Background

Multiple sclerosis (MS) and myasthenia gravis (MG) are autoimmune diseases affecting the central nervous system (CNS) and the neuromuscular junction (NMJ), respectively. These diseases are characterized by inflammation, immune dysregulation, and immune over activity [1, 2]. Defects in self-tolerance leading to autoimmunity distinguish MS from other well-known neuroinflammatory diseases including Parkinson’s disease, Alzheimer’s disease, and stroke episodes [3]. Likewise, the autoimmune component of MG distinguishes it from many other muscular dystrophies [2]. MS and MG affect 50/100,000 and 20/100,000 people, respectively, in the USA; this prevalence amounts to more than the collective prevalence of other neurologically relevant autoimmune diseases including optic neuritis, Guillain-Barre Syndrome (GBS), and acute disseminated encephalomyelitis [4,5,6,7,8]. Current treatments for MS and MG involve non-specific mechanisms of action, do not produce lifelong protection from flare-ups, and have undesirable side effects including fatalities [9,10,11,12]. Since T regulatory cells (Tregs) characterized as CD4+CD25+FoxP3+ (forkhead box P3) cells have emerged pivotal in suppressing autoimmune diseases like type 1 diabetes and others, this review is focused on evaluating the role of Treg defects or dysfunctions in MS- and MG-related autoimmune pathology [1, 13–17]. It also discusses the beneficial effects of Treg augmentation as a potential treatment strategy. It does so by summarizing recent data from MS and MG patients, as well as their pre-clinical models: experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune myasthenia gravis (EAMG), respectively.

Although MS and MG are distinct autoimmune diseases, some studies suggest a co-morbidity [18]. A retrospective analysis indicated that five out of 1718 patients with MS also had MG (0.29%; expected percentage (0.005–0.015%)) [18]. Notably, the severity of both MS and MG was mild and the MG symptoms were ocular with none of these patients having positive anti-acetylcholine receptor (AChR) antibodies [18]. In another study, MS preceded MG in five out of eight patients by 6 to 8 years [19]. Onset of MG in MS patients treated with interferon (IFN)-β and glatiramer acetate (GA) was reported in one [20], but not in another study [18]. Therefore, definitive evidence of co-morbidity between MS and MG requires further validation in large independent cohorts of patients.

Despite differences in pathologies of MS and MG, there are immunological similarities. Both MS and MG are considered largely T cell mediated [1, 21]. MS and MG patients have increased numbers of Th1 and Th17 cells along with their associated cytokines IL-1, IL-6, IL-17, IFN-γ, and tumor necrosis factor (TNF)-α (Table 1) [21,22,23,24,25,26]. Further, Tregs of these patients have numerous documented dysfunctionalities [1, 13, 27, 28]. T cells originate from the thymus, and evidence suggests the thymus as a key regulator in the pathogenesis MG, and less so in MS [18, 20, 29]. Thus, thymectomy is a treatment option from which few MS patients may benefit [30, 31]. Both MS and MG have altered recent thymic emigrants suggestive of lower thymic output (Table 1) [28, 29, 32]. Further, MG and MS thymuses have been found to contain clonally expanded B cell lines (Table 1), a characteristic not found in control patients, or patients with systemic lupus erythematosus (SLE), anther autoimmune disease [33]. Germinal centers appear in the pathogenesis of both diseases [34]. CXCR5, a receptor associated with germinal center (GC) migration, can be found expressed on both T and B cells, but is elevated on CD4+ cells in both diseases (Table 1) which correlates well with the disease severity [34]. Both MS and MG patients have evidence of antigen-specific T cells [35, 36]. While MG patients have definitive evidence of antigen-specific antibody-mediated damage to the NMJ, the presence of antigen-specific antibodies in MS is debatable [37,38,39]. Immunological similarities and differences become more evident with treatments using immune modulating agents. Rituximab, a B cell depleting anti-CD20 antibody, has proven beneficial in both diseases, indicating B cell importance for each [40, 41]. In contrast, MS and MG patients differ in their response to IFN-1 (i.e., alpha and beta) treatments (Table 1). Development of MG after IFN-α treatment has been previously reported, while treatment of MS with IFN-β has been shown to be beneficial [12, 42]. IFN-1 (IFN-β) has also led to increased autoantibody production, a possible reason for MG exacerbation upon IFN-1 (IFN-α) treatment [43]. Similarly, a patient with MS treated with GA was seen to develop MG [44]. Lastly, anti-TNF-α therapy has resulted in different effects in patients with MS and MG. In MS, anti-TNF-α therapy has been unsuccessful despite success in pre-clinical models [45]. Whereas a majority of MG patients receive benefit, although some receive no benefit or even disease exacerbation [45, 46]. These studies clearly indicate that the immunological perturbation in these two diseases may have some common features, but are dissimilar.

Table 1 Immunological Comparisons between multiple sclerosis and myasthenia gravis
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Autoimmune development may not only be influenced by inadequate Treg numbers or defective Treg function, but it is also influenced by effector T cells (Teff; CD4+FoxP3−) resistant to suppression [47]. Although this review focuses on restoring Treg numbers and deficits, Teff resistance should be briefly discussed. The local cytokine milieu of IL-2, IL-4, IL-6, IL-15, and TNF-α have all been shown to influence Teff resistance to suppression [48, 49]. In MS, a decrease in the frequency of Tregs and resistance of Teffs to suppression were noted [50,51,52]. Similarly, both Tregs and Teffs from MG patients were found to be defective in ex vivo studies [53]. Whereas FoxP3 inhibited Th17 differentiation via repression of transcription factor RORγt, exogenous provision of IL-6 supported the differentiation of Th17 cells, suggesting the plasticity of the T cell under appropriate conditions [54]. Genetic studies unraveled polymorphisms associated with molecules related to Treg function in MS and MG patients [55, 56]. Although these data suggest an intrinsic functional defect in Tregs (Table 1), it is not clear whether it is sufficient to impair the functionality of Tregs. However, the conversion of FoxP3+ Tregs derived from normal humans into Th17 cells under the influence of IL-1 and IL-2 ex vivo has been documented, supporting the plasticity of Tregs [57], also observed in mice [54]. This is also suggested from an experiment in EAMG noting that the Treg defects appear after disease induction but the disease itself can be suppressed upon adoptive transfer of ex vivo generated Tregs [58, 59]. Inasmuch as the Tregs appear to be defective in both MS and MG (Table 1), we have focused this review on both intrinsic and extrinsic factors affecting Treg function in these diseases [1, 13, 27, 28].

Main text

Implications of dysregulated Tregs in MS and MG

Tregs play a key role in maintaining self-tolerance, and their dysfunction is well documented in multiple autoimmune diseases including Type 1 diabetes, GBS, psoriasis, and others [1, 13–17]. Tregs regulate immune response in the periphery predominantly by suppressing Teff cells. Although significant differences in the number of circulating Tregs in MS or MG patients relative to healthy controls are not frequently reported, Tregs from these patients are reported to have lower suppressive capabilities [1, 13, 60, 61]. This suggests that functional deficits in Tregs may contribute to the pathogenesis of MS and MG. For example, defects in Treg suppressor molecules have been linked to MS, such as reduced IL-10 production and genetic variations in CD25 [27, 55]. Likewise, MG patients have documented dysregulation in cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression, IL-2 sensitivity, and the levels of transforming growth factor beta (TGF-β) gene expression [25, 62, 63]. Mechanistically, lower Treg suppressive capabilities may lead to enhanced production of pro-inflammatory cytokines such as IL-6, IL-17, and IFN-γ, as well as activation of autoantibody producing B cells. Normally, Tregs suppress the production of these pro-inflammatory cytokines through contact-dependent and contact-independent suppression of Teff cells known to produce IL-6, IL-17, and IFN-γ [64, 65]. Functional Tregs are also believed to directly eliminate both antigen-presenting and autoantibody-producing B cells in a contact-dependent manner through secretion of perforin and granzyme as seen in lupus prone mice (NZB/W) [66]. This mechanism may be pivotal for Treg-based amelioration of MG and perhaps MS [2, 66, 67]. The autoreactive B cells can produce autoantibodies against acetylcholine receptor (AChR) or muscarinic receptor, thus causing membrane damage via activation of the complement cascade in MG; likewise, it has been suggested that autoantibodies may also be involved in the pathogenesis of MS [68, 69]. Furthermore, activated B cells are found to be upregulated in the neuroinflammatory sites of the CNS in MS patients and in the blood of MG patients [55]. While CD127 is expressed highly on Teff, but minimally on Tregs, molecules such as GITR, OX40, Helios, CD49b are also expressed on Tregs [22, 28, 47, 77]. Furthermore, many MS patients have decreased FoxP3 expression in Tregs, decreased Treg suppressive function, and decreased levels of Tr1 [1, 27]. Additionally, recent research suggests that Tregs are unable to properly infiltrate the CNS during the course of the disease [104,105,106]. Brain biopsies from MS patients revealed that 30% of the lesions lacked FoxP3 expression [104]. Fas, a cellular apoptotic pathway receptor, is seen upregulated on Tregs in MS brain biopsies suggesting increased susceptibility to apoptosis [104]. Taken together, these findings suggest that Tregs might be restricted from migrating to neuroinflammatory sites or undergo apoptosis upon arrival. Treg dysfunction during the disease progression can also be noted through modulation of CD28 and CTLA-4 co-receptors mentioned earlier. Studies in EAE have shown that during relapse, there is increased expression of CD28, co-localizing with T cell receptor subunit CD3, in the CNS blood vessels and parenchyma [107]. During remission, CD28+ cells decrease while CTLA-4+ cells increase [107]. Considering Tregs constitutively express CTLA-4 while Teff do not, it is possible these cells are Tregs; however, the study did not look for co-expression of FoxP3 [107, 108]. Taken together, Treg abnormalities in MS appear to involve loss of suppressive capacity as well as defect in migration into the CNS.

Many treatments which increase Tregs in EAE have been found to be efficacious, while other treatments, although successful, showed no effect on Tregs [109]. Administration of an indoleamine 2,3-dioxygenase (IDO) metabolite which increases Treg number leads to significant amelioration of MOG-induced EAE [110]. Additionally, the effect of IL-10-based therapy has been evaluated in different EAE models; most resulted in reduced clinical scores, perhaps associated with an increase in Tr1 associated with IL-10 administration [111, 112]. Some of the most affirmative evidence advocating Treg augmenting treatments have utilized adoptive transfer techniques. Adoptive transfer of FoxP3− Treg cells that ectopically expressed the transcription factor FoxA1 (suggested marker for a novel Treg) ameliorated EAE in IFN-β knockout (KO) mice and these cells were increased in response to treatment of MS patients with IFN-β, suggesting a role for this Treg subset [113]. Although adoptive transfer of CD4+CD25+FoxP3+ cells reduced EAE, they differed in their efficacy. Adoptive transfer of CNS-derived KLRG1− and KLRG1+ cells modestly reduced the clinical score at the termination of the experiment without affecting peak response (KLRG1 is associated with natural killer cells but was tested due to its increased expression on Tregs when entering the CNS of EAE mice) [114]. Likewise, CNS-derived CD4+CD25+ cells reduced the peak clinical EAE score [115]. Finally, experiments knocking out known Treg proliferating molecules, such as IDO or IL-10, resulted in exacerbation of EAE [110, 116].

In contrast to the above findings, FoxP3gfp knock-in mice had an accumulation of Treg cells in the CNS, but it failed to control EAE [117]. Administration of cholesterol-reducing statins such as atorvastatin inhibited the development of EAE without increasing the frequency of FoxP3+ Treg cells or Th2 cells, yet there was a substantial increase in IL-10 expression [109]. Taken together, these data tend to suggest that modulation of FoxP3 Tregs may not always be necessary for the protective effect of certain treatment modalities for EAE. Inasmuch as administration of statins involve non-specific immunosuppression, it is hard to avoid undesirable consequences. Therefore, it appears that boosting the Treg cells may produce a desirable clinical outcome in MS.

Treg migratory receptor dysregulation may have implications in MS

Dysfunctional migration of Tregs to neuroinflammatory sites could have profound implications; for example, GCN2 (aids in CCL2-mediated migration) KO Tregs adoptively transferred into EAE mice were unable to enter the CNS and hence mice were unable to undergo remission [118]. Some chemoattractants (e.g., CCL17) or their receptors (e.g., CCR4 and CXCR3) appear to facilitate Treg homing to neuroinflammatory sites. Human CD4+CD25+ Treg cells express CCR4 which respond to mature dendritic cell-derived CCL17 and are thereby recruited to the site of inflammation. Butti et al. determined that CCR4 is indispensable for Treg recruitment into the cerebral spinal fluid (CSF) and that recruitment was necessary for EAE amelioration likely due to the increased CCL17 levels after administration of IL-4 gene therapy into the CSF [119]. Studies of malignant diseases also find a correlation between the levels of CCL17 and accumulation of FoxP3+ cells, implicating CCL17 as a Treg recruiter molecule [120, 121]. Therefore, CCL17 administration at specific sites may be utilized to recruit Tregs.

Molecules such as CXCR3 are important for Tr1 and Treg migration to the CNS [106, 122]. The chemokine receptor CXCR3 that promotes trafficking of activated T and natural killer cells in response to CXCL9, CXCL10, and CXCL11 is expressed heavily on Tr1 cells which can direct Tr1 to the CNS thereby implying a role in EAE [122]. Administration of CXCL10-Fc exacerbates EAE, whereas CXCL11-Fc leads to amelioration with subsequent polarization to Tr1 [122]. Since CXCR3 and CXCL10 are found elevated in neuroinflammatory sites in MS patients, over-recruitment of Teff may be occurring, thus use of CXCL11 could be a strategy to recruit additional Tr1 to these neuroinflammatory sites [123]. Additionally, CXCR3 KO mice have more severe EAE with reduced FoxP3+ cells in the CNS despite little change in total CD4+ infiltrates implying that CXCR3 may be crucial for Treg migration to the CNS [106]. Therefore, a strategy to increase CXCR3+ expression on FoxP3+ Tregs may be useful for recruitment to the CNS. Enhanced CXCR3 expression by Tregs has been seen with the use of IL-27, thus providing a treatment strategy to facilitate selective Treg migration [122, 124].

Therapeutics for multiple sclerosis which restore Treg abnormalities

Many common MS treatments could result in adverse effects revealing a need for new therapeutic strategies [9, 125]. Additionally, current treatments such as IFN-β (Avonex, Rebif, and Betaseron), glatiramer acetate (GA; Copaxone), fingolimod (Gilenya), teriflunomide (Aubagio), dimethyl fumarate (Tecfidera), Natalizumab (Tysabri), and mitoxantrone, although beneficial, are not curative [9]. Corticosteroids, which have been in use for over 75 years, are still the treatment of choice for acute MS symptoms [9, 126]. However, they lack specificity and tend to cause severe adverse effects such as abnormal weight gain, behavioral changes, oral thrush, and others [126]. Over two decades ago, glatiramer acetate became one of the first treatments for reducing relapse rates in MS patients; however, it had only a modest efficacy in reducing disease activity [127]. Many newer treatments, such as the ones indicated above, have been implemented since then, yet nearly all can cause significant side effects [9, 128, 129]. Therefore, there is a need for novel treatments which reduce disadvantages of current treatments while providing greater efficacy.

Most of the clinical treatments do not target Tregs as specifically as new pre-clinical treatments can [130]. Despite different targets, some treatments have been shown to increase Treg numbers such as IFN-β, GA, fingolimod, and teriflunomide, while others, such as dimethyl fumarate, natalizumab, mitoxantrone, and corticosteroids, have resulted in reduced, unaffected, or contradictive effects on Treg numbers [131,132,133,134,135,136,137,138,139,140]. Treatments such as the lipid-lowering drug atorvastatin mentioned earlier have no effect on the FoxP3 Treg population, indicating that increasing the Treg population, although may be desirable, may not always be necessary [109]. However, this review will discuss strategies to restore Treg abnormalities as therapeutic modalities that may be considered for further testing. These Treg abnormalities have been corrected using cytokines such as IL-10, IL-27, IL-35, bimolecular protein inhibitors (BPIs), indoleamine 2,3-dioxygenase (IDO), or the chemokine CXCL11 (Table 2; Fig. 1) [80, 106, 110, 111, 141]. Additionally, we propose modalities of treatment for further testing. These include Jagged-1/OX40L co-treatment and site-specific CCL17 administration for Treg recruitment (Table 2).

Table 2 Multiple sclerosis treatment approaches using Treg augmentation based on pre-clinical models
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Fig. 1
figure 1

MG and MS treatment schemes aimed at augmenting Tregs based on experimental models. AChR: acetylcholine receptor; APC: antigen-presenting cell; B7AP: B7 antisense peptide; Bimolecular peptide inhibitor; CNS: central nervous system; DC: dendritic cell; GM-CSF: granulocyte macrophage-colony stimulating factor; IDO: indoleamine 2,3-dioxygenase; iTr35: induced T-regulatory 35 cell; IFN-γ: interferon gamma; MG: myasthenia gravis; MS: multiple sclerosis; N3: Notch 3 receptor; IL: interleukin; Teff: effector T cell; TFH: follicular helper T cell; TFR: regulatory T follicular cell; TGF-β: transforming growth factor beta: Tr1: T-regulatory 1 cell; Treg: T-regulatory cell

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Administration of interleukins

IL-10 mediated suppression is regarded as a main mechanism of Treg suppression. IL-10 therapies have resulted in a reduction of symptoms in majority of the EAE models tested (reviewed in reference [111]) [142, 143]. Despite the success in pre-clinical models, it has not been as successful in clinical trials conducted in other autoimmune diseases such as Crohn’s disease and rheumatoid arthritis [144, 145]. Focus might instead be diverted toward IL-10 replenishing strategies which down modulates the Fas-mediated apoptosis both in Tregs and Bregs [111, 145]. Research shows increasing IL-10 expression may be done through upregulating IL-10 producing Tr1, increasing Treg number, or through induction of tolerogenic DCs (CD11c+CD8α−) [27, 67, 110]. Since many documented positive correlations between IL-10 and MS remission have been documented, and IL-10 secreting Tr1 usage in pre-clinical models appears useful, upregulation of Tr1 cells could be a useful IL-10 replenishment strategy which would also rectify Tr1 defects in MS [146, 147].

IL-27 and IL-35 are anti-inflammatory cytokines that can regulate Treg responses [148, 149]. IL-35 has been shown to play a key role in FoxP3+ Treg suppression and in the induction of iTr35 [80, 149]. Treatment with IL-35 has shown to lead to EAE amelioration with concomitant induction of iTr35 (Fig. 1) [80]. These iTr35 have also been shown to ameliorate other autoimmune models such as collagen-induced arthritis and IBD [150]. IL-35 is not produced by human FoxP3 Tregs, but ex vivo experiments on human T cells have been able to induce iTr35 suggesting that it may have a similar role in humans [150, 151]. IL-27 has both inflammatory and anti-inflammatory pathways. IL-27 activates Th1 response through T-bet signaling; however, it also inhibits Th17 development and induces IL-10 production [152]. IL-27 role in FoxP3 Tregs is less understood, yet it has been shown to induce Tr1 production in vivo leading to EAE amelioration [153]. Administration of exogenous IL-27 to EAE mice has also been shown to reduce IL-17 production, Th17 cells, and CNS infiltration [153, 11]. Although the mechanism is not understood, Tregs have been seen to modestly increase with the use of drugs not targeting Tregs, such as pyridostigmine, rituximab, azathioprine, and IVIG [190,191,192,193]. Corticosteroids lack specificity and importantly cause severe adverse effects such as bruising, abnormal weight gain, behavior changes, oral thrush, and others [194]. Pyridostigmine bromide, the most commonly used acetylcholinesterase inhibitor, is the first line for symptomatic treatment [195]. Yet it provides only short-term relief, it is not a disease-modifying therapeutic (DMT), and it can cause a hypersensitivity rash [195]. Azathioprine, a commonly used DMT, causes side effects in roughly 20% of patients and failed to prevent relapse in roughly 33% of patients in a long-term study of 117 MG patients [196]. Thymectomy has proven to be a beneficial DMT; however, it does not cure the underlying disease, and patients still suffer side effects from other treatments because thymectomized patients still require additional interventions such as corticosteroids or azathioprine [197]. Taken together, there is a need for more efficacious DMTs focused on destroying autoreactive B cells to reduce autoantibody production and NMJ destruction. Although anti-B cell therapies are in practice like rituximab (anti-CD20 antibody), Tregs’ ability to destroy autoreactive B cells may provide relatively better specificity [68, 92]. Treg-augmenting therapies which might accomplish this task or treat MG Treg abnormalities include GM-CSF, IL-2/mAb complexes, OX40L-Jagged-1 co-treatment, and TGF-β administration.

GM-CSF

GM-CSF treatment of MG has led to a reduction of clinical signs and symptoms while increasing Tregs [198]. In EAMG, we have determined there are numerous benefits from GM-CSF treatment, namely, increasing the number of Tregs, halting antigen-specific T cell proliferation, enhancing IL-10 production, suppressing B cell proliferation, reducing anti-AChR antibody production, and reducing expression of IL-6, IL-17, and IFN-γ [67, 199]. We have showed that GM-CSF can induce tolerogenic semi-mature DCs (CD11c+CD8a−) which cause expansion of Treg cells that suppress EAMG [200]. Such GM-CSF induced Tregs likely suppressed autoimmunity through the secretion of IL-10, as we have shown in an animal model of thyroiditis [201]. Ex vivo co-culture of GM-CSF exposed bone marrow DCs with CD4+ T cells induced selective expansion of Tregs via OX40L and Jagged-1-mediated signaling [200, 202]. Alternatively, it has also been shown that GM-CSF can directly bind to its receptor expressed on human Tregs leading to their expansion [203]. In all, GM-CSF treatment may rectify multiple Treg abnormalities (Table 3).

Table 3 Myasthenia gravis treatment approaches using Treg augmentation based on pre-clinical models
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Inhibit B and TFH cells at GCs

The thymus plays a critical role in anti-AChR antibody production and MG pathogenesis [204]. B cells are implicated not only because of autoantibodies but also because of increases in CXCL13, CCL21, and BAFF in the thymus of MG patients, all of which lead to B cell activation [205]. CXCL13 is utilized by B cells, TFH, and TRH cells to migrate to GCs using CXCR5. Thus, a therapeutic opportunity would be to increase Treg migration to the GC to suppress activated TFH and B cells. This may be achieved with IL-2/mAb complexes (monoclonal antibody greatly increases IL-2 half-life) that have ameliorated EAMG while reducing CD19+ cells, autoantibody levels, and IFN-γ expression [189]. The effects of IL-2/mAb complexes on Tregs and migration to GCs are better understood through an experiment using chronic graft-versus-host disease (cGVHD) in which administration of IL-2/mAb complexes increased splenic Tregs and TFR while ameliorating cGVHD without increasing TFH [94]. Adoptive transfer of wild type Tregs without IL-2/mAb administration caused a reduction in B cell number in the GC dependent upon CXCR5+ Tregs homing to GCs [94]. These findings in GVHD may be relevant to MG as GCs as well as B and T cells are also implicated in GVHD pathogenesis [206]. A mechanism by which IL-2/mAb may act on Tregs over Teff is through the use of IL-2Rα, CD25. The high affinity Tregs have for IL-2 through their CD25 expression will deplete IL-2 from the surrounding milieu restricting IL-2-dependent Teff activation [91]. It should be noted that IL-2/mAb complexes, when used in mice with acute GVHD, resulted in rapid death of mice within 4 days with concomitant increases in Teff, thus the efficacy of treatment may depend on the level of immune activation [94]. Therefore, using IL-2/mAb complexes may be an effective strategy to activate and proliferate Tregs and/or TFR causing suppression and reduction of TFH and B cells in the GC (Fig. 1; Table 3).

OX40L/Jagged-1 Co-treatment

As stated earlier, GM-CSF treatment induced Treg expansion via OX40L and Jagged-1 release from tolerogenic DCs (Fig. 1) [157, 200]. Considering OX40 is found to be upregulated in MG, and our recently published data shows that soluble OX40L and Jagged-1 co-treatment can delay autoimmune diabetes in NOD mice while increasing Tregs, EAMG could be a successful candidate for OX40L and Jagged-1 co-treatment [157, 158, 207]. More interestingly, CD46, a complement receptor expressed on many cell types, is found to co-stimulate with Jagged-1 leading to Treg activation as well as induction of Tr1 cells [161]. Although the role of Tr1 cells is unknown in MG, their suppressive capabilities have been well documented in other autoimmune models [147]. Thus, CD46 activation via Jagged-1 may provide some novel insights into MG treatment modalities [39]. In conclusion, OX40L and Jagged-1 treatment may lead to an increase in Tregs, or even Tr1 cells and rectify OX40 or CD46 dysregulation in MG (Table 3).

TGF-β

TGF-β is a potent activator of Tregs leading to iTreg generation [208]. Generation of iTregs ex vivo in the presence of TGF-β when cultured with B cells from lupus-prone mice (NZB/W) has been found to induce apoptosis of B cells, reduce B cell activation, and reduce autoantibody levels [208]. Additionally, adoptive transfer of iTregs resulted in a greater reduction of autoantibodies compared to nTregs (not exposed to TGF-β ex vivo) [208]. Ex vivo, the addition of TGF-β to mononuclear cells from MG patients induced suppression of cells autoreactive to AChR [209]. The blocking of complement receptors C3a and C5a has been shown to induce expression of TGF-β and IL-10 leading to generation of iTregs which when adoptively transferred potently suppressed the disease [210]. Other studies have also suggested a link between reduced complement expression with increased Treg numbers [211, 212]. Additionally, TGF-β has been shown to reduce iNOS production, again leading to concomitant Treg proliferation [213]. Taken together, it appears TGF-β could restore immunological imbalance in MG patients.

Conclusions

Current MS and MG treatments have modest long-term efficacy and are often associated with severe side effects that fail to treat the underlying disease. Although current treatments do not tend to act on Tregs, they provide a method for regulating autoimmune activation. The Treg augmentation therapies discussed herein focus on rectifying Treg abnormalities by enhancing Treg suppressive activity and/or numbers, increasing Treg migration, causing Treg-dependent B cell destruction or suppression, or enforcing tolerogenic signals from Tregs. Therapies which increase Treg suppressive activity and migration may be particularly useful for MS, whereas MG Treg augmenting therapies should focus on controlling autoreactive B cells. Finally, because these Treg therapeutics appear to act via different mechanisms (Fig. 1), it might be possible to provide synergistic benefits if combined with other tested pre-clinical treatments in MS or MG.