Foxp3 Expression In Tregs Research Articles

Unique function of B cells in immune regulation through CD86-mediated maintenance of Foxp3 (168.6)

Abstract In recent years, B cells have emerged as an important player in the immune regulatory network. Using the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we observed a lack of resolution of disease in B cell-deficient (μMT) mice. Whereas, their WT counterparts recovered, indicating a regulatory role for B cells in central nervous system (CNS) autoimmunity. CD4+Foxp3+ T regulatory cells (Tregs) also mediate resolution of EAE. Absence of CD80/CD86 (B7) on B cells correlated with late emergence of Tregs in the CNS during EAE, suggesting an interaction between B cells and Tregs through B7 molecules. Anti-CD20 mediated B cell depletion disrupted this interaction resulting in significant reduction in the number of Tregs and like μMT, manifested in unresolved EAE. Interestingly, in vitro studies identified a unique role for B cells in maintaining Treg Foxp3 expression, which was enhanced upon CD86 upregulation following anti-IgM activation. Adoptive transfer of B cells into μMT mice resulted in partial restoration of Treg numbers leading to resolution of EAE. When deficient in CD86, B cells had a significantly reduced ability to restore Treg numbers in μMT. These data identify a distinct role for B cells in immune regulation through CD86-mediated interaction with Tregs and maintenance of Foxp3. This novel immune regulatory property of B cells may be harnessed to develop therapies for autoimmune diseases.

Human induced CD4+CD25+FOXP3+ regulatory T cells are suppressive in vitro, but fail to suppress inflammation in vivo

BackgroundRegulatory T cells (Treg) are important to maintain immune homeostasis. Presence of Treg correlates with a favourable disease course in juvenile idiopathic arthritis (JIA) patients. A distinction within the Treg...

Molecular analysis of melanoma-induced sentinel lymph node immune dysfunction.

Sentinel lymph nodes (SLNs) of melanoma patients show evidence of tumor-induced immune dysfunction. Our previous works have shown that IL-10 and IFNγ co-regulate indoleamine-2,3-dioxygenase (IDO)-expressing immunosuppressive dendritic cells (DCs) in melanoma SLNs. The goal of this study is to examine the relationship between melanoma SLN tumor burden and the degree of SLN immune dysfunction as a model to study tumor-induced immune dysfunction. We hypothesize that SLN tumor burden correlates with the degree of SLN immune dysfunction. Patients undergoing SLN biopsy for clinical stages I and II melanomas were enrolled in the study under an IRB-approved protocol. During the SLN biopsy, non-hot and non-blue portion of the SLN was harvested, flash-frozen in liquid nitrogen, and mRNA was extracted. By using quantitative real-time PCR, gene expressions of cytokines (IL-4, IL-10, IFNγ, TGFβ, GM-CSF) and the surrogates of immunosuppressive regulatory and effector cells (IDO-expressing DCs and Foxp3-expressing T-regs, respectively) were measured and correlated against the SLN tumor burden (MART1) and against each other. The data were log transformed for normalization. Statistical test used Student's t-test and stepwise multivariate regression analysis. Statistical significance was determined at P<0.05. SLNs of 74 patients were analyzed in this analysis. Ten of seventy-four patients (13.5%) had tumor-positive SLNs. MART1 gene expression showed a significant difference between the SLN (+) and SLN (-) groups (P=0.04). Among the various cytokines, multivariate analysis showed that only IFNγ gene expression correlated independently with MART1 gene expression (P<0.0001, r=0.91). Similar multivariate analyses show that IFNγ (P<0.0001, r=0.78), IL-10 (P=0.0037, r=0.60), and TGFβ (P<0.0001, r=0.95) gene expressions correlated independently with IDO gene expression. IFNγ (P<0.0001, r=0.87) and GM-CSF (P=0.042, r=0.76) gene expressions correlated independently with Foxp3 gene expression. MART1 gene expression showed independent correlation with IDO (P=0.0002, r=0.75) and Foxp3 (P=0.0002, r=0.75) gene expressions. SLN tumor burden correlates with immunosuppressive IDO and Foxp3 expressions within the SLNs of melanoma patients. Our data are consistent with our theory that melanoma induces expressions of specific cytokines, which in turn, stimulate immune suppressors within the SLN. This study also supports our previous finding that IL-10 and IFNγ co-regulate IDO within the SLN. In our data, IFNγ is the sole cytokine that correlates with the SLN tumor burden and seems to play a central role in tumor-induced immunological changes in the SLN immune microenvironment.

Role of Circulating CD4+ CD25high Foxp3+ Regulatory T-Cells in Paediatric Asthma

Background: The role of T-Helper 2 (Th2) cells in the pathogenesis of allergy and asthma has been well described. However, the immunologic mechanisms that down modulate and protect against the development of these disorders are poorly characterized. A spectrum of CD4+ T cells, including, FOXP3-positive CD4+CD25+ T regulatory cells (Tregs) might play a critical role in regulating these diseases. Objective: To investigate the role of CD4+CD25high FoxP3 Tregs in the pathogenesis of pediatric asthma. Methods: The study included 24 asthmatic children, 12 had mild intermittent asthma and 12 were of severe persistent asthma . In addition, 12 healthy subjects were used as controls. All patients were subjected to clinical examination and laboratory investigations including complete blood count with differential leucocytic and absolute eosinophilic count, serum total IgE level by ELIZA and flow cytometry was used to study the frequency of Tregs in peripheral blood lymphocytes of all studied groups using specific markers: cell-surface CD25 and CD4 expression and cytoplasmic FoxP3 expression. Results: It was noticed a significant decrease in CD4+CD25+ % and CD4+CD25 high % in both mild intermittent cases and severe persistent asthmatic patients when compared to healthy controls. FoxP3 expression in Tregs was significantly lower in CD4+CD25high T-cells of mild asthmatic patients when compared to control group. While the FoxP3 expression in Tregs was non- significantly lower in CD4+CD25high T-cells of severe asthmatic patients .Tregs cells % was correlated significantly with mild asthma .While it did not show correlation with severe asthma . An inverse correlation between FoxP3 protein expression was revealed within CD4+CD25high T-cells and total serum IgE when analyzed for all subjects. However, when correlation analysis was performed in each studied group separately, no significant correlation was found between FoxP3 expression and total serum IgE levels and there was no correlation between FoxP3 protein expressions within CD4+CD25high T-cells and eosinophilic count was noticed. CONCLUSION: The correlation of CD4+CD25high FoxP3 Tregs with asthma pathogenesis indicates that it is important to evaluate Tregs in allergic asthmatic children. Greater understanding of the molecular and immunological mechanisms underlying the CD4+CD25high FoxP3 Tregs might contribute the development of treatment modalities to influence disease processes of bronchial asthma in children as a future therapeutic target.

Role of Circulating CD4+ CD25high Foxp3+ Regulatory T-Cells in Paediatric Asthma

Background: The role of T-Helper 2 (Th2) cells in the pathogenesis of allergy and asthma has been well described. However, the immunologic mechanisms that down modulate and protect against the development of these disorders are poorly characterized. A spectrum of CD4+ T cells, including, FOXP3-positive CD4+CD25+ T regulatory cells (Tregs) might play a critical role in regulating these diseases. Objective: To investigate the role of CD4+CD25high FoxP3 Tregs in the pathogenesis of pediatric asthma. Methods: The study included 24 asthmatic children, 12 had mild intermittent asthma and 12 were of severe persistent asthma . In addition, 12 healthy subjects were used as controls. All patients were subjected to clinical examination and laboratory investigations including complete blood count with differential leucocytic and absolute eosinophilic count, serum total IgE level by ELIZA and flow cytometry was used to study the frequency of Tregs in peripheral blood lymphocytes of all studied groups using specific markers: cell-surface CD25 and CD4 expression and cytoplasmic FoxP3 expression. Results: It was noticed a significant decrease in CD4+CD25+ % and CD4+CD25 high % in both mild intermittent cases and severe persistent asthmatic patients when compared to healthy controls. FoxP3 expression in Tregs was significantly lower in CD4+CD25high T-cells of mild asthmatic patients when compared to control group. While the FoxP3 expression in Tregs was non- significantly lower in CD4+CD25high T-cells of severe asthmatic patients .Tregs cells % was correlated significantly with mild asthma .While it did not show correlation with severe asthma . An inverse correlation between FoxP3 protein expression was revealed within CD4+CD25high T-cells and total serum IgE when analyzed for all subjects. However, when correlation analysis was performed in each studied group separately, no significant correlation was found between FoxP3 expression and total serum IgE levels and there was no correlation between FoxP3 protein expressions within CD4+CD25high T-cells and eosinophilic count was noticed. CONCLUSION: The correlation of CD4+CD25high FoxP3 Tregs with asthma pathogenesis indicates that it is important to evaluate Tregs in allergic asthmatic children. Greater understanding of the molecular and immunological mechanisms underlying the CD4+CD25high FoxP3 Tregs might contribute the development of treatment modalities to influence disease processes of bronchial asthma in children as a future therapeutic target.

High-risk corneal allografts and why they lose their immune privilege

Corneal allografts are routinely performed without HLA typing or systemic immunosuppressive drugs. However, certain conditions create high risks for immune rejection. This review discusses recent insights into the mechanisms that rob the corneal allograft of its immune privilege. Studies in mice have revealed that stimuli that induce new blood vessel growth in the cornea also elicit proliferation of lymph vessels. Lymph vessels facilitate migration of antigen-presenting cells to regional lymph nodes in which they induce alloimmune responses. The presence of blood vessels in the corneal graft bed creates a unique chemokine milieu that stimulates recruitment of sensitized lymphocytes into the corneal allograft. Other data indicate that although corneal allograft survival is closely associated with Foxp3 expression in CD4+CD25+Foxp3+ T regulatory cells (Tregs), reduced expression of Foxp3 in Tregs creates a high risk for graft rejection. Recent evidence indicates that allergic diseases have a profound impact on the immune response and produce a dramatic increase in corneal allograft rejection. Understanding the underlying mechanisms that create 'high-risk' hosts may provide important therapeutic targets for restoring immune privilege of corneal allografts and enhancing their survival.

Abstract 5321: Molecular analysis of melanoma-induced sentinel lymph node immune dysfunction

Abstract Introduction Sentinel lymph nodes (SLNs) of melanoma patients show evidence of tumor-induced immune dysfunction. Our previous works have shown that IL10 and IFNγ co-regulate indoleamine-2,3-dioxygenase (IDO)-expressing immunosuppressive dendritic cells (DCs) in melanoma SLNs. The goal of this study is to examine the relationship between melanoma SLN tumor burden and the degree of SLN immune dysfunction as a model to study tumor-induced immune dysfunction. We hypothesize that SLN tumor burden correlates with the degree of SLN immune dysfunction. Methods Patients undergoing SLN biopsy for clinical stages I and II melanomas were enrolled in the study under an IRB-approved protocol. During the SLN biopsy, non-hot and non-blue portion of the SLN was harvested, flash-frozen in liquid nitrogen, and mRNA was extracted. By using quantitative real-time PCR, gene-expressions of cytokines (IL4, IL10, IFNγ, TGFβ, GM-CSF), and the surrogates of immunosuppressive regulatory and effector cells (IDO-expressing DCs and Foxp3-expressing Tregs, respectively) were measured and correlated against the SLN tumor burden (MART1) and against each other. The data were log-transformed for normalization. Statistical test used Students’ t-test and stepwise multivariate regression analysis. Statistical significance was determined at p&amp;lt;0.05. Results Of 200 patients enrolled, SLNs of 74 patients were analyzed in this preliminary analysis. Ten of 74 patients (13.5%) had tumor-positive SLNs. MART1 gene-expression showed a significant difference between the SLN(+) and SLN(−) groups (p=0.04). Among the various cytokines, multivariate analysis showed that only IFNγ gene-expression correlated independently with MART1 gene-expression (p&amp;lt;0.0001, r=0.91). Similar multivariate analyses show that IFNγ (p&amp;lt;0.0001, r=0.78), IL10 (p=0.0037, r=0.60), and TGFβ (p&amp;lt;0.0001, r=0.95) gene-expressions correlated independently with IDO gene-expression. IFNγ (p&amp;lt;0.0001, r=0.87) and GM-CSF (p=0.042, r=0.76) gene-expressions correlated independently with Foxp3 gene-expression. MART1 gene-expression showed independent correlation with IDO (p=0.0002, r=0.75) and Foxp3 (p=0.0002, r=0.75) gene-expressions. Conclusion SLN tumor burden correlates with immunosuppressive IDO and Foxp3 expressions within the SLNs of melanoma patients. Our data are consistent with our theory that melanoma induces expressions of specific cytokines, which in turn, stimulate immune-suppressors within the SLN. This study also supports our previous finding that IL10 and IFNγ co-regulate IDO within the SLN. In our data, IFNγ is the sole cytokine that correlates with the SLN tumor burden and seems to play a central role in tumor-induced immunological changes in the SLN immune microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5321.

Is it necessary to deplete the lymphokine activated killers’ populations of CD4+CD25+ lymphocytes? Regulatory Foxp3-positive T cells within lymphokine activated killers

LAKs are used in cancer therapy. A common argument against LAK therapy is the probability of the activation of Tregs by IL-2 alongside with NK cells. In order to evaluate negative impact of Tregs, contents of Foxp3 +CD4 +CD25 + Tregs and their influence on LAKs’ cytotoxic activity in the samples of healthy volunteers’ PBMCs cultured with or without IL-2 were determined. A marked increase in the proportion of the CD4 +CD25 + T cells in the most of the PBMCs cultured with IL-2 was evident. Surprisingly, the contents of Foxp3 +CD4 +CD25 + Tregs showed variable response to IL-2 in blood samples of different people. Their proportions increased, remained on the same level or even decreased compared to PBMCs cultured without exogenous IL-2. Different methods of Treg isolation were compared regarding the purities of the resulting T-cell populations and their enrichment in the Foxp3-expressing Tregs, as well as their viability. Neither of the kits applied provided any enrichment in Foxp3-level in the purified cells compared to the CD4 +CD25 + T cells gated on applying flow-cytometric cell analysis in the non-separated LAKs. Moreover, the addition of the purified supposedly Tregs to LAKs never inhibited their cytotoxic reactions, even then they were added in great non-physiological excess. In some of the blood samples, there have been detected another non-identified type of the Foxp3-expressing cells, which lacked CD25, as well as CD4. They may be a new type of regulatory cells, which has not been described before.

Effects of epigallocatechin gallate on regulatory T cell number and function in obese v. lean volunteers

Obesity predisposes to an increased incidence of diabetes and CVD. Also, obesity is a pro-inflammatory state. Regulatory T cells (Tregs) are essential negative regulators of inflammation and are down-regulated in pro-inflammatory states. Animal models of obesity are associated with decreased Tregs. The dietary modulation of Tregs could be used as a therapeutic strategy to control inflammation. Epigallocatechin gallate (EGCG) is a potent anti-inflammatory agent and an active ingredient of green tea and is suggested to have a role as a preventive agent in obesity, diabetes and CVD. The role of EGCG in the modulation of Tregs has, however, not been studied. Thus, the aim of the present study was to determine the effect of EGCG on the number and function of Tregs in obese and lean human subjects in vitro, and to delineate its specific regulation mechanisms. Tregs were isolated from normal-weight and obese subjects. Tregs were cultured in the absence or presence of EGCG (20 mum) for 24 h. Foxp3-expressing Tregs were enumerated using flow cytometry. Histone deacetylase (HDAC) activity and nuclear NF-kappaBp65 level were measured by ELISA and Western blots. Obese subjects had lower Tregs and IL-10 production than lean subjects. EGCG treatment significantly enhanced the number of Foxp3-expressing Tregs and IL-10 production in vitro (P < 0.05) in both groups. Also, EGCG decreased NF-kappaB activity and increased HDAC activity and HDAC-2 expression in Tregs (P < 0.05) in both groups. Thus, in part, EGCG enhances the functionality of Tregs, i.e. IL-10 production and number by suppressing the NF-kappaB signalling pathway via inducing epigenetic changes.

Modulating T cell functions does not alleviate chronic inflammatory skin lesions in K5.TGFβ1 transgenic mice

To use mice with chronic hyperproliferative skin inflammation as psoriasis models, their thorough phenotypic and functional characterization is indispensable. Mice with keratin 5 promoter-controlled overexpression of latent human Transforming Growth Factor (TGF)beta1 within the basal epidermis (K5.TGF beta 1 mice) show a psoriasiform phenotype, but the underlying pathogenic mechanisms are not entirely clear. To elucidate the contribution of T lymphocytes to the pathogenesis in K5.TGF beta 1 mice, we used three complementary approaches: first, peripheral T cells were eradicated via systemic treatment with CD3- or CD4-depleting antibodies. However, this elimination did not alleviate the chronic inflammatory disorder. Second, bone marrow transplantation from transgenic mice into wildtype recipients and vice versa resulted in the expected reconstitution of both adaptive and innate immune system but had little effect on the cutaneous phenotype both in wildtype and transgenic chimeras. Third, based on the hypothesis that the disease course could be modulated by regulatory T cells (Tregs), we expanded Tregs in vivo using a superagonistic anti-CD28 antibody. While this treatment achieved a threefold increase in Foxp3-expressing Tregs, there was little, if any, effect on the chronic skin inflammation. We conclude from our findings that T cells play little, if any, role in the skin lesions of K5.TGF beta 1 mice.

Induction of Foxp3-Expressing Regulatory T-Cells by Donor Blood Transfusion Is Required for Tolerance to Rat Liver Allografts

BackgroundDonor-specific blood transfusion (DST) prior to solid organ transplantation has been shown to induce long-term allograft survival in the absence of immunosuppressive therapy. Although the mechanisms underlying DST-induced allograft tolerance are not well defined, there is evidence to suggest DST induces one or more populations of antigen-specific regulatory cells that suppress allograft rejection. However, neither the identity nor the regulatory properties of these tolerogenic lymphocytes have been reported. Therefore, the objective of this study was to define the kinetics, phenotype and suppressive function of the regulatory cells induced by DST alone or in combination with liver allograft transplantation (LTx).Methodology/Principal FindingsTolerance to Dark Agouti (DA; RT1a) rat liver allografts was induced by injection (iv) of 1 ml of heparinized DA blood to naïve Lewis (LEW; RT1l) rats once per week for 4 weeks prior to LTx. We found that preoperative DST alone generates CD4+ T-cells that when transferred into naïve LEW recipients are capable of suppressing DA liver allograft rejection and promoting long-term survival of the graft and recipient. However, these DST-generated T-cells did not express the regulatory T-cell (Treg) transcription factor Foxp3 nor did they suppress alloantigen (DA)-induced activation of LEW T-cells in vitro suggesting that these lymphocytes are not fully functional regulatory Tregs. We did observe that DST+LTx (but not DST alone) induced the time-dependent formation of CD4+Foxp3+ Tregs that potently suppressed alloantigen-induced activation of naïve LEW T-cells in vitro and liver allograft rejection in vivo. Finally, we present data demonstrating that virtually all of the Foxp3-expressing Tregs reside within the CD4+CD45RC− population whereas in which approximately 50% of these Tregs express CD25.Conclusions/SignificanceWe conclude that preoperative DST, in the absence of liver allograft transplantation, induces the formation of CD4+ T-cells that are not themselves Tregs but give rise directly or indirectly to fully functional CD4+CD45RC−Foxp3+Tregs when transferred into MHC mismatched recipients prior to LTx. These Tregs possess potent suppressive activity and are capable of suppressing acute liver allograft rejection. Understanding the mechanisms by which preoperative DST induces the generation of tolerogenic Tregs in the presence of alloantigens may lead to the development of novel antigen-specific immunological therapies for the treatment of solid organ rejection.

Foxp3-expressing T regulatory cells and mast cells in acute graft-versus-host disease of the skin

Acute graft-versus-host disease (aGVHD) limits the effectiveness of allogeneic hematopoietic stem cell transplantation. Foxp3 is required for the development and function of CD4+/CD25+ regulatory T cells (T-regs). Foxp3-expressing T-regs are thought to protect against GVHD. Mast cells are thought to be essential in CD4+/CD25+ regulatory T cell-dependent peripheral tolerance. Twenty biopsies of skin with grades I-III aGVHD were stained for Foxp3 and CD117. Inflammation was quantified by a 4 point scale, 0=no inflammation, 1=50%. T-regs and mast cells were quantified by a 4 point scale, 0=no cells per 20x field, 1=10 cells. T-regs were positively correlated with both inflammation and aGVHD grade. Twelve cases with low T-regs had mild inflammation and lower grades of aGVHD and 6 cases with high T-regs had dense inflammatory infiltrate and higher grades of aGVHD. The number of T-regs, mast cells and density of the inflammatory infiltrate were positively correlated only in cases with mild inflammation. In aGVHD of the skin, T-regs increased with the degree of inflammation and GVHD grade. Mast cells were present at the same density whether aGVHD was of lower or higher grade.

Defects in IL-2R Signaling Contribute to Diminished Maintenance of FOXP3 Expression in CD4+CD25+ Regulatory T-Cells of Type 1 Diabetic Subjects

OBJECTIVEIn humans, multiple genes in the interleukin (IL)-2/IL-2 receptor (IL-2R) pathway are associated with type 1 diabetes. However, no link between IL-2 responsiveness and CD4+CD25+FOXP3+ regulatory T-cells (Tregs) has been demonstrated in type 1 diabetic subjects despite the role of these IL-2–dependent cells in controlling autoimmunity. Here, we address whether altered IL-2 responsiveness impacts persistence of FOXP3 expression in Tregs of type 1 diabetic subjects.RESEARCH DESIGN AND METHODSPersistence of Tregs was assessed by culturing sorted CD4+CD25hi natural Tregs with IL-2 and measuring FOXP3 expression over time by flow cytometry for control and type 1 diabetic populations. The effects of IL-2 on FOXP3 induction were assessed 48 h after activation of CD4+CD25− T-cells with anti-CD3 antibody. Cytokine receptor expression and signaling upon exposure to IL-2, IL-7, and IL-15 were determined by flow cytometry and Western blot analysis.RESULTSMaintenance of FOXP3 expression in CD4+CD25+ Tregs of type 1 diabetic subjects was diminished in the presence of IL-2, but not IL-7. Impaired responsiveness was not linked to altered expression of the IL-2R complex. Instead, IL-2R signaling was reduced in Tregs and total CD4+ T-cells of type 1 diabetic subjects. In some individuals, decreased signal transducer and activator of transcription 5 phosphorylation correlated with significantly higher expression of protein tyrosine phosphatase N2, a negative regulator of IL-2R signaling.CONCLUSIONSAberrant IL-2R signaling in CD4+ T-cells of type 1 diabetic subjects contributes to decreased persistence of FOXP3 expression that may impact establishment of tolerance. These findings suggest novel targets for treatment of type 1 diabetes within the IL-2R pathway and suggest that an altered IL-2R signaling signature may be a biomarker for type 1 diabetes.

T‐cell‐specific deletion of gp130 renders the highly susceptible IL‐10‐deficient mouse resistant to intestinal nematode infection

Gp130 is the common receptor of the IL-6 family of cytokines and is involved in many biological processes, including acute phase response, inflammation and immune reactions. To investigate the role of gp130 under inflammatory conditions, T-cell-specific conditional gp130 mice were first bred to the IL-10-deficient background and were then infected with the gastrointestinal nematode Trichuris muris. While IL-10(-/-) mice were highly susceptible to T. muris, developed a mixed Th1/Th17 response and displayed severe inflammation of the caecum, infection of mice with an additional T-cell-specific deletion of gp130 signalling completely reversed the phenotype. These mice showed an accelerated worm expulsion that was associated with the rapid generation of a strong Th2 immune response and a significant increase in Foxp3-expressing Treg. Therefore, gp130 signalling in T cells regulates a switch between proinflammatory and pathogenic Th1/Th17 cells and regulatory Th2/Treg in vivo. Taken together, the data demonstrate that gp130 signalling in T cells is a positive regulator of inflammatory processes, favouring the Th1/Th17 axis.

Specific central nervous system recruitment of HLA‐G+ regulatory T cells in multiple sclerosis

We have recently described a novel population of natural regulatory T cells (T(reg)) that are characterized by the expression of HLA-G and may be found at sites of tissue inflammation (HLA-G(pos) T(reg)). Here we studied the role of these cells in multiple sclerosis (MS), a prototypic autoimmune inflammatory disorder of the central nervous system (CNS). Sixty-four patients with different types of MS, 9 patients with other neurological diseases, and 20 healthy donors were included in this study. Inflamed brain lesions from 5 additional untreated MS patients were examined. HLA-G(pos) T(reg) were analyzed in the cerebrospinal fluid (CSF) by flow cytometry and in inflammatory demyelinating lesions of MS brain specimens by immunohistochemistry. Functional capacity was accessed and transmigration was determined using an in vitro model of the human blood-brain barrier (BBB). HLA-G(pos) T(reg) were found enriched in the inflamed CSF of MS patients and in inflammatory demyelinating lesions of MS brain specimens. HLA-G(pos) T(reg) showed a strong propensity to transmigrate across BBB, which was vigorously driven by inflammatory chemokines, and associated with a gain of suppressive capacity upon transmigration. CSF-derived HLA-G(pos) T(reg) of MS patients represented a population of activated central memory activated T cells with an upregulated expression of inflammatory chemokine receptors and exhibiting full suppressive capacity. Unlike natural FoxP3-expressing T(reg), HLA-G(pos) T(reg) derived from peripheral blood were functionally unimpaired in MS. In MS, HLA-G(pos) T(reg) may serve to control potentially destructive immune responses directly at the sites of CNS inflammation and to counterbalance inflammation once specifically recruited to the CNS.

CD27 Expression on CD4+ T Cells Differentiates Effector from Regulatory T Cell Subsets in the Lung

Beryllium exposure in the workplace can result in chronic beryllium disease, a granulomatous lung disorder characterized by CD4(+) T cell alveolitis and progressive lung fibrosis. A large number of the CD4(+) T cells recruited to the lung in chronic beryllium disease recognize beryllium in an Ag-specific manner and express Th1-type cytokines following T cell activation. Beryllium-responsive CD4(+) T cells in the bronchoalveolar lavage (BAL) express an effector memory T cell phenotype and recognize beryllium in a CD28-independent manner. In this study, we show that the majority of beryllium-responsive CD4(+) T cells in BAL have lost CD27 expression, whereas a subset of beryllium-responsive cells in blood retains expression of this costimulatory molecule. In addition, loss of CD27 on BAL CD4(+) T cells inversely correlates with markers of lung inflammation. A small population of BAL CD4(+) T cells retains CD27 expression, and these CD4(+)CD27(+) T cells contain the FoxP3-expressing, naturally occurring regulatory T (T(reg)) cell subset. Coexpression of CD27 and CD25 identifies the majority of FoxP3-expressing T(reg) cells in blood and BAL, and these cells express potent suppressor function. Taken together, these findings suggest that CD27 is differentially expressed between effector T cells from the inflamed lung and can be used in conjunction with CD25 to isolate T(reg) cells and assess their functional capacity in an ongoing adaptive immune response in a target organ.

Ex Vivo-Generation of Gut-Homing Regulatory T-Cells: Characterization and Therapeutic Evaluation in Mice With Established Colitis (42.2)

Abstract The objective of this study was to develop an ex vivo method to generate large numbers of Foxp3-expressing Tregs that can be used to evaluate their therapeutic efficacy in a T-cell-dependent model of chronic colitis. We found that polyclonal activation of CD4+CD25- Foxp3- T-cells in the presence of small amounts of IL-2, TGFβ and all trans retinoic acid (RA) converts &amp;gt;90% of these T-cells to Foxp3-expressing Tregs (termed iTregs) as well as induces a 4-5-fold increase in proliferation following a 4 day incubation period in vitro. Furthermore, we found that the presence of RA enhances the surface expression of the gut-homing adhesion molecules α4β7 and CCR9. Finally, we observed that these iTregs are significantly more potent at suppressing T-cell activation in vitro and reversing established colitis in vivo. This protocol routinely generates 30-40 million iTregs from one mouse spleen assuming that the spleen yields approximately 10 million CD4+Foxp3- T-cells and that &amp;gt;90% of these T-cells convert to Foxp3-expressing iTregs as well as undergo a 4-fold proliferation during the 4 day incubation period. We conclude that this protocol generates sufficient numbers of iTregs that can be used in vitro and in vivo to assess the therapeutic efficacy and immunological mechanisms responsible for suppression of inflammation in different models of autoimmune disease (Supported by PO143785).

Induction of Regulatory T-cells by Donor-Specific Blood Transfusion in the Absence or Presence of Liver Allograft Transplantation (141.43)

Abstract Donor-specific blood transfusion (DST) has been shown to be effective at inducing tolerance and long-term survival in animals transplanted with MHC-mismatched tissue. The objective of this study was to ascertain how DST alone or in combination with liver allograft transplantation (LTx) affects the generation and function of regulatory T-cells in vitro and in vivo. We found that DST alone generates CD4+ T-cells that when transferred into naïve recipients suppress liver allograft rejection despite expressing little or no Foxp3 at the time of adoptive transfer. In addition, we present the novel finding that DST plus LTx (but not DST alone) increases the formation of Foxp3-expressing regulatory T-cells (Tregs) that suppress alloantigen-induced activation in vitro as well as induce long term tolerance to liver allografts when adoptively transferred into naïve recipients prior to LTx. Finally we demonstrate that virtually all Foxp3-expressing Tregs reside within the CD4+CD45RC- population whereas these Tregs are equally distributed between the CD4+CD25+ and CD4+CD25- populations. Taken together, our data suggest that DST administration, in the absence of allograft transplantation, induces the formation of CD4+ T-cells that are not Tregs themselves but develop into fully functional Foxp3+ Tregs or help to induce the formation of Foxp3+ Tregs following allograft transplantation.

Paclitaxel reduces regulatory T cell numbers and inhibitory function and enhances the anti-tumor effects of the TLR9 agonist PF-3512676 in the mouse.

The anti-tumor properties of Toll-like receptor (TLR) 9 agonist CpG oligodeoxynucleotides (ODN) are enhanced by combinations with several cytotoxic chemotherapy regimens. The mechanisms of this added benefit, however, remain unclear. We now report that, similar to the depletion of regulatory T cells (Treg) using anti-CD25, paclitaxel increased the anti-tumor effect of the TLR9 agonist PF-3512676 in a CD8(+) T cell-dependent fashion. Paclitaxel treatment decreased Treg numbers in a TLR4-independent fashion, and preferentially affected cycling Treg expressing high levels of FoxP3. The paclitaxel-induced reduction in Treg FoxP3 expression was associated with reduced inhibitory function. Adoptively transferred tumor-antigen specific CD8(+) T cells proliferated better in mice treated with paclitaxel and their recruitment in the tumor was increased. However, the systemic frequency of PF-3512676-induced tumor-antigen specific effector CD8(+) T cells decreased with paclitaxel, suggesting opposite effects of paclitaxel on the anti-tumor response. Finally, gene expression profiling and studies of tumor-associated immune cells revealed a complex modulation of the PF-3512676-induced immune response by paclitaxel, including a decrease of IL-10 expression and an increase in IL-17-secreting CD4(+) T cells. Collectively, these data suggest that paclitaxel combined with PF-3512676 may not only promote a better anti-tumor CD8(+) response though increased recruitment in the tumor, possibly through Treg depletion and suppression, but also exerts more complex immune modulatory effects.

TGF-β and Regulatory T Cell in Immunity and Autoimmunity

The immune response is controlled by several inhibitory mechanisms. These mechanisms include regulatory T cells, which exist in multiple classes. Notable among these are Foxp3-expressing regulatory T cells (Treg), NKT cells, and Tr1 cells. Common to these mechanisms are inhibitory cytokines such as interleukin-10 and transforming growth factor-beta (TGF-beta). TGF-beta and Foxp3-expressing Treg cells are critical in maintaining self-tolerance and immune homeostasis. The immune suppressive functions of TGF-beta and Treg cells are widely acknowledged and extensively studied. Nonetheless, recent studies revealed the positive roles for TGF-beta and Treg cells in shaping the immune system and the inflammatory responses. In this paper, we will discuss the role of these mechanisms in the control of immunity and autoimmunity and the mechanisms that underlie how these molecules control these responses.