Foxp3 Protein Expression Research Articles

Extraction of koumine from Gelsemium Elegans Benth. and its therapeutic effect on collagen-induced arthritis in mice

This study aimed to investigate the therapeutic effect of koumine on collagen-induced arthritis (CIA) in mice. Koumine was extracted from Gelsemium Elegans Benth. The CIA model was established in Balb/c mice. Forty successfully modeled mice were randomly divided into model group and 2, 4 and 8 mg/kg koumine groups, 10 mice in each group. Another 10 normal mice were selected as control group. The 2, 4 and 8 mg/kg koumine groups were treated with 2, 4 and 8 mg/kg koumine, respectively, for three successive weeks. At the end of treatment, compared with model group, in 4 and 8 mg/kg koumine groups the arthritis index was significantly decreased (P < 0.05), the peripheral blood interleukin-17 level and T helper 17 (Th17) cell percentage were significantly decreased (P < 0.05), the peripheral blood interleukin-10 level and regulatory T (Treg) cell percentage were significantly increased (P < 0.05), the spleen tissue RORγt protein expression level was significantly decreased (P < 0.05), and the spleen tissue Foxp3 protein expression level was significantly increased (P < 0.05). In conclusion, koumine has therapeutic effect on CIA in mice. The mechanism may be related to its regulating the RORγt/Foxp3 expressions, thus correcting the Th17/Treg immune imbalance.

Effects of astragaloside Ⅳ on inflammatory response and percentage of peripheral blood Th17 cells in mice with ulcerative colitis

This study aimed to investigate the anti-inflammatory effect of astragaloside Ⅳ in mice with ulcerative colitis(UC) and its effect on the percentage of peripheral blood T helper(Th17) cells. Following the establishment of UC mouse model with 2% sodium dextran sulfate(DSS), mice in the positive control group and low-and high-dose astragaloside Ⅳ groups were treated with corresponding drugs by gavage. Disease activity index(DAI) was calculated, and serum interleukin-17(IL-17), tumor necrosis factor-α(TNF-α), and transforming growth factor-β(TGF-β) levels were assayed by ELISA. The pathological changes in colon tissue were observed by HE staining, and Th17/regulatory T cells(Treg) ratio in the peripheral blood was determined by flow cytometry. Western blot was conducted for detecting the relative protein expression levels of forkhead box protein P3(Foxp3) and retinoic acid-related orphan nuclear receptor γT(ROR-γt). The findings demonstrated that in normal mice, the colonic structure was intact. The goblet cells were not reduced and the glands were neatly arranged, with no mucosal erosion, bleeding, or positive cell infiltration. In the model group, the colonic mucosal structure was seriously damaged, manifested as disordered arrangement or missing of glands, vascular dilatation, congestion, and massive inflammatory cell infiltration. The pathological injury of colon tissue was alleviated to varying degrees in drug treatment groups. Compared with the normal group, the model group exhibited elevated percentage of Th17 cells, increased IL-17 and TNF-α content, up-regulated relative ROR-γt protein expression, lowered TGF-β, reduced percentage of Treg cells, and down-regulated relative Foxp3 protein expression. The comparison with the model group showed that DAI score, pathological score, percentage of Th17 cells, IL-17 and TNF-α content, and relative ROR-γt protein expression in the positive control group, low-dose astragaloside Ⅳ group, and high-dose astragaloside Ⅳ group were decreased, while TGF-β content, percentage of Treg cells, and relative Foxp3 protein expression were increased. The DAI score, pathological score, percentage of Th17 cells, IL-17 and TNF-α content, and relative ROR-γt protein expression in the low-dose astragaloside Ⅳ group were higher than those in the positive control group, whereas the content of TGF-β, percentage of Treg cells, and relative Foxp3 protein expression were lower. DAI score, pathological score, percentage of Th17 cells, IL-17 and TNF-α content, relative ROR-γt protein expression in the high-dose astragaloside Ⅳ group declined in contrast to those in the low-dose astragaloside Ⅳ group, while the TGF-β content, percentage of Treg cells, and relative Foxp3 protein expression rose. There was no significant difference between the positive control group and the high-dose astragaloside Ⅳ group. Astragaloside Ⅳ is able to inhibit inflammatory response and diminish the percentage of Th17 cells in mice with UC.

Sodium houttuynia alleviates airway inflammation in asthmatic mice by regulating FoxP3/RORγT expression and reversing Treg/Th17 cell imbalance

Synthetized from a natural oil of Houttuynia cordata, sodium houttuynia was reported to have anti-inflammatory effects. The present study aimed to investigate whether sodium houttuynia could alleviate the characteristic airway inflammation and Treg/Th17 cell imbalance of asthma in vivo. Experimental mice with neutrophilic asthma were injected with sodium houttuynia or dexamethasone (alone or in combination) intraperitoneally. The airway reactivity was measured, and bronchoalveolar lavage fluid was collected for cell count. Hematoxylin/eosin and periodic acid–Schiff staining were performed to assess pulmonary inflammation and mucus hypersecretion. Immunohistochemical analysis was conducted to determine the expression of IL-10, IL-17A, FoxP3, and RORγT in the lung tissue, and the serum levels of IL-10 and IL-17A were analyzed by ELISA. The proportion of CD4+CD25+FoxP3+ Treg and Th17 cells within the CD4+ T cell subset of splenocytes was analyzed by flow cytometry. FoxP3 and RORγT mRNA and protein expressions in the lung were analyzed by real-time PCR and western blot, respectively. Overall, sodium houttuynia was found to ameliorate the Treg/Th17 cell imbalance and reduce the airway inflammation, hyperresponsiveness, and mucus hypersecretion by increasing the frequency of CD4+CD25+FoxP3+ Treg cells and the secretion of IL-10, while decreasing the proportion of Th17 cells and IL-17A production. Although the regulatory effect of sodium houttuynia was not as good as that achieved with dexamethasone, combination of the two compounds showed improved inhibitory effects on airway hyperresponsiveness, inflammation, and mucus hypersecretion. Hence, sodium houttuynia may be beneficial for the treatment of asthma.

MiRNA-221-5p suppressed the Th17/Treg ratio in asthma via ROR\u03b3t/Foxp3 by targeting SOCS1

BackgroundThis study was designed to investigate the mechanism and effects of miRNA-221-5p on the T-helper 17 (Th17)/T-regulatory (Treg) ratio in asthma.MethodsBALB/c mice were intranasally challenged with 100 µg OVA on 14 and 21 day. Mice were rechallenged with 2.5% OVA-PBS on 22 and 28 day. Mice were sacrificed using on day 30 under 35 mg/kg pentobarbital sodium. PBMCs were induced vitro model of asthma using 500 ng of lipopolysaccharides (LPS) for 4 h.ResultsThe expression of miRNA-221-5p was reduced in in vivo model, compared sham group. The vitro model of asthma treated with miRNA-221-5p mimic resulted in the reduction of IL-6, IL-17, IL-21 and IL-22 levels, and induction of IL-10, IL-35 and TGF-β levels. In addition, down-regulation of miRNA-221-5p induced the protein expression of suppressor of cytokine signaling 1 (SOCS1) and receptor-related orphan receptor-gamma-t (RORγt) and suppressed that of FOXP3 in in vitro model of asthma. Over-expression of miRNA-221-5p induced the protein expression of FOXP3, and suppressed that of SOCS1 and RORγt in in vitro model of asthma. The inhibition of SOCS1 or RORγt attenuated the effects of anti-miRNA-221-5p on Th17/Treg ratio in asthma.ConclusionmiRNA-221-5p may play critical roles in driving the differentiation of Th17/Treg ratio via RORγt/Foxp3 by Targeting SOCS1, reduced the function of Th17 cells by directly inhibiting RORγt/SOCS1 and promoted the function of Treg cells via Foxp3/ SOCS1 in asthma.

Epigenetic Alteration and its Association With Downregulated FOXP3 Gene in Indian Breast Cancer Patients.

Background: FOXP3 gene, known to be a potential tumor suppressor, has been identified to interact with HER2 in mammary cancer. Moreover, the high expression of FOXP3 serves as a good predictor of the survival of patients in breast cancer, prostate cancer, and gastric cancer. The expression and epigenetic alterations were evaluated in female breast cancer patients. Material and Methods: Expression studies at the mRNA level and protein level were conducted in 140 breast cancer cases by real-time PCR and immunohistochemistry, respectively. Epigenetic studies were also conducted by analyzing the methylation status at the promoter region of the gene using MS-PCR. Results: FOXP3 mRNA expression and protein expression were downregulated in breast cancer patients. The absence of FOXP3 protein expression is significantly associated with promoter methylation, where 70 methylated cases exhibited protein loss (70/95, 73.6%). Statistically, we also found a significant correlation between FOXP3 protein expression and TNM stage, promoter methylation, and histological grade. The methylated FOXP3 cases that did not express protein were also significantly associated with positive lymph node metastasis and HER-2 status. Conclusion: The expression profile of FOXP3 may serve as a prognostic factor. In short, FOXP3 may stand in the most crucial list of biomarkers for breast cancer, bringing compelling results in terms of treatment and management of the disease.

CD98-induced CD147 signaling stabilizes the Foxp3 protein to maintain tissue homeostasis

Regulatory T cell (Treg) stability is necessary for the proper control of immune activity and tissue homeostasis. However, it remains unclear whether Treg stability must be continually reinforced or is established during development under physiological conditions. Foxp3 has been characterized as a central mediator of the genetic program that governs Treg stability. Here, we demonstrate that to maintain Foxp3 protein expression, Tregs require cell-to-cell contact, which is mediated by the CD147-CD98 interaction. As Tregs are produced, CD147, which is expressed on their surface, is stimulated by CD98, which is widely expressed in the physiological environment. As a result, CD147’s intracellular domain binds to CDK2 and retains it near the membrane, leading to Foxp3 dephosphorylation and the prevention of Foxp3 degradation. In addition, the optimal distribution of Foxp3+ Tregs under both pathological and physiological conditions depends on CD98 expression. Thus, our study provides direct evidence that Foxp3-dependent Treg stability is reinforced in the periphery by the interaction between CD147 and CD98 in the surrounding environment. More importantly, Tregs with high CD147 expression effectively inhibit inflammatory responses and maintain Foxp3 stability, which has guiding significance for the application of Tregs in immunotherapy.

Co-Expression of FOXP3FL and FOXP3Δ2 Isoforms Is Required for Optimal Treg-Like Cell Phenotypes and Suppressive Function.

FOXP3 is the master transcription factor in both murine and human FOXP3+ regulatory T cells (Tregs), a T-cell subset with a central role in controlling immune responses. Loss of the functional Foxp3 protein in scurfy mice leads to acute early-onset lethal lymphoproliferation. Similarly, pathogenic FOXP3 mutations in humans lead to immunodysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) syndrome, which are characterized by systemic autoimmunity that typically begins in the first year of life. However, although pathogenic FOXP3 mutations lead to overlapping phenotypic consequences in both systems, FOXP3 in human Tregs, but not mouse, is expressed as two predominant isoforms, the full length (FOXP3FL) and the alternatively spliced isoform, delta 2 (FOXP3Δ2). Here, using CRISPR/Cas9 to generate FOXP3 knockout CD4+ T cells (FOXP3KOGFP CD4+ T cells), we restore the expression of each isoform by lentiviral gene transfer to delineate their functional roles in human Tregs. When compared to FOXP3FL or FOXP3Δ2 alone, or double transduction of the same isoform, co-expression of FOXP3FL and FOXP3Δ2 induced the highest overall FOXP3 protein expression in FOXP3KOGFP CD4+ T cells. This condition, in turn, led to optimal acquisition of Treg-like cell phenotypes including downregulation of cytokines, such as IL-17, and increased suppressive function. Our data confirm that co-expression of FOXP3FL and FOXP3Δ2 leads to optimal Treg-like cell function and supports the need to maintain the expression of both when engineering therapeutics designed to restore FOXP3 function in otherwise deficient cells.

Effect and Mechanism of Epimedium Polysaccharide on Bone Marrow Hematopoietic Function and Th17/Treg Balance in Aplastic Anemia

To observe the effects of Epimedium polysaccharides (EPS) on bone marrow hematopoietic function and Th17/Treg balance in aplastic anemia (AA) mice, and preliminarily explore its therapeutic mechanism. Forty BALB/C mice were randomly divided into control (control), model (model), stanozolol (stanozolol) and epimedium polysaccharide (EPS) group, with 10 mice in each group. Except for the control group, Acetophenazine, Gy irradiation and cyclophosphamide triple application were used to establish AA models for the other groups. After the model was established, the stanozolol group was intragastrically administered with 4 mg/kg stanozolol suspension, the EPS group was intragastrically administered with 100 mg/kg epimedium polysaccharide, while the control group and the model group were given an equal volume of 0.9% sodium chloride solution by gavage once a day, for 14 consecutive days. The automatic animal blood analyzer was used to detect the changes in peripheral blood hemoglobin (Hb), red blood cells (RBC), white blood cells (WBC) and platelets (PLT), flow cytometry was used to detect the proportion of Treg and Th17 cells, the levels of interleukin 2 (IL-2), interleukin 11 (IL-11) and tumor necrosis factor α (TNF-α) were detected by ELISA, the number of nucleated bone marrow cells was counted, HE staining and immunohistochemical staining were used to detect the number, the proliferation and apoptosis of bone marrow cells, Western blot was used to detect the expression of signal transducer and activator of transcription 3 (STAT3), retinoic acid receptor-related orphan receptor γ (RORγt), transducer and activator of transcription 5 (STAT5) and fork head transcription factor 3 (Foxp3). Compared with the model group, the levels of Hb, RBC, WBC and PLT in the peripheral blood of mice in stanozolol and EPS group significantly increased, the proportion of Th17 cells was significantly reduced, and the proportion of Treg cells significantly increased. The levels of IL-2 and TNF-α in serum were significantly reduced (P<0.05), the level of IL-11 significantly increased (P<0.05), the number of bone marrow nucleated cells significantly increased (P<0.05), the positive rate of Ki-67 significantly increased (P<0.05) and the positive rate of Caspase-3 was significantly reduced (P<0.05). At the same time, the protein expression of STAT3 and RORγt significantly decreased, and the protein expression of STAT5 and Foxp3 increased, the difference showed statistically significant (P<0.05). EPS can promote the recovery of bone marrow hematopoietic function in AA mice and improve Th17/Treg imbalance, the mechanism may be related to the inhibition of STAT3/RORγt expression and promotion of STAT5/Foxp3 expression.

Huganbuzure Granule Attenuates Concanavalin-A-Induced Immune Liver Injury in Mice via Regulating the Balance of Th1/Th2/Th17/Treg Cells and Inhibiting Apoptosis.

In Uygur medicine, Huganbuzure granule (HBG) is one of the classical prescriptions for liver protection. However, its role in immune liver injury remains unknown. This study evaluates the effect of HBG on concanavalin-A- (ConA-) induced immune liver injury and investigates its protective underlying mechanism. BALB/c mice were randomly divided into five groups (n = 24 mice per group): control, ConA, 1.6 g/kg HBG + ConA, 3.2 g/kg HBG + ConA, and 6 mg/kg prednisolone + ConA. HBG was intragastrically administrated once daily for ten consecutive days, prior to ConA (20 mg/kg) injection. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), superoxide dismutase (SOD), and malondialdehyde (MDA) in mouse serum were measured after ConA injection. Moreover, liver-related mRNA levels were evaluated by qPCR. The detection of liver-related proteins was assessed by immunohistochemistry and western blot analysis. Compared with the ConA group, HBG reduced the mRNA expression of IL-17A and IFN-γ and the protein expression of T-bet and ROR-γt. In addition, HBG increased the mRNA expression of IL-4 and TGF-β and protein expression of GATA3 and Foxp3, indicating that HBG regulated the balance of Th1/Th2 and Th17/Treg. Furthermore, HBG alleviated immune liver injury by reducing oxidative stress, inhibiting apoptosis, and decreasing the expression of p-JNK, p-ERK, p-p38, p-JAK1, p-STAT1, p-STAT3, and IRF1. Our data suggested that HBG attenuated ConA-induced immune liver injury by regulating the immune balance and inhibiting JAK1/STATs/IRF1 signaling, thereby reducing apoptosis induced by JNK activation. The findings indicate that HBG may be a promising drug for immune liver injury.

Regulatory T cells function in established systemic inflammation and reverse fatal autoimmunity.

The immunosuppressive function of regulatory T (Treg) cells is dependent on continuous expression of the transcription factor Foxp3. Foxp3 loss-of-function or induced ablation of Treg cells results in a fatal autoimmune disease featuring all known types of inflammatory responses with every manifestation stemming from Treg cell paucity, highlighting a vital function of Treg cells in preventing fatal autoimmune inflammation. However, a major question remains whether Treg cells can persist and effectively exert their function in a disease state, where a broad spectrum of inflammatory mediators can either inactivate Treg cells or render innate and adaptive pro-inflammatory effector cells insensitive to suppression. By reinstating Foxp3 protein expression and suppressor function in cells expressing a reversible Foxp3 null allele in severely diseased mice, we found that the resulting single pool of “redeemed” Treg cells normalized immune activation, quelled severe tissue inflammation, reversed fatal autoimmune disease, and provided long-term protection against them. Thus, Treg cells are functional in settings of established broad spectrum systemic inflammation and are capable of affording sustained reset of immune homeostasis.

The Expression of PD-L1 and B7-H4 in Thymic Epithelial Tumor and Its Relationship With Tumor Immune-Infiltrating Cells.

BackgroundPD-L1 and B7-H4 have been reported to be expressed in various malignancies and are considered as promising prognostic factors and potential immunotherapy targets.MethodsWe analyzed the correlation between the expression of PD-L1 and B7-H4 transcriptomes and clinicopathological characteristics in 121 TET patients from The Cancer Genome Atlas (TCGA) database. The immune-infiltration levels in the TET microenvironment were estimated using ssGSEA and quanTiseq algorithms. We collected 80 TET cases from 2008 to 2015. PD-L1、B7-H4、FOXP3 and CD163 protein expression in tumor tissues were detected by immunohistochemistry.ResultsTCGA database showed PD-L1 mRNA levels can predict the OS (P = 0.018) and DFS (P = 0.033) of TET patients. B7-H4 mRNA levels were positively related to the World Health Organization (WHO) pathological classification (P = 0.003) but not correlated with patient prognosis. Immune infiltration analysis showed PD-L1 is positively correlated with Tregs and M2 macrophages, B7-H4 is positively correlated with Tregs. Patients with high PD-L1 and Tregs or M2 macrophages, high B7-H4 and Tregs had a worse prognosis. Immunohistochemistry showed PD-L1 expression was positively correlated with the WHO pathological classification and Masaoka stage (P = 0.025, 0.003) and high PD-L1 expression can predict the poor OS of patients (P = 0.043); B7-H4 was also positively correlated with WHO pathological classification and Masaoka stage (P = 0.036, 0.049). However, B7-H4 expression did not correlate with patient prognosis. Evaluation of co-expression patterns showed TET patients with a high-grade WHO pathological classification harbored a 44.4% co-expression of PD-L1 and B7-H4. In addition, we found the expression level of PD-L1 is positively correlated with FOXP3 and CD163 (P = 0.004, P = 0.029) and B7-H4 is positively correlated with FOXP3 (P = 0.037). High PD-L1 combined with High FOXP3 and High CD163, High B7-H4 combined with High FOXP3 can be used to predict the poor prognosis of TET patients (P = 0.026, 0.031, 0.028, respectively).ConclusionPD-L1 and B7-H4 were related to the aggressiveness of TET and their expression level can indicate the suppressive immune microenvironment. Combined with FOXP3 and CD163, PD-L1 and B7-H4 can indicate a poor prognosis of TET.

Reduced frequency and functional potency of CD49d− T regulatory cells in patients with newly diagnosed type 2 diabetes mellitus

This study set out to check the quantitative and qualitative properties of peripheral CD4+CD25+CD49d− T regulatory (CD49d− Treg) cells in type 2 diabetes mellitus (T2DM) patients.This work comprised 35 newly diagnosed patients and 35 healthy controls (HCs). The frequency of FoxP3 expressing CD49d− Treg cells was determined by flow cytometry. The gene expression of FoxP3 and CD49d was assessed by real-time PCR. Suppression assays with purified CD49d− Treg cells and CD4+CD25− T conventional (Tconv) cells were done by flow cytometry. The supernatants of Tconv/CD49d− Treg co-cultures were tested for IFN-γ, IL-4, IL-17, and IL-10 using ELISA.The frequency of CD49d− Treg cells (by both CD4+CD25+CD49d− and CD4+CD25++CD49d− phenotypes) observed to be reduced in patients versus HCs. In the patients, decreased protein and gene expression of FoxP3 was seen in CD49d− Treg cells. Suppressive potency of CD49d− Treg cells to inhibit Tconv cells proliferation was diminished, and inversely related to fasting plasma glucose and hemoglobin A1c in the patients. Tconv cells from T2DM patients released higher amount of IL-17 and lower concentration of IL-10 versus HCs. In Tconv/CD49d− Tregs co-cultures, decreased IL-17 and increased IL-10 levels were seen in HCs, but not T2DM patients.CD49d− Treg cells from the patients have a fundamental defect and Treg cells fail to inhibit the aggressive inflammatory responses.

Nrf2 Is a Critical Mediator of CD4 T Cell-Induced Acute Graft-Versus-Host Disease

Recent studies have shown that, in contrast to typical proliferating lymphocytes, alloreactive T (allo-T) cells can be specifically killed by apoptosis through compounds that lead to generation of intracellular reactive oxygen species. Nuclear factor erythroid-derived 2-like 2 (Nrf2) is an ubiquitously expressed transcription factor well-known for its role in regulating the cellular redox pathway, but little is known about its involvement in T-cell biology. In this study, we examined the contribution of Nrf2 to allo-T cells in the context of an allogeneic hematopoietic cell transplant (allo-HCT).To study the role of Nrf2 in T cell function, we first assessed the expression pattern of Nrf2 in activated T cells. We found that total cellular and nuclear Nrf2 expression in T cells were significantly increased 24 hours after in vitro α-CD3/CD28 stimulation (p<0.001), as well as in vivo in donor allo-T cells on day 14 in a well-established MHC-disparate murine allo-HSC model (C57BL/6→BALB/c) (p<0.05) (Fig 1). We next examined the effects of Nrf2 deficiency on T cell alloreactivity by transplanting WT or Nrf2-/- donor T cells in MHC-disparate (C57BL/6→BALB/c), as well as MHC-matched, minor antigen mismatched (C57BL/6→LP) murine models. In both models, allo-HCT recipients of Nrf2-/- donor T cells had significantly less acute graft-versus-host disease (GVHD)-induced mortality (p<0.01), morbidity (p<0.001), and intestinal pathology (p<0.05) (Fig 2a). To test if Nrf2 differentially regulates CD4+ versus CD8+ T cell function, we mixed and matched WT versus Nrf2-/- CD4+ and CD8+ T cells for transplant. Compared to allo-HCT recipients of donor WT CD4+: WT CD8+ T cells, we observed similar GVHD mortality in allo-HCT recipients of donor WT CD4+: Nrf2-/- CD8+ T cells, while we saw reduced GVHD mortality in allo-HCT recipients of donor Nrf2-/- CD4+: WT CD8+ T cells (p<0.01) (Fig 2b). These data support the notion that Nrf2 predominately regulates donor CD4+ and is dispensable for CD8+ T cells during allo-activation and the development of GVHD.We assessed if Nrf2 could affect polarization of donor CD4 T cells after allo-HCT, and found a two-fold increase in the proportion and absolute numbers of CD25+Foxp3+ regulatory T cells (Tregs) in the spleens of donor Nrf2-/- T cell allo-HCT recipients on day 7 post transplant (p<0.001) (Fig 3a). We next examined if Nrf2-deficiency results in greater induction of Tregs (iTregs) or stabilization of natural Tregs (nTregs). While we found an equal fraction of WT and Nrf2-/- naïve CD4+ T cells induced towards Tregs in vitro, we saw a significantly greater percentage of donor Nrf2-/- Tregs expressing Helios, a marker for thymus-derived nTregs, compared to donor WT Tregs in allo-HCT recipients on day 7 (p<0.01) (Fig 3b). Recent studies have suggested that Helios is required to stabilize the expression of FoxP3, and thus, the abundance and inhibitory activity of Tregs in inflammatory settings. In keeping with this notion, we discovered an increase in the cellular FoxP3 protein expression within donor Nrf2-/- Tregs in allo-HCT recipients (p<0.01) (Fig 3c). To further elucidate the biological significance of Nrf2 function on nTregs, we removed CD4+CD25+ putative Tregs from the donor inoculum and transplanted only conventional T cells (Fig 3d). We found that the survival advantage with donor Nrf2-/- T cells was abolished when Tregs were depleted from the donor graft, suggesting that Nrf2 expression in donor T cells negatively regulates nTregs during allo-activation, most likely through downregulation of Helios and FoxP3 expression.Finally, to determine if Nrf2 was critical for T-cell mediated GVT activities, we challenged allo-HCT recipients with A20 (B-cell lymphoma) cells. Using bioluminescent imaging, we found that Nrf2-/- allo-T cells were equally efficient in clearing tumor burdens, and along with their mitigated GVHD activities, resulting in a significantly improved survival outcome.Taken together, we identified a novel role of Nrf2 in CD4+ T cells in the context of allo-HCT. Nrf2 negatively regulated CD4+ nTreg through Helios downregulation during GVHD, but appeared to be dispensable in CD8+ mediated antitumor responses, further highlighting the possibility of separating GVT from GVHD activities. Our findings suggest that targeted manipulation of Nrf2 activities in allo-T cells may offer a useful clinical strategy to mitigate GVHD while preserving GVT responses. [Display omitted] DisclosuresJenq:Seres: Research Funding. van den Brink:PureTech Health: Consultancy; Seres: Research Funding; Jazz Pharmaceuticals: Consultancy; Therakos Institute: Other: Speaking engagement.

Allicin regulates Treg/Th17 balance in mice with collagen-induced arthritis by increasing the expression of MEKK2 protein.

To study the role of Allicin in regulating Treg/Th17 ratio in splenic lymphocyte by increasing the expression of MEKK2 protein in MAPK signaling pathway, and to explore the mechanism of immune response in mice with collagen‐induced arthritis (CIA). Mouse CIA model was induced by chicken collagen type II, and experimental mice were randomly divided into NC group, Model group, and Allicin group. HE staining was used to compare the degree of joint pathological damage in mice of each group, and Masson staining to observe the proliferation of collagen tissue in each group. Flow cytometry detected Treg/Th17 ratio in splenic lymphocytes. Furthermore, RT‐PCR and WB were used to detect the mRNA and protein expression of related transcription factors and inflammatory factors Foxp3, ROR‐γt, and IL‐17A, as well as MEK2 protein expression in splenic lymphocytes. The results showed that Allicin treatment could reduce the severity of arthritis and the proliferation of collagen fibers on the surface of cartilage and bone joints in CIA mice. Compared with NC group, Treg decreased and Th17 increased in spleen lymphocyte of Model group (p < .01); after Allicin treatment, Treg increased while Th17 decreased significantly (p < .01). Meanwhile, MEKK2 protein expression in spleen lymphocyte of Model group decreased compared to that in NC group (p < .01), and MEK2 protein expression increased significantly after Allicin treatment (p < .01). To sum up, the present study suggests that MEKK2 protein plays an important role in the pathogenesis of CIA model. In terms of mechanism, Allicin may play a therapeutic role in rheumatoid arthritis (RA) by increasing the expression of MEKK2 protein and affecting Treg/Th17 ratio.

The imbalance of TNF and IL-6 levels and FOXP3 expression at the maternal-fetal interface is involved in adverse pregnancy outcomes in a susceptible murine model of congenital toxoplasmosis

Vertical transmission of Toxoplasma gondii leads to adverse pregnancy outcomes depending on the time at which the infection occurs and the immunological state of the mother. C57BL/6 and BALB/c mice have been described as susceptible and resistant mouse lineages to congenital T. gondii infection, respectively. This study aimed to elucidate the systemic and local cytokine profile of pregnant mice infected with T. gondii and whether the expression of the transcription factor FOXP3, related to T regulatory cells, is associated with the resistance/susceptibility of these lineages of mice in the context of experimental congenital toxoplasmosis. For this purpose, C57BL/6 and BALB/c females were orally infected with the T. gondii ME-49 strain on the day of vaginal plug detection or day 14 of gestation, examined 7 or 5 days later, respectively, as models of early and late pregnancy. Cytokine levels were measured systemically and in the uterus/placenta. Additionally, the uterus/placenta were evaluated macroscopically for resorption rates and histologically for parasite and FOXP3 immunostaining. The FOXP3 protein expression was also evaluated by western blotting assay. It was found that, during early pregnancy, the infection leads to high IFN-γ, TNF and IL-6 levels systemically, with the TNF levels being higher in C57BL/6 mice. At the maternal-fetal interface, the infection induced high levels of IFN-γ in both mouse lineages; however, higher levels were observed in BALB/c, while high TNF and IL-6 levels were found in C57BL/6, but not in BALB/c mice. In contrast, in late gestation, T. gondii interfered less strongly with the cytokine profile. In early pregnancy, a reduction of FOXP3 expression at the maternal-fetal interface of infected mice was also observed, and the reduction was larger in C57BL/6 compared with BALB/c mice. Additionally, the parasite was seldom found in the uterus/placenta. Thus, the worse pregnancy outcomes observed in C57BL/6 mice were associated with higher TNF systemically, and TNF and IL-6 at the maternal-fetal interface, with lower FOXP3 expression.

Advanced Molecular Characterization Using Digital Spatial Profiling Technology on Immunooncology Targets in Methylated Compared with Unmethylated IDH-Wildtype Glioblastoma

Introduction Glioblastoma (GBM) is the most common primary adult brain tumour with a median overall survival (OS) of 12–15 months. Molecular characterization of multiple immunooncology targets in GBM may help target novel immunotherapeutic strategies. We used NanoString GeoMx® Digital Spatial Profiling (DSP) to assess multiple immunooncology protein targets in methylated versus unmethylated IDH-wild-type glioblastoma. Methods NanoString GeoMx® DSP technology uses multiple primary antibodies conjugated to indexing DNA oligos with a UV photocleavable linker. Tissue regions of interest (ROIs) are selected with bound fluorescent antibodies; oligos are released via a UV-mediated linker and quantitated. We used DSP multiplex analysis of 31 immunooncology proteins and controls (CD4, CD14, CD68, CD8A, B7-H3, PD-L1, CD19, FOXP3, CD44, STAT3 (phospho Y705), CD45, Pan Cytokeratin, MS4A1/CD20, CD45RO, PD1, CD3, beta-2 microglobulin, VISTA, Bcl2, GZMB, PTEN, beta-catenin, CD56, Ki-67, STAT3, AKT, p-Akt, S6, Histone H3, IgG Rabbit control, and Mouse IgG control) from ROIs in a cohort of 10 IDH-wild-type glioblastomas (5 methylated and 5 unmethylated). An nCounter platform allowed quantitative comparisons of antibodies between ROIs in MGMT methylated and unmethylated tumours. Mean protein expression counts between methylated and unmethylated GBM were compared using technical and biological replicates. Results The analysis showed 10/27 immunooncology target proteins were significantly increased in methylated versus unmethylated IDH-wild-type glioblastoma tumour core (false discovery rate (FDR) <0.1 by Benjamini–Hochberg procedure). Conclusions NanoString GeoMx® DSP was used to analyse multiple immunooncology protein target expression in methylated versus unmethylated IDH-wild-type glioblastoma. In this small study, there was a statistical increase in CD4, CD14, CD68, CD8A, B7-H3, PDL-1, CD19, FOXP3, CD44, and STAT3 protein expression in methylated versus unmethylated GBM tumour core; however, this requires larger cohort validation. Advanced multiplex immunooncological biomarker analysis may be useful in identifying biomarkers for novel immunotherapeutic agents in GBMs.

Imbalance of Th17 and Tregs in thymoma may be a pathological mechanism of myasthenia gravis

An imbalance in Th17 cells and Tregs may be an important cause of the pathogenesis of thymoma with myasthenia gravis (MG). In this study, 30 patients with simple thymoma and 30 patients with thymoma with MG were analyzed. Flow cytometry analysis of Th17 and Tregs in peripheral blood revealed that the percentages of Th17 in thymoma were lower than those in thymoma with MG, while the percentages of Tregs were higher than those in simple thymoma. Serum cytokine ELISA assays showed that IL-6 levels in simple thymoma were lower than those in MG patients. Further, Th17 and Tregs levels were detected by immunohistochemical double staining of thymoma tissue; the number of positive Th17 cells in thymoma with MG was higher than that in simple thymoma, while positive Tregs showed the opposite results. RORγt protein and mRNA expression in thymoma with MG were both higher than those in simple thymoma. FOXP3 protein and mRNA expression in the thymoma with MG group were lower than those in simple thymoma. The results of coculture of thymoma cells and CD4 + T cells showed that thymoma cells could promote the differentiation of Th17 cells and inhibit the Tregs. Overall, Th17 cells and related transcription factors and cytokines in thymoma with MG patients were higher than those in thymoma patients, whereas, Tregs showed the opposite results, the mechanism may be that thymoma can secrete IL6 and IL21. These findings indicated that imbalances in Th17/Tregs may account for the pathogeny of thymoma with MG.

5-Aminoisoquinolinone, a PARP-1 Inhibitor, Ameliorates Immune Abnormalities through Upregulation of Anti-Inflammatory and Downregulation of Inflammatory Parameters in T Cells of BTBR Mouse Model of Autism.

Autism spectrum disorder (ASD) covers a range of neurodevelopmental disorders involving impairments in communication and repetitive and stereotyped patterns of behavior and reciprocal social interaction. 5-Aminoisoquinolinone (5-AIQ), a PARP-1 inhibitor, has neuroprotective and anti-inflammatory effects. We investigated the influence of 5-AIQ-treatment in BTBR T+ Itpr3tf/J (BTBR) mice as an autism model and used flow cytometry to assess the effect of 5-AIQ on FOXP3, Helios, GATA3, IL-9, IL-10 and IL-17A production by CXCR6+ and CD4+ T cells in the spleen. We also confirmed the effect of 5-AIQ treatment on expression of FOXP3, Helios, GATA3, IL-17A, IL-10, and IL-9 mRNA and protein expression levels in the brain tissue by quantitative PCR and western blotting. Our results demonstrated that 5-AIQ-treated BTBR mice had significantly increased numbers of CXCR6+FOXP3+, CXCR6+IL-10+, and CXCR6+Helios+ cells and decreased numbers of CD4+GATA3+, CD4+IL-9+, and CD4+IL-17A+ cells as compared with those in untreated BTBR mice. Our results further demonstrated that treatment with 5-AIQ in BTBR mice increased expression for FOXP3, IL-10, and Helios, and decreased expression for GATA3, IL-17A, and IL-9 mRNA. Our findings support the hypotheses that 5-AIQ has promising novel therapeutic effects on neuroimmune dysfunction in autism and is associated with modulation of Treg and Th17 cells.

Correlation between FOXP3, CD11c Protein Expression and Prognosis of Patients with Diffuse Large B-cell Lymphoma

To investigate the correlation between FOXP3, CD11c protein expression and the prognosis of patients with diffuse large B-cell lymphoma (DLBCL). This study included 48 patients with DLBCL who were admitted to Jiujiang No.1 People's Hospital and TCM-Integrated Hospital of Southern Medical University from January 2015 to January 2019. The DLBCL tissues removed during the operation were collected as test specimens. The expression of FOXP3 and CD11c protein were detected by immunohistochemistry. The deadline for postoperative follow-up was December 31, 2019, and the patient's short-term efficacy (complete remission, partial remission) and progression-free survival were recorded. FOXP3 protein was positively expressed in the nucleus, mostly focally or diffusely distributed, the FOXP3+ rate was 54.17% (26/48). While, the CD11c protein was positively expressed on the cell membrane, mostly diffusely distributed, and the CD11c+ rate was 60.47% (29/48). The expression of FOXP3 and CD11c protein was no significant relationship with age, sex, site of involvement, and lactate dehydrogenase level in DLBCL patients, but was a significant relationship with clinical stage and international prognostic index score, the difference was statistically significant (P<0.05). That was, the lower the clinical stage of DLBCL patients and international prognostic index score, the higher the positive expression rate of FOXP3 and CD11c protein (r=0.637, r=0.709). One year after surgery, the total effective rate of DLBCL patients with FOXP3+ or CD11c+ expression was significantly higher than that of patients with FOXP3- or CD11c-,expression (P<0.05). By the end of the follow-up, the median progression-free survival of DLBCL patients with FOXP3+ or CD11c+ expression was significantly longer than that of patients with FOXP3- or CD11c+ expression (P<0.05). In some patients with DLBCL, FOXP3 and CD11c expresse positively, and the positive expression rate is related to the clinical stage and international prognostic index score. The positive expression of FOXP3 and CD11c indicate a good prognosis.

Curcumin improves experimentally induced colitis in mice by regulating follicular helper T cells and follicular regulatory T cells by inhibiting interleukin-21.

To determine whether curcumin (Cur) can treat mice with experimentally-induced colitis by regulating follicular helper T cells (Tfh) and follicular regulatory T cells (Tfr) by inhibiting interleukin (IL)-21. In this study, 40 male C57BL/6 mice were randomly grouped into four groups, i.e., normal, trinitrobenzene sulfonic acid (TNBS), TNBS + curcumin, and TNBS + anti-IL-21. Mice with experimental colitis were induced by 100 mg/kg TNBS. The mice in the TNBS + Cur group were treated with 100 mg/kg curcumin for seven days, and mice in the TNBS + anti-IL-21 group were treated with anti-IL-21 (150 μg/mouse) once per week, intraperitoneally, starting on the second day after establishing the experimental colitis model. On day eight, the therapeutic effect of curcumin was evaluated by colon mucosa damage index (CMDI), histological examination, and disease activity index (DAI). Furthermore, the number of CD4 + CXCR5 + PD-1 + Tfh and CD4 + CXCR5 + FoxP3 + Tfr cells were measured by flow cytometry. The mRNA and protein expression of IL-21, Bcl-6, FOXP3, ICOS, and PD-1 in colonic mucosa was detected by reverse transcription polymerase chain reaction and the Western blot technique. Compared with the TNBS group, the DAI, CMDI, histological score, the number of CD4 + CXCR5 + PD-1 + Tfh cells, the expression of IL-21, Bcl-6, ICOS, and PD-1 were significantly decreased in the TNBS + curcumin group and TNBS + anti-IL-21 group; body weight, number of CD4 + CXCR5 + FoxP3 + Tfr cells, and the expression of FoxP3 were observably elevated in the TNBS + curcumin group (all P < 0.05). Curcumin may have a potential therapeutic effect on mice with colitis treated experimentally through regulation of the balance of Tfh and Tfr cells via inhibiting the synthesis of IL-21.