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IL-4 Modulation of CD4⁺CD25⁺ T Regulatory Cell-Mediated Suppression¹

Luigia Pace^*, Claudio Pioli and Gino Doria^2,*

^* Laboratory of Immunology, Department of Biology, University of Rome Tor Vergata, Italy; and Ente Nazionale Energie Alternative, Unit of Biotechnology, Section of Toxicology and Biomedicine, Rome, Italy

	Abstract

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Murine CD4⁺CD25⁺ T regulatory (Treg) cells were cocultured with CD4⁺CD25^– Th cells and APCs or purified B cells and stimulated by anti-CD3 mAb. Replacement of APCs by B cells did not significantly affect the suppression of CD4⁺CD25^– Th cells. When IL-4 was added to separate cell populations, this cytokine promoted CD4⁺CD25^– Th and CD4⁺CD25⁺ Treg cell proliferation, whereas the suppressive competence of CD4⁺CD25⁺ Treg cells was preserved. Conversely, IL-4 added to coculture of APCs, CD4⁺CD25^– Th cells, and CD4⁺CD25⁺ Treg cells inhibited the suppression of CD4⁺CD25^– Th cells by favoring their survival through the induction of Bcl-2 expression. At variance, suppression was not affected by addition of IL-13, although this cytokine shares with IL-4 a receptor chain. When naive CD4⁺CD25^– Th cells were replaced by Th1 and Th2 cells, cell proliferation of both subsets was equally suppressed, but suppression was less pronounced compared with that of CD4⁺CD25^– Th cells. IL-4 production by Th2 cells was also inhibited. These results indicate that although CD4⁺CD25⁺ Treg cells inhibit IL-4 production, the addition of IL-4 counteracts CD4⁺CD25⁺ Treg cell-mediated suppression by promoting CD4⁺CD25^– Th cell survival and proliferation.

	Introduction

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The adaptive immune response results from the interplay among APCs, CD4⁺CD25^– Th cells, and CD4⁺CD25⁺ T regulatory (Treg)³ cells. CD4⁺CD25⁺ Treg cells, a subset of CD4⁺ T cells expressing the IL-2R -chain (CD25) and the transcription factor Forkhead/winged helix transcription factor have recently become a major focus of interest. These cells contribute to maintain peripheral tolerance (1) and to prevent a number of immune-mediated diseases by suppressing immune responses to alloantigens (2) and autoantigens (3), including tumor antigens (4). CD4⁺CD25⁺ Treg cells arise in the thymus during ontogeny and constitute 5–10% of the peripheral CD4⁺ T cells in normal mice (5). Several lines of evidence suggest that CD4⁺CD25⁺ Treg cells control the expansion of the peripheral pool of CD4⁺CD25^– Th cells (6, 7). CD4⁺CD25⁺ Treg cells suppress TCR-induced proliferation of CD4⁺CD25^– Th cells in vitro by cell-to-cell interaction in the presence of APCs. CD4⁺CD25⁺ Treg cells are activated via TCR signals, whereas accessory molecules, such as CTLA-4, CD28, and glucocorticoid-induced TNF receptor family-related receptors, IL-2 and IL-6 cytokines, contribute to their activation and proliferation, hence tuning the intensity of suppression (8). Although a good deal of data demonstrates the negative effect of CD4⁺CD25⁺ Treg cells on CD4⁺CD25^– Th cell activation and function, the molecular mechanisms involved have not been elucidated.

The APC activation state influences CD4⁺CD25⁺ Treg cell-mediated suppression (9), and several observations suggest that immature DCs are involved in the control of autoimmune diseases and the maintenance of tolerance by promoting CD4⁺CD25⁺ Treg cell-mediated suppression (10, 11). Resting B cells are semiprofessional APCs that constitute the large majority of APCs in the spleen. Although experiments with µ-chain-deficient mice have suggested that B cells play a significant role in the regulation of the splenic CD4⁺CD25⁺ Treg cell pool in vivo (12), yet there is no direct evidence of B cell involvement in promoting CD4⁺CD25⁺ Treg cell proliferation and suppressive activity.

It is well established that IL-4 and IL-13 are associated with Th2-type responses, because they may promote humoral reactions and counteract cell-mediated immunity. These cytokines have broadly similar effects; they share a receptor chain (IL-4R -chain), but differ in the range of the target cells involved. IL-4 acts in the immune system using two receptor types. The type I receptor consists of the IL-4R -chain (IL-4R) in association with the common cytokine receptor -chain. The type II receptor is composed of IL-4R in association with IL-13R1. Although B cells, macrophages, and mast cells express both IL-4R types I and II, T cells express only IL-4R I. IL-4 is able to bind both receptors, whereas IL-13 signaling is restricted to IL-4R type II (13).

Successful (effective and not harmful) immune responses are achieved by an appropriate balance between activated CD4⁺CD25^– Th and CD4⁺CD25⁺ Treg cells, whereas their abnormal involvement is associated with pathology (8). Several studies suggest that inappropriate IL-4 production abrogates transplantation tolerance and worsens autoimmune diseases, implying that IL-4 may be involved in CD4⁺CD25⁺ Treg cell-mediated suppression. For instance, the use of mice genetically deficient in IL-4 (IL-4^–/–) has indicated the requirement for IL-4 in the regulatory mechanism of cardiac transplantation tolerance (14). Similarly, intragraft IL-4-gene transfer enhances the tolerogenic effects of systemic infusion of CD4⁺CD25⁺ Treg cells (15). Other studies, however, have provided conflicting results, suggesting that down-regulation of Th2 cytokines, including IL-4, is associated with prolonged cardiac allograft survival (16). Several studies have addressed the effects of IL-4 in various organ-specific autoimmune diseases. Although Th2 cytokines may inhibit autoimmune diseases, such as autoimmune diabetes (17), yet proteoglycan-induced arthritis (18) and, to some extent, systemic lupus erythematosus (SLE) and experimental allergic encephalomyelitis (19) are examples of Th2 cells contributing to the disease (20). In contrast, other studies demonstrate an irrelevant role of IL-4 to prevent or worsen autoaggressive T cell responses. In a mouse model of colitis, IL-4 appears to play no demonstrable role in either the development or the effector function of Treg cells, because CD4⁺CD45Rb^low Treg cells generated in IL-4^–/– mice were as potent as wild-type cells in preventing colitis (21). Furthermore, CD4⁺CD25⁺ Treg cells generated in IL-4^–/– mice were fully competent to inhibit CD4⁺CD25^– Th cell proliferation in vitro (22). These observations taken together suggest a conflicting involvement of IL-4 in transplantation tolerance and in some autoimmune diseases and indicate the need for additional investigations to understand the pivotal role of IL-4 in CD4⁺CD25⁺ Treg cell-mediated suppression.

In the present study we analyzed CD4⁺CD25⁺ Treg cell-suppressive activity on the survival and proliferation of CD4⁺CD25^– Th cells cocultured with APCs or purified B cells. With regard to the role of IL-4, this cytokine was found to act as a growth factor, favoring the survival of CD4⁺CD25^– Th cells through the induction of Bcl-2 expression. Actually, IL-4 added to the coculture assay inhibited suppression of CD4⁺CD25^– Th cell proliferation in a dose-dependent manner. Suppression was not affected if IL-4 was replaced by IL-13.

	Materials and Methods

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Mice

Eight- to 12-wk-old female C57BL/6 mice were obtained from Charles River Laboratories and maintained under pathogen-free conditions.

Abs and reagents

The following Abs and secondary reagents (PE-conjugated or biotinylated) were purchased from BD Pharmingen: anti-CD25 (7D4), anti-CD3 (145-2C11), anti-CD28 (37.51), anti-IL-4 (11B11), anti-IFN- (XMG1.2), anti-CD16/32 Fc Block (2.4G2), and isotype controls. Anti-IL-4R (sc-686) and anti-Bcl-2 (sc-492) were purchased from Santa Cruz Biotechnology. Anti--actin (AC-15) was obtained from Sigma-Aldrich. The following cytokines were purchased from R&D Systems: IL-4 (404-ML), IL-13 (413-ML), and IL-12 (419-ML). IL-2 (550069) was purchased from BD Pharmingen. All reagents used in cell culture were of no azide/low endotoxin quality.

Cell purification

All cell subsets were purified by immunomagnetic cell sorting (Miltenyi Biotec) according to the manufacturer’s instructions. RBC-lysed splenocytes were washed and enriched for B cells using anti-CD19 (130-052-201) microbeads and VarioMACS magnetic separation system. APCs were prepared by depletion of CD90⁺ cells using anti-CD90 microbeads (130-049-101). CD4⁺ T cell subsets were enriched using the MACS MultiSort kit (130-090-860). CD4⁺CD25⁺ Treg cells were purified after staining CD4⁺ T cells with biotinylated anti-CD25 and streptavidin microbeads (130-090-860). Naive CD4⁺ T cells were purified using anti-CD62L microbeads (130-049-701). Collected cells were found to be 95–99% pure by flow cytometry analysis.

Cell sorting

After 3 days of coculture, CFSE⁺CD4⁺CD25^– Th cells were isolated on a FACS Vantage cell sorter (BD Biosciences). The purity of FACS-sorted cells was 99.8%.

Proliferation assay

Along with mitomycin C-treated, T-depleted spleen cells or B cells (5 x 10⁴) as APCs, CD4⁺CD25^– Th cells (2.5 x 10⁴), purified as described above, were cultured for 3 days in the presence of increasing numbers of CD4⁺CD25⁺ Treg cells in 96-well, round-bottom plates (Corning; Costar) in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% FCS (Invitrogen Life Technologies), glutamine (Euroclone), penicillin and streptomycin (BioWhittaker), 50 µM 2-ME, and nonessential amino acids (Invitrogen Life Technologies) in the presence of 1 µg/ml anti-CD3 mAb. LPS (0.5 µg/ml; Difco; Escherichia coli 055:B5), IL-4, or IL-13 (10 ng/ml or as otherwise indicated) was added at the beginning of culture. [³H]TdR (1 µCi/well) incorporation by proliferating lymphocytes was measured after the last 4 h of culture. [³H]TdR incorporation was measured using a Matrix 96 Direct Beta Counter (Packard). The degree of suppression exerted by CD4⁺CD25⁺ Treg cells on CD4⁺CD25^– Th cells was defined as 100 x (cpm of CD4⁺CD25^– Th cells – (cpm of the mixed Th and CD4⁺CD25⁺ Treg cell populations)/cpm of CD4⁺CD25^– Th cells). The SE of the ratio between means was calculated as previously described (23).

To prepare Th1 and Th2 cells, naive CD4⁺ T cells were cultured in 24-well plates precoated with anti-CD3 mAb (10 µg/ml). Anti-CD28 mAb (1 µg/ml) was used in soluble form. In Th1-polarizing cultures, IL-12 (10 ng/ml) and anti-IL-4-blocking mAb (10 µg/ml) were added. In Th2-polarizing cultures IL-4 (5 ng/ml) and anti-IFN--blocking mAb (5 µg/ml) were added. After 4 days IL-12 plus IL-2 (300 U/ml) or IL-4 plus IL-2 (300 U/ml) were added to Th1 and Th2 cultures, respectively. After 7 days, polarized cells were collected, and blast cells were recovered by Percoll gradient (Amersham Biosciences) as previously described (24).

Cytokine titration

Cytokines were titrated in culture supernatants by ELISA (Endogen minikit). Avidin-peroxidase (A3151-Sigma; Sigma-Aldrich; 1/1000) was then added. Thereafter, ABTS substrate (Kirkegaard & Perry Laboratories; 506400) was added, and reactions were blocked by addition of 0.2 M citric acid. Absorbance was measured at 405 nm.

Analysis of cell death

CD4⁺CD25^– Th or CD4⁺CD25⁺ Treg cells were resuspended in complete RPMI 1640 medium at 2 x 10⁶/ml cell concentration. Cultures were set up in triplicate in 96-well, round-bottom culture plates (Corning; Costar) at 100 µl/well. IL-2 (5 ng/ml), IL-4 (10 ng/ml), and IL-13 (10 ng/ml) cytokines were added at the beginning of the culture period. Forty-eight hours later, cell samples were collected, stained with Annexin V^FITC and propidium iodide (PI; Trevigen), and analyzed by flow cytometry to determine the percentage of dead cells.

Flow cytometric analysis

In some experiments, cytokine production was assessed by intracellular staining as previously described (25). Briefly, cells were harvested and stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 5 h in the presence of brefeldin A (10 µg/ml; all from Sigma-Aldrich). Cells were washed, incubated with Fc Block anti-CD16/32, fixed with 1% paraformaldehyde, and permeabilized with 0.5% saponin and 0.5% BSA in PBS. Cytokine-producing cells were revealed by staining cells with PE-conjugated anti-IFN- mAb and PE-conjugated anti-IL-4 mAb. Isotype-matched mAbs were used as a control.

CD4⁺CD25^– Th or CD4⁺CD25⁺ Treg cells were CFSE-labeled with 5 µM CFSE (Molecular Probes) for 5 min at room temperature. Cells were washed and set up in culture in round-bottom, 96-well plates in triplicate. After 3 days, cells were pooled and analyzed by flow cytometry. Samples of viable 2.5 x 10⁵ cells were analyzed using a FACSCalibur (BD Biosciences), using CellQuest research software (BD Biosciences).

Western blot (WB)

Cells were lysed for 10 min on ice in lysis buffer (1 mM MgCl₂ 350 mM NaCl, 20 mM HEPES, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM Na₄P₂O₇, 1 mM PMSF, 1.5 mM aprotinin, 1.5 mM leupeptin, 1% phosphatase inhibitor mixture II (P5726; Sigma-Aldrich), 20% glycerol, and 1% Nonidet P-40). Cell lysates were clarified by centrifugation at 11,000 x g for 15 min. Supernatants were boiled for 10 min, separated on a 12% SDS-PAGE gel, and blotted onto Hybond-P transfer membrane (Amersham Biosciences). Membranes were blocked overnight in blocking reagent (Amersham Biosciences) at 4°C and probed with the indicated Abs for 60 min at room temperature. Membranes were washed and probed with alkaline phosphatase-conjugated anti-rabbit or anti-mouse Abs for 60 min at room temperature. Blots were visualized by chemiofluorescent labeling (Amersham Biosciences) according to the manufacturer’s protocol and acquired by the phosphor/fluorescence imager Storm 860 (Molecular Dynamics). The intensity of the bands was directly quantified by ImageQuant software (Molecular Dynamics), which gives rise to a volume report by integrating the area of the band and its intensity. Results are shown after normalization with -actin (26). Abs were subsequently stripped off from membranes for reprobing as described above.

	Results

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Suppression of CD4⁺CD25^– Th cell activation and proliferation by CD4⁺CD25⁺ Treg cells

CD4⁺CD25^– Th cells were stimulated in vitro with anti-CD3 mAb in the presence of APCs or B cells. The suppression of both CD4⁺CD25^– Th cell proliferation and IL-2 production increased with increasing CD4⁺CD25⁺ Treg cell number. Cell culture overgrowth was ruled out by the lack of suppression when CD4⁺CD25⁺ Treg cells were replaced with the same number of CD4⁺CD25^– Th cells (data not shown). Suppression of CD4⁺CD25^– Th cell proliferation was slightly more effective when B cells were used instead of APCs (Fig. 1A). Yet this difference was not evident for IL-2 production (Fig. 1C).

View larger version (10K): [in this window] [in a new window]   FIGURE 1. APCs and B cells promote CD4+CD25+ Treg cell-mediated suppression. CD4+CD25– Th cells were cultured in triplicate for 3 days with APCs () or B cells (•) in the presence of the indicated number of CD4+CD25+ Treg cells. A, [3H]TdR uptake was assessed 3 days later. Levels of proliferation, measured as cpm ± SE, were 3926 ± 194 for APCs and Th cells and 2257 ± 215 for B and Th cells. CD4+CD25+ Treg cell mediated-suppression of CD4+CD25– Th proliferative response is expressed as the percent inhibition of cell proliferation. B, The IL-2 concentration in culture supernatant was measured by ELISA. Data are the mean ± SE from one of three independent experiments. C, CD4+CD25+ Treg cell mediated-suppression of IL-2 production is expressed as the percent inhibition.

A large majority of CD4⁺CD25^– Th cells expresses a naive phenotype; 85% are CD45RB^highCD44^lowCD62L⁺. Both CD4⁺CD25^– Th and CD4⁺CD25⁺ Treg cells are resting cells, as determined by their small size and low CD69 expression (data not shown). When these cells were cultured in the absence of growth factors, they died by apoptosis. Using this approach, the ability of IL-2, IL-4, or IL-13 to protect CD4⁺CD25^– Th or CD4⁺CD25⁺ Treg cells from death was evaluated. Thus, a fixed number of purified CD4⁺CD25^– Th or CD4⁺CD25⁺ Treg cells were cultured in medium for 48 h with or without exogenous factors, subsequently stained with annexin V and PI, and examined by flow cytometry. The results demonstrate that IL-4 is an excellent survival factor for CD4⁺CD25^– Th cells and, to a lesser extent, for CD4⁺CD25⁺ Treg cells, whereas IL-13 has no effect on the survival of either cell type. IL-2, like IL-4, favors CD4⁺CD25⁺ Treg cell survival, as indicated by the percentage of living cells recovered (Fig. 2A).

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Role of IL-4 in CD4+ CD25– Th and CD4+CD25+ Treg cell survival and proliferation. A, CD4+ CD25– Th or CD4+CD25+ Treg cells were cultured in triplicate for 48 h in medium supplemented with IL-2, IL-4, or IL-13. Forty-eight hours later, T cells we re stained with annexin V-FITC and PI, and the mean percentage of dead cells was determined by flow cytometry. B, CD4+CD25– Th cells () or CD4+ CD25+ Treg cells () were cultured in triplicate for 2 days with APCs (left panel) or B cells (right panel) and stimulated with anti-CD3 mAb in the presence of anti-CD28 mAb, IL-2, IL-4, or IL-13 or were left unstimulated. Cell proliferation was determined by [3H]TdR incorporation. C, Purified resting B cells, APCs, CD4+CD25– Th cells, or CD4+ CD25+ Treg cells were lysed, and protein was resolved by SDS-PAGE. IL-4R -chain expression was detected by WB.

That the different responses of CD4⁺CD25^– Th and CD4⁺CD25⁺ Treg cells to IL-4 in terms of percent survival and cell proliferation were due to different expressions of IL-4R -chain was ruled out when extracts from resting CD4⁺CD25^– Th and CD4⁺CD25⁺ Treg cells were examined by WB. Results (Fig. 2C) show that the expression of IL-4R -chain is similar in CD4⁺CD25⁺ Treg cells, CD4⁺CD25^– Th cells, and APCs, but is significantly lower than that in B cells. These results suggest that although resting CD4⁺CD25⁺ Treg cells express IL-4R -chain, IL-4 exhibits a different ability to protect CD4⁺CD25^– Th and CD4⁺CD25⁺ Treg cells from apoptosis.

Suppressive activity of IL-4-pretreated CD4⁺CD25⁺ Treg cells

To assess whether IL-4 could affect CD4⁺CD25⁺ Treg cell inhibitory function, CD4⁺CD25⁺ Treg cells were stimulated with anti-CD3 mAb or anti-CD3 mAb and IL-4 in the presence of APCs. After 48 h, cells were harvested, and CD4⁺CD25⁺ Treg cells were recovered by magnetic cell sorting. Activated or freshly isolated CD4⁺CD25⁺ Treg cells were analyzed for their suppressive activity in the presence of APCs and CD4⁺CD25^– Th cells. The suppressive activity of anti-CD3 mAb- or anti-CD3 mAb- and IL-4-pretreated CD4⁺CD25⁺ Treg cells was found not only to be preserved, but to be even greater than that of freshly isolated CD4⁺CD25⁺ Treg cells (Fig. 3). This effect of IL-4 on the activity of CD4⁺CD25⁺ Treg cells has also been recently described (27). Thus, IL-4 promotes CD4⁺CD25⁺ Treg cell proliferation, whereas these cells maintain their suppressive activity when subsequently tested.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. IL-4-pretreated CD4+CD25+ Treg cells retain their suppressive activity. CD4+CD25+ Treg cells prestimulated for 2 days in the presence of APCs with anti-CD3 mAb or anti-CD3 mAb and IL-4 were washed and set up in culture with CD4+CD25– Th cells stimulated with anti-CD3 mAb along with fresh APCs for 3 days (•, anti-CD3 prestimulation; , anti-CD3/IL-4 prestimulation; , freshly isolated CD4+CD25+ Treg cells). Levels of proliferation, measured as cpm ± SE, were 3824 ± 241 for APCs and Th cells. CD4+CD25+ Treg cell mediated-suppression of the CD4+CD25– Th proliferative response is expressed as the percent inhibition of cell proliferation. Results from one of three independent experiments are shown.

As described above, IL-4, unlike IL-13, promotes CD4⁺CD25⁺ Treg cell proliferation and supports suppressive activity when subsequently tested. This result shows a critical involvement of IL-4 in the expansion of the CD4⁺CD25⁺ Treg cell pool, but does not reveal any effect of this cytokine on CD4⁺CD25⁺ Treg cell-mediated suppression. To determine whether IL-4 influences CD4⁺CD25⁺ Treg cell activity, IL-4 or IL-13 was added to the coculture assay. The CD4⁺CD25⁺ Treg cell assay was set up using CD4⁺CD25^– Th and CD4⁺CD25⁺ Treg cells in the presence of APCs or purified B cells and increasing concentrations of IL-4 or IL-13. The addition of IL-4 inhibited CD4⁺CD25⁺ Treg cell-mediated suppression in a dose-dependent manner in the presence of APCs or B cells (Fig. 4, A and B, left). Conversely, CD4⁺CD25⁺ Treg cells maintained their full competence to suppress CD4⁺CD25^– Th cell proliferation at each IL-13 concentration (Fig. 4, A and B, right). Thus, only IL-4 displayed a negative effect on CD4⁺CD25⁺ Treg cell-mediated suppression.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. Exogenous IL-4, but not IL-13, abrogates CD4+CD25+ Treg cell-mediated suppression. A, CD4+CD25– Th cells () or CD4+CD25– Th + CD4+CD25+ Treg cells (), mixed in an equal ratio with either APCs (upper panels) or B cells (lower panels), were stimulated for 3 days with anti-CD3 mAb and with IL-4 (left) or IL-13 (right) at the indicated concentrations. Cell proliferation was determined by [3H]TdR incorporation. B, CD4+CD25+ Treg cell-mediated suppression of the CD4+CD25– Th cell proliferative response in the presence of APCs () or B cells (•) is expressed as the percent inhibition vs total counts in the absence of CD4+CD25+ Treg cells for each condition examined. C, LPS reduces CD4+CD25+ Treg cell-mediated suppression of CD4+CD25– Th cell proliferation. CD4+CD25– Th cells were cultured for 3 days in the presence of APCs (left) or B cells (right). Cells were stimulated with anti-CD3 mAb () or were supplemented with LPS (), IL-4 (), or LPS plus IL-4 (•). Levels of proliferation, measured as cpm ± SE, for APCs and Th cells were: anti-CD3, 3,626 ± 271; anti-CD3 and LPS, 4,740 ± 214; anti-CD3 and IL-4, 6,182 ± 229; anti-CD3, LPS, and IL-4, 12,718 ± 274; and for B cells and Th cells were: anti-CD3, 2,415 ± 235; anti-CD3 and LPS, 3,423 ± 314; anti-CD3 and IL-4, 5,704 ± 289; anti-CD3, LPS, and IL-4, 10,565 ± 303. CD4+CD25+ Treg cell-mediated suppression of CD4+CD25– Th proliferative response is expressed as the percent inhibition of cell proliferation. Results from one of three independent experiments are shown.

Expression of Bcl-2 in CD4⁺CD25^– Th cells cocultured with CD4⁺CD25⁺ Treg cells

IL-4 is known to prevent the death of activated CD4⁺CD25^– Th cells, as it induces the antiapoptotic factor, Bcl-2 (29). This finding prompted us to examine whether the inhibition of CD4⁺CD25^– Th cell proliferation by CD4⁺CD25⁺ Treg cell mediated-suppression is linked to down-regulation of Bcl-2 expression.

Pretreatment of CD4⁺CD25^– Th cells with the cell division marker, CFSE, before culture offers the possibility of identifying this cell subset by CFSE staining. Whether the suppressed CD4⁺CD25^– Th cells fail to proliferate because of low expression of the survival factor, Bcl-2, was investigated in FACS-sorted CFSE⁺CD4⁺CD25^– Th cells after 3 days of coculture with APCs, with or without CD4⁺CD25⁺ Treg cells, in the presence or the absence of IL-4. The sorted CD4⁺CD25^– Th cells were lysed, and protein extracts were analyzed by WB for Bcl-2 expression. When CD4⁺CD25^– Th cells were activated in the presence of APCs and anti-CD3 mAb, Bcl-2 expression was up-regulated compared with that of freshly isolated CD4⁺CD25^– Th cells (Fig. 5). The addition of CD4⁺CD25⁺ Treg cells caused a decrease in the level of Bcl-2, but when the coculture was supplemented with IL-4, this negative effect of CD4⁺CD25⁺ Treg cells was prevented. Thus, the addition of IL-4 overcomes CD4⁺CD25⁺ Treg cell inhibition of Bcl-2 expression in CD4⁺CD25^– Th cells.

View larger version (25K): [in this window] [in a new window]   FIGURE 5. IL-4 prevents Bcl-2 inhibition in CD4+CD25– Th cells cocultured with CD4+CD25+ Treg cells. CFSE-labeled CD4+CD25– Th cells, either alone or mixed with an equal number of CD4+CD25+ Treg cells, were cultured in the presence of APCs and anti-CD3 mAb with or without IL-4 for 3 days, then FACS-sorted based on their CFSE signals. Freshly isolated CD4+CD25– Th cells served as a control. After sorting, cells were lysed, and protein extracts were resolved by SDS-PAGE. Bcl-2 expression was detected by WB analysis. Results from one of three independent experiments are shown.

The cytokine profiles of CD4⁺CD25^– Th cells, with or without the addition of IL-4, were analyzed in the presence or the absence of CD4⁺CD25⁺ Treg cells. CD4⁺CD25⁺ Treg and CFSE⁺-CD4⁺CD25^– Th cells were cocultured with APCs and anti-CD3 mAb, with or without IL-4. After 3 days of culture, cells were harvested and stimulated with PMA and ionomycin for 5 h in the presence of brefeldin A, then stained intracellularly for IL-4 or IFN- production. In the absence of IL-4, CD4⁺CD25^– Th cells mixed with CD4⁺CD25⁺ Treg cells showed reduced cell cycle progression (Fig. 6, left panel). The addition of IL-4 blocked CD4⁺CD25⁺ Treg cell mediated-suppression of CD4⁺CD25^– Th cell proliferation by restoring cell cycle progression to near-normal levels (Fig. 6, right panel). Flow cytometric analysis of CFSE⁺CD4⁺CD25^– Th cells stimulated with anti-CD3 mAb and APCs showed that they produce IFN- and barely detectable levels of IL-4. Addition of CD4⁺CD25⁺ Treg cells induced a decrease in IFN- production. Upon addition of IL-4, CD4⁺CD25^– Th cells produced IL-4, because 44% of CFSE⁺CD4⁺CD25^– Th cells expressed this cytokine. It should be noted that the addition of CD4⁺CD25⁺ Treg cells decreased the number of IL-4⁺CD4⁺CD25^– Th cells to 30%. The decrease in IL-4 production was associated with an increase in IFN- production. Thus, in vitro priming of CD4⁺CD25^– Th cells in the presence of CD4⁺CD25⁺ Treg cells supplemented with IL-4 resulted in the generation of a viable population of CD4⁺CD25^– Th cells that have cycled, but produced different cytokines.

View larger version (26K): [in this window] [in a new window]   FIGURE 6. CFSE and cytokine analysis of CD4+CD25– Th cells cultured in the presence or the absence of IL-4. CFSE-labeled CD4+CD25– Th cells, either alone or mixed with an equal number of CD4+CD25+ Treg cells, were cultured in the presence of APCs and anti-CD3 mAb with (right panels) or without (left panels) IL-4 for 3 days, then analyzed by flow cytometry for IL-4 (upper panels) or IFN- (lower panels) production. Polygonal gates are drawn around the upper limit of isotype mAb background staining. The percentage of cytokine-positive cells is indicated in the upper right corner. Results from one of three independent experiments are shown.

IL-4 is mainly produced by effector Th2 cells. The effect of CD4⁺CD25⁺ Treg cell-mediated suppression on IL-4-producing Th2 cells as well that as on IFN--producing Th1 cells were evaluated. Th1 and Th2 cells were obtained by culturing naive CD4⁺ T cells under in vitro polarizing conditions for 7 days. Differentiated CD4⁺CD25^– Th cells were collected, and blast cells were recovered. The suppressive activity of CD4⁺CD25⁺ Treg cells was evaluated on a fixed number of Th1 and Th2 cells and, as a control, freshly isolated naive CD4⁺CD25^– Th cells. The test was performed in the presence of APCs. Proliferative responses were almost completely inhibited when CD4⁺CD25^– Th cells instead of Th1 or Th2 cells were cocultured with CD4⁺CD25⁺ Treg cells. The proliferative responses of Th1 and Th2 cells were inhibited by the addition of high numbers of CD4⁺CD25⁺ Treg cells. Yet the maximal suppression observed was 75% with CD4⁺CD25^– Th cells and only 38% and 42% with Th1 and Th2 cells, respectively (Fig. 7).

View larger version (13K): [in this window] [in a new window]   FIGURE 7. CD4+CD25+ Treg cells exert lesser suppressive activity on Th1 and Th2 cells compared with Th cells, but are equally effective at suppressing IL-4 production. Naive CD4+ T cells were cultured under Th1- or Th2-polarizing conditions. After 7 days, Th1 blast cells, Th2 blast cells, or freshly isolated CD4+CD25– Th cells were collected, put in culture, and stimulated with anti-CD3 mAb in the presence of APCs. A, [3H]TdR uptake was assessed 72 h later on Th1 (), Th2 (•), or freshly isolated CD4+CD25– Th cells (). Levels of proliferation, measured as cpm ± SE, were 3571 ± 284 for APCs and Th cells, 5839 ± 316 for APCs and Th1 cells, and 6128 ± 412 for APCs and Th2 cells. CD4+CD25+ Treg cell-mediated suppression of the CD4+CD25– Th proliferative response is expressed as the percent inhibition of cell proliferation. IFN- (B) and IL-4 (C) concentrations in culture supernatant are indicated. , Th1 cells; , Th2 cells; , freshly isolated Th cells. Data are the mean ± SE from one of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
MHC molecules are necessary, but not sufficient, to induce efficient homeostatic proliferation of peripheral CD4⁺CD25^– Th cells; other signals are required for naive CD4⁺ T cell survival and proliferation (30). In vitro and in vivo experiments indicate that these signals are delivered by growth factors, such as IL-4, IL-6, and IL-7 (29, 31). The present results show that IL-4 protects resting naive CD4⁺CD25^– Th cells from death and contributes to CD4⁺CD25⁺ Treg cell survival. Upon TCR engagement in the presence of APCs, IL-4, like IL-2, plays a critical role in promoting CD4⁺CD25⁺ Treg cell proliferation. Furthermore, IL-4-pretreated CD4⁺CD25⁺ Treg cells maintain full competence to suppress CD4⁺CD25^– Th cell proliferation in the absence of exogenous IL-4. B cells were found to contribute to Treg cell proliferation less efficiently than total APCs. Because experiments with µ-deficient mice have suggested that B cells play a significant role in the regulation of the CD4⁺CD25⁺ Treg cell subset (12), our results suggest that factors other than B cells are involved in promoting Treg cell proliferation in vivo. Nonetheless, B cells promote Treg cell-suppressive activity.

The effect of IL-4 on CD4⁺CD25^– Th and CD4⁺CD25⁺ Treg cell activation led us to explore the role of this cytokine in CD4⁺CD25⁺ Treg cell-mediated suppression by adding increasing concentrations of this cytokine to the APC-Th cell-Treg cell coculture assay. Suppression was decreased in the presence of IL-4, but was unaffected if IL-4 was replaced with IL-13. This difference may be ascribed to the distinct effects of IL-4 and IL-13 on Th cell activation. Although IL-4R type II, the receptor that transduces the signal delivered by IL-13, is widely distributed, it is absent on T cells (13). This may account for the lack of responsiveness of CD4⁺CD25^– Th cells to IL-13, although it does not rule out that IL-4 and IL-13 promote the expression of different costimulatory molecules (32) during APC-Th cell-Treg cell interactions, which might interfere with the outcome of CD4⁺CD25⁺ Treg cell-mediated suppression. Similar results were obtained when APCs were substituted by B cells.

To generate an effective immune response against invading organisms, T cells need to be protected from Treg cell-mediated suppression to avoid the pathological consequences of an inefficient immune response. The results presented in this report confirm LPS inhibition of CD4⁺CD25⁺ Treg cell-mediated suppression (10, 28), hence contributing to the rescue process of CD4⁺CD25^– Th cells as induced by IL-4. However, it has been demonstrated that pretreatment with LPS could directly activate not only APCs, but also CD4⁺CD25⁺ Treg cells (33). As a matter of fact, the addition of LPS to coculture may induce APC maturation (34) and CD4⁺CD25^– Th cell survival (35) through the induction of IL-6 and other soluble mediators (28), leading to protection of CD4⁺CD25^– Th cells from CD4⁺CD25⁺ Treg cell-mediated suppression.

We hypothesized that in the presence of IL-4, CD4⁺CD25^– Th cells might exhibit a protective mechanism that counteracts inhibition of CD4⁺CD25^– Th cell proliferation by CD4⁺CD25⁺ Treg cells. As reported previously (29), the ability of IL-4 to protect T cells from death and to promote cell proliferation depends on its ability to induce the expression of Bcl-2, a protein known to protect Th cells from apoptosis. The results indicate that CD4⁺CD25⁺ Treg cells significantly reduced Bcl-2 expression in coactivated CD4⁺CD25^– Th cells. This observation agrees with the recent finding that CD4⁺CD25⁺ Treg cells induce apoptosis in CD4⁺CD25^– Th cells (36). Yet the suppressive activity of CD4⁺CD25⁺ Treg cells on Bcl-2 expression in CD4⁺CD25^– Th cells could be inhibited by the addition of IL-4.

We found that the addition of IL-4 protects CD4⁺CD25^– Th cell proliferation from suppression. Yet a negative effect of CD4⁺CD25⁺ Treg cells on CD4⁺CD25^– Th cell ability to produce IL-4 could not be excluded. We found, indeed, that CD4⁺CD25⁺ Treg cells down-regulated IL-4 production in CD4⁺CD25^– Th cells. This finding suggests a negative feedback of CD4⁺CD25⁺ Treg cells on IL-4 production. Hence, inhibition of IL-4 production may subsequently favor suppression of CD4⁺CD25^– Th cell proliferation. It should be noted that under such conditions, a reduction of IL-4 production was associated with an increase in IFN- production; this cytokine is a major driver for Th1 cell responses. However, IFN- also plays a significant role in promoting activation-induced T cell death (37). A strict association between IFN- and CD4⁺CD25⁺ Treg cells to mediate tryptophan catabolism and therefore the production of proapoptotic metabolites in DCs has recently been demonstrated (38). However, at the time point examined in our experiment (Fig. 5), IFN--induced CD4⁺CD25^– Th cell death is ruled out, because CD4⁺CD25^– Th cells cocultured with CD4⁺CD25⁺ Treg cells expressed similar levels of Bcl-2 in the presence of IL-4. Alternatively, the reversal of IFN- and IL-4 production observed in the presence of IL-4 and CD4⁺CD25⁺ Treg cells may be explained by the action of IL-4 on IL-12 production by APCs and thus on the Th1 cell-driving potential (39).

To better understand the role of CD4⁺CD25⁺ Treg cells in IL-4- and IFN--production, we examined IL-4-producing Th2 cells and IFN--producing Th1 cells. When Th1 and Th2 cells were used instead of naive CD4⁺CD25^– Th cells, CD4⁺CD25⁺ Treg cell-mediated suppression was significantly reduced. This is probably due to the different activation state of naive CD4⁺CD25^– Th cells compared with those of Th1 and Th2 cells, suggesting that CD4⁺CD25⁺ Treg cells are more efficient at inhibiting priming than at inhibiting the effector phase of immune responses. Nonetheless, in the presence of CD4⁺CD25⁺ Treg cells, IL-4 production by Th2 cells is reduced. The experiments with Th2 cells confirm the ability of CD4⁺CD25⁺ Treg cells to inhibit IL-4 production. Inhibition of cytokine production in Th2 cells appears to be more sensitive to the suppressive activity of CD4⁺CD25⁺ Treg cells. The higher degree of sensitivity of Th2 cells compared with Th1 cells to suppression by CD4⁺CD25⁺ Treg cells was also apparent in a model of colitis induced by Th1 or Th2 cells (40). At variance, Cosmi et al. (41) found that human CD25⁺ regulatory thymocyte clones produce lesser suppression of Th2 vs Th1 clones. In this model, however, Th2 clones were stimulated with anti-CD3 mAb and allogeneic PBMCs. These Th2 clones were able to produce IL-4 even in the presence of CD25⁺ regulatory thymocytes, thus leading to inhibition of CD25⁺ regulatory thymocyte-mediated suppression. Conversely, in our experimental system, Th2 cells activated only by anti-CD3 mAb in the presence of syngeneic APCs failed to produce IL-4 and subsequently showed reduced proliferation as did Th1 cells in the presence of CD4⁺CD25⁺ Treg cells. It should be pointed out that human CD25⁺ regulatory thymocyte clones isolated by these researchers were quite different from murine peripheral CD4⁺CD25⁺ Treg cells, because they did not proliferate in response to IL-4 and were able to exert suppression also in the presence of IL-2 (8).

The results presented herein indicate that IL-4 is involved in CD4⁺CD25^– Th and CD4⁺CD25⁺ Treg cell survival and proliferation and demonstrate that IL-4, unlike IL-13, negatively modulates Treg cell-mediated suppression of the CD4⁺CD25^– Th cell proliferative response by activating an antiapoptotic mechanism. Thus, IL-4 is expected to reduce tolerance and favor autoimmunity under conditions in which Treg cell-mediated suppression plays a critical physiopathological role. For instance, the relevance of IL-4 in promoting systemic autoimmunity is supported by the observation that some IL-4 transgenic mice develop SLE-like antinuclear Abs, hemolytic anemia, and immune-mediated renal disease (42). These results are supported by the finding that µ-chain-deficient and IL-4 transgenic mice develop advanced progression of kidney disease independently of any effect of IL-4 on Ab nephritogenicity (43). These observations may be explained by the protective effect of transgenic IL-4 on the survival and proliferation of autoreactive T cells. It should be noted that resistance to apoptosis is considered a trait contributing to the lupus phenotype, because Bcl-2 expression has generally been found to be elevated in PBMCs from SLE patients and may be selectively expressed in T cells (44, 45).

The immune response is the result of a fine equilibrium between aggressive and regulatory cell components, because the activation kinetics and survival times of the lymphocyte subsets involved in the process will determine the outcome. The present results, pointing to the multiple effects of IL-4 on separate CD4⁺CD25^– Th and CD4⁺CD25⁺ Treg cell survival and proliferation as well as on CD4⁺CD25^– Th cell survival in the presence of CD4⁺CD25⁺ Treg cells, shed light on the unclear role of IL-4 in transplantation tolerance and autoimmunity. The observation that IL-4 could directly expand the CD4⁺CD25⁺ Treg cell subset provides a new mechanism to prevent the induction of autoimmunity and may explain those experimental models in which IL-4 successfully hampers the emergence of pathological signs. It is conceivable that regulation of autoimmunity proceeds through expansion of the CD4⁺CD25⁺ Treg cell subset, which might subsequently control autoreactive T cells. Nevertheless, under conditions characterized by aberrant autoreactive T cell activation, IL-4 may promote CD4⁺CD25^– Th cell expansion by protecting autoreactive T cells from CD4⁺CD25⁺ Treg cell-mediated suppression, hence leading to the dangerous effects of IL-4 observed under some pathological conditions. In conclusion, the aforementioned considerations suggest that the site and/or the stage of the immune response may be critical determinants of whether the dominant effect of IL-4 is to increase the CD4⁺CD25⁺ Treg cell subset or to protect CD4⁺CD25^– Th cells from CD4⁺CD25⁺ Treg cell-mediated suppression. Additional investigations are needed to analyze the effects of IL-4 on APC-activating function, CD4⁺CD25^– Th cell sensitivity to suppression, and CD4⁺CD25⁺ Treg cell-suppressive activity.

	Acknowledgments

 
We thank Dr. Vanessa Ubaldi for advice and Dr. Rita Carsetti and Ezio Giorda for cell sorting.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
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 Progetto N.1AN/F5 from Istituto Superiore di Sanità.

² Address correspondence and reprint requests to Dr. Gino Doria, Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, Italy. E-mail address: gino.doria{at}uniroma2.it

³ Abbreviations used in this paper: Treg, regulatory T cell; PI, propidium iodide; SLE, systemic lupus erythematosus; WB, Western blot.

Received for publication December 15, 2004. Accepted for publication March 25, 2005.

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