Human anergic/suppressive CD4(+)CD25(+) T cells: a highly differentiated and apoptosis-prone population

Anergic/suppressive CD4(+)CD25(+) T cells exist in animal models but their presence has not yet been demonstrated in humans. We have identified and characterized a human CD4(+)CD25(+) T cell subset, which constitutes 7-10 % of CD4(+) T cells in peripheral blood and tonsil. These cells are a CD45RO(+)CD45RB(low) highly differentiated primed T cell population that is anergic to stimulation. Depletion of this small subset from CD4(+) T cells significantly enhances proliferation by threefold in the remaining CD4(+)CD25(-) T cells, while the addition of isolated CD4(+)CD25(+) T cells to CD4(+)CD25(-) T cells significantly inhibits proliferative activity. Blocking experiments suggest that suppression is not mediated via IL-4, IL-10 or TGF-beta and is cell-contact dependent. Isolated CD4(+)CD25(+) T cells are susceptible to apoptosis that is associated with low Bcl-2 expression, but this death can be prevented by IL-2 or fibroblast-secreted IFN-beta. However, the anergic/suppressive state of these cells is maintained after cytokine rescue. These human regulatory cells are therefore a naturally occurring, highly suppressive, apoptosis-prone population which are at a late stage of differentiation. Further studies into their role in normal and pathological situations in humans are clearly essential.     

CD5 plays an inhibitory role in the suppressive function of murine CD4(+) CD25(+) T(reg) cells

A subset of CD4(+) T cells, the CD4(+) CD25(+) regulatory T (T(reg)) cells in the lymphoid organs and peripheral blood are known to possess suppressive function. Previous in vitro and in vivo studies have indicated that T cell receptor (TCR) signal is required for development of such 'natural regulatory (T(reg)) cells' and for activation of the effector function of CD4(+) CD25(+) regulatory T cells. CD5 is a cell surface molecule present on all T cells and a subtype of B lymphocytes, the B-1 cells, primarily localized to coelomic cavities, Peyer's patches, tonsils and spleen. CD5 acts as a negative regulator of T cell and B cell signaling via recruitment of SHP-1. Here, we demonstrate that T(reg) cells obtained from CD5(-/-) mice are more potent than those from wild type mice in suppressing the in vitro cell proliferation of anti-CD3 stimulated CD4(+) CD25(-) responder T cells. This phenomenon was cell contact and GITR dependent. Lack of CD5 expression on T(reg) cells (from spleen, lymph node and thymus) did not affect the intracellular levels of Foxp3. However, CD5(-/-) T(reg) thymocytes were able to elicit a higher Ca(2+) response to TCR + co-stimulatory signals than the wild type cells. CD5(-/-) mice expressed more Foxp3 mRNA in the colon than wild type mice, and additionally, the severity of the dextran sulfate sodium (DSS)-induced colitis in CD5(-/-) mice was less than the wild type strain. We suggest that manipulation of CD5 expression or the downstream signaling components of CD4(+) CD25(+) T(reg) cells as a potential strategy for therapeutic intervention in cases of auto-immune disorders.     

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.     

Role of CD4(+)CD25(+) T regulatory cells in IL-2-induced vascular leak

T regulatory cells (CD4(+)CD25(+)) play an important role in the regulation of the immune response. However, little is known about the ability of T regulatory cells to regulate endothelial cell (EC) damage following activation of lymphocytes with IL-2. Therefore, in the current study, we examined the role of T regulatory cells and the subsequent T(h)1/T(h)2 bias in IL-2-mediated EC injury using the well-characterized C57BL/6 (T(h)1-biased) and BALB/c (T(h)2-biased) models. Following IL-2 treatment, BALB/c mice were less susceptible to IL-2-induced vascular leak syndrome (VLS) compared with C57BL/6 mice. Splenocytes from BALB/c mice displayed less cytotoxicity against ECs compared with those from C57BL/6 mice. Interestingly, BALB/c mice had significantly higher numbers of CD4(+)CD25(+) T regulatory cells, which proliferated more profoundly following IL-2 treatment, compared with CD4(+)CD25(+) T regulatory cells from C57BL/6 mice. In addition, T regulatory cells from naive BALB/c mice were more potent suppressors of anti-CD3 mAb-stimulated proliferation of T cells than similar cells from C57BL/6 mice. Depletion of T regulatory cells in both BALB/c and C57BL/6 mice led to a significant increase in IL-2-induced VLS. Together, the results from this study suggest that CD4(+)CD25(+) T regulatory cells play an important role in the regulation of IL-2-induced EC injury.     

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.     

Cat allergen peptide immunotherapy reduces CD4(+) T cell responses to cat allergen but does not alter suppression by CD4(+) CD25(+) T cells: a double-blind placebo-controlled study

BACKGROUND: We have previously described both modification of allergen immunotherapy using peptide fragments, and reduced regulation of allergen stimulated T cells by CD4(+) CD25(+) T cells from allergic donors when compared with nonallergic controls. It has been suggested that allergen immunotherapy induces regulatory T cell activity: we hypothesized that allergen peptide immunotherapy might increase suppressive activity of CD4(+) CD25(+) T cells. OBJECTIVE: To examine cat allergen-stimulated CD4 T cell responses and their suppression by CD4(+) CD25(+) T cells before and after cat allergen peptide immunotherapy in a double-blind placebo-controlled study. METHODS: Peripheral blood was obtained and stored before and after peptide immunotherapy or placebo treatment. CD4(+) and CD4(+) CD25(+) were then isolated by immunomagnetic beads and cultured with allergen in vitro. RESULTS: Comparing cells from blood taken before with that after peptide immunotherapy there was a significant reduction in both proliferation and IL-13 production by allergen-stimulated CD4+ T cells, whereas no change was seen after placebo. CD4(+) CD25(+) T cells suppressed both proliferation and IL-13 production by CD4(+) CD25(-) T cells before and after therapy but peptide therapy was not associated with any change in suppressive activity of these cells. CONCLUSION: Allergen peptide immunotherapy alters T cell response to allergen through mechanisms other than changes in CD4(+) CD25(+) T cell suppression.     

Control of T cell hyperactivation in IL-2-deficient mice by CD4(+)CD25(-) and CD4(+)CD25(+) T cells: evidence for two distinct regulatory mechanisms

In IL-2-deficient mice, antigen-activated CD4 T cells accumulate and cause lethal immune pathology. Wild-type cells of hematopoietic origin present in the same animal are able to prevent this hyperactivation of T cells, but the mechanisms and cells controlling the IL-2-deficient cells are unknown. Here we show that IL-2(-) CD4 cells with an ovalbumin-specific transgenic TCR (IL-2(-) OVAtg) undergo both clonal expansion and clonal contraction when transferred to euthymic recipients and challenged with antigen, but continuously expand in athymic hosts. Cotransfer of wild-type CD4 T cells prevents the accumulation of IL-2-deficient cells. On the residual IL-2(-) TCRtg cells CD69 and CD25 are up-regulated, suggesting that activation per se is not suppressed and that the cells had received an IL-2 signal. Since IL-2 is able to restore the defective antigen-induced cell death (AICD) of IL-2-deficient T cells in vitro, paracrine IL-2 provided by the wild-type CD4 cells may thus be able to allow clonal contraction of IL-2-deficient cells also in vivo. Interestingly however, regulatory CD4(+)CD25(+) cells also efficiently contain the clone size of antigen-stimulated IL-2-deficient T cells. Since CD4(+)CD25(+) cells do not produce IL-2, this suggests a mechanism of suppression distinct from paracrine IL-2 delivery. In keeping with this, the residual IL-2(-) TCRtg cells recovered after cotransfer of regulatory CD4(+)CD25(+) cells do not show increased CD25 or CD69 expression, suggesting that they had not received paracrine IL-2 and that clonal containment occurred at the level of initial activation rather than clonal contraction by AICD. IL-2 deficiency therefore may upset T cell homeostasis by two distinct mechanisms: the failure to program expanding T cells for apoptosis, and the failure to generate functional CD4(+)CD25(+) regulatory 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.     

4-1BB co-stimulation enhances human CD8(+) T cell priming by augmenting the proliferation and survival of effector CD8(+) T cells

Interactions between 4-1BB and its ligand, 4-1BBL, enhance CD8(+) T cell-mediated antiviral and antitumor immunity in vivo. However, mechanisms regulating the priming of CD8(+) T cell responses by 4-1BB remain unclear, particularly in humans. The 4-1BB receptor was undetectable on naive or resting human CD8(+) T cells and induced in vitro by TCR triggering. Naive cord blood cells were therefore primed in vitro against peptides or cellular antigens and then co-stimulated with 4-1BBL or agonistic antibodies. Co-stimulation enhanced effector function such as IFN-gamma production and cytotoxicity by augmenting numbers of antigen-specific and effector CD8(+) T cells. OKT3 responses also showed reduced cell death and revealed that the proliferation of CD8(+) T cells required two independently regulated events. One, the induction of IL-2 production, could be directly triggered by 4-1BB engagement on CD8(+) T cells in the absence of accessory cells. The other, expression of CD25, was induced with variable efficacy by accessory cells. Thus, suboptimal accessory cells and 4-1BB co-stimulation combined their effects to enhance IL-2 production and proliferation. Reduced apoptosis observed after co-stimulation in the presence of accessory cells correlated with increased levels of Bcl-X(L) in CD8(+) T cells, while Bcl-2 expression remained unchanged. Altogether, 4-1BB enhanced expansion, survival and effector functions of newly primed CD8(+) T cells, acting in part directly on these cells. As 4-1BB triggering could be protracted from the TCR signal, 4-1BB agonists may function through these mechanisms to enhance or rescue suboptimal immune responses.     

Differential cytokine requirements for regulation of autoimmune gastritis and colitis by CD4(+)CD25(+) T cells

Murine autoimmune gastritis, induced by neonatal thymectomy or the injection of CD25-depleted lymphocytes into nu/nu recipients, is characterized by an inflammatory infiltrate into the gastric mucosa, parietal cell destruction and circulating anti-parietal cell antibodies. Using RAG-2(-/-)mice as recipients, we determined that the induction of disease relies on CD4(+)CD25(-)effector cells and prevention relies on CD4(+)CD25(+)regulatory cells; neither requires participation of CD8 cells or B cells. The severity of gastritis was dependent on the cytokine repertoire of CD4(+)CD25(-)effector T cells. Recipients of IL-4(-/-)T cells developed more severe gastritis and recipients of INF-gamma(-/-)T cells developed milder disease than recipients of wildtype or IL-10(-/-)effector T cells. Gastritis did not develop in the absence of IL-12. Protection from gastritis does not require either IL-4 or IL-10 because CD4(+)CD25(+)cells from IL-4(-/-)or IL-10(-/-)mice completely abrogated the disease process. CD4(+)CD25(+)cells also protected RAG-2(-/-)recipients from colitis and inhibitory activity was partially dependent on IL-10 expression. These findings highlight the critical role of CD4(+)CD25(+)regulatory T cells in protection from several autoimmune syndromes and delineate the differential contribution of IL-10 to CD4(+)CD25(+)Treg activity in the settings of gastritis and colitis.     

Dietary n-3 polyunsaturated fatty acids suppress splenic CD4(+) T cell function in interleukin (IL)-10(-/-) mice

Our laboratory has demonstrated that down-regulation of proliferation and cytokine synthesis by CD4(+) T cells in mice fed diets rich in n-3 polyunsaturated fatty acids (PUFA) is highly dependent on the involvement of the co-stimulatory molecule, CD28. It has been reported that the inhibitory cytokine interleukin (IL)-10 acts directly on T cells which up-regulate IL-10 receptor (IL-10R) expression following stimulation via CD28 by efficiently blocking proliferation and cytokine production. Thus, it was hypothesized that dietary n-3 PUFA would suppress T cell function through the effects of IL-10. The proliferation of purified splenic CD4(+) T cells activated in vitro with anti-CD3 and anti-CD28 (alphaCD3/CD28) from conventional mice (C57BL/6) fed either a control corn oil (CO)-enriched diet devoid of n-3 PUFA, docosahexaenoic acid (DHA; 22 : 6) or eicosapentaenoic acid (EPA; 20 : 5) for 14 days was suppressed by dietary DHA and EPA. Surprisingly, a similar trend was seen in IL-10 gene knock-out (IL-10(-/-)) mice fed dietary n-3 PUFA. IL-10R cell surface expression was also significantly down-regulated on CD4(+) T cells from both the C57BL/6 and IL-10(-/-) mice fed dietary n-3 PUFA after 72 h of in vitro stimulation with alphaCD3/CD28. Enzyme-linked immunosorbent assay (ELISA) measurements revealed that C57BL/6 mice fed DHA had significantly reduced interferon (IFN)-gamma and IL-10 levels 48 h post-activation. However, CD4(+) T cells from IL-10(-/-) mice fed dietary n-3 PUFA produced significantly greater levels of IFN-gamma than the CO-fed group. Our data suggest that in the absence of IL-10, CD4(+) T cells from n-3 PUFA-fed mice may up-regulate IFN-gamma. Suppressed CD4(+) T cells from n-3 PUFA-fed C57BL/6 mice may use mechanisms other than IL-10 to down-regulate T cell function.     

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.     

Functional alteration of peripheral CD25(+)CD4(+)immunoregulatory T cells in a transgenic rat model of autoimmune diseases

Transgenic rats carrying the env-pX gene of human T cell leukemia virus type-I (env-pX rats) develop various collagen vascular diseases. Since autoantibodies are present in their sera, env-pX rats are considered to be a prototype model for autoimmune diseases. Adoptive transfers of spleen cells from syngenic non-transgenic rats decreased the incidence of diseases in env-pX rats, thus suggesting that normal spleen contains cells, which suppress autoimmune diseases. Murine peripheral CD25(+)CD4(+)T cells play roles in maintaining immunological self-tolerance. To examine if alterations of immunoregulatory cells may be evident in env-pX rats, quantitative and qualitative analyses of splenic CD25(+)CD4(+)T cells were done before these rats developed autoimmune diseases. Env-pX and non-transgenic rats had equivalent number of CD25(+)CD4(+)T cells. However, CD25(+)CD4(+)T cells from env-pX rats did not suppress proliferation of T cells stimulated by anti-CD3 antibodies (Ab) in vitro, whereas those from non-transgenic rats did. Additionally, env-pX CD25(+)CD4(+)T cells showed autologous and anti-CD3 Ab-mediated proliferation, in contrast to the anergic features in non-transgenic rats. These findings appear to be the first evidence that CD25(+)CD4(+)immunoregulatory T cells are altered in animal models, which naturally develop autoimmune diseases.     

Interleukin-7 matures suppressive CD127(+) forkhead box P3 (FoxP3)(+) T cells into CD127(-) CD25(high) FoxP3(+) regulatory T cells

We have identified a novel interleukin (IL)-7-responsive T cell population [forkhead box P3 (FoxP3(+) ) CD4(+) CD25(+) CD127(+) ] that is comparably functionally suppressive to conventional FoxP3(+) CD4(+) CD25(+) regulatory T cells (T(regs) ). Although IL-2 is the most critical cytokine for thymic development of FoxP3(+) T(regs) , in the periphery other cytokines can be compensatory. CD25(+) CD127(+) T cells treated with IL-7 phenotypically 'matured' into the known 'classical' FoxP3(+) CD4(+) CD25(high) CD127(-) FoxP3(+) T(regs) . In freshly isolated splenocytes, the highest level of FoxP3 expression was found in CD127(+) CD25(+) T cells when compared with CD127(-) CD25(+) or CD127(+) CD25(-) cells. IL-7 treatment of CD4(+) CD25(+) T cells induced an increase in the accumulation of FoxP3 in the nucleus in vitro. IL-7-mediated CD25 cell surface up-regulation was accompanied by a concurrent down-regulation of CD127 in vitro. IL-7 treatment of the CD127(+) CD25(+) FoxP3(+) cells also resulted in up-regulation of cytotoxic T lymphocyte antigen 4 without any changes in CD45RA at the cell surface. Collectively, these data support emerging evidence that FoxP3(+) T cells expressing CD127 are comparably functionally suppressive to CD25(+) CD127(-) FoxP3(+) T cells. This IL-7-sensitive regulation of FoxP3(+) T(reg) phenotype could underlie one peripheral non-IL-2-dependent compensatory mechanism of T(reg) survival and functional activity, particularly for adaptive T(regs) in the control of autoimmunity or suppression of activated effector 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.     

Human CD4+CD25+ T cells derived from the majority of atopic donors are able to suppress TH1 and TH2 cytokine production

BACKGROUND: Recently, it has been established that CD4(+)CD25(+) T cells with regulatory capacity are present in human peripheral blood, inhibiting allogeneic proliferation and cytokine production of preactivated CD4(+)CD25(-) respond-er T cells. OBJECTIVE: The aim of this study was to analyze in an allergen-specific setting whether such regulatory CD4(+)CD25(+) T cells also exist and function normally in atopic individuals, especially concerning the inhibition of T(H)2 cytokines. METHODS: For this purpose, CD4(+)CD25(-) or CD4(+)CD25(+) T cells from donors allergic to grass or birch pollen (mainly with rhinitis) or from healthy nonatopic donors were stimulated in the presence of autologous, mature, monocyte-derived, allergen-pulsed dendritic cells, and the preactivated CD4(+)CD25(+) T cells were added to CD4(+)CD25(-) T cells during restimulation. RESULTS: CD4(+)CD25(+) T cells from the nonatopic donors and from the majority of the patients investigated proliferated poorly, produced fewer cytokines, and inhibited the proliferation and T(H)1 (IFN-gamma) and T(H)2 (IL-4 and IL-5) cytokine production of CD4(+)CD25(-) T cells but not IL-10 production. The suppression of CD4(+)CD25(-) T cells by CD4(+)CD25(+) T cells was at least partially antigen unspecific and not reversible with anti-IL-10, anti-transforming growth factor beta, or anti-cytotoxic T lymphocyte-associated antigen 4 mAb but was reversible with IL-2. In some atopic patients preactivated CD4(+)CD25(+) T cells reproducibly showed strong proliferative responses, produced higher amounts of IL-4 and IL-10 than CD4(+)CD25(-) T cells, and suppressed only the IFN-gamma production of CD4(+)CD25(-) T cells. CONCLUSION: These data indicate that regulatory CD4(+)CD25(+) T cells are present and functional in most atopic patients with allergic rhinitis and are able to inhibit T(H)1, as well as T(H)2, cytokine production.     

Glucocorticoid amplifies IL-2-dependent expansion of functional FoxP3(+)CD4(+)CD25(+) T regulatory cells in vivo and enhances their capacity to suppress EAE

IL-2 is crucial for the production of CD4(+)CD25(+) T regulatory (Treg) cells while important for the generation of effective T cell-mediated immunity. How to exploit the capacity of IL-2 to expand Treg cells, while restraining activation of T effector (Teff) cells, is an important and unanswered therapeutic question. Dexamethasone (Dex), a synthetic glucocorticoid steroid, has been reported to suppress IL-2-mediated activation of Teff cells and increase the proportion of Treg cells. Thus, we hypothesized that glucocorticoids may be useful as costimulants to amplify IL-2-mediated selective expansion of Treg cells. We show in this study that short-term simultaneous administration of Dex and IL-2 markedly expanded functional suppressive Foxp3(+)CD4(+)CD25(+) T cells in murine peripheral lymphoid tissues. In a myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) mouse model, we observed that splenic CD4(+)CD25(+) T cells failed to suppress the proliferation of CD4(+)CD25(-) T cells. Pretreatment with Dex/IL-2 remarkably increased the proportion of CD4(+)FoxP3(+) cells and partially restored the function of splenic CD4(+)CD25(+) T cells, and inhibited the development of EAE. Therefore, the combination of glucocorticoid and IL-2, two currently used therapeutics, may provide a novel approach for the treatment of autoimmune diseases, transplant rejection and graft-vs.-host disease.     

大鼠CD4+ CD25+ T调节细胞的分离培养及其功能分析

目的:研究大鼠CD4 CD25 T调节细胞(Tr)的分离培养,并对其功能进行初步分析。方法:无菌条件下切取大鼠脾脏分离脾淋巴细胞。用免疫磁珠细胞分离系统(MACS)分选CD4 CD25 T细胞,并以流式细胞术检测其纯度后,对其进行扩增。采用混合淋巴细胞反应研究CD4 CD25 Tr细胞对CD4 CD25-T细胞的免疫抑制作用。用ELISA法检测培养上清中IL-2、IFN-γ及IL-10水平的差异。结果:MACS分离的CD4 CD25 T细胞的纯度达86%~93%。该细胞与CD4 CD25-T细胞相比能特异性地表达Foxp3基因。体外培养中能明显抑制效应T细胞增殖及其分泌IFN-γ、IL-2,但其自身能分泌Th2型细胞因子IL-10。结论:采用MACS系统阴性加阳性分选,可高效快速的获得理想纯度和免疫抑制功能的大鼠CD4 CD25 T调节细胞,该细胞对CD4 CD25-T细胞具有明显的免疫抑制作用,并能特异性的表达Foxp3基因。