Chemokine responsiveness of CD4+ CD25+ regulatory and CD4+ CD25− T cells from atopic and nonatopic donors

Background:  Allergic inflammation is associated with Th2-type T cells, which can be suppressed by CD4+ CD25+ regulatory T cells (Tregs). Both express chemokine receptors (CCR) 4 and CCR8, but the dynamics of expression and effect of atopic status are unknown.
Objective:  To examine the expression of chemokine receptors by CD4+ CD25+ and CD4+ CD25− T cells from atopic and nonatopic donors, and document response to allergen stimulation in vitro .
Methods:  Chemokine receptor expression was examined by flow cytometry and quantitative PCR of CD4+ CD25hi and CD4+ CD25− T cells from atopics and nonatopics. Responsiveness to chemokines was by actin polymerization. Dynamics of chemokine receptor expression in 6-day allergen-stimulated cultures was analysed by carboxyfluoroscein succinimidyl ester labelling.
Results:  CD4+ CD25hi Tregs preferentially expressed CCR3, CCR4, CCR5, CCR6 and CCR8. CD4+ CD25hi Tregs responded to the chemokine ligands for CCR4, CCR6 and CCR8 (CCL17, 22, 20 and 1 respectively), with no differences between atopic and nonatopic donors. Over 6-day allergen stimulation, CD4+ CD25+ T-cells downregulated CCR4 and upregulated CCR7, in contrast to CD4+ CD25− effector cells, which downregulated CCR7 and upregulated CCR4.
Conclusions:  CCR4, CCR6 and CCR8 have potential roles in localization of both CD4+ CD25+ regulatory and CD4+ CD25− effector T cells to sites of allergic inflammation. Upregulation of CCR7 and downregulation of CCR4 upon allergen stimulation of Tregs may allow their recirculation from sites of inflammation, in contrast to retention of effector T cells.     

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.     

Regulatory activity of human CD4 CD25 T cells depends on allergen concentration, type of allergen and atopy status of the donor

Regulatory CD4+ CD25+ FoxP3-positive T cells (Treg) are functional in most atopic patients with allergic rhinitis and are able to inhibit T helper type 1 (Th1) and Th2 cytokine production of CD4+ CD25- T cells. This study was designed to analyse the following additional aspects: influence of allergen concentration, influence of the type of allergen, and influence of the atopy status of the donor on the strength of the regulatory activity. CD4+ CD25- T cells from healthy non-atopic controls or from grass-pollen-allergic or wasp-venom-allergic donors were stimulated alone or in the presence of Treg with autologous mature monocyte-derived dendritic cells which were pulsed with different concentrations of the respective allergens. Treg from grass-pollen-allergic donors failed to inhibit proliferation but not cytokine production of CD4+ CD25- T cells at high antigen doses while Treg from non-atopic donors did not fail at these allergen concentrations. Proliferative responses and cytokine production of CD4+ CD25- T cells from most of the examined wasp-venom-allergic patients were not inhibited at any concentration of wasp venom. The use of wasp venom- or phospholipase A2-pulsed dendritic cells for stimulation of CD4+ CD25- T cells from donors who were not allergic to wasp stings only resulted in an inhibited proliferation and Th2 cytokine production by Treg at 10-fold lower than the optimal concentration, while interferon-gamma production was inhibited at all concentrations investigated. These data demonstrate that in allergic diseases the function of Treg is dependent on the concentration and the type of the respective allergen with different thresholds for individual allergens and patients.     

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.     

IFN-gamma inhibits the proliferation of allergen-activated T lymphocytes from atopic, asthmatic patients by inducing Fas/FasL-mediated apoptosis

The defect in interferon-gamma (IFN-gamma) production that results in a T helper cell type 2-dominated response may be responsible for a decrease in the apoptosis of allergen-activated T cells in asthma. We investigated the effect of recombinant IFN-gamma on proliferation, Fas/Fas ligand (FasL) expression, and apoptosis in allergen-stimulated peripheral blood mononuclear cells obtained from atopic, asthmatic patients and nonatopic, control subjects. The addition of IFN-gamma at the start of cultures markedly inhibited the proliferative response to a specific allergen in cells from all asthmatic patients, whereas no change was observed in cells from nonatopic, control subjects. IFN-gamma induced an increase in the expression of Fas and FasL by allergen-stimulated CD4+ T cells from asthmatic patients and caused the apoptosis of these cells. A Fas-blocking monoclonal antibody prevented the inhibitory effect of IFN-gamma on allergen-induced proliferation. These results suggest that IFN-gamma inhibits the proliferation of allergen-stimulated CD4+ T cells from atopic, asthmatic patients by inducing the surface expression of Fas and FasL, which in turn triggers their apoptotic program. The defect in IFN-gamma production involved in the allergic, immune response may therefore be responsible for a decrease in apoptosis of allergen-activated T lymphocytes in the airways of atopic, asthmatic patients.     

Depletion of CD4+CD25+CD127lo regulatory T cells does not increase allergen‐driven T cell activation

Background It has been suggested that allergic diseases are caused by defective suppression of allergen‐specific Th2 cells by CD4⁺CD25⁺ regulatory T cells. However, such studies have been hampered by the difficulty in distinguishing regulatory T cells from CD25‐expressing activated T cells. Recently, it was shown that conventional T cells expressed high levels of CD127, whereas regulatory T cells were CD127^lo, allowing discrimination between these distinct T cell subpopulations. Objective The aim of this study was to study whether the putative regulatory subset defined as CD4⁺CD25⁺CD127^lo was involved in grass pollen‐reactive T cell responses. Methods Peripheral blood mononuclear cells (PBMCs) were obtained from allergic donors and non‐atopic controls out of season. Grass pollen‐induced cytokine production and proliferation were compared in cultures of undepleted cells and cells depleted of CD4⁺CD25⁺, CD4⁺CD25⁺CD127^hi or CD4⁺CD25⁺CD127^lo T cells. Results Undepleted cell cultures from allergic patients showed significantly increased proliferation and Th2 cytokine production compared with non‐atopic controls. Depletion of all CD25⁺ T cells did not increase cytokine production or proliferation, and more importantly, no increase in Th2 cytokine production or proliferation was observed in cell cultures depleted of CD4⁺CD25⁺CD127^lo cells (putative regulatory T cells) compared with undepleted PBMCs in both the allergic and the non‐atopic group. Conclusion Our study showed that T cells from grass pollen‐allergic patients and non‐atopic controls responded very differently to grass pollen extract, but this difference could not be explained by differences in regulatory T cell function. Further studies are needed to understand the importance of regulatory T cells in allergy.     

Immune regulation by CD4+CD25+ T cells and interleukin-10 in birch pollen-allergic patients and non-allergic controls

BACKGROUND: CD4+CD25+ regulatory T (Treg) cells and the cytokines IL-10 or TGF-beta play key roles in the maintenance of T cell homeostasis and tolerance to infectious and non-infectious antigens such as allergens. OBJECTIVE: To investigate the regulation of immune responses to birch pollen allergen compared with influenza antigen by Treg cells obtained from birch pollen-allergic patients and non-allergic controls. METHODS: Peripheral blood was collected from 10 birch pollen-allergic patients and 10 non-allergic healthy controls. CD4+CD25+ and CD4+CD25- cells isolated by magnetic-activated cell sorting were co-cultured and stimulated with birch pollen extract or influenza vaccine in the absence or presence of anti-IL-10 or soluble TGF-betaRII. RESULTS: CD4+CD25+ cells from non-allergic controls were able to suppress influenza antigen and birch pollen stimulated effector cell proliferation, whereas CD4+CD25+ cells from allergic patients suppressed influenza antigen-, but not birch pollen-stimulated proliferation. The production of Th1 cytokines, but not Th2 cytokines, was suppressed by CD4+CD25+ cells from both allergic patients and controls, upon stimulation with birch pollen extract. Neutralization of IL-10 led to significantly increased production of IFN-gamma in cultures with CD4+CD25- T effector cells. In addition, six-fold higher concentrations of TNF-alpha were detected after neutralization of IL-10 in both CD4+CD25- and CD4+CD25+ cell cultures from allergic patients and controls. CONCLUSION: We demonstrate that the allergen-specific suppressive function of CD4+CD25+ cells from allergic patients is impaired compared with non-allergic controls. Moreover, neutralization of IL-10 enhances the production of TNF-alpha, suggesting counter-acting properties of IL-10 and TNF-alpha, where IL-10 promotes tolerance and suppression by Treg cells and TNF-alpha promotes inflammatory responses.     

Demonstration of circulating allergen-specific CD4+CD25highFoxp3+ T-regulatory cells in both nonatopic and atopic individuals

BACKGROUND: CD4(+)CD25(+)Foxp3(+) T-regulatory (Treg) cells play a fundamental role in the control of autoimmunity. Whether human CD4(+)CD25(+)Foxp3(+) Treg cells that recognize foreign antigens also exist is less clear. OBJECTIVE: To investigate the existence in humans of circulating Treg cells able to recognize exogenous antigens, including allergens. METHODS: CD4(+)CD25(high)Foxp3(+) and CD4(+)CD25(-)Foxp3(-) cells were purified from human peripheral blood and cultured for 15 days with autologous dendritic cells (DCs), unloaded, or loaded with Der p 1 allergen or the bacterial antigen streptokinase (SK). RESULTS: CD4(+)CD25(high)Foxp3(+) circulating T cells obtained from healthy nonatopic subjects and cultured with Der p 1-loaded DCs, but not those cultured with either unloaded or SK-loaded DCs, suppressed the proliferative response to Der p 1 of autologous Der p 1-specific T cells generated from the CD4(+)CD25(-)Foxp3(-) subset. The antigen specificity of either Der p 1-CD4(+)CD25(high)Foxp3(+) or SK-CD4(+)CD25(high)Foxp3(+) T cells was confirmed even at clonal level. Finally, under the same experimental conditions, functionally active Der p 1-specific Treg cells were obtained from the pool of circulating CD4(+)CD25(high)Foxp3(+) T cells of Der p 1-sensitive, atopic individuals. CONCLUSION: These data provide undoubted demonstration of the existence of human CD4(+)CD25(high)Foxp3(+) circulating Treg cells specific for exogenous antigens, including the Der p 1 allergen, and indicate that CD4(+)CD25(high)Foxp3(+) Treg cells specific for Der p 1 are present and functionally active in both nonatopic and Der p 1-sensitive, atopic individuals. CLINICAL IMPLICATIONS: Caution should be advised in interpreting allergic disorders as simply resulting from defective Treg cell activity.     

CD4+CD25+ T lymphocytes in human tonsils suppress the proliferation of CD4+CD25- tonsil cells

Animal studies define CD4+CD25+ T cells as a subset that protect against autoimmune inflammation. We wanted to investigate whether CD4+CD25+ T cells from patients with recurrent tonsillitis could suppress the proliferation of other tonsil cells, in vitro, as this immunological tissue also may serve as a model for chronic inflammation. Tonsil CD4+CD25+ cells markedly suppressed the proliferation of CD4+CD25- T cells in Concanavalin A-stimulated cocultures compared with cultures containing CD4+CD25- T cells only. The suppression exerted by the CD4+CD25+ cells was abrogated if these cells were irradiated before coculture or if interleukin (IL)-2 was added to the culture medium. CD4+CD25+ T cells proliferated poorly in response to mitogen, when cultured alone. Substitution with CD4+CD25+ T cells isolated from peripheral blood, enriched by similar methods, did not downregulate the proliferation of CD4+CD25- responder cells from tonsils. The augmented suppressive ability of tonsil CD4+CD25+ T cells compared with cells of this phenotype from blood, on CD4+CD25- responder cells from tonsils, suggests that there may be a functional difference between CD25+ cells from the two locations. In conclusion, CD4+CD25+ T cells from inflamed tonsils distinctly suppressed T-cell responses to mitogen in vitro, pointing to a regulatory role for CD4+CD25+ cells retrieved from inflammatory reactions in humans.     

Induction of IL-10+CD4+CD25+ T cells by grass pollen immunotherapy

BACKGROUND: Immunotherapy involves the modulation of allergen-specific T-cell responses, either T(H)2-to-T(H)1 immune deviation or, in bee venom-treated patients, induction of IL-10 production by CD4+CD25+ T cells. IL-10-producing CD4+CD25+ regulatory T cells have emerged as potential mediators of immune tolerance in numerous murine models of immunopathology. OBJECTIVE: The aim of this study was to evaluate the role of IL-10 production and CD4+CD25+ T cells in the response to grass pollen immunotherapy. METHODS: PBMCs were isolated from patients after 1 year of grass pollen immunotherapy and from matched untreated atopic and healthy control subjects. After 6 days of in vitro stimulation with Phleum pratense, production of IL-10, IL-5, IL-4, and IFN-gamma and proliferation and numbers of CD4+CD25+ T cells were measured. T cells were then stimulated for a further 5 hours with phorbol 12-myristate 13-acetate and ionomycin and assessed for intracellular IL-10 by means of flow cytometry. RESULTS: Patients undergoing immunotherapy produced significantly more IL-10 than atopic control subjects (patients undergoing immunotherapy, 116 +/- 21 pg/mL [n = 11]; atopic patients, 30 +/- 5 pg/mL [n = 11]; P <.001), and the number of CD4+CD25+ cells identified after allergen stimulation was also greater in the immunotherapy group. The numbers of CD4+CD25+ T cells correlated positively with activation as measured by proliferation in both of the control groups but not in the immunotherapy group. Moreover, only T cells from patients undergoing immunotherapy were positive for intracellular IL-10, and these were almost exclusively CD4+CD25+ cells. CONCLUSION: Grass pollen immunotherapy results in a population of circulating T cells that express the IL-10(+) CD4+CD25+ phenotype in response to allergen restimulation.     

T lymphocytes expressing CCR3 are increased in allergic rhinitis compared with non-allergic controls and following allergen immunotherapy

BACKGROUND: In T cell-associated allergic inflammation, homing of T-helper 2 (Th2) effector cells to mucosal sites may be influenced by chemokine receptor expression. Previous studies have identified CCR3 and CCR4 as putative markers of Th2 cells and CCR5 and CXCR3 as markers of Th1 cells. The aim of this study was to assess differential chemokine receptor expression from symptomatic atopic grass pollen-sensitive subjects, compared with patients on high-dose allergen injection immunotherapy (IT) and healthy controls. METHODS: We examined chemokine receptor expression (CCR1-7 and CXCR1-4) by flow cytometry of peripheral blood CD4+ and CD8+ T cells. We also depleted peripheral blood mononuclear cell (PBMC) populations of CCR3+ CD4+ cells by magnetic bead separation and cells were stimulated with grass pollen allergen for 6 days. Cytokine production was measured by enzyme-linked immunosorbent assay. RESULTS: On freshly isolated PBMC, atopic individuals exhibited increased numbers of CCR3+ CD4+ cells compared with normal controls (P < 0.01). CCR3 expression in IT patients was reduced compared with matched atopic rhinitic controls (P < 0.05) and comparable with that observed in normal subjects. Depletion of CCR3+ CD4+ cells from allergen-stimulated PBMC cultures resulted in decreased interleukin (IL)-5 production compared with whole CD4+ populations (P < 0.05). Freshly isolated CCR3+ CD4+ cells have significantly higher intracellular IL-4 and lower IFN-gamma levels than CCR3- CD4+ cells. CD4+ T cells cultured from both peripheral cells and nasal biopsies demonstrated increased expression of CCR3 in the presence of IL-4 (P < 0.05). CONCLUSION: CCR3+ CD4+ T cells are increased in allergic rhinitis, are reduced by allergen IT, have a Th2 phenotype and contribute to allergen-specific responses. Strategies against CCR3+ T cells may be effective in human allergic diseases.     

人CD4+CD25+调节性T细胞系的建立与功能分析

目的：建立可在体外长期培养的人CD4^ CD25^ 调节性T细胞系并研究其免疫生物学特性。方法：用流式分选的方法从健康人外周血淋巴细胞中得到CD4^ CD25^ T细胞，并对其进行体外长期培养、扩增；淋巴细胞转化实验分析其免疫抑制功能，流式细胞法分析其表型。结果：经对人CD4^ CD25^ T细胞的长期培养扩增，获得具有免疫抑制功能的调节性T细胞系。该T细胞系对经TCR的刺激不敏感，且能抑制同一来源或同种异型CD4^ CD25^ T细胞的活化，大剂量IL－2可以逆转其抑制功能。长期培养的CD4^ CD25^ T细胞，膜表面CD25和CTLA－4分子持续高表达，而CD4^ CD25^ T细胞CD25和CTLA－4分子的表达呈周期性变化。结论：将CD4^ CD25^ T细胞体外扩增培养成系，其功能和表型与同一来源的CD4^ CD25^ T细胞显著不同。     

Regulatory CD4+CD25+ T lymphocytes in peripheral blood from patients with atopic asthma

The aim of this study was to analyze whether regulatory CD4+CD25+ T lymphocytes exist and function normally in patients with atopic asthma. Our data showed that a significant increase in CD4+CD25+ cell numbers was seen in atopic asthmatics during acute exacerbation, but not in those stable asthmatics, atopic nonasthmatics, and normal subjects. The mean inhibition values of the proliferation response of CD4+CD25- cells by CD4+CD25+ cells from normal controls and asthmatics were almost the same. There was no difference in inhibitory effects on both Th1 and Th2 cytokine production of CD4+CD25- cells by CD4+CD25+ cells in the two groups. These data demonstrated that although CD4+CD25+ cells increase in atopic asthma during exacerbation, these regulatory T cells appear to function normally with regard to suppression of T-cell proliferation as well as Th1-Th2 cytokine production.     

The functional insufficiency of human CD4+CD25 high T-regulatory cells in allergic asthma is subjected to TNF-alpha modulation

Background:  Natural CD4⁺CD25^highFoxp3⁺ regulatory T (nTreg) cells are important in maintaining immunologic tolerance, but their role in the pathogenesis of allergic asthma is unclear. We studied the function of nTreg cells in allergic asthmatic children and assessed the factors which may relate to the functional insufficiency of nTreg cells.
Methods:  The percentage of CD4⁺CD25^high Treg cells, the expression of Foxp3, and the cell-induced suppressive activity of nTreg cells isolated from nonatopic controls, allergic asthmatics, and allergen-specific immunotherapy (AIT)-treated asthmatic patients were studied.
Results:  Although the percentage of nTreg in peripheral blood mononuclear cells was increased, the expression of Foxp3 and its cell-induced suppressive activity were significantly lower in Dermatophagoides pteronyssinus (Der p)-sensitive asthmatic children when compared to nonatopic controls. In contrast, the expression of Foxp3 and the functional activity of nTreg cells were reversed in allergic asthmatics who received AIT. The addition of recombinant tumor necrosis factor (TNF)-α directly downregulated Foxp3 expression and abrogated the cell-induced suppressive function of Treg cells. The anti-TNF-α reagent, etanercept, restored the functional activity and Foxp3 expression of CD4⁺CD25^high Treg derived from allergic asthmatics.
Conclusions:  The functional insufficiency of nTreg cells in patients with allergic asthma may be related to the enhanced production of TNF-α and its effect on the Foxp3 expression. These results may explain, in part, the effectiveness of anti-TNF-α therapy in the treatment of allergic asthma.     

The presence of cytokine-suppressive CD4+CD25+ T cells in the peripheral blood and synovial fluid of patients with rheumatoid arthritis

CD4+CD25+ T cells have been shown to play a regulatory or suppressive role in the immune response and are possibly relevant to the pathogenesis of autoimmune diseases. In the present study, we attempted to investigate the levels of CD4+CD25+ T cells in the peripheral blood (PB) and synovial fluid (SF) of patients with rheumatoid arthritis (RA) and the effects of CD4+CD25+ T cells on the in vitro cytokine production by stimulated SF mononuclear cells (SFMC). The results showed that RA patients had similar frequencies of CD4+CD25+ T cells in PB, expressed as a percentages of the lymphocyte population, as did healthy subjects (mean +/- SD: 10.52 +/- 5.87% versus 11.11 +/- 4.58%., respectively). But in contrast to PB, the SF of RA patients contained significantly higher levels of CD4+CD25+ T cells (17.77 +/- 7.92% versus 10.52 +/- 5.87%, respectively. P < 0.001). When cocultured in vitro with SFMC, CD4+CD25+ T cells purified from either PB or SF were found to exert a considerable suppressive effect on the production of cytokines including TNF-alpha, IFN-gamma and interleukin-10 (IL-10). The percentages of inhibition of each cytokines ranged from 41.8 to 98.4% (mean, 80.0%) for TNF-alpha, 42.8 to 98.9% (mean, 83.2%) for IFN-gamma and 59.3 to 96.6% (mean, 80.0%) for IL-10. Because both pro-inflammatory and anti-inflammatory cytokines were suppressed by CD4+CD25+ T cells, whether CD4+CD25+ T cells might play a beneficial role in the suppression of sustained inflammation in rheumatoid synovium remains to be elucidated.     

CD4+CD25+ T cells as immunoregulatory T cells in vitro

We have further characterized the in vitro phenotype and function of anergic and suppressive CD4(+)25(+) T cells. Following TCR ligation, DO.11.10 CD4(+)25(+) T cells suppress the activation of OT-1 CD8(+)25(-) T cells in an antigen nonspecific manner. Although suppression was seen when using a mixture of APC from both parental strains, it was very much more marked when using F1 APC. APC pretreated with, and then separated from CD4(+)25(+) T cells did not have diminished T cell costimulatory function, suggesting that APC are not the direct targets of CD4(+)25(+) T cell regulation. CTLA-4 blockade failed to abrogate suppression by CD4(+)25(+) T cells in mixing experiments. Although CD4(+)25(+) T cells failed to respond following cross-linking of TCR, they could be induced to proliferate following the addition of exogenous IL-2, allowing the generation of a T cell line from CD4(+)25(+) T cells. After the first in vitro restimulation, CD4(+)25(+) T cells were still anergic and suppressive following TCR engagement. However, after three rounds of restimulation, their anergic and suppressive status was abrogated.     

Identification of a CD4+CD25+ T cell subset committed in vivo to suppress antigen-specific T cell responses without additional stimulation

Naturally occurring CD4+ regulatory T cells can be identified on the basis of expression of CD25 and suppression of T cell responses in vitro after TCR triggering. Here, we demonstrate that a CD134+ subset of CD4+CD25+ T cells in naive rats suppresses antigen-specific T cell responses in vitro without additional TCR stimulation. In contrast, CD4+CD25+CD134- regulatory T cells and total CD4+CD25+ regulatory T cells have suppressive activity only during simultaneous activation of responder and regulatory T cells or after in vitro pre-activation. Furthermore CD4+CD25+CD134+ T cells have a more activated phenotype than CD4+CD25+CD134- T cells, as based on the expression of CD62L, CD45RC, and MHC class II. We propose that the CD134+ regulatory T cells contain an in vivo activated and highly suppressive regulatory T cell subset. CD4+CD25+CD134+ T cells can be found in several compartments of the immune system, including spleen, lymph nodes, and blood. Interestingly though, the relative amounts of these cells within the CD4+ population and their CD134 expression levels are highest in mucosa-draining lymph nodes and lowest in blood. This suggests that the presence of CD4+CD25+CD134+ T cells indicates sites of active immune suppression.     

CD4+ CD30+ T cells perpetuate IL-5 production in Dermatophagoides pteronyssinus allergic patients

BACKGROUND: Airway allergic diseases are regulated by interleukin (IL)-5, which causes infiltration of eosinophils into the bronchial epithelium, and by IL-4 which increases serum immunoglobulin E (IgE) production and promotes CD30 expression on Th cells. CD30 generates a costimulatory signal involved in apoptosis or cell proliferation, depending on the microenvironment. Our aims were: (i) to analyze if CD4+ CD30+ T cells from allergic patients proliferate in response to Dermatophagoides pteronyssinus, and (ii) if upon stimulation this cell population produces IL-4 and IL-5. METHODS: Peripheral blood mononuclear cell (PBMC) from 17 allergic rhinitis and mild allergic asthma patients and 12 healthy nonallergic individuals were stimulated with allergen in the presence or absence of anti-IL-4, anti-IL-5 or anti-IL-4Ralpha monoclonal antibodies (mAbs). TdT-mediated dUTP nick end-labeling (TUNEL) assay, 7-aminoactinomycin-D (7-AAD) intercalation, and flow cytometry were used to determine the CD4+ CD30+ blasts percentage, cell proliferation, apoptosis, and intracellular cytokines after 7 culture days. RESULTS: Cell proliferation induced with allergen showed that 90% of the allergen-stimulated blasts were CD4+, 50% of which were CD30+. Allergen-stimulated PBMC showed a progressive increase (mean: from 7% to 23%) of CD4+ CD30+IFN-gamma+ and CD4+ CD30+IL-4+ blasts which diminished (mean: 6%) after 5 culture days. In contrast, CD4+ CD30+IL-5+ blasts showed a continuous progression (from 12% to 24%) that maintained after 7 culture days. The vast majority of CD4+ CD30+ blasts were negative to 7-AAD or TUNEL. Additionally, a significant decrease (34%) was observed in the number of CD4+ CD30+ blasts when IL-4 was neutralized. CONCLUSIONS: These data suggest that specific allergen stimulation of PBMC isolated from allergic patients generates a nonapoptotic CD4+ CD30+ blast subset that produces IL-5.