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Cutting Edge: IL-4-Induced Protection of CD4⁺CD25^– Th Cells from CD4⁺CD25⁺ Regulatory T Cell-Mediated Suppression¹

Luigia Pace^*, Stefania Rizzo^*, Cecilia Palombi^*, Frank Brombacher and Gino Doria^2,*

^* Department of Biology, University of Rome Tor Vergata, Rome, Italy; and Institute for Infectious Diseases and Molecular Medicine, and Division of Immunology, Health Science Faculty, University of Cape Town, South Africa

	Abstract

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CD4⁺CD25⁺ T regulatory (Treg) cells are a CD4⁺ T cell subset involved in the control of the immune response. In vitro, murine CD4⁺CD25⁺ Treg cells inhibit CD4⁺CD25^– Th cell proliferation induced by anti-CD3 mAb in the presence of APCs. The addition of IL-4 to cocultured cells inhibits CD4⁺CD25⁺ Treg cell-mediated suppression. Since all cell types used in the coculture express the IL-4R chain, we used different combinations of CD4⁺CD25^– Th cells, CD4⁺CD25⁺ Treg cells, and APCs from wild-type IL-4R^+/+ or knockout IL-4R^–/– mice. Results show that the engagement of the IL-4R chain on CD4⁺CD25^– Th cells renders these cells resistant to suppression. Moreover, the addition of IL-4 promotes proliferation of IL-4R^+/+CD4⁺CD25⁺ Treg cells, which preserve full suppressive competence. These findings support an essential role of IL-4 signaling for CD4⁺CD25^– Th cell activation and indicate that IL-4-induced proliferation of CD4⁺CD25⁺ Treg cells is compatible with their suppressive activity.

	Introduction

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Efficient and protective immune responses are induced by APCs and result from the balanced participation of T cells with antithetic functions, such as CD4⁺CD25^– Th cells and CD4⁺CD25⁺ T regulatory (Treg)³ cells, whereas their abnormal involvement is associated with pathology. CD4⁺CD25⁺ Treg cells naturally arise in the thymus or are Ag-induced in the peripheral tissues and constitute 5–10% of the peripheral pool of CD4⁺ T cells in mice and humans. CD4⁺CD25⁺ Treg cells contribute to maintain peripheral tolerance and to prevent a number of immune-mediated diseases by suppressing immune responses to allo- and autoantigens, including tumor Ags (1).

Upon naive Th cells activation, the pattern of cytokines produced plays an important role in the regulation of the immune response. IL-4 is a pleiotropic cytokine that participates in major regulatory mechanisms of the immune response. Yet, the effects of IL-4 on autoimmune diseases have generated controversial results. In systemic lupus erythematosus, experimental allergic encephalomyelitis, cartilagen-induced arthritis, and proteoglycan-induced arthritis, IL-4 may promote the expansion of the autoreactive Th cell population (2, 3). This cytokine supports survival and proliferation of both CD4⁺CD25^– Th and CD4⁺CD25⁺ Treg cells in vitro (4). The cell survival induced by IL-4 results from the ability of this cytokine to sustain the expression of the anti-apoptotic factor Bcl-2 (5). Our recent studies indicate that CD4⁺CD25⁺ Treg cells inhibit the expression of Bcl-2 in cocultured CD4⁺CD25^– Th cells. The addition of IL-4 at the time of in vitro priming of CD4⁺CD25^– Th cells cocultured with CD4⁺CD25⁺ Treg cells profoundly affects the overall expansion of the responding CD4⁺CD25^– Th cell population by favoring the survival and proliferation of these cells through the induction of Bcl-2 expression (4).

Since the IL-4R chain is expressed on different cell types, the possibility of molecular and cellular cross-regulation cannot be excluded. The IL-4R chain, indeed, is expressed by CD4⁺CD25^– Th cells, CD4⁺CD25⁺ Treg cells, and APCs (4). It follows that treatment of cell cocultures with IL-4 yields results of equivocal interpretation as to which cell type is affected by this cytokine. To overcome this difficulty, the present work analyzed the effect of IL-4 on APC-activating function, CD4⁺CD25^– Th cell sensitivity to suppression, and CD4⁺CD25⁺ Treg cell suppressive activity in coculture experiments using combinations of CD4⁺CD25^– Th cells, CD4⁺CD25⁺ Treg cells, and APCs from wild-type IL-4R^+/+ or knockout IL-4R^–/– mice. Results show that IL-4 preserves CD4⁺CD25^– Th cell proliferation in the presence of CD4⁺CD25⁺ Treg cells and demonstrate that IL-4 promotes CD4⁺CD25⁺ Treg cell proliferation without interfering with their suppressive activity.

	Materials and Methods

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Mice

All mice were of the C57BL/6 strain. IL-4R^–/– mice (6) were backcrossed for nine generations to the C57BL/6 strain. Mice were housed at the Instituto Superiore di Santà facilities under pathogen-free conditions. Eight- to 12-wk-old females were used.

Cell purification

All cell subsets were purified by immunomagnetic cell sorting (Miltenyi Biotec). APCs were prepared by depletion of CD90⁺ cells. CD4⁺ T cells were enriched by using the MACS MultiSort kit. CD4⁺CD25⁺ Treg cells were purified after staining CD4⁺ T cells with biotinylated anti-CD25 (7D4; BD Pharmingen) and streptavidin microbeads. Collected cells were found to be 95–99% pure by flow cytometry.

Cell sorting

CD4⁺CD25^– Th or CD4⁺CD25⁺ Treg cells were labeled with 5 µM CFSE (Molecular Probes) for 5 min at room temperature. Cells were washed and set up in culture. After 3 days, cells were analyzed by flow cytometry. CFSE⁺ Th cells were isolated by a FACSVantage cell sorter (BD Biosciences). Purity of FACS-sorted cells was 99%.

Proliferation assay

Along with mitomycin C (MMC)-treated T-depleted spleen cells (5 x 10⁴) as APCs, CD4⁺CD25^– Th cells (2.5 x 10⁴) were cultured for 3 days in the presence of CD4⁺CD25⁺ Treg cells in 96-well round-bottom plates in the presence of 1 µg/ml anti-CD3 mAb (145-2C11; BD Pharmingen). IL-4 (10 ng/ml, 404-ML; R&D Systems) or IL-2 (2 ng/ml; BD Pharmingen) was added at the beginning of culture. [³H]TdR (1 µCi/well) incorporation was measured after the last 4 h of culture by a Packard Matrix 96 Direct Beta Counter. The degree of CD4⁺CD25^– Th cell proliferation in the presence of CD4⁺CD25⁺ Treg cells was defined as 100 x [(cpm of the mixed CD4⁺CD25^– Th and CD4⁺CD25⁺ Treg cell populations)/cpm of CD4⁺CD25^– Th cells]. SE of the ratio between means was calculated as described elsewhere (4).

Cytokine titration

IL-2 was titrated in culture supernatants by ELISA (Endogen). Avidin-peroxidase (Sigma-Aldrich) was then added. Thereafter, ABTS substrate (Kirkegaard and Perry Laboratories) was added and absorbance was measured at 405 nm.

	Results

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IL-4-mediated inhibition of suppression requires IL-4R chain expression by CD4⁺CD25^– Th cells

Since the IL-4R chain is expressed on all cell types involved in the in vitro coculture assay, the mechanism of suppression inhibition by IL-4 has been disentangled by using CD4⁺CD25^– Th cells, CD4⁺CD25⁺ Treg cells, and APCs from IL-4R^+/+ or IL-4R^–/– mice, in mix and match experiments, according to a matrix of eight IL-4-treated or -untreated cell combinations. CD4⁺CD25^– Th and CD4⁺CD25⁺ Treg cells were normally distributed in peripheral lymphoid tissues regardless of the IL-4R chain expression (data not shown). Results reported in Fig. 1 (upper left panel) represent percentages of total cell proliferation by comparing the ability of CD4⁺CD25⁺ Treg cells from IL-4R^+/+ or IL-4R^–/– mice to suppress IL-4R^+/+CD4⁺CD25^– Th cells cocultured with IL-4R^+/+ APCs in the presence or absence of added IL-4. CD4⁺CD25⁺ Treg cells from both types of mice exerted comparable suppressive activity. The addition of IL-4 to cocultures of IL-4R^+/+CD4⁺CD25^– Th cells, IL-4R^+/+CD4⁺CD25⁺ Treg cells, and IL-4R^+/+APCs resulted in increased cell proliferation. Moreover, when IL-4R^+/+CD4⁺CD25^– Th cells were cocultured with IL-4R^–/–CD4⁺CD25⁺ Treg cells, the addition of IL-4 led to enhancement of T cell proliferation similar to that seen when both subsets expressed the IL-4R chain.

View larger version (29K): [in this window] [in a new window]   FIGURE 1. IL-4R chain expression by CD4+CD25– Th but not CD4+CD25+ Treg cells is required to inhibit suppression. CD4+CD25– Th cells from IL-4R+/+ (upper panels) or IL-4R–/– (lower panels) mice were cocultured in triplicate with anti-CD3 mAb in the presence of IL-4R+/+ APCs (left panels) or IL-4R–/– APCs (right panels) in the absence ( and ) or presence (• and ) of IL-4. The indicated number of CD4+CD25+Treg cells from IL-4R+/+ (• and ) or IL-4R–/– ( and ) mice were added to the coculture. [3H]TdR uptake was assessed 3 days later. Results are expressed as percentage of T cell proliferation. Results from one of three independent experiments. wt, Wild type; ko, knockout.

Furthermore, results in Fig. 1 (upper and lower right panels) exclude the role of the IL-4R chain on APCs in IL-4-mediated inhibition of CD4⁺CD25⁺ Treg cell suppressive activity.

IL-4 promotes proliferation of CD4⁺CD25⁺ Treg cells without interfering with their suppressive activity

As described above, IL-4 protects CD4⁺CD25^– Th cells from CD4⁺CD25⁺ Treg cell-mediated suppression, since, in the presence of IL-4, IL-4R^+/+CD4⁺CD25^– Th cells are refractory to the suppressive activity of IL-4R^–/–CD4⁺CD25⁺ Treg cells.

Unexpectedly, T cell proliferation was observed when IL-4R^–/–CD4⁺CD25^– Th cells were cocultured with IL-4R^+/+CD4⁺CD25⁺ Treg cells in the presence of IL-4. As IL-4 promotes IL-4R^+/+CD4⁺CD25⁺ Treg cell proliferation (4), in these cocultures suppression is not appraised by the only measurement of [³H]TdR incorporation, and the results remain of equivocal interpretation. This is why we then examined the relative contribution of each cell subset to the total cell proliferation by analysis of the cell division marker CFSE. Before coculture only one cell subset (CD4⁺CD25^– Th or CD4⁺CD25⁺ Treg) was stained with CFSE. Experiments were performed using IL-4R^–/– APCs (Fig. 2). In the absence of IL-4, CFSE-labeled IL-4R^+/+CD4⁺CD25^– Th (CFSE⁺ Th) cells mixed with CFSE^–CD4⁺CD25⁺ Treg cells showed reduced cell cycle progression (Fig. 2A, left). At variance, the addition of IL-4 blocked suppression of IL-4R^+/+CFSE⁺ Th cells and restored cell cycle progression to near normal levels (Fig. 2A, right).

View larger version (33K): [in this window] [in a new window]   FIGURE 2. Effects of IL-4 on CD4+CD25– Th cell proliferation and CD4+CD25+ Treg cell suppressive activity. IL-4R+/+ or IL-4R–/–CD4+CD25– Th (A and C) or CD4+CD25+ Treg (B and D) cells were labeled with CFSE before coculture. The cell subset labeled with CFSE is marked with an asterisk. For each panel, we indicated the cocultured cell subsets (left side). IL-4R+/+ (A and B) IL-4R–/– (C and D) CD4+CD25– Th cells either alone or mixed with an equal number of IL-4R+/+ or IL-4R–/–CD4+CD25+ Treg cells were cultured in the presence of IL-4R–/– APCs and anti-CD3 mAb with (right panels) or without (left panels) addition of IL-4 for 3 days, and then analyzed by flow cytometry. Results of one from three independent experiments. wt, Wild type; ko, knockout.

We also performed CFSE analysis on IL-4R^+/+ or IL-4R^–/–CD4⁺CD25⁺ Treg (CFSE⁺ Treg) cells cocultured with CFSE^–IL-4R^+/+ or IL-4R^–/–CD4⁺CD25^– Th cells (Fig. 2, B and D). In the presence of IL-4, IL-4R^+/+CFSE⁺ Treg cells proliferate. IL-4R^+/+CFSE⁺ Treg cell proliferation was found relatively greater when these cells were cocultured in the presence of IL-4R^–/–CD4⁺CD25^– Th cells. Thus, IL-4 promotes proliferation of IL-4R^+/+CD4⁺CD25⁺ Treg cells without interfering with their suppressive activity.

CD4⁺CD25⁺ Treg cells inhibit IL-2 production by CD4⁺CD25^– Th cells in the presence of IL-4

Beside suppressing CD4⁺CD25^– Th cell proliferation, CD4⁺CD25⁺ Treg cells have been shown to inhibit IL-2 production. Because suppression of IL-2 production has been observed also in the presence of proliferating CD4⁺CD25^– Th cells and CD4⁺CD25⁺ Treg cells (7), we subsequently analyzed IL-2 production in culture supernatants collected from the cocultures described in Fig. 1. Results in Fig. 3 show that in all cocultures, IL-2 production was inhibited.

View larger version (29K): [in this window] [in a new window]   FIGURE 3. In the presence of IL-4, CD4+CD25+ Treg cells inhibit IL-2 production. IL-2 concentration was measured in supernatants collected from cocultures described in Fig. 1 legend. CD4+CD25– Th cells from IL-4R+/+ (upper panels) or IL-4R–/– (lower panels) mice were cocultured in triplicate with anti-CD3 mAb in the presence of IL-4R+/+APCs (left panels) or IL-4R–/– APCs (right panels) in the presence of IL-4R+/+ Treg cells ( ); IL-4R+/+ Treg cells plus IL-4 (); IL-4R–/– Treg cells (); and IL-4R–/– Treg cells plus IL-4 (). Treg cells alone (2.5 x 104), cultured with APCs and anti-CD3 mAb, were used as control. Data are the mean ± SE from one of three independent experiments. wt, Wild type; ko, knockout.

IL-4R^+/+ but not IL-4R^–/–CD4⁺CD25^– Th cells escape from the CD4⁺CD25⁺ Treg cell-induced unresponsiveness

Our results demonstrate that, although IL-4R^+/+CD4⁺CD25^– Th cells cocultured with CD4⁺CD25⁺ Treg cells proliferate in the presence of IL-4, CD4⁺CD25⁺ Treg cells still retain their suppressive activity to inhibit IL-2 production. Several studies sustain a central role of IL-2 in T cell responses, as this cytokine may control the development and subsequent contraction of Ag-specific cell proliferation. However, more recent data demonstrate that Th cells may proliferate also in a IL-2R- independent manner (8). With regard to CD4⁺CD25⁺ Treg cell suppressive function, the inhibition of IL-2 production should not represent the only mechanism by which suppressor cells interfere with immunity. Since CD4⁺CD25⁺ Treg cells can suppress the autoimmune response of IL-2^–/– or IL-2R-deficient T cells (9, 10), it excludes the possibility that competition for IL-2 or suppression of IL-2 transcription represents an essential mechanism of suppression in vivo (11). Based on these considerations, the analysis of IL-2 production alone may be insufficient to account for the suppressive activity of CD4⁺CD25⁺ Treg cells. This is why we decided to analyze in more detail the effect of CD4⁺CD25⁺ Treg cells on CD4⁺CD25^– Th cell proliferation. We evaluated CD4⁺CD25^– Th cell responsiveness to secondary culture after an initial coincubation with CD4⁺CD25⁺ Treg cells with or without the addition of IL-4. To this end, CD4⁺CD25^– Th cells were stained with the cell division marker CFSE (CFSE⁺ Th cells) before coculture. Thereafter, IL-4R^+/+ or IL-4R^–/–CFSE⁺ Th cells were coincubated with IL-4R^+/+ or IL-4R^–/–CFSE^–CD4⁺CD25⁺ Treg cells and IL-4R^–/–CFSE^– APCs in medium supplemented or not with IL-4 as indicated in Fig. 4. After 3 days of primary coculture, CFSE⁺ Th cells were FACS sorted based on their CFSE signal and analyzed for their proliferative response to TCR and/or IL-2 stimulation, in the presence of MMC-treated IL-4R^+/+ splenocytes as APCs. If the major effect of CD4⁺CD25⁺ Treg cells was to suppress IL-2 production by CD4⁺CD25^– Th cells, thus leading to inhibition of cell proliferation, than sorted CFSE⁺ Th cells should normally proliferate upon secondary restimulation with anti-CD3 mAb alone or combined with IL-2. Relevant results are reported in Fig. 4. The data show that FACS-sorted CFSE⁺ Th cells primed in the presence of APCs normally proliferate in response to IL-2 and/or TCR stimulation. Conversely, IL-4R^–/–CFSE⁺ Th cells cocultured with either IL-4R^+/+ or IL-4R^–/– CD4⁺CD25⁺ Treg cells in medium supplemented or not with IL-4 were profoundly impaired in their ability to proliferate in response to anti-CD3 mAb and/or IL-2 as compared with IL-4R^+/+ or IL-4R^–/–CFSE⁺ Th cells cultured in the presence of IL-4 during priming. Similar results were obtained when IL-4R^+/+CFSE⁺ Th cells were cocultured with IL-4R^–/–CD4⁺CD25⁺ Treg cells and subsequently restimulated. Thus, addition of IL-2 or TCR stimulation failed to recover the CFSE⁺ Th cell proliferative response, suggesting that responsiveness to both TCR and IL-2 stimulations are blocked after the CD4⁺CD25⁺ Treg cell encounter. Moreover, although IL-4 promotes proliferation of CD4⁺CD25⁺ Treg cells, these cells retain their ability to suppress IL-4R^–/–CFSE⁺ Th cells, which are refractory to restimulation.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. IL-4 protects IL-4R+/+ but not IL-4R–/–CD4+CD25– Th cells from CD4+CD25+ Treg cell-induced unresponsiveness. CFSE+IL-4R–/– Th cells alone or mixed with an equal number of CFSE–IL-4R+/+ or IL-4R–/– CD4+CD25+ Treg cells, or CFSE+ IL-4R+/+ Th cells alone or mixed with an equal number of CFSE–IL-4R–/–CD4+CD25+ Treg cells were cocultured in the presence of IL-4R–/– APCs and anti-CD3 mAb with or without IL-4 as indicated in the primary culture conditions. After 3 days, CFSE+ Th cells were FACS sorted. Thereafter, FACS-sorted CFSE+ Th cells were cultured in triplicate for 3 days in the presence of MMC-treated IL-4R+/+ splenocytes as APCs, with anti-CD3 mAb (), IL-2 ( ), or anti-CD3 mAb and IL-2 (). Levels of proliferation are measured as cpm ± SE. Results of one of six independent experiments. wt, Wild type; ko, knockout.

These findings suggest that inhibition and subsequent deprivation of IL-2 production by Th cells cannot be considered as the only mechanism of inhibition of Th cell proliferation, but other mechanisms induced by CD4⁺CD25⁺ Treg cells should be responsible for the block of the Th cell proliferative response (12).

	Discussion

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We have previously shown that IL-4 inhibits CD4⁺CD25⁺ Treg cell-mediated-suppression, but the mechanism and detailed cell analysis of the IL-4-mediated block of suppression remained elusive (4). Because IL-4R^–/– mice lack activation by IL-4 signaling, the use of cells from IL-4R^–/– mice provided the opportunity to identify the cellular target in IL-4-induced protection from CD4⁺CD25⁺ Treg cell-mediated suppression. We used different combinations of CD4⁺CD25^– Th cells, CD4⁺CD25⁺ Treg cells, or APCs from either wild-type or knockout mice at the IL-4R locus, cocultured in the presence or absence of added IL-4. The present results demonstrate that IL-4 protects IL-4R^+/+ but not IL-4R^–/–CD4⁺CD25^– Th cells from CD4⁺CD25⁺ Treg cell-mediated suppression, and the former cells proliferate to normal levels when subsequently restimulated. At variance, CD4⁺CD25^– Th cells cocultured with CD4⁺CD25⁺ Treg cells in the absence of IL-4 are unable to proliferate upon secondary stimulation. Nonetheless, CD4⁺CD25⁺ Treg cells retain full competence to inhibit IL-2 production. As previously demonstrated (4), the addition of CD4⁺CD25⁺ Treg cells to CD4⁺CD25^– Th cells cultured in the presence of IL-4 also decreased the number of cultured IL-4-producing Th cells, with an increase in IFN- production. Based on these findings, at least two distinct pathways of suppression have been identified: 1) the inhibition and subsequent block of Th cell proliferation, which is prevented by the addition of IL-4; and 2) the suppression of IL-2 and IL-4 production, which occurs also in the presence of IL-4. The inhibition of these cytokines might represent a feedback mechanism exerted by CD4⁺CD25⁺ Treg cells to counteract the mitogenic effects exerted by exogenous IL-4.

Because IL-4 promotes CD4⁺CD25⁺ Treg cell proliferation (4), we were interested in investigating whether, in the presence of IL-4, CD4⁺CD25⁺ Treg cell suppressive activity was preserved. Our data reveal that upon IL-4 stimulation, CD4⁺CD25⁺ Treg cells proliferate without loosing their ability to suppress IL-4R^–/–CD4⁺CD25^– Th cell proliferation. Thus, proliferation and suppressive function of CD4⁺CD25⁺ Treg cells are not always mutually exclusive in vitro, confirming results already obtained in vivo (11).

It is well known that some cytokines may be involved in lymphoid homeostasis by modulating cell survival, growth, differentiation, and apoptosis. Namely, IL-2, IL-4, IL-7, and IL-15, that signal through receptors sharing the common -chain (_c), have been shown to promote lymphoid cell proliferation and development in vivo (5). Of note, similar to IL-4, IL-2 and IL-7 also have been described as interfering with CD4⁺CD25⁺ Treg cell suppressive function (13). Noteworthily, among the _c cytokine family, IL-2 is necessary to maintain peripheral CD4⁺CD25⁺ Treg cells. In the absence of IL-2 signal, IL-2^–/– or IL-2R^–/– mice have reduced frequencies of peripheral foxp3⁺ Treg cells. Conversely, no foxp3⁺ Treg cells are detectable in _c-deficient mice, indicating that _c signals are absolutely required for CD4⁺CD25⁺ Treg cell development and that other _c family cytokines are able to partially compensate for the absence of IL-2 in IL-2^–/– or IL-2R^–/– mice. The functional competence of IL-2^–/– or IL-2R^–/–CD4⁺CD25⁺ Treg cells is provided by a less severe lymphoproliferative autoimmune syndrome and greatly extended life span of these mice as compared with foxp3^–/– mice (14). The _c family cytokines able to compensate for IL-2 are unknown. Our data suggest IL-4 as a possible candidate in vitro (4). Since IL-4^–/– and IL-4R^–/– mice normally develop CD4⁺CD25⁺ Treg cells, it is clear that the major contribution of IL-4 to maintenance and expansion of CD4⁺CD25⁺ Treg cells should become relevant after immune activation and better evidenced in type-2 responses, because under these conditions the effect of IL-4 is dominant.

The results presented herein stress the role of IL-4 as growth factor since IL-4 enhances TCR-induced cell proliferation and protects CD4⁺CD25^– Th cells from suppression. These results are relevant to questions concerning the contribution of IL-4 to some autoimmune diseases, as IL-4 could have opposite effects on the pathogenesis of the autoimmune disease according to the disorder stage and cells involved (2). The first critical evidence for the role of IL-4 in the pathogenesis of autoimmune disease stems from the observation that constitutive expression of IL-4 causes autoimmune-type disorders in murine multiple organs (15). This finding was followed by more detailed studies. There is evidence suggesting that IL-4 has limited capacity to inhibit IL-4NODBDC2.5 double-transgenic cells to develop insulin-dependent diabetes mellitus (16). Moreover, contrary to expectation, lack of the IL-4R chain exerted a very significant protective effect on the frequency of insulin-dependent diabetes mellitus (17). Other works on both cartilagen-induced arthritis (3) and experimental allergic encephalomyelitis (18) models have shown that Th2 responses may exacerbate the autoimmune disease, rather than displaying a protective function. These findings suggest that IL-4 could activate the autoreactive T cell repertoire, triggering the effector phase of autoimmune diseases and worsen the clinical outcome.

So far there is a widespread acceptance of the immunomodulatory role of CD4⁺CD25⁺ Treg cells as a major tolerance-inducing mechanism that can suppress the response of other pathogenic immune cells (1). Yet, the relative contribution of CD4⁺CD25^– Th and CD4⁺CD25⁺ Treg cells to immunity or tolerance is also influenced by a fine tuning of the local cytokine environment at the site of Ag recognition. Upon activation, leukocytes synthesize and secrete cytokines, including IL-4. These cytokines are growth and differentiation factors, so that naive T cells proliferate and differentiate into effector T cells. However, in some pathological conditions characterized by aberrant T cell activation, IL-4 might promote autoreactive CD4⁺CD25^– Th cell expansion by favoring their proliferation and protecting them from CD4⁺CD25⁺ Treg cell-mediated suppression, thus leading to the dangerous effects of IL-4 described in the aforementioned autoimmune diseases. Likewise, the IL-4 inhibition of CD4⁺CD25⁺ Treg cell-mediated suppression is also expected to worsen allergic diseases.

Altogether, our results demonstrate a multiplicity of IL-4 activities on CD4⁺CD25^– Th cell proliferation and CD4⁺CD25⁺ Treg cell-mediated suppression after TCR stimulation. These findings of the effects of IL-4 on the regulatory mechanism of Ag-driven T cell proliferation need to be examined in vivo to fully understand the clinical relevance of IL-4 manipulation in autoimmunity and tolerance.

	Acknowledgments

 
We are grateful to R. Carsetti and E. Giorda for cell sorting.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Project 1AN/F11 from Istituto Superiore de Sanité.

² Address correspondence and reprint requests to Prof. Gino Doria, Department of Biology, University of Rome Tor Vergata, Via Ricerca Scientifica, 00133 Rome, Italy. E-mail address: gino.doria{at}uniroma2.it

³ Abbreviations used in this paper: Treg, regulatory T; MMC, mitomycin C; _c, common -chain; foxp3, forkhead box protein P3.

Received for publication November 4, 2005. Accepted for publication February 1, 2006.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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