Treg Cells In Patients Research Articles

O73. The role of Th17/Treg imbalance in normal pregnancy and pre-eclampsia

Introduction The etiology and pathogenesis of pre-eclampsia are not yet fully understood. Many of the findings support a hypothesis that an inappropriate activation of immune system is associated with the development of this syndrome. Objectives The aim of the study was to estimate the prevalence of CD3 + CD4 + T lymphocytes producing IL-17, IL-2, IFN-and IL-4, as well as CD4 + CD25 + FoxP3 + Tregulatory cells (Tregs) in peripheral blood of patients with preeclampsia and healthy women in the third trimester of normal pregnancy. Furthermore, the purpose of our study was to assess the immunosuppressive activity of Treg cells of patients with preeclampsia in comparison with the controls. Methods Thirty four patients with preeclampsia and 27 healthy women in third trimester of pregnancy were included to the study. The percentage of CD4 + CD25 + FoxP3 + Treg cells and CD3 + CD4 + T lymphocytes with intracellular expressions of cytokines was estimated using monoclonal antibodies and flow cytometry. The in vitro functional assays were performed with the use of Treg Cells Isolation Kit and 3 H-thimidine. Results The percentages of T CD3 + CD4 + lymphocytes producing IL-17A were significantly higher in preeclampsia when compared to healthy normotensive pregnant women in the third trimester of normal pregnancy ( p + CD25 + FoxP3 + Treg cells was significantly lower in the study when compared to the control group ( p + CD4 + CD25 − T lymphocytes of patients with preeclampsia during in vitro assay without Treg cells and after the addition of autologous Tregs. In normal pregnancy the stimulation index of CD3 + CD4 + CD25 − T lymphocytes was significantly higher without Treg cells when compared to this response after addition of autologous Tregs ( p Conclusions The results obtained suggest the up-regulation of Th17 immune response in preeclampsia. It seems that the decreased number and function of Treg cells may be responsible for the activation of inflammatory response in this disorder. In preeclampsia the predominance of Th17 immunity can act through the modulation of Th1/Th2 immune response.

Docosahexaenoic Acid Alleviates Atopic Dermatitis in Mice by Generating T Regulatory Cells and M2 Macrophages

Han et al. (this issue) describe a novel mechanism by which docosahexaenoic acid (DHA) may suppress atopic dermatitis symptoms in mice. They find that DHA induces FoxP3 T regulatory cells in vivo, M2 macrophages drive transforming growth factor-β and IL-10 conversion of CD4 T cells to CD4 FoxP3 T regulatory cells in vitro, and DHA-treated M2 macrophages suppress atopic dermatitis in mice.

Analysis of expression and function of the co-stimulatory receptor SLAMF1 in immune cells from patients with systemic lupus erythematosus (SLE).

The signaling lymphocytic activation molecule SLAMF1 (CD150) is a co-stimulatory molecule that is expressed by most immune cells, including T regulatory (Treg) lymphocytes. Since different abnormalities have been reported regarding the number and function of Foxp3+ Treg cells in patients with systemic lupus erythematosus (SLE), we decided to analyze the expression and function of CD150 in these regulatory lymphocytes in this condition. We isolated peripheral blood mononuclear cells from 20 patients with SLE, and 20 healthy controls. The expression of SLAMF1 was determined by multi-parametric flow cytometry and the suppressive function of CD4+CD25+ lymphocytes, upon engagement or not of CD150 with an agonistic monoclonal antibody, was analyzed by an assay of inhibition of cell proliferation. We observed a significantly increased expression of SLAMF1 by CD3+CD4+ helper T cells and CD19+ B cells in patients with SLE and active disease. However, similar levels of SLAMF1 expression were detected in Foxp3+ Treg cells from patients and controls. In contrast, a higher proportion of SLE patients increased their suppressive function of Treg cells upon CD150 engagement compared to healthy controls. Our data suggest that SLAMF1 is another significant piece in the intricate defective immune-regulatory function of patients with SLE.

Increased regulatory T cells in patients with liver cirrhosis correlated with hyperbilirubinemia and predict bacterial complications.

Patients with liver cirrhosis (LC) were regarded as immunocompromised status with high incidence of bacterial infection. Regulatory T cell (Treg cell) is known as an immune suppressor and also plays an important role in patients with sepsis. This paper aims to study the role of Treg cells in patients with liver cirrhosis and their correlations to bacterial complications. Thirty-three normal controls (NC) and 82 cirrhotic patients were enrolled for the case-control study. The Treg cells, defined as CD4+ CD25+ Foxp3+ T cells, in peripheral blood of these patients were evaluated. The percentage of Treg cells increased significantly in patients with liver cirrhosis when compared with normal volunteers. Furthermore, this increase of Treg cells was mainly memory phenotype defined as CD45RO+ Treg cells and was significantly correlated with serum bilirubin levels as evaluated by multiple linear regression analysis. In addition, the tumor necrosis factor (TNF)-α receptor II (TNFRII) expression also significantly increased on Treg cells in these patients. Interestingly, these membranous TNFRII would be shed and released into supernatant. Lastly, this increased percentage of Treg cells in cirrhotic patients correlate well with and predict subsequent bacterial complications. The Treg cells, mainly with memory phenotype and with high TNFRII expression, increased significantly in patients with liver cirrhosis and significantly correlated with the serum bilirubin levels. Furthermore, this increased Treg cells correlate with and predict subsequent bacterial complications in cirrhotic patients.

The Activation of NF-κB in Infiltrated Mononuclear Cells Negatively Correlates with Treg Cell Frequency in Oral Lichen Planus.

Oral lichen planus (OLP) is a T cell-mediated chronic inflammatory mucosal disease with persistent accumulation of T cells in the lamina propria. Nuclear factor-kappa B (NF-κB) is a major regulator of immune responses, and NF-κB-dependent cytokines and pro-inflammatory mediators can be detected in higher levels in the saliva and serum from patients with OLP. CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells play an important role in the prevention of autoimmune pathology by regulating the immune response. To explore the correlation between NF-κB p65 activation and accumulation of Treg cells in patients with OLP, 40 ethnic Chinese patients with OLP and 10 healthy volunteers were recruited. The nuclear expression of NF-κB p65 in infiltrated mononuclear cells and Treg cells in the OLP lesion and the normal oral mucosa (NOM) was analyzed by immunohistochemistry assay. Our results showed that both the nuclear expression of NF-κB p65 and the number of Foxp3(+) Treg were higher in the OLP lesions. Furthermore, the frequency of Treg cells was negatively correlated with NF-κB nuclear expression in subepithelial lymphocytic infiltrate of the OLP lesion. This finding provides a new insight into the pathogenesis of OLP and may contribute to novel therapeutic strategies for the treatment of OLP by modulating the immune system.

Modulation of STAT3 and STAT5 activity rectifies the imbalance of Th17 and Treg cells in patients with acute coronary syndrome

The signal transducer and activator of transcription (STAT) activity plays an important role in the differentiation and imbalance of Th17 and Treg cells in acute coronary syndrome (ACS) patients. We determined that the basal STAT3 phosphorylation level was significantly increased and exhibited a positive relationship with Th17 cells but was negatively correlated with Treg cells in ACS patients. Opposite effects were observed for STAT5 activity. Using the pharmaceutical inhibitor TG101348 or knockdown of STAT3 reduced the number of Th17 cells while promoting the number and function of Treg cells via the Janus kinase2 (JAK2)/STAT3 pathway in ACS patients. Significantly more STAT5 bound to the Foxp3 locus when STAT3 was knocked down, and overexpression of STAT5 led to an increased number of Treg cells but a decreased number of Th17 cells in ACS patients. Our findings demonstrate that modulation of STAT3/STAT5 activity rectifies the imbalance of Th17/Treg cells in ACS patients.

Correlation between Treg/Th17 and Traditonal Chinese Medicine syndrome differentiation classification in patients with immune thrombocytopenia

Objective To explore the role of Treg/Th17 cell ratio imbalance in the pathogenesis of idiopathic thrombocytopenic purpura(ITP)patients with different Traditional Chinese Medicine(TCM)syndrome differentiation classifications:bleeding due to blood-heat,Yin deficiency with fire hyperactivity and Qi deficiency-caused bleeding.Methods A total of 92 patients were divided into the bleeding due to blood-heat group(n=30),Yin deficiency with fire hyperactivity group(n=31)and Qi deficiency-caused bleeding group(n=31)according to the TCM syndrome differentiation classification.The peripheral blood samples were obtained from the patients and 30 volunteers served as healthy controls.The percentages of Treg cells and Th17 cells in the blood samples were analyzed by flow cytometry,and the mRNA levels of Foxp3 and ROR-γt were analyzed by RT-PCR.Results The percentages of Treg cells in the peripheral blood of 3different TCM syndrome ITP groups were significantly lower than those of control group(P0.05),that of the bleeding due to blood-heat group was significantly lower than that of Yin deficiency with fire hyperactivity group and Qi deficiency-caused bleeding group(P0.05),and that of Qi deficiency-caused bleeding group was signficantly lower than that of Yin deficiency with fire hyperactivity group(P0.05).The percentages of Th17 cells in peripheral blood of 3different TCM syndrome ITP groups were significantly higher than that of control group(P0.05),and that of Yin deficiency with fire hyperactivity group was signficanlty higher than that of Qi deficiency-caused bleeding group(P0.05).The ratios of Treg/Th17 of the 3different TCM syndrome ITP groups were significantly lower than that of control group(P0.05),that of the bleeding due to blood-heat group was significantly lower than that of Qi deficiency-caused bleeding group and Yin deficiency with fire hyperactivity group(P0.05),and that of Qi deficiency-caused bleeding group was significantly lower than that of Yin deficiency with fire hyperactivity group(P0.05).The Foxp3 mRNA levels of 3different TCM syndrome ITP groups were significantly lower than that of control group(P0.05),and there were significant differences between each two groups by the pairwise comparison(P0.05).While ROR-γmRNA levels of the 3different TCM syndrome ITP groups were significantly higher than that of the control group(P0.01).Conclusion The decreased Treg cells in patients with ITP contributes to the development and progression of ITP.The imbalance of Treg/Th17 ratio may play a critical role in the pathogenesis of ITP.The distribution of the percentages of Treg cells,the ratio of Treg/Th17 and the mRNA level of Foxp3 in a increasing order is:bleeding due to blood-heat group Qi deficiency-caused bleeding group Yin deficiency with fire hyperactivity group.

Rapamycin Can Reduce Apoptosis of CD4+CD25+ Regulatory T Cells from Mouse Model of Severe Aplastic Anemia

CD4+CD25+ regulatory T cells (Tregs) are believed to control the development and progression of autoimmunity by suppressing autoreactive T cells and decreased numbers of these cells are associated with impaired immune homeostasis and development of aplastic anemia (AA). We have developed a novel mouse model of severe aplastic anemia (SAA) induced by interferon-gamma in combination with Busulphan, in which excessive apoptotic CD4+CD25+ Treg cells was found in our previous experiments. It might be one of the reasons for reduction of CD4+CD25+ Treg cells in patients with AA. Rapamycin (RAPA) is a macrolide antibiotic produced by streptomyces hygroscopicus and is considered as a potential immunosuppressant in the treatment of AA, while the effect of RAPA on apoptosis of CD4+CD25+ Tregs from patients with AA is not clear. In this study, the SAA model female mice were intraperitoneal injection with RAPA at daily dose of 0.5 mg/kg for 5 days (the RAPA-treated group, n=60), the SAA group (n=60) and the un-treated group (n=60) were as control. All mice selected to set up the SAA model developed the typical clinical and pathological patterns of SAA from day 10 post dosing. Most of them presented bleedings in association with anemia and infections. In order to explore the effect of RAPA on apoptosis of CD4+CD25+ Tregs from the novel mouse SAA model, the mononuclear cells of the peripheral blood and spleen were subjected to assess the intracellular Foxp3 expression in CD4+CD25+ Tregs by flow cytometry (FCM). In the SAA group, the FCM analysis showed down-expression of Foxp3 in CD4+CD25+ Tregs compared with the un-treated group (P<0.05). These results were in accordance with our previous observations. However, after treatment with RAPA, the expression of Foxp3 in CD4+CD25+ Tregs was increased (P<0.05). In addition, after being pured by immunomagnetic beads, the splenic CD4+CD25+ Tregs was subjected to assess apoptosis by FCM and the Akt and Stat3 phosphorylation using western blot. Compared with the un-treated group, increased CD4+CD25+ Tregs apoptosis with increased Akt phosphorylation accompanied by increased Stat3 phosphorylation was found in SAA group (P<0.05, respectively). On the contrary, RAPA-treated group exhibited CD4+CD25+ Tregs with a reduction in apoptosis rate, Akt phosphorylation and Stat3 phosphorylation compared with the SAA group (P<0.05, respectively). These results indicated that RAPA may increase the expression of Foxp3 by down-regulation the levels of Akt and Stat3 phosphorylation in splenic CD4+CD25+ Tregs from the mice model of SAA, through which might RAPA reduce apoptosis of the CD4+CD25+ Tregs from this model. DisclosuresNo relevant conflicts of interest to declare.

Changes of regulatory T cells in peripheral blood samples from patients with lung cancer and their correlation with clinicopathologic features of lung cancer

Objective To investigate the changes of CD 4^＋CD25^＋CD127^low regulatory T （ Treg ） cells in peripheral blood samples from patients with lung cancer and their correlation with clinicopathologic features of lung cancer .Methods Flow cytometry was used to measure the percentages of CD 4, CD8, nat-ural killer （ NK） and Treg cells in peripheral blood samples collected from 160 patients with lung cancer and 60 healthy subjects .The correlations between the levels of Treg cells and clinicopathologic features of lung cancer were analyzed .The percentages of Treg cells in 60 patients with lung cancer before and after surgery were compared .Results The percentages of CD 4＋and NK cells and the ratios of CD 4＋/CD8＋cells in pa-tients with lung cancer were significantly lower than those in healthy control , while the percentages of Treg cells in patients with lung cancer were decreased as compared with those in healthy subjects .The percenta-ges of Treg cells in patients with advanced cancer were significantly higher than those in patients at early stage （P〈0.05）, which dropped significantly after surgery （P〈0.01）.Conclusion The results of this study indicated that Treg cells in patients with lung cancer might inhibit antitumor immune responses and correlate with the progression of cancer .It would be worthwhile to check Treg cells in patients with lung cancer for monitoring the prognosis and treatment effects . Key words: Regulatory T cells; Lung cancer; Flow cytometry; CD4^＋CD25^＋CD127^low

Identification of novel immune phenotypes for allergic and nonallergic childhood asthma

Childhood asthma is classified into allergic asthma (AA) and nonallergic asthma (NA), yet both are treated identically, with only partial success. We sought to identify novel immune phenotypes for childhood AA and NA. The Clinical Asthma Research Association cohort study includes 275 steroid-naive 4- to 15-year-old German children (healthy control subjects [HCs], patients with AA, and patients with NA). In PBMCs both quantitative and functional analysis of regulatory T (Treg) and TH17 cells (flow cytometry/Treg cell suppression) before/after anti-CD3/CD28, lipid A, and peptidoglycan stimulation were performed. Cytokines and gene expression, as assessed by using Luminex or transcriptomics/quantitative real-time RT-PCR, were analyzed by means of regression analysis. Linear discriminant analysis was applied to discriminate between phenotypes. The 3 phenotypes were immunologically well discriminated by means of microarray and protein analysis with linear discriminant analysis. Patients with AA were characterized by increased Treg cells compared with those in HCs but not those in patients with NA. Treg cells from patients with AA, but not patients with NA, significantly suppressed IL-5, IL-13, and IFN-γ secretion. Patients with AA had decreased expression of chloride intracellular channel 4 (CLIC4) and tuberous sclerosis 1 (TSC1), important innate immunity regulators. Patients with NA were characterized by increased proinflammatory IL-1β levels, neutrophil counts, and IL-17-shifted immunity. In parallel, expressions of anti-inflammatory IL37, proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2), and the neutrophil-associated genes CD93, triggering receptor expressed on myeloid cells 1 (TREM1), and regulator of G-protein signaling 13 (RGS13) were increased in patients with NA. Ashared TH2 immunity was present in both asthma phenotypes. Novel immune-regulatory mechanisms in childhood asthma identified increased Treg cells in patients with AA compared with those in HCs but not those in NA and decreased innate immunity genes for patients with AA, the first potentially indicating a counterregulatory mechanism to suppress cytokines yet not sufficient to control allergic inflammation. Very distinctly, patients with NA showed an IL-17-shifted proinflammatory immunity, promoting neutrophil inflammation and less functional Treg cells. Identification of these unique pathways provides a profound basis for future strategies for individualized prediction of asthma development, disease course, and prevention.

Persistent improper upregulation of Th17 and TReg cells in patients with juvenile idiopathic arthritis.

Juvenile idiopathic arthritis (JIA) is the most common childhood rheumatic disease. A break in the balance between Th17 and TReg cells has been reported as an important factor in the development of autoimmune diseases. This study aimed to analyze peripheral Th17 and TReg cell levels in patients with JIA. The balance of Th17 and TReg cells among active and inactive JIA patients and normal control subjects were compared. Human peripheral blood mononuclear cells were isolated from the patients and controls. Surface and intracellular staining for CD4, CD25, Foxp3, IL-17, Th17, and TReg were analyzed. Twenty-eight JIA patients, including 12 with active JIA and 16 with inactive JIA, and 20 health controls were analyzed. Patients with active JIA had higher Th17 (1.85±1.15 vs. 1.05±0.72, p = 0.008) and TReg cells (1.1±0.8 vs. 0.6±0.7, p = 0.04) levels than those with inactive JIA. Among active JIA patients, remission days were highly correlated with the CD4(+)IL17A(+) T cell percentage, 276.5 ± 137.40 days (range, 130 ∼ 525 days), p < 0.01. There were no differences in Th17/TReg percentage between JIA patients and controls in the peripheral blood. Th17 and TReg cell levels are elevated in patients with active JIA and there is no Th17/TReg imbalance. The higher Th17 level predicted longer period to reach disease inactive stage. Improper Th17 up-regulation might contribute to JIA activation.

Decreased expression of microRNA-21 correlates with the imbalance of Th17 and Treg cells in patients with rheumatoid arthritis.

The imbalance of Th17/Treg cell populations has been suggested to be involved in the regulation of rheumatoid arthritis (RA) pathogenesis; however, the mechanism behind this phenomenon remains unclear. Recent studies have shown how microRNAs (miRNAs) are important regulators of immune responses and are involved in the development of a variety of inflammatory diseases, including RA. In this study, we demonstrated that the frequencies of CD3+CD4+IL-17+Th17 cells were significantly higher, and CD4+CD25+FOXP3+ Treg cells significantly lower in peripheral blood mononuclear cells from RA patients. Detection of cytokines from RA patients revealed an elevated panel of pro-inflammatory cytokines, including IL-17, IL-6, IL-1β, TNF-α and IL-22, which carry the inflammatory signature of RA and are crucial in the differentiation and maintenance of pathogenic Th17 cells and dysfunction of Treg cells. However, the level of miR-21 was significantly lower in RA patients, accompanied by the increase in STAT3 expression and activation, and decrease in STAT5/pSTAT5 protein and Foxp3 mRNA levels. Furthermore, lipopolysaccharide stimulation up-regulated miR-21 expression from healthy controls, but down-regulated miR-21 expression from RA patients. Therefore, we speculate that miR-21 may be part of a negative feedback loop in the normal setting. However, miR-21 levels decrease significantly in RA patients, suggesting that this feedback loop is dysregulated and may contribute to the imbalance of Th17 and Treg cells. MiR-21 may thus serve as a novel regulator in T-cell differentiation and homoeostasis, and provides a new therapeutic target for the treatment of RA.

Impaired function of regulatory T-cells in patients with chronic obstructive pulmonary disease (COPD)

Anti-inflammatory pathways affecting chronic obstructive pulmonary disease (COPD) are poorly understood. Regulatory T-cells (Tregs) are important negative regulators of T-cell activity and hence were investigated in COPD patients in this study. We hypothesised that functional defects in Tregs may promote increased inflammation contributing to the pathogenesis of COPD.Peripheral blood mononuclear cells (PBMC) were isolated from patients with stable COPD and age-matched non-smoking controls. Treg-mediated suppression of memory non-Treg (Foxp3−CD45RO+) CD4+ T-cell activation was analysed by comparing PBMC responses to staphylococcal enterotoxin-B (SEB) pre- and post-depletion of Tregs (CD25+CD127lowCD4+ T-cells) by fluorescence-activated cell sorting (FACS). Activation of T-cells was assessed by HLA-DR expression. Levels of secreted cytokines were measured by ELISA.Depletion of Tregs increased SEB-induced activation of Foxp3−CD45RO+ CD4+ T-cells in samples from 15/15 healthy controls (demonstrating Treg-mediated suppression) and 9/14 COPD patients (Fisher's test, p=0.017). A screen of clinical data associated a failure of Treg-mediated suppression in the remaining five COPD patients with a higher body mass index (BMI) (33–38kg/m2) compared to patients with unimpaired Treg function (20–32kg/m2).In conclusion, we demonstrate impaired Treg-mediated suppression of CD4+ T-cell activation in a subset of COPD patients, all of whom had high BMI. Obesity and/or perturbed homeostasis of Treg subsets may explain this defect and therefore contribute to increased inflammation observed in COPD.

The alteration and clinical significance of Th1/Th2/Th17/Treg cells in patients with multiple myeloma.

Immunological T cells and associated cytokines have been shown to be involved in the pathogenesis of multiple myeloma (MM). However, the abnormal immune imbalance of T lymphocyte subsets on MM remains unknown. We investigate the proportions of T helper 1 (Th1)/Th2/Th17/T regulatory (Treg) cells in peripheral blood mononuclear cells (PBMCs) by flow cytometry (FCM), and serum levels of relevant cytokines in MM patients and controls were detected by enzyme-linked immunosorbent assay (ELISA). The messenger RNA (mRNA) expression of T-bet, STAT6, RORgammat, and Foxp3 was measured by real-time quantitative polymerase chain reaction (PCR). The CD4+ Th1 and CD4+ Th17 cells in patients with MM were significantly higher than those in health controls as well as the expression of T-bet and RORgammat mRNA. Furthermore, serum levels of interferon gamma (IFN-γ), IL-6, and IL-17A in MM group were greatly increased and significantly associated with each other. Significant differences on Th cells, cytokines, and transcription factors were observed on MM patients. The imbalance of T lymphocyte subsets was thought to contribute to the pathogenesis and underlying mechanisms of MM.

The changes and its significance of Th17 and Treg cells and related cytokines in patients with tuberculosis pleurisy.

BackgroundTuberculous pleurisy is a kind of tuberculosis, it is well known that Th1 lymphocytes play a key role in the treatment of tuberculosis infection. However, latest studies show that Th17 lymphocyte may also play an important role tuberculosis infection. There is close relationship between Treg and Thl7 cells, and changes in the number or the function of the two kinds of cells may lead to diseases. The current researches on Thl7 and Treg cells maily focus on autoimmune diseases, however, reports about their role in tuberculosis are limited. In this study, we investigate the function of th17 and Treg cells and the above cytokines in the pathogenesis of tuberculosis pleurisy; by determining the expression of Th17 and Treg cells in peripheral CD4 T cells and the related cytokines in patients with tuberculous compared with healthy people.ResultsTh17 cells in patients were higher than that in the Healthy control group, expression of Treg cells in patients were lower than that in the healthy group; IL-17, IL-23 levels in peripheral blood and hydrothorax from the patients were higher than that in the healthy group; IL-17, IL-23 and IL-6 levels in hydrothorax were higher than that in peripheral blood. There was no difference in IL-6 level in peripheral blood between the patients and healthy control; TGF- β level in peripheral blood from the healthy group was higher than that in peripheral blood and hydrothorax from the patients. And there were no differences in TGF- β level between peripheral blood and hydrothorax. Th17 cells were negatively correlated with Treg cells ,but were positive correlation with IL-17, IL-23, IL-6 levels in peripheral blood; TGF- β level was positive correlation with Treg cells in the peripheral blood, but no correlation with Th17 cells.ConclusionTh17 and Treg cells may be involved in the immune pathological mechanism of tuberculous pleurisy and changes of related cytokines may be involved in the differentiation of Th17 and Treg cells and inflammatory response. Thus, Th17 and Treg cells and related cytokines may be important immunopathogenesis for tuberculous pleurisy.

Evaluation of CD25+CD4+ Regulatory T-Lymphocyte Subpopulations in Coronary Artery Diseases Patients

Background. The development of atherosclerosis may be associated with a deficiency in the regulatory T-cells, which should serve a protective function and inhibit the accumulation of lymphocytes and macrophages. The aim of this study was the analysis of the T-lymphocyte subpopulations, particularly CD4+CD25+ regulatory T-cells in patients with different form of coronary artery disease.Materials and Methods. In the study 30 patients with stable coronary heart disease and 30 patients with unstable coronary heart disease take part. Lymphocytes subpopulations were measured with flow cytometry technique. The analysis of the treated cells parameters was performed with the use of CellQuest program.Results. We have observed statistically significant increase in activated lymphocytes subpopulations in patients with unstable coronary artery disease in comparison to stable group and significant decrease in CD25+, CD25/CD3+, and CD25/CD4+ subpopulations in unstable patients comparing to stable patients group.Conclusions. A strong interest in regulatory lymphocytes is due to their possible therapeutic use as a factor in modifying the immune response in various diseases. Questions regarding the role of regulatory T-cells in the development of atherosclerosis remain unclear. Mechanisms of the regulatory T-cells impact on suppression of atherosclerosis need more experiments to be done.

Alteration of Th17 and Treg cells in patients with unexplained recurrent spontaneous abortion before and after lymphocyte immunization therapy.

BackgroundSeveral types of T cells have been associated with the pathogenesis of unexplained recurrent spontaneous abortion (URSA), including Th1/Th2/Th17/Tregs cell. It has been appreciated that immunotherapy with paternal or third party lymphocytes is an effective method of treatment for URSA patients. The balance of Th1/Th2 cells could be maintained and an increase of Treg cells would be beneficial after immunotherapy; however, the mechanism by which the Th17/Treg balance affects URSA has not yet been fully elucidated.MethodsHere, we used flow cytometry, liquid chip technology and quantitative real-time PCR (qPCR) methods to characterize Th17/Treg cell populations after immunotherapy. We found that after immunotherapy in URSA patients, the percentage of Th17 cells decreased and the percentage of Treg cells in peripheral blood mononuclear cells (PBMC) increased, as detected by flow cytometry.ResultsImmunotherapy may induce a decrease in the Th17/Treg ratio and the Treg bias, which may be beneficial for the maintenance of pregnancy. The expression level of ROR gamma t, a transcription factor found in Th17 cells, decreased and the expression of the Treg-specific transcription factor Foxp3 increased in peripheral blood as detected by qPCR. Immunotherapy may induce a decrease in the ratio of ROR gamma t to Foxp3 and a Treg cell bias, which would be beneficial for pregnancy maintenance. The secretion of the Treg-associated cytokine TGF-beta, as well as Th2 cytokines, was increased in serum, while the secretion of Th17-associated cytokine IL-17A and Th1 cytokine production was decreased. The Th1/Th2 cytokine ratio significantly decreased. Similarly, the Th17/Treg ratio significantly decreased in the total patient after immunotherapy.ConclusionsThese results indicate that in patients with URSA, immunotherapy with mononuclear cells derived from the baby’s father could affect both Th1/Th2 and Th17/Treg balance, and we found that the Th2 and Treg bias would be beneficial for pregnancy, which may lead to a balancing of the Th17/Treg ratio in URSA patients after immunotherapy.

Signaling lymphocytic activation molecule (SLAM)/SLAM-associated protein pathway regulates human B-cell tolerance

Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) can mediate the function of SLAM molecules, which have been proposed to be involved in the development of autoimmunity in mice. We sought to determine whether the SLAM/SAP pathway regulates the establishment of human B-cell tolerance and what mechanisms of B-cell tolerance could be affected by SAP deficiency. We tested the reactivity of antibodies isolated from single B cells from SAP-deficient patients with X-linked lymphoproliferative disease (XLP). The expressions of SAP and SLAM family members were assessed in human bone marrow-developing B cells. We also analyzed regulatory T (Treg) cell function in patients with XLP and healthy control subjects. We found that new emigrant/transitional B cells from patients with XLP were enriched in autoreactive clones, revealing a defective central B-cell tolerance checkpoint in the absence of functional SAP. In agreement with a B cell-intrinsic regulation of central tolerance, we identified SAP expression in a discrete subset of bone marrow immature B cells. SAP colocalized with SLAMF6 only in association with clustered B-cell receptors likely recognizing self-antigens, suggesting that SLAM/SAP regulate B-cell receptor-mediated central tolerance. In addition, patients with XLP displayed defective peripheral B-cell tolerance, which is normally controlled by Treg cells. Treg cells in patients with XLP seem functional, but SAP-deficient T cells were resistant to Treg cell-mediated suppression. Indeed, SAP-deficient T cells were hyperresponsive to T-cell receptor stimulation, which resulted in increased secretion of IL-2, IFN-γ, and TNF-α. SAP expression is required for the counterselection of developing autoreactive B cells and preventstheir T cell-dependent accumulation in the periphery.

The Percentage of FoxP3+Helios+ Treg Cells Correlates Positively With Disease Activity in Systemic Lupus Erythematosus

To assess the use of Helios in combination with FoxP3 as a superior method for identifying non-cytokine-producing human Treg cells in patients with systemic lupus erythematosus (SLE) and to determine if FoxP3+Helios+ Treg cells are maintained at normal levels in patients with clinically active disease. Peripheral blood mononuclear cells (PBMCs) were purified from the blood of healthy volunteer donors and from 52 consecutive patients with SLE of varying clinical activity (Systemic Lupus Erythematosus Disease Activity Index scores of 0, 2-4, and ≥ 5). PBMCs (either fresh or after 4 hours of stimulation for cytokine production) were then analyzed by flow cytometry for the expression of cell surface markers (CD4, CD25, CD127, and CD45RA) and transcription factors (FoxP3 and Helios), as well as for the production of cytokines (interleukin-2 and interferon-γ). FoxP3+Helios+ Treg cells were found to be non-cytokine producing in both SLE patients and healthy controls. Patients with clinically active SLE had higher percentages of FoxP3+Helios+ Treg cells than did patients with inactive SLE or healthy controls. When corrected for the total CD4 cell count, the absolute numbers of FoxP3+Helios+ Treg cells in patients with moderately-to-highly active SLE were normal. Previous reports of a deficiency in Treg cell number or function in SLE are limited by their use of CD25, either alone or in combination with other markers, to identify human Treg cells. Helios in combination with FoxP3 is a superior method for detecting all non-cytokine-producing Treg cells, irrespective of CD25 or CD45RA expression. Using this method, we showed that FoxP3+Helios+ Treg cell numbers are not reduced in patients with clinically active SLE.

Enhanced Activity of Akt in Teff Cells From Children With Lupus Nephritis Is Associated With Reduced Induction of Tumor Necrosis Factor Receptor–Associated Factor 6 and Increased OX40 Expression

The breakdown of peripheral tolerance mechanisms is central to the pathogenesis of systemic lupus erythematosus (SLE). Although true Treg cells in patients with SLE exhibit intact suppressive activity, Teff cells are resistant to suppression. The underlying mechanisms are incompletely understood. This study was undertaken to examine the Akt signaling pathway and molecules that may alter its activity in T cells in lupus patients. The Akt pathway and its regulators were analyzed in Teff and Treg cells from children with lupus nephritis and controls using flow cytometry and real-time quantitative polymerase chain reaction. T cell proliferation was assessed by analysis of 5,6-carboxyfluorescein succinimidyl ester dilution. CD4+CD45RA-FoxP3(low) and FoxP3- Teff cells from children with lupus nephritis expressed high levels of activated Akt, resulting in the down-regulation of the proapoptotic protein Bim and an enhanced proliferative response. The induction of tumor necrosis factor receptor-associated factor 6 (TRAF6) was impaired, and TRAF6 levels inversely correlated with Akt activity. Although the expression of OX40 was enhanced on Teff cells from children with lupus nephritis compared to controls, OX40 stimulation failed to significantly increase TRAF6 expression in cells from patients, in contrast to those from healthy controls, but resulted in further increased Akt activation that was reversed by blockade of OX40 signaling. Moreover, inhibition of Akt signaling markedly decreased the proliferation of Teff cells from lupus patients. Our findings indicate that hyperactivation of the Akt pathway in Teff cells from children with lupus nephritis is associated with reduced induction of TRAF6 and up-regulation of OX40, which may cause Teff cell resistance to Treg cell-mediated suppression.