CD25 Foxp3 Research Articles

Silencing of Foxp3 delays the growth of murine melanomas and modifies the tumor immunosuppressive environment.

Forkhead box p3 (Foxp3) expression was believed to be specific for T-regulatory cells but has recently been described in non-hematopoietic cells from different tissue origins and in tumor cells from both epithelial and non-epithelial tissues. The aim of this study was to elucidate the role of Foxp3 in murine melanoma. The B16F10 cell line Foxp3 silenced with small interference Foxp3 plasmid transfection was established and named B16F10.1. These cells had lower levels of Foxp3 mRNA (quantitative real-time reverse transcription-polymerase chain reaction [0.235-fold]), protein (flow cytometry [0.02%]), CD25+ expression (0.06%), cellular proliferation (trypan blue staining), and interleukin (IL)-2 production (enzyme-linked immunosorbent assay [72.35 pg/mL]) than those in B16F10 wild-type (WT) cells (P<0.05). Subcutaneous inoculation of the B16F10.1 cell line into C57BL/6 mice delayed the time of visible tumor appearance, increased the time of survival, and affected the weight of tumors, and also decreased the production of IL-10, IL-2, and transforming growth factor beta compared with mice inoculated with the B16F10 WT cell line. The B16F10.1 cells derived from tumors and free of T-cells (isolated by Dynabeads and plastic attachment) expressed relatively lower levels of Foxp3 and CD25+ than B16F10 WT cells (P<0.05) in a time-dependent manner. The population of tumor-infiltrating lymphocytes of T CD4+ cells (CD4+, CD4+CD25+, and CD4+CD25+Foxp3+) increased in a time-dependent manner (P<0.05) in tumors derived from B16F10 WT cells and decreased in tumors derived from B16F10.1 cells. Similar data were obtained from spleen cells. These results suggest that, in melanomas, Foxp3 partly induces tumor growth by modifying the immune system at the local and peripheral level, shifting the environment toward an immunosuppressive profile. Therapies incorporating this transcription factor could be strategies for cancer treatment.

Abstract A026: Validation of an assay to identify regulatory T cells by flow cytometry in global clinical trials

Abstract The measurement of regulatory T cells (Treg) by flow cytometry provides important information in the assessment of new anti-tumor drug candidates, particularly in regards to an immuno-oncology mechanism of action. An assay was validated using a fit-for-purpose approach to characterize an optimized multi-parameter flow cytometry protocol for the identification and quantification of Treg from whole blood for use in global clinical trials, using standardized central laboratory sample testing. The method established at ILS-New York detects Treg by using fluorochrome-conjugated monoclonal antibodies which bind the cellular surface marker proteins CD45, CD3, CD4, CD25, and intracellular FOXP3. This method was determined to yield low non-specific labeling and increased Treg resolution with limited cell loss. Validation of the Treg assay included titration of monoclonal antibodies, assessment of stability of collected samples, inter- and intra- assay precision determinations, and analyst-to-analyst and instrument-to-instrument comparisons. In addition, global harmonization of the procedure was conducted across three ILS laboratories in New York, Dublin and Singapore. Samples were acquired on a BD FACSCanto II flow cytometer and results averaged across five donors. Samples, assessed at 0, 24, 48 and 72 hours post-collection, were stable up to 72 hours post-collection. The percent coefficient of variation calculated for intra- and inter- assay precision assessments, in the measurement of the percentage of CD25+FOXP3+ cells, was found to be &amp;lt;10%. Comparisons of variability between analysts and instruments indicated, for the percentage of CD25+FOXP3+ cells, a percent difference of &amp;lt;20%. Global laboratory assessments of the Treg phenotype were made between New York and Dublin and also between New York and Singapore, using aliquots from the same donor samples; the percent differences between laboratories were 13.43% and 24.19%, respectively. In conclusion, in clinical trials analyzing cancer immunotherapy candidates that modulate lymphocyte immune responses, it is informative to have an established method for Treg analysis that is robust and reproducible. This is especially important as Treg are a low frequency subpopulation, ranging from 1% to 7% of CD4+ T lymphocytes in normal peripheral blood, and assessment of differences between low frequency populations can lead to increased variability. Despite the low frequency of these cells, our data indicate that a validated Treg assay allows for clinical trials to be carried out with multiple subjects that can be assessed at multiple time points. Importantly, this method has been validated at different sites around the world, with the ability to obtain comparable data from North America, Europe, and Asia. Applying complex scientific techniques such as multicolor flow cytometry, in a globally harmonized manner, will provide important data for drug development of immuno-oncology therapeutic candidates and ultimately serve to accelerate new therapeutic options to patients. Citation Format: Li Zhou, Krista D. Buono, Christina D. Swenson, Michelle Brien, Thomas W. Mc Closkey. Validation of an assay to identify regulatory T cells by flow cytometry in global clinical trials. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A026.

Activation of mTOR (mechanistic target of rapamycin) in rheumatic diseases.

Mechanistic target of rapamycin (mTOR, also known as mammalian target of rapamycin) is a ubiquitous serine/threonine kinase that regulates cell growth, proliferation and survival. These effects are cell-type-specific, and are elicited in response to stimulation by growth factors, hormones and cytokines, as well as to internal and external metabolic cues. Rapamycin was initially developed as an inhibitor of T-cell proliferation and allograft rejection in the organ transplant setting. Subsequently, its molecular target (mTOR) was identified as a component of two interacting complexes, mTORC1 and mTORC2, that regulate T-cell lineage specification and macrophage differentiation. mTORC1 drives the proinflammatory expansion of T helper (TH) type 1, TH17, and CD4(-)CD8(-) (double-negative, DN) T cells. Both mTORC1 and mTORC2 inhibit the development of CD4(+)CD25(+)FoxP3(+) T regulatory (TREG) cells and, indirectly, mTORC2 favours the expansion of T follicular helper (TFH) cells which, similarly to DN T cells, promote B-cell activation and autoantibody production. In contrast to this proinflammatory effect of mTORC2, mTORC1 favours, to some extent, an anti-inflammatory macrophage polarization that is protective against infections and tissue inflammation. Outside the immune system, mTORC1 controls fibroblast proliferation and chondrocyte survival, with implications for tissue fibrosis and osteoarthritis, respectively. Rapamycin (which primarily inhibits mTORC1), ATP-competitive, dual mTORC1/mTORC2 inhibitors and upstream regulators of the mTOR pathway are being developed to treat autoimmune, hyperproliferative and degenerative diseases. In this regard, mTOR blockade promises to increase life expectancy through treatment and prevention of rheumatic diseases.

Increased numbers of FoxP3-expressing CD4+ CD25+ regulatory T cells in peripheral blood from dogs with atopic dermatitis and its correlation with disease severity.

Atopic dermatitis (AD) is a common chronic inflammatory skin disease of humans and dogs. Regulatory T cells (Tregs) are essential controllers of immune homeostasis and have been shown to play a key role in human AD, even though frequencies of Tregs in atopic human patients vary greatly. Only two studies have reported Treg numbers in the peripheral blood of dogs with canine AD (CAD). This study aimed to assess the numbers of circulating Tregs in healthy and atopic dogs, and to determine whether Treg numbers correlate with age, sex, disease severity or pre-treatment. Client-owned dogs including 14 healthy dogs and 35 dogs with CAD. Expression of Tregs in peripheral blood mononuclear cells was evaluated by flow cytometry. Tregs were phenotypically identified as T cells triple positive for CD4, CD25 and FoxP3. The percentage of circulating CD4(+) CD25(+) FoxP3(+) Tregs in atopic dogs was increased significantly compared to healthy dogs (mean 2.1% versus 1%, P=0.002) and correlated with disease severity (Pruritus Scale: r=0.48, P=0.003; CADESI-04: r=0.34, P=0.044). No significant differences in age or sex were found in either group and pre-treatment had no influence on results for atopic dogs. Data suggest that, as in humans, CD4(+) CD25(+) FoxP3(+) Tregs may contribute to the pathogenesis of CAD as indicated by an association between Treg frequency and disease severity. Further investigation is required to improve the understanding of the role of Tregs in atopic dogs.

Mechanisms for T cell tolerance induced with granulocyte colony-stimulating factor.

Granulocyte colony-stimulating factor (G-CSF) has been widely accepted as a mediator of T cell tolerance. The immune modulatory effect of G-CSF on T cells is believed to be mediated exclusively through other effector cells, such as monocytes, tolerogenic dendritic cells (DC), and myeloid-derived suppressor cells. Recent advances confirmed the direct effects of G-CSF in inducing immune tolerance of T cells through the G-CSF-G-CSF receptor pathway and related molecular mechanisms. This review aims to summarize the findings associated with the direct and indirect mechanisms for T cell tolerance induced with G-CSF. The role of G-CSF in preventing graft-versus-host disease (GVHD) and in treating autoimmune diseases (ADs) is also discussed. It is conceivable that G-CSF and immune cell compositions, such as tolerogenic DC and CD4(+)CD25(+)Foxp3(+) T cells, modulated by G-CSF could become an integral part of the immunomodulatory therapies against GVHD and ADs in the future.

γδ T Cell-Dependent Regulatory T Cells Prevent the Development of Autoimmune Keratitis.

To prevent potentially damaging inflammatory responses, the eye actively promotes local immune tolerance via a variety of mechanisms. Owing to trauma, infection, or other ongoing autoimmunity, these mechanisms sometimes fail, and an autoimmune disorder may develop in the eye. In mice of the C57BL/10 (B10) background, autoimmune keratitis often develops spontaneously, particularly in the females. Its incidence is greatly elevated in the absence of γδ T cells, such that ∼80% of female B10.TCRδ(-/-) mice develop keratitis by 18 wk of age. In this article, we show that CD8(+) αβ T cells are the drivers of this disease, because adoptive transfer of CD8(+), but not CD4(+), T cells to keratitis-resistant B10.TCRβ/δ(-/-) hosts induced a high incidence of keratitis. This finding was unexpected because in other autoimmune diseases, more often CD4(+) αβ T cells, or both CD4(+) and CD8(+) αβ T cells, mediate the disease. Compared with wild-type B10 mice, B10.TCRδ(-/-) mice also show increased percentages of peripheral memory phenotype CD8(+) αβ T cells, along with an elevated frequency of CD8(+) αβ T cells biased to produce inflammatory cytokines. In addition, B10.TCRδ-/- mice have fewer peripheral CD4(+) CD25(+) Foxp3(+) αβ regulatory T cells (Tregs), which express lower levels of receptors needed for Treg development and function. Together, these observations suggest that in B10 background mice, γδ T cells are required to generate adequate numbers of CD4(+) CD25(+) Foxp3(+) Tregs, and that in B10.TCRδ(-/-) mice a Treg deficiency allows dysregulated effector or memory CD8(+) αβ T cells to infiltrate the cornea and provoke an autoimmune attack.

High sugar and butter (HSB) diet induces obesity and metabolic syndrome with decrease in regulatory T cells in adipose tissue of mice.

The purpose of the study was to develop a novel diet based on standard AIN93G diet that would be able to induce experimental obesity and impair immune regulation with high concentrations of both carbohydrate and lipids. To compare the effects of this high sugar and butter (HSB) diet with other modified diets, male C57BL/6 mice were fed either mouse chow, or AIN93G diet, or high sugar (HS) diet, or high-fat (HF) diet, or high sugar and butter (HSB) diet for 11 weeks ad libitum. HSB diet induced higher weight gain. Therefore, control AIN93G and HSB groups were chosen for additional analysis. Regulatory T cells were studied by flow cytometry, and cytokine levels were measured by ELISA. Although HF and HSB diets were able to induce a higher weight gain compatible with obesity in treated mice, HSB-fed mice presented the higher levels of serum glucose after fasting and the lowest frequency of regulatory T cells in adipose tissue. In addition, mice that were fed HSB diet presented higher levels of cholesterol and triglycerides, hyperleptinemia, increased resistin and leptin levels as well as reduced adiponectin serum levels. Importantly, we found increased frequency of CD4(+)CD44(+) effector T cells, reduction of CD4(+)CD25(+)Foxp3(+) and Th3 regulatory T cells as well as decreased levels of IL-10 and TGF-β in adipose tissue of HSB-fed mice. Therefore, HSB represents a novel model of obesity-inducing diet that was efficient in triggering alterations compatible with metabolic syndrome as well as impairment in immune regulatory parameters.

Graft Versus Host Disease (GvHD) Is Attenuated By Administration of Pregnancy Specific Glycoproteins through Induction of Immune Tolerance

Hematopoietic stem cell transplantation is curative for many disorders; however, it can be associated with significant morbidity and mortality, often as a result of graft versus host disease (GvHD). GvHD is an immune mediated reaction in which donor T-cells recognize the host antigens as foreign, causing donor T-cells to proliferate and attack host tissues. Establishment of a tolerogenic immune environment while preserving immune response to infectious agents and malignancy is required for successful bone marrow transplantationand GvHD presents a significant obstacle to this.Pregnancy specific glycoproteins (PSGs) are synthesized by the placenta at the onset of pregnancy and are believed to play a role in maintaining a tolerogenic immune environment to prevent rejection of the fetus by the maternal immune system. Two specific PSG proteins, PSG1 and PSG9, are of particular note as they have both been shown to be involved in pathways devoted to induction of immune tolerance. Both PSG1 and PSG9 are involved in activation of transforming growth factor-β (TGFβ), a cytokine essential to suppression of inflammatory T-cells and important for differentiation of tolerance inducing CD4+ CD25+ FoxP3+ regulatory T-cells (Tregs), a cell population shown to be important in the prevention of GvHD. We thus hypothesized that PSG1 and PSG9 (PSG1/9) could be used to treat or prevent GvHD by inducing immune tolerance through TGFβ.Using surface plasmon resonance (SPR) analysis, we have shown that PSG1/9 bind directly to the latency associated peptide (LAP), a protein that confers latency to mature TGFβ, effectively blocking binding of TGFβ to its receptors. Further, bioassays and ELISA data show an increase in the levels of bioactive TGFβ after treatment with PSG1/9, indicating that these proteins have a role in TGFβ activation through their interaction with LAP. In vitro data using naïve mouse T-cells showed that upon treatment with PSG1, there was a significant increase of CD4+ CD25+ cells expressing FoxP3 (18%±1.5; n=3) compared to only 2% in untreated controls (2%±0.4; n=6) (p<0.0001). Similarly, PSG9 treatment also increased FoxP3 expression (11%±2.8; n=3) over controls (p<0.0001). The increase in FoxP3 expression was also observed upon treatment of primary naïve human T-cells with both PSG1 and PSG9 (PSG1, p=0.0073; PSG9, p=0.0028). When a TGFβ receptor inhibitor was added to cell culture, the increase in FoxP3 expression was effectively blocked, further supporting the hypothesis that PSG1/9 induce expression of FoxP3+ Tregs through regulation of TGFβ. As IL-2 is important for the stability and differentiation of Tregsin vivo and its transcription is suppressed by TGFβ, we performed bioassays on CD4+ naïve T-cells with no added IL-2. We observed that in the absence of added IL-2, treatment with PSG1/9 resulted in an inhibition of IL-2 secretion by activated CD4+ T-cells, and there was no increase in the number and percentage of FoxP3 expressing cells.SBE-luc mice, which express luciferase in response to activation of the TGFβ-SMAD pathway, exhibited significantly increased luciferase expression in the abdomen when injected intraperitoneally with PSG1. This supports the idea that PSG1 induces expression of FoxP3 through the TGFβ receptor pathway in vivo. Finally, using a murine model of GvHD, we observed that mice receiving PSG1 had reduced numbers of infiltrating inflammatory CD3+ T-cells in the colon and showed a marked improvement physically and histologically over untreated controls. In addition, PSG1 treated mice had significantly higher expression of FoxP3 in CD4+ CD25+ splenic cells (30%±9.6; n=4) when compared to untreated GvHD controls (8.6%±3.2; n=3) (p=0.0287).Currently, we are studying the cytokine profiles of mice treated with PSG to determine the efficacy of PSG1/9 in reducing the release of pro-inflammatory cytokines during GvHD. Overall, our data suggests that PSG1/9 induce immune tolerance in GvHD and may be a future treatment option for these patients. DisclosuresNo relevant conflicts of interest to declare.

Combination Therapy Using IL-2/IL-2 Monoclonal Antibody Complexes, Rapamycin, and Islet Autoantigen Peptides Increases Regulatory T Cell Frequency and Protects against Spontaneous and Induced Type 1 Diabetes in Nonobese Diabetic Mice.

Regulatory T cells (Treg) play a crucial role in the maintenance of self-tolerance. In this study, we sought to expand Ag-specific Tregs in vivo and investigate whether the expanded Tregs can prevent or delay the development of type 1 diabetes (T1D) in the NOD mouse model. NOD mice were treated with a combination of IL-2/anti-IL-2 Ab complex, islet Ag peptide, and rapamycin. After the combined treatment, CD4(+)CD25(+)Foxp3(+) Tregs were significantly expanded in vivo, they expressed classical Treg markers, exerted enhanced suppressive functions in vitro, and protected against spontaneous development of T1D in NOD mice. Moreover, treated mice were almost completely protected from the adoptively transferred, aggressive form of T1D caused by in vitro-activated cytotoxic islet Ag-specific CD8 T cells. Protection from T1D was transferrable by Tregs and could be attributed to reduced islet infiltration of immune cells as well as the skewing of the immune response toward a Th2 cytokine profile. This new method of peripheral immune regulation could potentially contribute to development of novel immunotherapeutic strategies to prevent the development of T1D or to promote tolerance to islet transplants without using immunosuppressive drugs for long terms.

Interferon-α induces marked alterations in circulating regulatory T cells, NK cell subsets, and dendritic cells in patients with JAK2V617F-positive essential thrombocythemia and polycythemia vera.

Long-term therapy with IFN-α2 is associated with sustained major molecular remissions in JAK2-positive ET and PV. The efficacy of IFN-α2 may be partly mediated by modulation of immune cells, which was investigated in twenty patients with ET (n=6) and PV (n=14). The frequency of CD4(+) CD25(+) Foxp3(+) T cells was significantly increased during IFN-α2 treatment in all patients (P<0.0001). A significant expansion of the CD56(bright) NK cells (P=0.0002) and a concomitant decrease in the frequency of CD56(dim) NK cells (P<0.0001) were also detected. Myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) were studied in nine patients, and decreased frequencies of both cell types were observed during the course of treatment. On both mDCs and pDCs, HLA-ABC expression was upregulated (P=0.003), but decreasing expression levels of HLA-DR was detected on mDCs. The expression of CD40 (P=0.002), CD83 (P=0.03), and CD86 (P=0.01) increased, but was confined to pDCs. Furthermore, PD-L1 expression was reduced on mDC (P=0.003) and increased on pDCs (P=0.02). No significant correlations were found between the changes in immune cells and hematological or molecular responses achieved in our cohort of patients. So forth, it remains to be revealed whether the profound changes in circulating immune cells contribute to the beneficial effects of long-term IFN-α2 treatment in some patients.

Immunomodulation of endothelial differentiated mesenchymal stromal cells: impact on T and NK cells.

Wharton's jelly mesenchymal stromal cells (WJ-MSCs) are promising candidates for tissue engineering, as their immunomodulatory activity allows them to escape immune recognition and to suppress several immune cell functions. To date, however, few studies have investigated the effect of differentiation of the MSCs on this immunomodulation. To address this question, we sought to determine the impact of differentiation toward endothelial cells on immunoregulation by WJ-MSCs. Following differentiation, the endothelial-like cells (ELCs) were positive for CD31, vascular endothelial cadherin and vascular endothelial growth factor receptor 2, and able to take up acetylated low-density lipoproteins. The expression of HLA-DR and CD86, which contribute to MSCs immunoprivilege, was still weak after differentiation. We then co-cultured un- and differentiated MSCs with immune cells, under conditions of both direct and indirect contact. The proliferation and phenotype of the immune cells were analyzed and the mediators secreted by both ELCs and WJ-MSCs quantified. Interleukin (IL)-6, IL-1β, prostaglandin E2 and in particular indoleamine-2,3-dioxygenase expression were upregulated in ELCs on stimulation by T and NK cells, suggesting the possible involvement of these factors in allosuppression. ELCs co-cultured with T cells were able to generate CD25(+) T cells, which were shown to be of the CD4(+)CD25(+)FoxP3(+) regulatory subset. Direct contact between NK cells and ELCs or WJ-MSCs decreased the level of NK-activating receptor natural-killer group 2, member D. Moreover, direct co-culturing with ELCs stimulates CD73 acquisition on NK cells, a mechanism which may induce adenosine secretion by the cells and lead to an immunosuppressive function. Taken together, our results show that ELCs obtained following differentiation of WJ-MSCs remain largely immunosuppressive.

Intranasal immunization with heat shock protein 60 induces CD4(+) CD25(+) GARP(+) and type 1 regulatory T cells and inhibits early atherosclerosis.

Atherosclerosis is an autoimmune inflammatory disease involving both innate and adaptive immune mechanisms. Immune tolerance induction may have therapeutic potential for the suppression of atherosclerosis. Current interest is directed towards mucosal tolerance induction, especially nasal tolerance. Previous studies have shown that heat shock protein 60 (HSP60) is recognized as an important autoantigen in atherosclerosis, and nasal or oral HSP60 can induce tolerance and ameliorate atherosclerosis by inducing several subsets of regulatory T cells (Tregs ) such as latency-associated peptide (LAP)(+) and forkhead box transcription factor 3 (FoxP3)(+) Tregs. However, little is known regarding the detailed mechanisms of nasal tolerance. Here, we again investigated the impact of nasal HSP60 on atherosclerosis and the mechanisms underlying the anti-atherosclerosis responses. We found that nasal HSP60 caused a significant 33·6% reduction in plaque size at the aortic root in the early stages of atherosclerosis (P < 0·001). Notably, a significant increase in activated CD4(+) CD25(+) glycoprotein A repetitions predominant (GARP)(+) Tregs, type 1 Tregs (Tr1 cells), and CD4(+) CD25(+) FoxP3(+) Tregs, as well as a marked decrease in the numbers of type 1 and 17 T helper cells was detected in the spleens and cervical lymph nodes of HSP60-treated mice. Moreover, nasal HSP60 increases the production of transforming growth factor (TGF)-β and interleukin (IL)-10 and decreases the secretion of IFN-γ and IL-17. Interestingly, the atheroprotective role of nasal HSP60 treatment was abrogated partly by the neutralization of IL-10. Our findings show that nasal administration of HSP60 can attenuate atherosclerotic formation by inducing GARP(+) Tregs, Tr1 cells and FoxP3(+) Tregs, and that these Tregs maintain immune homeostasis by secreting IL-10 and TGF-β.

Role of immune tolerance in BALB/c mice with anaphylactic shock after Echinococcus granulosus infection.

This study tested the hypothesis that immune tolerance mediated by regulatory T (Treg) cells is protective against cystic echinococcosis (CE)-induced anaphylactic shock. BALB/c mice were inoculated with protoscoleces of Echinococcus granulosus. After 3 months, the presence of cysts in the peritoneal cavity was confirmed after which a subset of mice was sensitized using a cyst fluid suspension to induce anaphylactic shock. While IgE levels were significantly higher in both groups inoculated with E. granulosus as compared to the healthy control group (both P < 0.01), sensitized mice had higher IgE levels as compared with those with E. granulosus alone (P < 0.05). Mice inoculated with E. granulosus alone and sensitized mice both had significantly higher histamine levels as compared to the healthy controls. The proportion of CD4(+)CD25(+)Foxp3(+) Treg cells relative to CD4(+) cells was significantly higher in mice inoculated with E. granulosus alone (P < 0.0167); significantly higher interleukin-10 (IL-10) and tumor growth factor-β (TGF-β1) levels were also noted in this group (all P < 0.01). In contrast, IL-13 and IL-17A levels were significantly higher in the sensitized mice (both P < 0.05). Taken together, these data suggest that the biphasic changes in Treg cell and cytokine levels may be associated with anaphylactic shock induced by CE.

Food-allergic infants have impaired regulatory T-cell responses following in vivo allergen exposure.

Regulatory T cells (Tregs) are critical for development of oral tolerance, and studies suggest that dysfunction of Tregs may lead to food allergy. However, to date, no study has investigated Treg responses following in vivo exposure to peanut or egg allergens in humans. To examine changes in peripheral blood CD4(+) CD25(+) Foxp3(+) Treg populations (total, activated and naive) in food-allergic, food-sensitized but tolerant, and healthy (non-sensitized non-allergic) patients over time following in vivo allergen exposure. A subset of infants from the HealthNuts study with egg or peanut allergy (n = 37), egg or peanut sensitization (n = 35), or who were non-sensitized non-allergic (n = 15) were studied. All subjects underwent oral food challenge (OFC) to egg or peanut. PBMCs were obtained within 1 h of OFC (in vivo allergen exposure), and Treg populations enumerated ex vivo on day 0, and after 2 and 6 days rest in vitro. Non-allergic infants showed stable total Treg frequencies over time; food-sensitized infants had a transient fall in Treg percentage with recovery to baseline by day 6 (6.87% day 0, 5.27% day 2, 6.5% day 6); and food-allergic infants showed persistent reduction in Treg (6.85% day 0, 5.4% day 2, 6.2% day 6) following in vivo allergen exposure. Furthermore, food-allergic infants had a significantly lower ratio of activated Treg:activated T cells (10.5 ± 0.77) at day 0 compared to food-sensitized (14.6 ± 1.24) and non-allergic subjects (16.2 ± 1.23). Our data suggest that the state of allergen sensitization is associated with depletion of Treg following allergen exposure. Impaired capacity to regenerate the Treg pool following allergen exposure may be an important factor that determines clinical allergy vs. sensitization without allergic reaction.

Depletion of regulatory T-cells leads to moderate B-cell antigenicity in respiratory syncytial virus infection.

The regulation of the immunopathology of respiratory syncytial virus (RSV) by regulatory T-cells (CD4(+)CD25(+)Foxp3(+); Tregs) is not understood. To deduce the same, Tregs were depleted in BALB/c mice by injecting anti-CD25 antibody followed by RSV infection (anti-CD25-RSV mice). In this model, a decrease in anti-fusion (F) antibody and neutralizing activity, and an increase in anti-nucleocapsid (N) antibody in serum, were seen. Decreased antibody-dependent cell-mediated cytotoxicity (ADCC) activity, increased IgG2a, and an influx of activated CD8(+) T-cells into the lungs were also observed. Co-culture of splenic CD45RA(+) B-cells from RSV-infected normal mice with CD4(+) cells isolated from anti-CD25-RSV mice (B/CD4) increased anti-F antibody secretion. The inclusion of CD25(+) Tregs isolated from isotype Ig-RSV mice into the B/CD4 co-culture substantially enhanced the frequency of anti-F antibody production. However, the same effect was not seen in the co-culture of CD45RA(+) B-cells with dendritic cells (DCs) (B/DCs) or CD8(+) cells (B/CD8) that were obtained from anti-CD25-RSV mice. The transfer of enriched B-cells from anti-CD25-RSV mice into RSV-infected SCID mice increased severe lung inflammation associated with the increased viral load and eosinophil number. These results indicate that Tregs modulate B-cell activity, particularly in producing F-specific neutralizing antibodies, to regulate RSV-mediated exacerbated diseases.

Suppression of CD4+ Effector Responses by Naturally Occurring CD4+ CD25+ Foxp3+ Regulatory T Cells Contributes to Experimental Cerebral Malaria.

The role of naturally occurring CD4(+) CD25(+) Foxp3(+) regulatory T cells (nTreg) in the pathogenesis of cerebral malaria (CM), which involves both pathogenic T cell responses and parasite sequestration in the brain, is still unclear. To assess the contribution and dynamics of nTreg during the neuropathogenesis, we unbalanced the ratio between nTreg and naive CD4(+) T cells in an attenuated model of Plasmodium berghei ANKA-induced experimental CM (ECM) by using a selective cell enrichment strategy. We found that nTreg adoptive transfer accelerated the onset and increased the severity of CM in syngeneic C57BL/6 (B6) P. berghei ANKA-infected mice without affecting the level of parasitemia. In contrast, naive CD4(+) T cell enrichment prevented CM and promoted parasite clearance. Furthermore, early during the infection nTreg expanded in the spleen but did not efficiently migrate to the site of neuroinflammation, suggesting that nTreg exert their pathogenic action early in the spleen by suppressing the protective naive CD4(+) T cell response to P. berghei ANKA infection in vivo in both CM-susceptible (B6) and CM-resistant (B6-CD4(-/-)) mice. However, their sole transfer was not sufficient to restore CM susceptibility in two CM-resistant congenic strains tested. Altogether, these results demonstrate that nTreg are activated and functional during P. berghei ANKA infection and that they contribute to the pathogenesis of CM. They further suggest that nTreg may represent an early target for the modulation of the immune response to malaria.

Persistence of cirrhosis is maintained by intrahepatic regulatory T cells that inhibit fibrosis resolution by regulating the balance of tissue inhibitors of metalloproteinases and matrix metalloproteinases.

Fibrosis is the result of the abnormal accumulation of the extracellular matrix and ineffective clearance of fibroplasia. CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are immunosuppressive lymphocytes that are highly expressed in the fibrotic tissues and peripheral blood of patients with cirrhosis or hepatocellular carcinoma. The role of Tregs in the progression of liver fibrosis is not well understood. Our experiments reveal that abundant of Tregs was scattered around sites of fibroplasia. Conversely, the depletion of Tregs promoted the resolution of liver fibrosis. As a consequence of Tregs depletion, the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) was altered; mmp9 and timp1 were reduced, whereas mmp2 and mmp14 were enhanced. The mmp9/timp1, mmp13/timp1, and mmp14/timp2 ratios were significantly increased in association with fibrosis resolution. Kupffer cells (KCs) are the main source of MMP. We observed that when KCs were cocultured with Tregs, the Tregs were able to inhibit MMP expression of KCs even at a low ratio; and anti-transforming growth factor-β (TGF-β) significantly reversed the inhibition of Tregs on MMP. Meanwhile, we also found that after Tregs depletion, TGF-β levels decreased in the mice liver, unlike in fibrosis. Furthermore, double depletion of both KCs and Tregs did not cause fiber resolution in mice. Thus, our results demonstrate that the persistence of liver cirrhosis is maintained by increased Tregs in the sites of fibroplasia and the subsequent regulation of the MMP/TIMP balance and that the suppression of KC-mediated MMP expression contributed to the regulatory process.

Transforming growth factor-β1 sustains the survival of Foxp3(+) regulatory cells during late phase of oropharyngeal candidiasis infection.

As CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) play crucial immunomodulatory roles during infections, one key question is how these cells are controlled during antimicrobial immune responses. Mechanisms controlling their homeostasis are central to ensure efficient protection against pathogens, as well as to control infection-associated immunopathology. Here we studied how their viability is regulated in the context of mouse oropharyngeal candidiasis (OPC) infection, and found that these cells show increased protection from apoptosis during late phase of infection and reinfection. Tregs underwent reduced cell death because they are refractory to T cell receptor restimulation-induced cell death (RICD). We confirmed their resistance to RICD, using mouse and human Tregs in vitro, and by inducing α-CD3 antibody-mediated apoptosis in vivo. The enhanced viability is dependent on increased transforming growth factor-β1 (TGF-β1) signaling that results in upregulation of cFLIP (cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein) in Tregs. Protection from cell death is abrogated in the absence of TGF-β1 signaling in Tregs during OPC infection. Taken together, our data unravel the previously unrecognized role of TGF-β1 in promoting Treg viability, coinciding with the pronounced immunomodulatory role of these cells during later phase of OPC infection, and possibly other mucosal infections.

Regulatory T cells in cardiovascular diseases.

Inflammation is essential in the initial development and progression of many cardiovascular diseases involving innate and adaptive immune responses. The role of CD4(+)CD25(+)FOXP3(+) regulatory T (TREG) cells in the modulation of inflammation and immunity has received increasing attention. Given the important role of TREG cells in the induction and maintenance of immune homeostasis and tolerance, dysregulation in the generation or function of TREG cells can trigger abnormal immune responses and lead to pathology. A wealth of evidence from experimental and clinical studies has indicated that TREG cells might have an important role in protecting against cardiovascular disease, in particular atherosclerosis and abdominal aortic aneurysm. In this Review, we provide an overview of the roles of TREG cells in the pathogenesis of a number of cardiovascular diseases, including atherosclerosis, hypertension, ischaemic stroke, abdominal aortic aneurysm, Kawasaki disease, pulmonary arterial hypertension, myocardial infarction and remodelling, postischaemic neovascularization, myocarditis and dilated cardiomyopathy, and heart failure. Although the exact molecular mechanisms underlying the cardioprotective effects of TREG cells are still to be elucidated, targeted therapies with TREG cells might provide a promising and novel future approach to the prevention and treatment of cardiovascular diseases.

Manipulating regulatory T cells: a promising strategy to treat autoimmunity.

CD4(+)CD25(+)Foxp3(+)regulatory T cells (Treg cells) are extremely important in maintaining immune tolerance. Manipulation of Treg cells, especially autoantigen-specific Treg cells is a promising approach for treatments of autoimmune disease since Treg cells may provide the advantage of antigen specificity without overall immune suppression. However, the clinical application of Treg cells has long been limited due to low numbers of Treg cells and the difficulty in identifying their antigen specificity. In this review, we summarize studies that demonstrate regression of autoimmune diseases using Treg cells as therapeutics. We also discuss approaches to generate polyclonal and autoantigen-specific Treg cells in vitro and in vivo. We also discuss our recent study that describes a novel approach of generating autoantigen-specific Treg cells in vivo and restoring immune tolerance by two steps apoptosis-antigen therapy.