Interferon-γ Promotes Inflammation and Development of T-Cell Lymphoma in HTLV-1 bZIP Factor Transgenic Mice

Human T-cell leukemia virus type 1 (HTLV-1) is an etiological agent of several inflammatory diseases and a T-cell malignancy, adult T-cell leukemia (ATL). HTLV-1 bZIP factor (HBZ) is the only viral gene that is constitutively expressed in HTLV-1-infected cells, and it has multiple functions on T-cell signaling pathways. HBZ has important roles in HTLV-1-mediated pathogenesis, since HBZ transgenic (HBZ-Tg) mice develop systemic inflammation and T-cell lymphomas, which are similar phenotypes to HTLV-1-associated diseases. We showed previously that in HBZ-Tg mice, HBZ causes unstable Foxp3 expression, leading to an increase in regulatory T cells (Tregs) and the consequent induction of IFN-γ-producing cells, which in turn leads to the development of inflammation in the mice. In this study, we show that the severity of inflammation is correlated with the development of lymphomas in HBZ-Tg mice, suggesting that HBZ-mediated inflammation is closely linked to oncogenesis in CD4⁺ T cells. In addition, we found that IFN-γ-producing cells enhance HBZ-mediated inflammation, since knocking out IFN-γ significantly reduced the incidence of dermatitis as well as lymphoma. Recent studies show the critical roles of the intestinal microbiota in the development of Tregs in vivo. We found that even germ-free HBZ-Tg mice still had an increased number of Tregs and IFN-γ-producing cells, and developed dermatitis, indicating that an intrinsic activity of HBZ evokes aberrant T-cell differentiation and consequently causes inflammation. These results show that immunomodulation by HBZ is implicated in both inflammation and oncogenesis, and suggest a causal connection between HTLV-1-associated inflammation and ATL.     

HTLV-1 bZIP Factor Impairs Anti-viral Immunity by Inducing Co-inhibitory Molecule,T Cell Immunoglobulin and ITIM Domain (TIGIT)

Human T-cell leukemia virus type 1 (HTLV-1) infects CD4⁺ T cells and induces proliferation of infected cells in vivo, which leads to the onset of adult T-cell leukemia (ATL) in some infected individuals. The HTLV-1 bZIP factor (HBZ) gene, which is encoded in the minus strand of HTLV-1, plays critical roles in pathogenesis. In this study, RNA-seq and ChIP-seq analyses using HBZ transduced T cells revealed that HBZ upregulates the expression and promoter acetylation levels of a co-inhibitory molecule, T cell immunoglobulin and ITIM domain (TIGIT), in addition to those of regulatory T cells related genes, Foxp3 and Ccr4. TIGIT was expressed on CD4⁺ T cells from HBZ-transgenic (HBZ-Tg) mice, and on ATL cells and HTLV-1 infected CD4⁺ T cells of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in vivo. Expression of Blimp1 and IL-10 was upregulated in TIGIT⁺CD4⁺ cells of HBZ-Tg mice compared with TIGIT^-CD4⁺ T cells, suggesting the correlation between TIGIT expression and IL-10 production. When CD4⁺ T cells from HBZ-Tg mice were stimulated with TIGIT’s ligand, CD155, their production of the inhibitory cytokine IL-10 was enhanced. Furthermore, dendritic cells from HBZ-Tg mice produced high levels of IL-10 after stimulation. These data suggest that HBZ alters immune system to suppressive state via TIGIT and IL-10. Importantly, TIGIT suppressed T-cell responses to another HTLV-1 virus protein, Tax, in vitro. Blocking of TIGIT and PD-1 slightly increased anti-Tax T-cell activity in some HAM/TSP patients. These results suggest that HBZ-induced TIGIT on HTLV-1 infected cells impairs T-cell responses to viral antigens. This study shows that HBZ-induced TIGIT plays a pivotal role in attenuating host immune responses and shaping a microenvironment favorable to HTLV-1.     

Abnormally High Levels of Virus-Infected IFN-γ+CCR4+CD4+CD25+ T Cells in a Retrovirus-Associated Neuroinflammatory Disorder

Background

Human T-lymphotropic virus type 1 (HTLV-1) is a human retrovirus associated with both HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which is a chronic neuroinflammatory disease, and adult T-cell leukemia (ATL). The pathogenesis of HAM/TSP is known to be as follows: HTLV-1-infected T cells trigger a hyperimmune response leading to neuroinflammation. However, the HTLV-1-infected T cell subset that plays a major role in the accelerated immune response has not yet been identified.

Principal Findings

Here, we demonstrate that CD4⁺CD25⁺CCR4⁺ T cells are the predominant viral reservoir, and their levels are increased in HAM/TSP patients. While CCR4 is known to be selectively expressed on T helper type 2 (Th2), Th17, and regulatory T (Treg) cells in healthy individuals, we demonstrate that IFN-γ production is extraordinarily increased and IL-4, IL-10, IL-17, and Foxp3 expression is decreased in the CD4⁺CD25⁺CCR4⁺ T cells of HAM/TSP patients as compared to those in healthy individuals, and the alteration in function is specific to this cell subtype. Notably, the frequency of IFN-γ-producing CD4⁺CD25⁺CCR4⁺Foxp3⁻ T cells is dramatically increased in HAM/TSP patients, and this was found to be correlated with disease activity and severity.

Conclusions

We have defined a unique T cell subset—IFN-γ⁺CCR4⁺CD4⁺CD25⁺ T cells—that is abnormally increased and functionally altered in this retrovirus-associated inflammatory disorder of the central nervous system.     

Induction of Regulatory T Cells by High-Dose gp96 Suppresses Murine Liver Immune Hyperactivation

Immunization with high-dose heat shock protein gp96, an endoplasmic reticulum counterpart of the Hsp90 family, significantly enhances regulatory T cell (Treg) frequency and suppressive function. Here, we examined the potential role and mechanism of gp96 in regulating immune-mediated hepatic injury in mice. High-dose gp96 immunization elicited rapid and long-lasting protection of mice against concanavalin A (Con A)-and anti-CD137-induced liver injury, as evidenced by decreased alanine aminotransaminase (ALT) levels, hepatic necrosis, serum pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-6), and number of IFN-γ ⁺ CD4⁺ and IFN-γ ⁺ CD8⁺ T cells in the spleen and liver. In contrast, CD4⁺CD25⁺Foxp3⁺ Treg frequency and suppressive function were both increased, and the protective effect of gp96 could be generated by adoptive transfer of Treg cells from gp96-immunized mice. In vitro co-culture experiments demonstrated that gp96 stimulation enhanced Treg proliferation and suppressive function, and up-regulation of Foxp3, IL-10, and TGF-β1 induced by gp96 was dependent on TLR2- and TLR4-mediated NF-κB activation. Our work shows that activation of Tregs by high-dose gp96 immunization protects against Con A- and anti-CD137-induced T cell-hepatitis and provides therapeutic potential for the development of a gp96-based anti-immune hyperactivation vaccine against immune-mediated liver destruction.     

Differential Effects of IL-12 on Tregs and Non-Treg T Cells: Roles of IFN-γ, IL-2 and IL-2R

Complex interactions between effector T cells and Foxp3⁺ regulatory T cells (Treg) contribute to clinical outcomes in cancer, and autoimmune and infectious diseases. Previous work showed that IL-12 reversed Treg-mediated suppression of CD4⁺Foxp3⁻ T cell (Tconv) proliferation. We and others have also shown that Tregs express T-bet and IFN-γ at sites of Th1 inflammation and that IL-12 induces IFN-γ production by Tregs in vitro. To investigate whether loss of immunosuppression occurs when IFN-γ is expressed by Tregs we treated mouse lymphocyte cultures with IL-12. IFN-γ expression did not decrease the ability of Tregs to suppress Tconv proliferation. Rather, IL-12 treatment decreased Treg frequency and Foxp3 levels in Tregs. We further showed that IL-12 increased IL-2R expression on Tconv and CD8 T cells, diminished its expression on Tregs and decreased IL-2 production by Tconv and CD8 T cells. Together, these IL-12 mediated changes favored the outgrowth of non-Tregs. Additionally, we showed that treatment with a second cytokine, IL-27, decreased IL-2 expression without augmenting Tconv and CD8 T cell proliferation. Notably, IL-27 only slightly modified levels of IL-2R on non-Treg T cells. Together, these results show that IL-12 has multiple effects that modify the balance between Tregs and non-Tregs and support an important role for relative levels of IL-2R but not for IFN-γ expression in IL-12-mediated reversal of Treg immunosuppression.     

CD4+CD62L+ Central Memory T Cells Can Be Converted to Foxp3+ T Cells

The peripheral Foxp3⁺ Treg pool consists of naturally arising Treg (nTreg) and adaptive Treg cells (iTreg). It is well known that naive CD4⁺ T cells can be readily converted to Foxp3⁺ iTreg in vitro, and memory CD4⁺ T cells are resistant to conversion. In this study, we investigated the induction of Foxp3⁺ T cells from various CD4⁺ T-cell subsets in human peripheral blood. Though naive CD4⁺ T cells were readily converted to Foxp3⁺ T cells with TGF-β and IL-2 treatment in vitro, such Foxp3⁺ T cells did not express the memory marker CD45RO as do Foxp3⁺ T cells induced in the peripheral blood of Hepatitis B Virus (HBV) patients. Interestingly, a subset of human memory CD4⁺ T cells, defined as CD62L⁺ central memory T cells, could be induced by TGF-β to differentiate into Foxp3⁺ T cells. It is well known that Foxp3⁺ T cells derived from human CD4⁺CD25^- T cells in vitro are lack suppressive functions. Our data about the suppressive functions of CD4⁺CD62L⁺ central memory T cell-derived Foxp3⁺ T cells support this conception, and an epigenetic analysis of these cells showed a similar methylation pattern in the FOXP3 Treg-specific demethylated region as the naive CD4⁺ T cell-derived Foxp3⁺ T cells. But further research showed that mouse CD4⁺ central memory T cells also could be induced to differentiate into Foxp3⁺ T cells, such Foxp3⁺ T cells could suppress the proliferation of effector T cells. Thus, our study identified CD4⁺CD62L⁺ central memory T cells as a novel potential source of iTreg.     

BDC12-4.1 T-Cell Receptor Transgenic Insulin-Specific CD4 T Cells Are Resistant to In Vitro Differentiation into Functional Foxp3+ T Regulatory Cells

The infusion of ex vivo-expanded autologous T regulatory (Treg) cells is potentially an effective immunotherapeutic strategy against graft-versus-host disease (GvHD) and several autoimmune diseases, such as type 1 diabetes (T1D). However, in vitro differentiation of antigen-specific T cells into functional and stable Treg (iTreg) cells has proved challenging. As insulin is the major autoantigen leading to T1D, we tested the capacity of insulin-specific T-cell receptor (TCR) transgenic CD4⁺ T cells of the BDC12-4.1 clone to convert into Foxp3⁺ iTreg cells. We found that in vitro polarization toward Foxp3⁺ iTreg was effective with a majority (>70%) of expanded cells expressing Foxp3. However, adoptive transfer of Foxp3⁺ BDC12-4.1 cells did not prevent diabetes onset in immunocompetent NOD mice. Thus, in vitro polarization of insulin-specific BDC12-4.1 TCR transgenic CD4⁺ T cells toward Foxp3⁺ cells did not provide dominant tolerance in recipient mice. These results highlight the disconnect between an in vitro acquired Foxp3⁺ cell phenotype and its associated in vivo regulatory potential.     

Prevention or Early Cure of Type 1 Diabetes by Intranasal Administration of Gliadin in NOD Mice

Induction of long-term tolerance to β-cell autoantigens has been investigated both in animal models and in human type 1 diabetes (T1D) in order to prevent the disease. As regards external compounds, the dietary plant protein fraction has been associated with high penetrance of the disease, whereas gluten-free diets prevent T1D in animal models. Herewith we investigated whether intranasal (i.n.) administration of gliadin or gluten may arrest the diabetogenic process. I.n. administration of gliadin to 4-week-old NOD mice significantly reduced the diabetes incidence. Similarly, the insulitis was lowered. Intranasal gliadin also rescued a fraction of prediabetic 13-week-old NOD mice from progressing to clinical onset of diabetes compared to OVA-treated controls. Vaccination with i.n. gliadin led to an induction of CD4⁺Foxp3⁺ T cells and even more significant induction of γδ T cells in mucosal, but not in non-mucosal lymphoid compartments. This prevention strategy was characterized by an increased proportion of IL-10 and a decreased proportion of IL-2, IL-4 and IFN-γ-positive CD4⁺Foxp3⁺ T cells, and IFN-γ-positive γδ T cells, preferentially in mucosal lymphoid organs. In conclusion, i.n. vaccination with gliadin, an environmental antigen with possible etiological influence in T1D, may represent a novel, safer strategy for prevention or even early cure of T1D.     

Conventional and Regulatory CD4+ T Cells That Share Identical TCRs Are Derived from Common Clones

Results from studies comparing the diversity and specificity of the TCR repertoires expressed by conventional (Tconv) and regulatory (Treg) CD4⁺ T cell have varied depending on the experimental system employed. We developed a new model in which T cells express a single fixed TCRα chain, randomly rearranged endogenous TCRβ chains, and a Foxp3-GFP reporter. We purified CD4⁺Foxp3^- and CD4⁺Foxp3⁺ cells, then performed biased controlled multiplex PCR and high throughput sequencing of endogenous TCRβ chains. We identified >7,000 different TCRβ sequences in the periphery of 5 individual mice. On average, ~12% of TCR sequences were expressed by both conventional and regulatory populations within individual mice. The CD4⁺ T cells that expressed shared TCR sequences were present at higher frequencies compared to T cells expressing non-shared TCRs. Furthermore, nearly all (>90%) of the TCR sequences that were shared within mice were identical at the DNA sequence level, indicating that conventional and regulatory T cells that express shared TCRs are derived from common clones. Analysis of TCR repertoire overlap in the thymus reveals that a large proportion of Tconv and Treg sharing observed in the periphery is due to clonal expansion in the thymus. Together these data show that there are a limited number of TCR sequences shared between Tconv and Tregs. Also, Tconv and Tregs sharing identical TCRs are found at relatively high frequencies and are derived from common progenitors, of which a large portion are generated in the thymus.     

Foxp3+ Regulatory T Cells among Tuberculosis Patients: Impact on Prognosis and Restoration of Antigen Specific IFN-γ Producing T Cells

CD4⁺CD25⁺Foxp3⁺ Regulatory T cells (Treg) and programmed death-1 (PD-1) molecules have emerged as pivotal players in immune suppression of chronic diseases. However, their impact on the disease severity, therapeutic response and restoration of immune response in human tuberculosis remains unclear. Here, we describe the possible role of Treg cells, their M. tuberculosis driven expansion and contribution of PD-1 pathway to the suppressive function of Treg cells among pulmonary tuberculosis (PTB) patients. Multicolor flow cytometry, cell culture, cells sorting and ELISA were employed to execute the study. Our results showed significant increase in frequency of antigen-reactive Treg cells, which gradually declined during successful therapy and paralleled with decline of M. tuberculosis–specific IL-10 along with elevation of IFN-γ production, and raising the IFN-γ/IL-4 ratio. Interestingly, persistence of Treg cells tightly correlated with MDR tuberculosis. Also, we show that blocking PD-1/PD-L1 pathway abrogates Treg-mediated suppression, suggesting that the PD-1/PD-L1 pathway is required for Treg-mediated suppression of the antigen-specific T cells. Treg cells possibly play a role in dampening the effector immune response and abrogating PD-1 pathway on Treg cells significantly rescued protective T cell response, suggesting its importance in immune restoration among tuberculosis patients.     

Activation of natural killer T cells inhibits the development of induced regulatory T cells via IFNγ

Recent reports have provided evidence for cross-talk between regulatory T (Treg) cells and natural killer T (NKT) cells. However, it is unclear whether NKT cells play a role in the differentiation of Treg cells. By employing NKT cell-abundant Vα14 TCR transgenic (Tg) and NKT cell-deficient CD1d knock-out (KO) mice, we examined the effects of NKT cells on the in vitro differentiation of induced Treg (iTreg) cells with IL2 and TGFβ. We found that iTreg induction from CD1d KO mice was significantly increased compared to the control. Also, the addition of isolated NKT cells from Vα14 TCR Tg mice to naïve CD4⁺ T cells from CD1d KO mice during iTreg differentiation caused a remarkable reduction of iTreg cells. Through IFNγ neutralization, we showed that this reduction was mediated by IFNγ. Furthermore, the main source of IFNγ during iTreg differentiation was NK1.1⁻CD4⁺Foxp3⁻ T cells. This finding implied that early-activated NKT cells induced Th1-type cells and subsequently underwent apoptosis. Taken together, our results suggest that NKT cells inhibit the in vitro development of iTreg cells by increasing IFNγ.     

Regulatory Function of a Novel Population of Mouse Autoantigen-Specific Foxp3? Regulatory T Cells Depends on IFN-γ, NO,and Contact with Target Cells

Background

Both naturally arising Foxp3⁺ and antigen-induced Foxp3⁻ regulatory T cells (Treg) play a critical role in regulating immune responses, as well as in preventing autoimmune diseases and graft rejection. It is known that antigen-specific Treg are more potent than polyclonal Treg in suppressing pathogenic immune responses that cause autoimmunity and inflammation. However, difficulty in identifying and isolating a sufficient number of antigen-specific Treg has limited their use in research to elucidate the mechanisms underlying their regulatory function and their potential role in therapy.

Methodology/Principal Findings

Using a novel class II MHC tetramer, we have isolated a population of CD4⁺ Foxp3⁻ T cells specific for the autoantigen glutamic acid decarboxylase p286–300 peptide (NR286 T cells) from diabetes-resistant non-obese resistant (NOR) mice. These Foxp3⁻ NR286 T cells functioned as Treg that were able to suppress target T cell proliferation in vitro and inhibit type 1 diabetes in animals. Unexpected results from mechanistic studies in vitro showed that their regulatory function was dependent on not only IFN-gamma and nitric oxide, but also on cell contact with target cells. In addition, separating NR286 Treg from target T cells in transwell assays abolished both production of NO and suppression of target T cells, regardless of whether IFN-γ was produced in cell cultures. Therefore, production of NO, not IFN-gamma, was cell contact dependent, suggesting that NO may function downstream of IFN-gamma in mediating regulatory function of NR286 Treg.

Conclusions/Significance

These studies identified a unique population of autoantigen-specific Foxp3⁻ Treg that can exert their regulatory function dependent on not only IFN-γ and NO but also cell contact with target cells.     

SA-4-1BBL Costimulation Inhibits Conversion of Conventional CD4(+) T Cells into CD4(+)FoxP3(+) T Regulatory Cells by Production of IFN-γ

Tumors convert conventional CD4⁺ T cells into induced CD4⁺CD25⁺FoxP3⁺ T regulatory (iTreg) cells that serve as an effective means of immune evasion. Therefore, the blockade of conventional CD4⁺ T cell conversion into iTreg cells represents an attractive target for improving the efficacy of various immunotherapeutic approaches. Using a novel form of 4-1BBL molecule, SA-4-1BBL, we previously demonstrated that costimulation via 4-1BB receptor renders both CD4⁺and CD8⁺ T effector (Teff) cells refractory to inhibition by Treg cells and increased intratumoral Teff/Treg cell ratio that correlated with therapeutic efficacy in various preclinical tumor models. Building on these studies, we herein show for the first time, to our knowledge, that signaling through 4-1BB inhibits antigen- and TGF-β-driven conversion of naïve CD4⁺FoxP3⁻ T cells into iTreg cells via stimulation of IFN-γ production by CD4⁺FoxP3⁻ T cells. Importantly, treatment with SA-4-1BBL blocked the conversion of CD4⁺FoxP3⁻ T cells into Treg cells by EG.7 tumors. Taken together with our previous studies, these results show that 4-1BB signaling negatively modulate Treg cells by two distinct mechanisms: i) inhibiting the conversion of CD4⁺FoxP3⁻ T cells into iTreg cells and ii) endowing Teff cells refractory to inhibition by Treg cells. Given the dominant role of Treg cells in tumor immune evasion mechanisms, 4-1BB signaling represents an attractive target for favorably tipping the Teff:Treg balance toward Teff cells with important implications for cancer immunotherapy.     

Essential Role of NK Cells in IgG Therapy for Experimental Autoimmune Encephalomyelitis

Intravenous immunoglobulin has long been used in treating autoimmune diseases, although mechanisms remain uncertain. Activating Fcγ receptors are receptors of IgG and reported to be essential in intravenous immunoglobulin (IVIG) therapy. Therefore, we hypothesized natural killer (NK) cells, which express abundant activating Fcγ receptors, are the potential cellular target. In experimental autoimmune encephalomyelitis (EAE), we demonstrated that IgG suppressed disease development in intact, but not in NK cell depleted mice. Adoptive transfer of IgG-treated NK cell could protect mice against EAE, and suppressed interferon γ and interleukin 17 production. The percentage of CD4⁺Foxp3⁺ regulatory T cells was significantly increased. The increase of regulatory T cells was also observed in IgG-treated EAE mice but not in NK cell depleted mice. In vitro experiments confirmed that IgG-treated NK cells enhanced regulatory T cell induction from naïve CD4⁺ T cells. Interestingly, cells from draining lymph nodes produced more interleukin 2 after the adoptive transfer of IgG-treated NK cells. We neutralized interleukin 2 and the induction of CD4⁺Foxp3⁺ T cells by IgG-treated NK cells was significantly reduced. To our knowledge, we identified for the first time the critical role of NK cells in the mechanism of IgG-induced induction of Treg cells in treatment of autoimmunity.     

CD8+ T Cells Mediate Female-Dominant IL-4 Production and Airway Inflammation in Allergic Asthma

The prevalence and severity of bronchial asthma are higher in females than in males after puberty. Although antigen-specific CD8⁺ T cells play an important role in the development of asthma through their suppressive effect on cytokine production, the contribution of CD8⁺ T cells to sex differences in asthmatic responses remains unclear. In the present study, we investigated the sex-specific effect of CD8⁺ T cells in the suppression of asthma using an ovalbumin mouse model of asthma. The number of inflammatory cells in bronchoalveolar lavage (BAL) fluid, lung type 2 T-helper cytokine levels, and interleukin-4 (IL-4) production by bronchial lymph node cells were significantly higher in female wild-type (WT) mice compared with male mice, whereas no such sex differences were observed between male and female cd8α-disrupted mice. The adaptive transfer of male, but not female, CD8⁺ T cells reduced the number of inflammatory cells in the recovered BAL fluid of male recipient mice, while no such sex difference in the suppressive activity of CD8⁺ T cells was observed in female recipient mice. Male CD8⁺ T cells produced higher levels of IFN-γ than female CD8⁺ T cells did, and this trend was associated with reduced IL-4 production by male, but not female, CD4⁺ T cells. Interestingly, IFN-γ receptor expression on CD4⁺ T cells was significantly lower in female mice than in male mice. These results suggest that female-dominant asthmatic responses are orchestrated by the reduced production of IFN-γ by CD8⁺ T cells and the lower expression of IFN-γ receptor on CD4⁺ T cells in females compared with males.     

An Oriental Medicine,Hyungbangpaedok-San Attenuates Motor Paralysis in an Experimental Model of Multiple Sclerosis by Regulating the T Cell Response

The preventive and therapeutic mechanisms in multiple sclerosis are not clearly understood. We investigated whether Hyungbangpaedok-san (HBPDS), a traditional herbal medicine, has a beneficial effect in experimental autoimmune encephalomyelitis (EAE) mice immunized with myelin oligodendrocyte glycoprotein peptide (MOG_35-55). Onset-treatment with 4 types of HBPDS (extracted using distilled water and 30%/70%/100% ethanol as the solvent) alleviated neurological signs, and HBPDS extracted within 30% ethanol (henceforth called HBPDS) was more effective. Onset-treatment with HBPDS reduced demyelination and the recruitment/infiltration and activation of microglia/macrophages in the spinal cord of EAE mice, which corresponded to the reduced mRNA expression of pro-inflammatory cytokines (TNF-α, IL–6, and IL–1β), iNOS, and chemokines (MCP–1, MIP–1α, and RANTES) in the spinal cord. Onset-treatment with HBPDS inhibited changes in the components of the blood-brain barrier such as astrocytes, adhesion molecules (ICAM–1 and VCAM–1), and junctional molecules (claudin–3, claudin–5, and zona occludens–1) in the spinal cord of EAE mice. Onset-treatment with HBPDS reduced the elevated population of CD4⁺, CD4⁺/IFN-γ⁺, and CD4⁺/IL–17⁺ T cells in the spinal cord of EAE mice but it further increased the elevated population of CD4⁺/CD25⁺/Foxp3⁺ and CD4⁺/Foxp3⁺/Helios⁺ T cells. Pre-, onset-, post-, but not peak-treatment, with HBPDS had a beneficial effect on behavioral impairment in EAE mice. Taken together, HBPDS could alleviate the development/progression of EAE by regulating the recruitment/infiltration and activation of microglia and peripheral immune cells (macrophages, Th1, Th17, and Treg cells) in the spinal cord. These findings could help to develop protective strategies using HBPDS in the treatment of autoimmune disorders including multiple sclerosis.