CD4+CD25+ T regulatory cells control anti-islet CD8+ T cells through TGF-beta-TGF-beta receptor interactions in type 1 diabetes

Pancreatic lymph node-derived CD4+CD25+ T regulatory (Treg) cells inhibit in situ differentiation of islet-reactive CD8+ T cells into cytotoxic T lymphocytes, thereby preventing diabetes progression. The mechanism by which these Treg cells suppress anti-islet CD8+ T cells is unknown. Here, we show by using a CD8+ T cell-mediated model of type 1 diabetes that transforming growth factor (TGF)-beta-TGF-beta receptor signals are critical for CD4+CD25+ Treg cell regulation of autoreactive islet-specific cytotoxic T lymphocytes. Transgenic expression of tumor necrosis factor alpha from birth to 25 days of age in the islets of B6 mice that constitutively express CD80 on their beta cells results in accumulation of CD4+CD25+TGF-beta+ cells exclusively in the islets and pancreatic lymph nodes, which delays diabetes progression. In contrast, expression of tumor necrosis factor alpha until 28 days of age prevents islet accumulation of CD4+CD25+TGF-beta+ Treg cells, resulting in acceleration to diabetes. Furthermore, adoptive transfer experiments demonstrated that CD4+CD25+ Treg cells could not control na?ve or activated islet-reactive CD8+ T cells bearing a dominant negative TGF-beta receptor type II. Our data demonstrate that, in vivo, TGF-beta signaling in CD8+ T cells is critical for CD4+CD25+ Treg cell suppression of islet-reactive CD8+ T cells in type 1 diabetes.     

IL-10-dependent infectious tolerance after the treatment of experimental allergic encephalomyelitis with redirected CD4+CD25+ T lymphocytes

How small numbers of CD4+CD25+ regulatory T cells suppress autoimmune responses in vivo is unclear. In this report we analyze the immunomodulatory activity of CD4+CD25+ T cells that are antigen-specifically redirected against myelin basic protein (MBP)89-101-specific autoreactive T cells by a MBP89-101-IA(s)-zeta chimeric receptor. We have previously shown that these redirected regulatory T cells are highly potent in treating a model autoimmune disease, experimental allergic encephalomyelitis. We show here that they have only limited effect in vivo on autoreactive T cell proliferation and therefore do not act by deleting or suppressing the expansion of pathologic effector cells. Rather, the redirected CD4+CD25+ T cells divert the pathologic T helper 1 self-specific T cell response to one characterized by high IL-10 and lower IL-4 production. Significantly, when isolated from the inducing CD4+CD25+ regulatory T cells, these self-specific T cells can independently suppress the autoreactive T cell response and experimental allergic encephalomyelitis development in an IL-10-dependent manner. These results provide evidence that CD4+CD25+ regulatory T cells can manipulate the adaptive immune response in vivo through the infectious induction of tolerance, specifically by promoting the formation of antigen-specific, IL-10-secreting regulatory T cells.     

Dendritic cells with TGF-beta1 differentiate naive CD4+CD25- T cells into islet-protective Foxp3+ regulatory T cells

CD4(+)CD25(+)Foxp3(+) regulatory T cells (T regs) are important for preventing autoimmune diabetes and are either thymic-derived (natural) or differentiated in the periphery outside the thymus (induced). Here we show that beta-cell peptide-pulsed dendritic cells (DCs) from nonobese diabetic (NOD) mice can effectively induce CD4(+)CD25(+)Foxp3(+) T cells from na?ve islet-specific CD4(+)CD25(-) T cells in the presence of TGF-beta1. These induced, antigen-specific T regs maintain high levels of clonotype-specific T cell receptor expression and exert islet-specific suppression in vitro. When cotransferred with diabetogenic cells into NOD scid recipients, T regs induced with DCs and TGF-beta1 prevent the development of diabetes. Furthermore, in overtly NOD mice, these cells are able to significantly protect syngeneic islet grafts from established destructive autoimmunity. These results indicate a role for DCs in the induction of antigen-specific CD4(+)CD25(+)Foxp3(+) T cells that can inhibit fully developed autoimmunity in a nonlymphopoenic host, providing an important potential strategy for immunotherapy in patients with autoimmune diabetes.     

Diversity of regulatory CD4+T cells controlling distinct organ-specific autoimmune diseases

Depletion of selected regulatory CD4+ T cell subsets induces the spontaneous onset of various immune or autoimmune disorders. It is not clear, however, whether a given subset, notably CD4+CD25+ regulatory T cells, protects from a wide spectrum of immune disorders, or whether specialized subsets of regulatory T cells control each given disease or group of diseases. We report here, using diabetes prone nonobese diabetic (NOD) mice, that depending on the regulatory T cells that are depleted, i.e., CD25+, CD62L+, or CD45RB(low), distinct immune diseases appear after transfer into NOD severe combined immunodeficiency (SCID) recipients. Thus, reconstitution of NOD SCID mice with CD25- T cells induces major gastritis and late-onset diabetes, but no or mild colitis. Reconstitution with CD62L- T cells induces fulminant diabetes with no colitis or gastritis. Reconstitution with CD45RB(high) T cells induces major colitis with wasting disease and no or very moderate gastritis and diabetes. Major differences among the three regulatory T cell subsets are also seen in vitro. The bulk of suppressor cells inhibiting the proliferation of CD4+CD25- T cells in coculture is concentrated within the CD25+ but not the CD62L+ or CD45RB(low) T cell subsets. Similarly, cytokine production patterns are significantly different for each regulatory T cell subset. Collectively, these data point to the diversity and organ selectivity of regulatory T cells controlling distinct autoimmune diseases whatever the underlying mechanisms.     

IL-10 and TGF-beta induce alloreactive CD4+CD25- T cells to acquire regulatory cell function

We previously reported that interleukin-10 (IL-10) and transforming growth factor (TGF)-beta treatment of primary mixed lymphocyte reaction (MLR) cultures resulted in secondary alloantigen-specific hyporesponsiveness and protection from graft-versus-host disease (GVHD) lethality. Here, we report that CD4+ T cells recovered from the IL-10- and TGF-beta-treated primary MLR cultures have immunoregulatory function. Tolerized cells significantly inhibited proliferation of naive alloreactive CD4+ T cells in a primary MLR. Inhibition of the naive alloresponse was observed with as few as 1 tolerized cell to 10 naive responder cells. Tolerized cells were able to significantly reduce GVHD lethality when injected with naive alloreactive CD4+ T cells into major histocombatibility class (MHC) II disparate recipients. Rigorous CD25 depletion of the primary MLR had no effect on generation of a regulatory capacity, suggesting that the regulatory cells likely originated from CD4+CD25- T cells. Immune suppression was mediated independently of IL-10 and TGF-beta production, as neutralizing antibodies for IL-10, IL-10R, and TGF-beta were unable to revert suppression, and IL-10- deficient CD4+ T cells were able to mediate in vitro and in vivo suppression. The generation of immunoregulatory cells from a CD4+CD25- population during tolerization with IL-10 and TGF-beta provides an additional mechanism to prevent GVHD lethality by T cells that may escape full tolerance induction.     

Vasoactive intestinal peptide induces CD4+,CD25+ T regulatory cells with therapeutic effect in collagen-induced arthritis

OBJECTIVE: CD4+,CD25+ T regulatory cells (Treg) control the immune response to a variety of antigens, including self antigens, and may offer opportunities to intervene in the course of autoimmune diseases. Several models support the idea of the peripheral generation of CD4+,CD25+ Treg from CD4+,CD25- T cells, but little is known about the endogenous factors and mechanisms controlling the peripheral expansion of CD4+,CD25+ Treg. We undertook this study to investigate the capacity of the vasoactive intestinal peptide (VIP), an immunosuppressive antiarthritic neuropeptide, to induce functional Treg in vivo during the development of collagen-induced arthritis (CIA). METHODS: We measured the number of CD4+,CD25+ Treg following VIP administration to CIA mice, and we characterized their phenotype and their ability to suppress activation of autoreactive T cells. We determined the capacity of VIP to induce Treg in vitro as well as the use of Treg in the treatment of CIA, measuring the clinical evolution and the inflammatory and autoimmune components of the disease. RESULTS: The administration of VIP to arthritic mice resulted in the expansion of CD4+,CD25+,Foxp3+ Treg in the periphery and joints, which inhibited autoreactive T cell activation/expansion. VIP induced more efficient suppressors on a per-cell basis. The VIP-generated CD4+,CD25+ Treg transfer suppressed and significantly ameliorated the progression of the disease. CONCLUSION: These results demonstrate the involvement of the generation of Treg in the therapeutic effect of VIP on CIA. The generation of highly efficient Treg by VIP ex vivo could be used as an attractive therapeutic tool in the future, avoiding the administration of the peptide to patients with rheumatoid arthritis.     

Transforming growth factor beta induced FoxP3+ regulatory T cells suppress Th1 mediated experimental colitis

BACKGROUND AND AIMS: The imbalance between effector and regulatory T cells plays a central role in the pathogenesis of inflammatory bowel diseases. In addition to the thymus, CD4+CD25+ regulatory T cells can be induced in the periphery from a population of CD25- T cells by treatment with transforming growth factor beta (TGF-beta). Here, we analysed the in vivo function of TGF-beta induced regulatory T (Ti-Treg) cells in experimental colitis. METHODS: Ti-Treg cells were generated in cell culture in the presence or absence of TGF-beta and tested for their regulatory potential in experimental colitis using the CD4+CD62L+ T cell transfer model. RESULTS: Ti-Treg cells significantly suppressed Th1 mediated colitis on CD4+CD62L+ T cell transfer in vivo, as shown by high resolution endoscopy, histology, immunohistochemistry, and cytokine analysis. Further analysis of in vivo and in vitro expanded Ti-Treg cells showed that exogenous interleukin 2 (IL-2) was crucial for survival and expansion of these cells. CONCLUSION: Our data suggest that regulatory Ti-Treg cells expand by TGF-beta and exogenous IL-2 derived from effector T cells at the site of inflammation. In addition to Tr1 and thymic CD4+CD25+ T cells, peripheral Ti-Treg cells emerge as a class of regulatory T cells with therapeutic potential in T cell mediated chronic intestinal inflammation.     

Suppression of myasthenogenic responses of a T cell line by a dual altered peptide ligand by induction of CD4+CD25+ regulatory cells

Myasthenia gravis is a T cell-dependent, antibody-mediated autoimmune disease. A dual altered peptide ligand (APL) that is composed of the tandemly arranged two single amino acid analogs of two myasthenogenic peptides, p195-212 and p259-271, was demonstrated to down-regulate in vitro and in vivo myasthenia gravis-associated autoreactive responses. The aims of this study were to demonstrate the suppressive properties and to elucidate the mechanism of action of the dual APL on a T cell line specific to the myasthenogenic peptide p195-212. We demonstrate here that incubation of cells of the line with the dual APL resulted in the inhibition of proliferation and secretion of IL-2 and IFN-gamma triggered by p195-212. In contrast, secretion of TGF-beta and IL-10 was upregulated. The dual APL induced the generation of CD4+CD25+ cells that were characterized by the expression of CD45Rb(low), cytotoxic T lymphocyte-associated antigen-4, TGF-beta, CD62L, Foxp3, and neuropilin. In addition, the dual APL-treated cells were capable of inhibiting the proliferation response of the line when the two sets of cells were cocultured. The role of CD4+CD25+ cells was further confirmed by demonstrating that the suppression was abrogated by blocking/neutralization of CD25. Thus, the dual APL acts by inducing the formation of CD4+CD25+ regulatory cells. By using a T cell line, we could show that the immunosuppressive CD4+CD25+ cells were indeed induced by the dual APL and are not part of the naturally occurring regulatory cells.     

CD4+ CD25+ 调节性T细胞与自身免疫性甲状腺疾病

CD4+ CD25+ 调节性T细胞为新近发现的一群功能成熟的T细胞亚群.其特征性表达叉头盒蛋白3(Foxp3)分子,专职免疫无能和免疫抑制,在维持外周免疫耐受,防止自身免疫性疾病发病中起着极为关键的作用.CD4+ CD25+ 调节性T细胞在自身免疫性甲状腺疾病(AITD)发病中的作用引起了人们的关注.动物实验发现CD4+ CD25+ 调节性T细胞存在与否决定了实验动物是否发生实验性自身免疫性甲状腺炎(EAT)和Graves病.人体研究发现CD4+ CD25+ 调节性T细胞数目和功能异常与人AITD发生密切相关.这些研究结果提示,CD4+ CD25+ 调节性T细胞可能在AITD发病中起重要作用.     

Induction of allopeptide-specific human CD4+CD25+ regulatory T cells ex vivo

Although CD4+CD25+ regulatory T cells are pivotal in the prevention of autoimmunity and appear to mediate transplantation tolerance, little is known concerning their antigen specificity. Here we describe the induction of a human CD4+CD25+ regulatory T-cell line specific for a defined peptide alloantigen (human leukocyte antigen A2 [HLA-A2] 138-170) by priming purified CD4+CD25+ cells ex vivo. The regulatory cells were anergic and retained their ability to suppress antigen-driven responses of CD4+CD25- cells. They inhibited not only interleukin 2 (IL-2) secretion by CD4+CD25- T cells specific for the same peptide but also direct alloresponse of naive CD4+CD25- T cells stimulated by semiallogeneic dendritic cells (DCs) in the presence of the peptide ("linked suppression"). They also suppressed the response of CD4+ T cells specific for viral and bacterial antigens. The suppressive T-cell line showed sustained high CD25 expression. These findings suggest that peripheral CD4+CD25+ regulatory cells are a precommitted cell lineage from which cells with specificity for non-self-peptides can be selected. This may pave the way for inducing and expanding peptide antigen-specific regulatory T cells ex vivo for cell therapy in transplantation, allergy, and autoimmune disease.     

Circulating CD4+CD25+ Regulatory T Cells in the Pathobiology of Ulcerative Colitis and Concurrent Primary Sclerosing Cholangitis

Background

Immunopathogenetic features of primary sclerosing cholangitis (PSC) in ulcerative colitis (UC) still remains unclear. Peripheral blood CD4+CD25+ regulatory T cells have a key role in the induction and maintenance of peripheral self-tolerance and inhibit several organ-specific autoimmune diseases. Therefore, CD4+CD25+ T cells are believed to play an essential role in autoimmune diseases. The aim of the present study is to analyze the role of CD4+CD25+ T cells in the pathogenesis of UC-associated PSC.

Methods

This study evaluated the levels of CD4+CD25+ T cells in peripheral blood mononuclear cells (PBMC) of 27 UC patients with PSC and 20 UC patients as controls. CD4+CD25+ T cells were isolated from PBMC with a direct immunofluorescence technique, using mice monoclonal antibodies namely FITC-labeled anti-CD4 and PE-labeled anti-CD25. In each patient, CD4+CD25+ T cells percentage in PBMC were studied by flow cytometry, and then the number of CD4+CD25+ T cells were calculated.

Results

Twenty-seven UC patients with PSC and 20 UC patients without PSC as controls were enrolled in the present study. The percentage of CD4+CD25+ regulatory T cells among PBMC were significantly elevated in UC + PSC patients compared with UC patients without PSC (p = 0.04).

Conclusions

CD4+CD25+ T cells were found to be elevated in UC patients with PSC suggesting a partial role of activated T cell response in the disease pathophysiology. Our findings imply that CD4+CD25+ regulatory T cells may play a key role in the immunopathogenesis of UC-associated PSC and may affect the therapeutic management of these diseases.     

CD4+CD25+ cell depletion from the normal CD4+ T cell pool prevents tolerance toward the intestinal flora and leads to chronic colitis in immunodeficient mice

BACKGROUND: CD4+CD25+ regulatory T cells have been shown to prevent immune-mediated colitis in mice; however, it is unclear whether the absence of CD4+CD25+ in the normal CD4+ T cell pool is responsible for the development of chronic colitis. Using the T cell-deficient Tgepsilon26 mouse model, we show that CD4+CD25- cells but not CD4+CD25+ cells induce a severe intestinal inflammation. Transfer of CD4+CD25+ cells, together with CD4+CD25- cells, ameliorated intestinal inflammation, and reconstitution with the whole mesenteric lymph node cell pool did not induce colitis in recipients. Transferred CD4+CD25- cells were found mainly in the mesenteric lymph nodes, where they showed an activated TH1-like phenotype. In the absence of regulatory CD4+CD25+ T cells, recipient CD4 cells secreted IFN-gamma in response to stimulation with intestinal bacterial antigen that was prevented in vivo and in vitro by regulatory CD4+CD25+ cells. These studies suggest that CD4+CD25- cells have a strong colitogenic effect in the Tgepsilon26 colitis model and that CD4+CD25+ cells may be the main regulators that prevent or downregulate the proinflammatory effect of colitogenic T cells in the Tgepsilon26 mouse model.     

自身免疫性甲状腺疾病和CD4~+CD25~+调节性T细胞

自身免疫性甲状腺疾病(AITD)的发生及发展与CD4~+CD25~+调节性T细胞(Treg细胞)的数量和功能密切相关.动物实验证明Treg细胞可抑制AITD的发生.如果清除动物体内的该类细胞,可导致AITD发病或使原有的甲状腺疾病加重,Treg细胞通过抑制效应性T细胞的激活而发挥对AITD的影响作用.无论是胸腺还是外周,不同诱导体系来源的Treg细胞均对AITD有影响作用.     

自身免疫性甲状腺疾病和CD4~+CD25~+调节性T细胞

自身免疫性甲状腺疾病(AITD)的发生及发展与CD4~+CD25~+调节性T细胞(Treg细胞)的数量和功能密切相关.动物实验证明Treg细胞可抑制AITD的发生.如果清除动物体内的该类细胞,可导致AITD发病或使原有的甲状腺疾病加重,Treg细胞通过抑制效应性T细胞的激活而发挥对AITD的影响作用.无论是胸腺还是外周,不同诱导体系来源的Treg细胞均对AITD有影响作用.     

CD28 disruption exacerbates inflammation in Tgf-beta1-/- mice: in vivo suppression by CD4+CD25+ regulatory T cells independent of autocrine TGF-beta1

Tgf-beta1-/- mice develop a progressive, lethal, inflammatory syndrome, but mechanisms leading to the spontaneous activation of Tgf-beta1-/- T cells remain unclear. Here we show the disruption of CD28 gene expression accelerates disease in Tgf-beta1-/- mice, and we link this increase in severity to a reduction in the number of CD4+CD25+ regulatory T cells. CD4+CD25+ T cells develop normally in Tgf-beta1-/- mice and display characteristic expression of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), alpha(E)beta7 integrin, and Foxp3. Adoptive transfer of Tgf-beta1-/- splenocytes to Tgf-beta1+/+/Rag2-/- mice induced an autoimmune inflammatory disease with features similar to those of the Tgf-beta1-/- phenotype, and disease transfer was accelerated by the depletion of Tgf-beta1-/- CD4+CD25+ T cells from donor splenocytes. Cotransfer of Tgf- beta1-/- CD4+CD25+ T cells clearly attenuated disease in Rag2-/- recipients of CD25+-depleted Tgf-beta1-/- spleen and lymph node cells, but suppression was incomplete when compared with Tgf-beta1+/+ CD4+CD25+ T cells. These data demonstrate that CD4+CD25+ regulatory T cells develop in complete absence of endogenous transforming growth factor-beta1 (TGF-beta1) expression and that autocrine TGF-beta1 expression is not essential for these cells to suppress inflammation in vivo.     

自身免疫性甲状腺疾病和CD4~+CD25~+调节性T细胞

自身免疫性甲状腺疾病(AITD)的发生及发展与CD4~+CD25~+调节性T细胞(Treg细胞)的数量和功能密切相关.动物实验证明Treg细胞可抑制AITD的发生.如果清除动物体内的该类细胞,可导致AITD发病或使原有的甲状腺疾病加重,Treg细胞通过抑制效应性T细胞的激活而发挥对AITD的影响作用.无论是胸腺还是外周,不同诱导体系来源的Treg细胞均对AITD有影响作用.     

Functional role of CD4+CD25+ regulatory T cells and transforming growth factor-beta1 in childhood immune thrombocytopenic purpura

CD4+CD25+ regulatory T cells (Tregs) are critical in maintaining self tolerance and preventing organ specific autoimmune diseases. Their role in childhood immune thrombocytopenic purpura (ITP), an immune disorder in which the production of platelet autoantibodies might be caused by cytokine network dysregulation, has not been clearly defined. Transforming Growth Factor-beta1 (TGF-beta1) has been suggested to mediate Tregs suppressive function. The aim of this study was to assess cell populations of CD4+CD25+ T regulatory cells as well as mRNA expression of TGF-beta1 level in peripheral blood of children with ITP and evaluate their possible role in prediction of disease severity and response to therapy. Thirty-three children with ITP (13 acute and 20 chronic cases) and 10 healthy controls were studied. CD4+CD25+ regulatory T cells were assessed in peripheral blood by flowcytometry. Expression of TGF-beta1 mRNA was examined by real-time quantitative polymerase chain reaction assay. The frequency of CD4+CD25+ Tregs was significantly decreased in acute and chronic ITP patients as compared to controls. Acute ITP patients had significantly lower percentage of Tregs compared with chronic ITP patients. Higher frequency of CD4+CD25+ Tregs was detected in chronic ITP patients with platelet count >100 x 10(9)/L compared with patients with platelet count <100 x 10(9)/L and in steroid responsive patients compared with steroid resistant patients. The expression of mRNA TGF-beta1 level was significantly lower in acute and chronic ITP patients compared with controls and in chronic ITP patients with pltc <100 x 10(9)/L in comparison with patients with pltc > 100 x l0(9)/L. A significant positive correlation was found between percentage of CD4+CD25+ Tregs and mRNA expression of TGF-beta1 in chronic ITP patients. In conclusion, immune regulation of TGF-beta1 by Tregs may play a fundamental role in the pathogenesis of childhood immune thrombocytopenic purpura. This might form a base for future specific immunomodulatory therapies for ITP.     

Vasoactive intestinal peptide induces CD4+,CD25+ T regulatory cells with therapeutic effect in collagen‐induced arthritis

Objective

CD4+,CD25+ T regulatory cells (Treg) control the immune response to a variety of antigens, including self antigens, and may offer opportunities to intervene in the course of autoimmune diseases. Several models support the idea of the peripheral generation of CD4+,CD25+ Treg from CD4+,CD25− T cells, but little is known about the endogenous factors and mechanisms controlling the peripheral expansion of CD4+,CD25+ Treg. We undertook this study to investigate the capacity of the vasoactive intestinal peptide (VIP), an immunosuppressive antiarthritic neuropeptide, to induce functional Treg in vivo during the development of collagen‐induced arthritis (CIA).

Methods

We measured the number of CD4+,CD25+ Treg following VIP administration to CIA mice, and we characterized their phenotype and their ability to suppress activation of autoreactive T cells. We determined the capacity of VIP to induce Treg in vitro as well as the use of Treg in the treatment of CIA, measuring the clinical evolution and the inflammatory and autoimmune components of the disease.

Results

The administration of VIP to arthritic mice resulted in the expansion of CD4+,CD25+,Foxp3+ Treg in the periphery and joints, which inhibited autoreactive T cell activation/expansion. VIP induced more efficient suppressors on a per‐cell basis. The VIP‐generated CD4+,CD25+ Treg transfer suppressed and significantly ameliorated the progression of the disease.

Conclusion

These results demonstrate the involvement of the generation of Treg in the therapeutic effect of VIP on CIA. The generation of highly efficient Treg by VIP ex vivo could be used as an attractive therapeutic tool in the future, avoiding the administration of the peptide to patients with rheumatoid arthritis.

     

Regulatory T cells in human autoimmune thyroid disease

CONTEXT: T regulatory cells have a key role in the pathogenesis of autoimmune diseases in different animal models. However, less information is available regarding these cells in human autoimmune thyroid diseases (AITD). OBJECTIVE: The objective of the study was to analyze different regulatory T cell subsets in patients with AITD. DESIGN: We studied by flow cytometry and immunohistochemistry different T regulatory cell subsets in peripheral blood mononuclear cells (PBMCs) and thyroid cell infiltrates from 20 patients with AITD. In addition, the function of T(REG) lymphocytes was assessed by cell proliferation assays. Finally, TGF-beta mRNA in thyroid tissue and its in vitro synthesis by thyroid mononuclear cells (TMCs) was determined by RNase protection assay and quantitative PCR. RESULTS: PBMCs from AITD patients showed an increased percent of CD4+ lymphocytes expressing glucocorticoid-induced TNF receptor (GITR), Foxp3, IL-10, TGF-beta, and CD69 as well as CD69+CD25(bright), CD69+TGF-beta, and CD69+IL-10+ cells, compared with controls. TMCs from these patients showed an increased proportion of CD4+GITR+, CD4+CD69+, and CD69+ cells expressing CD25(bright), GITR, and Foxp3, compared with autologous PBMCs. Furthermore, a prominent infiltration of thyroid tissue by CD69+, CD25+, and GITR+ cells, with moderate levels of Foxp3+ lymphocytes, was observed. The suppressive function of peripheral blood T(REG) cells was defective in AITD patients. Finally, increased levels of TGF-beta mRNA were found in thyroid tissue, and thyroid cell infiltrates synthesized in vitro significant levels of TGF-beta upon stimulation through CD69. CONCLUSIONS: Although T regulatory cells are abundant in inflamed thyroid tissue, they are apparently unable, in most cases, to downmodulate the autoimmune response and the tissue damage seen in AITD.     

Foxp3+ CD25- CD4 T cells constitute a reservoir of committed regulatory cells that regain CD25 expression upon homeostatic expansion

Expression of the IL-2 receptor alpha chain (CD25) by peripheral CD4 T cells follows cellular activation. However, CD25 expression by CD4 cells is widely used as a marker to identify regulatory T cells (T(R)), although cells with regulatory properties are also found in the CD4+CD25- subset. By using in vivo functional assays and Foxp3 expression as a faithful marker of T(R) differentiation, we have evaluated the requirements for CD25 expression by peripheral T(R). We first show that in vivo depletion of CD25+ cells prevents the development of spontaneous encephalomyelitis in recombination-activating gene (RAG)-deficient anti-myelin basic protein T cell antigen receptor (TCR) transgenic mice, and allows disease induction in otherwise healthy RAG-competent transgenic mice. Similar treatment in normal thymectomized animals is followed by the fast recovery of a normal number of CD25+ T(R). Consistently, Foxp3-expressing T(R) encompassed in the CD25- cell population convert to CD25+ after homeostatic expansion and are selectable by IL-2 in vitro. Surface expression of CD25 on T(R) is controlled by the activity of conventional CD4 cells and is fully labile because it can be lost and regained without affecting the functional potential of the cells. These findings reveal that Foxp3-expressing CD25- cells constitute a peripheral reservoir of differentiated T(R), recruited to the CD25+ pool upon homeostatic expansion and/or activation. This analysis, together with the notion that physiological commitment of T(R) takes place exclusively in the thymus should help for the interpretation of experiments assessing peripheral T(R) differentiation from naive CD4 T cells, defined as CD25-.