Application of iTreg cell in preparation of medicine for preventing or treating autoimmune encephalomyelitis

Abstract

The invention belongs to the technical field of biological medicines, and relates to application of iTreg cells in preparation of a medicine for preventing or treating autoimmune encephalomyelitis, in particular to application of iTreg cells in preparation of a medicine for preventing or treating autoimmune encephalomyelitis with aggravated high-salt diet. The iTregs inhibit pro-inflammatory DCs and promote the pro-inflammatory DCs to be transformed into anti-inflammatory phenotypes through a membrane-bound TGF-beta signal (but not an IL-10R signal), and the function transformation of the DCs may be caused by the fact that the iTreg inhibits an AKT / mTOR signal channel in a DC cell; according to the invention, different mechanisms of the Treg subgroup for regulating and controlling the DCs function are clarified, a new insight is provided for Treg cell treatment in a complex environment, and the wider potential of iTregs in treatment of autoimmune diseases is highlighted.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of iTreg cells in the preparation of drugs for preventing or treating autoimmune encephalomyelitis. Background Art

[0002] Multiple Sclerosis (MS) and its animal model Experimental Autoimmune Encephalomyelitis (EAE) are neurodegenerative diseases characterized by inflammatory cell infiltration and myelin loss caused by auto-activated immune cells acting on the Central Nervous System (CNS). The clinical symptoms of MS mainly include blurred vision, limb numbness, quadriplegia, etc. It mostly occurs in the adolescent population and is difficult to cure, with a high disability rate. It is currently distributed worldwide, and more than millions of people are suffering from this disease. Its incidence in China is also increasing year by year, and it is known as the "cancer that does not die".

[0003] Regulatory T cells (Tregs) are crucial for maintaining immune tolerance and homeostasis, but their stability remains controversial in Treg cell therapy. Thymus-derived regulatory T cells (tTregs) can inhibit the progression of Experimental Autoimmune Encephalomyelitis (EAE). However, in the EAE model aggravated by a high-salt diet (HSD), the inhibitory effect mediated by tTregs is significantly impaired. The reason is that the high-salt environment will weaken the immunosuppressive ability of tTreg cells. Sodium chloride (NaCl), as a basic component of the daily diet, its excessive intake has been proven to promote the differentiation of Th17 cells, ultimately forming a highly pathogenic phenotype, thus exacerbating the condition of EAE. In addition, high-salt intake can also activate the complement system and inflammasomes, leading to the occurrence of salt-sensitive hypertension. The study also found that high salt can induce lipid oxidation in dendritic cells (DCs), further aggravating the symptoms of hypertension. At the same time, the high-salt environment will promote tTregs to secrete IFNγ, impairing their immunosuppressive function, thus exacerbating the condition of experimental graft-versus-host disease. Existing Treg therapies do not distinguish the functional differences between tTregs and iTregs and cannot precisely intervene in salt-sensitive inflammation. In addition, tTregs are prone to transform into other effector T cell subsets, including IFNγ-secreting (Th1) and IL-17A-secreting (Th17) T cells, in the EAE model aggravated by a high-salt diet (HSD) and other disease microenvironments with abnormal autoimmunity, accompanied by the degradation of Foxp3 and the weakening of its immunosuppressive function, resulting in poor treatment effects.

[0004] Therefore, a method is needed to suppress experimental autoimmune encephalomyelitis, especially experimental autoimmune encephalomyelitis exacerbated by a high-salt diet, to solve the above technical problems. Summary of the Invention

[0005] The present invention provides the following technical solutions:

[0006] The application of iTreg cells in the preparation of drugs for preventing or treating autoimmune encephalomyelitis.

[0007] Preferably, the autoimmune encephalomyelitis includes autoimmune encephalomyelitis exacerbated by a high-salt diet.

[0008] The present invention also discloses a drug for preventing or treating autoimmune encephalomyelitis, which contains iTreg cells.

[0009] Preferably, the autoimmune encephalomyelitis includes autoimmune encephalomyelitis exacerbated by a high-salt diet.

[0010] Preferably, the drug uses iTregs as the active ingredient and is added with one or more pharmaceutically acceptable excipients; the excipients include conventional diluents, excipients, fillers, binders, wetting agents, absorption promoters, surfactants, lubricants, stabilizers, flavoring agents, sweetening agents, and pigments in the pharmaceutical field.

[0011] More preferably, the active ingredients of the drug include: iTregs, tolDCs, sirolimus, or SRI-011381; the excipients of the drug include: cryoprotectant, PBS buffer.

[0012] More preferably, the cryoprotectant includes: 10% DMSO + 90% fetal bovine serum.

[0013] Preferably, the drug is made into a pharmaceutically acceptable dosage form, and the dosage form includes: tablets, capsules, granules, oral liquids, sustained-release preparations, nano-preparations, injections, freeze-dried powder preparations.

[0014] The beneficial effects of the present invention are:

[0015] 1. The present invention discloses the application of iTregs in alleviating EAE and the brain inflammation of EAE exacerbated by a high-salt diet (HSD). iTregs inhibit pro-inflammatory DCs and promote their transformation into an anti-inflammatory phenotype through membrane-bound TGF-β signaling (rather than IL-10R signaling). This functional transformation of DCs may be mediated by the inhibition of the AKT / mTOR signaling pathway induced by iTregs. Notably, under high-salt conditions, this regulatory effect is only seen in iTregs, while tTregs instead upregulate the AKT / mTOR of DCs; in addition, the present invention elucidates the different mechanisms by which Treg subsets regulate the function of DCs, provides new insights for Treg cell therapy in complex environments, and highlights the broader potential of iTregs in treating autoimmune diseases.

[0016] 2. The present invention discloses that iTregs cells can effectively resist the inflammation environment induced by high salt. iTreg can resist the transformation of IL-6-induced Th17 cells, thereby alleviating the disease progression of EAE exacerbated by a high-salt diet and reducing side effects; the present invention breaks through the limitations of Treg therapy in salt-sensitive diseases such as EA induced by a high-salt diet. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a comparison chart of IFNγ and IL-17A expression between iTreg cells and tTreg cells of the embodiments of the present invention in high-salt patients in vitro;

[0018] Figure 2 It is a comparison chart of IFNγ and IL-17A expression between iTreg cells and tTreg cells of the present invention in vivo after high-salt pretreatment, a comparison chart of the in vivo stability between iTreg cells and tTreg cells; and a chart of the difference in the inhibition of effector T cell proliferation after their high-salt pretreatment;

[0019] Figure 3 It is a chart of the difference in the therapeutic effect of iTreg cells and tTreg cells of the embodiments of the present invention on EAE;

[0020] Figure 4 It is a chart showing the differences in the antigen-presenting phenotype and anti-inflammatory phenotype of DCs between iTreg cells and tTreg cells of the embodiments of the present invention in the treatment of EAE;

[0021] Figure 5 、 Figure 6 It is a schematic diagram of the therapeutic effect of iTregs of the present invention on EAE and the transformation of the DC antigen-presenting phenotype into an immune tolerance phenotype in EAE. This transformation mainly depends on TGF-β signaling rather than IL-10R signaling;

[0022] Figure 7Schematic diagram of the induction of the transformation of DCs from an antigen - presenting phenotype to an immune - tolerant phenotype by iTregs regulating the AKT / mTOR pathway in the present invention;

[0023] Figure 8 Comparison diagram of different biological characteristics between iTreg cells and tTreg cells of the present invention under high - salt environment and inflammatory conditions. Detailed implementation manners

[0024] The following will clearly and completely describe the related technologies in the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0025] As Figures 1 to 8 shown, the treatment methods and steps in this embodiment include:

[0026] 1. In vitro preparation and function enhancement of iTregs;

[0027] 1.1 Cell source: Isolate CD4 + CD25 + T cells from the peripheral blood of patients or healthy donors.

[0028] For the generation of iTreg cells, naive CD4 + CD44 - CD62L + T cells (purity > 95%) from B6 mice were stimulated with anti - CD3 / CD28 magnetic beads (cell: magnetic bead = 5:1) in the presence of rhTGF - β (2 ng / ml) and rh - IL - 2 (50 U / ml) for 72 hours and cultured in X - VIVO 15 medium. The generated iTregs were further sorted into high - purity iTregs (FoxP3+ > 90%). Then, a total of 0.5x10^6 purified iTregs were intravenously injected into EAE mice induced by high salt.

[0029] 1.2 Function enhancement:

[0030] Add an AKT / mTOR inhibitor (such as sirolimus 1 mg / kg / day) to inhibit the AKT / mTOR level and enhance the tolerance ability of DCs.

[0031] Or add a TGF - β receptor agonist (such as SRI - 011381, dissolved in physiological saline, and intraperitoneally injected into EAE mice at a dose of 30 mg / kg once every 2 days) to enhance the tolerance ability of DCs and synergistically inhibit the expression of Th17 cells.

[0032] 1.3 Drug combination: combination of iTregs and tolerogenic dendritic cells (tolDCs): co - infusing iTregs and tolDCs at a ratio of 1:1 to enhance the therapeutic effect.

[0033] 2. Administration regimen:

[0034] 2.1 Intravenous infusion of iTregs (1×10^6 cells / kg);

[0035] 2.2 Combined intravenous infusion of iTregs (1×10^6 cells / kg) and tolDCs (1×10^6 cells / kg);

[0036] 2.3 Enhancing the therapeutic effect: on the basis of cell therapy, intraperitoneal injection of the AKT / mTOR inhibitor sirolimus (1 mg / kg / day) or the TGF - β receptor agonist (SRI - 011381, dissolved in physiological saline, at 30 mg / kg / 2 days)

[0037] 3. Pharmaceutical composition;

[0038] 3.1 Components

[0039] Active ingredients: iTregs, tolDCs, sirolimus (or SRI - 011381);

[0040] Excipients: cryoprotectant (10% DMSO + 90% fetal bovine serum), PBS buffer;

[0041] Dosage form: suspension for intravenous injection or freeze - dried powder preparation;

[0042] Examples

[0043] 1.1 iTreg can resist inflammation in patients induced by high salt and improve the condition of EAE.

[0044] This experiment confirmed that iTregs cells (rather than tTregs) can effectively resist the inflammatory environment induced by high salt. iTreg can resist the transformation of Th17 cells induced by IL - 6, thus alleviating the disease progression of EAE exacerbated by high - salt diet and reducing side effects.

[0045] 1.2 The combined use of iTregs and DCs can improve EAE.

[0046] It is known that pro-inflammatory DCs (tolDCs) exacerbate the progression of EAE, while tolerogenic DCs play a regulatory role in restoring immune balance and alleviating the pathogenesis of EAE. This experiment demonstrated that iTregs are more capable than tTregs of inducing immune tolerance in DCs, further proving that iTregs are more effective than tTregs in treating EAE. In addition, this experiment also demonstrated that DCs treated with iTregs have a powerful effect in inducing naive T cells to transform into Treg cells. Based on this, it can be speculated that the combined use of Tregs and DCs can be used to treat EAE and improve its condition.

[0047] 1.3 Based on the results of this experiment, it can be speculated that the combined use of iTreg and AKT or mTOR inhibitors can treat EAE caused by high salt.

[0048] This experiment has confirmed that the levels of p-AKT and p-P70S6K in spleen DCs treated with the iTreg subset are significantly decreased, verifying that iTregs may promote the tolerance function of DCs through the AKT / mTOR pathway.

[0049] 2. Materials and Methods:

[0050] 2.1 Mice

[0051] In this experiment, male C57BL / 6J (B6) mice aged 6 - 8 weeks were purchased from the Lanzhou Veterinary Research Institute. B6CCAR2- / - and B6 Foxp3-GFP mice were kindly provided by Professor Song from the Institute of Cell and Gene Therapy, Shanghai Jiao Tong University School of Medicine. B6 Rag1- / - mice were purchased from Cyagen Biosciences Inc. All mice were housed in the SPF-class animal facility of the School of Medicine, Lanzhou University, and all experimental operations complied with the regulations of the Experimental Animal Ethics Committee of Lanzhou University.

[0052] 2.2 Establishment of experimental autoimmune encephalomyelitis (EAE) model and high-salt diet

[0053] After anesthetizing the mice, 250 μg of myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide (Proteimax) was subcutaneously injected into the upper and lower sides of the back. The peptide was completely emulsified in an equal volume of complete Freund's adjuvant (CFA, Sigma, St Louis, MO, USA), and the adjuvant contained 4 mg / ml heat-inactivated Mycobacterium tuberculosis H37Ra (Difco Laboratories, Detroit, MI, USA). At approximately 0 hours and 48 hours after immunization, 500 ng of pertussis toxin (Alexis, San Diego, USA) was intraperitoneally injected. The clinical scores of the mice were evaluated daily, and the average score of each mouse was recorded every three days. In some experiments, two weeks before EAE induction, the mice were fed a high-salt diet containing 6% sodium chloride (ssniff, Germany; Xietong, Jiangsu, China) and were allowed to freely drink tap water containing 1% NaCl. During the entire experimental period, there were no significant differences in the body weight changes of the mice in each group (data not shown), indicating that the food and water intakes of each group were similar. The high-salt diet continued until the mice were sacrificed.

[0054] 2.3 Adoptive T cell transfer experiment

[0055] C57BL / 6 EAE model mice were randomly divided into three groups and were respectively injected with iTregs cells, tTregs cells (both 1×10^6 cells per mouse) or normal saline (as a control) via the tail vein on the 4th day after immunization. To study the immunosuppressive mechanism of Treg subsets in vivo, anti-IL-10 receptor antibody or isotype control IgG1 antibody (0.25 mg / kg body weight), or ALK5 inhibitor (LY-364947, 0.5 mg / mouse; Sigma) was intraperitoneally injected once a week for 3 consecutive times, and the first injection was performed one day after cell infusion. Most experiments included 5 mice in each group, and the experiments were repeated at least twice with consistent results. On the 30th day after MOG injection, all the mice (n = 5 in each group) were sacrificed, and spleen and brain tissue cells were collected. The proportions of Treg cells, Th17 cells, Th1 cells, and CD11c+ dendritic cells (DCs) in the spleen and brain tissue were detected by flow cytometry.

[0056] 2.4 Tissue sampling and immune cell isolation

[0057] On the 30th day, the animals were euthanized by perfusion with 4% (w / v) paraformaldehyde under deep anesthesia (10% chloral hydrate, 0.2 ml / mouse, intraperitoneal injection), and then perfused transcardially with cold phosphate-buffered saline. Subsequently, the brain, spleen, and lymph nodes were collected for further processing. Specifically, the spleen and lymph nodes were placed in a cell strainer (70 μm) and homogenized using a syringe plunger, and then washed with RPMI-1640 containing 2% NCS. The brain tissue was minced and then digested with 2 mg / ml type IV collagenase (Sigma Aldrich) in 1640 containing 2% NCS by stirring at 37 °C for 60 minutes. After digestion, the brain tissue was filtered and then separated by centrifugation (800×g, 20 minutes) using a 30% / 70% Percoll solution (Cytiva). Then the separated cell pellet was collected for staining and flow cytometry procedures.

[0058] 2.5 Histology

[0059] Each group of mice was perfused with 4% (w / v) paraformaldehyde under deep anesthesia induced by intraperitoneal injection of 10% chloral hydrate (0.2 ml / mouse). Subsequently, the brain tissue was carefully extracted and embedded in paraffin, and these sections were dissected and stained with hematoxylin and eosin (H&E) according to the established protocol, and the degree of inflammation was blindly evaluated according to the published criteria. The inflammation score was graded in a semi-quantitative manner: 0, no inflammatory cell infiltration; 1, scattered cell infiltration was visible throughout the field of view; 2, organization of perivascular infiltration, or mild cell infiltration into the parenchyma; perivascular cuffing infiltration and extension to adjacent tissues.

[0060] 2.6 tTreg Isolation, iTreg Generation, and In Vitro and In Vivo Treg Subset Conversion

[0061] Thymus-derived Tregs (tTregs) were isolated from B6 WT, B6 CCAR2− / −, or B6 Foxp3-GFP mice by gating on CD4+CD25− or CD4+GFP+ cells, with a purity of 95 - 99%. Subsequently, these cells were stimulated with anti-CD3 / CD28-coated beads (Invitrogen, Carlsbad, CA) and rhIL-2 (100 U / ml; R&D Systems, Minneapolis, MN) at a ratio of 1 bead:2 cells for 72 hours for expansion. All tTregs used in the in vitro experiments of this study were pre-expanded.

[0062] For the generation of iTreg cells, naive CD4+CD44-CD62L+ T cells (purity > 95%) from WT, CCAR2− / −, or Foxp3-GFP B6 mice were stimulated with anti-CD3 / CD28 dynabeads (cell: bead = 5:1, Invitrogen) in the presence of rhTGF-β (2 ng / ml) and rh-IL-2 (50 U / ml) (both from R&D System) for 72 h. Both Treg subsets were cultured in X-VIVO 15 medium (LONZA) in the presence or absence of NaCl (40 mM, Sigma-Aldrich), IL-6 (100 ng / ml, R&D System), or a combination of both.

[0063] For in vivo Treg subset conversion, freshly isolated tTregs or generated iTregs from Foxp3-GFP reporter mice were further sorted into CD4+GFP+ cells with a purity of 99%. Then, a total of 0.5x106 purified tTregs or iTregs were injected intravenously into Rag1− / − mice. In some groups, tTregs or iTregs were pretreated with 40 mM NaCl and 100 ng / ml IL-6 for 48 h before sorting and adoptive transfer. Mice were sacrificed on days 5 and 10, and T cells from mesenteric lymph nodes (MLN) were stained to detect IFNγ and IL-17A.

[0064] 2.7 In vitro Treg suppression assay

[0065] Initially, tTregs and iTregs were pretreated with or without 40 mM NaCl and 100 ng / ml IL-6 in the presence of anti-CD3 / CD28 beads (5 cells per bead) for 72 h. Subsequently, the pretreated cells were co-cultured with 5×10^5 naive CD4+CD44-CD62L+ T cells (from WT B6 mice) labeled with 2 μM CFSE (BioLegend) at various indicated ratios. The cell mixture was simultaneously stimulated with soluble anti-CD3 (0.5 μg / ml) for 3 days. The division index was calculated by assessing the rate and intensity of CFSE dilution using flow cytometry.

[0066] 2.8 Co-culture of naive T cells with dendritic cell (DC) subsets

[0067] CD11c+ DCs were isolated from the spleens of mice that had been treated with PBS, iTregs, or tTregs on day 30 to induce experimental autoimmune encephalomyelitis (EAE). DC isolation was performed using a mouse CD11c+ cell-specific magnetic bead sorting kit (Miltenyi). To evaluate DC-mediated T cell proliferation, carboxyfluorescein succinimidyl ester (CFSE, 2 μM)-labeled naive T cells from wild-type (WT) B6 strain were co-cultured with different DC subsets and stimulated with anti-CD3 antibody (0.1 μg / ml; BD Pharmingen) for 3 days. T cell proliferation was evaluated by analyzing the dilution of CFSE by flow cytometry (FACS). To evaluate DC-induced Treg cell differentiation, WT CD4+CD44-CD62L+ naive T cells were sorted from B6 Foxp3-GFP reporter mice and co-cultured with different DC subsets in the presence of anti-CD3 antibody (0.5 μg / ml) and recombinant human interleukin 2 (rh-IL2, 50 U / ml) for 3 days. The expression of green fluorescent protein (GFP) was evaluated by flow cytometry.

[0068] 2.9 Co-culture of Treg cells with splenic DCs

[0069] Spleen CD11c+ DCs from WT B6 mice with a purity greater than 90% were cultured in medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF, 500 U / ml) and interleukin 4 (IL-4, 200 U / ml) for 72 hours. Then the DCs were collected, washed, and resuspended in RPMI 1640 medium containing 10% fetal calf serum (FCS) and seeded in 48-well plates. Subsequently, the DCs were co-cultured with WT-tTregs, WT-iTregs, WT-tTregs pretreated with sodium chloride (NaCl), and WT-iTregs pretreated with NaCl at a ratio of 5:1 (Tregs:DCs) and stimulated with anti-CD3 antibody (0.5 μg / ml, BD Pharmingen) for 24 hours. Then, the DCs from each group were collected and subjected to Western blot (WB) analysis.

[0070] 2.10 Flow cytometry analysis

[0071] The collected cells were resuspended in ice-cold phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA), and then stained with labeled antibodies against CD3, CD4, and CD25 (eBioscience) or corresponding isotype control antibodies at 4 °C for 15 - 20 minutes. To evaluate Foxp3 expression, cells were first surface stained and then treated with fixation buffer (Biolegend). Subsequently, the cells were permeabilized using the Foxp3 / Transcription Factor Staining Buffer Kit (eBioscience) and kept at 4 °C for 1 hour. After that, they were incubated with FITC-labeled anti-mouse Foxp3 antibody (FJK-16s, eBioscience) for FACS analysis.

[0072] For intracellular cytokine staining, cultured cells were stimulated in vitro with phorbol ester (PMA) and ionomycin (both at 0.25 μg / ml; Sigma-Aldrich) for 5 hours (37 °C), and brefeldin A (5 μg / ml; BioLegend) was added during the last 4 hours. After surface staining, the cells were fixed and permeabilized using the Transcription Factor Staining Buffer Kit (ThermoFisher Scientific), and then intracellular staining was performed using anti-mouse IFNγ-PE and anti-mouse IL-17A-APC (TC11-18H10) antibodies.

[0073] For DC staining, total splenocytes were pretreated with anti-CD16 / CD32 antibody (BD Biosciences) and then stained with surface antibodies including anti-CD11c, anti-MHCII, anti-CD80, anti-CD86, anti-LAP, anti-B7H4 antibodies (all from eBioscience). To evaluate the expression of phosphorylated Akt (p-Akt) and phosphorylated p70 S6 kinase (p-P70S6K) in DCs, cells stained for surface CD11c were first fixed, then permeabilized, and incubated with monoclonal rabbit antibody against p-Akt (Ser473, Cell Signaling Technology) or phosphorylated-p70 S6 kinase (Thr389) antibody (649704, BioLegend). The production of interleukin 10 (IL-10) in DCs was measured by stimulating total splenocytes with 10 ng / ml PMA and 100 μg / ml ionomycin for 6 hours in the presence of 5 μg / ml monensin. After surface CD11c staining, cells were fixed, permeabilized, and then intracellularly stained using anti-mouse IL-10-APC (JES5-16E3, Biolegend) antibody. All flow cytometry analyses were performed using the following isotype controls: PE Rat IgG2a, Rat IgG2k; APC Rat IgG2a, RatIgG1; PerCP-Cy5.5 Rat IgG2b. Data were analyzed and acquired using FlowJo 10 software.

[0074] 2.11 Western blotting

[0075] DCs co-cultured with different Treg subsets were collected and lysed in pre-warmed RPMI 1640 medium. Briefly, total proteins of cultured CD11c+ cells were extracted and transferred onto PVDF membranes. Subsequently, the PVDF membranes were blocked in TBST buffer containing 5% BSA or 5% non-fat milk for 1 hour. Then, the membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies were against phospho-Akt (Ser473), total AKT, phospho-P70S6 kinase (Thr389), total P70S6 kinase, and GAPDH, and all primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The next day, the membranes were incubated with HRP-conjugated secondary antibodies for 1 hour. Protein bands were developed using ECL luminescence reagent (Beyotime, Shanghai, China).

[0076] 2.12 Statistical methods

[0077] All statistical analyses in this example were performed using GraphPad Prism software (Version 5), and the results were expressed as mean ± standard error of the mean (SEM). Inter-group differences were evaluated using two-tailed Student's t-test. Comparison among multiple groups was performed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Statistical significance was defined as p < 0.05 (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant).

[0078] 3. Conclusion:

[0079] 3.1 Under high-salt conditions and pro-inflammatory cytokine stimulation, iTregs showed stronger functional stability and immunosuppressive ability than tTregs.

[0080] CD4+GFP+ cells were sorted from B6 Foxp3-GFP mouse thymocytes (tTregs), and iTreg cells (iTregs) were cultured with tTregs and iTregs cells in the presence of NaCl (40 mM) or IL-6 (100 ng / mL) for 3 days, and then cells producing IFNγ or IL-17A were detected by flow cytometry. Under 40 mM NaCl stimulation, the tTreg subset showed higher levels of IFNγ and IL-17A expression than the iTreg subset ( Figure 1 A, Figure 1 B). In addition, when exposed to IL-6, tTregs also expressed more IL-17A than iTregs ( Figure 1 A, Figure 1 C). In the presence of anti-IFNγ, when each Treg subset was co-stimulated with IL-6 and NaCl, the production of IL-17A in the tTreg subset increased significantly, while the iTreg subset still completely failed to express IL-17A ( Figure 2 A, Figure 2 B). Subsequently, the in vivo stability of the two Treg subsets was compared. Both tTregs and iTregs were pretreated with IL-6 and NaCl for 48 hours and then adoptively transferred into Rag1- / - recipients at a dose of 0.5 × 106 per mouse to evaluate their stability for 10 consecutive days under lymphohematopoietic conditions. GFP-expressing cells in the MLN were quantified on day 5 and day 10 respectively. As Figure 2 C and Figure 2As shown in Figure 2 E Figure 2 F, there was some loss of Foxp3 in both Treg populations, but at day 5 after adoptive transfer, the loss of Foxp3 in iTregs was slightly less than that in tTregs. However, by day 10, approximately 75% of Foxp3 expression was retained in iTregs, especially in iTregs pretreated with NaCl and IL-6. In contrast, the Foxp3 expression of pretreated tTregs was significantly reduced compared with that of untreated tTregs. Along with the change in Foxp3 expression, both tTregs and pretreated tTregs showed strong expression of IL-17A and IFNγ, and the production of these two cytokines by pretreated tTregs was significantly higher than that of untreated tTregs( Figure 2 E、 Figure 2 F). Consistent with the in vitro results, no significant expression of IL-17A and IFNγ was observed in iTregs or pretreated iTregs( Figure 2 E、 Figure 2 F). Subsequently, in this example, carboxyfluorescein (CFSE)-labeled WT B6 mouse spleen CD4+ T cells were co-cultured with Treg subsets at different ratios of T responder cells and Treg cells, with or without pretreatment with (NaCl + IL-6) for 3 days. After adding iTregs or tTregs to T responder cells, CFSE labeling showed that proliferation was significantly inhibited, showing a dose-dependent inhibitory effect. However, the inhibitory function of pretreated tTregs was significantly reduced, while pretreated iTregs maintained their inhibitory ability( Figure 2 G、 Figure 2 H). In addition, the inhibitory effect of pretreated iTregs on T cell proliferation far exceeded that of pretreated tTregs( Figure 2 G、 Figure 2 H). In summary, these results indicate that under the condition of the combination of inflammatory cytokines and sodium chloride, iTreg cells are more stable than tTreg cells and show stronger functionality.

[0081] 3.2 Compared with tTregs, iTregs show superior efficacy in high-salt-induced EAE

[0082] Mice showed disease manifestations on day 12 after EVE induction, initially presenting with tail weakness and subsequently hindlimb and / or forelimb paralysis. Both iTregs and tTregs had significant functional inhibitory effects on the clinical manifestations of EAE, delaying the onset and improving the symptoms( Figure 3 A). H&E staining showed that compared with the disease model group, the level of lymphocyte infiltration in the tTreg or iTreg treatment group was significantly reduced, and the remission of lymphocyte infiltration in the iTreg group was more obvious than that in the tTreg group( Figure 3B). In addition, in this example, it was observed that iTregs were more effective than tTregs in inhibiting lymphocyte infiltration, which was related to the clinical symptoms described in Figure 2 A. In addition, this example analyzed the changes in Th1 and Th17 cells in each group because these subsets represent key pathogenic immune cells during the development of EAE. Flow cytometry analysis showed that compared with the disease model control group, Th17 cells in the spleen ( Figure 3 E, F) and brain ( Figure 3 G, H) of the two treatment groups were significantly inhibited. In particular, compared with the mice treated with tTregs, the Th17 cell population in the brains of the mice treated with iTregs was significantly reduced (p<0.05) ( Figure 3 E and Figure 3 G), indicating an obvious difference in treatment between iTregs and tTregs. Subsequently, compared with the model group, the frequency of Th1 cells in the spleen and brain treated with iTregs was significantly reduced, while the frequency of Th1 cells in the spleen and brain treated with tTregs was not significantly reduced ( Figure 3 F, Figure 3 H). Specifically, compared with iTreg administration, tTreg administration only inhibited the development of Th1 cells in the brain, and the effect was not obvious ( Figure 3 H). Given the key role of DC priming of T lymphocytes in the pathogenesis of EAE, this example analyzed the spleen CD11c+DCs in each group. Both the tTreg and iTreg subsets reduced the frequency of spleen CD11c+DCs, and iTregs showed a more superior therapeutic effect ( Figure 3 I, Figure 3 J). These findings together demonstrate that both tTregs and iTregs can reduce the severity of EAE, and iTregs exhibit stronger inhibitory activity, and it is DCs that are involved in this effect.

[0083] 3.3 iTregs inhibit pro-inflammatory DC phenotypes and promote tolerogenic activity in the EAE environment

[0084] The immunogenicity of dendritic cells (DCs) reflects the balance between pro-inflammatory and anti-inflammatory signals in EAE. Pro-inflammatory DCs exacerbate the progression of EAE, while tolerogenic DCs play a regulatory role in restoring immune balance and alleviating the pathogenesis of EAE: Subsequently, this example evaluated the immunophenotypes and functions of spleen CD11c+DCs in three groups of EAE mice treated with PBS (model-DCs, m-DCs), tTregs (t-DCs), and iTregs (i-DCs). Compared with m-DCs and t-DCs, only the mean fluorescence intensity (MFI) of the co-stimulatory molecule CD80 in i-DCs was significantly reduced ( Figure 4 A,Figure 4 B). Similarly, compared with the control group and mice treated with tTregs, the mean fluorescence intensity (MFI) of another co-stimulatory molecule, CD86, in CD11c+ DCs of mice treated with the iTreg subset was also significantly decreased. The level of CD86 expressed by i-DCs was much lower than that of t-DCs ( Figure 4 A, Figure 4 C). In addition, compared with the model group, the MFI of major histocompatibility complex class II molecules (I-Ad) and activation molecules such as CD40 and CD83 in the Treg treatment group also decreased, but did not reach statistical significance (data not shown) ( Figure 4 A, Figure 4 B). Studies on well-known tolerogenic DC cells have also found that latent-associated peptide (LAP) expressed by i-DCs ( Figure 4 A, Figure 4 D) and the level of intracellular IL-10 ( Figure 4 E) were significantly higher than those of m-DCs, expressed as MFI or frequency, respectively. In t-DCs, significant expression of these two tolerogenic markers was not observed in this example, only an increasing trend was observed ( Figure 4 A, Figure 4 D, Figure 4 E). These findings demonstrate that iTregs are more capable of inducing DC immune tolerance than tTregs, which helps to explain why the iTreg subset is more effective in treating EAE than tTregs.

[0085] When the i-DC subset was co-cultured with CFSE-labeled naive T cells (CD4+CD62L+CD44-), CFSE proliferation was significantly inhibited compared with co-culture with m-DCs or t-DCs ( Figure 3 F, 3G), indicating that i-DCs can inhibit T cell proliferation and promote Treg cell differentiation. Similarly, i-DCs also showed a significant ability to induce the development of Treg cells. Approximately 10% of naive T cells became CD25+GFP+ regulatory T cells ( Figure 4 H, Figure 4 I). This was not observed in the PBS-DC co-culture system. Although t-DCs also significantly produced a large number of Foxp3+ cells, i-DCs had a stronger ability to induce naive T cells to transform into Treg cells compared with t-DCs ( Figure 4 I). Therefore, these results indicate that iTreg-modified DCs have low reactivity and anti-inflammatory functions.

[0086] 3.4 iTregs endow DCs with a tolerogenic phenotype and function mainly through TGF-β rather than IL-10 signaling

[0087] In this example, TGF-β receptor I inhibitor (ALK5) and anti-IL-10R antibody were used in the EAE model. As Figure 2 shown in A, EAE induction and iTreg cell infusion were performed. In addition, some mice receiving iTreg cell treatment were also injected with neutralizing anti-IL-10R antibody (isotype-matched IgG1 antibody as a control) or ALK5 inhibitor (ALK5i). The research results of this example showed that the infusion of iTreg cells had a significant inhibitory effect on the treatment of EAE, manifested as a decrease in clinical score ( Figure 5 A) and brain tissue inflammation ( Figure 5 B). Compared with the model group, the administration of DMSO or IgG1 alone did not affect the disease progression of EAE mice (data not shown). According to the clinical score, the blockade of IL-10 signaling had a significant statistical effect on the efficacy of iTregs. However, the neuroinflammation score remained unchanged after anti-IL-10R blockade, indicating that the role of IL-10 signaling in the immunosuppressive ability of iTreg cell treatment of EAE may be limited ( Figure 5 B). On the contrary, both the clinical score and the pathological inflammation score of the iTreg + ALK5i group were completely reversed compared with the iTreg-only group, indicating that blocking the activity of TGF-β receptor I obviously weakened the protective effect of iTreg cells ( Figure 5 A, Figure 5 B). These results preliminarily indicated that both TGF-β and IL-10 signaling played roles in the inhibitory ability of iTreg cells, and TGF-β signaling was more critical. In this example, FACS was also used to detect the presence of Th1 and Th17 cells in the brain. The infusion of iTreg significantly inhibited the differentiation of Th17 cells in the brain. However, compared with the iTreg infusion group, the combined administration of ALK5i restored the Th17 frequency in the brain to the EAE level. At the same time, only in the anti-IL-10R antibody injection group in this example, the inhibition of the iTregs treatment on the Th17 cell frequency showed a reverse trend ( Figure 5 C, Figure 5 D). In addition, there was also a statistically significant decrease in Th1 cells producing IFNγ in the brains treated with iTreg. On the contrary, neither ALK5i nor anti-IL-10R could significantly reverse the inhibition of Th1 differentiation by iTreg infusion ( Figure 5 C, Figure 5 E). These experimental observations indicated that TGF-β signaling played a dominant role in the immunosuppressive function of iTregs against EAE, while IL-10R signaling, even if it played a role, only played a partial role.

[0088] In this example, the frequencies of splenic DCs in each group of EAE mice were detected on the 30th day after immunization. ALK5i could completely alleviate this phenomenon, but anti-IL-10R could not ( Figure 5 F). In splenocytes treated with PBS, CD11c+ DCs expressed baseline levels of CD80, CD86, and LAP. DCs from mice treated with iTregs showed downregulation of CD80 and CD86 ( Figure 6 A, Figure 6 B), as well as upregulation of LAP expression ( Figure 6 A, Figure 6 C). Compared with splenocytes treated with iTregs, co-administration of ALK5i could significantly upregulate the expression of CD80 and CD86 ( Figure 6 A, Figure 6 B), while reducing the expression of LAP ( Figure 6 A, Figure 6 C). However, no statistically significant changes in these molecules were observed in the group co-administered with anti-IL-10R ( Figure 6 A- Figure 6 C). Next, this example conducted a series of DC-T cell co-culture experiments to evaluate the effects of ALK5i and anti-IL10R on the tolerogenic activity of these in vivo-modified DCs. On the 30th day, CD11c+ DCs were sorted from the spleens (splenocytes) of each EAE group. Using the same Treg polarization conditions as Figure 3 described, these DCs were co-cultured with naive B6 T cells. As Figure 5 shown in G, compared with DCs sorted from EAE mice treated with PBS, iTreg-DCs induced up to 10% of naive T cells to start expressing Foxp3. However, splenic DCs in the group co-administered with ALK5i significantly lost their tolerogenic activity, resulting in almost no formation of CD4+Foxp3+ cells. However, DCs in the anti-IL10-treated group showed a certain degree of tolerogenic function, although the generation of CD4+Foxp3+ cells was statistically lower compared with iTreg-DCs ( Figure 5 G, 5H). Another approach was to evaluate CFSE dilution using a co-culture system. The proliferation of WT B6 naive T cells co-cultured with iTreg-DCs was much lower than that of cells co-cultured with PBS-DCs. Similarly, naive T cells co-cultured with DCs co-administered with ALK5i also showed significant proliferative ability, with a level similar to that of naive T cells co-cultured with PBS-DCs. In contrast, compared with iTreg-DCs, anti-IL10R-co-administered DCs retained some immunosuppressive function, but this was not statistically significant, as reflected in the inhibition of CFSE proliferation ( Figure 5 I and Figure 5J). In summary, these data revealed that iTregs induced the formation of tolerogenic DCs mainly through membrane-bound TGF-β signaling rather than IL-10 signaling.

[0089] 3.5 iTreg cells acquire the ability to tolerate DCs by inhibiting the AKT / mTOR pathway

[0090] The AKT / mTOR signaling pathway has been well established to be involved in the inflammatory and maturation processes of DCs. P70S6K is one of the most important substrates of mTOR signal transduction, contributing to the regulation of the production of inflammatory cytokines in DCs and participating in its antigen presentation and pro-inflammatory functions. To investigate whether iTregs alter the immunosuppressive ability of splenic DCs through the AKT / mTOR signal, the levels of total AKT (t-AKT), phosphorylated AKT (p-AKT), total P70S6K (t-P70S6K), and phosphorylated P70S6K (p-P70S6K) in splenic DCs treated with iTregs and tTregs were compared. By WB detection, the expressions of total AKT and P70S6K in DC cells treated with iTregs or tTregs were similar ( Figure 7 A, Figure 7 B). However, p-AKT and p-P70S6K decreased significantly only in the iTreg-treated group, and these differences were statistically significant ( Figure 7 A, Figure 7 C). Given that high salt and / or inflammatory cytokines can significantly affect the stability and function of the two Treg subsets in vitro and in vivo, in this example, the phenotypes of DC cells co-cultured with Treg subsets, which were pre-treated with NaCl and IL-6 in combination, were immediately evaluated. Compared with pre-treated tTregs, pre-treated iTregs could significantly inhibit the expressions of p-Akt and p-P70S6K on DC cells ( Figure 7 A, Figure 7 E). No statistical differences in t-Akt and t-P70S6K were found between the two groups in this example ( Figure 7 A, Figure 7 D). In summary, these findings suggest that the AKT / mTOR pathway may play an important role in mediating the inhibitory effect of iTregs on the immunomodulatory activity of DCs. In addition, compared with the tTreg subset, the iTreg subset showed stronger inhibitory activity in promoting the tolerogenic function of DCs. This excellent inhibitory ability may be achieved by inhibiting the AKT / mTOR signaling pathway.

[0091] 3.6 iTregs (rather than tTregs) have a therapeutic effect on EAE mice by reducing brain inflammation caused by a high-salt diet

[0092] A high-salt diet exacerbates the progression of EAE. Previous studies have identified a population of pro-inflammatory tTreg cells that are altered by high salt. These cells are characterized by the secretion of IFNγ and have been found to be dysfunctional both in vitro and in vivo. However, this example demonstrates that the TGF-β-induced iTreg population is highly stable and functional under high-salt conditions. Therefore, to determine the different immunosuppressive capacities of these two Treg subsets in regulating EAE mice fed a high-salt diet, this example conducted an adoptive transfer study using a treatment protocol. As Figure 8 shown in A, this example observed that iTregs played a significant inhibitory role in treating high-salt-fed EAE, thereby improving the clinical score. However, the inhibitory effect of tTregs on the disease progression of high-salt-fed EAE mice was significantly reduced ( Figure 8 A). Similarly, the results of detecting IL-17A+ and IFNγ+ in CD4+ GFP-T cells in the brain showed that iTregs significantly reduced the frequencies of Th1 and Th17 cells, while tTregs failed to inhibit these two inflammatory T effector cells ( Figure 8 B, Figure 8 C). This example also detected the frequencies and phenotypes of splenic DCs in each group. Compared with the PBS control group, CD11c+ DCs in the group receiving iTreg administration were significantly reduced. However, administering tTreg only had a slight effect on the frequency of splenic DCs ( Figure 8 D, Figure 8 E). At the same time, iTregs induced a decrease in the expression of CD80 and CD86 on splenic dendritic cells (p<0.05). In contrast, the MFI indices of these two DC maturation markers returned to levels similar to those of the PBS control group after treatment with tTregs ( Figure 8 F and Figure 8 G). In addition, the expression of LAP was enhanced in splenic DCs in the iTreg treatment group, while it was not in the tTreg treatment group ( Figure 8 F, Figure 8 G). In summary, based on the head-to-head comparison experiment, this example verified that the two Treg subsets exhibited different biological characteristics in complex environments such as high-salt environments and inflammatory conditions. Therefore, TGF-β-induced Treg cells may have certain advantages in clinical applications.

[0093] In summary, the present invention elucidates the different mechanisms by which Treg subsets regulate the functions of DCs, provides new insights into Treg cell therapy in complex environments, and highlights the broader potential of iTregs in treating autoimmune diseases.

[0094] It should be emphasized that the above are only the preferred embodiments of the present invention, and there is no limitation to the present invention in any form. Any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. Application of iTreg cells in the preparation of drugs for preventing or treating autoimmune encephalomyelitis.

2. Use of the iTreg cells according to claim 1 in the preparation of a drug for preventing or treating autoimmune encephalomyelitis, characterized in that: The autoimmune encephalomyelitis includes autoimmune encephalomyelitis aggravated by a high-salt diet.

3. A drug for preventing or treating autoimmune encephalomyelitis, characterized in that: The medicament comprises iTreg cells.

4. A drug for preventing or treating autoimmune encephalomyelitis according to claim 3, characterized in that: The autoimmune encephalomyelitis includes autoimmune encephalomyelitis aggravated by a high-salt diet.

5. The drug for preventing or treating autoimmune encephalomyelitis according to claim 3, characterized in that: The drug uses iTregs as an active ingredient and is added with one or more pharmaceutically acceptable excipients; the excipients include conventional diluents, excipients, fillers, adhesives, wetting agents, absorption promoters, surfactants, lubricants, stabilizers, flavoring agents, sweeteners and pigments in the pharmaceutical field.

6. A drug for preventing or treating autoimmune encephalomyelitis according to claim 5, characterized in that: The active ingredients of the drug include: iTregs, tolDCs, sirolimus or SRI-011381; the auxiliary materials of the drug include: cryoprotectant and PBS buffer.

7. A drug for preventing or treating autoimmune encephalomyelitis according to claim 6, characterized in that: The cryoprotectant includes: DMSO+fetal bovine serum.

8. A drug for preventing or treating autoimmune encephalomyelitis according to any one of claims 3 to 7, characterized in that: The drug is prepared into pharmaceutically acceptable dosage forms, including tablets, capsules, granules, oral liquids, sustained-release preparations, nano preparations, injections, and freeze-dried powder preparations.

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