The different effects of indirubin on effector and CD4+CD25+ regulatory T cells in mice: potential implication for the treatment of autoimmune diseases

CD4(+)CD25(+) regulatory T (Treg) cells play an essential role in the induction and maintenance of peripheral self-tolerance. Indirubin, a traditional Chinese medicine, was clinically used in the treatment of chronic myelocytic leukemia as well as some autoimmune diseases, including Alzheimer's disease, diabetes, and so on. The effects of indirubin on CD4(+)CD25(+)Treg cells, which play a critical role in controlling autoimmunity, have not been addressed. In the present study, we observed the cell levels, phenotypes, and immunoregulatory function of CD4(+)CD25(+)Treg cells in indirubin-treated mice. Treatment with indirubin significantly enhanced the ratios of CD4(+)CD25(+)Treg cells or CD4(+)CD25(+)Foxp3(+)Treg cells to CD4(+)T cells in peripheral blood, lymph nodes, and spleens (P < 0.01 compared with control mice). CD4(+)CD25(+)Foxp3(+)Treg cells to CD4 single positive cells in the thymi of indirubin-treated mice were significantly higher than those in control mice. Furthermore, splenic CD4(+)CD25(+)Treg cells in indirubin-treated mice showed immunosuppressive ability on the immune response of T effector cells to alloantigens or mitogen as efficiently as the control CD4(+)CD25(+)Treg cells in vitro. The present studies indicate that CD4(+)CD25(+)Treg cells are more resistant to indirubin than effector T cells in vivo. The selectively enhanced CD4(+)CD25(+)Treg cell levels by indirubin made host to be more favorable for immune tolerance induction, which opened one possibility for indirubin to treat autoimmune diseases.     

Age-related changes in the occurrence and characteristics of thymic CD4(+) CD25(+) T cells in mice

Natural regulatory CD4(+) CD25(+) T cells play an important role in preventing autoimmunity by maintaining self-tolerance. They express CD25 constitutively and are produced in the thymus as a functionally mature T-cell population. Changes in the potential of these cells to regulate the activity of conventional effector lymphocytes may contribute to an increased susceptibility to infection, cancer and age-associated autoimmune diseases. In this study we demonstrated that the thymi of aged mice are populated by a higher percentage of CD4(+) CD25(+) thymocytes than in young animals. The expression of several surface markers (CD69, CD5, CD28, CTLA-4, CD122, FOXP3), usually used to characterize the phenotype of CD4(+) CD25(+) T regulatory cells, was compared between young and aged mice. We also examined the ability of sorted thymus-deriving regulatory T cells of young and aged BALB/c mice to inhibit the proliferation of lymph node lymphocytes activated in vitro. Natural regulatory T cells isolated from the thymi of young mice suppress the proliferation of responder lymph node cells. We demonstrated that thymus-deriving CD4(+) CD25(+) T cells of old mice maintain their potential to suppress the proliferation of activated responder lymphocytes of young mice. However, their potential to inhibit the proliferation of old responder T cells is abrogated. Differences in the occurrence and activity of CD4(+) CD25(+) thymocytes between young and old animals are discussed in relation to the expression of these surface markers.     

Origin and T cell receptor diversity of Foxp3+CD4+CD25+ T cells

Foxp3(+)CD4(+)CD25(+) regulatory T cells can differentiate from Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) naive T cells. However, the impact of these two processes on size and composition of the peripheral repertoire of regulatory T cells is unclear. Here we followed the fate of individual Foxp3(+)CD4(+)CD25(+) thymocytes and T cells in vivo in T cell receptor (TCR) transgenic mice that express a restricted but polyclonal repertoire of TCRs. By utilizing high-throughput single-cell analysis, we showed that Foxp3(+)CD4(+) peripheral T cells were derived from thymic precursors that expressed a different TCRs than Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) T cells. Furthermore, the diversity of TCRs on Foxp3(+)CD4(+) regulatory T cells exceeded the diversity of TCRs on Foxp3(-)CD4(+) naive T cells, even in mice that lack expression of tissue-specific antigens. Our results imply that higher TCR diversity on Foxp3(+) regulatory T cells helps these cells to match the specificities of autoreactive and naive T cells.     

Expansion and activation of CD4(+)CD25(+) regulatory T cells in Heligmosomoides polygyrus infection

Regulatory T cell responses to infectious organisms influence not only immunity and immunopathology, but also responses to bystander antigens. Mice infected with the gastrointestinal nematode parasite Heligmosomoides polygyrus show an early Th2-dominated immune response (days 7-14), but by day 28 a strongly regulatory profile is evident with antigen-specific IL-10 release and elevated frequency of CD4(+) T cells bearing surface TGF-beta. CD4(+)CD25(+) T cells from infected mice show enhanced capacity to block in vitro effector T cell proliferation. CD4(+)CD25(+) cell numbers expand dramatically during infection, with parallel growth of both CD25(+)Foxp3(+) and CD25(+)Foxp3(-) subsets. CTLA-4 and glucocorticoid-induced tolerance-associated receptor, also associated with regulatory T cell function, become more prominent, due to both expanded CD25(+) cell numbers and increased expression among the CD25(-) population. Both intensity and frequency of CD103 expression by CD4(+) T cells rise significantly, with greatest expansion among CD25(+)Foxp3(+) cells. While TGF-beta expression is observed among both CD25(+)Foxp3(+) and CD25(+)Foxp3(-) subsets, it is the latter population which shows higher TGF-beta staining following infection. These data demonstrate in a chronic helminth infection that Foxp3(+) regulatory T cells are stimulated, increasing CD103 expression in particular, but that significant changes occur to other populations including expansion of CD25(+)TGF-beta(+)Foxp3(-) cells, and induction of CTLA-4 on CD25(-) non-regulatory lymphocytes.     

IL-10 is involved in the suppression of experimental autoimmune encephalomyelitis by CD25+CD4+ regulatory T cells

CD25(+)CD4(+) regulatory T cells inhibit the activation of autoreactive T cells in vitro and in vivo, and suppress organ-specific autoimmune diseases. The mechanism of CD25(+)CD4(+) T cells in the regulation of experimental autoimmune encephalomyelitis (EAE) is poorly understood. To assess the role of CD25(+)CD4(+) T cells in EAE, SJL mice were immunized with myelin proteolipid protein (PLP)(139-151) to develop EAE and were treated with anti-CD25 mAb. Treatment with anti-CD25 antibody following immunization resulted in a significant enhancement of EAE disease severity and mortality. There was increased inflammation in the central nervous system (CNS) of anti-CD25 mAb-treated mice. Anti-CD25 antibody treatment caused a decrease in the percentage of CD25(+)CD4(+) T cells in blood, peripheral lymph node (LN) and spleen associated with increased production of IFN-gamma and a decrease in IL-10 production by LN cells stimulated with PLP(130-151) in vitro. In addition, transfer of CD25(+)CD4(+) regulatory T cells from naive SJL mice decreased the severity of active EAE. In vitro, anti-CD3-stimulated CD25(+)CD4(+) T cells from naive SJL mice secreted IL-10 and IL-10 soluble receptor (sR) partially reversed the in vitro suppressive activity of CD25(+)CD4(+) T cells. CD25(+)CD4(+) T cells from IL-10-deficient mice were unable to suppress active EAE. These findings demonstrate that CD25(+)CD4(+) T cells suppress pathogenic autoreactive T cells in actively induced EAE and suggest they may play an important natural regulatory function in controlling CNS autoimmune disease through a mechanism that involves IL-10.     

Schistosoma japonicum egg antigens stimulate CD4 CD25 T cells and modulate airway inflammation in a murine model of asthma

A number of epidemiological and clinical studies have suggested an inverse association between allergy and helminth infection, such as Schistosomiasis. Therefore, we hypothesize that Schistosoma japonicum egg antigens, a type of native antigen, can induce production of CD4(+) CD25(+) T cells with regulatory activity, modulating airway inflammation and inhibiting asthma development. The frequency of CD4(+) CD25(+) T cells was determined by flow cytometry for mice treated with ovalbumin (OVA), CD25(+) depletion/OVA, schistosome egg antigens, schistosome egg antigens/OVA and for control mice. The ability of CD25(+) T cells from these mice to suppress T-cell proliferation and cytokine production was investigated both in vivo and in vitro. Results showed that the CD4(+) CD25(+) T cells of OVA-treated mice exhibited impaired control of dysregulated mucosal T helper 2 responses compared to the controls (P < 0.05). Depletion of CD25(+) cells accelerated OVA-induced airway inflammation and increased the expression of interleukin (IL)-5 and IL-4. Treatment with schistosome egg antigens increased the number and suppressive activity of CD4(+) CD25(+) T cells, which made IL-10, but little IL-4. In a murine model of asthma, S. japonicum egg antigens decreased the expression of Th2 cytokines, relieved antigen-induced airway inflammation, and inhibited asthma development. Thus, we provided evidence that S. japonicum egg antigens induced the production of CD4(+) CD25(+) T cells, resulting in constitutive immunosuppressive activity and inhibition of asthma development. These results reveal a novel form of protection against asthma and suggest a mechanistic explanation for the protective effect of helminth infection on the development of allergy.     

Phenotypic characterization of regulatory CD4+CD25+ T cells in rats

CD25 has become widely used as a marker for a subset of regulatory CD4(+) T cells present in the thymus and periphery of mice, rats and humans. However, CD25 is also expressed on conventionally activated T cells that are not regulatory and not all peripheral regulatory T cells express CD25. The identification of a stable and unique marker for regulatory T cells would therefore be valuable. This study provides a detailed account of the phenotype of CD4(+)CD25(+) regulatory T cells in rats. In the thymus, CD4(+)CD8(-)CD25(+) cells were found to have a more mature phenotype than the corresponding CD4(+)CD8(-)CD25(-) cells with respect to expression of Thy1 (CD90), CD53 and CD44, suggesting that CD25 expression, and perhaps commitment to regulatory function, might be a late event in thymocyte development. CD4(+)CD25(+) cells in both the thymus and periphery were found to have enriched and heterogeneous expression of activation markers such as OX40 (CD134) and OX48 (an antibody determined in this study to be specific for CD86). CD4(+)CD25(+) T cells were also found to have enriched expression of CD80, at both the mRNA and protein level. However, functional studies in vitro and in vivo showed that neither OX40 or CD86 were useful markers for the further subdivision of regulatory T cells. Our studies indicate that, at present, CD25 remains the most useful marker to enrich for regulatory CD4(+) T cells in rats and no further subdivision of the regulatory component of CD4(+)CD25(-)CD45RC(low) T cells has yet been achieved.     

The development and functions of CD4(+) T cells expressing a transgenic TCR specific for an MHC-I-restricted tumor antigenic epitope

It has been reported that the ratio of CD4(+) to CD8(+) T cells has no bias in a few class I major histocompatibility complex (MHC-I)-restricted T-cell receptor (TCR)-transgenic mice specific for alloantigens or autoantigens, in which most CD4(+) T cells express an MHC-I-restricted TCR. In this study, we further showed that more than 50% of CD4(+) T cells in MHC-I-restricted P1A tumor antigen-specific TCR (P1ATCR)-transgenic mice could specifically bind to MHC-I/P1A peptide complex. P1A peptide could stimulate the transgenic CD4(+) T cells to proliferate and secrete both type 1 helper T cell and type 2 helper T cell cytokines. The activated CD4(+) T cells also showed cytotoxicity against P1A-expressing tumor cells. The analysis of TCR α-chains showed that these CD4(+) T cells were selected by co-expressing endogenous TCRs. Our results show that CD4(+) T cells from P1ATCR transgenic mice co-expressed an MHC-I-restricted transgenic TCR and another rearranged endogenous TCRs, both of which were functional.     

Detectable expression of IL-35 in CD4+ T cells from peripheral blood of chronic hepatitis B patients

Epstein-Barr virus-induced gene 3 (Ebi3) and the p35 subunit of IL-12 have been reported to form a heterodimeric cytokine, named IL-35, in human and mouse. In mice, IL-35 has been shown to be constitutively expressed by CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) and suggested to contribute to their suppressive activity. However, human CD4(+)CD25(+)Foxp3(+) Tregs do not constitutively express detectable amounts of IL-35 in both mRNA and protein levels. Circulating CD4(+)CD25(+) Treg frequency of chronic Hepatitis B patients significantly correlates with serum viral load. In this study, we investigated whether IL-35 expression could be detected in CD4(+) T cells from peripheral blood of chronic Hepatitis B patients. Using both RT-PCR and immunoprecipitation plus Western blot analysis, we demonstrated that IL-35 expression could be detected in the CD4(+) T cells from peripheral blood of Chronic Hepatitis B patients.     

Reduction of forkhead box P3 levels in CD4+CD25high T cells in patients with new-onset systemic lupus erythematosus

The aim of this study was to quantify and evaluate the forkhead box P3 (FoxP3) expression regulatory T cells in new-onset systemic lupus erythematosus (SLE) patients before and after treatment. Forty-four newly diagnosed and untreated SLE patients, including 24 with active disease (SLEDAI > or = 10) and 20 with inactive disease (SLEDAI < 5), were enrolled in this study. Twenty-one age- and sex-matched healthy volunteers were also included as controls. Peripheral blood samples were collected and mononuclear cells isolated. The expression of CD25 and FoxP3 in CD4(+) T cells were analysed with flow cytometry. CD4(+)CD25(+) (3.95-13.04%) and CD4(+)CD25(high) (0.04-1.34%) T cells in peripheral blood in untreated patients with new-onset active lupus were significantly lower than that in the patients with inactive lupus (7.27-24.48%, P < 0.05 and 0.14-3.07% P < 0.01 respectively) and that in healthy controls (5.84-14.84%, P < 0.05). Interestingly, the decrease in CD4(+)CD25(high) T cells was restored significantly in patients with active lupus after corticosteroid treatment. There was, however, a significantly higher percentage of CD4(+)FoxP3(+) T cells in patients with active (5.30-23.00%) and inactive (7.46-17.38%) new-onset lupus patients compared with healthy control subjects (2.51-12.94%) (P < 0.01). Intriguingly, CD25 expression in CD4(+)FoxP3(+) T cells in patients with active lupus (25.24-62.47%) was significantly lower than that in those patients with inactive lupus (30.35-75.25%, P < 0.05) and healthy controls (54.83-86.38%, P < 0.01). Most strikingly, the levels of FoxP3 expression determined by mean fluorescence intensity in CD4(+)CD25(high) cells in patients with active SLE were significantly down-regulated compared with healthy subjects (130 +/- 22 versus 162 +/- 21, P = 0.012). CD4(+)CD25(high) T cells are low in new-onset patients with active SLE and restored after treatment. Despite that the percentage of CD4(+)FoxP3(+) T cells appear high, the levels of FoxP3 expression in CD4(+)CD25(high) T cells are down-regulated in untreated lupus patients. There is a disproportional expression between CD25(high) and FoxP3(+) in new-onset patients with active SLE.     

Natural CD4 CD25(+) regulatory T cells control the burst of superantigen-induced cytokine production: the role of IL-10.

In normal mice a subpopulation of CD4 T cells constitutively expresses the IL-2 receptor alpha chain (CD25). This natural CD4 CD25(+) subset is thymus-born, constitutively expresses IL-10 mRNA,does not produce IL-2 and is resistant to apoptosis. These cells behave as regulatory T cells in the control of self-tolerance, inflammatory reactions and T cell homeostasis. The mechanisms by which natural CD4 CD25(+) cells control the immune response is unclear. We examined CD25-deficient mice, which over-express various cytokines, including proinflammatory molecules, after bacterial superantigen stimulation in vivo. We observed that this abnormal cytokine production could be controlled by the injection of natural CD4 CD25(+) T cells and that IL-10 production is needed, as CD4 CD25(+) T cells from IL-10 knockout mice do not correct cytokine over-production in vivo. As the circulating IL-10 produced by CD25-deficient mice was ineffective, we deduced that the key source of IL-10 was the regulatory T cell population. IL-10 is also involved in the control of cytokine production by normal T cells. However, the target of IL-10 in this control is undefined. Whether it acts directly on the effector T cells or on the regulatory CD4 CD25(+) T cells themselves to induce their functional maturation has to be clarified.     

Daily subcutaneous injections of peptide induce CD4+ CD25+ T regulatory cells

Peptide immunotherapy is being explored to modulate varied disease states; however, the mechanism of action remains poorly understood. In this study, we investigated the ability of a subcutaneous peptide immunization schedule to induce of CD4(+) CD25(+) T regulatory cells. DO11.10 T cell receptor (TCR) transgenic mice on a Rag 2(-/-) background were injected subcutaneously with varied doses of purified ovalbumin (OVA(323-339)) peptide daily for 16 days. While these mice have no CD4(+) CD25(+) T regulatory cells, following this injection schedule up to 30% of the CD4(+) cells were found to express CD25. Real-time quantitative polymerase chain reaction (QPCR) analysis of the induced CD4(+) CD25(+) T cells revealed increased expression of forkhead box P3 (FoxP3), suggesting that these cells may have a regulatory function. Proliferation and suppression assays in vitro utilizing the induced CD4(+) CD25(+) T cells revealed a profound anergic phenotype in addition to potent suppressive capability. Importantly, co-injection of the induced CD4(+) CD25(+) T cells with 5,6-carboxy-succinimidyl-fluorescence-ester (CFSE)-labelled naive CD4(+) T cells (responder cells) into BALB/c recipient mice reduced proliferation and differentiation of the responder cells in response to challenge with OVA(323-339) peptide plus adjuvant. We conclude that repeated subcutaneous exposure to low-dose peptide leads to de novo induction of CD4(+) CD25(+) FoxP3(+) T regulatory cells with potent in vitro and in vivo suppressive capability, thereby suggesting that one mechanism of peptide immunotherapy appears to be induction of CD4(+) CD25(+) Foxp3(+) T regulatory cells.     

Alterations of peripheral CD4+CD25+Foxp3+ T regulatory cells in mice with STZ-induced diabetes

Complications arising from abnormal immune responses are the major causes of mortality and morbidity in diabetic patients. CD4+CD25+T regulatory cells (Tregs) play pivotal roles in controlling immune homeostasis, immunity and tolerance. The effect of hyperglycemia on CD4+CD25+Tregs has not yet been addressed. Here we used streptozotocin (STZ)-induced diabetic mice to study the effects of long-term hyperglycemia on CD4+CD25+Tregs in vivo. Four months after the onset of diabetes, the frequency of CD4+CD25+Foxp3+ T regulatory cells was significantly elevated in the spleen, peripheral blood lymphocytes (PBLs), peripheral lymph nodes (pLNs) and mesenteric LNs (mLNs). CD4+CD25+Tregs obtained from mice with diabetes displayed defective immunosuppressive functions and an activated/memory phenotype. Insulin administration rescued these changes in the CD4+CD25+ Tregs of diabetic mice. The percentage of thymic CD4+CD25+ naturally occurring Tregs (nTregs) and peripheral CD4+Helios+Foxp3+ nTregs were markedly enhanced in diabetic mice, indicating that thymic output contributed to the increased frequency of peripheral CD4+CD25+Tregs in diabetic mice. In an in vitro assay in which Tregs were induced from CD4+CD25- T cells by transforming growth factor (TGF)-β, high glucose enhanced the efficiency of CD4+CD25+Foxp3+ inducible Tregs (iTregs) induction. In addition, CD4+CD25- T cells from diabetic mice were more susceptible to CD4+CD25+Foxp3+ iTreg differentiation than those cells from control mice. These data, together with the enhanced frequency of CD4+Helios-Foxp3+ iTregs in the periphery of mice with diabetes, indicate that enhanced CD4+CD25+Foxp3+ iTreg induction also contributes to a peripheral increase iCD4+CD25+Tregs in diabetic mice. Our data show that hyperglycemia may alter the frequency of CD4+CD25+Foxp3+ Tregs in mice, which may result in late-state immune dysfunction in patients with diabetes.     

Dendritic cells partially abrogate the regulatory activity of CD4+CD25+ T cells present in the human peripheral blood

The factors that influence the functionality of human CD4(+)CD25(+) regulatory T cells are not well understood. We sought to characterize the effects of dendritic cells (DCs) on the in vitro regulatory activity of CD4(+)CD25(+) T cells obtained from peripheral blood of healthy human donors. Flow cytometry showed that a higher proportion of CD4(+)CD25(+(High)) T cells expressed surface glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) and CTL-associated antigen 4 than CD4(+)CD25(-) or CD4(+)CD25(+(Med-low)) T cells. Intracellular Foxp3 was equivalently expressed on CD4(+)CD25(+(All)), CD4(+)CD25(+(High)), CD4(+)CD25(+(Med-low)) and CD4(+)CD25(-) T cell populations, irrespective of GITR and CTL-associated antigen 4 expression. CD4(+)CD25(+) T cells were isolated and then cultured in vitro with CD4(+)CD25(-) responder T cells and stimulated with anti-CD3 antibodies, and immature dendritic cells (iDCs), mature dendritic cells (mDCs), PBMCs or PBMCs plus anti-CD28 antibodies to provide co-stimulation. In addition, secretion of the T(h)1 cytokine IFN-gamma, IL-2 and the immunoregulatory cytokines, IL-10 and transforming growth factor (TGF)-beta, were also assessed in these cultures. We found that iDCs and mDCs were capable of reversing the suppression of proliferation mediated by CD4(+)CD25(+) regulatory T cells. However, the reversal of suppression by DCs was not dependent upon the increase of IFN-gamma and IL-2 production or inhibition of IL-10 and/or TGF-beta production. Therefore, DCs are able to reverse the suppressive effect of regulatory T cells independent of cytokine production. These results suggest for the first time that human DCs possess unique abilities which allow them to influence the functions of regulatory T cells in order to provide fine-tuning in the regulation of T cell responses.     

Total glucosides of paeony induces regulatory CD4(+)CD25(+) T cells by increasing Foxp3 demethylation in lupus CD4(+) T cells

Total glucosides of paeony (TGP), an active compound extracted from Paeony root, has been used in therapy for autoimmune diseases. However the molecular mechanism of TGP in the prevention of autoimmune response remains unclear. In this study, we found that TGP treatment significantly increased the percentage and number of Treg cells in lupus CD4(+) T cells. Further investigation revealed that treatment with TGP increased the expression of Foxp3 in lupus CD4(+) T cells by down-regulating Foxp3 promoter methylation levels. However, we couldn't observe similar results in healthy control CD4(+) T cells treated by TGP. Moreover, our results also showed that IFN-γ and IL-2 expression was enhanced in TGP-treated lupus CD4(+) T cells. These findings indicate that TGP inhibits autoimmunity in SLE patients possibly by inducing Treg cell differentiation, which may in turn be due to its ability to regulate the methylation status of the Foxp3 promoter and activate IFN-γ and IL-2 signaling.     

CD4(+) and CD8(+) cells are key participants in the development of recurrent herpetic stromal keratitis in mice

Ocular herpes simplex virus (HSV) infection results in an immune-mediated inflammation of the corneal stroma known as herpetic stromal keratitis (HSK). Recurrent HSK is a common cause of virus-induced corneal blindness in humans. The role of CD4(+) and CD8(+) T cell subsets in the disease pathogenesis is ill defined and varies with the virus strain and host genetic background. To examine the contribution of T cell subsets to corneal disease, we studied the development of recurrent HSK in CD4 or CD8 gene knockout (KO) mice ocularly infected with HSV-1 McKrae strain. Following UV-B induced viral reactivation, corneal opacity in latently infected BALB/c (HSV sensitive) CD4 and CD8 KO mice was reduced compared to infected BALB/c mice with normal genotype. In contrast, opacity in C57BL/6 (HSV resistant) CD4 and CD8 KO latent mice did not differ from genetically normal latent mice. Virus-induced corneal opacity was not demonstrable in C57BL/6 CD4/CD8 double KO mice. Increased viral shedding, measured by reactivation rate, days shedding or viral titers, occurred in CD4 KO mice of both strains. Our findings indicate that both CD4(+) and CD8(+) cells play a role in the immunopathogenesis of recurrent HSK, and their role is dependent upon the host genetic profile.     

Induction and maintenance of self tolerance: the role of CD4+CD25+ regulatory T cells

The immune system responds vigorously to invading pathogens (non-self, foreign), while remaining unresponsive (tolerant) to the body's own components and circulating constituents (self). This indifference to self components is a result of finely orchestrated events of thymic negative selection (central tolerance) of developing T cells that are autoaggressive combined with those operative in the periphery (peripheral tolerance) to control the activity of potentially autoreactive T cells that escaped thymic tolerance. Recently, autoimmune regulator expressed in the thymus has been identified as a critical mediator of central tolerance towards tissue-specific antigens. In the periphery, a variety of regulatory T cells are involved in effecting tolerance. There is immense interest and excitement about the newly identified subset of CD4(+)CD25(+) T cells. This is a unique subset of CD4(+) T cells that bear CD25 (IL-2Ralpha chain) on the cell surface in the na?ve state and express FoxP3 as a unique marker. These cells suppress the activity of autoreactive effector T cells primarily via cell-cell contact. The deficiency and/or altered function of CD4(+)CD25(+) T cells is associated with autoimmunity. Mice deficient in FoxP3 (scurfy mice) bear an autoimmune phenotype, and human males with mutations in the corresponding gene express the phenotype of wide-spread autoimmunity, the immune dysregulation, polyendocrinopathy and enteropathy, and X-linked syndrome. In vitro expansion of antigen-specific CD4(+)CD25(+) T cells and their adoptive transfer into patients suffering from autoimmunity is emerging as a promising new therapeutic approach for these debilitating disorders.     

Increase of circulating CD4+CD25+ T cells in myasthenia gravis patients with stability and thymectomy

BACKGROUND: CD4(+)CD25(+) regulatory T cells are key controllers of peripheral immunological self-tolerance and suppress various autoimmune diseases in animal models, but few studies have been done to define their roles in myasthenia gravis (MG) so far. OBJECTIVE: To investigate frequencies and dynamic changes of blood CD4(+)CD25(+) T cells from MG patients. METHODS: The peripheral blood CD4(+)CD25(+) T cells of 29 MG patients and 23 healthy controls were detected by three-color flow cytometry. RESULTS: Myasthenic patients with symptomatically uncontrollable disease showed slightly lower percentages of CD4(+)CD25(+) T cells (mean = 3.79 +/- 1.40%; P = 0.12), whereas MG patients with clinically stable disease had significantly increased CD4(+)CD25(+) T cells (mean = 8.45 +/- 1.96%, P = 0.0001), as compared with healthy controls (mean = 4.53 +/- 0.96%). In addition, thymectomized MG patients had significantly higher percentages of CD4(+)CD25(+) T cells (mean = 8.44 +/- 2.39%), as compared with both non-thymectomized MG patients (mean = 5.88 +/- 2.89%, P = 0.038) and healthy controls (P = 0.003). CONCLUSIONS: Our observations indicate that increased percentages of CD4(+)CD25(+) T cells in MG patients may be related to disease stability and that thymectomy in patients with MG resulted in augmented CD4(+)CD25(+) T cells.     

Naturally anergic and suppressive CD25(+)CD4(+) T cells as a functionally and phenotypically distinct immunoregulatory T cell subpopulation

A CD4(+) T cell subpopulation defined by the expression levels of a particular cell surface molecule (e.g. CD5, CD45RB, CD25, CD62L or CD38) bears an autoimmune-preventive activity in various animal models. Here we show that the expression of CD25 is highly specific, when compared with other molecules, in delineating the autoimmune-preventive immunoregulatory CD4(+) T cell population. Furthermore, although CD25 is an activation marker for T cells, the following findings indicate that immunoregulatory CD25(+)CD4(+) T cells are functionally distinct from activated or anergy-induced T cells derived from CD25(-)CD4(+) T cells. First, the former are autoimmune-preventive in vivo, naturally unresponsive (anergic) to TCR stimulation in vitro and, upon TCR stimulation, able to suppress the activation/proliferation of other T cells, whereas the latter scarcely exhibit the in vivo autoimmune-preventive activity or the in vitro suppressive activity. Second, such activated or anergy-induced CD25(-) spleen cells produce various autoimmune diseases when transferred to syngeneic athymic nude mice, whereas similarly treated normal spleen cells, which include CD25(+)CD4(+) T cells, do not. Third, upon polyclonal T cell stimulation, CD25(+)CD4(+) T cells express CD25 at higher levels and more persistently than CD25(-)CD4(+) T cell-derived activated T cells; moreover, when the stimulation is ceased, the former revert to the original levels of CD25 expression, whereas the latter lose the expression. These results collectively indicate that naturally anergic and suppressive CD25(+)CD4(+) T cells present in normal naive mice are functionally and phenotypically stable, distinct from other T cells, and play a key role in maintaining immunologic self-tolerance.