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Objective: To carry out a preliminary analysis on the Treg lymphocyte counts present in the peripheral blood of allergic asthmatic children from the city of Cartagena, Colombia, compared to healthy controls., Methods: We compared cytometry counts of ten asthmatic patients (age 7-16 years) and seven healthy controls (6-12 years), recruited in the city of Cartagena. Peripheral blood samples were stained using Cytek's 14-color cFluor Immunoprofiling kit (Cytek ® cFluor ® Immunoprofiling Kit 14 Color RUO kit), and analyzed on a Northern Lights™ spectral cytometer (Cytek ® Biosciences, Fremont, CA, USA), to read 50.000 events per sample. The data obtained were analyzed in SpectroFlo ® and FlowJo. The study was approved by the ethics committee of the University of Cartagena (SGR, Grant BPIN2020000100405)., Results: The frequency of CD3+, CD4+, CD25+, CD127- Tregs was 11% of all CD4+ T cells, with a range of minimum 8,1% and maximum 17,7%. There was no significant difference in the proportion of Tregs between allergic asthmatic patients and healthy controls (P = 0,2)., Conclusions: With this preliminary sample size, no significant differences were found in the Treg lymphocyte population between allergic asthmatic patients and healthy controls. The 14-color multiplexed panel is a useful tool not only to count CD3+ and CD4+ populations, but also to obtain the percentage of regulatory T cells using cell surface markers.

Human regulatory T (Treg) cells play a central role in controlling allergic inflammation in the airways. A reduced number of peripheral Treg cells and decreased suppressive function have been previously reported in the pathogenesis of allergic asthma. However, the characteristic role of specific Treg cell subsets and their mechanisms in the pathogenesis of allergic asthma remain unclear. In this study, we examined the proportion of different Treg cell subsets in both healthy subjects and patients with allergic asthma using flow cytometry and single-cell RNA sequencing. The migration function of the cells was compared using cell sorting and Transwell experiments. Furthermore, two allergen-challenged mouse models and a cell transfer experiment were used to examine the role of these Treg subsets. We found that the proportion of CD25 + Foxp3 + CD127 - Treg cells in the peripheral blood of patients with allergic asthma was lower than in those of healthy subjects. Furthermore, the circulating Treg cells expressed lower levels of CCR6 and IL-17 compared with healthy subjects. The chemokine from the airway mucosa, CCL20, was abundantly expressed, and Transwell experiments further proved that this chemokine promoted CCR6 + Treg cell migration in vitro . A mouse model induced by house dust mite (HDM) revealed that the number of CCR6 + Treg cells in the lung tissue increased remarkably. The incidence of allergic asthma may be related to an increase in Treg cells secreting IL-17 in the lung tissue. Recruited CCR6 + Treg cells are likely to differentiate into Th17-like cells under the Th17 environment present in the lungs. IL-17 derived from Th17-like cells could be associated with the pathology of allergic asthma by promoting Th17 responses, thereby favoring HDM-induced asthma exacerbations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Shen, Zhang, Xie, Chen, Li, Zheng, Chai, Wang, Zang and He.)

Background: Differences in the expression of regulatory T cells (Tregs) have been suggested to explain why some smokers develop COPD and some do not. Upregulation of Tregs in response to smoking would restrain airway inflammation and thus the development of COPD; while the absense of such upregulation would over time lead to chronic inflammation and COPD. We hypothesized that-among COPD patients-the same mechanism would affect rate of decline in lung function; specifically, that a decreased expression of Tregs would be associated with a more rapid decline in FEV 1 ., Methods: Bronchoscopy with BAL was performed in 52 subjects recruited from the longitudinal OLIN COPD study; 12 with COPD and a rapid decline in lung function (loss of FEV 1  ≥ 60 ml/year), 10 with COPD and a non-rapid decline in lung function (loss of FEV 1  ≤ 30 ml/year), 15 current and ex-smokers and 15 non-smokers with normal lung function. BAL lymphocyte subsets were determined using flow cytometry., Results: The proportions of Tregs with regulatory function (FoxP3 + /CD4 + CD25 bright ) were significantly lower in COPD subjects with a rapid decline in lung function compared to those with a non-rapid decline (p = 0.019). This result was confirmed in a mixed model regression analysis in which adjustments for inhaled corticosteroid usage, smoking, sex and age were evaluated. No significant difference was found between COPD subjects and smokers or non-smokers with normal lung function., Conclusions: COPD subjects with a rapid decline in lung function had lower proportions of T cells with regulatory function in BAL fluid, suggesting that an inability to suppress the inflammatory response following smoking might lead to a more rapid decline in FEV 1 . Trial registration Clinicaltrials.gov identifier NCT02729220.

Background: CXCL4 plays an essential role in the regulation of multiple immune diseases. However, the underlying role of CXCL4 is still not clear in sepsis. Aim: In the present study, we aimed to investigate the function of CXCL4 in sepsis. Methods: Sepsis model was constructed on mouse. Flow cytometry was used to determine the ratio of CD4 + CD25 + FOXP3 + Treg cells. ELISA assays were used to determine the levels of CXCL4, IL-6, IL-10, and TNF-α respectively. Western blot was used to examine protein contents. Results: Our results suggested that the serum level of CXCL4 was upregulated in patients with sepsis and positively associated with the ratio of human CD4 + CD25 + FOXP3 + Treg cells. To further examine the role of CXCL4 in sepsis, we constructed the mouse sepsis model. Our results indicated that the mouse antibody of CXCL4 treatment reduced the expression of urine creatinine and urea nitrogen in sepsis model. Moreover, the frequency of CD25 + FOXP3 + mouse regulatory T cells (Tregs) cells was decreased in mouse CD4 + T cells in the presence of mouse CXCL4 antibody. Further, the mouse recombinant protein CXCL4 was used to culture normal mouse CD4 + T cells in vitro . Our finding indicated that the recombinant protein CXCL4 promoted the percentage of mouse CD25 + FOXP3 + Treg cells and enhanced the phosphorylation of STAT5 in mouse CD4 + T cells in a dose-dependent manner. However, these effects were significantly reversed by the STAT5 inhibitor ( p  < .001). Conclusion: our findings not only indicated the function and signalling pathway of CXCL4 in CD4 + T cells but also provided novel insight and target in sepsis treatment.

Experimental autoimmune neuritis (EAN) induced by peripheral nerve myelin (PNM) is self-limiting and re-immunization with PNM does not re-activate disease. This study showed inhibition of EAN by CD4 + CD25 + T cells both from sensitized hosts or from naïve hosts after ex-vivo activation by PNM and rIL-2. Transfer of naïve CD4 + CD25 + T cells has no effect on EAN, nor did naïve CD4 + CD25 + T cells activated with rIL-2 and renal tubular antigen. Culture of naive CD4 + CD25 + Treg with rIL-2 and PNM induced mRNA for the IFN-gamma receptor. We showed naïve CD4 + CD25 + T cells activated by specific auto-antigen and rIL-2 produced more potent antigen-specific Treg that may have therapeutic potential., Competing Interests: Declaration of Competing Interest SJH and BMH hold patents related to activation of T regulatory cells. All other authors have no conflicts of interest., (Crown Copyright © 2020. Published by Elsevier B.V. All rights reserved.)

Background: Adaptive immune cells, including CD4 + CD69 + and CD4 + CD25 + FoxP3 + regulatory T (Treg) cells, are important for maintaining immunological tolerance. In human systemic lupus erythematosus (SLE), CD4 + CD25 + FoxP3 + Treg cells are reduced, whereas CD69 expression is increased, resulting in a homeostatic immune imbalance that may intensify autoreactive T cell activity. To analyze the mechanisms implicated in autotolerance failure, we evaluated CD4 + CD69 + and CD4 + CD25 + FoxP3 + T cells and interleukin profiles in a pristane-induced SLE experimental model., Methods: For lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p < 0.05 considered significant., Results: Compared with the controls, SLE-induced animals presented increased numbers of CD4 + CD69 + T cells in the blood on T90 and T120 (p = 0.022 and p = 0.008) and in the spleen on T120 (p = 0.049), but there were decreased numbers in the PL (p = 0.049) on T120. The percentage of Treg was lower in blood (p < 0.005 and p < 0.012) on T90 and T120, in spleen (p = 0.043) on T120 and in PL (p = 0.001) on T90. Increased numbers of CD4 + CD69+ T cells in the PL were positively associated with high IL-2 (p = 0.486) and IFN-γ (p = 0.017) levels, whereas reduced Treg cells in the blood were negatively correlated with TNFα levels (p = 0.043) and positively correlated with TGFβ1 (p = 0.038)., Conclusion: Increased numbers of CD4 + CD69 + T cells and reduced numbers of CD4 + CD25 + FoxP3 + Treg cells with an altered interleukin profile suggests loss of autotolerance in pristane-induced lupus mice, which is similar to human lupus. Therefore, this model is useful in evaluating mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in human SLE.

Regulatory T cells (Tregs) are CD4+ T cells that express CD25 and transcription factor Forkhead box P3 (FOXP3), and Tregs play a central role in regulation of tumor immunity. FOXP3 immunohistochemistry has been widely used to study Tregs in paraffin embedded tissue, and flow cytometry using fresh tissue has been used to identify FOXP3+/CD25+ double positive Tregs. In our study, we validated the FOXP3/CD25 double staining antibody cocktails for detecting Tregs in paraffin-embedded tissue. Tissue microarrays (TMA) included 115 malignant tumors, 3 ovarian mucinous borderline tumors and 15 benign tissues. Digital image analysis was performed using ImageJ software. Our results showed that FOXP3/CD25 double positive lymphocytes, a subset of FOXP3+ lymphocytes, accounted for variable percentage of the total FOXP3+ lymphocytes and they were positively correlated with FOXP3+ cell counts. Tumors from different sites had variable FOXP3+/CD25+ and FOXP3+ lymphocyte counts. Tumors from lung, head & neck and colon had more and renal cell carcinoma had minimal FOXP3+/CD25+ and FOXP3+ lymphocytes. In conclusion, FOXP3/CD25 double staining antibody cocktails can be easily applied to paraffin-embedded tissue, and FOXP3+/CD25+ Tregs count was positively correlated with FOPX3+ Tregs count but they were not interchangeable. We recommend using CD25/FOXP3 double staining for studying Tregs in tumor tissue., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Regulatory T cells (Tregs) play a major role in tumor escape from immunosurveillance by suppressing effector cells. The number of Tregs is increased in tumor sites and peripheral blood of breast cancer patients. However, the data regarding phenotypic and functional heterogeneity of Treg subpopulations in breast cancer are limited. The present study aimed to investigate the number and suppressive potential of Tregs that possess natural naïve-(N nTregs), effector/memory-like (EM nTregs), and Tr1-like phenotypes in breast cancer patients and healthy women., Methods: The study included 10 HW and 17 primary breast cancer patients. Numbers of CD4 + CD25 + FoxP3 + CD45RA + N nTregs, CD4 + CD25 + FoxP3 + CD45RA - EM nTregs, and CD4 + IL-4 - IL-10 + Tr1 subsets and the expression of CTLA-4, CD39, GITR, LAP, and IL-35 by these Treg subsets were measured in freshly obtained peripheral blood by flow cytometry., Results: Herein, we demonstrate that the percentages of N nTregs, EM nTregs, CD25 + and FoxP3 + Tr1 cells are elevated in the peripheral blood of breast cancer patients, but do not correlate with cancer stages. Nevertheless, the frequency of CD25 + Tr1 cells was associated with nodal involvement, while the number of EM nTregs correlated with clinical outcome. The expression of CTLA-4 and IL-35 by all assessed Treg subsets was increased throughout all tumor stages (I-III)., Conclusions: Collectively, the current study shows phenotypic alterations in suppressive receptors of Treg subsets, suggesting that breast cancer patients have increased activity of N nTregs, EM nTregs and Tr1 cells; and EM nTregs and CD25 + Tr1 cells represent prospective markers for assessing disease prognosis.

The gut microbiota in chicken has long been studied, mostly from the perspective of growth performance. However, there are some immunological studies regarding gut homeostasis in chicken. Although CD4 + CD25 + T cells are reported to act as regulatory T cells (Tregs) in chicken, there have been no studies showing the relationship between gut microbiota and Tregs. Therefore, we established a model for 'antibiotics (ABX)-treated chickens' through administration of an antibiotic cocktail consisting of ampicillin, gentamycin, neomycin, metronidazole, and vancomycin in water for 7 days. CD4 + CD8 - CD25 + and CD4 + CD8 + CD25 + T cells in cecal tonsils were significantly decreased in this model. Gram-positive bacteria, especially Clostridia, was responsible for the changes in CD4 + CD8 - CD25 + or CD4 + CD8 + CD25 + T cells in cecal tonsils. Feeding ABX-treated chickens with acetate recovered CD4 + CD8 - CD25 + and CD4 + CD8 + CD25 + T cells in cecal tonsils. GPR43, a receptor for acetate, was highly expressed in CD4 + CD8 - CD25 + T cells. In conclusion, our study demonstrated that the gut microbiota can regulate the population of CD4 + CD8 - CD25 + and CD4 + CD8 + CD25 + T cells, and that acetate is responsible for the induction of CD4 + CD8 - CD25 + T cells in cecal tonsils via GPR43.

The characteristics and functions of CD4 + CD25 + regulatory T cells (Tregs) have been well defined in murine and human systems. However, the interaction or crosstalk between CD4 + CD25 + Tregs and dendritic cells (DCs) remains controversial. In this study, the effects of chronic hepatitis B (CHB) CD4 + CD25 + Tregs on the maturation and function of monocyte-derived DCs were examined. The results showed that CD4 + CD25 + render the DCs inefficient as antigen-presenting cells (APCs) despite prestimulation with CD40 ligand. This effect was marginally reverted by applying neutralizing antibodies (Abs) to IL-10 and TGF-β. There were an increased IL-10 and TGF-β secretion and reduced expression of costimulatory molecules in DC. Thus, in addition to a direct suppressor effect on CD4 + T cells, CD4 + CD25 + may modulate the immune response through DCs in CHB patients., (© 2018 John Wiley & Sons Ltd.)

Background: The diagnosis of drug-induced autoimmune hepatitis (DIAIH) and its differentiation from idiopathic autoimmune hepatitis (AIH) is challenging. This study aimed to differentiate DIAIH from AIH by comparing the biochemical changes, histological features, and frequencies of CD4 + Foxp3 + CD25 +/- regulatory T cells (Tregs) in liver tissues or peripheral blood lymphocytes., Methods: A total of 15 DIAIH patients and 24 AIH patients who underwent liver biopsies at initial presentation were enrolled in this study. The liver histological changes were assessed by HE staining. The phenotypic recognition and distribution of CD4 + Foxp3 + CD25 +/- Tregs in liver tissues were evaluated by single/double immunostains in serial sections. The CD4 + Foxp3 + CD25 +/- Tregs in peripheral blood were analyzed by flow cytometry., Results: The median values of ALT and AST were 404.50 U/L and 454.10 U/L in DIAIH patients and 309.50 U/L and 315.00 U/L in AIH patients, respectively. More importantly, for the first time we found that patients with DIAIH had higher levels of serum ALT and AST, more severe degree of lobular inflammation, higher frequencies of zone 3 necrosis and higher number of lobular CD4 + Foxp3 + CD25 - Tregs compared with AIH (P < 0.05). Furthermore, there were positive correlations in DIAIH between the degree of lobular inflammation and either the AST/ALT level or the number of lobular CD4 + Foxp3 + CD25 - Tregs (P < 0.05). However, the frequency of peripheral blood CD4 + Foxp3 + CD25 +/- Tregs were not significantly different between DIAIH and AIH., Conclusions: The differences of ALT, AST and the number of lobular CD4 + Foxp3 + CD25 - Tregs between patients with DIAIH and those with AIH are clinically helpful in differentiating these two diseases in their early stage., (Copyright © 2018. Published by Elsevier B.V.)

Bilirubin (BR), a metabolite with increased concentrations in plasma during viral hepatitis, has been recognized as a potential immune-modulator. We recently reported that conjugated BR (CB) augments regulatory T cell (Treg) suppressor activity during acute hepatitis A virus (HAV) infection. However, the mechanisms related to the effects of CB on Treg function in the course of hepatotropic viral diseases have not been elucidated. T cell immunoglobulin domain and mucin domain 3 (TIM-3), via its interactions with galectin-9 (GAL-9), is a receptor associated with enhanced Treg function. Thus, TIM-3 expression may be related to the crosstalk between CB and Tregs during HAV infection. Herein, in vitro treatment with high concentrations of CB upregulated TIM-3 expression on Tregs from healthy donors. CB treatment in vitro did not induce de novo Treg generation, and in vitro stimulation with TGF-β, which shows increased secretion during HAV infection, resulted in a trend toward increased TIM-3 expression on Tregs and CD4 + T lymphocytes (TLs) from healthy donors. Interestingly, an upregulation of TIM-3 expression on CD4 + CD25 + T cells and an increase in the proportion of CD4 + TLs expressing GAL-9 were found in HAV-infected patients with abnormal CB values relative to healthy controls. In addition, a statistically significantly reduction in IL-17F production was observed after treatment of CD4 + TLs from healthy donors with high doses of CB in vitro. In summary, our results suggest that CB might regulate Treg activity via a TIM-3-mediated mechanism, ultimately leading to an anti-inflammatory hepatoprotective effect.

Regulatory T cells (Tregs) play crucial role in maintenance of peripheral tolerance. Recent clinical trials confirmed safety and efficacy of Treg treatment of deleterious immune responses. However, Tregs lose their characteristic phenotype and suppressive potential during expansion ex vivo. Therefore, multiple research teams have been studding Treg biology in aim to improve their stability in vitro. In the current paper, we demonstrate that mild hypothermia of 33 °C induces robust proliferation of Tregs, preserves expression of FoxP3, CD25 and Helios, and prevents TSDR methylation during culture in vitro. Tregs expanded at 33 °C have stronger immunosuppressive potential and remarkably anti-inflammatory phenotype demonstrated by the whole transcriptome sequencing. These observations shed new light on impact of temperature on regulation of immune response. We show that just a simple change in temperature can preserve Treg stability, function and accelerate their proliferation, responding to unanswered question- how to preserve Treg stability in vitro.

Signal transducer and activator of transcription 3 (STAT3) is a key signaling protein in the skeletal system as well as in the immune system. Accumulating evidence demonstrates that the inflammatory response is deeply involved in the healing process of bone fractures, but how the immune system is regulated during this process is unclear. In this study, we examined STAT3-mediated regulation of immunity in adult patients with closed tibia fracture. In all patients, the expression and activation of STAT3 peaked at around day 7 to day 14 after surgery, and gradually decreased during the rest of the healing period. At day 7 (peak STAT3 expression and phosphorylation), the CD4 + CD25 + T cells from bone fracture patients presented the highest level of STAT3 activation among lymphocyte subsets. Therefore, we investigated the role of STAT3 in CD4 + CD25 + T cells. The level of FOXP3 expression by CD4 + CD25 + T cells was directly correlated with the level of STAT3 phosphorylation in these cells. The level of STAT3 phosphorylation in CD4 + CD25 + T cells was also inversely correlated with the level of IFN-γ and TNF-α secretion in peripheral blood mononuclear cells. Inhibition of STAT3 significantly suppressed FOXP3 and IL-10 expression by CD4 + CD25 + T cells, as well as the ability of CD4 + CD25 + T cells to suppress T-cell IFN-γ and TNF-α secretion. Furthermore, early healers patients presented significantly higher STAT3 expression and phosphorylation than late healers, possibly due to the higher IL-6 and IL-10 levels in the serum of early healing patients. Together, these data demonstrated that STAT3 was beneficial to bone fracture healing, possibly by enhancing Treg-mediated suppression of counteracting inflammations, and suggested that STAT3 could be used as a prognostic marker to identify otherwise undistinguishable patients at risk of developing delayed union or nonunion., (© 2017 APMIS. Published by John Wiley & Sons Ltd.)

We have previously found that ex vivo expanded human CD4 + CD25 + Treg cells suppress proliferation of lymphoma B-cell lines. Here we demonstrate that the immunomodulatory drug lenalidomide potentiates suppression of lymphoma B-cell proliferation by freshly isolated CD4 + CD25 + Tregs, as well as suppression by Tregs expanded polyclonally in the presence of rapamycin from CD4 + CD25 + T cells or CD4 + CD25 + CD127 lo T cells. The regulation of lymphoma cell proliferation by Tregs pre-expanded with "third-party" allogeneic MoDCs in the presence of rapamycin was also potentiated by lenalidomide. Lenalidomide contributed to the suppression exerted by Tregs despite concomitant downregulation of Treg proliferation. Lenalidomide did not reduce the suppression of conventional T cells by expanded Tregs. The exposure of polyclonally expanded Tregs to lenalidomide did not significantly alter their phenotype. There was no uniform pattern of lenalidomide effect on Treg-mediated regulation of lymphoma B cells freshly isolated from patients. Freshly isolated lymphoma cells activated with multimeric CD40L and IL-4 to support their survival in vitro varied in their sensitivity to lenalidomide, and the regulatory effect of Tregs on such lymphoma cells ranged from suppression to help in individual patients. Lenalidomide potentiated or attenuated Treg effects on the survival of freshly isolated lymphoma cells. A combination of lenalidomide treatment with adoptive transfer of CD4 + CD25 + Tregs or CD4 + CD25 + CD127 lo Tregs expanded ex vivo could be used to suppress proliferation of residual lymphoma in select patients with lymphoma responsive to the regulation by Tregs and sensitive to lenalidomide.

Objectives: Randomised-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 disease is unknown. Moreover, the role of regulatory T (Treg) cells in the pathogenesis of GCA remains underexplored. Given the plasticity of Tregs and the importance of IL-6 in their biology, we hypothesised that TCZ might modulate the Treg response in GCA. We therefore characterised the Treg compartment of patients with GCA treated with TCZ., Methods: We classified 41 patients with GCA into three groups: active disease (aGCA, n=11), disease remission on corticosteroids (rGCA-CS, n=19) and disease remission on TCZ (rGCA-TCZ, n=11). Healthy controls (HCs) were included for comparison. We determined the frequency, phenotype and function of peripheral blood Tregs., Results: Patients with aGCA demonstrated a hypoproliferating Treg compartment enriched in IL-17-secreting Tregs (IL-17 + Tregs). Tregs in patients with aGCA disproportionally expressed a hypofunctional isoform of Foxp3 that lacks exon 2 (Foxp3Δ2). Foxp3Δ2-expressing Tregs coexpressed CD161, a marker commonly associated with the Th17 linage, significantly more often than full-length Foxp3-expressing Tregs. Compared with those of HCs, GCA-derived Tregs demonstrated impaired suppressor capacity. Treatment with TCZ, in contrast to CS therapy, corrected the Treg abnormalities observed in aGCA. In addition, TCZ treatment increased the numbers of activated Tregs (CD45RA - Foxp3 high ) and the Treg expression of markers of trafficking (CCR4) and terminal differentiation (CTLA-4)., Conclusions: TCZ may exert its therapeutic effects in GCA by increasing the proliferation and activation of Tregs, and by reverting the pathogenic Treg phenotype seen during active disease., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

The goal of this study was to observe the pathological characteristics of atherosclerotic plaques in the aortic walls of ApoE -/- and C57BL/6J mice and the changes of CD4 + CD25 + regulatory T cells (Tregs) in atherosclerotic mice. Twenty ApoE -/- mice were split into high-fat diet (AH) and normal diet (AN) groups and 10 C57BL/6J male mice were designated as the control group (BN). The serum concentrations of IL-10 and TGF-β1 were detected by enzyme-linked immunosorbent assay; paraffin sections of the aorta were stained with hematoxylin & eosin, and morphometric parameters were measured using the Image Pro Plus 6.0 system. Verhoeff stain was used to observe the distribution of elastic fibers, and immunohistochemical staining was performed to verify the phenotype of the forkhead box protein 3 (Foxp3 + ) CD25 + cells in the atherosclerotic tissue. The proportion of CD4 + CD25 + Tregs in the spleen was calculated by flow cytometry. The thickness of the intima, the intima/media ratio, the plaque area, and the plaque/lumen ratio of mice in AN group were significantly larger than those of mice in BN group. The thickness of the intima, the plaque area, and the plaque/lumen ratio of the mice in AH group were significantly increased compared with those of the AN group mice. The serum concentrations of IL-10 and TGF-β1 and the percentage of splenic CD4 + CD25 + Tregs in AN group mice were significantly decreased compared with the control group. The serum concentrations of IL-10 and TGF-β1 and the percentage of splenic CD4 + CD25 + Tregs in the mice in AH group were significantly decreased compared with those in AN group. The proportions of Foxp3 + and CD25 + cells within the total lymphocyte population were significantly decreased in AH group mice compared with those in AN group mice. Atherosclerosis in an experimental mouse model was correlated with Treg depletion in the lymphoid tissues and plaques, indicating the important antiatherosclerotic role of CD4 + CD25 + Tregs. Impact statement In this article, we conclude that Tregs decreased with atherosclerosis (AS) as determined in ApoE knockout mice fed a high fat diet. It is an important matter for understanding the AS pathology.

Previous studies established that GM-CSF-deficient (Csf2-deficient) mice exhibit profound resistance to experimental autoimmune encephalomyelitis. This study addressed whether the resistance of Csf2-deficient mice was a result of a requirement for GM-CSF in controlling the functional balance between effector and regulatory T cell subsets during experimental autoimmune encephalomyelitis. The main observation was that treatment with the anti-CD25 mAb PC61 rendered Csf2-deficient mice fully susceptible to severe, chronic experimental autoimmune encephalomyelitis, with disease incidences and severities equivalent to that of C57BL/6 mice. When both donors and recipients were treated with PC61 in a passive model of experimental autoimmune encephalomyelitis, adoptive transfer of myelin-specific Csf2-deficient T cells into Csf2-deficient recipients resulted in a nonresolving chronic course of severe paralytic experimental autoimmune encephalomyelitis. The peripheral Csf2-deficient T cell repertoire was marked by elevated CD3 + T cell frequencies that reflected substantial accumulations of naïve CD44 null-low CD4 + and CD8 + T cells but essentially normal frequencies of CD4 + CD25 + forkhead box P3 + T cells among the CD3 + T cell pool. These findings suggested that Csf2-deficient mice had secondary deficiencies in peripheral T cell sensitization to environmental antigens. In accordance, myelin oligodendrocyte glycoprotein 35-55/CFA-sensitized Csf2-deficient mice exhibited deficient peripheral sensitization to myelin oligodendrocyte glycoprotein, whereas pretreatment of Csf2-deficient mice with PC61 enabled the robust induction of myelin oligodendrocyte glycoprotein-specific T cell responses in the draining lymphatics. In conclusion, the experimental autoimmune encephalomyelitis resistance of Csf2-deficient mice, at least in part, reflects a deficient induction of effector T cell function that cannot surmount normal regulatory T cell barriers. Experimental autoimmune encephalomyelitis effector responses, however, are unleashed upon depletion of regulatory CD25 + T cells., (© Society for Leukocyte Biology.)

Donor lymphocyte infusions are used to treat relapse after allogeneic hematopoietic stem cell transplantation, but responses are inadequate. In addition to effector cells, infusions contain CD25 + regulatory T cells (Treg) that may suppress graft-versus-tumor responses. We undertook a phase I study of donor lymphocyte infusions depleted of CD25 + T cells in patients with hematologic malignancies who had relapsed after transplantation. Twenty-one subjects received CD25/Treg-depleted infusions following removal of CD25 + cells using antibody-conjugated magnetic beads. Sixteen subjects received prior cytoreductive therapy. Four were in complete remission at the time of infusion. Two dose levels were administered: 1×10 7 (n=6) and 3×10 7 CD3 + cells/kg (n=15). A median 2.3 log-depletion of CD4 + CD25 + FOXP3 + Treg was achieved. Seven subjects (33%) developed clinically significant graft-versus-host disease by 1 year, including one patient who died. At dose level 1, five subjects had progressive disease and one had stable disease. At dose level 2, nine subjects (60%) achieved or maintained responses (8 complete responses, 1 partial response), including seven with active disease at the time of infusion. A shorter period between relapse and infusion was associated with response at dose level 2 (P=0.016). The 1-year survival rate was 53% among patients treated with dose level 2. Four of eight subjects with acute myeloid leukemia remained in remission at 1 year. When compared to unmodified donor lymphocyte infusions in 14 contemporaneous patients meeting study eligibility, CD25/Treg depletion was associated with a better response rate and improved event-free survival. Circulating naïve and central memory CD4 + T cells increased after CD25/Treg-depleted infusion, but no immunophenotypic signature for response was noted. CD25/Treg-depleted donor infusion appears feasible and capable of inducing graft-versus-tumor responses without excessive graft-versus-host disease. (ClinicalTrials.gov NCT#00675831)., (Copyright© Ferrata Storti Foundation.)

Signaling lymphocytic activation molecule family 3 (SLAMF3/Ly9) is a coregulatory molecule implicated in T-cell activation and differentiation. Systemic lupus erythematosus (SLE) is characterized by aberrant T-cell activation and compromised IL-2 production, leading to abnormal regulatory T-cell (Treg) development/function. Here we show that SLAMF3 functions as a costimulator on CD4(+) T cells and influences IL-2 response and T helper cell differentiation. SLAMF3 ligation promotes T-cell responses to IL-2 via up-regulation of CD25 in a small mothers against decapentaplegic homolog 3 (Smad3)-dependent mechanism. This augments the activation of the IL-2/IL-2R/STAT5 pathway and enhances cell proliferation in response to exogenous IL-2. SLAMF3 costimulation promotes Treg differentiation from naïve CD4(+) T cells. Ligation of SLAMF3 receptors on SLE CD4(+) T cells restores IL-2 responses to levels comparable to those seen in healthy controls and promotes functional Treg generation. Taken together, our results suggest that SLAMF3 acts as potential therapeutic target in SLE patients by augmenting sensitivity to IL-2., Competing Interests: The authors declare no conflict of interest.

Cell-mediated immunity (CMI) plays a critical role in the control of brucellosis. Regulatory T cells (Tregs) have a functional character in modulating the balance between host immune response and tolerance, which can eventually lead to chronic infection or relapse. The aim of this study was to assess the alteration of Tregs in cases of brucellosis before and after treatment. Thirty cases of acute brucellosis with the mean age of 41.03±15.15 years (case group) and 30 healthy persons with the mean age of 40.63±13.95 years (control group) were selected and assessed. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of all individuals. We analyzed the alteration of Treg cell count using flow cytometry for CD4, CD25, and FoxP3 markers. The level of CD4+ CD25+ FoxP3+ Treg cells was increased in active patients compared with controls (2.5±0.99% vs 1.6±0.84%, p= 0.0004), but it had declined in the treated cases (1.83±0.73%, p=0.02). The level of Tregs was elevated in three relapsed cases. The frequency of Tregs and Treg/Teff (effector T cell) ratio was correlated with inverse serum agglutination test (SAT) and, 2-mercaptoethanol (2-ME) titers as markers of treatment in brucellosis. Based on our findings, we suggest that regulatory cells, such as CD4+ CD25+ FoxP3+ Treg cells, may contribute to the development of infection processes involving immune responses in brucellosis, and evaluation of regulatory T-cell levels may be a potential diagnostic strategy for the treatment outcome in chronic and relapsed cases of brucellosis.

Soluble fibrinogen-like protein 2, sfgl2, is the new effector of CD4(+)CD25(+)FOXP3(+) regulatory T cell (Treg) and exerts immunosuppressive activity. We design this study to investigate the possible role of sfgl2 in atherosclerosis. A total of 58 acute coronary syndrome (ACS) patients, together with 22 stable angina (SA) patients and 31 normal coronary artery (NCA) people were enrolled in our study. Serum level of sfgl2 and plasma level of Treg were respectively measured. In line with the change of Treg, serum level of sfgl2 in ACS (8.70 ng/mL) was significantly decreased (P = 0.003), compared with that in SA (11.86 ng/mL) and NCA (17.55 ng/mL). Both sfgl2 and Treg level were obviously decreased in ACS; Sfgl2 may play a protective role in atherosclerosis., (© 2016 by the Society for Experimental Biology and Medicine.)

Background: Robust evidence for the efficacy of rituximab monotherapy in pemphigus is lacking. The effects of rituximab on T-regulatory cells (Tregs) in pemphigus have not been studied., Objective: The primary objective was to assess the efficacy of rituximab monotherapy in severe pemphigus vulgaris. The secondary objectives were to assess whether counts of different subsets of Tregs in the peripheral blood correlate with baseline clinical severity and whether clinical response in severe pemphigus is associated with an alteration in the Treg count., Methods: Eighteen eligible subjects with severe pemphigus vulgaris were recruited and were treated with 1 g of intravenous rituximab on days 0 and 15. Efficacy was assessed in terms of disease control, time to disease control, complete remission off therapy, and relapse. Flow cytometric analysis of CD4+CD25+FoxP3, IL-10-secreting Tr1, and TGF-β secreting Th3 regulatory cells was performed. Clinical evaluation and flow cytometric analysis of Tregs was performed periodically until follow-up at 26 weeks., Results: Rituximab monotherapy was able to induce complete remission in all but 5 (68.75%) patients and was well tolerated. No direct relationship between clinical severity and CD4+CD25+FoxP3 cell counts was found. There were inverse correlations between serially measured values of the cutaneous and mucosal Autoimmune Bullous Skin Disorder Intensity Score (ABSIS) and Th3 cell count., Conclusion: Rituximab is a safe and effective monotherapy option for severe pemphigus. As the immunological findings were somewhat different from those observed in other autoimmune conditions treated with rituximab, further studies are required to substantiate the findings of our study in pemphigus patients., (© 2016 S. Karger AG, Basel.)

Jorge Lobo's disease (JLD) is a chronic granulomatous mycosis described in various Latin American countries. The main objective of the present study was to investigate the possible role of Th17 and Foxp3+ Treg cells in the pathogenesis of Jorge Lobo's disease. Human skin biopsies were submitted to an immunohistochemistry protocol to detect Foxp3, interleukin (IL)-1beta, CD25, IL-6, IL-17, and IL-23. The epidermis presented acanthosis, hyperkeratosis, and frequent presence of fungi. The dermis presented inflammatory infiltrate comprising macrophages, lymphocytes, epithelioid and multinucleated cells, and an intense number of fungi. Foxp3+ Treg cells and IL-17+ cells were visualized in lymphocytes in the inflammatory infiltrate. IL-1, IL-2R (CD25), IL-6, and IL-23 were visualized in the dermis, intermingled with fungal cells, permeating or participating of the granuloma. Following IL-17, the most prominent cytokine was IL-6. IL-23 and cells expressing CD25 were present in fewer number. The comparative analysis between IL-17 and Foxp3 demonstrated a statistically significant increased number of IL-17+ cells. Th17 cells play a role in the immune response of JLD. IL-1beta and IL-6 added to the previously described increased number of TGF-beta would stimulate such pattern of response. Th17 cells could be present as an effort to modulate the local immune response; however, high levels of a Th17 profile could overcome the role of Treg cells. The unbalance between Treg/Th17 cells seems to corroborate with the less effective immune response against the fungus., (© The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Profilins are actin-binding proteins that regulate the polymerization of actin filaments. In apicomplexan parasites, they are essential for invasion. Profilins also trigger the immune response of the host by activating TLRs on dendritic cells (DCs), inducing the production of pro-inflammatory cytokines. In this study we characterized for the first time the immune response and protection elicited by a vaccine based on Neospora caninum profilin in mice. Groups of eight BALB/c mice received either two doses of a recombinant N. caninum profilin expressed in Escherichia coli. (rNcPRO) or PBS, both formulated with an aqueous soy-based adjuvant enriched in TLR-agonists. Specific anti-profilin antibodies were detected in rNcPRO-vaccinated animals, mainly IgM and IgG3, which were consumed after infection. Splenocytes from rNcPRO-immunized animals proliferated after an in vitro stimulation with rNcPRO before and after challenge. An impairment of the cellular response was observed in NcPRO vaccinated and infected mice following an in vitro stimulation with native antigens of N. caninum, related to an increase in the percentage of CD4+CD25+FoxP3+. Two out of five rNcPRO-vaccinated challenged mice were protected; they were negative for parasite DNA in the brain and showed no histopathological lesions, which were found in all PBS-vaccinated animals. As a whole, our results provide evidence of a regulatory response elicited by immunization with rNcPRO, and suggest a role of profilin in the modulation and/or evasion of immune responses against N. caninum., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Visceral leishmaniasis (VL), caused by a protozoan parasite Leishmania donovani, is still a threat to mankind due to treatment failure, drug resistance and coinfection with HIV. The limitations of first-line drugs have led to the development of new strategies to combat this dreaded disease. Recently, we have shown the immunomodulatory property of Ara-LAM, a TLR2 ligand, against leishmanial pathogenesis. In this study, we have extended our study to the effect of Ara-LAM on regulatory T cells in a murine model of VL. We observed that Ara-LAM-treated infected BALB/c mice showed a strong host-protective Th1 immune response due to reduced IL-10 and TGF-β production, along with marked decrease in CD4(+) CD25(+) Foxp3(+) GITR(+) CTLA4(+) regulatory T cell (Treg) generation and activation. The reduction in Foxp3 expression was due to effective modulation of TGF-β-induced SMAD signaling in Treg cells by Ara-LAM. Moreover, we demonstrated that Ara-LAM-induced IRF1 expression in the Treg cells, which negatively regulated foxp3 gene transcription, resulting in the reduced immunosuppressive activity of Treg cells. Interestingly, irf1 gene knockdown completely abrogated the effect of Ara-LAM on Treg cells. Thus, these findings provide detailed mechanistic insight into Ara-LAM-mediated modulation of Treg cells, which might be helpful in combating VL., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The pro-inflammatory S100 calcium binding protein A8 (S100A8) is elevated in the serum of patients with Plasmodium falciparum malaria, but its function in Plasmodium vivax malaria is not yet clear. This function was investigated in P. vivax-infected patients in this study., Methods: The level of S100A8 in the serum was measured with ELISA. Full amino acids of S100A8 were synthesized to verify the functions for maturation of immature dendritic cell (iDC) and evaluation of CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) generation by mature DC (mDC)., Results: A higher amount of S100A8 was detected in vivax-infected patients (141.2 ± 61.849 ng/ml, n = 40) compared with normal control group (48.1 ± 27.384 ng/ml, n = 40). The level of S100A8 did not coincide with that of anti-malarial antibody measured by indirect fluorescent antibody test (IFAT) using parasite-infected red blood cells as antigen. Programmed death-ligand 1 (PD-L1) was up-regulated on the surface of iDCs following treatment with synthetic S100A8, not with synthetic MSP-1, AMA-1 and CSP, as compared to the expression seen for non-treated iDCs. The addition of red blood cells of infected patients to iDCs also elevated their surface expression of CD86. However, the serum levels of S100A8 decreased with increase in parasitaemia. DCs matured by sera containing S100A8 generated Treg cells from naïve T cells. The ratio of Treg cells generated was inversely proportional to the concentration of S100A8 in sera., Conclusions: Treg cells suppress the activity of cytotoxic T cells, which kill malaria parasites; therefore, the up-regulation of S100A8 in malaria patients may contribute to pathogen immune escape or tolerance.

Regulatory T cells (Tregs) play a pivotal role in suppressing specific antiviral immune responses during the progression of chronic hepatitis B virus infection (CHB) as well as tumorigenesis. Programmed death-1 ligand-1 (PD-L1) expressed on Tregs can transduce an inhibitory signal into effector T cells through interacting with programmed death-1 (PD-1). However, in CHB patients, the clinical significance of PD-L1 expression on Tregs has not been clearly described. This study investigated the frequency of circulating Tregs and PD-L1 expression on Tregs and analyzed their correlations with clinical parameters. The data show that both the frequency of CD4+CD25+FoxP3+ Tregs and PD-L1 expression on Tregs in the peripheral blood increased significantly in CHB patients when compared with healthy controls. At the same time, it is shown that PD-L1 expression on Tregs was positively correlated with the percentage of Tregs in CHB patients. Moreover, the results demonstrated that both Treg frequency and PD-L1 expression on Tregs positively correlated with the levels of alanine aminotransaminase (ALT) and aspartate aminotransferase (AST), both of which are indicators of the extent of liver injury. Taken together, these findings suggest that PD-L1 on Tregs might contribute to progression of hepatitis B virus infection through mediating the inhibitory function of Tregs. Thereby, blockade of interaction between Treg-expressing PD-L1 and PD-1 on effector T cells may be adopted as a potential therapeutic approach in CHB.

Background: Previous studies showed controversial results regarding CD4(+) CD25(high) FoxP3(+) T-regulatory cells (Tregs) in atopic dermatitis (AD) and effect of therapy., Methods: Circulating CD4(+) CD25(high) FoxP3(+) Tregs were assessed by flow cytometry in 20 controls and 20 patients with AD at baseline and after narrowband ultraviolet B with assessment of disease severity., Results: Patients showed higher pretreatment T-effector cells (Teffs) (%) and lower pretreatment Tregs FoxP3 expression% than controls (P = 0.003 and 0.01, respectively). Mild AD showed a lower Tregs/Teffs ratio compared to controls (P = 0.013), while moderate group showed higher Teffs%, and lower Tregs FoxP3 expression% and Tregs/Teffs compared to controls (P = 0.016, 0.007, and 0.009 respectively). The severe group had higher Tregs% and Teffs%, yet with a lower Tregs FoxP3 expression% compared to controls (P < 0.001, P = 0.043, P = 0.044, respectively). There was significant reduction of severity after narrowband ultraviolet B (P = 0.007), with overall significant elevation of Tregs FoxP3 expression% in patients (P = 0.004). All patients' post-treatment laboratory findings were statistically matched to each other and to controls whatever their previous severity or therapeutic response. The improvement of severity score correlated with the change in both Tregs% and Tregs/Teffs., Conclusions: Significant reduction in AD disease severity is correlated with the change in Tregs% and Tregs/Teffs., (© 2015 The International Society of Dermatology.)

CD83 is a widely recognized surface marker for mature dendritic cells, which are essential for priming naïve CD4+ T cells into effector cells. However, CD83 is also expressed on activated CD4+ T cells, which remains an enigma in T‑cell mediated immunity. Therefore, the identification of the biological features and regulation of the expression of CD83 on activated CD4+ T cells is important in understanding the function of CD83 in the adaptive immune response. The present study revealed a time‑dependent manner of the expression of CD83 on anti‑CD3/CD28‑stimulated human CD4+ T cells, which is characterized by the maximum expression at day 2 and a significant decrease at day 3. The reduced expression is not a result of a reduced rate of cell proliferation. The activation of interleukin‑2 and secretion of interferon‑γ accumulated progressively from day 1 to 3. Of note, sustained expression of CD83 was observed when CD4+ T cells were induced by transforming growth factor-β to differentiate into CD4+CD25+ forkhead box P3+ regulatory T (iTreg) cells. Confocal immunofluorescence microscopy analysis demonstrated that CD83 was highly co‑localized with CD25 on activated CD4+ T cells. In conclusion, the findings of the present study suggested that the continuous expression of CD83 on activated human CD4+ T cells is correlated with their differentiation into iTreg cells.

HCV is a global health problem with an estimated 230 million chronically infected people worldwide. It has been reported that a 17-kd protein translated from core-encoding genomic region can contribute to immune-mediated mechanisms associated with the development of the chronic disease. Also, Treg cells can be contributed to an inadequate response against the viruses, leading to chronic infection. Here we evaluated the ability of protein F to modulate the frequency of CD4+CD25+FoxP3+T and IL-10+T cells in patients with chronic HCV infection. F gene was amplified and cloned in the expression vector. The protein was purified and used for stimulation of PBMCs in the HCV chronic patients and the control groups. The frequency of CD4+CD25+FoxP3+ T cell-like populations and IL-10-producing CD4+CD25+ T cells was assessed in the HCV-infected patients and in the healthy controls by flow cytometry, which showed an increase of both CD4+CD25+FoxP3+ T cell-like population and IL-10-producing CD4+CD25+ T cells in the HCV-infected patients positive for anti-F antibody. Our results suggest the potential involvement of F and core antigens in increasing the frequency of CD4+CD25+FoxP3+ T cell-like population and IL-10-producing CD4+CD25+ T cells which may be associated with HCV-persistent infection., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

With the increasing interest in clinical trials with regulatory T cells (Tregs), immunological profiling of prospective target groups and standardized procedures for Treg isolation are needed. In this study, flow cytometry was used to assess peripheral blood lymphocyte profiles of young healthy individuals and patients undergoing haemodialysis treatment. Tregs obtained from the former may be used in haematopoietic stem cell transplantation and Tregs from the latter in the prevention of kidney transplant rejection. FOXP3 mRNA expression with accompanying isoform distribution was also assessed by the quantitative reverse transcriptase polymerase chain reaction. Flow-cytometric gating strategies were systematically analysed to optimize the isolation of Tregs. Our findings showed an overall similar immunological profile of both cohorts in spite of great differences in both age and health. Analysis of flow-cytometric gating techniques highlighted the importance of gating for both CD25high and CD127low expression in the isolation of FOXP3-positive cells. This study provides additional insight into the immunological profile of young healthy individuals and uraemic patients as well as in-depth analysis of flow-cytometric gating strategies for Treg isolation, supporting the development of Treg therapy using cells from healthy donors and uraemic patients., (© 2015 John Wiley & Sons Ltd.)

Background: The roles of CD4(+)CD25(+) regulatory T cells (Treg) in chronicity of hepatitis B virus (HBV) infection have been confirmed. We aimed to explore alteration of Treg in patients with HBV-related acute-on-chronic liver failure (ACLF)., Methods: Thirty-two HBV-related ACLF patients, 44 chronic hepatitis B patients, and 41 healthy controls were recruited. We detected frequencies of peripheral Treg and intrahepatic forkhead winged helix transcription factor (Foxp3)(+) cells. Inhibitory activity of Treg was assessed by functional suppression assays. Serum interferon-γ and interleukin-10 were also determined., Results: Peripheral Treg and intrahepatic Foxp3(+) cells were more markedly increased in ACLF than chronic hepatitis B and controls (all p < 0.001), and the Foxp3(+) cells located predominantly in the portal areas. The Treg frequency was positively correlated with HBV DNA load, international normalized ratio, model of end stage liver disease score, and serum interleukin-10 level in ACLF patients. Functional assays in vitro demonstrated that ACLF patients exhibited higher suppressive effects of Treg on proliferations of autologous CD4(+)CD25(-) T cells than controls. On logistic regression, prolonged international normalized ratio and higher peripheral Treg frequency predicted 30-day survival of ACLF., Conclusion: The patients with HBV-related ACLF exhibit increased amounts of Treg, of which redistribution from periphery to liver seems to modulate liver inflammation. Higher Treg amounts are associated with more severe liver disease in ACLF, and its level in combination with international normalized ratio may assist prediction of short-term outcomes of HBV-related ACLF., (Copyright © 2013. Published by Elsevier B.V.)

Aim: To compare the number of regulatory T-cells (Tregs) measured by flow cytometry with those obtained using a real-time quantitative PCR (qPCR) method in patients suffering from inflammatory bowel disease (IBD)., Methods: Tregs percentages obtained by both flow cytometry and qPCR methods in 35 adult IBD patients, 18 out of them with Crohn´s disease (CD) and 17 with ulcerative colitis (UC) were compared to each other as well as to scores on two IBD activity questionnaires using the Harvey Bradshaw Index (HBI) for CD patients and the Simple Colitis Clinical Activity Index (SCCAI) for UC patients. The Treg percentages by flow cytometry were defined as CD4(+)CD25(high)CD127(low)FOXP3(+) cells in peripheral blood mononuclear cells, whereas the Treg percentages by qPCR method were determined as FOXP3 promoter demethylation in genomic DNA., Results: We found an average of 1.56% ± 0.78% Tregs by using flow cytometry, compared to 1.07% ± 0.53% Tregs by using qPCR in adult IBD patients. There were no significant correlations between either the percentages of Tregs measured by flow cytometry or qPCR and the HBI or SCCAI questionnaire scores in CD or UC patients, respectively. In addition, there was no correlation between Treg percentages measured by qPCR and those measured by flow cytometry (r = -0.06, P = 0.73; Spearman Rho). These data suggest that, either Treg-related immune function or the clinical scores in these IBD patients did not accurately reflect actual disease activity. Until the cause(s) for these differences are more clearly defined, the results suggest caution in interpreting studies of Tregs in various inflammatory disorders., Conclusion: The two methods did not produce equivalent measures of the percentage of total Tregs in the IBD patients studied which is consistent with the conclusion that Tregs subtypes are not equally detected by these two assays.

CD4(+)CD25(+)FOXP3(+) regulatory T cells have long been shown to mediate susceptibility to Leishmania infection, mainly via interleukin 10 production. In this work, we showed that the main sources of interleukin 10 in peripheral blood mononuclear cells (PBMCs) from patients with cutaneous leishmaniasis due to Leishmania braziliensis are CD4(+)CD25(-)CD127(-/low)FOXP3(-) cells. Compared with uninfected controls, patients with CL had increased frequencies of circulating interleukin 10-producing CD4(+)CD25(-)CD127(-/low) cells, which efficiently suppressed tumor necrosis factor α production by the total PBMC population. Also, in CL lesions, interleukin 10 was mainly produced by CD4(+)CD25(-) cells, and interleukin 10 messenger RNA expression was associated with interleukin 27, interleukin 21, and interferon γ expression, rather than with FOXP3 or transforming growth factor β expressions. Active production of both interleukin 27 and interleukin 21, together with production of interferon γ and interleukin 10, was also detected in the lesions. Since these cytokines are associated with the differentiation and activity of Tr-1 cells, our results suggest that this cell population may play an important role in the immunomodulation of CL. Therefore, development of treatments that interfere with this pathway may lead to faster parasite elimination., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Conventional regulatory T cells (Tregs) can suppress human immunodeficiency virus type 1 (HIV-1)-specific immune responses but cannot control immune activation in primary HIV infection. Here, we characterized Treg subsets, using recently defined phenotypic delineation, and analyzed the relative contribution of cell subsets to the production of immunosuppressive cytokines in primary HIV infection., Methods: In a longitudinal prospective study, ex vivo phenotyping of fresh peripheral blood mononuclear cells from patients with primary HIV infection was performed at baseline and month 6 of follow-up to characterize Treg subsets, immune activation, and cytokine production in isolated CD4(+) T cells., Results: The frequency of CD4(+)CD25(+)CD127(low) Tregs and the distribution between the naive, memory, and activated/memory Treg subsets was similar in patients and healthy donors. However, Tregs from patients with primary HIV infection showed peculiar phenotypic profiles, such as elevated FoxP3, ICOS, and CTLA-4 expression, with CTLA-4 expression strikingly increased in all Treg subsets both at baseline and month 6 of follow-up. The great majority of interleukin 10 (IL-10)-producing CD4(+) T cells were FoxP3(neg) (ie, Tr1-like cells). In contrast to conventional Tregs, Tr1-like cells were inversely correlated with immune activation and not associated with lower effector T-cell responses., Conclusion: FoxP3(neg) Tr1-like cells-major contributors to IL-10 production-may have a beneficial role by controlling immune activation in early HIV infection., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Leprosy is characterized histologically by a spectrum of different granulomatous skin lesions, reflecting patients' immune responses to Mycobacterium leprae. Although CD4+CD25+ FoxP3+ T regulatory cells are pivotal in the immuneregulation, presence, frequency, and distribution of Tregs in leprosy, its reactional states have been investigated in few studies., Objectives: This study aimed to verify the frequency and distribution of regulatory T cells in different clinical forms and reactional states of leprosy., Methods: We performed an immunohistochemical study on 96 leprosy cases [Indeterminate (I): 9 patients; tuberculoid tuberculoid: 13 patients; borderline tuberculoid: 26 patients; borderline borderline: 3 patients; borderline lepromatous: 8 patients; lepromatous lepromatous: 27 patients; reversal reaction: 8 patients; and erythema nodosum leprosum: 2 patients]., Results: FoxP3-positive cells were present in 100% of the cases with an average density of 2.82% of the infiltrate. Their distribution was not related to granulomatous structures or special locations. There was a statistically significant increment of FoxP3 expression in patients with leprosy reversal reactions when compared with patients presenting with type I leprosy (P= 0.0228); borderline tuberculoid leprosy (P = 0.0351) and lepromatous leprosy (P = 0.0344)., Conclusions: These findings suggest that Tregs play a relevant role in the etiopathogenesis of leprosy, mainly in type I leprosy reaction.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease with complex genetic predisposing factors involved. Ets-1 transcription factor plays an important role in the suppressive activity of CD4(+)CD25(+) Treg cells and stable expression of FOXP3. To find its potential role in the pathogenesis of SLE, we investigate the mRNA expression of Ets-1 and FOXP3 mRNA in CD4(+)CD25(+) Treg cells from patients with SLE. Real-time transcription-polymerase chain reaction analysis was used to determine the expression of Ets-1 and FOXP3 mRNA in CD4(+)CD25(+) Treg cells from 36 patients with SLE and 18 sex-and-age-matched healthy controls. The Ets-1 mRNA expression level was decreased in patients with SLE [0.225 (0.135, 0.337)] than healthy controls [0.528 (0.303, 0.681)] (P < 0.001). The expression levels of FOXP3 mRNA were lower in SLE patients [0.608 (0.272, 1.164)] than healthy controls [0.919 (0.690, 1.223)], but the difference was not significant (P = 0.106). Significant reduction in Ets-1 and FOXP3 expression was also found in new-onset SLE subgroup when compared with healthy controls (P < 0.001). The level of Ets-1 and FOXP3 mRNA was not significantly different in hyperactive and lower active SLE group when compared with inactive SLE group, respectively (P > 0.05). There were no significant differences between SLE with lupus nephritis (LN) and SLE without LN either (P > 0.05). Associations of Ets-1 and FOXP3 mRNA expression levels with major clinical and laboratory parameters of SLE patients were also analyzed. However, no significant association was found. Significant positive correlation was found between Ets-1 and FOXP3 mRNA expression in CD4(+)CD25(+) Treg cells from SLE patients (r = 0.698, P < 0.001). Our results found that the expression levels of Ets-1 mRNA were decreased in SLE patients and Ets-1 expression was positively correlated with the expression of FOXP3. It indicated that Ets-1 may play an important role in the stable expression of FOXP3 in CD4(+)CD25(+) Treg cells.

Background: Actinic cheilitis (AC) is an oral potentially malignant lesion which is the counterpart of actinic keratosis of the skin and has potential to develop into squamous cell carcinoma. Regulatory T cells (Tregs) have a critical role in modulating the antitumor immune responses. The presence of regulatory T cells in potentially malignant lesions has not been described. We chose investigate the involvement of regulatory T cells in potentially malignant lesions., Methods: The frequency, phenotype, and activity of CD4+CD25+ T cells isolated from blood and lesion of AC patients were analyzed by flow cytometry. Cytokines were quantified by ELISA. Data were compared with samples from healthy subjects., Results: The frequency and suppressor activity of circulating CD4+CD25+ T cells was similar in AC patients and control subjects. However, the frequencies of IL-10-positive Tregs were higher in AC patients, and these cells inhibited interferon-gamma (IFN-γ) and increased interleukin (IL)-10 productions in co-cultures. Furthermore, CD4+CD25+ T cells accumulate in AC lesions. Lesions-derived regulatory T cells suppressed lymphocyte proliferation and pro-inflammatory cytokine production. Moreover, high levels of IL-10 and transforming growth factor-β (TGF-β), and low IFN-γ were detected in the potentially malignant lesions., Conclusion: Therefore, our data show that Tregs accumulate in AC lesions, and these cells could be suppressing immune responses in a potentially malignant microenvironment., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Regulatory T (TREG) cells play an important role in maintaining immune tolerance and avoiding autoimmunity. We analyzed the expression of membrane molecules in TREG and effector T cells in systemic lupus erythematosus (SLE). TREG and effector T cells were analyzed for the expression of CTLA-4, PD1, CD28, CD95, GITR, HLA-DR, OX40, CD40L, and CD45RO in 26 patients with active disease, 31 with inactive disease, and 26 healthy controls. TREG cells were defined as CD25+/high CD127 Ø/low FoxP3+, and effector T cells were defined as CD25+CD127+FoxP3 Ø. The ratio of TREG to effector T cells expressing GITR, PD1, HLA-DR, OX40, CD40L, and CD45RO was determined in the three groups. The frequency of TREG cells was similar in patients with SLE and controls. However, SLE patients had a decreased frequency of CTLA-4+TREG and CD28+TREG cells and an increased frequency of CD40L+TREG cells. There was a decrease in the TREG/effector-T ratio for GITR+, HLA-DR+, OX40+, and CD45RO+ cells, and an increased ratio of TREG/effector-T CD40L+ cells in patients with SLE. In addition, CD40L+TREG cell frequency correlated with the SLE disease activity index (P=0.0163). In conclusion, our findings showed several abnormalities in the expression of functionally critical surface molecules in TREG and effector T cells in SLE that may be relevant to the pathogenesis of this disease.

CD4(+) T cells expressing the immunotolerizing molecule HLA-G have been described as a unique human thymus-derived regulatory T (tTreg) cell subset involved in immunoregulation and parenchymal homeostasis during infectious and autoimmune inflammation. We compared properties and molecular characteristics of human CD4(+)HLA-G(+) with those of CD4(+)CD25(+)FoxP3-expressing tTreg cells using in vitro studies of T-cell receptor (TCR) signaling, single-cell electrophysiology, and functional in vivo studies. Both tTreg populations are characterized by alterations in proximal-signaling pathways on TCR stimulation and a hyperpolarization of the plasma membrane when compared to conventional CD4(+) T cells. However, both clearly differ in phenotype and pattern of secreted cytokines, which results in distinct mechanisms of suppression: While CD4(+)HLA-G(+) cells secrete high levels of inhibitory molecules (IL-10, soluble HLA-G, IL-35), CD4(+)CD25(+)FoxP3(+) cells express these molecules at significantly lower levels and seem to exert their function mainly in a contact-dependent manner via cyclic adenosine-monophosphate. Finally we demonstrate that human CD4(+)HLA-G(+) tTreg cells significantly ameliorated graft-versus-host disease in a humanized mouse model as a first proof of their in vivo relevance. Our data further characterize and establish CD4(+)HLA-G(+) cells as a potent human tTreg population that can modulate polyclonal adaptive immune responses in vivo and thus being a promising candidate for potential clinical applications in the future., (© FASEB.)

An immune role of neural stem/progenitor cells (NSPCs) has been proposed in many recent studies; however much still remains to be elucidated. In the current investigation, we report that NSPCs possess the ability to convert encephalitogenic T cells into CD4(+)-CD25(+)-FOXP3(+) regulatory T cells (T(reg)). Encephalitogenic and nonencephalitogenic T cells isolated from sham and Japanese encephalitis virus (JEV) infected animals were co-cultured with mouse NSPCs. Post co-culture, significant increase in the number of T(regs) was observed from encephalitogenic T cells co-cultured with NSPCs. This increased conversion was found to be dependent on direct contact between T cells and NSPCs. Neutralization of TGF-β and IFN-γ in NSPC cultures abrogated this increased conversion of encephalitogenic T cells into T(regs). Flow cytometric, quantitative RT-PCR, and immunoblot analysis of both T cells and NSPCs revealed surface and intracellular changes post co-culture. Co-stimulatory molecules (B7) and ICAM-1 were increased on NSPCs post co-culture; levels of TGFβ, IFNγ, and TGFβR1 were also increased in NSPCs. This study provides a basic insight into the interaction between CNS-infiltrating encephalitogenic T cells and NSPCs during viral encephalitis. Conversion of encephalitogenic T cells into CD4(+)-CD25(+)-FOXP3(+) T(regs) through interaction with NSPCs indicates an attempt in regulation of excessive inflammation in the CNS.

Background: Although regulatory T cells (Tregs) are affected in several autoimmune skin diseases, only two studies have been performed in patients with bullous pemphigoid (BP) with contrasting results., Objective: To characterize Tregs and to determine the serum levels of regulatory cytokines in patients with BP., Methods: In BP lesional skin, immunohistochemistry and confocal microscopy were performed for CD4(+) , CD25(+) , forkhead/winged helix transcription factor (FOXP3)(+) , transforming growth factor (TGF)-β(+) and interleukin (IL)-10(+) cells. In addition, the number of CD4(+) CD25(++) FOXP3(+) Tregs in peripheral blood was assessed by flow cytometry, and the levels of TGF-β and IL-10 were determined in serum samples by enzyme-linked immunosorbent assay before and after steroid therapy. Controls included patients with psoriasis, atopic dermatitis (AD) and healthy donors., Results: The frequency of FOXP3(+) cells was significantly reduced in skin lesions from patients with BP (P < 0.001) compared with psoriasis and AD. Moreover, the number of IL-10(+) cells was lower in BP than in psoriasis (P < 0.001) and AD (P = 0.002), while no differences were observed in the number of TGF-β(+) cells. CD4(+) CD25(++) FOXP3(+) Treg in the peripheral blood of patients with BP was significantly reduced compared with healthy controls (P < 0.001), and augmented significantly after steroid therapy (P = 0.001). Finally, TGF-β and IL-10 serum levels were similar in patients with BP compared with healthy controls. However, after therapy, BP patients showed significantly higher IL-10 serum levels than before therapy (P = 0.01)., Conclusions: These data suggest that the depletion of Tregs and of IL-10 in patients with BP may be an important factor in the pathogenesis of the disease., (© 2013 The Authors Journal of the European Academy of Dermatology and Venereology © 2013 European Academy of Dermatology and Venereology.)

Background: We and others have previously reported that cell membrane-bound TGFβ (mTGFβ) on activated T regulatory (Treg) cells mediates suppressor function. Current findings suggest that a novel protein known as Glycoprotein A Repetitions Predominant (GARP) anchors mTGFβ to the Treg cell surface and facilitates suppressor activity. Recently, we have described that GARP+TGFβ+ Treg cells expand during the course of FIV infection. Because Treg cells are anergic and generally exhibit poor proliferative ability, we asked how Treg homeostasis is maintained during the course of feline immunodeficiency virus (FIV) infection., Results: Here, we report that Treg cells from FIV+ cats express GARP and mTGFβ and convert T helper (Th) cells into phenotypic and functional Treg cells. Th to Treg conversion was abrogated by anti-TGFβ or anti-GARP treatment of Treg cells or by anti-TGFβRII treatment of Th cells, suggesting that Treg cell recruitment from the Th pool is mediated by TGFβ/TGFβRII signaling and that cell-surface GARP plays a major role in this process., Conclusions: These findings suggest Th to Treg conversion may initiate a cascade of events that contributes to the maintenance of virus reservoirs, progressive Th cell immunosuppression, and the development of immunodeficiency, all of which are central to the pathogenesis of AIDS lentivirus infections.

Background: Lepromatous leprosy caused by Mycobacterium leprae is associated with antigen specific T cell unresponsiveness/anergy whose underlying mechanisms are not fully defined. We investigated the role of CD25(+)FOXP3(+) regulatory T cells in both skin lesions and M.leprae stimulated PBMC cultures of 28 each of freshly diagnosed patients with borderline tuberculoid (BT) and lepromatous leprosy (LL) as well as 7 healthy household contacts of leprosy patients and 4 normal skin samples., Methodology/principle Findings: Quantitative reverse transcribed PCR (qPCR), immuno-histochemistry/flowcytometry and ELISA were used respectively for gene expression, phenotype characterization and cytokine levels in PBMC culture supernatants. Both skin lesions as well as in vitro antigen stimulated PBMC showed increased percentage/mean fluorescence intensity of cells and higher gene expression for FOXP3(+), TGF-β in lepromatous (p<0.01) as compared to tuberculoid leprosy patients. CD4(+)CD25(+)FOXP3(+) T cells (Tregs) were increased in unstimulated basal cultures (p<0.0003) and showed further increase in in vitro antigen but not mitogen (phytohemaglutinin) stimulated PBMC (iTreg) in lepromatous as compared to tuberculoid leprosy patients (p<0.002). iTregs of lepromatous patients showed intracellular TGF-β which was further confirmed by increase in TGF-β in culture supernatants (p<0.003). Furthermore, TGF-β in iTreg cells was associated with phosphorylation of STAT5A. TGF-β was seen in CD25(+) cells of the CD4(+) but not that of CD8(+) T cell lineage in leprosy patients. iTregs did not show intracellular IFN-γ or IL-17 in lepromatous leprosy patients., Conclusions/significance: Our results indicate that FOXP3(+) iTregs with TGF-β may down regulate T cell responses leading to the antigen specific anergy associated with lepromatous leprosy.

Regulatory T cells (Treg) are important mechanisms that regulate autoimmunity and CD4+CD25+Foxp3+ regulatory T cells (CD25+Treg) have been extensively investigated. Recently, we have identified CD4+CD25-LAG3+ regulatory T cells (LAG3+Treg) that express an anergy associated transcription factor Egr2. Egr2 regulates IL-10 production in response to IL-27, and Egr2-deficiency in T cells and B cells results in systemic autoimmunity with increased IL-17 production. Moreover, addition of Egr3 deficiency to Egr2-deficient mice significantly accelerates systemic autoimmunity without functional impairment of CD25+Treg, indicating cooperative autoimmune-regulation by Egr2 and Egr3. The linkage between Egr2 and systemic autoimmunity is also suggested by the fact that stimulation with Ly108, a candidate lupus susceptibility gene in lupus-prone NZM2410 mice, induces Egr2 expression in T cells. Collectively, LAG3+Treg and Egr2 are the unique regulators of autoimmunity and further examination may help to understand and control immune responses.

The influence of CD25(+)CD4(+) regulatory T cells (Treg) on acute and chronic viral infection of the central nervous system (CNS) was examined using a glial tropic murine coronavirus. Treg in the CNS were highest during initial T cell mediated virus control, decreased and then remained relatively stable during persistence. Anti-CD25 treatment did not affect CNS recruitment of inflammatory cells. Viral control was initially delayed; however, neither the kinetics of viral control nor viral persistence were affected. By contrast, the absence of Treg during the acute phase resulted in increased demyelination during viral persistence. These data suggest that CNS inflammation, progression of viral control and viral persistence are relatively independent of CD25(+)CD4(+) Treg. However, their absence during acute infection alters the ability of the host to limit tissue damage., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)