Treg Expression Research Articles

Oroxylin A inhibits the generation of Tregs in non-small cell lung cancer.

Oroxylin A (OA), a naturally occurring monoflavonoid isolated from Scutellariae radix, has previously been reported to inhibit the proliferation of several cancer cell lines. CD4+CD25+Foxp3+ regulatory T cells (Tregs) play an important role in maintenance of immunologic self-tolerance. Tregs also increase in cancer and take part in suppressing antitumor immune responses. Here, we explored how OA affected the Tregs in lung cancer environment and the involved underlying mechanism. It is found that OA reversed the generation of Tregs induced by H460 lung cancer cells co-culture. Furthermore, in vivo, OA reduced tumor formation rate and attenuated Foxp3 expression in tumor-infiltrating lymphocytes. We also found that transforming growth factor-β1 (TGF-β1) neutralizing antibody reversed the enhancement of Treg number and expression of p-Smad3ˎ p-p38ˎp-JNKˎp-ERK1/2 in the co-culture model. Moreover, OA reduced the secretion of TGF-β1 and down-regulated the activation of NF-κB signaling in H460 cells. OA also inhibited Treg activity by a direct inhibition of the T cells' response to TGF-β1. In conclusion, our study demonstrated that OA inhibits the generation of Tregs in lung cancer environment by inhibiting the T cells' response to TGF-β1 and decreasing the secretion of TGF-β1 in lung cancer cells via NF-κB signaling.

Lifelong training improves anti-inflammatory environment and maintains the number of regulatory T cells in masters athletes.

The purpose of this study was to quantify and characterize peripheral blood regulatory T cells (Tregs), as well as the IL-10 plasma concentration, in Masters athletes at rest and after an acute exhaustive exercise test. Eighteen Masters athletes (self-reported training: 24.6 ± 1.83years; 10.27 ± 0.24months and 5.45 ± 0.42h/week per each month trained) and an age-matched control group of ten subjects (that never took part in regular physical training) volunteered for this study. All subjects performed an incremental test to exhaustion on a cycle ergometer. Blood samples were obtained before (Pre), 10min into recovery (Post), and 1h after the test (1h). Absolute numbers of Tregs were similar in both groups at rest. Acute exercise induced a significant increase in absolute numbers of Tregs at Post (0.049 ± 0.021 to 0.056 ± 0.024 × 109/L, P = 0.029 for Masters; 0.048 ± 0.017 to 0.058 ± 0.020 × 109/L, P = 0.037 for control) in both groups. Treg mRNA expression for FoxP3, IL-10, and TGF-β in sorted Tregs was similar throughout the trials in both groups. Masters athletes showed a higher percentage of subjects expressing the FoxP3 (100% for Masters vs. 78% for Controls, P = 0.038) and TGF-β (89% for Masters vs. 56% for Controls, P = 0.002) after exercise and a higher plasma IL-10 concentration (15.390 ± 7.032 for Masters vs. 2.411 ± 1.117 for control P = 0.001, ES = 2.57) at all timepoints. KLRG1 expression in Tregs was unchanged. Our findings showed that Masters athletes have elevated anti-inflammatory markers and maintain the number of Tregs, and may be an adaptive response to lifelong training.

Fasciola hepatica reinfection potentiates a mixed Th1/Th2/Th17/Treg response and correlates with the clinical phenotypes of anemia.

BackgroundFascioliasis is a severe zoonotic disease of worldwide extension caused by liver flukes. In human fascioliasis hyperendemic areas, reinfection and chronicity are the norm and anemia is the main sign. Herein, the profile of the Th1/Th2/Th17/Treg expression levels is analyzed after reinfection, correlating them with their corresponding hematological biomarkers of morbidity.Methodology/Principal findingsThe experimental design reproduces the usual reinfection/chronicity conditions in human fascioliasis endemic areas and included Fasciola hepatica primo-infected Wistar rats (PI) and rats reinfected at 8 weeks (R8), and at 12 weeks (R12), and negative control rats. In a cross-sectional study, the expression of the genes associated with Th1 (Ifng, Il12a, Il12b, Nos2), Th2 (Il4, Arg1), Treg (Foxp3, Il10, Tgfb, Ebi3), and Th17 (Il17) in the spleen and thymus was analyzed. After 20 weeks of primary infection, PI did not present significant changes in the expression of those genes when compared to non-infected rats (NI), but an increase of Il4, Arg1 and Ifng mRNA in the spleen was observed in R12, suggesting the existence of an active mixed Th1/Th2 systemic immune response in reinfection. Foxp3, Il10, Tgfb and Ebi3 levels increased in the spleen in R12 when compared to NI and PI, indicating that the Treg gene expression levels are potentiated in chronic phase reinfection. Il17 gene expression levels in R12 in the spleen increased when compared to NI, PI and R8. Gene expression levels of Il10 in the thymus increased when compared to NI and PI in R12. Ifng expression levels in the thymus increased in all reinfected rats, but not in PI. The clinical phenotype was determined by the fluke burden, the rat body weight and the hemogram. Multivariate mathematical models were built to describe the Th1/Th2/Th17/Treg expression levels and the clinical phenotype. In reinfection, two phenotypic patterns were detected: i) one which includes only increased splenic Ifng expression levels but no Treg expression, correlating with severe anemia; ii) another which includes increased splenic Ifng and Treg expression levels, correlating with a less severe anemia.Conclusions/SignificanceIn animals with established F. hepatica infection a huge increase in the immune response occurs, being a mixed Th2/Treg associated gene expression together with an expression of Ifng. Interestingly, a Th17 associated gene expression is also observed. Reinfection in the chronic phase is able to activate a mixed immune response (Th1/Th2/Th17/Treg) against F. hepatica but T and B proliferation to mitogens is strongly suppressed in all infected rats vs control in the advanced chronic phase independently of reinfection The systemic immune response is different in each group, suggesting that suppression is mediated by different mechanisms in each case. Immune suppression could be due to the parasite in PI and R8 rats and the induction of suppressive cells such as Treg in R12. This is the first study to provide fundamental insight into the immune profile in fascioliasis reinfection and its relation with the clinical phenotypes of anemia.

Altered Treg and IL-1A Expression in the Immune Microenvironment of Lung Squamous-cell Cancer after EGFR Blockade

背景与目的精确靶向表皮生长因子受体（epidermal growth factor receptor, EGFR）的治疗在肺鳞癌、口腔和肠胃癌中取得了一定疗效，但会引发系统性炎症。本实验旨在探究EGFR抑制剂治疗癌引发的肿瘤内部免疫变化。方法我们通过含H-ras基因逆转录病毒转染EGFR基因缺失或野生型小鼠角质细胞，将改造的细胞同源移植至小鼠以成瘤，吉非替尼治疗荷瘤小鼠，流式细胞仪检测T细胞比例与程序性死亡受体1（programmed death 1, PD-1）表达，RT-PCR检测细胞因子与趋化因子的表达。结果敲除EGFR基因形成的肿瘤较野生型小，并且肿瘤微环境中浸润FoxP3+调节性T细胞（regulatory cells, Treg）细胞较少，FoxP3 RNA较少，程序性死亡受体1（programmed death 1, PD-1）阳性CD4+细胞比例降低。表明肿瘤细胞可以自主调节肿瘤微环境。野生型成瘤模型使用吉非替尼治疗1周显示，相对于对照组肿瘤较小；在这短期的药理模型中，同样观察到FoxP3+细胞、FoxP3 RNA减少的趋势，同时IL-1A/IL-1RA比例明显升高，表明相对短暂的系统性抑制EGFR信号通路可改变靶向肿瘤的免疫微环境。结论肿瘤细胞自发（基因）或系统性（药理作用）的抑制EGFR信号通路可减少肿瘤的生长和肿瘤微环境中Treg的渗透。EGFR依赖性Treg细胞增强肺鳞癌的生长，是EGFR抑制剂治疗的靶标。

Age-related changes in CD4+CD25+FOXP3+ regulatory T cells and their relationship with lung cancer.

ObjectivesCD4+CD25+FOXP3+ regulatory T cells (Treg) inhibit the anti-tumour immune response and reduce the effect of cancer immunotherapy. Although studies have demonstrated that the number and suppressive activity of Treg increase with age, it is not clear whether these changes correlate with a higher incidence of tumours in the elderly. This study was designed to explore the relationship between increase in CD4+CD25+FOXP3+ Treg and the higher risk of lung cancer in the elderly.MethodsSeventy lung cancer patients and 60 sex- and age-matched controls were recruited. Both groups were divided into three subgroups based on their age (young, middle-aged, or elderly). The proportion of CD4+CD25+FOXP3+ /CD4+ T cells was detected using flow cytometry, and the level of FOXP3 mRNA in the peripheral blood was examined with real-time RT-PCR.ResultsThe levels of CD4+CD25+FOXP3+/CD4+ T cells and FOXP3 mRNA were significantly higher in lung cancer patients than in healthy controls (t = 7.16, P < 0.01 and t = 3.65, P < 0.01, respectively). Within the healthy groups, the elderly group had larger proportion of CD4+CD25+FOXP3+ Treg (F = 32.54, P < 0.01) and higher FOXP3 mRNA expression (F = 4.76, P < 0.01) than their younger counterparts. Among the six subgroups, the elderly lung cancer patients exhibited the highest levels of both CD4+CD25+FOXP3+ Treg (11.81 ± 2.40%) and FOXP3 mRNA (3.14 ± 1.30).ConclusionsThe accumulation of CD4+CD25+FOXP3+ Treg with age correlates well with the increasing incidence of lung cancer in the elderly.

Cathepsin K Deficiency Ameliorates Systemic Lupus Erythematosus-like Manifestations in Faslpr Mice.

Cysteinyl cathepsin K (CatK) is expressed in osteoclasts to mediate bone resorption, but is also inducible under inflammatory conditions. Faslpr mice on a C57BL/6 background develop spontaneous systemic lupus erythematosus-like manifestations. Although normal mouse kidneys expressed negligible CatK, those from Faslpr mice showed elevated CatK expression in the glomeruli and tubulointerstitial space. Faslpr mice also showed elevated serum CatK levels. CatK deficiency in Faslpr mice reduced all tested kidney pathologies, including glomerulus and tubulointerstitial scores, glomerulus complement C3 and IgG deposition, chemokine expression and macrophage infiltration, and serum autoantibodies. CatK contributed to Faslpr mouse autoimmunity and pathology in part by its activity in TLR-7 proteolytic processing and consequent regulatory T (Treg) cell biology. Elevated TLR7 expression and proteolytic processing in Faslpr mouse kidneys and Tregs showed significantly reduced levels in CatK-deficient mice, leading to increased spleen and kidney Treg content. Purified CD4+CD25highFoxp3+ Tregs from CatK-deficient mice doubled their immunosuppressive activity against T effector cells, compared with those from CatK-sufficient mice. In Faslpr mice, repopulation of purified Tregs from CatK-sufficient mice reduced spleen sizes, autoantibody titers, and glomerulus C3 and IgG deposition, and increased splenic and kidney Treg contents. Tregs from CatK-deficient mice had significantly more potency than CatK-sufficient Tregs in reducing spleen sizes, serum autoantibody titers, and glomerulus C3 deposition, and in increasing splenic and kidney Treg content. This study established a possible role of CatK in TLR7 proteolytic activation, Treg immunosuppressive activity, and lupus autoimmunity and pathology.

Significance of Foxp3+CD4+ regulatory T cells in the peripheral blood of Uygur patients in the acute and chronic phases of pigeon breeder's lung.

Pigeon breeder's lung (PBL) is a type of lung inflammatory disease associated with the immune response to repeated pigeon-derived antigen exposure. The pathogenesis of PBL remains unclear. In this study, peripheral blood samples were collected from Uygur acute - and chronic-phase PBL patients and healthy subjects with pigeon contact. Foxp3+CD4+ regulatory T cell (Treg) activity in different phases of PBL was characterized by changes in Foxp3+CD4+ Treg, CD4+CD25+ T cell, and T lymphocyte subsets. Based on hypersensitivity pneumonitis (HP) diagnosis criteria, 32 PBL cases from January 2012 to December 2013 in the People's Hospital of Xinjiang Uygur Autonomous Region Respiratory Department were included. Lung high-resolution computed tomography was performed, and the cases were classified based on the HP phase into 15 acute-phase and 17 chronic-phase cases. The control group included 30 healthy subjects with Uygur pigeon contact. Blood samples were collected, and the T cell subsets were analyzed via flow cytometry. In both PBL groups, the Foxp3+CD4+ Treg and CD4+CD25+ and CD4+CD3+ T cell percentages and CD4+/CD8+ ratios were significantly lower than in the control group (p < 0.01). In the PBL groups, particularly the acute-phase group, the CD8+CD3+ T lymphocyte percentage was significantly higher than in the control group (p < 0.01). There were no significant differences in CD4+CD25+ cells between the PBL groups. In peripheral blood from the PBL groups, the CD4+/CD8+ ratio was positively correlated with the Foxp3+CD4+ Treg (r = 0.864, p < 0.05) and CD4+/CD25+ cell (r = 0.34, p < 0.05) percentages. Low Foxp3+CD4+ Treg expression or overconsumption may be a pathogenic factor in PBL.

Expressions of Immune Negative Regulator FoxP3+Treg and PD-L1 Protein in the Immune Microenvironment of Cervical Lesion.

Objective To explore the expression patterns of immune negative regulator FoxP3+Treg and PD-L1 protein in cervical carcinoma and its precancerous lesions. Methods The expression patterns of FoxP3+Treg and PD-L1 protein in 45 cases of cervical lesions tissues of the cervix and 20 cases of normal cervix tissues by using immunohistochemistry (IHC). Results Compared with the normal cervix,the expressions of FoxP3+Treg (H=43.211,P=0.000) and PD-L1 protein (t=213.00,P=0.001) were significantly increased in cervical lesions. Compared with the low-grade squamous cell carcinoma,the invasiveness of FoxP3+Treg was increased in high-grade squamous cell carcinoma (H=28.307,P=0.000),along with increased expression of PD-L1 protein (t=239.000,P=0.028). The FoxP3+Treg expression was positively correlated with PD-L1 protein expression in abnormally differentiated cells (rs=0.364,P=0.003). Conclusion Along with the progression of cervical cancer,the FoxP3+Treg amount increases in the local microenvironment,along with enhanced PD-L1 protein expression in abnormally differentiated cells.

Histidine decarboxylase (HDC)-expressing granulocytic myeloid cells induce and recruit Foxp3+ regulatory T cells in murine colon cancer

ABSTRACTThe colorectal tumor microenvironment contains a diverse population of myeloid cells that are recruited and converted to immunosuppressive cells, thus facilitating tumor escape from immunoediting. We have identified a genetically and functionally distinct subset of dynamic bone marrow myeloid cells that are characterized by histidine decarboxylase (HDC) expression. Lineage tracing in Hdc-CreERT2;R26-LSL-tdTomato mice revealed that in homeostasis, there is a strong bias by HDC+ myeloid cells toward the CD11b+Ly6Ghi granulocytic lineage, which was accelerated during azoxymethane/dextran sodium sulfate (AOM/DSS)-induced colonic carcinogenesis. More importantly, HDC+ myeloid cells strongly promoted colonic tumorigenesis, and colon tumor progression was profoundly suppressed by diphtheria toxin A (DTA)-mediated depletion of HDC+ granulocytic myeloid cells. In addition, tumor infiltration by Foxp3+ regulatory T cells (Tregs) was markedly impaired following HDC+ myeloid cell depletion. We identified an HDC+ myeloid-derived Cxcl13/Cxcr5 axis that mediated Foxp3 expression and Treg proliferation. Ablation of HDC+ myeloid cells or disruption of the Cxcl13/Cxcr5 axis by gene knockdown impaired the production and recruitment of Tregs. Cxcl13 induction of Foxp3 expression in Tregs during tumorigenesis was associated with Stat3 phosphorylation. Overall, HDC+ granulocytic myeloid cells affect CD8+ T cells directly and indirectly through the modulation of Tregs and thus appear to play key roles in suppressing tumoricidal immunity.

Regulatory T and B Cells in Asthmatic Women: Variations From Pregnancy to Postpartum.

Allergic asthma and rhinitis are common in pregnancy. The immune mechanisms underlying the effects of asthma on pregnancy and vice versa are not completely understood. The aim of this study was to investigate changes in regulatory T and B cells in asthmatic women from late pregnancy to postpartum. Four groups of women were enrolled for this study: asthmatic (n=23) and healthy (n=43) third trimester-pregnant women and asthmatic (n=33) and healthy (n=35) nonpregnant women. Pregnant women were also evaluated postpartum (>6 weeks after delivery). Blood samples were taken from each woman and flow cytometry was used to characterize circulating regulatory T cells (Tregs) and regulatory B cells (Bregs). Foxp3 expression was assessed in CD4DimCD25Hi Tregs. Tregs did not vary significantly from pregnancy to postpartum in asthmatic or healthy women, but CD24HiCD38Hi Bregs decreased in pregnancy and increased significantly postpartum. Foxp3 expression in Tregs was also impaired during pregnancy in both asthmatic and healthy women, but recovered postpartum. Asthmatic pregnant women had higher Foxp3 expression levels than healthy pregnant women (P=.007), probably due to the use of control medication. Women with controlled asthma showed variations in regulatory cell subsets during pregnancy and postpartum. A similar pattern was observed for Foxp3 expression and CD24HiCD38Hi Bregs during this period, corroborating the interaction between Tregs and Bregs in immune responses. Considering the immunomodulatory potential of these immune mediators, more studies are needed to evaluate their relationship with asthma and rhinitis complications in pregnancy.

New strategies for cancer immunotherapy: targeting regulatory T cells

The immunosuppressive action of regulatory T (Treg) cells is one mechanism attributed to the limited success of cancer immunotherapies with checkpoint blockers. Two recent studies report distinct transcriptional profiles of tumor-infiltrating Treg cells and expression of specific molecules, suggesting novel strategies to overcome resistance to cancer immunotherapy.

Periodic elevation of regulatory T cells on the day of embryo transfer is associated with better in vitro fertilization outcome

Treg cells have been shown to be important in maintaining maternofetal tolerance, but the expression of Tregs in assisted reproductive technology (ART) in women on the day of embryo transfer (D0), 5days (D5) and 14days after ET (D14); the related factors influencing the expression levels of Tregs; the proliferation ability and the relevant cytokine epression by Tregs on D14 have not been investigated. In this study, 124 women undergoing in vitro fertilization-intracytoplasmic sperm injection (IVF/ICSI) were enrolled. Early morning fasting blood samples were obtained for the measurement of Tregs and other relevant indicators on the D0, D5and D14days after ET. we showed that the Tregs were increased on D0 and D14 in pregnant women, while there was no obvious fluctuation in non-pregnant women. IL-10 and TGF-β levels and the expansion of Tregs were significantly higher in successfully pregnant women than in non-pregnant women on D14. The levels of E2, P did not significantly differ between the groups. We suggest that periodic elevation of Tregs on the day of ET was associated with higher embryo implantation rate after ART.

RORγt expression in Tregs promotes systemic lupus erythematosus via IL-17 secretion, alteration of Treg phenotype and suppression of Th2 responses.

Systemic lupus erythematosus (SLE) is a common autoimmune disorder with a complex and poorly understood immunopathogenesis. However, a pathogenic role for the T helper type 17 (Th17) axis was demonstrated by many studies, while regulatory T cells (Tregs ) were shown to mediate protection. Recently, we and others characterized a novel and independent T cell population expressing both the Treg characteristic transcription factor forkhead box protein 3 (FoxP3) and the Th17-defining retinoic acid receptor-related orphan nuclear receptor γt (RORγt). Studies in a model of acute glomerulonephritis unveiled potent regulatory, but also proinflammatory, functions of RORγt+ FoxP3+ Tregs . This bi-functional nature prompted us to suggest the name 'biTregs '. Importantly, the pathogenic biTreg effects were dependent upon expression of RORγt. We thus aimed to evaluate the contribution of RORγt+ FoxP3+ biTregs to pristane-induced SLE and explored the therapeutic potential of interference with RORγt activation. Our analyses revealed expansion of IL-17 producing biTregs in a distinctive time-course and organ-specific pattern, coincident with the development of autoimmunity and tissue injury. Importantly, specific ablation of RORγt activation in endogenous biTregs resulted in significant amelioration of pristane-induced pulmonary vasculitis and lupus nephritis. As potential mechanisms underlying the observed protection, we found that secretion of IL-17 by biTregs was abrogated completely in FoxP3Cre ×RORCfl/fl mice. Furthermore, Tregs showed a more activated phenotype after cell-specific inactivation of RORγt signalling. Finally, and remarkably, biTregs were found to potently suppress anti-inflammatory Th2 immunity in a RORγt-dependent manner. Our study thus identifies biTregs as novel players in SLE and advocates RORγt-directed interventions as promising therapeutic strategies.

Response Gene to Complement-32 Promotes the Imbalance of Treg/Th17 in Patients with Dilated Cardiomyopathy

Background/Aims: The imbalance of Treg/Th17 cells plays important role in the pathogenesis of dilated cardiomyopathy (DCM). Response gene to complement (RGC)-32 is a cell cycle regulator that plays an important role in cell proliferation. We evaluated whether the upregulation of RGC-32 was implicated in the homeostasis of Treg/Th17 cells in DCM. Methods: The levels of plasma RGC-32, IL-17 and TGF-β1, and the frequencies of circulating CD4⁺ RGC-32⁺ T cells, Th17 and Treg cells in patients with DCM were determined by Cytokine-specific sandwich ELISA and the flow cytometer (FCM), respectively. Results: A significant elevation of plasma RGC-32 in patients with DCM compared with healthy control (HC) subjects was observed. This upregulation was associated with an increase in frequency of Th17 and a decrease in frequency of Treg cells. To further assessed the role of RGC-32, we investigated the effects of RGC-32 up- or down-regulation on frequencies of Th17 and Treg cells in peripheral blood mononuclear cells (PBMCs) from subjects. Importantly, overexpression of RGC-32 was accompanied by an augmentation of Th17 and a reduction of Treg expression. Conclusion: In summary, our study demonstrated the up-regulation of RGC-32 contributed to the imbalance of Treg/Th17 cells in patients with DCM.

CD4+IL-21+T cells are correlated with regulatory T cells and IL-21 promotes regulatory T cells survival during HIV infection

IntroductionIL-21 enhances T and natural killer cells survival and antiviral functions without promoting T cell activation during HIV infection, which makes it a better adjuvant in anti-HIV immunotherapy. Due to the pleiotropy and redundancy of cytokines, it is vital to have a comprehensive knowledge of the role of IL-21 in the regulation of immune responses. Regulatory T cells (Tregs) play an important role in immune regulation and are a determinant of immune therapeutic efficacy in certain circumstances. In this study, we explored the direct effect of IL-21 on Tregs during HIV infection, which has not been addressed before. MethodsThirty-four HIV treatment-naïve patients were enrolled and the relationship between CD4+IL-21+T cells and Tregs were studied. The effects of IL-21 on CD4+CD25+CD127low Tregs’ apoptosis, proliferation, and CTLA-4 and TGF-β expression in HIV-infected patients was investigated and compared with the effect of other common γ-chain cytokines. ResultsWe found the percentage and absolute numbers of CD4+IL-21+T cells were positively related to the frequency or absolute numbers of CD4+CD25+ or CD4+CD25+CD127low Tregs. Compared with the media-alone control, IL-21, IL-7, and IL-15 could significantly reduce apoptosis of Tregs (p<0.05). IL-21 did not promote the proliferation of Tregs as compared with media alone, while IL-2, IL-7, and IL-15 could significantly increase the proliferation of Tregs (p<0.05). IL-21 enhanced CTLA-4 expression by Tregs (p<0.05), but could not induce TGF-β secretion of Tregs from HIV infected patients. There were no significant differences of the fold induction of apoptosis, proliferation, or CTLA-4 and TGF-β expression by Tregs from HIV-infected patients and normal controls after IL-21 treatment. In vitro experiment showed that pretreatment with IL-21 significantly enhanced the suppressive effect of Tregs on CD8+ T cells’ IFN-γ expression. ConclusionWe conclude that IL-21 promotes the survival and CTLA-4 expression of Tregs and enhanced the suppressive capacity of Tregs during HIV infection. These results broaden the understanding of HIV pathogenesis and provide critical information for HIV interventions.

Tumor-derived CD4 + CD25 + regulatory T cells inhibit dendritic cells function by CTLA-4

PurposeCD4+CD25+regulatoryT cells (Tregs) play an important role in anti-tumor immune responses. Poor prognosis and declining survival rates have intimate connection with high Treg expression in cancer patients. Cytotoxic T Lymphocyte-associated protein (CTLA-4) is one of the most prominent molecules on Treg. In our previous research, we have demonstrated that HCC-derived Tregs can interfere with Dendritic cells (DCs) function and down-modulate CD80/CD86 on DCs in vitro in a cell-contact dependent way. However the mechanism of how HCC-derived Treg affect DC phenotype are not very clear. Therefore, we investigated the function of CTLA-4 in anti-tumor immune responses. Materials and methodsWe established BABL/C mouse with hepatocellular carcinoma model, and tumor-derived Tregs were purified by magnetic cell sorting using mouse CD4+CD25+regulatoryT cell isolation kit. Splenic DCs were enriched using CD11c-conjugated microbeads. Then splenic DCs co-cultured with tumor-derived Tregs and antibody-blocking experiments was performed. ResultsIn our research, we found the down-modulation of CD80/CD86 on DCs was inhibited by blocking CTLA-4. HCC-derived Tregs down-modulated CD80/CD86 on DCs in a CTLA-4-dependent way. Blockade of CTLA-4 can lead to increase DC-mediated immunity. ConclusionCTLA-4 play a vital role in Treg-mediated immnue inhibition and this discovery can open up new ideas for the development of therapeutic strategies.

An expanded population of pathogenic regulatory T cells in giant cell arteritis is abrogated by IL-6 blockade therapy

ObjectivesRandomised-controlled trials have recently proven the efficacy of the interleukin (IL)-6 receptor antagonist tocilizumab (TCZ) in giant cell arteritis (GCA). However, the mechanism of action of IL-6 blockade in this...

Imbalance of Th17 and Treg in peripheral blood mononuclear cells of active tuberculosis patients

ObjectiveMaintaining a right balance between Th17 and Treg might be critical to the immunopathogenesis of active tuberculosis (TB). This study aimed to assess whether the Th17/Treg balance is altered in active TB patients. Methods250 study subjects (90 active TB patients, 80 latent TB subjects, and 80 healthy controls) were recruited for the study. The expression of Th17 and Treg in peripheral blood mononuclear cells (PBMCs) in the 250 subjects was investigated by flow cytometry. Plasma levels of cytokines IL-17 and IL-10, which are related to Th17 and Treg, respectively, were determined by ELISA. ResultsThe percentages of Th17 and Treg in PBMCs from active TB patients were significantly higher than those from latent TB or control groups (Th17: 4.31±1.35% vs. 1.58±0.71% or 1.15±0.49%, p<0.05; Treg: 11.44±2.69% vs. 7.54±1.56% or 4.10±0.99%, p<0.05). The expression of IL-17 and IL-10 was significantly increased in active TB patients in comparison to that in latent TB or control groups (IL-17: 16.85±9.68 vs. 7.23±5.19 or 8.21±5.51pg/mL, p<0.05; IL-10: 28.70±11.27 vs. 20.25±8.57 or 13.94±9.00pg/mL, p<0.05). ConclusionsOur study demonstrated an altered balance of Treg/Th17 in active TB patients, with higher percentages of Th17 and Treg in PBMCs. Further research on this imbalance may offer a new direction for TB treatment.

Granzyme B Expression in T-Regulatory Cells Is a Strong Predictor of Acute Graft-Versus-Host Disease after Day +30 in Patients with "Classic" Immunosuppression after Allo-HSCT

Introduction. Granzyme B is a serine protease commonly found in the granules of cytotoxic lymphocytes and natural killer cells. It is secreted with the pore forming protein perforin and mediates apoptosis in target cells. Granzyme B mediated cytolysis is one of the regulatory mechanisms (together with IL-2 receptors (CD25)) by which T-regulatory cells (T-regs) influence on T-effectors cells. Despite the well-known fact that T-regs participate in pathogenesis of aGVHD and their amount after allo-HSCT inversely correlates with the probability of aGVHD incidence, data about functional status of T-regs and aGVHD is still limited.Patients and methods. Peripheral blood samples were collected in EDTA-tubes at day +30 after allo-HSCT. We use PBMC from 29 patients with hematological malignancies obtained by density gradient media. This method was used due to lymphopenia in this group of patients. Group with no aGVHD consist of 22 patients (AML=12, ALL n=5, LPD=1, CML=2, AA=1, MDS=1) after allo-HSCT n=17 from MUD, n=5 from mismatch unrelated donor. MAC conditioning regimen was used in 17 cases, 5 patients receive RIC. The group of aGVHD after day +30 include 7 patients. (AML=4, ALL n=3) after allo-HSCT from MUD (n=5), mismatch unrelated donor (n=1) and n=1 from sibling HLA-identical donor. Two patients receive MAC and 5 patients receive RIC conditioning regimen. All patients in both groups receive standard immunosuppression (MMF+CSA+ATG). All patients developed II-IV grade aGVHD (II (n=1); III (n=4); IV (n=2)) with a median time onset on day +50 (34-150).The anti-CD4-APC-Cy7, anti-CD25-APC, anti-CD127-FITC and anti-Granzyme B-PE (Becton Dickinson, USA) antibodies were used to determine T-regulatory cells population. A Bland-Altman plot (difference plot) was used in analyzing the agreement between the two different methods of T-regs assay (CD4+CD25high and CD4+CD25highCD127low) - differences were not found (p=0,942). Due to this fact in our experiments CD4+CD25high cells were identified as T-reg cells (Gregg et al., 2005). 30000 of CD4+ cells were analyzed on a BD FACSCanto II to achieve sufficient statistical power (Becton Dickinson, USA).Results. As we can see on chart 1 level of Granzyme B was higher in patients who never developed aGVHD. In accordance with chart 2 percentage of Granzyme B positive T-regs in group of patients who never developed aGVHD was 7,26±1,89% in comparison with 2,04±0,93% in group who developed aGVHD after day +30 (p=0,02*). We should note that according to our previous experiments bacterial or viral infection (e.g CMV) and HLA-disparity does not affect the level of granzyme B expression in T-regs. Using ROC- curve analysis (see Chart 3) we obtained area under curve (AUC) 0,74.Conclusion. Our data shows that Granzyme B in T-reg cells after allo-HSCT in patients with standard IST may predict aGVHD onset after day +30 with satisfactory test results (AUC=0,74, «cut-off» - 4,15%; sensitivity - 85,71%; specificity - 45,45%).). According to data of our transplant center (for 2006-2016), "granzyme B-based strategy" can help us to predict up to 47,3% of all aGVHD.Disclosures: No relevant conflicts of interest to declare.Chart 1: Example of Granzyme B expression (right; shown on horizontal axis) in T-regs (left; upper right quadrant) on day +30 after allo-HSCT in group with standard immunosuppressionChart 2: Percentage of Granzyme B positive T-reg cells in patient without aGVHD and patients who develop aGVHD after day +30.Chart 3: ROC curve analysis of Granzyme B in T-regs as predictor of aGVHD. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract A067: PD-1 blockade enhances OX40 expression on regulatory T-cells and decreases suppressive function through induction of phospho-STAT3 signaling

Abstract Blockade of the co-inhibitory receptor PD-1 has had unprecedented success in the treatment of metastatic melanoma, with response rates of approximately 40% in previously untreated patients. However, since the majority of patients fail to respond, a better understanding of the mechanisms of action involved in response/resistance and novel approaches to overcome resistance are needed. We evaluated T-cells present in peripheral blood samples of resected stage III/IV melanoma patients treated with adjuvant nivolumab. Data from intra-patient, paired pre-treatment/post-treatment samples showed a significant (p&amp;lt;0.05) increase in phosphorylated STAT3 (pSTAT3) in patients without relapse, but not in patients relapsing. Increases in regulatory T-cells (Tregs) (p&amp;lt;0.05) in relapsers, but not in non-relapsers, and OX40 expression on Tregs (p&amp;lt;0.05) in both relapsers and non-relapsers were also observed. Based on these data we evaluated the ability of PD-1 blocking antibodies to induce pSTAT3 signaling in vitro. Culturing with anti-PD-1 significantly increased levels of pSTAT3 in T-cells, including Tregs, both at the basal state (p&amp;lt;0.05) and post-CD3/CD28 activation (p&amp;lt;0.01). Additionally, culturing T-cells with PD-1 blocking antibodies increased the production of the STAT3 regulated cytokine IL-10 (p&amp;lt;0.05), the number of Tregs (p&amp;lt;0.01) and Treg expression of OX40 (p&amp;lt;0.05). Increases in all these markers/populations were abrogated with the addition of a STAT3 inhibitor (p&amp;lt;0.01). Similar increases in the number of Tregs and the expression of OX40 were also found when T-cells were treated in vitro with recombinant IL-10. Addition of IL-10 neutralizing antibodies ameliorated upregulation of Treg numbers and OX40 expression resulting from PD-1 blockade. Additionally, serum levels of IL-10 were found to be significantly elevated in non-relapsing patients relative to relapsers (p&amp;lt;0.05). RNA-seq analysis comparing Tregs from relapsers to non-relapsers further supported the observed increase in Tregs resulting from nivolumab treatment, with pathway analyses demonstrating significantly increased cell cycle, DNA replication, mitosis and other proliferation related pathways in Tregs from non-relapsed patients. Next we evaluated changes in the function of patient Tregs in an allogenic mixed lymphocyte suppression assay. Tregs from non-relapsers had a significant reduction in suppressive capacity post-nivolumab treatment in paired analyses (p&amp;lt;0.05), while Tregs from relapsers had no significant change. Post-treatment samples from relapses were also found to be significantly more suppressive (p&amp;lt;0.01) than those of non-relapsers. Additionally, decreased percentages of CCR4+CD45RA+ Tregs, previously described as having enhanced suppressive capacity, were found in non-relapsers post-treatment, but not in relapsers. A similar decrease in CCR4+CD45RA+ Tregs was achieved in vitro using OX40 agonist antibodies. Therefore, we propose that PD-1 blockade triggers STAT3 signaling, a hereunto unknown effect. These data support a model in which STAT3 induction by PD-1 blockade results in IL-10 production, which induces proliferation of Tregs with reduced suppressive capacity and triggers OX40 expression. Based on these results and previous findings of others demonstrating that ligation of OX40 expressed on Tregs hinders suppressive capacity, combining OX40 agonist antibodies may enhance the effectiveness of PD-1 blockade by reducing Treg suppression. Citation Format: David M. Woods, Rupal Ramakrishnan, Andressa L. Sodré, Anders Berglund, Jeffrey Weber. PD-1 blockade enhances OX40 expression on regulatory T-cells and decreases suppressive function through induction of phospho-STAT3 signaling [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A067.