scholarly journals TGF-β Regulates Hepatocellular Carcinoma Progression by Inducing Treg Cell Polarization

2015 ◽  
Vol 35 (4) ◽  
pp. 1623-1632 ◽  
Author(s):  
Yinan Shen ◽  
Yongpeng Wei ◽  
Zhouchong Wang ◽  
Yingying Jing ◽  
Haiguan He ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Mouse Model ◽  
Treg Cell ◽  
Flow Cytometric Analysis ◽  
Foxp3 Expression ◽  
Treg Cells ◽  
Cell Polarization ◽  
Flow Cytometric ◽  
Β Receptor

Background/Aims: TGF-β plays a key role in the progression of various tumors. The main objective of our study was to investigate whether TGF-β is able to regulate N-nitrosodiethylamine (DEN)-induced hepatocellular carcinoma (HCC) progression in a mouse model by inducing Treg cell polarization. Methods: HCC progression, TGF-β and Foxp3 expression levels, serum TGF-β, IL10 and GP73 levels as well as percentage of Treg cells were analyzed in healthy, HCC and HCC+SM-16 mouse groups. The effect of TGF-β on Treg cell polarization in vitro was measured by flow cytometric analysis. The expression of TGF-β and IL10 was identified by IHC in HCC patients and the correlation between TGF-β and IL10 was also assessed. Results: TGF-β expression is up-regulated in a DEN-induced HCC mouse model. TGF-β can promote the differentiation of Foxp3+CD4+ T cells (Treg cells) in vitro. However, blocking the TGF-β pathway with a specific TGF-β receptor inhibitor, SM-16, reduced HCC progression and the percentage of Treg cells in liver tissue. The correlation between TGF-β and Treg cells was also confirmed in HCC patients and the expression of both TGF-β and IL-10 was shown to be associated with HCC progression. Conclusion: TGF-β is necessary for HCC progression, acting by inducing Treg cell polarization.

scholarly journals Androgen receptor modulates Foxp3 expression in CD4+CD25+Foxp3+ regulatory T-cells

2015 ◽  
Vol 26 (15) ◽  
pp. 2845-2857 ◽  
Author(s):  
Magdalena Walecki ◽  
Florian Eisel ◽  
Jörg Klug ◽  
Nelli Baal ◽  
Agnieszka Paradowska-Dogan ◽  
...  
Keyword(s):  
T Cells ◽  
Androgen Receptor ◽  
Treg Cell ◽  
Foxp3 Expression ◽  
Treg Cells ◽  
Strong Increase ◽  
Histone H4 ◽  
And Function

CD4+CD25+Foxp3+ regulatory T (Treg) cells are able to inhibit proliferation and cytokine production in effector T-cells and play a major role in immune responses and prevention of autoimmune disease. A master regulator of Treg cell development and function is the transcription factor Foxp3. Several cytokines, such as TGF-β and IL-2, are known to regulate Foxp3 expression as well as methylation of the Foxp3 locus. We demonstrated previously that testosterone treatment induces a strong increase in the Treg cell population both in vivo and in vitro. Therefore we sought to investigate the direct effect of androgens on expression and regulation of Foxp3. We show a significant androgen-dependent increase of Foxp3 expression in human T-cells from women in the ovulatory phase of the menstrual cycle but not from men and identify a functional androgen response element within the Foxp3 locus. Binding of androgen receptor leads to changes in the acetylation status of histone H4, whereas methylation of defined CpG regions in the Foxp3 gene is unaffected. Our results provide novel evidence for a modulatory role of androgens in the differentiation of Treg cells.

scholarly journals Elevated FOXC2 Expression Promotes Invasion of HCC Cell Lines and is Associated with Poor Prognosis in Hepatocellular Carcinoma

2017 ◽  
Vol 44 (1) ◽  
pp. 99-109 ◽  
Author(s):  
Fang Yang ◽  
Lizhi Lv ◽  
Kun Zhang ◽  
Qiucheng Cai ◽  
Jianyong Liu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometric Analysis ◽  
Vital Role ◽  
Migration And Invasion ◽  
Flow Cytometric ◽  
Kaplan Meier ◽  
Proliferation Assays ◽  
The Relationship

Background/Aims: Increasing evidence has indicated that Forkhead box protein C2 (FOXC2) plays an important role in carcinogenesis. However, the expression and the role of FOXC2 in hepatocellular carcinoma (HCC) have not been extensively studied. Methods: FOXC2 expression was analyzed by quantitative real-time polymerase chain reaction, Western blot analysis and immunohistochemistry in HCC tissue and cells. The relationship between FOXC2 expression and patient clinical significance and survival were assessed by Pearson’s correlation and Kaplan-Meier analysis, respectively. Cell proliferation assays, colony formation assays, flow cytometric analysis and Transwell assays were employed to measure the effects of FOXC2 on HCC cells in vitro. Results: The expression of FOXC2 was increased in HCC tissue, and high FOXC2 expression was associated with worse patient survival. Knockdown of FOXC2 inhibited HCC cell growth, migration, and invasion in vitro, as well as tumor growth. Furthermore, we found that activation of AKT-mediated MMP-2 and MMP-9 was involved in FOXC2 promoting an aggressive phenotype. Conclusions: Taken together, these findings demonstrate that FOXC2 is upregulated in HCC tissue and is associated with tumor size, vascular invasion and advanced TNM stage. Further investigation suggested that FOXC2 may play a vital role in promoting proliferation and invasion in HCC and serves as a novel therapeutic target in HCC.

scholarly journals Indole hydrazide compound ZJQ-24 inhibits angiogenesis and induces apoptosis cell death through abrogation of AKT/mTOR pathway in hepatocellular carcinoma

2020 ◽  
Vol 11 (10) ◽  
Author(s):  
Jing Liu ◽  
Ying Liu ◽  
Jianqiang Zhang ◽  
Dan Liu ◽  
Yafeng Bao ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Tumor Growth ◽  
Scientific Evidence ◽  
Flow Cytometric Analysis ◽  
Xenograft Model ◽  
Mtor Pathway ◽  
Flow Cytometric ◽  
M Phase

Abstract Angiogenesis and the activation of AKT/mTOR pathway are crucial for hepatocarcinoma development and progression, the activation of mTORC1/2 and relevant substrates have been confirmed in clinical hepatocarcinoma samples. Therefore, AKT/mTOR pathway represents the major targets for anti-cancer drugs development. Here, we investigated the anti-proliferative activity and mechanisms of ZJQ-24 in hepatocellular carcinoma, both in vivo and in vitro. A hepatocellular carcinoma xenograft model showed that ZJQ-24 significantly inhibited tumor growth with few side effects. MTT assays, flow cytometric analysis, Western blotting and immunohistochemistry identified that ZJQ-24 effectively suppressed hepatocellular carcinoma cell proliferation via G2/M phase arrest and caspase-dependent apoptosis but had no cytotoxic on normal cells. Furthermore, ZJQ-24 significantly blocked AKT/mTOR signaling by down-regulation of mTORC1 molecules, including phospho-p70S6K (Thr389) and phospho-4EBP-1 (Ser65, Thr37/46, Thr70) and phospho-AKT (Ser473) in HCC cells. It is very important that the ZJQ-24 did not induce the mTORC1-depdent PI3K/Akt feedback activation through JNK excitation. Moreover, ZJQ-24 inhibited the cap-dependent translation initiation by impairing the assembly of the eIF4E/eIF4G complex. Immunohistochemistry further confirmed ZJQ-24 inhibited the tumor growth through suppression of VEGF and AKT/mTOR pathways in vivo. Thus, the present study is the first to illustrate that ZJQ-24 triggers antiangiogenic activity and apoptosis via inhibiting the AKT/mTOR pathway in hepatocellular carcinoma cells, providing basic scientific evidence that ZJQ-24 shows great potential function as inhibitor of angiogenesis and tumor growth in hepatocellular carcinoma.

scholarly journals The relationship between human thymus-derived regulatory T cells, TGF-β-induced regulatory T cells and conventional CD4+ T cells as revealed by proteomics

2021 ◽  
Author(s):  
Mark Mensink ◽  
Ellen Schrama ◽  
Maartje van den Biggelaar ◽  
Derk Amsen ◽  
Jannie Borst ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Protein Expression ◽  
Treg Cell ◽  
Ex Vivo ◽  
Foxp3 Expression ◽  
Treg Cells ◽  
Cell Populations ◽  
The Relationship

The CD4+ regulatory T (Treg) cell lineage, as defined by FOXP3 expression, comprises thymus-derived (t)Treg cells and peripherally induced (p)Treg cells. In human, naive tTreg cells can be purified from blood, but occur in low abundance, while effector pTreg and tTreg cell populations cannot be purified for lack of discriminating cell surface markers. Therefore, studies often employ TGF-β-induced (i)Treg cells that are generated from CD4+ conventional T (Tconv) cells in vitro. Here, we describe the relationship of iTreg cells to tTreg and Tconv cells, as optimally purified from human blood. Global proteomic analysis revealed that iTreg, tTreg and Tconv cell populations each have a unique protein expression pattern. We next used as a benchmark a previously defined proteomic signature that discerns ex vivo naive and effector phenotype Treg cells from Tconv cells and reflects unique Treg cell properties. This Treg cell core signature was largely absent from iTreg cells, while clearly present in simultaneously analyzed tTreg cells. In addition, we used a proteomic signature that distinguishes ex vivo effector Treg cells from Tconv cells and naive Treg cells. This effector Treg cell signature was partially present in iTreg cells. Thus, iTreg cells are distinct from tTreg cells and largely lack the common Treg cell proteomic signature. However, they do have certain protein expression features in common with ex vivo effector Treg cells. These data demonstrate the utility of the core and effector Treg cell signatures as tools to define Treg cell populations and encourage the use of ex vivo Treg cells for functional analyses.

Role of the Hypoxic Bone Marrow Microenvironment in Multiple Myeloma Tumor Progression.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2348-2348
Author(s):  
Kewal Asosingh ◽  
Hendrik De Raeve ◽  
Mark de Ridder ◽  
Guy A. Storme ◽  
Angelo Willems ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Mouse Model ◽  
Tumor Progression ◽  
Flow Cytometric Analysis ◽  
Surrogate Marker ◽  
Hypoxic Conditions ◽  
Flow Cytometric

Abstract Recently we reported that pre-clinical myeloma disease progression in the 5T2MM mouse model is characterized by predominant CD45+ MM-cells in the early, pre-angiogenic stage stage of slow tumor progression, followed by expansion of CD45− MM-cells during the subsequent angiogenic stage of progressive tumor growth. Unlike other cancer cells, multiple myeloma (MM) cells have to survive and to grow in a microenvironment which is already hypoxic by nature. This hypoxic bone marrow (BM) microenvironment is essential for normal hematopoiesis. However, the role of BM hypoxia in myeloma tumor progression is not known. Herein we addressed this topic in the 5T2MM mouse model. Flow cytometric analysis of control mice and 5T2MM diseased mice injected with pimonidazole hypoxyprobe indicated that both normal BM and myeloma infiltrated BM are hypoxic. However, in myelomatous BM the hypoxia was significantly decreased. Analysis of HIF-1a expression, a surrogate marker of hypoxia, by flow cytometry also demonstrated significantly lower levels of hypoxia in myeloma infiltrated BM. HIF-1a expression was found in 5T2MM-cells and was significantly higher compared to the non-tumor cell fraction. In vitro culturing of 5T2MM cells under hypoxic conditions, indicated increased activation of apoptosis inducing caspase-3 in the CD45− MM-fraction, but not in the CD45+ 5T2MM-cells, suggesting that native BM hypoxia selects the tumor population for tumor initiating CD45+ 5T2MM-cells. Although angiogeneic switch and angiogeneic heterogeneity has been reported in MM, the role of myeloma associated angiogensis is remains unclear. The decreased hypoxia in myeloma infiltrated BM adds strength to the hypothesis that myeloma associated neovascularization is functional by increasing BM oxygenation. The data also suggest that the angiogenesis allows expansion of CD45− 5T2MM-cells by decreasing BM hypoxia. All together, these findings suggest an important role of BM hypoxia in myeloma tumor progression.

scholarly journals Decreased Treg Cell and TCR Expansion Are Involved in Long-Lasting Graves’ Disease

2021 ◽  
Vol 12 ◽  
Author(s):  
Ziyi Chen ◽  
Yufeng Liu ◽  
Shiqian Hu ◽  
Meng Zhang ◽  
Bingyin Shi ◽  
...  
Keyword(s):  
T Cell ◽  
Treg Cell ◽  
Flow Cytometric Analysis ◽  
Autoimmune Disorder ◽  
Cell Receptor ◽  
Treg Cells ◽  
Thyroid Stimulating Hormone ◽  
Graves Disease ◽  
Rna Seq ◽  
Flow Cytometric

Graves’ disease (GD) is a T cell-mediated organ-specific autoimmune disorder. GD patients who have taken anti-thyroid drugs (ATDs) for more than 5 years with positive anti-thyroid stimulating hormone receptor autoantibodies value were defined as persistent GD (pGD). To develop novel immunotherapies for pGD, we investigated the role of T cells in the long-lasting phase of GD. Clinical characteristics were compared between the pGD and newly diagnosed GD (nGD) (N = 20 respectively). Flow cytometric analysis was utilized to determine the proportions of Treg and Th17 cells (pGD, N = 12; nGD, N = 14). T cell receptor sequencing (TCR-seq) and RNA sequencing (RNA-seq) were also performed (pGD, N = 13; nGD, N = 20). Flow cytometric analysis identified lower proportions of Th17 and Treg cells in pGD than in nGD (P = 0.0306 and P = 0.0223). TCR-seq analysis revealed a lower diversity (P = 0.0025) in pGD. Specifically, marked clonal expansion, represented by an increased percentage of top V-J recombination, was observed in pGD patients. Interestingly, pGD patients showed more public T cell clonotypes than nGD patients (2,741 versus 966). Meanwhile, RNA-seq analysis revealed upregulation of the inflammation and chemotaxis pathways in pGD. Specifically, the expression of pro-inflammatory and chemotactic genes (IL1B, IL13, IL8, and CCL4) was increased in pGD, whereas Th17 and Treg cells associated genes (RORC, CARD9, STAT5A, and SATB1) decreased in pGD. Additionally, TCR diversity was negatively correlated with the expression of pro-inflammatory or chemotactic genes (FASLG, IL18R1, CCL24, and CCL14). These results indicated that Treg dysregulation and the expansion of pathogenic T cell clones might be involved in the long-lasting phase of GD via upregulating chemotaxis or inflammation response. To improve the treatment of pGD patients, ATDs combined therapies, especially those aimed at improving Treg cell frequencies or targeting specific expanded pathogenic TCR clones, are worth exploring in the future.

Loss of FOXP3 Expression and Emergence of Cytokine-Producing Cells after In Vitro Expansion of Human CD4+CD25+CD127 - Regulatory T Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 63-63 ◽  
Author(s):  
Petra Hoffmann ◽  
Ruediger Eder ◽  
Tina J. Boeld ◽  
Jochen Huehn ◽  
Stefan Floess ◽  
...  
Keyword(s):  
T Cells ◽  
Treg Cell ◽  
Cell Cultures ◽  
Methylation Status ◽  
Foxp3 Expression ◽  
Treg Cells ◽  
Culture Conditions ◽  
Cell Therapies ◽  
Over Time

Abstract We and others previously showed that the adoptive transfer of donor-type CD4+CD25+ regulatory T (Treg) cells protects from graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT) in animal models. Exploring this strategy in human SCT, we currently perform a first Phase I clinical trial using freshly isolated Treg cells. For future trials requiring large Treg cell numbers for repetitive treatments, we described in vitro culture conditions that permit a more than 3-log expansion of polyclonal Treg cells (Blood 104:895; 2004). A highly enriched starting population proved to be crucial for the generation of pure Treg cell products and we could show that the naive, CD45RA+ subpopulation of CD4+CD25high T cells fulfills this criterion (Blood108:4260; 2006). A recently proposed alternative approach for the isolation of pure Treg cells relies on the exclusion of CD127+ cells, as activated (i.e. CD25+) conventional T cells express high levels of CD127 while CD4+CD25+ Treg cells show no or only weak expression levels (Seddiki et al. and Liu et al., JEM203:1693; 2006). To directly compare these two approaches, we isolated CD4+CD25+CD127low/neg T cells (CD127-Treg) and CD45RA+CD4+CD25high T cells (RA+ Treg) from the same leukapheresis products and analyzed the cells after 2 and 3 weeks of expansion. Whereas both populations were > 94% FOXP3+ upon isolation, only RA+ Treg maintained this high level of FOXP3+ cells throughout the expansion period (93% (range: 78 to 97%; n=11) FOXP3+ after 2 and 87% (range: 71 to 97%; n=9) FOXP3+ after 3 weeks). In contrast, the proportion of FOXP3+ cells in CD127-Treg cultures was already reduced after 2 weeks (82% (range: 56 to 96%; n=11)) and highly variable and significantly lower than that of RA+ Treg cultures after 3 weeks (57% (range: 18 to 93%; n=9; p=0.006)). When further subdivided into CD45RA+ and CD45RA- subpopulations before expansion, cultures of CD45RA-CD127-Treg cells lost FOXP3 expression and comprised substantial numbers of FOXP3- cytokine producing cells (on average 54% and 38% IL-2 and IFN-γ producers, respectively) after 3 weeks, whereas CD45RA+ CD127- Treg behaved similar to RA+ Treg cells, as they maintained FOXP3 expression over time and contained only low numbers of cytokine producers. Furthermore, when we analyzed the DNA methylation status of Treg cells, we found the Treg-specific CpG demethylation pattern within the FOXP3 gene (EJI 37, 2007) in RA+ Treg cell lines, while CD127- Treg cell cultures showed increased methylation over time and even more so RA- Treg cell cultures. Based on these findings, we suggest that isolation and expansion of CD45RA+CD4+CD25high T cells at present represents the best strategy for adoptive cell therapies requiring in vitro expanded Treg cells.

scholarly journals Efficient IL-2R signaling differentially affects the stability, function, and composition of the regulatory T-cell pool

2021 ◽  
Author(s):  
Marc Permanyer ◽  
Berislav Bošnjak ◽  
Silke Glage ◽  
Michaela Friedrichsen ◽  
Stefan Floess ◽  
...  
Keyword(s):  
T Cell ◽  
Treg Cell ◽  
Cell Function ◽  
Interleukin 2 ◽  
Foxp3 Expression ◽  
Treg Cells ◽  
Receptor Expression ◽  
Spontaneous Mutant ◽  
Cell Pool ◽  
And Function

AbstractSignaling via interleukin-2 receptor (IL-2R) is a requisite for regulatory T (Treg) cell identity and function. However, it is not completely understood to what degree IL-2R signaling is required for Treg cell homeostasis, lineage stability and function in both resting and inflammatory conditions. Here, we characterized a spontaneous mutant mouse strain endowed with a hypomorphic Tyr129His variant of CD25, the α-chain of IL-2R, which resulted in diminished receptor expression and reduced IL-2R signaling. Under noninflammatory conditions, Cd25Y129H mice harbored substantially lower numbers of peripheral Treg cells with stable Foxp3 expression that prevented the development of spontaneous autoimmune disease. In contrast, Cd25Y129H Treg cells failed to efficiently induce immune suppression and lost lineage commitment in a T-cell transfer colitis model, indicating that unimpaired IL-2R signaling is critical for Treg cell function in inflammatory environments. Moreover, single-cell RNA sequencing of Treg cells revealed that impaired IL-2R signaling profoundly affected the balance of central and effector Treg cell subsets. Thus, partial loss of IL-2R signaling differentially interferes with the maintenance, heterogeneity, and suppressive function of the Treg cell pool.

scholarly journals Tolerogenic Splenic IDO+Dendritic Cells from the Mice Treated with Induced-Treg Cells Suppress Collagen-Induced Arthritis

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Jie Yang ◽  
Yiming Yang ◽  
Huahua Fan ◽  
Hejian Zou
Keyword(s):  
Dendritic Cells ◽  
Immune Responses ◽  
Foxp3 Expression ◽  
Treated Group ◽  
Treg Cells ◽  
Therapeutic Effects ◽  
Dependent Manner ◽  
Collagen Induced Arthritis

TGF-β-induced regulatory T cells (iTregs) retain Foxp3 expression and immune-suppressive activity in collagen-induced arthritis (CIA). However, the mechanisms whereby transferred iTregs suppress immune responses, particularly the interplay between iTregs and dendritic cells (DCs)in vivo, remain incompletely understood. In this study, we found that after treatment with iTregs, splenic CD11c+DCs, termed “DCiTreg,” expressed tolerogenic phenotypes, secreted high levels of IL-10, TGF-β, and IDO, and showed potent immunosuppressive activityin vitro. After reinfusion with DCiTreg, marked antiarthritic activity improved clinical scores and histological end-points were observed. The serological levels of inflammatory cytokines and anti-CII antibodies were low and TGF-βproduction was high in the DCiTreg-treated group. DCiTregalso induced new iTregsin vivo. Moreover, the inhibitory activity of DCiTregon CIA was lost following pretreatment with the inhibitor of indoleamine 2,3-dioxygenase (IDO). Collectively, these findings suggest that transferred iTregs could induce tolerogenic characteristics in splenic DCs and these cells could effectively dampen CIA in an IDO-dependent manner. Thus, the potential therapeutic effects of iTregs in CIA are likely maintained through the generation of tolerogenic DCsin vivo.

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