The Profile of CD4+CD25+CD127low/- Regulator T Cell and the Associated Expression of Cytokines and Transcription Factors in Immune Thrombocytopenia Murine Model

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4208-4208
Author(s):  
Liping Ma
Keyword(s):  
Transcription Factors ◽  
Mrna Expression ◽  
Murine Model ◽  
Peripheral Blood ◽  
Immune Thrombocytopenia ◽  
Mononuclear Cells ◽  
Treg Cells ◽  
Intracellular Cytokines ◽  
Proliferation And Differentiation ◽  
Daily Intraperitoneal Injection

Abstract Background Regulator T cell (Treg cell) may be associated with immune thrombocytopenia (ITP). Our previous study has showed that the profile of CD4+CD25+CD127low/-Treg cells presented significantly decreased in ITP patients and weakened immunosuppressive activity. This cellular defect about immune modulation may play important roles in the pathogenesis of ITP. The precise reason for these abnormalities remains being elucidated. To further investigate the role of Treg cells in ITP，we detected the profiles of Treg cells and the associated mRNA expression of cytokines and Transcription factors in CD4+CD25+ Treg cells in ITP murine model . Methods 1. The profiles of Treg cell in peripheral blood in ITP patients were examined with flow cytometry through intracellular cytokines analysis. Treg cells were identified as those that were CD4+CD25+CD127low/-. 2. ITP was induced by daily intraperitoneal injection of anti-platelet membrane CD41 antibody（MWReg30）into BALB/c murine and the controls were daily intraperitoneal injection of IgG antibodies. 3. The proportion of Treg cells in peripheral blood and spleen mononuclear cells were measured by FCM analysis. Treg cells were identified as those that were CD4+CD25+CD127low/- 4. The mRNA expression of Treg cells associated transcription factors (Foxp3, Smad7, STAT5 and Akt-1) and cytokines (IL-10, TGF-β) in CD4+CD25+ T cells which were enriched from spleen mononuclear cells were measured by real-time PCR. 5. Statistical analysis. All values were analyzed using SPSS version 18.0 software. We used the Kolmogorov-Smirnov goodness-of-fit model to assess the normality of the data. Because of the data did not show a normal distribution, the values were presented as median (range) and statistical significance was evaluated using a Wilcoxon rank-sum test. Spearman’s test was used for correlation analysis. Differences were considered significant at P <0.05. Results 1. We detected the expression of intracellular cytokines. In peripheral blood of ITP patients, the median of Treg cells was 3.9 % (3.1–6.3 %), respectively, significantly lower than the controls of which that was 6.0 % (5.3–7.85 %, p <0.05). 2. The percentage of CD4+CD25+CD127low/-Treg cells was significantly lower in both splenocyte and peripheral blood of ITP murine as compared with that in normal controls (p<0.05). 3. ITP murine had lower mRNA expression of IL-10, TGF-β and Foxp3 in splenocyte CD4+CD25+T cells (p<0.05). The expression of Smad7 mRNA was significantly higher than the controls. No significantly difference of the STAT5 and Akt-1 mRNA expression were observed between ITP murine and controls both in splenocyte and peripheral blood. Conclusions Our study data supported a low Treg polarization of the immune response in ITP murine model. The lower mRNA expressions of IL-10 and TGF-β indicated that the indirect immunosuppressive effect of Treg cells had impaired. The lower mRNA expressions of TGF-β and Foxp3 accelerated effects on the proliferation and differentiation of Treg cells, and the higher mRNA expression of Smad 7 inhibited the proliferation and differentiation of Treg cells. Results indicated that disorder proliferation and differentiation of Treg cells involved in the pathogenesis of ITP. Disclosures No relevant conflicts of interest to declare.

scholarly journals Transcription Factor Assay of Peripheral Blood T cells in Different Groups of Rheumatoid Arthritis Patients

2020 ◽  
Vol 8 (4) ◽  
pp. 153-162
Author(s):  
Saeid Taghiloo ◽  
◽  
Abolghasem Ajami ◽  
Mohsen Tehrani ◽  
Arezou Abbasi ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Transcription Factors ◽  
Mrna Expression ◽  
Peripheral Blood ◽  
Treg Cells ◽  
The Body ◽  
Th2 Cells ◽  
Healthy Controls ◽  
Relative Expression ◽  
Significant Difference

Background: Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints and other tissues and organs of the body. Previous reports have demonstrated the imbalance of T helper (Th) subsets and Treg activity in the development, progression, and remission of RA. Here, we investigated the mRNA expression of four major transcription factors T-bet (Th1), GATA (Th2), RORc (Th17), and Foxp3 (Treg) in peripheral blood of different groups of RA patients. Materials and methods: In this case-control study, 60 patients with RA, including 20 newly diagnosed, 20 under treatment, and 20 in remission, as well as 20 patients with osteoarthritis, and 20 age- and the sex-matched healthy individual were enrolled. Diagnosis and classification of patients were done according to the American College of Rheumatology criteria. The relative mRNA expression of transcription factors, including T-bet, GATA, RORc, and Foxp3, was measured using qRT-PCR. Results: The relative expression of T-bet in RA patients was significantly increased in healthy controls (P = 0.002), while the relative expression of Foxp3 in RA patients was significantly decreased in healthy controls (P < 0.0001). There was no significant difference in the expression of GATA3 or RORc among RA patients, healthy controls, and osteoarthritis group. Conclusions: The results indicate the importance of Th1 and Treg cells in RA; however, the role of Th17 cells appear to be of little importance in these patients. It seems that Th2 cells do not interfere with RA development.

scholarly journals Function of regulatory T-cells improved by dexamethasone in Graves' disease

2012 ◽  
Vol 166 (4) ◽  
pp. 641-646 ◽  
Author(s):  
Yun Hu ◽  
Wei Tian ◽  
Ling-Ling Zhang ◽  
Hao Liu ◽  
Guo-Ping Yin ◽  
...  
Keyword(s):  
T Cells ◽  
Mrna Expression ◽  
Treg Cell ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Cell Function ◽  
Treg Cells ◽  
Th2 Cells ◽  
Graves Disease ◽  
Treg Cell Function

ObjectiveIntrathyroid injection of dexamethasone (DEX) has been used to treat Graves' disease (GD); however, the mechanism of this treatment remains poorly understood. The objective of this study was to investigate the effects of DEX on the function of regulatory T (Treg) cells (CD4+CD25+T cells) in patients with GD.MethodsPeripheral blood was obtained from 20 patients with GD, and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll–Hypaque density gradient separation. CD4+CD25–/CD4+CD25+T cells were isolated by immunomagnetic selection and DEX was co-cultured with PBMCs or isolated T-cells for 72 h. Treg cell function was analyzed using the proliferation rate of CD4+CD25–T cells.ResultsThe proportion of Treg cells and the transcription factor forkhead box P3 (FOXP3) mRNA expression in PBMCs decreased in GD patients compared with healthy subjects, and Treg cell function was impaired in patients with GD. Although the proportion of Treg cells and FOXP3 mRNA expression in PBMCs did not increase, the function of Treg cells improved after the treatment with DEX. Moreover, the proportion of T-helper 2 (Th2) cells was decreased by the DEX treatment.ConclusionsDEX could effectively improve the function of Treg cells and set up a new balance of Th1/Th2 in GD patients. This study might help to further understand the immune mechanism of the intrathyroid injection of DEX in the treatment of GD and facilitate the potential use of this therapy.

Analysis of Peripheral Blood CD4+/CD25high Treg Cells and FOXP3 mRNA Expression in Patients Treated with Ipilimumab (Monoclonal Human Anti-CTLA4, MDX-010) for Relapse of Malignancy Following Allogeneic Hematopoietic Transplantation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1735-1735
Author(s):  
Jiehua Zhou ◽  
Rui-kun Zhong ◽  
Iveta Kalcheva ◽  
Bridget Medina ◽  
Edward D. Ball ◽  
...  
Keyword(s):  
T Cells ◽  
Mrna Expression ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Treg Cells ◽  
Negative Regulator ◽  
Clinical Effects ◽  
Foxp3 Mrna ◽  
Hematopoietic Stem ◽  
Significant Change

Abstract CTLA-4 is expressed upon activation of T-cells,and serves as an important negative regulator of their effector function. It is also expressed constitutively on CD4+/CD25+ regulatory T cells (Treg), where its function is not clear. Following allogeneic hematopoietic stem cell transplantation (allo-HCT), CTLA-4 function may be involved in suppression of alloreactive T cells that mediate the graft-versus malignancy effect and GVHD. We have studied the administration of a single dose of Ipilimumab (MDX-010), a fully human monoclonal anti-CTLA-4 antibody, in a dose escalation trial in patients with relapse/progression of malignancy following allo-HCT. Here we report effects of ipilimumab on peripheral CD4+/CD25+ cell levels and FOXP3 mRNA expression in these patients. Seventeen patients with a variety of malignancies were enrolled in this study. Ipilimumab was given intravenously at a dose level of 0.1, 0.33, 0.66, 1, and 3mg/kg. The blood samples were obtained prior and after infusion at day 7, 14 and 30. The immunophynotyping of peripheral blood mononuclear cells (PBMC) was analyzed by flow cytometry. CD4+/CD25+ cells from nine patients at day 0, 7, and five normal donors were separated using a Dynal CD4+/CD25+ Treg kit. FOXP3 mRNA expression on CD4+/CD25+ cells were analyzed by a quantitative RT-PCR. Expression level of FOXP3 was normalized to 18S rRNA. Within CD4+ cell population, the percentage of CD4+/CD25high cells was significantly higher in patients at day 0 (11.6±6.7%, n=17), compared with normal donors (3.8±1.6%, n=12; P<0.001). There was no significant change in CD4+/CD25high Treg cells in 17 patients after infusion. However, there was a transient 55±24% decrease of CD4+/CD25high Treg cells in 6 patients at day 7. FOXP3 mRNA expression in CD4+/CD25+ cells was significantly higher in patients at day 0 (2451±1731, n=9) compared with normal donors (918±348, n=5; P<0.001). After ipilimumab infusion, 3/9 patients showed a greater than 50% decrease, 4/9 patients showed no significant change, and 2/9 patients showed a 3–10 fold increase of FOXP3 mRNA expression at day 7. There was no correlation between the dose of ipilimumab and the percentage of CD4+/CD25high cells, or the FOXP3 mRNA expression. We did not observe a correlation of CD4+/CD25high Treg cells and FOXP3 mRNA expression in patients who had a clinical response or immune breakthrough adverse events in response to ipilimumab. These data suggest that in-vivo CTLA-4 blockade does not consistently impact the number of CD4+ CD25high Treg cells and that the clinical effects observed are probably related to the effects of ipilimumab upon activated effector T-cells.

Sensitivity and reliability of zinc transporter and metallothionein gene expression in peripheral blood mononuclear cells as indicators of zinc status: responses to ex vivo zinc exposure and habitual zinc intake in humans

2020 ◽  
pp. 1-8
Author(s):  
Stephen R. Hennigar ◽  
Alyssa M. Kelley ◽  
Bradley J. Anderson ◽  
Nicholes J. Armstrong ◽  
Holly L. McClung ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Human Subjects ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Essential Nutrient ◽  
Zn Status

Abstract Zn is an essential nutrient for humans; however, a sensitive biomarker to assess Zn status has not been identified. The objective of this study was to determine the reliability and sensitivity of Zn transporter and metallothionein (MT) genes in peripheral blood mononuclear cells (PBMCs) to Zn exposure ex vivo and to habitual Zn intake in human subjects. In study 1, human PBMCs were cultured for 24 h with 0–50 µm ZnSO4 with or without 5 µm N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), and mRNA expression of SLC30A1-10, SLC39A1-14, MT1 subtypes (A, B, E, F, G, H, L, M and X), MT2A, MT3 and MT4 mRNA was determined. In study 2, fifty-four healthy male and female volunteers (31·9 (sd 13·8) years, BMI 25·7 (sd 2·9) kg/m2) completed a FFQ, blood was collected, PBMCs were isolated and mRNA expression of selected Zn transporters and MT isoforms was determined. Study 1: MT1E, MT1F, MT1G, MT1H, MT1L, MT1M, MT1X, MT2A and SLC30A1 increased with increasing concentrations of Zn and declined with the addition of TPEN. Study 2: Average daily Zn intake was 16·0 (sd 5·3) mg/d (range: 9–31 mg/d), and plasma Zn concentrations were 15·5 (SD 2·8) μmol/l (range 11–23 μmol/l). PBMC MT2A was positively correlated with dietary Zn intake (r 0·306, P = 0·03) and total Zn intake (r 0·382, P < 0·01), whereas plasma Zn was not (P > 0·05 for both). Findings suggest that MT2A mRNA in PBMCs reflects dietary Zn intake in healthy adults and may be a component in determining Zn status.

scholarly journals Role of T cells in intrauterine administration of activated peripheral blood mononuclear cells in recurrent implantation failure.

2021 ◽  
Author(s):  
Guillaume Ricaud ◽  
Cathy Vaillancourt ◽  
Veronique Blais ◽  
Marjorie Disdier ◽  
Fabien Joao ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Treg Cells ◽  
Implantation Failure ◽  
Recurrent Implantation Failure ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

Intrauterine administration of autologous peripheral blood mononuclear cells (PBMC) has been recently proposed as new immunotherapy for patients with unexplained recurrent implantation failure (RIF). In these patients, administration of activated PBMC 24-h or 72-h before embryo transfer resulted in a 3-fold increase in biochemical pregnancy rate. In this study we evaluated the role of T cells to promotes human endometrial receptivity. On the day of ovulation, PBMC were isolated from and activated with T cells mitogen, the phytohemagglutinin (PHA) and hCG for 48-h in a conditioned culture medium. Distributions of CD4+ T cells were characterized in 157 patients by flow cytometry before and after PHA/hCG activation. Cytokine production was analyzed by cytometric beads array. We observed in RIF patients a significant decrease in Th2 and natural Treg cells before activation with PHA/hCG and an increase of Th17 cells after activation compared to intrauterine sperm insemination (IUI) and in vitro fertilization (IVF) groups. Furthermore, the hCG/PHA treatment increases anti-inflammatory T cells (Th2 and Treg cells) compared to non-treated T cells. Principal component analysis (PCA) performed on CD4 T cell subtypes revealed a different cellular profile in the RIF compared to the IUI and IVF groups. This inflammatory state change could explain how endometrium immunomodulation by hCG-activated PBMC helps patients with unexplained RIF to reach implantation.

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