Induce T-cell Differentiation Research Articles

Short-chain fatty acids ameliorate allergic airway inflammation via sequential induction of PMN-MDSCs and Treg cells.

Reinforcement of the immune-regulatory pathway is a feasible strategy for prevention and therapy of allergic asthma. The short-chain fatty acids (SCFAs) acetate, propionate, and butyrate are pleiotropic microbial fermentation products known to induce regulatory T (Treg) cells and exert an immune-regulatory effect. The cellular mechanism underlying SCFA immune regulation in asthma is not fully understood. We investigated the role of myeloid-derived suppressor cells (MDSCs) and Treg cells, the immune-regulatory cells of innate and adaptive origin, respectively, in SCFA-elicited protection against allergic airway inflammation. BALB/c mice were given SCFA-containing drinking water before being rendered asthmatic in response to ovalbumen. When indicated, mice were given a GR1-depleting antibody to investigate the function of MDSCs in allergic inflammation of the airways. MDSCs were sorted to examine their immunosuppressive function and interaction with T cells. The mice receiving SCFAs developed less severe asthma that was accompanied by expansion of PMN-MDSCs and Treg cells. Mice depleted of PMN-MDSCs exhibited aggravated asthma, and the protective effect of SCFAs was abrogated after PMN-MDSC depletion. SCFAs were able to directly induce T-cell differentiation toward Treg cells. Additionally, we found that PMN-MDSCs enhanced Treg cell expansion in a cell contact-dependent manner. Whilst membrane-bound TGF-β has been shown to induce Treg cell differentiation, we found that MDSCs upregulated surface expression of TGF-β after coculture with T-cells and that MDSC-induced Treg cell differentiation was partially inhibited by TGF-β blockage. Although previous studies revealed Treg cells as the effector mechanism of SCFA immune regulation, we found that SCFAs ameliorate allergic airway inflammation by relaying immune regulation, with sequential induction of PMN-MDSCs and Treg cells.

Orally Administered Brain Protein Combined With Probiotics Increases Treg Differentiation to Reduce Secondary Inflammatory Damage Following Craniocerebral Trauma.

Craniocerebral trauma is caused by external forces that can have detrimental effects on the vasculature and adjacent nerve cells at the site. After the mechanical and structural primary injury, a complex series of secondary cascades of injury exacerbates brain damage and cognitive dysfunction following mechanical and structural primary injury. Disruption of the blood-brain barrier and exposure of brain proteins following craniocerebral trauma, recognition by the immune system triggering autoimmune attack, and excessive secondary inflammatory responses causing malignant brain swelling, cerebral edema, and subsequent brain cell apoptosis provide a new direction for the suppression of brain inflammatory responses in the treatment of craniocerebral trauma. We observed that CD4+T/CD8+T in peripheral blood T cells of craniocerebral trauma rats were significantly higher than those of normal rats, and the ratio of CD4+CD25+Foxp3 (Foxp3)+Regulatory T cell (Treg) was significantly lower than that of normal rats and caused increased secondary inflammation. We constructed a rat model of post-surgical brain injury and orally administered brain protein combined with probiotics, which was observed to significantly reduce CD4+T/CD8+T and induce T-cell differentiation into CD4+CD25+Foxp3+Treg, thus, reducing secondary inflammatory responses following craniocerebral trauma. However, collecting intestinal stool and small intestinal tissues for broad target metabolomics, 16s rRNA bacteriomics, and the combined analysis of intestinal tissue proteomics revealed that oral administration of brain protein combined with probiotics activates glycerophospholipid and vitamin B6 metabolic pathways to promote the production of CD4+CD25+Foxp3+Treg. Therefore, we propose the novel idea that oral administration of brain protein combined with probiotics can induce immune tolerance by increasing Treg differentiation, thus, reducing secondary inflammatory injury following craniocerebral trauma.

Abstract 1785: Inhibition of the IDO-Kyn-AhR cascade optimizes virotherapy in glioblastoma and depends on CD4 T-cell activation

Abstract Immune enhancement of virotherapy by reshaping the tumor immune landscape may improve its success rates. IDO, an IFNγ inducible tryptophan catabolizing enzyme, is upregulated in glioblastoma, correlating with poor prognoses. IDO-mediated tryptophan depletion in the tumor-microenvironment decreases viral replication and diminishes cell proliferation and induces apoptosis of surrounding effector T-cells. Furthermore, kynurenine (kyn), a metabolite of tryptophan, induces T-cell differentiation into Tregs. Excess kynurenine also elicits AhR-mediated lymphocyte dysfunction and immunosuppression. The immune stimulating effect of oncolytic-virus, D24-RGDOX, clinically known as DNX-2440, triggers IFNγ production contributing to a positive IDO-Kyn-AhR feedback loop. Thus, we hypothesized that combining D24-RGDOX with IDO inhibitors will synergize to treat glioblastoma. We studied the IDO-Kyn-AhR cascade in response to D24-RGDOX infection in human and murine glioblastoma cells. Orthotopic syngeneic glioblastoma mouse models were used to determine efficacy and tumor immune effects of D24-RGDOX and IDO inhibitors as single agents or as a combination treatment. We characterized IDO and AhR in D24-RGDOX infected cancers using immunofluorescence, qRT-PCR, and flow cytometry and found increased expression of both proteins in vitro and in vivo compared to mock infected controls. D24-RGDOX also increased activity of AhR in cancer cells as indicated by an AhR responsive elements transcription assay. Furthermore, we showed that viral protein expression and viral activity was enhanced in D24-RGDOX infected cultures under IDO inhibition. To test the therapeutic efficacy of combining D24-RGDOX with IDO inhibitors, we used a murine immunocompetent glioblastoma model; the combination produced 30% more long-term survivors compared D24-RGDOX alone (P=0.03, log-rank test). Next, lymphocytic depletion studies illustrated that this anticancer effect was dependent on CD4+ T-cell activation. We observed 100% survival in the re-challenged long-term glioblastoma survivors, indicating the establishment of immune memory by the combination. Functional studies showed significant increases in anti-tumor activity of splenocytes from combination-treated mice compared to D24-RGDOX-treated mice (P=0.009, student t-test). Flow cytometry studies revealed that combination-treated mice yielded the highest levels of chronically activated T-cells and lowest levels of Tregs and MDSCs compared to D24-RGDOX alone (P≤0.05 in all populations, student t-test). This microenvironment remodeling correlated with complete tumor elimination. Altogether, D24-RGDOX activates the IDO-Kyn-AhR cascade in gliomas, identifying new targets, which when inhibited have the potential to enhance the anti-glioma effect of oncolytic-viruses by reversing tumor immunosuppression. Citation Format: Teresa T. Nguyen, Dong Ho Shin, Hong Jiang, Derek A. Wainwright, Sagar Sohoni, Sumit Gupta, Ashley Ossimetha, Frederick Lang, Marta Alonso, Candelaria Gomez-Manzano, Juan Fueyo. Inhibition of the IDO-Kyn-AhR cascade optimizes virotherapy in glioblastoma and depends on CD4 T-cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1785.

EXTH-45. DELTA-24-RGDOX ACTIVATION OF THE IDO-KYN-AHR CASCADE IN GLIOBLASTOMA: OLD TARGETS FOR A NEW THERAPY

Abstract Immune enhancement of virotherapy by reshaping the tumor immune landscape may improve its success rates. IDO, an IFNγ inducible tryptophan catabolizing enzyme, is upregulated in glioblastoma, correlating with poor prognoses. IDO-mediated tryptophan depletion in the tumor-microenvironment decreases proliferation and induces apoptosis of surrounding effector T-cells. Kynurenine, a metabolite of tryptophan, induces T-cell differentiation into immunosuppressive Tregs. Excess kynurenine elicits AhR-mediated lymphocyte dysfunction and immunosuppression. The immune stimulating effect of oncolytic-virus, Delta-24-RGDOX, triggers IFNγ production contributing to a positive IDO-Kynurenine-AhR feedback loop. We hypothesized that combining Delta-24-RGDOX with IDO inhibitors will synergize to effectively treat glioblastoma. We characterized IDO and AhR in Delta-24-RGDOX infected cancers using immunofluorescence, qRT-PCR, and flow cytometry and found increased expression of both proteins in vitro and in vivo. We also observed induction of AhR in CD4+ and CD8+ T-cells by Delta-24-RGDOX in vivo. Delta-24-RGDOX also increased activity of AhR in cancer cells as indicated by an AhR responsive elements transcription assay. We used a murine glioblastoma model to test the efficacy of combining Delta-24-RGDOX with IDO inhibitor, 1MT/indoximod; the combination produced 30% more long-term survivors compared Delta-24-RGDOX alone (P=0.03), which we showed, through lymphocytic depletion studies, was dependent on CD4+ T-cell activation. We observed 100% survival in the re-challenged long-term glioblastoma survivors, indicating the establishment of immune memory by the combination. Functional studies showed significant increases in anti-tumor activity of splenocytes from combination-treated mice compared to Delta-24-RGDOX-treated mice (P=0.009). Flow cytometry studies revealed that combination-treated mice yielded the highest levels of chronically activated T-cells and lowest levels of Tregs and myeloid derived suppressor cells compared to Delta-24-RGDOX single treatment (P≤0.05). This microenvironment remodeling correlated with complete tumor elimination. Altogether, Delta-24-RGDOX activates the IDO-Kyn-AhR cascade in gliomas, identifying new targets, which when inhibited have the potential to enhance the anti-glioma effect of oncolytic-viruses by reversing tumor immunosuppression.

Construction of genetically modified Lactococcus lactis that produces bioactive anti-interleukin-4 single-chain fragment variable.

Interleukin 4 (IL-4) is a cytokine that induces T-cell differentiation and the production of antibodies from B cells, and plays a crucial role in the allergic response. Therefore, development of a therapeutic approach against IL-4 signaling is expected to prevent or control Th2-related allergic diseases. IL-4 single-chain fragment variable (scFv), which is a recombinant protein consisting of the Fv region of an IL-4 antibody connected to a flexible peptide linker, is expected to be an inhibitor of IL-4 signaling. In this study, recombinant IL-4 scFv was produced by genetically modified lactic acid bacteria (gmLAB); this system is gaining attention as a type of microbial therapeutics. Recombinant gene expression was confirmed with western blotting, and the IL-4 recognition ability of IL-4 scFv produced by gmLAB was examined with an enzyme-linked immunosorbent assay. The macrophage cell line, Raw264.7, and peritoneal macrophages isolated from C57BL/6 mice were employed for an in vitro IL-4 signaling inhibition assay. IL-4 stimulation increased the mRNA expression of arginase-1, a biomarker of IL-4 signaling in macrophages, but arginase-1 expression was suppressed by IL-4 scFv produced by gmLAB, indicating that IL-4 scFv has IL-4 signaling inhibitory activity. gmLAB that produces bioactive IL-4 scFv that was constructed in this study could be an attractive approach for treating allergic disorders.

Human inflammatory dendritic cells in malignant pleural effusions induce Th1 cell differentiation

Dendritic cells are crucial for the initiation and regulation of immune responses against cancer and pathogens. DCs are heterogeneous and highly specialized antigen-presenting cells. Human DCs comprise several subsets with different phenotypes and functional properties. In the steady state, human DC subsets have been well studied. However, the components of DC subsets and their immune functions during the inflamed setting are poorly understood. We identified and characterized DC subsets in the malignant pleural effusions of NSCLC patients. We analyzed the capacity of these DC subsets to induce T-cell differentiation. We observed the presence of inflammatory DCs (infDCs) and macrophages in the malignant pleural effusions of NSCLC patients, as identified by the CD11C+HLA-DR+CD16−BDCA1+ and CD11C+HLA-DR+CD16+BDCA1− phenotypes, respectively. InfDCs represented approximately 1% of the total light-density cells in the pleural effusion and were characterized by the expression of CD206, CD14, CD11b, and CD1α, which were absent on blood DCs. InfDCs also expressed CD80, although at a low level. As infDCs did not express CD40, CD83 and CD275, they remained functionally immature. We found that TLR agonists promoted the maturation of infDCs. Compared with macrophages, infDCs had a weaker capacity to phagocytose necrotic tumor cell lysates. However, only infDCs induced autologous memory CD4+ T-cell differentiation into Th1 cells. For the first time, we found that infDCs were present in the malignant pleural effusions of NSCLC patients. We conclude that infDCs represent a distinct human DC subset and induce Th1 cell differentiation in the presence of TLR agonists.

Roles of Factor XII in Innate Immunity.

Factor XII (FXII) is the zymogen of serine protease, factor XIIa (FXIIa). FXIIa enzymatic activities have been extensively studied and FXIIa inhibition is emerging as a promising target to treat or prevent thrombosis without creating a hemostatic defect. FXII and plasma prekallikrein reciprocally activate each other and result in liberation of bradykinin. Due to its unique structure among coagulation factors, FXII exerts mitogenic activity in endothelial and smooth muscle cells, indicating that zymogen FXII has activities independent of its protease function. A growing body of evidence has revealed that both FXII and FXIIa upregulate neutrophil functions, contribute to macrophage polarization and induce T-cell differentiation. In vivo, these signaling activities contribute to host defense against pathogens, mediate the development of neuroinflammation, influence wound repair and may facilitate cancer maintenance and progression. Here, we review the roles of FXII in innate immunity as they relate to non-sterile and sterile immune responses.

Ikaros tumor suppressor function includes induction of active enhancers and super-enhancers along with pioneering activity

Ikaros encodes a transcription factor that functions as a tumor suppressor in T-cell acute lymphoblastic leukemia (T-ALL). The mechanisms through which Ikaros regulates gene expression and cellular proliferation in T-ALL are unknown. Re-introduction of Ikaros into Ikaros-null T-ALL cells resulted in cessation of cellular proliferation and induction of T-cell differentiation. We performed dynamic, global, epigenomic, and gene expression analyses to determine the mechanisms of Ikaros tumor suppressor activity. Our results identified novel Ikaros functions in the epigenetic regulation of gene expression: Ikaros directly regulates de novo formation and depletion of enhancers, de novo formation of active enhancers and activation of poised enhancers; Ikaros directly induces the formation of super-enhancers; and Ikaros demonstrates pioneering activity by directly regulating chromatin accessibility. Dynamic analyses demonstrate the long-lasting effects of Ikaros DNA binding on enhancer activation, de novo formation of enhancers and super-enhancers, and chromatin accessibility. Our results establish that Ikaros’ tumor suppressor function occurs via global regulation of the enhancer and super-enhancer landscape and through pioneering activity. Expression analysis identified a large number of novel signaling pathways that are directly regulated by Ikaros and Ikaros-induced enhancers, and that are responsible for the cessation of proliferation and induction of T-cell differentiation in T-ALL cells.

Effects of Pim-1 inhibitor on mouse model of inflammatory bowel disease induced by TNBS

To explore the role of Pim-1 in the pathology of inflammatory bowel disease and the potential effect of Pim-1 inhibitor on treating such disease. Methods: Forty-five BALB/c mice were randomly divided into 5 groups (n=9): A normal control group, a inflammatory bowel disease group, two different dose of Pim-1 inhibitor treatment groups, and steroidhormone treatment group. The model of inflammatory bowel disease was induced by intracolonic administration of 2, 4, 6-trinitrobenzenestdfonic acid (TNBS) and ethanol mixture. Mice were treated with Pim-1 inhibitor [intraperitoneal inject, 5 or 10 mg/(kg.d)] for 5 days and prednisone (intragastric administration, 0.1 mg/d) for 5 days. The DAI, colon length, gross score and pathological grade were evaluated. The expressions of T cell master transcription factors T-box expressed in T cells (T-bet), GATA binding protein 3 (GATA-3), RA orphan receptorγ (RORγt) and forkhead box P3 (Foxp3) were measured by Real-time PCR and Western blot, respectively. Results: Pim-1 inhibitor and prednisone showed therapeutic effect on acute TNBS colitis in vivo. GATA3 and RORγt were significantly up-regulated in acute TNBS colitis (P<0.05). In contrast, the expression of Foxp3 was suppressed in the inflammatory bowel disease group, whereas it did not cause any significant change in T-bet expression (P>0.05). Administration of Pim-1 inhibitor and prednisone resulted in suppression of GATA3, RORγt expression, and the increase of Foxp3 expression (P<0.05). Administration of Pim-1 inhibitor and prednisone resulted in inhibition of T-bet mRNA expression (P<0.05), but only prednisone could inhibit T-bet protein expression (P>0.05). Conclusion: Pim-1 inhibitor significantly suppresses Th2- and Th17-type immune responses. Furthermore, Pim-1 inhibitor could induce T-cell differentiation towards a Treg phenotype. Pim-1 inhibitor has therapeutic effect on acute TNBS colitis.

Inhibiting IL-2 Signaling and the Regulatory T-Cell Pathway Using Computationally Designed Novel Peptides

Regulatory T-cells (TREG) are the gateway to immune function and typically regulate immune cell activation. Cytokines, including interleukin-2 (IL-2), induce T-cell differentiation and promote a regulatory phenotype. Once activated via the IL-2 receptor (IL-2R), a cascade of events in T-cells initiate signal transducer and activator of transcription 5 (STAT5) and Forkhead box P3 (FOXP3) activation which appear to function as important regulators of this immunologic pathway and promote the development and function of TREG cells. In non-Hodgkin lymphoma (NHL), we have found that intratumoral TREG cells are increased in number and suppress immune function. In previous work, we have found that TREG cells inhibit T-cell proliferation, suppress cytokine production and limit effector cell cytotoxicity. We have also shown that increased serum levels of soluble sIL-2Rα is a prognostic factor in NHL and that sIL-2Rα can bind to IL-2 and promote its signaling thereby increasing TREG cell numbers. In this study, we developed a strategy to inhibit the binding of IL-2 to sIL-2Rα with the goal of suppressing the induction of FOXP3 and decreasing TREG cell numbers. To do this, we developed peptides designed to disrupt the interaction between IL2 and sILRα.In collaboration with CMDBioscienceSM, we developed and analyzed 22 peptide compounds derived by structure-based computational design. Initially, we screened each peptide at increasing concentrations using an ELISA assay to test the inhibition of IL-2/IL-2Rα binding by the solubilized peptide. Candidate peptides were then further tested using upregulation of pSTAT5 and FOXP3 in T-cells measured by flow cytometry as a measure of inhibition of IL-2 signaling. The peptides were developed according to different design hypotheses and grouped into different families; the screening ELISA results indicated 4 promising peptides that inhibited IL2/IL2Rα binding (up to 100% inhibition; max peptide concentration of 100uM). These peptides were then used to determine their effect on STAT5 and FOXP3 expression. A lead candidate peptide consistently reduced the expression of FOXP3 and STAT5 expression compared to cells not exposed to peptide. Use of the peptide to disrupt IL-2 signaling inhibited the development of cells with a TREG phenotype.We conclude that structure-based peptide design can be used to identify novel peptide inhibitors that block IL-2/IL-2Rα signaling and inhibit STAT5 and FOXP3 upregulation. These peptides could be used as new therapeutic agents to limit immune suppression by TREG cells and promote a more effective anti-tumor immune response in NHL. DisclosuresNo relevant conflicts of interest to declare.

A plasma microRNA signature as a biomarker for acquired aplastic anemia.

Aplastic anemia is an acquired bone marrow failure characterized by marrow hypoplasia, a paucity of hematopoietic stem and progenitor cells, and pancytopenia of the peripheral blood, due to immune attack on the bone marrow. In aplastic anemia, a major challenge is to develop immune biomarkers to monitor the disease. We measured circulating microRNAs in plasma samples of aplastic anemia patients in order to identify disease-specific microRNAs. A total of 179 microRNAs were analyzed in 35 plasma samples from 13 aplastic anemia patients, 11 myelodysplastic syndrome patients, and 11 healthy controls using the Serum/Plasma Focus microRNA Polymerase Chain Reaction Panel. Subsequently, 19 microRNAs from the discovery set were investigated in the 108 plasma samples from 41 aplastic anemia patients, 24 myelodysplastic syndrome patients, and 43 healthy controls for validation, confirming that 3 microRNAs could be validated as dysregulated (>1.5-fold change) in aplastic anemia, compared to healthy controls. MiR-150-5p (induction of T-cell differentiation) and miR-146b-5p (involvement in the feedback regulation of innate immune response) were elevated in aplastic anemia plasma, whereas miR-1 was decreased in aplastic anemia. By receiver operating characteristic curve analysis, we developed a logistic model with these 3 microRNAs that enabled us to predict the probability of a diagnosis of aplastic anemia with an area under the curve of 0.86. Dysregulated expression levels of the microRNAs became normal after immunosuppressive therapy at 6 months. Specifically, miR-150-5p expression was significantly reduced after successful immunosuppressive therapy, but did not change in non-responders. We propose 3 novel plasma biomarkers in aplastic anemia, in which miR-150-5p, miR-146b-5p, and miR-1 can serve for diagnosis and miR-150-5p for disease monitoring. Clinicaltrials.gov identifiers:00260689, 00217594, 00961064.

Multiparameter monitoring for optimal T-cell adoptive therapy

Adoptive transfer of ex vivo -expanded antigen-specific T cells is a promising therapeutic approach for the treatment of cancer and infectious diseases. Clinical studies have proven the feasibility and potency of this procedure but several limitations need to be overcome before this form of immunotherapy reach its full potential. For instance, the efficacy of the transferred cells is often impeded by terminal effector differentiation and exhaustion acquired during in vitro expansion. This would notably explain the lack of further in vivo proliferation and persistence in many trials. However, the factors that induce T-cell differentiation and functional impairment in culture remain poorly defined. Using the model antigen HA-1, we determined that phenotypic and functional features indicating T-cell exhaustion/dysfunction may not be detected simultaneously and depend on the method of expansion as well as the antigenic repertoire stimulated. Thus, our study has defined critical parameters to monitor in order to optimally differentiate and expand antigen-specific T cells in culture prior to adoptive transfer.

Defining novel parameters for the optimal priming and expansion of minor histocompatibility antigen-specific T cells in culture.

BackgroundAdoptive transfer of minor histocompatibility antigen (MiHA)-specific T cells is a promising therapy for patients with hematological cancers. However, the efficacy of the transferred cells is hampered by the acquisition of terminal effector differentiation and exhaustion features during expansion in vitro thus preventing their function and persistence in vivo. Yet, the factors that induce T-cell differentiation and functional impairment in culture remain poorly defined and are likely to vary depending on the method used for expansion.MethodsUsing the clinically relevant HLA-A0201-restricted MiHA HA-1 as well as reagents and procedures that are readily transferable to a clinical environment, we designed a novel culture protocol and defined how exhaustion features appeared in function of time. The optimal time points for the expansion of “fit” MiHA-specific T cells were delineated using phenotypic and functional assessments including KLRG-1 and PD-1 surface markers as well as Ki67 staining and cytokine secretion assays.ResultsFollowing a priming phase, an enrichment step and a rapid expansion stage, our method generates MiHA-specific T-cell lines. Evidence of phenotypic and functional dysfunction appear in function of culture duration, but display different characteristics following the extension of the priming or rapid expansion phases. While repeated antigen exposure during the priming phase induced the decline of the antigen-specific population and the expression of PD-1 and KLRG-1 on antigen-specific CD8+ T cells, the prolongation of an antigen-free expansion phase induced proliferation arrest and the relative loss of antigen-specific cells without impairing polyfunctional cytokine secretion or inducing PD-1 and KLRG-1 expression. A similar pattern was also observed after stimulating a virus-specific memory repertoire, except for the more rapid acquisition of exhaustion features upon repeated antigen exposure.ConclusionOur results offer novel insights on the impact of culture duration on the acquisition of T-cell exhaustion features. Using a new clinical-compliant protocol, we define critical parameters to monitor in order to optimally differentiate and expand MiHA-specific T cells in culture prior to adoptive transfer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0495-z) contains supplementary material, which is available to authorized users.

Circulating miRNAs in myasthenia gravis: miR-150-5p as a new potential biomarker.

ObjectiveMyasthenia gravis (MG) is a chronic autoimmune disorder where autoantibodies target the nicotinic acetylcholine receptors (AChR+) in about 85% of cases, in which the thymus is considered to play a pathogenic role. As there are no reliable biomarkers to monitor disease status in MG, we analyzed circulating miRNAs in sera of MG patients to find disease-specific miRNAs.MethodsOverall, 168 miRNAs were analyzed in serum samples from four AChR+ MG patients and four healthy controls using Exiqon Focus miRNA polymerase chain reaction (PCR) Panel I + II. Specific accumulation pattern of 13 miRNAs from the discovery set was subsequently investigated in the sera of 16 AChR+ MG patients and 16 healthy controls. All patients were without immunosuppressive treatment. Selected specific miRNAs were further analyzed in the serum of nine MG patients before and after thymectomy to assess the effect of thymus removal on the accumulation of the candidate miRNAs in patient sera.ResultsThree miRNAs were specifically dysregulated in AChR+ MG patient sera samples. Hsa-miR150-5p, which induces T-cell differentiation, as well as hsa-miR21-5p, a regulator of Th1 versus Th2 cell responses, were specifically elevated in MG sera. Additionally, hsa-miR27a-3p, involved in natural killer (NK) cell cytotoxicity, was decreased in MG. Hsa-miR150-5p levels had the highest association with MG and were significantly reduced after thymus removal in correlation with disease improvement.InterpretationWe propose that the validated miRNAs: hsa-miR150-5p, hsa-miR21-5p, and hsa-miR27a-3p can serve as novel serum biomarkers in AChR+ MG. Hsa-miR-150-5p could be a helpful marker to monitor disease severity.

Ovatodiolide inhibits the maturation of allergen-induced bone marrow-derived dendritic cells and induction of Th2 cell differentiation

Ovatodiolide was a unique macrocyclic diterpenoid isolated from the traditional Chinese medicinal herb Anisomeles indica. The present study attempted to examine the ovatodiolide effects on dendritic cell (DC) maturation and immuno-stimulatory activities. The effects of ovatodiolide on DC surface molecule expression, cytokine production, and capacity to induce T-cell differentiation were examined in ovalbumin (OVA)/thymic stromal lymphopoietin (TSLP)-stimulated DCs. Ovatodiolide attenuated the expression of DC surface molecules CD80, CD86, histocompatibility complex (MHC) class II, and Th2 subset of CD4(+) T cells co-stimulatory molecule-OX40 ligand production. Additionally, ovatodiolide suppressed the CD4(+) T cells proliferation, and production of inflammatory cytokines interleukin (IL)-4, IL-5, and tumor necrosis factor (TNF)-α. This study may be useful to develop ovatodiolide as a therapeutic adjuvant.

18β-Glycyrrhetinic acid from licorice root impairs dendritic cells maturation and Th1 immune responses

18β-Glycyrrhetinic acid (β-GA) is a natural triterpenoid compound derived from licorice root. β-GA has been demonstrated to exert antiviral and antitumor effects. However, the effects of the maturation and immunostimulatory functions of dendritic cells (DCs) remain to be clearly elucidated. In this study, we attempted to determine whether β-GA could influence DCs surface molecule expression, antigen uptake capacity, cytokine production and capacity to induce T-cell differentiation. The DCs used in this study were derived from murine bone marrow cells, and were used as immature or lipopolysaccharide (LPS)-stimulated mature DCs. The DCs were then assessed with regard to surface molecules expression, cytokine production, capacity to induce T-cell differentiation and proliferation. β-GA was shown to significantly suppress the expression of surface molecules CD80, CD86, major histocompatibility complex (MHC) class I and MHC class II as well as the levels of interleukin-12 production in LPS-stimulated DCs. Moreover, β-GA-treated DCs showed an impaired induction of the T helper type 1 immune response. These findings provide important understanding of the immunopharmacological functions of β-GA and have ramifications for the development of therapeutic adjuvants for the treatment of DCs-related acute and chronic diseases.

Human Dendritic Cells Stimulated with a Novel Peanut Protein Express High Levels of RALDH2 and Induce RA-Sensitive Genes in Naïve T Cells

Dendritic cells instruct naïve T-cells to differentiate into various effector cells determining immune responses such as allergy or tolerance. Our objective was to identify peanut extract (PE)-induced changes in gene expression in human myeloid dendritic cells (mDC), and assess the role of differentially expressed genes in the induction of T-cell differentiation.

Baicalin From Scutellaria baicalensis Impairs Th1 Polarization Through Inhibition of Dendritic Cell Maturation

Baicalin from Scutellaria baicalensis is a major flavonoid constituent found in the traditional Chinese medicinal herb Baikal skull cap. It has been widely used for the treatment of various diseases such as pneumonia, diarrhea, and hepatitis. Recent studies have demonstrated that baicalin possesses a wide range of pharmacological and biological activities, including anti-inflammatory, anti-microbial, anti-oxidant, and anti-tumor properties. Specifically, its antiinflammatory activity has been estimated in various animal models of acute and chronic inflammation; however, its effects on dendritic cells (DCs) maturation and immuno-stimulatory activities are still unknown. In this study, we attempted to determine whether baicalin could influence DC surface molecule expression, antigen uptake capacity, cytokine production, and capacity to induce T-cell differentiation. Baicalin was shown to significantly suppress the expression of surface molecules CD80, CD86, major histocompatibility complex (MHC) class I, and MHC class II as well as the levels of interleukin-12 production in lipopolysaccharide stimulated DCs. Moreover, baicalin-treated DCs showed an impaired induction of the T helper type 1 immune response and a normal cell-mediated immune response. These findings provide important understanding of the immunopharmacological functions of baicalin and have ramifications for the development of therapeutic adjuvants for the treatment of DCs-related acute and chronic diseases.

The Indoleamine 2,3-dioxygenase Inhibitor 1-methyl-tryptophan Suppresses Mitochondrial Function, Induces Aerobic Glycolysis and Decreases Interleukin-10 Production in Human Lymphocytes

Background: Indoleamine 2,3-dioxygenase (IDO) suppresses adaptive immunity. It is known that IDO induces T-cell differentiation to regulatory T-cells (Treg) through tryptophan depletion and/or kynurenine pathway products. CD4+ effector T-cells require distinct metabolic programs in order to support their function as compared to Treg cells. Furthermore, glucose metabolism is also known to affect B-cell survival and function. The effect of IDO on glucose metabolism of lymphocytes was evaluated by using its inhibitor 1-methyl-DL-tryptophan (1-MT). Methods: Ten healthy volunteers vaccinated against tetanus. Peripheral blood mononuclear cells (PBMC) were cultured with or without tetanus toxoid and/or 1-MT. Cell proliferation was assessed by optical microscopy, glucose uptake by measuring its concentration in the supernatant, aerobic glycolysis by assessing lactate concentration in the supernatant, mitochondrial function by XTT assay, and finally production of Tregs’ signature cytokine IL-10 by means of ELISA. Results: Primarily, IDO decreases glucose uptake by stimulated lymphocytes. Secondly, IDO increases mitochondrial function in stimulated lymphocytes. In addition, IDO decreases aerobic glycolysis in stimulated lymphocytes. Finally, IDO induces the production of the immunosuppressive cytokine IL-10 by stimulated lymphocytes. Conclusion: Considering that cell metabolism plays a significant role in lymphocyte differentiation and function, IDO may exert its immunomodulatory effect by interfering with cell metabolism.

Positive and Negative Regulation of Cellular Immune Responses in Physiologic Conditions and Diseases

The immune system has evolved to allow robust responses against pathogens while avoiding autoimmunity. This is notably enabled by stimulatory and inhibitory signals which contribute to the regulation of immune responses. In the presence of a pathogen, a specific and effective immune response must be induced and this leads to antigen-specific T-cell proliferation, cytokines production, and induction of T-cell differentiation toward an effector phenotype. After clearance or control of the pathogen, the effector immune response must be terminated in order to avoid tissue damage and chronic inflammation and this process involves coinhibitory molecules. When the immune system fails to eliminate or control the pathogen, continuous stimulation of T cells prevents the full contraction and leads to the functional exhaustion of effector T cells. Several evidences both in vitro and in vivo suggest that this anergic state can be reverted by blocking the interactions between coinhibitory molecules and their ligands. The potential to revert exhausted or inactivated T-cell responses following selective blocking of their function made these markers interesting targets for therapeutic interventions in patients with persistent viral infections or cancer.