Plasmodium yoelii infection of BALB/c mice results in expansion rather than induction of CD4+ Foxp3+ regulatory T cells

Recently, we demonstrated elevated numbers of CD4(+) Foxp3(+) regulatory T (Treg) cells in Plasmodium yoelii‐infected mice contributing to the regulation of anti‐malarial immune response. However, it remains unclear whether this increase in Treg cells is due to thymus‐derived Treg cell expansion or induction of Treg cells in the periphery. Here, we show that the frequency of Foxp3(+) Treg cells expressing neuropilin‐1 (Nrp‐1) decreased at early time‐points during P. yoelii infection, whereas percentages of Helios(+) Foxp3(+) Treg cells remained unchanged. Both Foxp3(+) Nrp‐1(+) and Foxp3(+) Nrp‐1(−) Treg cells from P. yoelii‐infected mice exhibited a similar T‐cell receptor Vβ chain usage and methylation pattern in the Treg‐specific demethylation region within the foxp3 locus. Strikingly, we did not observe induction of Foxp3 expression in Foxp3(−) T cells adoptively transferred to P. yoelii‐infected mice. Hence, our results suggest that P. yoelii infection triggered expansion of naturally occurring Treg cells rather than de novo induction of Foxp3(+) Treg cells

Regulatory T cell proliferative potential as novel marker to investigate immune tolerance and clinical progression in Multiple Sclerosis

In autoimmune disorders such as Multiple Sclerosis (MS) one of the determining alteration is the breakdown of self-antigen immune-tolerance. Peripheral immune tolerance is maintained, at least in part, by Regulatory T cells (Treg). Several studies have shown that either defects in the frequency or the suppressive capacity of Treg cells can contribute to the development of break of self-tolerance, and that in animal models of autoimmunity, adoptive transfer of Treg cells was able to stop disease process. Treg cells are known to be anergic in vitro to T cell receptor-induced (TCR) stimulation and this state correlates with their in vitro suppressive capacity. It has been reported that there are differences in the number of Treg cells in MS patients when compared with healthy controls. However there is also extensive evidence indicating a defect in the suppressive function of Treg cells from MS patients. In previous studies we showed that Treg cells produce an higher amount of leptin when compared with effector T cells and that leptin acts as a negative signal for the proliferation of Treg cells. In vitro leptin neutralization results in Treg cells proliferation. Although in last few years several studies have been performed to understand the molecular mechanism leading to autoimmune disorders development, there are no surrogate markers to predict the clinical progression of autoimmune diseases and the clinical response to the classical therapeutic regimes

Biased Treg/Th17 balance away from regulatory toward inflammatory phenotype in relapsed multiple sclerosis and its correlation with severity of symptoms

The opposing immune functions of Treg and Th17 lymphocytes and the plasticity of Treg/Th17 differentiation, has led us to investigate the effects of their fluctuations and counterbalance in autoimmune condition of multiple sclerosis (MS). Evaluation of Treg and Th17 frequency in peripheral blood of a group of relapsed MS patients, showed a decrease in Treg/Th17 ratio compared to that of healthy controls. A reverse correlation between these subsets was observed in controls but not in patient groups. Both Treg frequency and Treg/Th17 ratio were negatively correlated with severity of symptoms. There was shown to be an enduring increase in Treg frequency associated with MS disease

Inhaled corticosteroid use is associated with increased circulating tregulatory cells in children with asthma

BACKGROUND: T regulatory (Treg) cells are important in balancing immune responses and dysregulation of Treg cells has been implicated in the pathogenesis of multiple disease states including asthma. In this study, our primary aim was to determine Treg cell frequency in the peripheral blood of children with and without asthma. The secondary aim was to explore the association between Treg cell frequency with allergen sensitization, disease severity and medication use. METHODS: Peripheral blood mononuclear cells from healthy control subjects (N = 93) and asthmatic children of varying disease severity (N = 66) were characterized by multi-parameter flow cytometry. RESULTS: Our findings demonstrate that children with asthma had a significantly increased frequency of Treg cells compared to children without asthma. Using a multivariate model, increased Treg cell frequency in children with asthma was most directly associated with inhaled corticosteroid use, and not asthma severity, allergic sensitization, or atopic status of the asthma. CONCLUSION: We conclude that low dose, local airway administration of corticosteroids is sufficient to impact the frequency of Treg cells in the peripheral blood. These data highlight the importance of considering medication exposure when studying Treg cells and suggest inhaled corticosteroid use in asthmatics may improve disease control through increased Treg cell frequency

Imbalanced expression of functional surface molecules in regulatory and effector T cells in systemic lupus erythematosus

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+/highCD127Ø/lowFoxP3+, and effector T cells were defined as CD25+CD127+FoxP3Ø. 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.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de MedicinaUniversidade Católica de Goiás Departamento de BiomedicinaUniversidade de São Paulo Faculdade de Medicina Departamento de Clínica MédicaUNIFESP, EPM, Depto. de MedicinaSciEL

Low frequency of CD4+CD25+ Treg in SLE patients: a heritable trait associated with CTLA4 and TGFβ gene variants

<p>Abstract</p> <p>Background</p> <p>CD4⁺CD25⁺regulatory T cells play an essential role in maintaining immune homeostasis and preventing autoimmunity. Therefore, defects in Treg development, maintenance or function have been associated with several human autoimmune diseases including Systemic Lupus Erythematosus (SLE), a systemic autoimmune disease characterized by loss of tolerance to nuclear components and significantly more frequent in females.</p> <p>Results</p> <p>To investigate the involvement of Treg in SLE pathogenesis, we determined the frequency of CD4⁺CD25⁺CD45RO⁺T cells, which encompass the majority of Treg activity, in the PBMC of 148 SLE patients (76 patients were part of 54 families), 166 relatives and 117 controls. SLE patients and their relatives were recruited in several Portuguese hospitals and through the Portuguese Lupus Association. Control individuals were blood donors recruited from several regional blood donor centers. Treg frequency was significantly lower in SLE patients than healthy controls (z = -6.161, <it>P </it>< 0.00001) and intermediate in the relatives' group. Remarkably, this T cell subset was also lower in females, most strikingly in the control population (z = 4.121, <it>P </it>< 0.001). We further ascertained that the decreased frequency of Treg in SLE patients resulted from the specific reduction of <it>bona fide </it>FOXP3⁺CD4⁺CD25⁺Treg. Treg frequency was negatively correlated with SLE activity index (SLEDAI) and titers of serum anti-dsDNA antibodies. Both Treg frequency and disease activity were modulated by IVIg treatment in a documented SLE case. The segregation of Treg frequency within the SLE families was indicative of a genetic trait. Candidate gene analysis revealed that specific variants of <it>CTLA4 </it>and <it>TGFβ </it>were associated with the decreased frequency of Treg in PBMC, while <it>FOXP3 </it>gene variants were associated with affection status, but not with Treg frequency.</p> <p>Conclusion</p> <p>SLE patients have impaired Treg production or maintenance, a trait strongly associated with SLE disease activity and autoantibody titers, and possibly resulting from the inability to convert FOXP3⁺CD25^-into FOXP3⁺CD25⁺T cells. Treg frequency is highly heritable within SLE families, with specific variants of the <it>CTLA4 </it>and <it>TGFβ </it>genes contributing to this trait, while <it>FOXP3 </it>contributes to SLE through mechanisms not involving a modulation of Treg frequency. These findings establish that the genetic components in SLE pathogenesis include genes related to Treg generation or maintenance.</p

Rorc restrains the potency of ST2+ regulatory T cells in ameliorating intestinal graft-versus-host disease

Soluble stimulation-2 (ST2) is increased during graft-versus-host disease (GVHD), while Tregs that express ST2 prevent GVHD through unknown mechanisms. Transplantation of Foxp3- T cells and Tregs that were collected and sorted from different Foxp3 reporter mice indicated that in mice that developed GVHD, ST2+ Tregs were thymus derived and predominantly localized to the intestine. ST2-/- Treg transplantation was associated with reduced total intestinal Treg frequency and activation. ST2-/- versus WT intestinal Treg transcriptomes showed decreased Treg functional markers and, reciprocally, increased Rorc expression. Rorc-/- T cells transplantation enhanced the frequency and function of intestinal ST2+ Tregs and reduced GVHD through decreased gut-infiltrating soluble ST2-producing type 1 and increased IL-4/IL-10-producing type 2 T cells. Cotransfer of ST2+ Tregs sorted from Rorc-/- mice with WT CD25-depleted T cells decreased GVHD severity and mortality, increased intestinal ST2+KLRG1+ Tregs, and decreased type 1 T cells after transplantation, indicating an intrinsic mechanism. Ex vivo IL-33-stimulated Tregs (TregIL-33) expressed higher amphiregulin and displayed better immunosuppression, and adoptive transfer prevented GVHD better than control Tregs or TregIL-33 cultured with IL-23/IL-17. Amphiregulin blockade by neutralizing antibody in vivo abolished the protective effect of TregIL-33. Our data show that inverse expression of ST2 and RORγt in intestinal Tregs determines GVHD and that TregIL-33 has potential as a cellular therapy avenue for preventing GVHD

Differential Responses of Human Regulatory T Cells (Treg) and Effector T Cells to Rapamycin

Background: The immunosuppressive drug rapamycin (RAPA) promotes the expansion of CD4+ CD25highFoxp3+ regulatory\ud T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-c chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA.\ud Methodology/Principal Findings: CD4+CD25+ and CD4+CD25neg T cells were isolated from PBMC of normal controls (n = 21)\ud using AutoMACS. These T cell subsets were cultured in the presence of anti-CD3/CD28 antibodies and 1000 IU/mL IL-2 for 3 to 6 weeks. RAPA (1–100 nM) was added to half of the cultures. After harvest, the cell phenotype, signaling via the PI3K/ mTOR and STAT pathways, expression of survival proteins and Annexin V binding were determined and compared to values obtained with freshly-separated CD4+CD25high and CD4+CD25neg T cells. Suppressor function was tested in co-cultures with autologous CFSE-labeled CD4+CD25neg or CD8+CD25neg T-cell responders. The frequency and suppressor activity of Treg were increased after culture of CD4+CD25+ T cells in the presence of 1–100 nM RAPA (p,0.001). RAPA-expanded Treg were largely CD4+CD25highFoxp3+ cells and were resistant to apoptosis, while CD4+CD25neg T cells were sensitive. Only Treg upregulated anti-apoptotic and down-regulated pro-apoptotic proteins. Treg expressed higher levels of the PTEN protein than CD4+CD25neg cells. Activated Treg6RAPA preferentially phosphorylated STAT5 and STAT3 and did not utilize the PI3K/ mTOR pathway.\ud Conclusions/Significance: RAPA favors Treg expansion and survival by differentially regulating signaling, proliferation and sensitivity to apoptosis of human effector T cells and Treg after TCR/IL-2 activation

Regulatory T Cell Suppression of Gag-Specific CD8+ T Cell Polyfunctional Response After Therapeutic Vaccination of HIV-1-Infected Patients on ART

We tested the hypothesis that therapeutic vaccination against HIV-1 can increase the frequency and suppressive function of regulatory, CD4+ T cells (Treg), thereby masking enhancement of HIV-1-specific CD8+ T cell response. HIV-1-infected subjects on antiretroviral therapy (N = 17) enrolled in a phase I therapeutic vaccine trial received 2 doses of autologous dendritic cells (DC) loaded with HIV-1 peptides. The frequency of CD4+CD25hiFOXP3+ Treg in blood was determined prior to and after vaccination in subjects and normal controls. Polyfunctional CD8+ T cell responses were determined pre- and post-vaccine (N = 7) for 5 immune mediators after in vitro stimulation with Gag peptide, staphylococcal enterotoxin B (SEB), or medium alone. Total vaccine response (post-vaccine–pre-vaccine) was compared in the Treg(+) and Treg-depleted (Treg-) sets. After vaccination, 12/17 subjects showed a trend of increased Treg frequency (P = 0.06) from 0.74% to 1.2%. The increased frequency did not correlate with CD8+ T cell vaccine response by enzyme linked immunosorbent assay for interferon γ production. Although there was no significant change in CD8+ T cell polyfunctional response after vaccination, Treg depletion increased the polyfunctionality of the total vaccine response (P = 0.029), with a >2-fold increase in the percentage of CD8+ T cells producing multiple immune mediators. In contrast, depletion of Treg did not enhance polyfunctional T cell response to SEB, implying specificity of suppression to HIV-1 Gag. Therapeutic immunization with a DC-based vaccine against HIV-1 caused a modest increase in Treg frequency and a significant increase in HIV-1-specific, Treg suppressive function. The Treg suppressive effect masked an increase in the vaccine-induced anti-HIV-1-specific polyfunctional response. The role of Treg should be considered in immunotherapeutic trials of HIV-1 infection

Statins increase the frequency of circulating CD4+ FOXP3+ regulatory T cells in healthy individuals

RESUMEN: Las estatinas se han demostrado para modular el número y la función supresora de células T CD4 + FOXP3 (Treg) en condiciones inflamatorias. Sin embargo, no está bien establecido si estatinas también podría afectar Treg en ausencia de inflamación. Para abordar esta cuestión, dieciocho sujetos masculinos normocolesterolémicos fueron tratados con lovastatina o atorvastatina al día durante 45 días. La frecuencia y el fenotipo de circulación Treg se evaluaron en los días 0, 7, 30, y 45. Los niveles de ARNm de FOXP3, IDO, TGF-β, e IL-10 se midieron en las células T CD4 +. Se encontró que tanto los que aumenta significativamente los niveles de ARNm y de frecuencia de Treg FOXP3 en el día 30. En el día 45, los números de Treg volvieron a los valores basales; Sin embargo, el TGF-β y los niveles de ARNm de FOXP3 se mantuvo alta, acompañada por el aumento de los porcentajes de CTLA-4 y GITR que expresan Treg. Treg expresión de Ki-67 se redujo tras el tratamiento con estatinas. la frecuencia de Treg correlacionó positivamente con los niveles plasmáticos de colesterol de lipoproteínas de alta densidad (HDL-C), lo que sugiere un papel para el HDL-C en la homeostasis de Treg. Por lo tanto, las estatinas parecen tener efectos inmunomoduladores inflamación independiente. Por lo tanto, el aumento de la frecuencia de Treg células probablemente contribuye a inmunomoduladores efecto de las estatinas, incluso en individuos sanos.ABSTRACT: Statins have been shown to modulate the number and the suppressive function of CD4+FOXP3+ T cells (Treg) in inflammatory conditions. However, it is not well established whether statin could also affect Treg in absence of inflammation. To address this question, eighteen normocholesterolemic male subjects were treated with lovastatin or atorvastatin daily for 45 days. The frequency and phenotype of circulating Treg were evaluated at days 0, 7, 30, and 45. mRNA levels of FOXP3, IDO, TGF-β, and IL-10 were measured in CD4+ T cells. We found that both statins significantly increased Treg frequency and FOXP3 mRNA levels at day 30. At day 45, Treg numbers returned to baseline values; however, TGF-β and FOXP3 mRNA levels remained high, accompanied by increased percentages of CTLA-4- and GITR-expressing Treg. Treg Ki-67 expression was decreased upon statin treatment. Treg frequency positively correlated with plasma levels of high-density lipoprotein cholesterol (HDL-c), suggesting a role for HDL-c in Treg homeostasis. Therefore, statins appear to have inflammation-independent immune-modulatory effects. Thus, the increase in Treg cells frequency likely contributes to immunomodulatory effect of statins, even in healthy individual