Expression Levels Of Foxp3 mRNA Research Articles

An Increase of Treg Cells in the Peripheral Blood is Associated with a Better In Vitro Fertilization Treatment Outcome

The objective of this study was to determine whether there was any association between the peripheral blood CD4(+) CD25(+) Foxp3(+) regulatory T cells (Treg cells) and implantation success in patients undergoing in vitro fertilization (IVF) treatment. Prospective observational study of 101 randomly selected women who underwent IVF treatment for tubal factor from May 2011 to June 2011. The percentage of peripheral blood Treg cells and the expression levels of Foxp3 and CTLA4 mRNA in peripheral blood mononuclear cells (PBMCs) were recorded and their relations to IVF treatment outcomes were analyzed. Treg cells were significantly elevated in the pregnant group ( P = 0.03). The expression level of Foxp3 mRNA in PBMCs from pregnant group also significantly increased ( P = 0.02). A receiver operating characteristic analysis (area under curve = 0.631) found that those women with Treg cells >0.6%, the pregnancy rate and live birth rate were much higher as compared to women with Treg cells below this level ( P < 0.05). An increase of Treg cells in the peripheral blood was associated with a better IVF treatment outcome (OR 4.3, 95% CI = 1.76-10.48), with a sensitivity of 64%, specificity of 71%. An elevated level of circulating Treg cells was associated with increased rates of pregnancy and live birth in IVF treatment.

Kirenol exerts a potent anti-arthritic effect in collagen-induced arthritis by modifying the T cells balance

Rheumatoid arthritis is characterized by the imbalance of T cells, which leads to increased pro-inflammatory and reduced anti-inflammatory cytokines. Modulating the balance among T cells is crucial for the treatment of RA. Kirenol is a major diterpenoid components of Herba Siegesbeckiae, which has been applied for arthritic therapy for centuries. Since prior research showed Kirenol exhibited anti-inflammatory effect in rats, in this study we have evaluated the effect and mechanism of bioactive Kirenol in a rat model of collagen-induced arthritis (CIA) on modulation of T cells. After immunization with bovine type II collagen (CII), Wistar rats were orally administered saline (CIA group), 2mg/kg Kirenol or 2mg/kg prednisolone daily for 30 days. The severity of arthritis was clinically and histologically assessed. The numbers of CD4+CD25+Foxp3+ T regulatory cells (Tregs) and IFNγ+CD4+ and IL4+CD4+ T cells were determined by flow cytometry, the mRNA expression level of Foxp3 was quantified by RT-PCR, cytokine levels were measured by ELISA and CII-induced cell proliferation was quantified in vitro. Kirenol significantly delayed the occurrence and reduced the disease severity of CIA. Histological analysis confirmed Kirenol suppressed joint inflammation and inhibited cartilage and bone destruction, compared to the CIA group. Kirenol also upregulated the mRNA expression of Foxp3, increased the numbers of CD4+CD25+Foxp3+ and IL4+CD4+ T cells, and reduced the number of IFNγ+CD4+ T cells. Kirenol reduced the levels of TNF-α, IL-17A and IL-6 in synovial fluid and TNF-α, IL-17A and IFN-γ in serum, and increased the serum levels of IL-4, IL-10 and TGF-β1. In addition, Kirenol inhibited the ability of CII to induce splenocyte, PBMC and lymph node cell proliferation in vitro, compared to cells from CIA rats. In conclusion, these results suggest that Kirenol may be a potential immunosuppressant for the treatment for rheumatoid arthritis.

Expressions of RORγt and Foxp3 in peripheral blood of children with atopic dermatitis

Objective To detect the mRNA expression levels of RORγt and Foxp3 genes in peripheral blood mononuclear cells(PBMCs) of children with atopic dermatitis(AD),and to explore their possible roles in the development of AD.Methods Sixty-three children with AD and 54 age-matched normal children were eligible for this study.Real-time fluorescence-based quantitative PCR was carried out to measure the mRNA expressions of RORγt and Foxp3 genes in PBMCs of these subjects.Results The relative expression levels of Foxp3 and RORγt mRNA were significantly higher in children with AD compared with the normal controls (2.4060 ± 0.3355 vs.1.1852 ± 0.4189,t =17.50,P＜ 0.01; 6.9130 ± 0.2046 vs.2.7501 ± 0.2518,t =98.63,P ＜ 0.01 ).A statistical increase was observed in the level of RORγt mRNA in children with severe AD compared with children with mild and moderate AD(7.1203 ± 0.1056 vs.6.8046 ± 0.1731 and 6.8655 ± 0.3671,t =6.61,5.23,both P ＜ 0.01).No significant difference was noted in the expression of RORγt mRNA between children with mild and moderate AD,or in the Foxp3 mRNA expression among children with mild,moderate and severe AD.The severity of AD was positively correlated with the level of RORγt mRNA (r =0.62,P ＜ 0.01 ),but uncorrelated with the level of Foxp3 mRNA(r =0.04,P ＞ 0.05).Conclusions The high expressions of RORγt and Foxp3 genes may be involved in the pathogenesis of AD,and the severity of AD is positively correlated with the expression intensity of RORγt gene. Key words: Dermatitis, atopic; CD4-positive T-lymphocytes; RORγt gene; Foxp3 gene

The role of T-regulatory cells in the pathogenesis of psoriasis

T-regulatory cells control the strength and duration of immune response and play an important role in the pathogenesis of psoriasis. The goal of the study was to examine the content of T-regulatory cells in peripheral blood and affected skin of psoriasis patients, the level of FOXP3 mRNA expression in psoriatic skin. Determination of subpopulations of T-regulatory lymphocytes in peripheral blood was performed using flow cytometry. The median level of FOXP3 mRNA expression was determined in biopsies of the skin by PCR in real time. Immunohistochemistry was performed with monoclonal anti- FOXP3 antibodies. Revealed an increase in the relative content of T-regulatory cells in the peripheral blood of patients with psoriasis in the progressive period, compared with healthy people. The level of FOXP3 mRNA expression in psoriatic foci was 3.1 times higher than in healthy individuals. Was noted a 10-fold increase in the number of T-regulatory lymphocytes in the skin of psoriasis patients in the progressive period, compared with skin from healthy donors.

The imbalance of Th17, CD4+ CDhigh25 Treg cells and the related cytokines in patients with systemic lupus erythematosus

Objective To analyze the levels of Th17,CD4+ CDhigh25and the related cytokines (IL-17,IL-21,TGF-β,IL-10)in the plasma of patients with SLE.Methods The percentage of Th17 and CD4+ CDhigh25 Treg cells in peripheral blood were examined by flow cytometry.QRTPCR was used to detect the mRNA expression level of RORγt and Foxp3 of 56 SLE patients (26 patients are inactive and 30 patients are active) and 28 healthy controls.Plasma cytokines levels were measured by ELISA.Results The percentage of Th17 cells in patients with SLE was higher than that of healthy controls [ (3.44 ± 0.96) ％ vs (2.42 ± 0.52) ％,t =5.38,P =0.000 ].The percentage of CD4+ CDhigh25 Treg cells in patients with SLE was lower than that of healthy controls [ ( 1.32 ± 0.57 ) ％ vs ( 2.07 ± 0.67 ) ％,t =3.28,P =0.034].The expressions of RORγt mRNA was increased compared with healthy controls[ (0.219 ±0.063) vs (0.087 ± 0.045 ),t =6.41,P =0.000 ],but the expressions of Foxp3 mRNA was decreased compared with healthy controls [ (0.063 ±0.045) vs (0.128 ±0.056),t =5.28,P =0.000].Plasma levelsof IL-17,IL-21 and IL-10 in SLE patients were 122.4 ( 60.5 - 188.3 ),167.2 ( 128.7 - 871.4 ),51.3(20.9 - 123.7 ) ng/L,respectively,and in the control group were 27.1 ( 18.1 - 86.2 ),31.0 ( 31.0 -424.5),33.5( 16.4 - 54.1 ) ng/L,respectively,they were significantly different between the two groups (Z =3.83,4.54,1.87,all P ＜ 0.05 ).However,TGF-β levels were markedly decreased in SLE patients compared with controls [ 31.0 ( 31.0 - 168.6 ) ng/L vs 159.8 ( 63.4 - 389.7 ) ng/L,Z =4.87,P ＜0.05.IL-17 was positive correlation with Th17 cells,SLEDAI score,dsDNA antibody titers ( r =0.621,0.581,0.512,respectively,all P ＜ 0.05 ),but was negative correlation with C3 ( r =- 0.543,P ＜ 0.05 ) in SLE patients.Although the percentage of CD4 + CDhigh25 Treg cells were negatively correlated with SLEDAI score ( r =- 0.423,P ＜ 0.05 ),it was positively correlated with C3 ( r =0.511,P ＜ 0.05 ).Conclusion This study suggests that the imbalance of Th17 cells and CD4+ CDhigh25 Treg cells and the disorder of related cytokines are important in the pathogenesis of SLE patients. Key words: Lupus erythematosus,systemic; T-Lymphocytes; Cytokines

The Treg/Th17 Imbalance in Toxoplasma gondii‐Infected Pregnant Mice

To evaluate whether impaired Treg/Th17 balance exists in the pregnant mice infected with Toxoplasma gondii. Regulatory T (Treg) and T-helper type 17 (Th17) cells were measured in both placenta and spleens of the pregnant mice infected with and without T. gondii by flow cytometry. The mRNA and protein expression levels of transforming growth factor-β (TGF-β) and interleukin-17A (IL-17A) were analyzed using real-time PCR and enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of forkhead box P3 (Foxp3), retinoic acid-related orphan receptor γt (RORγt), and IL-6 were also analyzed using real-time PCR. The correlations of the ratio of Treg/Th17 to the mRNA or protein expression level of those factors were analyzed by Spearman's correlation analysis. Data were analyzed by unpaired t-test and paired t-test. The proportion of Tregs or Th17 cells in the placenta and spleens of the T. gondii-infected pregnant mice was significantly lower or higher than in those of non-infected mice, respectively. Upregulation of TGF-β and downregulation of IL-17A were found in the placenta of T. gondii-infected pregnant mice. The ratio of Treg to Th17 was significantly lower in the infected mice than that in the non-infected mice (P<0.01).The ratio of Treg to Th17 positively or negatively correlated with the protein expression level of TGF-β (r=0.6204, P<0.05) or IL-17A (r=-0.6296, P<0.05), respectively. The ratio also positively correlated with the mRNA expression level of Foxp3 (r=0.7985, P<0.01), but negatively correlated with the mRNA expression level of RORγt (r=-0.6153, P<0.05), and IL-6 (r=-0.7492, P<0.01). TheTreg/Th17 imbalance exists in the pregnant mice infected with T. gondii, which is associated with the expression of related cytokine and key transcription factors. This result suggests that the embryo loss caused by this parasite may be associated with a reduced ratio of Treg to Th17 cell number.

Cutting Edge: Immunological Consequences and Trafficking of Human Regulatory Macrophages Administered to Renal Transplant Recipients

Regulatory macrophages (M regs) were administered to two living-donor renal transplant recipients. Both patients were minimized to low-dose tacrolimus monotherapy within 24 wk of transplantation and subsequently maintained excellent graft function. After central venous administration, most M regs remained viable and were seen to traffic from the pulmonary vasculature via the blood to liver, spleen, and bone marrow. By 1 y posttransplantation, both patients displayed patterns of peripheral blood gene expression converging upon the IOT-RISET signature. Furthermore, both patients maintained levels of peripheral blood FOXP3 and TOAG-1 mRNA expression within the range consistent with nonrejection. It is concluded that M regs warrant further study as a potential immune-conditioning therapy for use in solid-organ transplantation. The results of this work are being used to inform the design of The ONE Study, a multinational clinical trial of immunomodulatory cell therapy in renal transplantation.

Maternal Foxp3 Expressing CD4+ CD25+ and CD4+ CD25− Regulatory T‐Cell Populations are Enriched in Human Early Normal Pregnancy Decidua: A Phenotypic Study of Paired Decidual and Peripheral Blood Samples

Citation Dimova T, Nagaeva O, Stenqvist A‐Christin, Hedlund M, Kjellberg L, Strand M, Dehlin E, Mincheva‐Nilsson L. Maternal Foxp3 Expressing CD4+ CD25+ and CD4+ CD25− Regulatory T‐Cell Populations are Enriched in Human Early Normal Pregnancy Decidua: A Phenotypic Study of Paired Decidual and Peripheral Blood Samples. Am J Reprod Immunol 2011; 66 (Suppl. 1): 44–56Problem Regulatory T cells (Treg cells), a small subset of CD4+ T cells maintaining tolerance by immunosuppression, are proposed contributors to the survival of the fetal semiallograft. We investigated Treg cells in paired decidual and peripheral blood (PB) samples from healthy women in early pregnancy and PB samples from non‐pregnant women.Method of study Distribution, location, cytokine mRNA, and phenotype were assessed in CD4+ CD25+ Treg cells from paired samples using immunohistochemistry, immunofluorescence, flow cytometry, and real‐time quantitative RT‐PCR.Results The presence and in situ distribution of CD4+ Foxp3+ Treg cells in decidua are hereby demonstrated for the first time. Three Foxp3+ cell populations, CD4+ CD25++ Foxp3+, CD4+ CD25+ Foxp3+, and CD4+ CD25− Foxp3+, were enriched locally in decidua. In contrast, no statistically significant difference in numbers of circulating Treg cells between pregnant and non‐pregnant women was found. The Foxp3+ cells expressed the surface molecules CD45RO, CTLA‐4, CD103, Neuropilin‐1, LAG‐3, CD62L, and TGFβ1 mRNA consistent with Treg phenotype. The population of CD4+ CD25− Foxp3+ cells, not described in human decidua before, was enriched 10‐fold compared with PB in paired samples. Their cytokine expression was often similar to Th3 profile, and the Foxp3 mRNA expression level in CD4+ CD25− cells was stable and comparable to that of CD4+ CD25+ Treg cells implying that the majority of CD4+ CD25− Foxp3+ cells might be naïve Treg cells.Conclusion (i) There is a local enrichment of Treg cells in decidua (ii) The exclusive accumulation of decidual CD4+ CD25− Foxp3+ cells suggests an additional reservoir of Foxp3+ naïve Treg cells that can be converted to ‘classical’ Treg cells in uterus.

The pretreatment of triptolide could prevent the liver ischemia-reperfusion injury by inducing regulatory T cells in mice

Objective To investigate the effect and related mechanism of triptolide pretreatment to prevent from ischemia/reperfusion (I/R) injury in mice liver. Methods Sixty male C57BL/6 mouse were randomized into four groups (15/group): A:sham group with saline , B: sham group with triptolide, C: saline I/R group, D: triptolide I/R group. The mice were pretreated with either saline or triptolide (0. 1 mg/kg/d) through intraperitoneal (ip) injection for one week. The mouse partial liver model of I/R injury was established, and samples were collected at 24 h after the I/R injury. Results Serum ALT and AST levels were significantly decreased and histological damage was significantly alleviated in the triptolide I/R group as compared with the saline I/R group (P＜0.05), the concentration of MDA in the triptolide groups was significantly decreased, while SOD activity was significantly increased compared with that of the saline I/R group (P＜0.05). The percentages of CD4+ CD25+ regulatory T cells (Tregs) cells among CD4+ T cells in groups A, B, C, and D were(7. 55 ± 1.87)%, (12. 59±3. 87)%,(7. 85±1.07)%, and(12. 02±3. 16)% in liver tissue, respectively. The expression levels of Foxp3 mRNA were significantly higher in the triptolide I/R group than those of saline I/R group (P＜0. 05). ELISA showed that triptolide could significantly inhibit the levels of IL-6, IL-Iβ and TNF-αand promoted the level of IL-10 in the serum (P＜0.05). Conclusion Pretreatment with triptolide could effectively prevent from liver I/R injury, which may be related to the induction of Treg cells by triptolide, the increase in the level of IL-10 in serum, and the inhibition of IL-6, IL-1β and TNF-α production in serum. Key words: Triptolide; CD4+CD25+regulatory T cells; Ischemia/reperfusion; Liver injury

Expansion of CD4+CD25+ regulatory T cells from cord blood CD4+ cells using the common γ-chain cytokines (IL-2 and IL-15) and rapamycin

Rapamycin has important roles in the modulation of regulatory T cells. We tried to expand CD4(+)CD25(+) regulatory T cells (Treg cells) from umbilical cord blood (CB) CD4-positive cells using interleukin (IL)-15 or IL-2 with transforming growth factor (TGF)-β and rapamycin. We were able to obtain more than 500-fold expansion of CD4(+)CD25(+) cells from CB CD4(+) cells using IL-15 and TGF-β with rapamycin. These expanded CD4(+)CD25(+) cells expressed forkhead box P3 (FoxP3) mRNA at a level about 100-fold higher and could suppress allogeneic mixed lymphocyte culture (MLC) by more than 50%. Early after rapamycin stimulation, CB CD4(+) cells showed increased expression of FoxP3 and a serine/threonine kinase Pim2 and sustained expression of negative phosphoinositide 3-kinase regulator phosphatase and tensin homolog deleted on chromosome 10 (PTEN). On the other hand, CD4(+)CD25(+) cells expanded with rapamycin for 8 days showed much higher levels of FoxP3 mRNA expression and decreased expression of PTEN. A comparison of IL-15 stimulation and IL-2 stimulation showed slightly higher efficiency of IL-15 for expansion of CD4(+)CD25(+) cells, and for FoxP3 expression, IL-15 also showed significantly higher efficacy for inhibition of MLC. The combination of the common γ-chain cytokine IL-15, TGF-β, and rapamycin may be a useful means for expanding Treg cells. Pim2 expression early after stimulation with rapamycin may be important for conferring rapamycin resistance for growth of Treg cells. IL-15 is not less useful than IL-2 for expansion of Treg cells.

Intraperitoneal Echinococcus multilocularis infection in mice modulates peritoneal CD4+ and CD8+ regulatory T cell development

Intraperitoneal proliferation of the metacestode stage of Echinococcus multilocularis in experimentally infected mice is followed by an impaired host immune response favoring parasite survival. We here demonstrate that infection in chronically infected mice was associated with a 3-fold increase of the percentages of CD4+ and CD8+ peritoneal T (pT) cells compared to uninfected controls. pT cells of infected mice expressed high levels of IL-4 mRNA, while only low amounts of IFN-γ mRNA were detected, suggesting that a Th2-biased immune response predominated the late stage of disease. Peritoneal dendritic cells from infected mice (AE-pDCs) expressed high levels of TGF-β mRNA and very low levels of IL-10 and IL-12 (p40) mRNA, and the expression of surface markers for DC-maturation such as MHC class II (Ia) molecules, CD80, CD86 and CD40 was down-regulated. In contrast to pDCs from non-infected mice, AE-pDCs did not enhance Concanavalin A (ConA)-induced proliferation when added to CD4+ pT and CD8+ pT cells of infected and non-infected mice, respectively. In addition, in the presence of a constant number of pDCs from non-infected mice, the proliferation of CD4+ pT cells obtained from infected animals to stimulation with ConA was lower when compared to the responses of CD4+ pT cells obtained from non-infected mice. This indicated that regulatory T cells (Treg) may interfere in the complex immunological host response to infection. Indeed, a subpopulation of regulatory CD4+ CD25+ pT cells isolated from E. multilocularis-infected mice reduced ConA-driven proliferation of CD4+ pT cells. The high expression levels of Foxp3 mRNA by CD4+ and CD8+ pT cells suggested that subpopulations of regulatory CD4+ Foxp3+ and CD8+ Foxp3+ T cells were involved in modulating the immune responses within the peritoneal cavity of E. multilocularis-infected mice.

Low responder T cell susceptibility to the suppressive function of regulatory T cells in patients with dilated cardiomyopathy

ObjectiveThe pathogenesis of dilated cardiomyopathy (DCM) is closely connected with dysfunction of the autoimmune system, and CD4+CD25highCD127low/− regulatory T (Treg) cells have a vital role in maintaining self-tolerance. In this...

1-MT enhanced potency of dendritic cells pulsed with tumor cell lysate by downregulation of CD4 ~+ CD25~+ treg

Objective To determine whether 1-methyl-tryptophan (1-MT, an IDO inhibitor) might reduce CD4~+ CD25~+ Treg and improve the anti-tumor effects of dendritic cells pulsed with tumor cell lysate in the murine pancreas adenocarcinoma model. Methods Pan02 cell line was injected into syngeneic C57BL/6 mice. 1-MT was administered to pancreas adenocarcinoma mice and its effect on the prevalence of regulatory T cells (Treg) in tumor draining lymph nodes (TDLNs) and spleen was investigated. The prevalence of Treg in the TDLNs and tumor spleen (TS) was detected by using flow cytometry. The expression level of Foxp3 mRNA was detected in the spleen and TDLN by real-time PCR. To prepare the DC vaccine,dendritic cells were pulsed with tumor cell lysate. This DC vaccine, as a single agent or in combination with 1-MT, was administered to pancreas adenocarcinoma mice. The anti-tumor effects in different treatment were deter mined by regular observation of tumor development. Results There was statistically significant difference in the percentage of CD4~+ CD25~+ T lyraphocytessignificant between 1-MT-treated group [TDLNs (16.01 ± 2.21)% and TS (13. 11 ± 1. 93)%] than PBS control group [TDLNs (25. 00 ± 2. 16)% and TS (22.14 ±2.33)%] (P<0.05,P<0.01). In 1-MT-treated group,the Foxp3 expression was significantly lower in TDLN and TS than in control LN and spleen. Furthemore,in 1-MT + DC vaccine-treated group,the tumor growth rate was significantly slower than in groups of DC vaccine, 1-MT and PBS (the tumor volume was (789.0 ±111.0), (1768.0 ±251. 3), (1854.0 ±192. 1),(1899.0 ±201.2) mmJ at the 36th day af ter tumor challenge, respectively (P < 0. 01). Conclusion 1-MT can enhance anti-tumor effects of den dritic cells pulsed with tumor cell lysate by downregulating the expression of CD4~+ CD25~+ Treg. Key words: Pancreas adenocarcinoma; Dendritic cells; Vaccine

ORIGINAL ARTICLE: Imbalance of T‐cell Transcription Factors Contributes to the Th1 Type Immunity Predominant in Pre‐eclampsia

Problem Extensive studies have demonstrated that Th1 type immunity is predominant in pre‐eclampsia, but there is little concern with regard to the intracellular mechanisms behind this initial T‐cell polarization. In this study, we investigated whether the imbalance of the T‐cell transcription factors contributes to it.Method of study A total of 15 pre‐eclamptic patients and 15 healthy pregnant women were enrolled in this study. The expression levels of transcription factors for Th1 (T‐bet), Th2 (GATA3), Th17 (RORc) and Treg (FOXP3) cells, together with the Th1/Th2 status, were simultaneously investigated in both peripheral blood mononuclear cells (PBMCs) and decidua.Results The expression levels of FOXP3 mRNA were decreased in both PBMCs and decidua from pre‐eclamptic patients compared with healthy pregnant women (P &lt; 0.05), and T‐bet mRNA and RORc mRNA were significantly increased (P &lt; 0.05), while Th1/Th2 balance shifted toward the Th1 immunity. Furthermore, there was a negative correlation between FOXP3 mRNA and Th1 cells (P &lt; 0.05), and the expression level of T‐bet mRNA correlated strongly with Th1 cells (P &lt; 0.05).Conclusion Decreased expression of FOXP3 mRNA and increased expression of T‐bet mRNA may contribute to Th1 type immunity predominant in pre‐eclampsia.

Activation of the Adenosine A2A Receptor Promotes the Development of Donor-Derived T Regulatory Cells in a Graft-Versus Host Disease Mouse Model.

Abstract 1339Poster Board I-361 IntroductionGraft versus host disease (GVHD) remains a significant complication of allogenic stem cell transplantation and is a considerable cause of transplant morbidity and mortality. The recognition of the role of Foxp3+ regulatory T cells in immunomodulation has given rise to interest in using these cells to abrogate or modify the severity of graft versus host disease as well as in methods to increase their development during transplant. A less well known avenue of research is the targeting of the adenosine A2A receptor (A2AR). In ischemia models, activation of the Gs-coupled adenosine receptors play a role in terminating inflammation and improving survival of damaged and/or transplanted organs by directly down regulating the activity of the receptor bound T cell. We have shown previously that the use of a specific A2AR agonist known as ATL146e decreases the incidence and severity of GVHD as well as improves survival of mice in a GVHD transplant model (paper in submission). MethodsIn order to further understand the role of the agonist in GVHD abrogation we performed studies looking at the possible role of T regulatory cells in relation to the use of the agonist. Using a parental into irradiated F1 offspring transplant model (C57BL/6J [B6, H-2b] → B6D2F1/J [BDF1, H-2b/d]) we can induce GVHD manifested by weight loss and mortality in 100% of mice by infusing an additional 10 million donor T cells into mice previously engrafted with 10 million bone marrow donor cells and 800cGy of radiation. We administered the ATL146e or a PBS control by osmotic mini pumps resulting in continuous subcutaneous infusion for 14days starting one day before the donor T cell infusion. Mice that received only the congenic donor bone marrow transplant and no donor T cells did not develop GVHD and served as an additional control. Post transplant, splenocytes and peripheral blood cells were collected and stained for CD4, CD25 and FoxP3 and were analyzed by flow cytometry. The level of Foxp3 mRNA expression in lymphocytes was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and the concentration of IL-10 in serum was measured by enzyme-linked immunosorbent assay (ELISA). To identify the origin of activated T regulatory cells, we also performed transplants used a B6.PL-Thy1/CyJ (B6-Thy1.1, H-2b) mouse strain as the donor. ResultsFrom these studies we confirmed that the A2AR agonist, ATL146e, inhibited the weight loss and mortality associated with acute GVHD progression and seen in the PBS treated controls. More notably, treatment with ATL146e resulted in a 7 fold increase in CD4+CD25+FoxP3+ T regulatory cells in both the spleen and peripheral blood compared to our PBS treated group at days 14 to 20 after hematopoietic stem cell transplantation. From our Thy-1 disparate transplants we determined that the increased T regulatory cells were of donor origin. We also found that the expression of Foxp3 mRNA in splenocytes and the level of IL-10 in the serum was increased 3 fold and 2.4 fold respectively in the ATL146e treated mice. ATL146e agonist activity is very specific to A2AR. When using an alternate adenosine agonist less specific to A2AR, we did not see the same increases of Foxp3 or IL-10, nor did we see any decrease in the severity of the graft versus host disease. ConclusionsThus we believe that the specific activation of A2AR inhibits acute GVHD through the increase of donor-derived CD4+ CD25+ FoxP3+ immunosuppressive T regulatory cells. Our observation provides an additional mechanistic basis for the anti-inflammatory capacity of A2AR agonist in acute GVHD. Additional studies are ongoing to elucidate further the mechanism of the agonist's ability to increase the T regulatory population as well as the effects of combination therapies. DisclosuresNo relevant conflicts of interest to declare.

Expression of indoleamine 2,3-dioxygenase and increased prevalence of regulatory T cells in pancreas adenocarcinoma-bearing mice

Objective To investigate the expression of indoleamine 2,3-dioxygenase (IDO) and the prevalence of regulatory T cells (Treg) in tumor draining lymph nodes (TDLNs) and the spleen in a murine pancreas adenocarcinoma model, and study the mechanism by which tumors escaped from immune recognition. Methods Pan02 cell line was injected into syngeneic C57BL/6 mice (n=24) and the IDO expression level in the TDLNs was detected by fluorescence quantitative RT-PCR and Western blot. The prevalence of Treg in the TDLNs and tumor spleen (TS) was assayed by using flow cytometry. The expres-sion level of Foxp3 mRNA was measured in the spleen and TDLNs. Results The expression levels of IDO mRNA and protein in tumor-bering mice (1.670±0.099 and 1. 401±0.079) were significantly higher than in controls (1.123±0. 243 and 0. 872±0.193). The percentage of CD25~+CD4~+T lymphocytes reached (25.00±2.16)% of CD4~+T cells in TDLNs and (22.14±2.33)% in TS. In contrast,the per-eentage of CD25~+ CD4~+ T cells in the lymphocyte nodes (LN) of non-tumor-bearing mice remained (11.02±1.31) % and (12.11±0.93) % respectively. Foxp3 expression was considerably higher in TDLNs and TS than in control LN and spleen. Conclusion The up-regulation of the IDO expression in TDLNs and increased prevalence of Treg in TDLNs and TS are induced by pancreas adenocarcinoma. Key words: Pancreas adenocarcinoma; Indoleamine 2,3-dioxygenase; CD4; CD25

Urinary FOXP3 mRNA in patients with lupus nephritis--relation with disease activity and treatment response

Regulatory T lymphocytes (Tregs) probably play an important role in the pathogenesis of SLE. We quantified messenger RNA (mRNA) expression of FOXP3, a critical regulator for the development and function of Tregs, in the urinary sediment of 25 subjects with active lupus nephritis (LN), 17 with inactive lupus and 7 healthy subjects. We found that the expression level of FOXP3 was significantly higher in urine from patients with active LN than from subjects with inactive lupus and healthy controls (24.5 +/- 45.8 vs 0.8 +/- 1.0 vs 0.6 +/- 0.8 copy; P < 0.001). In the active group, urinary FOXP3 mRNA expression level was higher in patients with proliferative LN than non-proliferative nephritis (34.6 +/- 56.3 vs 2.7 +/- 2.1 copy; P = 0.019). Urinary FOXP3 mRNA level significantly correlated with SLEDAI (r = 0.668; P < 0.001) and proteinuria (r = 0.414; P = 0.006). In the active group, urinary FOXP3 mRNA level also significantly correlated with histological activity index (r = 0.541; P = 0.009) and marginally with intra-renal FOXP3 mRNA level (r = 0.360; P = 0.08). Urinary FOXP3 mRNA in patients with no response to therapy was higher than those with partial response or complete response (57.6 +/- 69.8 vs 2.4 +/- 1.9 copies; P = 0.02). We concluded that urinary FOXP3 mRNA is markedly up-regulated in patients with active LN, and the level of expression is closely correlated with the clinical and histological disease activity. A high urinary FOXP3 mRNA in LN predicts a poor therapeutic response. Measurement of FOXP3 mRNA in urine sediment may be a non-invasive biomarker for assessing the severity and risk stratification in LN.

Analysis of CD4+CD25+Foxp3+ regulatory T cells in patients of haemorrhagic fever with renal syndrome

To investigate the percentage and significance of CD4(+)CD25(+)Foxp3(+) regulatory T cells in the patients with haemorrhagic fever with renal syndrome (HFRS). Peripheral blood samples were collected from 27 HFRS patients, and 19 healthy controls to examine the proportion of the CD4(+)CD25(+)Foxp3(+) cells. RT-PCR was used to detect the Foxp3 mRNA expression in the mononuclear cells in peripheral blood. Biopsy was conducted on the HFRS patients during oliguria stage. Peripheral blood samples and morning urine samples were collected in the stages and in the very day of renal biopsy to examine the urine protein, urine creatinine (Cr), blood Cr, and creatinine clearance rate (Ccr). The percentages of CD4(+)CD25(+)Foxp3(+) in the HFRS patients in the hypotension, oliguria, polyuria, and convalescent stages were 5.0% +/- 1.2%, 3.1% +/- 0.8%, 4.2% +/- 0.9%, and 5.0% +/- 1.1% respectively, all significantly lower than that of the normal control group (6.5% +/- 2.1%, all P < 0.01). The proportion of CD4(+)CD25(+)Foxp3(+)T cells in the CD4(+)T cells in the oliguria stage was significantly lower than those in other stages and that of the normal controls (P < 0.01). The percentage of CD4(+)CD25(+)Foxp3(+) in the patients with HFRS was significantly correlated with the renal injury index (r = 0.51, P < 0.01), but not significantly correlated with urine protein and renal function changes (r = 0.11 and 0.18, both P > 0.05). The expression level of Foxp3 mRNA in peripheral mononuclear cells of the HEFS patients in the oliguria stage was (503 +/- 64), significantly lower than that of the normal controls (3102 +/- 409, P < 0.01) and those of the HFRS patients in the hypotension, polyuria, and convalescent stages (1805 +/- 291, 1869 +/- 320, and 2697 +/- 420, all P < 0.01). The proportion of CD4(+)CD25(+)Foxp3(+) regulatory T cells decreases in patients with HFRS, which may contribute to the pathological process in such disease.

Reduction of natural regulatory T cells in thymomas accompanying myasthenia gravis and its possible association with Foxp3 and thymic stromal lymphopoietin

Objective To investigate the expression of both thymic regulatory T cells (CD4 + CD25 + Foxp3 + cells, Treg) and thymic stromal lymphopoietin (TSLP) in thymomas accompanying myasthenia gravis. Methods We used immunohistochemistry and real-time reverse trancription polymerase chain reaction (real-time RT-PCR) techniques to determine Foxp3 + Treg counts and the expression levels of Foxp3 mRNA and TSLP mRNA in thymomas of 23 MG patients and thymuses of 4 healthy controls. Results The CD4 + Foxp3 + nTreg (natural regulatory T cells) counts in thymomas were significantly lower than those in normal thymuses ( P<0.01), and the expression levels of Foxp3 mRNA and TSLP mRNA were also lower in thymomas( P<0.01). Among the thymoma types, type B1 thymoma had the highest Foxp3 + nTreg count and standard values of Foxp3 mRNA and TSLP mRNA. There was a strong positive correlation between the mRNA transcriptional levels of Foxp3 and TSLP. Conclusion The insufficient expression of Foxp3 in thymoma, which may be caused by decreased transcription of TSLP, may result in the reduction of Tregs and cause autoimmune disorders.

Quantification and molecular characterization of regulatory T cells in connective tissue diseases

The aim of our study was to investigate and characterize regulatory T cells (Treg) in peripheral blood of patients with connective tissue diseases (Systemic lupus erythematosus, systemic sclerosis, Sjögren's syndrome, poly- and dermatomyositis) as compared with blood from healthy controls. Treg cells were quantified and phenotypically characterized by flow cytometry while the expression level of Foxp3 mRNA was evaluated by real time PCR. A reduced percentage of peripheral blood Treg cells was found in patients than in controls, irrespective of the type of connective tissue disease. Treg cells, especially those expressing one of the phenotypical markers, seemed to differ not only between patients and healthy controls but also among types of diseases. Additionally, the presence of autoantibodies as well as disease activity appeared to be correlated with particular Treg cell populations, especially those expressing one of the examined phenotypical markers. Correlations with therapy suggested that glucocorticoids plus antimalarial or other immunosuppressor drugs diminished the percentage of Treg cells, especially of those with memory phenotype. These findings indicated dysregulations at the level of Treg cells and suggested an involvement of these cells in the pathology of connective tissue diseases. Moreover, our data are in agreement with the suggestion that Treg cells could be therapeutic targets for some autoimmune diseases.