Sodium Selenite Diminished the Regulatory T Cell Differentiation In Vitro.

Sodium selenite modulates the activity of lymphocytes. It negatively regulates the suppressive activity of cells and increases the immune response. In this study, we evaluated whether the regulatory T cell differentiation was modulated by sodium selenite. The percentages of CD4+CD25+Foxp3+, CD4+CD25+, and CD4+CTLA-4+ cells in CD4+ T cells cultures stimulated with IL-2 and TGF-ß in the presence or absence of selenium, in the form of sodium selenite (2.0×10-6M), were evaluated by flow cytometry. The mRNA expression of TET2/3 enzymes and IL-10 was analyzed by RT-qPCR and the levels of IL-10 were measured by an ELISA. We observed a decrease in CD4+CD25+Foxp3+ and CD4+CTLA-4+ cells in presence of selenium. However, normal percentages were reached again after selenium removal. An increase in CD4+CTL4-4+ cells was detected in selenium-primed cell cultures in absence of IL-2 and TGF-ß. In addition, we observed a decrease in TET3 in presence of selenium. Finally, we observed an augment in IL-10 transcription and protein levels and relative expression of TET2 in cultures exposed to selenium. We suggest that selenium reversibly affects the regulatory T cell differentiation in vitro. Likewise, selenium may modulate Treg percentages promoting optimal immune responses and, at the same time, the expression of specific suppressor molecules.

Immunosuppressive activity of non-psychoactive Cannabis sativa L. extract on the function of human T lymphocytes.

BACKGROUND: Cannabis sativa L. extracts (CSE) are used for treating inflammatory conditions, but little is known about their immunomodulatory effects. We investigated a novel CSE with high (14%) CBD and low (0.2%) THC concentration in comparison with pure CBD on primary human lymphocytes. METHODS: Proliferation, cell cycle distribution, apoptosis/necrosis and viability were analysed with standard methods. Genotoxicity was evaluated with the comet-assay. The effect on T lymphocyte activation was evaluated via CD25/CD69 marker expression, degranulation assays and the production of cytokines. The influence on the transcription factors was analysed using Jurkat reporter cell lines. Specific CB2 receptor antagonist SR144528 and TRPV1 receptor antagonist A78416B were used to study the involvement of CB2 or TRPV1 receptors. RESULTS: CSE inhibited the proliferation of activated T lymphocytes in a dose-dependent manner without inducing apoptosis, necrosis, or affecting cell viability and DNA integrity. The inhibitory effect was mediated via the suppression of T lymphocytes activation, particularly by the suppression of CD25 surface marker expression. Furthermore, CSE interferes with the functionality of the T lymphocytes, as indicated by inhibition of degranulation, IL-2, and IFN-γ production. AP-1-and-NFAT-reporter activation was reduced implicating an AP-1-and-NFAT-mediated mode of action. The effects were in part reversed by SR144528 and A78416B, showing that the effects were mainly mediated by CB2 and TRPV1 receptors. CONCLUSION: CSE and CBD have immunomodulatory effects and interfere with the activation and functionality of T lymphocytes. A comparison between CSE and CBD suggests that the immunosuppressive effect of CSE is mostly due to the effect of CBD.

Ginsenoside Rg3 Alleviates Complete Freund's Adjuvant-Induced Rheumatoid Arthritis in Mice by Regulating CD4+CD25+Foxp3+Treg Cells.

Ginsenoside Rg3 (GRg3) is one of the major bioactive ingredients of ginseng, which is not only used as a herbal medicine but also used as a functional food to support body functions. In this study, the beneficial effects of GRg3 on rheumatoid arthritis (RA) mice was evaluated from anti-inflammatory and immunosuppressive aspects. The footpad swelling rate, pathological changes of the ankle joint, and levels of tumor necrosis factor α, interleukin 6, interleukin 10, and tumor necrosis factor ß were used to assess the anti-inflammatory effect of GRg3 on RA mice. Flow cytometric analysis of CD4+CD25+Foxp3+Treg cell percentage and metabolomic analysis based on gas chromatography-tandem mass spectrometry were used to assess the immunosuppressive effect and underlying mechanisms. GRg3 exhibited anti-inflammatory and immunosuppressive effects on RA mice. The potential mechanisms were related to regulate the pathways of oxidative phosphorylation and enhance the function of CD4+CD25+Foxp3+Treg cells to maintain peripheral immune tolerance of RA mice. These findings can provide a preliminary experimental basis to exploit GRg3 as a functional food or an effective complementary for the adjuvant therapy of RA.

CD25 as an adverse prognostic factor in elderly patients with acute myeloid leukemia.

OBJECTIVES: CD25 has been reported to be highly expressed in leukemia stem cells and correlated with adverse outcomes in young patients with acute myeloid leukemia (AML). However, the significance of CD25 expression in elderly patients with AML has not yet been investigated. METHODS: We retrospectively analyzed 154 newly diagnosed AML patients aged 60 years or over by flow cytometry. RESULTS: CD25-positive AML was characterized by high white blood cell counts, secondary AML, rare favorable karyotypes, and positivity for CD34 and CD7 antigens, compared with CD25-negative AML. CD25 positivity was significantly correlated with an inferior complete remission (CR), event-free survival (EFS), and overall survival. Multivariate analysis showed CD25 positivity to be a significant prognostic predictor of CR and EFS. A regimen of low-dose cytarabine and aclarubicin combined with granulocyte-colony-stimulating factor (CAG) led to higher CR rates in the CD25-positive AML patients than intensive chemotherapies. CD25 expression was increased at relapse and in the development of leukemic status from myelodysplastic syndrome or myeloproliferative neoplasm. DISCUSSION: An effective treatment strategy for elderly patients with CD25-positive AML has not been established. Further studies are needed to evaluate the effect of a CAG regimen and allogenic stem cell transplantation in patients. CONCLUSION: CD25 is an independent prognostic factor in elderly AML patients. Alternative therapies for CD25-positive elderly AML patients are needed.

Yerba mate (Ilex paraguariensis) inhibits lymphocyte activation in vitro.

Yerba mate (YM) has been shown to have anti-inflammatory properties in several studies. However, this effect has been found mainly in obesity-related inflammation. The aim of this work was to study the effect of YM on cultured peripheral blood mononuclear cells to see whether it has anti-inflammatory properties. We stimulated peripheral blood mononuclear cells in vitro with phytohemagglutinin (PHA) in the presence of yerba mate and determined their activation by measuring the expression of CD25 by flow cytometry. We observed that YM treatment produced a dose-dependent reduction in PBMC activation (CD25 positive cells) when they were stimulated with PHA. This effect was also observed in T cells' (CD3 positive) subpopulation. Microarray analysis revealed the differential expression of 128 genes in YM-treated cells. According to a protein-protein interaction database, these genes were highly connected and they are involved in the inflammatory response. In summary, it was demonstrated that YM produces a reduction in the amount of activated cells under the stimulation of PHA. Therefore, it might be used in diseases with an inflammatory component.

ADCT-301, a Pyrrolobenzodiazepine (PBD) Dimer-Containing Antibody-Drug Conjugate (ADC) Targeting CD25-Expressing Hematological Malignancies.

Despite the many advances in the treatment of hematologic malignancies over the past decade, outcomes in refractory lymphomas remain poor. One potential strategy in this patient population is the specific targeting of IL2R-α (CD25), which is overexpressed on many lymphoma and leukemic cells, using antibody-drug conjugates (ADC). ADCT-301 is an ADC composed of human IgG1 HuMax-TAC against CD25, stochastically conjugated through a dipeptide cleavable linker to a pyrrolobenzodiazepine (PBD) dimer warhead with a drug-antibody ratio (DAR) of 2.3. ADCT-301 binds human CD25 with picomolar affinity. ADCT-301 has highly potent and selective cytotoxicity against a panel of CD25-expressing human lymphoma cell lines. Once internalized, the released warhead binds in the DNA minor groove and exerts its potent cytotoxic action via the formation of DNA interstrand cross-links. A strong correlation between loss of viability and DNA cross-link formation is demonstrated. DNA damage persists, resulting in phosphorylation of histone H2AX, cell-cycle arrest in G2-M, and apoptosis. Bystander killing of CD25-negative cells by ADCT-301 is also observed. In vivo, a single dose of ADCT-301 results in dose-dependent and targeted antitumor activity against both subcutaneous and disseminated CD25-positive lymphoma models. In xenografts of Karpas 299, which expressed both CD25 and CD30, marked superiority over brentuximab vedotin (Adcetris) is observed. Dose-dependent increases in DNA cross-linking, γ-H2AX, and PBD payload staining were observed in tumors in vivo indicating a role as relevant pharmacodynamic assays. Together, these data support the clinical testing of this novel ADC in patients with CD25-expressing tumors. Mol Cancer Ther; 15(11); 2709-21. ©2016 AACR.

Integrative epigenomic analysis identifies biomarkers and therapeutic targets in adult B-acute lymphoblastic leukemia.

UNLABELLED: Genetic lesions such as BCR-ABL1, E2A-PBX1, and MLL rearrangements (MLLr) are associated with unfavorable outcomes in adult B-cell precursor acute lymphoblastic leukemia (B-ALL). Leukemia oncoproteins may directly or indirectly disrupt cytosine methylation patterning to mediate the malignant phenotype. We postulated that DNA methylation signatures in these aggressive B-ALLs would point toward disease mechanisms and useful biomarkers and therapeutic targets. We therefore conducted DNA methylation and gene expression profiling on a cohort of 215 adult patients with B-ALL enrolled in a single phase III clinical trial (ECOG E2993) and normal control B cells. In BCR-ABL1-positive B-ALLs, aberrant cytosine methylation patterning centered around a cytokine network defined by hypomethylation and overexpression of IL2RA(CD25). The E2993 trial clinical data showed that CD25 expression was strongly associated with a poor outcome in patients with ALL regardless of BCR-ABL1 status, suggesting CD25 as a novel prognostic biomarker for risk stratification in B-ALLs. In E2A-PBX1-positive B-ALLs, aberrant DNA methylation patterning was strongly associated with direct fusion protein binding as shown by the E2A-PBX1 chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq), suggesting that E2A-PBX1 fusion protein directly remodels the epigenome to impose an aggressive B-ALL phenotype. MLLr B-ALL featured prominent cytosine hypomethylation, which was linked with MLL fusion protein binding, H3K79 dimethylation, and transcriptional upregulation, affecting a set of known and newly identified MLL fusion direct targets with oncogenic activity such as FLT3 and BCL6. Notably, BCL6 blockade or loss of function suppressed proliferation and survival of MLLr leukemia cells, suggesting BCL6-targeted therapy as a new therapeutic strategy for MLLr B-ALLs. SIGNIFICANCE: We conducted the first integrative epigenomic study in adult B-ALLs, as a correlative study to the ECOG E2993 phase III clinical trial. This study links for the first time the direct actions of oncogenic fusion proteins with disruption of epigenetic regulation mediated by cytosine methylation. We identify a novel clinically actionable biomarker in B-ALLs: IL2RA (CD25), which is linked with BCR-ABL1 and an inflammatory signaling network associated with chemotherapy resistance. We show that BCL6 is a novel MLL fusion protein target that is required to maintain the proliferation and survival of primary human adult MLLr cells and provide the basis for a clinical trial with BCL6 inhibitors for patients with MLLr.

The effect of Astragaloside IV on immune function of regulatory T cell mediated by high mobility group box 1 protein in vitro.

High mobility group box 1 protein (HMGB1), a potent pro-inflammatory cytokine, contributes to the pathogenesis of diverse inflammatory and infectious disorders. Some studies have illustrated the potential effect of HMGB1 on regulatory T cells (Tregs). Astragaloside IV (AST IV) isolated from a Chinese herb, Astragalus mongholicus, is known to have a variety of immunomodulatory activities. However, it is not yet clear whether AST IV possesses potential regulatory effect on the pro-inflammatory ability of HMGB1 with subsequent activation of Tregs. This study was carried out to investigate the antagonistic effects of different doses of AST IV on the immune function of Tregs mediated by HMGB1 in vitro. Tregs isolated from the spleens of mice were co-cultured with HMGB1 and/or AST IV. Cell phenotypes of Tregs were analyzed, and the contents of various cytokines in the cell supernatants as a result of co-culture and the proliferation of CD4(+)CD25(-) T cells were determined. Results showed that HMGB1 stimulation resulted in significantly down-regulation of expressions of Tregs cell phenotypes. However, AST IV can rival the suppressing effect of HMGB1 on immune function of Tregs with a dose-dependent in vitro. These results indicate that AST IV has the potential therapeutic action on inflammation augmented by HMGB1.

Correction of interleukin-2 gene expression by in vitro zinc addition to mononuclear cells from zinc-deficient human subjects: a specific test for zinc deficiency in humans.

A nutritional deficiency of zinc in humans is widespread in the developing world, and a conditioned zinc deficiency is observed in many diseased states, the elderly population, and pregnant women of both developed and developing nations. It was recently reported that zinc is required for Nuclear Factor-kappaB (NF-kappaB) activation and gene expressions of both interleukin-2 (IL-2) and interleukin-2 receptor alpha (IL-2Ralpha) and beta in HUT-78, a Th0 human malignant lymphoblastoid cell line. In this study, it has been reported for the first time that zinc is also required for gene expression of IL-2 and IL-2Ralpha in primary cells. Isolated peripheral blood mononuclear cells (MNCs) from zinc-deficient elderly subjects was used for this study. NF-kappaB activation was shown to have decreased in the MNCs from zinc-deficient subjects, which was corrected by in vivo zinc supplementation. It was further shown that either in vivo zinc supplementation or the addition of zinc in vitro to MNCs from zinc-deficient subjects results in correction of the gene expression of IL-2 and IL-2Ralpha. Therefore, it was proposed that in vitro addition of zinc to MNCs for correction of gene expression of IL-2 in humans may be used as a specific test for zinc deficiency. Although currently no known specific laboratory test exists for the diagnosis of zinc deficiency in humans, the use of correction of IL-2 messenger RNA (mRNA) with in vitro zinc addition to MNCs from zinc-deficient subjects may be very useful.

In vitro anti-allergic activities of a newly concocted traditional Chinese medicine--the wheeze-relief formula.

Asthma is one of the most common chronic diseases worldwide. Western medications such as glucocorticoids are effective therapeutic agents but may be associated with side effects. Traditional Chinese medicine (TCM) has been used for treating allergic diseases with observable clinical benefits. The present study investigated whether a novel TCM concoction, the wheeze-relief formula (WRF), possesses in vitro anti-allergic activities. We measured the effects of WRF on the release of eosinophil cationic protein (ECP) by human eosinophils using fluorescence enzyme immunoassay, expression of chemokine receptor CCR3 and adhesion molecule CD49d on eosinophils using immunophenotyping, cytokine induction from peripheral blood mononuclear cells (PBMC) using cytometric bead array (CBA), and the gene expression of cytokines and cytokine receptors using cDNA expression array. Results demonstrated that WRF dose-dependently and significantly: (1) suppressed ECP release from eosinophils activated with granulocyte macrophage-colony stimulating factor (GM-CSF) and platelet activating factor (PAF); (2) inhibited the expression of CCR3 and CD49d on PAF-activated eosinophils; and (3) attenuated the production of tumor necrosis factor alpha and gene expression of IL-2 receptor chain alpha (CD25) on house dust mite (Der p 1) activated PBMC. The above results suggest a possible anti-allergic role of WRF and provide a biochemical basis for further clinical trial on human subjects.