days every 28 days for a treatment cycle lasting 18 months. The primary endpoint was survival at day 640 (approximately 21 months) after treatment, and secondary endpoints included safety assessments, changes in ALSFRS-R scores, and changes in the dynamics of a range of biomarkers, such as the number of peripheral blood Tregs, cerebrospinal fluid phosphorylated neurofilament heavy chain (CSF-pNFH), and levels of C-C motif chemokine ligand 2 (CCL2) in plasma and CSF. CSF CCL2 and CSF-pNFH were used as markers for neuronal damage and have been reported as prognostic factors for ALS survival. The results of the study showed a 19% reduction in the risk of death in the ld-IL-2 group before adjustment for baseline prognostic covariates, but did not reach statistical significance. In contrast, after correction for all prognostic covariates measured at the time of randomization, the risk of death was significantly reduced in the ld-IL-2 treatment group (hazard ratio 0.32, 95% CI: 0.14–0.73, p = 0.007). In addition, ld-IL-2 had a favorable safety profile, consistently and significantly increasing the number of peripheral blood Tregs throughout the treatment period and decreasing plasma levels of CCL2, further supporting its potential therapeutic value in modulating neuroinflammation. Stratification based on CSF-pNFH levels measured at randomization revealed that in 70% of patients with low CSF-pNFH levels (750–3700 pg/mL), ld-IL-2 treatment significantly reduced the risk of death by 48% (hazard ratio 0.52, p = 0.016). However, in 21% of patients with high CSF-pNFH levels (> 3700 pg/mL), no significant difference was observed (hazard ratio 1.37, p = 0.38). Patients with low CSF-pNFH levels appeared to have a more robust immune response and responded better to ld-IL-2 treatment. In contrast, patients with high CSF-pNFH levels may be in a more aggressive phase of disease progression, with immune system changes potentially leading to a reduced response to ld-IL-2. Therefore, in future ALS treatments targeting IL-2, CSF-pNFH could serve as an important biomarker for patient stratification, enabling the identification of those most likely to benefit, and facilitating personalized treatment for ALS [50], https://clinicaltrials.gov/ct2/show/NCT03039673. Abatacept is an FDA-approved drug often used as a single agent or in combination with other anti-inflammatory drugs for the treatment of autoimmune diseases. Recently, an open-label study evaluated the safety of IL-2 in combination with Abatacept (CTLA4-Ig) in ALS and its impact on immune and oxidative stress-related markers. A total of 4 ALS patients were enrolled in the study and received 5 consecutive days of ld-IL-2 (1 × 10⁶ IU/dose/day) subcutaneously every 2 weeks and simultaneous subcutaneous injections of CTLA4-Ig (125 mg/mL) on day 1 of each cycle. During the study period, disease progression and treatment-associated adverse events (TEAEs) were monitored by the ALSFRS-R, and phenotypic changes in T cell populations and serum levels of oxidative stress markers were dynamically assessed. The results showed that the combination regimen was safe and well tolerated, with an increase in the number and immunosuppressive function of Tregs, a rise in their peripheral blood ratio during treatment, and a return to baseline levels 6 weeks after the end of treatment, accompanied by a decrease in the levels of biomarkers related to oxidative stress, neuroinflammation, and neuronal degeneration [48], https://clinicaltrials.gov/ct2/show/NCT06307301. Overall, ld-IL-2 has a favorable safety profile and immunomodulatory potential in ALS patients, selectively amplifying Tregs, improving the inflammatory microenvironment, and showing a trend toward delaying disease progression in some studies.

Rapamycin

Rapamycin is a highly effective inhibitor of mechanistic target of rapamycin (mTOR), which has been widely used in immunosuppressive therapy and organ transplantation. mTOR signaling pathway is an important regulatory node in the regulation of the metabolism and fate of T cells, which mainly consists of two complexes: mTORC1 and mTORC2. The activation of mTORC1 promotes the differentiation and function of Teffs, such as Th1 and Th17, while inhibiting Treg generation. mTORC1 is highly sensitive to rapamycin. After entering cells, rapamycin can bind to FKBP12 and inhibit the activity of mTORC1, which in turn promotes the expression of FOXP3, induces the differentiation of Tregs and enhances their immunosuppressive function [63, 64]. Therefore, targeting the mTOR pathway has emerged as a potential strategy to regulate the function of Tregs. In recent years, several studies have begun to explore the immunomodulatory effects of rapamycin in ALS, especially by targeting Tregs to ameliorate the immune imbalance.

Alsuliman et al. established a safe, controlled and reproducible Good Manufacturing Practice (GMP) process to isolate CD4⁺ CD25⁺ Tregs from peripheral blood of ALS patients, and further used a culture system containing rapamycin and IL-2 to expand Tregs in vitro for 25 days, which resulted in 25- to 200-fold increase in the number of cells, and a significant restoration of their immunosuppressive capacity to a level comparable to the healthy controls, laying a solid foundation for the implementation of Tregs overlay cell therapy in ALS patients [65]. Mandrioli et al. carried out a multicenter, randomized, double-blind clinical trial enrolling a total of 63 patients with ALS who were randomly assigned in a 1:1:1 ratio to receive treatment with rapamycin at 2 mg/m²/day, 1 mg/m²/day, or placebo. The primary endpoint of the study was the proportion of patients with an increase in the number of Tregs of more than 30% from baseline to the end of treatment. However, the primary endpoint was not met due to a reduction in the number of analyzable samples at week 18. Nonetheless, the results of the study showed that rapamycin was well tolerated in patients with ALS and no serious adverse events were observed. The trial confirmed the safety of low-dose rapamycin treatment in patients with ALS, but failed to demonstrate a significant effect on Treg levels [51], https://clinicaltrials.gov/ct2/show/NCT03359538. Rapamycin as a Treg modulator has demonstrated a good biological basis and safety, and although clinical data are still limited at this stage, its potential in ALS immune intervention deserves further in-depth exploration.

RNS60

RNS60 is a novel experimental therapeutic drug, which is essentially an oxygen supersaturated 0.9% sodium chloride solution containing oxygenated charge-stabilized nanostructures (CSN), which are generated by a proprietary process under high oxygen pressure conditions using a modified Taylor-Couette-Poiseuille (TCP) flow generation [66]. RNS60 has been investigated as a potential therapeutic strategy in a variety of neurodegenerative disease models, showing promising anti-inflammatory and anti-apoptotic properties, which are effective in reducing tissue damage [67]. The anti-inflammatory effect of RNS60 is partly attributed to its targeting of Tregs. It was found that RNS60 significantly increased the number of peripheral CD4⁺ /FOXP3⁺ Tregs and exerted neuroprotective effects by enhancing protective glial cell responses and preserving peripheral nerve architecture in a SOD1 mutant ALS mouse model [68]. In addition, in an experimental autoimmune encephalomyelitis (EAE) model, RNS60 has also been shown to delay disease progression by increasing Treg numbers and inhibiting autoimmune Th17 cells [69].

Based on the promising results achieved with RNS60 in preclinical models of ALS, Paganoni et al. conducted a preliminary open-label trial enrolling 16 patients to assess the feasibility, safety, and tolerability of long-term administration of RNS60 in patients with ALS. Patients received weekly intravenous infusions and daily nebulized inhalation therapy. The primary outcome indicators were safety (measured by the number and severity of all adverse events) and tolerability (defined as the ability to complete 23 weeks of study drug therapy). Secondary outcome indicators included plasma IL-17 levels and whole blood FOXP3 mRNA expression levels. The results showed that long-term use of RNS60 was safe and well tolerated, with no serious adverse events associated with RNS60 observed and no subjects withdrawn from the trial due to drug-related adverse effects. However, no significant changes in blood levels of both FOXP3 mRNA and IL-17 were seen during the treatment period, which may be related to the small sample size [52], https://clinicaltrials.gov/ct2/show/NCT02525471. Beghi et al. conducted a randomized, double-blind, placebo-controlled phase II trial in patients with ALS, enrolling 147 participants (74 assigned to the RNS60 group and 73 to the placebo group), to evaluate the effects of RNS60 on multiple candidate biomarkers and clinical outcomes. The primary objective of the study was to investigate the effect of RNS60 treatment on candidate markers related to inflammation and neurodegeneration in the peripheral blood of patients with ALS; secondary objectives included assessing its impact on dysfunction, respiratory function, quality of life, self-care, and survival through relevant scales, as well as assessing its safety and tolerability (through the occurrence of adverse events). Subjects were randomly assigned to receive either RNS60 or placebo on top of Riluzole for a 24-week course of treatment. The results showed that RNS60 had no significant effect on inflammation and neurodegeneration-related biomarkers, ALSFRS-R score, or survival. Treg-related markers, such as FOXP3 and CD25 mRNA expression, also showed no significant differences between groups [53], https://clinicaltrials.gov/ct2/show/NCT03456882. However, subsequent follow-up analyses suggest that subjects randomized to RNS60 may have prolonged survival compared to those randomized to placebo in a subset of patients, particularly in subgroups with slower decline in respiratory function or lower levels of specific biomarkers (e.g., NfL, MCP-1) [70]. Although the current clinical study failed to validate the significant improvement of RNS60 on Treg function or disease progression in the overall population, the possible survival benefit of RNS60 in specific subgroups suggests its potential therapeutic value, and further validation of its efficacy and mechanism based on larger sample size and fine stratification is necessary in the future.

Dimethyl fumarate

Dimethyl fumarate (DMF), trade name Tecfidera, is a fumarate ester compound that has been approved by FDA and the European Medicines Agency for the treatment of relapsing-remitting multiple sclerosis [71]. In recent years, multiple studies have shown that DMF can exert immunomodulatory effects by targeting Tregs. In one study, DMF was found to activate the Nrf2 signaling pathway in Treg cells and promote FOXP3 expression, thereby upregulating Treg numbers and effectively suppressing inflammatory responses in an asthma model [72]. In addition, DMF has been shown to increase the ratio of Treg to Th17 cells in the peripheral blood of patients with psoriasis and multiple sclerosis [73]. By modulating Treg function, DMF has shown promising results in remodeling immune homeostasis and is therefore considered as one of the potential disease-modifying drug candidates in ALS.

Vucic et al. conducted the phase II TEALS trial, a multicenter, randomized, double-blind, placebo-controlled study enrolling 107 patients with ALS, to evaluate the efficacy and safety of DMF. The results of the study showed that DMF did not demonstrate a significant advantage in the primary endpoint, the ALSFRS-R score, and there were no significant differences in the secondary endpoints of neuroinflammation, survival, and respiratory function compared to placebo. Nonetheless, the study confirmed that DMF has a favorable safety and tolerability profile in patients with sporadic ALS. Unfortunately, the trial did not perform T-cell profiling, so the lack of clinical efficacy may be related to the failure of DMF to effectively modulate T-cell populations in the current population of ALS patients [55], and its potential therapeutic value needs to be further validated in additional clinical studies, https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?ACTRN=12618000534280.

Vitamin D

Vitamin D is a steroid hormone, with its active form being 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), which is involved in various physiological processes. Recent studies have suggested that vitamin D may have neuroprotective effects in various neurological disorders, including Parkinson’s disease (PD), multiple sclerosis, cognitive impairment, and neurovascular diseases [74]. Its mechanisms of action may involve alleviating neuroinflammation and oxidative stress, promoting the production of neurotrophic factors, regulating neurotransmitter synthesis, and protecting the blood-brain barrier, thereby improving clinical symptoms and slowing disease progression [75]. In addition, upon binding to the vitamin D receptor (VDR), 1,25(OH)₂D₃ promotes the direct interaction of VDR with vitamin D response elements (VDREs) located within the conserved noncoding sequence of the first intron of the FOXP3 gene, thereby upregulating FOXP3 expression in CD4⁺ T cells and inducing their differentiation into immunosuppressive Tregs [76]. In this context, growing attention has been directed toward the potential association between vitamin D and ALS. A randomized, double-blind, placebo-controlled clinical study found that in vitamin D-deficient PD patients, supplementation with vitamin D₃ significantly up-regulated the levels of Treg cells in the peripheral blood (from 3.25% to 4.52%), while down-regulating the levels of Th17 cells (from 4.62% to 3.25%), which was accompanied by a significant improvement in Unified Parkinson’s Disease Rating Scale (UPDRS) score. It is suggested that vitamin D may delay the progression of motor dysfunction in PD patients by modulating immune homeostasis [77].

Regarding the potential association between Vitamin D levels and disease progression or survival in patients with ALS, the results of the available studies are inconsistent. Camu et al. found that the active form of 1,25(OH)₂D₃, by enhancing the expression of several neurotrophic factors (e.g., GDNF, BDNF, CNTF), could significantly increase the survival of mouse embryonic motor neurons. On the clinical side, ALSFRS-R scores of severely vitamin D deficient individuals declined approximately four times faster than those of normal individuals and median survival was significantly shorter, suggesting that low vitamin D levels are an independent risk factor for accelerated progression of ALS and an elevated risk of death [78]. Karam et al. conducted a retrospective analysis of serum vitamin D levels and supplementation effects in ALS patients. The results showed that 81% of patients had vitamin D deficiency (< 30 ng/mL). Daily supplementation with 2000 IU of vitamin D for 9 months did not cause any significant adverse reactions and may have had a beneficial effect on ALSFRS-R scores [79]. However, some studies have also reported different findings. Blasco et al. found that higher serum vitamin D levels were instead associated with a poor prognosis in a cohort of ALS patients [80]; and Yang et al. found no significant correlation between vitamin D concentrations and ALS survival [81]. In addition, a prospective randomized controlled study conducted by Trojsi et al. included 48 ALS patients with vitamin D insufficiency who were given three doses of vitamin D₃ supplementation, 50,000 IU, 75,000 IU, and 100,000 IU per month, and showed that vitamin D supplementation did not significantly improve motor function in patients with ALS after a 6-month intervention progress [82]. A clinical study called VITALS is further exploring the immunomodulatory role of vitamin D in ALS. Using an open-label, non-randomized assignment design, the study plans to enroll 70 ALS patients and 27 healthy controls to examine the composition of peripheral blood T-cell subsets, including Treg, Th17, Th1, Th2, and CD8⁺ T-cells, and to analyze their association with plasma vitamin D levels and prognostic markers of ALS, such as the rate of muscle atrophy. For ALS patients with vitamin D deficiency, a vitamin D intervention will be given for 6 months to dynamically assess changes in T cell phenotype. The results of this study are not yet publicly available, https://clinicaltrials.gov/ct2/show/NCT02756104.

Fecal microbial transplantation

A growing body of research suggests that an imbalance in gut microbiota is closely associated with the onset and progression of ALS. Gut microecology may affect neurological homeostasis by regulating the gut-brain axis, and the mediating mechanisms may include gut barrier dysfunction, changes in microbial metabolites, and activation of the peripheral immune system [83]. In view of this, it has been proposed to improve the immune-inflammatory status of ALS patients by intervening in the gut microbiota, and fecal microbiota transplantation (FMT) has emerged as a potential intervention strategy [84]. Mandrioli et al. initiated a multicenter, randomized, double-blind clinical trial in 2019 to evaluate the therapeutic potential of FMT in patients with ALS. The study plans to enroll a total of 42 patients with early-stage ALS, assigned in a 2:1 ratio to the FMT intervention and control groups, with a 12-month follow-up period for each patient. The study design includes three bowel biopsy endoscopies at baseline, month 6 and month 12; fresh feces from healthy donors will be infused at patients in the intervention arm at baseline and month 6. The primary observation will be the change in the number of Tregs between the intervention and control groups at month 6. Secondary observations included changes in immune cell profiles and inflammatory status, central and peripheral biomarkers of ALS, and a comprehensive analysis of gut, salivary, and fecal microbiota. In addition, safety, quality of life, and validated clinical outcome measures for ALS (e.g., survival, spirometry, and modified ALSFRS-R score) were assessed. To date, the results of this study have not been published [54], https://clinicaltrials.gov/ct2/show/NCT0376632.

Directions for Treg cell therapy strategies

Despite a large number of studies suggesting that neuroimmune inflammation plays a key role in the pathogenesis of ALS, most clinical trials targeting immunosuppression have failed to achieve the desired results, mainly due to the lack of selectivity of the action of these drugs on the immune system, which often leads to systemic immunosuppression, limiting their clinical application. In contrast, targeting Tregs to increase their numbers or improve their ratio to Teffs offers greater specificity and safety, reduces side effects associated with systemic immunosuppression, and induces long-term immune tolerance, making it a promising option for clinical applications [85]. Studies have shown that targeted cellular drugs exhibit good potential for development, with superior targeting ability, higher biocompatibility, and longer in vivo circulation times.

Adequate and stable Tregs are critical for achieving cell therapy efficacy, and their main sources include umbilical cord blood (UCB), adult peripheral blood (APB), and neonatal thymus [86]. Among them, APB is the most commonly used source in current clinics, but the peripheral-derived Treg pTreg from APB are prone to phenotypic instability and may convert to effector T cells in inflammatory environments. With the establishment of cord blood banking and the improvement of the third-party donor system, UCB is gradually gaining attention as an allogeneic source of Treg cells. Studies have shown that UCB-derived Treg cells have higher purity, stronger expansion capacity, more significant immunosuppressive effects, as well as lower plasticity and more stable phenotypic characteristics [87]. Currently, clinical trials have been conducted using UCB-derived Tregs for overt metastatic therapy and have shown favorable immunomodulatory effects [13]. Treg therapeutic strategies commonly used in clinical practice include polyclonal Treg cells, TCR-Tregs and chimeric antigen receptor Tregs (CAR-Tregs) [61]. Among them, polyclonal Treg cells are the most commonly used type, which are derived from Treg cells in patients or healthy donors, and are prepared into therapeutic cells that can be infused back into the patient’s body by means of in vitro culture, expansion and antigen stimulation. Stimulation with anti-CD3/CD28 magnetic beads under conditions of supplementation with low doses of IL-2 and the mTOR pathway inhibitor rapamycin significantly enhances the functional stability of Tregs, allowing them to grow preferentially over other T cell subsets during expansion, thereby maximizing Treg yield and purity [88]. Although polyclonal Treg therapies perform well in terms of expansion efficiency and safety, their lack of antigenic specificity makes it difficult to accurately target specific focal regions and carries the risk of nonspecific immunosuppression, and thus may have limited therapeutic efficacy in the disease [89]. To enhance therapeutic specificity, researchers have explored the in vitro generation of antigen-specific Tregs using antigen-presenting cells (APCs). Compared with polyclonal Tregs, these cells require fewer numbers to exert potent local and targeted immunosuppression. However, the induction and expansion of antigen-specific Tregs remain technically complex and yield-limited, restricting their clinical translation [90]. To overcome these limitations, recent advances have focused on engineered regulatory T cells (engineered Tregs), which combine the scalability of polyclonal Tregs with the precision of antigen-specific Tregs. Two principal approaches have emerged: TCR-Tregs and CAR-Tregs. TCR-Tregs are generated by introducing antigen-specific T cell receptors into Tregs via lentiviral or retroviral transduction, enabling recognition of specific peptide–MHC complexes and mediating targeted immunosuppression [91]. In contrast, CAR-Tregs are engineered to express chimeric antigen receptors, typically comprising an antibody-derived single-chain variable fragment (scFv), a transmembrane domain, and an intracellular signaling domain [92, 93]. CAR-Tregs can directly recognize intact proteins expressed on the surface of target tissues, independent of antigen processing and MHC restriction, thereby conferring a broader recognition spectrum and greater targeting flexibility [94]. Studies have demonstrated that CAR-Tregs not only retain the immunosuppressive properties of conventional Tregs but also precisely target disease-associated antigens, showing promising therapeutic potential in a range of immune-mediated disorders, including multiple sclerosis, neuromyelitis optica, and myasthenia gravis [95, 96]. Notably, TCR-Tregs can be activated at low antigen expression levels, whereas CAR-Tregs generally require higher antigen density to elicit a response, suggesting that the selection of either approach should be guided by the antigen expression profile of the target tissue [97]. Furthermore, CAR-Tregs are limited to recognizing cell-surface antigens, whereas TCR-Tregs can detect peptide antigens derived from multiple intracellular compartments, including the cytoplasm, nucleus, and membrane protein degradation products [98]. Thus, CAR-Tregs are better suited for mediating targeted immunosuppression against surface antigens of specific tissues or organs, whereas TCR-Tregs can recognize a broader range of intracellularly derived epitopes and are therefore more applicable in diseases with complex antigenic profiles (Fig. 3A).

Fig. 3.

Treg cell therapy: manufacturing workflow and strategies to improve function. A Treg cell manufacturing and infusion workflow. Tregs are isolated from peripheral blood, thymus, or umbilical cord blood. After isolation, they are expanded ex vivo using APCs or anti-CD3/CD28 beads under IL-2 and rapamycin, and can be further engineered with lentiviral vectors to generate polyclonal, antigen-specific, CAR or TCR-Treg cells, which are subsequently re-infused into patients; B Improving Treg function: stability and delivery. Approaches to enhance Treg stability include CRISPRi/a, IL-2 + TGF-β, TSDR demethylation, and SSO-induced splicing, while delivery strategies involve liposomes, exosomes, and microneedle patches. APCs: Antigen-presenting cells; CAR-Tregs: Chimeric antigen receptor Tregs; CRISPRi/a: CRISPR interference/activation; IL-2: Interleukin-2; TGF-β: Transforming growth factor-β; TSDR: Treg-specific demethylated region; SSOs: Splice-switching oligonucleotides. The figure was created by Biorender.com

In addition, many studies have been devoted to enhancing the phenotypic stability of Treg cells through novel techniques [23]. One strategy is to utilize the CRISPR interference/activation (CRISPRi/a) system to restore the stable expression and function of FOXP3 in Treg cells by targeting the silencing of X-inactive specific transcript (XIST) and activating the expression of endogenous FOXP3 gene [99]. Meanwhile, the addition of cytokines such as IL-2 and TGF-β during the in vitro expansion process also helps to maintain FOXP3 expression and enhance cell stability [100, 101]. Regulation at the epigenetic level has also received attention, for example, acting on the TSDR by methyltransferases can stabilize the active state of the FOXP3 promoter, thus consolidating the phenotypic characteristics of Treg [102]. In addition, a regulatory strategy for FOXP3 splice variants has also been proposed, in which researchers use splice-switching oligonucleotides (SSOs) to induce splicing to the functionally intact full-length form, which is expected to further enhance the functional stability of Treg [103]. Compared to the regulation of Treg cell intrinsic stability, functional enhancement strategies based on exogenous induction or delivery systems have also gained attention in recent years. For example, liposomal delivery systems can co-deliver antigenic peptides with active vitamin D3 to dendritic cells in draining lymph nodes, inducing antigen-specific Treg differentiation and thus effectively suppressing effector T cell responses, which has shown promising efficacy in animal models of rheumatoid arthritis and vasculitis [104]. In addition, exosomes isolated from Treg cells also exhibit similar immunosuppressive and tolerance-inducing abilities as Treg, and have the potential to be an alternative therapy to Treg [105]. Another emerging strategy is a topical delivery system based on hydrogel microneedle patches, which releases Treg chemokine CCL22 and survival factor IL-2 to amplify endogenous Treg locally in the skin, and has been used for the treatment of autoimmune dermatological diseases such as pemphigus vulgaris, with high drug loading capacity and storage stability (Fig. 3B) [106].Treg cell therapy shows great potential and broad prospects in many immune-related diseases, and it is expected to further promote its clinical translation and wide application through technical optimization and mechanism breakthroughs in the future.

Limitations and future challenges

Although the potential of Treg-targeted therapy in ALS continues to be revealed, there are several limitations in the current research that must be addressed in future studies (Fig. 4). Firstly, a primary constraint is the predominant reliance on the mutant SOD1 mouse model in preclinical studies; although this model provides valuable insights into the pathology of SOD1-linked familial ALS, it captures only a subset of familial ALS cases. The predominant sporadic cases of ALS and diseases driven by other genotypes (such as C9orf72, TARDBP, FUS) may involve distinct molecular pathways and immune microenvironments. Additionally, most current studies employ a single administration route (e.g., intravenous injection), with relatively limited research on alternative delivery methods (e.g., local administration, intrathecal injection, genetically engineered Treg delivery). Although techniques like intrathecal injection enhance Treg distribution within the central nervous system, precisely targeting Tregs to all affected neurons remains challenging. This may result in insufficient penetration of some Tregs, leading to inadequate drug concentrations at local pathological sites and consequently compromising therapeutic efficacy. Furthermore, current research often fails to fully consider patient stratification. ALS patients exhibit significant heterogeneity in genetic backgrounds, disease subtypes, and immune status. Variations in disease onset site (e.g., spinal vs. bulbar ALS), age at onset (e.g., young-onset vs. late-onset), causative factors (e.g., SOD1 vs. C9orf72), and disease progression rate (e.g., stable vs. rapidly progressive) could all impact the effectiveness of treatment. Stratifying patients as part of clinical trial inclusion criteria is becoming an important consideration for evaluating drug efficacy. Timing of administration is equally pivotal, with earlier treatment potentially yielding superior immunomodulatory effects.

Fig. 4.

Limitations and Future Directions of Treg-Based Therapies in ALS. The figure illustrates the key limitations and future directions for Treg-based therapies in ALS. On the left side, the limitations are listed, including the reliance on SOD1 mouse models, the focus on intravenous injection as the sole administration route, significant patient heterogeneity, suboptimal IL-2 dosage, and concerns regarding exogenous Treg-induced GVHD and immune imbalance. On the right, future directions highlight the need for more diverse ALS animal models, precise patient stratification, optimized treatment timing, and long-term treatment evaluation, alongside the development of standardized CAR/TCR-Treg production and storage systems. The figure also emphasizes exploring the combined application of Tregs with stem cells and neuroprotective drugs, as well as the importance of rigorous preclinical safety evaluations and long-term immune monitoring. CAR: Chimeric antigen receptor; GVHD: Graft-versus-host disease; IL-2: Interleukin-2; TCR: T cell receptor.The figure was created by Biorender.com

Treg therapy shows great promise in ALS. While IL-2 is widely used for Treg expansion, it may induce non-specific T cell proliferation, necessitating further research to optimize IL-2 dosage and delivery methods. Moreover, the therapeutic effect of IL-2 in ALS patients has been shown to correlate significantly with CSF-pNFH levels, which can serve as an important biomarker for patient screening. Further research is needed to explore how to combine CSF-pNFH with other potential biomarkers to guide personalized treatment. Additionally, the use of in vitro expanded natural Tregs or genetically engineered Tregs offers the advantage of precisely controlling the dose, phenotype, and specificity of the infused cells. However, this approach faces two major challenges: the risk of graft-versus-host disease (GVHD) and the potential for rapid recognition and clearance by the host immune system, which limits its sustained action in vivo. Treg cell therapy itself may also carry certain risks, including infection, tumorigenesis, and immune imbalance. Therefore, strict quality control and functional evaluation must be performed prior to cell infusion to ensure their stability and safety. Specifically, during in vitro expansion, Tregs are prone to plasticity, with the risk of converting into effector T cells. If not properly controlled, this may worsen inflammation or trigger autoimmune diseases. Furthermore, the application of Treg therapy in central nervous system diseases still faces many unresolved mechanistic issues, such as whether Tregs can cross the blood-brain barrier, their migration and homing abilities within the central nervous system, and the impact of the local microenvironment on their immunoregulatory functions. And these aspects require further investigation. Therefore, long-term monitoring of immune responses after Treg therapy is critical, to prevent excessive immune suppression or autoimmune risks.

Overall, Treg-targeted therapy offers new hope for ALS treatment, but its widespread adoption requires overcoming challenges related to individual variability, technical feasibility, and cost. Future efforts should prioritize patient stratification, tailoring treatments based on immune phenotypes, disease phenotypes, and genetic information to enhance efficacy. Consideration should also be given to conducting stratified trials across different stages of ALS to further evaluate the optimal timing of intervention in early versus late disease phases. Furthermore, future research should explore additional ALS animal models to comprehensively evaluate Treg therapy efficacy across diverse immune response patterns. Diversifying delivery methods is another critical research direction, comparing the impact of different routes on treatment outcomes to optimize therapeutic regimens. Current clinical trials predominantly focus on short-term outcomes, while the long-term survival and sustained immunoregulatory capacity of Tregs in vivo remain incompletely understood. Research is needed to explore methods for maintaining the long-term activity and function of Tregs. Whether expanded Tregs can persist stably in vivo, and whether they might “lose control” and convert into Teffs, remain urgent questions to address. Future key directions include optimizing the engineering design of CAR/TCR-Tregs, establishing standardized production and preservation systems, and exploring combination therapies involving Tregs, stem cells, and neuroprotective drugs. Therefore, the development of Treg products must include rigorous preclinical safety evaluations and establish long-term immune monitoring in clinical trials to dynamically assess patients’ immune status.

Conclusion

Tregs play a critical immunoregulatory role in the pathogenesis and progression of ALS, with dynamic changes in their abundance and function closely linked to the rate of disease progression. Current studies have identified Tregs as an important target for ALS immunotherapy, and enhancing their number or improving their function is believed to have the potential to slow down the disease process. Despite initial progress in relevant basic and clinical studies, Treg-mediated therapeutic strategies still face a series of key challenges, such as insufficient cellular stability, limited migration and homing ability, and the optimal timing of treatment has yet to be clarified. In particular, engineered Treg therapy still needs more evidence to support in terms of safety and feasibility. Preclinical research should explore a wider variety of ALS animal models and administration routes, while clinical studies must focus more on patient stratification. Future efforts should rely on multicenter, large-scale clinical trials to systematically evaluate the efficacy and safety of Treg-based therapies in ALS. Stratification based on patients’ clinical characteristics will be crucial for developing personalized treatment plans. Furthermore, optimizing cell sources, engineering methods, and treatment protocols, along with exploring combination therapies with existing or emerging treatments, is essential to provide ALS patients with more precise, feasible, and sustained therapeutic interventions.

Abbreviations

1,25(OH)₂D₃

1α,25-dihydroxyvitamin D₃

ALS

Amyotrophic lateral sclerosis

ALSFRS-R

ALS Functional Rating Scale–Revised

APB

Adult peripheral blood

APCs

Antigen-presenting cells

CAR-Tregs

Chimeric antigen receptor Tregs

CCL2

C-C motif chemokine ligand 2

CNS

Central nervous system

CRISPRi/a

CRISPR interference/activation

CSF-pNFH

Cerebrospinal fluid phosphorylated neurofilament heavy chain

CSNs

Charge-stabilized nanostructures

CTLA-4

Cytotoxic T-lymphocyte antigen 4

DCs

Dendritic cells

DMF

Dimethyl fumarate

EAE

Experimental autoimmune encephalomyelitis

EGFR

Epidermal growth factor receptor

engineered Tregs

Engineered regulatory T cells

Teffs

Effector T cells

FDA

Food and Drug Administration

FMT

Fecal microbiota transplantation

FOXP3

Transcription factor Forkhead box protein P3

GMP

Good Manufacturing Practice

GVHD

Graft-versus-host disease

IDO

Indoleamine 2,3-dioxygenase

IL-2

Interleukin-2

IL-2Rα

Interleukin-2 receptor alpha chain

IL-4

Interleukin-4

IL-10

Interleukin-10

IL-35

Interleukin-35

iTregs

Induced regulatory T cells

LAG-3

Lymphocyte activation gene 3

ld-IL-2

Low-dose interleukin-2

LTB4

Leukotriene B4

MHC-II

Major histocompatibility complex class II

mTOR

Mechanistic target of rapamycin

NF-κB

Nuclear factor kappa-light-chain-enhancer of activated B cells

NK cells

Natural killer cells

NO

Nitric oxide

nTregs

Natural regulatory T cells

PD

Parkinson’s disease

PGE2

Prostaglandin E2

PPAR-γ

Peroxisome proliferator-activated receptor gamma

pTregs

Peripheral regulatory T cells

RCT

Randomized controlled trial

ROS

Reactive oxygen species

scFv

Single-chain variable fragment

SSOs

Splice-switching oligonucleotides

STAT5

Signal transducer and activator of transcription 5

TCP

Taylor–Couette–Poiseuille

TCR

T cell receptor

TEAEs

Treatment-associated adverse events

TGF-β

Transforming growth factor-β

TIGIT

T cell immunoglobulin and ITIM domains

Tregs

Regulatory T cells

TSDR

Treg-specific demethylated region

UCB

Umbilical cord blood

UPDRS

Unified Parkinson’s Disease Rating Scale

VDR

Vitamin D receptor

VDREs

Vitamin D response elements

Authors’ contributions

YPC designed and supervised the project. NZ selected the articles, extracted and cross-checked the data, contributed to the statistical analysis and wrote the first draft of the manuscript. WMS, TC, YW, BC and QZ selected the articles and extracted and cross-checked the data. NZ and YPC wrote, revised and discussed the final edition. All authors read and approved the final version of the manuscript.

Funding

This study was supported by the National Natural Science Fund of China (Grant no. 82371422 and no. 81971188 to Y.P.C.), the National Key Research and Development Program of China (Grant no. 2022YFC2703101 to Y.P.C.), and the 1·3·5 project for disciplines of excellence Clinical Research Fund, West China Hospital, Sichuan University (Grant no. 2023HXFH032 to Y.P.C.).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not Applicable.

Competing interests

The authors declare no competing interests.