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ARTICLE |
CORRESPONDENCE Clare M. Lloyd: c.lloyd{at}imperial.ac.uk
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Asthma, which affects
Although the ability of CD4+CD25+ regulatory T cells to suppress Th1 cellmediated diseases has been extensively studied, their role in controlling Th2 cellmediated allergic responses is the subject of more recent investigation and have thus far been limited to in vitro and complex in vivo studies. It has been shown that CD4+CD25+ regulatory T cells isolated from hay fever patients have an impaired ability to suppress proliferation and IL-5 production of CD4+CD25 T cells compared with healthy controls (9). Mouse studies have shown that Th2 cellpolarized CD4+ T cells depleted of the CD4+CD25+ population resulted in increased airway eosinophilia compared with unfractionated T cells after adoptive transfer of the Th2 cells (10). Moreover, double transgenic mice with T cells that express the T cell receptor for OVA (DO11.10), as well as OVA targeted to the lung epithelium, demonstrated pulmonary infiltrates containing CD4+CD25+ T cells (11). These cells were able to prevent OVA-induced proliferation of CD4+CD25 cells in vitro. In addition, the mice were shown to be tolerant to OVA when spleen cells were removed and challenged ex vivo. However, if exogenous OVA was given to the mice, AHR still developed, although lung eosinophilia and ex vivo Th2 responses were reduced. In a different model, cotransfer of CD4+CD25+ T cells with CD4+CD25 T cells from mice sensitized to OVA into T celldeficient Rag/ hosts inhibited both allergen-induced airway inflammation and IL-4 expression (12). Although these studies imply that CD4+CD25+ regulatory T cells may have a beneficial role in suppressing allergen-induced airway inflammation in vivo, this has not yet been clearly demonstrated.
In the present study, we have determined the effect of allergen-specific CD4+CD25+ regulatory T cells on the development of allergen-induced pulmonary inflammation using a well-characterized in vivo murine model involving systemic sensitization and airway challenge with antigen. We have demonstrated that transfer of allergen-specific CD4+CD25+ regulatory T cells from DO11.10 transgenic mice inhibited the classical pathology associated with allergic asthma, namely AHR, lung eosinophilia, and Th2 cytokine production. Blocking experiments determined that this effect is dependent on IL-10. Moreover, we have shown that CD4+CD25+ regulatory T cells exerted their suppressive effects in vivo independently of their capacity to produce IL-10 but induced IL-10 production from recipient CD4+ T cells.
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Results |
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Allergen-specific CD4+CD25+ regulatory cells were transferred into BALB/c mice 24 h before airway allergen challenge. Flow cytometric analysis of bronchoalveolar lavage (BAL), lung tissue and draining lymph nodes confirmed that transferred DO11.10 CD4+CD25+ cells were present in the airway lumen, lung, and lymph nodes of OVA-sensitized mice after allergen challenge (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20051166/DC1). Transferred cells were not detected in the sham-sensitized mice that received alum/PBS.
CD4+CD25+ regulatory T cells suppress AHR and leukocyte recruitment in vivo
AHR is characteristic of the pulmonary response to inhaled antigen in sensitized animals. Transfer of CD4+CD25+ regulatory T cells significantly decreased AHR in OVA-sensitized mice at a dose of 30 mg/ml methacholine (P < 0.05; Fig. 1 A). These results were confirmed by direct measurement of lung resistance (RL) and dynamic compliance (Cdyn; Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20051166/DC1). In contrast, transfer of OVA-specific CD4+CD25 T cells had no effect on increased AHR after allergen challenge (unpublished data).
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Th2 response to allergen challenge is modulated by transfer of CD4+CD25+ regulatory T cells
Th2 cells are important effector cells in the pulmonary allergic response, and, therefore, differences in Th2 cell numbers could potentially account for the decreases in AHR and eosinophilia mediated by transfer of CD4+CD25+ regulatory T cells. Th2 cell numbers were determined in the BAL and lung tissue by costaining cells with CD4 and the Th2 cellspecific marker T1/ST2 (13). There was an increase in Th2 (CD4+T1/ST2+) cell numbers in the airway lumen and lung of OVA-sensitized and -challenged mice compared with unsensitized mice 24 h after the final OVA challenge, and this was significantly reduced by transfer of CD4+CD25+ regulatory T cells (P < 0.05; Fig. 2, A and B). Furthermore, antigen-induced increases in BAL IL-5 and IL-13 were significantly reduced by transfer of CD4+CD25+ regulatory T cells in OVA-sensitized mice (P < 0.05; Fig. 2 C). Similarly, lung homogenate IL-5 and IL-13 were also decreased by transfer of CD4+CD25+ regulatory T cells (Fig. 2 D). In contrast, production of the Th1-specific cytokine IFN- was unaffected by transfer of CD4+CD25+ regulatory T cells. Lung IFN-
levels were increased in OVA-sensitized mice to 843 ± 94 pg/ml from 620 ± 53 pg/ml in alum controls. However, there was no substantial difference between OVA-sensitized mice and mice that received CD4+CD25+ cells (838 ± 106 pg/ml).
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Similarly, transfer of CD4+CD25+ T regulatory cells had no effect on serum IgE or OVA-specific IgE levels in OVA-sensitized mice (Fig. S5, available at http://www.jem.org/cgi/content/full/jem.20051166/DC1).
CD4+CD25+ T cell transfer increases pulmonary IL-10 expression
The regulatory activity of CD4+CD25+ T cells has been attributed to IL-10 and TGF-ß in some models (6, 7, 8). Therefore, these cytokines were measured in BAL fluid and lung homogenate supernatant 24 h after the final allergen challenge. The concentration of IL-10 was significantly increased in the BAL and lung of OVA-sensitized mice that received CD4+CD25+ regulatory T cells compared with OVA-sensitized mice that received PBS (P < 0.05; Fig. 2, E and F). In contrast, levels of TGF-ß were unaffected by transfer of CD4+CD25+ regulatory T cells (Fig. 2, E and F).
Suppression of allergic inflammation and AHR by CD4+CD25+ regulatory T cells is IL-10 dependent
Because suppression of Th2 responses after transfer of OVA-specific CD4+CD25+ T cells in OVA-sensitized mice was associated with increased expression of IL-10, we investigated the role of IL-10 in regulation. To this end, we administered a neutralizing antibody against the IL-10 receptor during the allergen challenge phase. AntiIL-10R treatment completely abrogated the suppression of AHR and lung eosinophil infiltration by CD4+CD25+ regulatory cells (Fig. 3, A and B). Moreover, CD4+CD25+ regulatory T cells were unable to reduce Th2 cell numbers and IL-5 and IL-13 levels in the lung in the presence of antiIL-10R (Fig. 3, C and D, and not depicted). Interestingly, antiIL-10R treatment had no effect on the increased lung IL-10 levels associated with CD4+CD25+ regulatory T cells (Fig. 3 E).
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Discussion |
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Previous studies have determined that CD4+CD25+ regulatory T cells are able to regulate autoimmune immunopathologies, which are generally of a Th1 cell type (7, 15, 16), although a recent study using a Leishmania major infection model in SCID mice confirmed that both Th1 and Th2 cell function can be suppressed by naturally occurring CD4+ CD25+ T regulatory cells (17). Other studies have implied that CD4+CD25+ T cells may be able to suppress some aspects of allergic pathology, but these have all involved complex transgenic models or cell transfer into T celldeficient recipient mice (1012). We have shown that transfer of CD4+CD25+ T regulatory cells into a classical model of systemic allergen sensitization in immunocompetent mice reduces multiple pathophysiological parameters, including both allergen-induced airway inflammation and AHR. These findings support our conclusions from human studies in vitro, which argue that an imbalance between suppression and activation exists in patients with allergic disease (9).
Transfer of allergen-specific CD4+CD25+ T cells into OVA-sensitized mice reduced eosinophilia and AHR, together with Th2 cell numbers and Th2 cytokine production. This implies that there is an effect on either Th2 cell trafficking or in local expansion of allergen-specific Th2 cells. However, the lack of effect on Th2-attracting chemokines such as CCL22, CCL17, and CCL11 suggests that Th2 cell recruitment can still occur. Although we saw decreases in Th2 cytokine secretion after transfer of CD4+CD25+ T cells, there was no accompanying reduction in serum IgE production. This might seem surprising because CD4+ CD25+ T cells have previously been reported to suppress IgE in a transgenic model of hyper IgE (18). In addition, patients with immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome who lack CD4+CD25+ regulatory T cells because of a mutation in Foxp3 exhibit elevated serum IgE levels (19). However, IL-10 has been shown to actually increase IgE production from B cells that have already switched but inhibits germline class switch (20). The lack of change in IgE in our model probably reflects the comparably short time course of the model and the timing of CD4+CD25+ T cell transfer.
The mechanism of suppression by allergen-specific CD4+ CD25+ T regulatory cells is controversial. In vitro, the phenomenon is contact dependent but can be observed in the absence of either IL-10 or TGF-ß (9, 21, 22). In contrast, in vivo experiments in a colitis model suggest that IL-10 and TGF-ß are involved in the suppression mediated by these cells (15). However, the effect may be model dependent because it was not observed in a gastritis model (23). We show that IL-10 but not TGF-ß is up-regulated in the lung after transfer of allergen-specific CD4+CD25+ T regulatory cells and that this is observed together with an abrogation of AHR and allergic inflammation. Moreover, we highlight the importance of this IL-10 by showing that neutralizing antiIL-10R antibody treatment abolished the suppressive effect of the CD4+CD25+ regulatory T cells so that AHR, eosinophilia, and Th2 cytokine levels were comparable to those seen in the absence of regulatory cells. IL-10 has previously been reported to be a potential regulatory factor in allergen-induced airway inflammation because transfer of engineered IL-10producing T cells reduced AHR and inflammation in a murine model (24). Although this study showed that neutralization of IL-10 in the lung worsened allergic inflammation, we did not find that antiIL-10R treatment had any effect on OVA-sensitized mice in the absence of transferred CD4+CD25+ regulatory T cells. This may be because of differences in the time of IL-10 neutralization. Oh et al. administered antiIL-10 during both sensitization and challenge phases of allergen-induced airway inflammation, whereas we have blocked during the challenge phase only (24).
We have shown that the suppression by CD4+CD25+ regulatory T cells was not dependent on IL-10 production from allergen-specific T regulatory cells themselves, because the suppressive effect was preserved in CD4+CD25+ cells isolated from OVA-transgenic IL-10 / mice. Interestingly, Asseman et al. previously reported that IL-10/ CD4+ CD25+ T cells could inhibit colitis induced by transfer of naive but not antigen-experienced T cells (25). In our model, suppression was seen after transfer of IL-10/ CD4+ CD25+ T cells into mice that had been sensitized to OVA and, thus, presumably OVA-experienced Th2 cells were present. The differential dependence on IL-10 may reflect differences in the degree of inflammation and T cell activation in the gut in the colitis model compared with our lung model but is in keeping with data from a gastritis model (23). IL-10 expression in the airway was still elevated after transfer of IL-10/ CD4+CD25+ T cells, suggesting that the regulatory T cells may induce IL-10 production in another cell population within the lung. These could be CD4+CD25 T cells, which, in humans, have been reported to acquire a regulatory phenotype dependent on IL-10 production after in vitro co-culture with CD4+CD25+ T cells (26). In addition, a recent report also suggests that CD4+CD25+ T cells might not be the only source of IL-10 in a model of infectious tolerance with CD4+CD25+ T cells during experimental allergic encephalomyelitis (27). Here, we show for the first time that the percentage of pulmonary CD4+IL-10producing cells is increased after transfer of CD4+CD25+ regulatory T cells in vivo. Although we cannot exclude the possibility that other cells contribute to IL-10 production, we did not find expression in macrophages, dendritic cells, or granulocytes. Interestingly, we found IL-10 production by CD4+ cells was similarly increased after transfer of IL-10deficient CD4+CD25+ regulatory T cells. This implies that CD4+CD25+ regulatory T cells suppress inflammation via induction of IL-10 from CD4+ T cells and that production of IL-10 from the regulatory cells themselves is not required. Thus, our data may reconcile the apparent contradiction in previous in vitro and in vivo reports because suppression in vivo may rely on the induction of IL-10 from recipient CD4+ T cells.
The data presented in this paper suggest that induction of IL-10 in vivo by T regulatory cells may represent a novel treatment for allergic asthma. Indeed, it has been previously demonstrated that nonallergic individuals have a higher percentage of allergen-specific IL-10producing cells, whereas allergic individuals are characterized by a higher percentage of allergen-specific IL-4producing cells (8). Strategies for inducing IL-10 may include expansion of the naturally occurring CD4+CD25+ population or enhancement of regulatory T cell function by drugs such as corticosteroids, as shown previously in vitro (28). Alternatively it may be possible to induce populations of IL-10producing or TGF-ß surface-positive T regulatory cells in vivo, as has been described in several different murine systems using mycobacterial exposure, airway allergen delivery before sensitization and challenge, or in vitro derivation by stimulation in the presence of immunosuppressive drugs (29, 30, 31, 16). Allergen immunotherapy has been used for many years to control symptoms in a variety of allergic diseases, and current data suggest that this may induce IL-10producing regulatory T cell populations (32, 33). Peptide therapy induced IL-10producing regulatory cells in mice (34), and recent data suggest that allergen-derived T cell peptides may also reduce features of asthma in humans through induction of IL-10 (35).
In conclusion, we provide for the first time direct evidence to show that transfer of allergen-specific CD4+ CD25+ T regulatory cells to sensitized mice abrogates the features of allergic airway disease in vivo. These include reduced eosinophil and Th2 cell recruitment, AHR, and Th2 cytokine production. Down-regulation of inflammation was observed with a concomitant increase in pulmonary IL-10 production from CD4+ T cells. Blocking studies showed that the suppressive effect was dependent on IL-10, but, importantly, the effect was not dependent on IL-10 production from the CD4+CD25+ regulatory T cells themselves. Thus, our data suggest that strategies designed to maximize the function of T regulatory cells in vivo could be of benefit to allergic asthmatic patients in the future.
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Materials and Methods |
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Isolation of CD4+CD25+ regulatory T cells
DO11.10 and DO11.10 IL-10deficient mice were killed by cervical dislocation, and OVA-specific CD4+CD25+ cells were isolated from the spleen and peripheral lymph nodes using a CD4+CD25+ regulatory T cell isolation kit according to the manufacturer's protocol (Miltenyi Biotec). Purity was assessed by flow cytometric analysis (as described in Staining of BAL...) and was typically >90%.
In vitro assessment of regulatory capacity
BALB/c mice were sensitized using OVA (Sigma-Aldrich) at a concentration of 0.1 mg/mouse in 0.2 ml alum (Au-Gel-S; SERVA Electrophoresis) i.p. 1 wk later, mice were killed by cervical dislocation. Spleens were recovered from mice, and single-cell suspensions were prepared. Splenocytes were cultured in triplicate at 5 x 105 splenocytes/well in flat-bottomed 96-well plates in the presence of complete media alone or with 100 µg/ml OVA for 48 h. Complete media consisted of RPMI 1640 (Sigma-Aldrich) containing 10% FCS (Invitrogen), 5mM L-glutamine (Invitrogen), and 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich). The cells were incubated for a further 18 h in the presence of 1 µCi/well [3H]thymidine (MP Biochemicals) to quantify cell proliferation. To assess the suppressive capacity of freshly isolated CD4+CD25+ regulatory T cells, these cells were isolated from DO11.10 spleen and peripheral lymph nodes and added at 1.25 x 105 cells/well. To assess the proliferative capacity of isolated CD4+CD25+ cells alone, splenocytes were irradiated at 3,000 rad and cultured with CD4+CD25+ cells. [3H]thymidine incorporation data in cpm was converted to stimulation index (fold increase = cpm with OVA / cpm of medium only condition).
Induction of AHR and eosinophilia
BALB/c mice were sensitized using OVA at a concentration of 0.1 mg/mouse in 0.2 ml alum on day 0. Control mice received the same volume of PBS in alum. On day 6, mice received either 5 x 105 CD4+CD25+ cells intravenously in PBS or the same volume of PBS as a control. In some experiments, OVA-specific CD4+CD25 T cells were injected as a further control. All groups of mice were challenged daily with 5% OVA (aerosolized for 20 min) via the airways between days 7 and 12. In some experiments, mice were dosed with 250 µg antiIL-10R (1B1.3) 30 min before the first, third, and fifth OVA challenge. Mice were killed by exsanguination under terminal anesthesia at 24 h after the final OVA challenge.
Measurement of AHR
Airway responsiveness was measured in mice 24 h after the final OVA challenge by recording respiratory pressure curves by whole-body plethysmography (Buxco Electronics) in response to inhaled methacholine (Sigma-Aldrich) at concentrations of 330 mg/ml for 1 min, as described previously (38). Results are shown for Penh after allergen challenge. Penh results were confirmed by direct measurements of RL and Cdyn in anaesthetized and tracheostomized mice in response to inhaled methacholine at concentrations of 3100 mg/ml in a Buxco system (Buxco Electronics) in a modified version of previously described methods (39).
Cell recovery
Airway lumen.
BAL was performed as previously described (3). In brief, the airways of the mice were lavaged three times with 0.4 ml PBS via a tracheal cannula. BAL fluid was centrifuged at 200 g for 5 min at 4°C. Cells were counted and pelleted onto glass slides by cytocentrifugation (5 x 104 cells/slide). Differential cell counts were performed on Wright-Giemsastained (Thermo) cytospins. Percentages of eosinophils, lymphocyte/mononuclear cells, neutrophils, and macrophages were determined by counting their number in eight randomly selected high power fields (40x; total area = 0.5 mm2/area) and dividing this number by the total number of cells counted. To obtain absolute numbers of each leukocyte subtype, these percentages were multiplied by the total number of cells obtained in the lavage fluid. All differential counts were performed blind and in a randomized order at the end of the study by the same observer (J. Kearley).
Lung parenchyma.
To disaggregate the cells from the lung tissue, one lobe (100 mg) of lung was incubated at 37°C for 1 h in digest reagent (0.15 mg/ml collagenase [type D; Roche], 25 µg/ml DNase [type 1; Roche]) in complete media. The recovered cells were filtered through a 70-µm nylon sieve (Falcon), washed twice, resuspended in complete media, and counted in a hemocytometer (VWR). Cytocentrifuge preparations were prepared and Wright-Giemsa stained, and differential counts were performed as for BAL.
Draining lymph nodes.
Draining lymph nodes were removed, weighed, and filtered through a 70-µm nylon sieve. The resulting single cell suspension was centrifuged at 200 g for 5 min at 4°C, resuspended in complete media, and counted in a hemocytometer.
Lung tissue histopathology
Lungs were fixed in 10% normal buffed formalin. 4-µm paraffin-embedded sections were stained with hemotoxylin and eosin.
Staining of BAL, lung, and lymph node cells for flow cytometric analysis
Suspensions of BAL and lung tissue and lymph node cells were stained in PBS containing 1% FCS and 0.01% sodium azide. To reduce nonspecific binding, cells were incubated with rabbit serum (Sigma-Aldrich) for 15 min before staining. The antibodies used were antimouse CD4, antimouse CD11b, antimouse CD11c, antimouse B220, antimouse CD8, antimouse IL-10 and its isotype control (all obtained from BD Biosciences), antimouse T1/ST2 (Morwell Diagnostics), and antimouse KJ1-26 and its isotype control (Caltag). Cells were stained for 20 min on ice, washed twice, and fixed in cellfix (Becton Dickinson). For intracellular cytokine staining, cells were stimulated with PMA/ionomycin (Merck) in the presence of Brefeldin A (Sigma-Aldrich) for 6 h before extracellular staining. After staining and fixing, cells were permeabilized using 0.5% saponin (Sigma-Aldrich) and stained with antiIL-10 antibody or the appropriate isotype control. Flow cytometric analysis was performed using a FACSCalibur (Becton Dickinson).
Cytokine analysis
Cytokines were analyzed in BAL samples and lung-tissue homogenate supernatants. Lung tissue was homogenized at 50 mg/ml in HBSS (Invitrogen) and centrifuged at 800 g for 10 min and the supernatant wascollected. Paired antibodies for murine IFN- and TGF-ß (BD Biosciences), IL-5 (Endogen), eotaxin/CCL11, and TARC/CCL17 (R&D Systems) were used in standardized sandwich ELISAs according to the manufacturer's protocol. MDC/CCL22 levels were measured using a sandwich ELISA generated by coating ELISA plates with antimouse MDC (a gift from ICOS Corp., Bothell, WA) and detecting bound antibody with biotinylated antimouse MDC (R&D Systems) against a standard curve generated using recombinant MDC (R&D Systems) as previously described (40). ELISA kits to measure IL-13 and IL-10 were purchased from R&D Systems and eBioscience, respectively, and were used according to the manufacturer's protocol.
IgE
Levels of total IgE were measured in serum by ELISA using paired antibodies according to the manufacturer's instructions (BD Biosciences). Levels of anti-OVA IgE were measured in serum and lung homogenate supernatant by ELISA as described previously (41).
Data analysis
Data are expressed as medians unless otherwise stated. Statistical significance between groups was tested using a Mann-Whitney U Test. A p-value of <0.05 was considered significant. Graph generation and statistical analysis were performed by using Prism software (version 4.00; GraphPad).
Online supplemental material
Fig. S1 shows that allergen-specific CD4+CD25+ regulatory T cells suppress allergen-induced proliferation in vitro. Fig. S2 shows that allergen-specific CD4+CD25+ regulatory T cells are able to migrate to the airway lumen, lung tissue, and draining lymph nodes in vivo. Fig. S3 shows that transfer of CD4+CD25+ regulatory T cells suppresses changes in RL and Cdyn. Fig. S4 shows that proinflammatory chemokine levels in the lung are unaffected by transfer of CD4+CD25+ regulatory T cells. Fig. S5 shows that transfer of CD4+CD25+ regulatory T cells has no effect on serum IgE levels.
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Acknowledgments |
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This work was supported by the Wellcome Trust (057704) and AstraZeneca. J. Kearley is supported by a Biotechnology and Biological Sciences Research Council/Cooperative Award in Science and Engineering studentship, D.S. Robinson is supported by a Wellcome Trust Research Leave Award for Clinical Academics, and C.M. Lloyd is supported by a Wellcome Senior Fellowship in Basic Biomedical Sciences.
The authors have no conflicting financial interests.
Submitted: 9 June 2005
Accepted: 18 October 2005
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References |
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