Abstract
Type I IFNs are central to a vast array of immunological functions. Their early induction in innate immune responses provides one of the most important priming mechanisms for the subsequent establishment of adaptive immunity. The outcome is either promotion or inhibition of these responses, but the conditions under which one or the other prevails remain to be defined. The main objective of the current study was to determine the involvement of IFN-α on murine CD4+CD25− Th cell activation, as well as to define the role played by this cytokine on CD4+CD25+ regulatory T (Treg) cell proliferation and function. Although IFN-α promotes CD4+CD25− Th cells coincubated with APCs to produce large amounts of IL-2, the ability of these cells to respond to IL-2 proliferative effects is prevented. Moreover, in medium supplemented with IFN-α, IL-2–induced CD4+CD25+ Treg cell proliferation is inhibited. Notably, IFN-α also leads to a decrease of the CD4+CD25+ Treg cell suppressive activity. Altogether, these findings indicate that through a direct effect on APC activation and by affecting CD4+CD25+ Treg cell-mediated suppression, IFN-α sustains and drives CD4+CD25− Th cell activation.
Type I IFNs encompass a large group of closely related cytokines, such as IFN-α and -β (IFN-α/β), that exert their activity through a common receptor, the type I IFN receptor (IFNAR) (1). Type I IFNs represent one of the most important classes of cytokines, with a dominant role in defining the qualitative and quantitative features of innate and adaptive immune processes. The major role of IFN-α/β is the induction of the priming state through the production and regulation of other mediators, including cytokines (i.e., IL-6 and IL-15), the upregulation of MHC and costimulatory molecules, chemokines, and chemokine receptors (1–3). In mice, IFN-α/β prevent activated T cell death during inflammatory responses (4), and the IFN-α signaling is critical for the generation of effector and memory CD8+ T cells following viral infections (5) as well as for prolonging expansion of Ag-specific T CD8+ cells following cross-priming (6, 7). IFN-α also stimulates CD4+ T cells to enhance Ag-specific B cell responses (8). Moreover, type I IFNs have been described to enhance the adaptive immune response, and these cytokines have also been associated with several autoimmune diseases. Increased levels of type I IFNs have been observed in synovial fluid of patients affected by rheumatoid arthritis, and type I IFN activity has also been found in the serum of patients suffering from several other autoimmune diseases (9). Studies in lupus-prone mice have confirmed the critical role of type I IFNs in systemic lupus erythematosus (SLE) pathogenesis; in IFNAR-deficient mice, the lupus-like disease is reduced (10). Of note, the therapeutic use of type I IFNs in patients with cancer and hepatitis has often been associated with the induction of signs of autoimmunity other than SLE. Tyroiditis is the most common autoimmune manifestation, but diabetes and autoimmune dermatitis have also been described previously (9).
In contrast to the well-established function of IFN-α on CD8+ T cell activation, little is known about the role of this cytokine on CD4+ T cell activation. Previous results demonstrated that IFN-α reduces the proliferative response of polyclonally stimulated CD4+ T cell subset, as well as the endogenous production of IL-2 (11–14), but these observations were not subsequently developed in more details. Nonetheless, these scattered data have often led to confusing and incomplete interpretations of the effects of IFN-α on the CD4+ T cell-mediated adaptive immune response.
Natural arising CD4+CD25+Foxp3+ regulatory T (Treg) cells are a distinct CD4+ T cell subset involved in the maintenance of self-tolerance and immune homeostasis by suppressing aberrant immune responses, such as in autoimmune diseases (15). IL-2, as well as IL-4 (16, 17), increases the fitness and proliferation of peripheral CD4+CD25+ Treg cells, as well as their function. High doses of IL-2 together with antigenic stimulation have been used to expand Ag-specific CD4+CD25+ Treg cells that exhibit more potent Ag-specific suppression. Moreover, IL-2 (18, 19), IL-4 (16, 17), IL-12 (20), and IL-6 combined with other TLR-induced cytokines (21) can also abrogate CD4+CD25+ Treg cell-mediated suppressive activity. Thus, various cytokines can exert several effects on CD4+CD25+ Treg cells, thereby contributing to tuning the magnitude of suppression (22). The function of CD4+CD25+ Treg cells is manifested at various aspects of T cell activation, including cytokine production, proliferation, and inflammation in target tissues. Although several observations have indicated that cognate interactions between CD4+CD25+ Treg and effector T cells are essential for immune suppression, the critical molecular interactions, which occur during Treg cell suppression, are still to be defined. This is an important open issue, because strategies that clonally expand Ag-specific natural CD4+CD25+ Treg cells while inhibiting the activation and expansion of effector T cells might be useful to strengthen or re-establish self-tolerance in autoimmune diseases, organ transplantation, and allergy. Conversely, strategies addressed to decrease CD4+CD25+ Treg suppressive activity might be helpful in the treatment of cancer (23).
As type I IFNs are good candidates for therapeutic intervention, the present research aims at investigating and characterizing the involvement of these innate immune mediators in the control and establishment of CD4+CD25− Th cell activity, with emphasis on CD4+CD25+ Treg cell immune regulation. The results show that IFN-α induces CD4+CD25− Th cells coincubated with APCs to produce large amounts of IL-2, but at the same time, their ability to respond to its proliferative effects is prevented. IL-2–induced CD4+CD25+ Treg cell proliferation is also inhibited. Moreover, IFN-α decreases the suppressive activity. Altogether, these findings indicate a pivotal role of IFN-α on CD4+CD25− Th cell activity by a direct control on APC activation and by an indirect effect on CD4+CD25+ Treg cell-mediated suppression.
Materials and Methods
Mice
All mice were of C57BL/6 strain. Eight- to 10-wk-old female C57BL/6J mice were obtained from Charles River Laboratories (Charles River Laboratories, Calco, Italy) and maintained under pathogen-free conditions. IFNAR−/− C57BL/6 mice were provided by U. Kalinke (Paul-Ehrlich Institut, Langen, Germany) (24). Mice were housed at the Università di Roma La Sapienza and Istituto Superiore di Sanita’ facilities under pathogen-free condition. All mice were treated in accordance with the European Community guidelines.
Abs and reagents
The following Abs and secondary reagents (PE, FITC, PE-Cy5, APC, or biotinylated) were purchased from BD Pharmingen (San Diego, CA): anti-CD25 (7D4), anti-CD4 (RM4-5) anti-CD3ε (145-2C11), anti-CD28 (37.51), anti–IL-2 (JES6-5H4), anti-Ly6C (104-2.1), anti–IL-6 (MP5-20F3). IFN-α and IL-2 (550069) were purchased, respectively, from PBL (Piscataway, NJ) and BD Pharmingen. Anti-IFNAR1 (H-M, sc7391) and anti-IFNAR2 (V-20, sc20218) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and anti–β-actin (AC-15) from Sigma-Aldrich (St. Louis, MO). Anti-mouse or anti-rabbit alkaline phosphatase conjugates were purchased from Amersham Biosciences (Piscataway, NJ). Anti-goat/sheep IgG (GT-34)-peroxidase conjugate was from Sigma-Aldrich. All reagents used in cell culture were of no azide/low endotoxin quality quality.
Cell purification
All cell subsets were purified by immunomagnetic cell sorting (Miltenyi Biotec, Auburn, CA), according to the manufacturer’s instructions, and VarioMACS magnetic separation system. RBC-lysed splenocytes were washed and enriched for CD4+ T cells using the MACS MultiSort kit (130-090-860). CD4+CD25+ Treg cells were purified after staining CD4+ T cells with biotinylated anti-CD25 mAb and streptavidin microbeads (130-090-860). APCs were prepared by depletion of CD90+ cells using anti-CD90 microbeads (130-049-101). Collected cells were found to be 95–99% pure by flow cytometry analysis. In some experiments, CD4+CD25−CD69− Th and CD4+CD25+CD69− Treg cells were isolated on a FACS Vantage cell sorter (BD Biosciences, San Jose, CA). Purity of FACS-sorted cells was 99.8%.
Proliferation assay
Along with mitomycin C-treated T-depleted spleen cells (5 × 104) as APCs, CD4+CD25− Th cells (2.5 × 104) purified as described above were cultured for 3 d in the presence of CD4+CD25+ Treg cells (2.5 × 104) in 96-well round-bottom plates (Corning Glass, Corning, NY; Costar, Cambridge, MA) in RPMI 1640 medium (Life Technologies, Rockville, MD) supplemented with 10% FCS (Life Technologies), glutamine (Life Technologies), penicillin and streptomycin (BioWhittaker, Walkersville, MD), 50 μM 2-ME, and nonessential amino acids (Life Technologies) in the presence of 1 μg/ml anti-CD3 mAb. IFN-α (5 × 103 U/ml, or otherwise indicated) and/or IL-2 (5 ng/ml) were added at the beginning of culture. [3H]Thymidine deoxyribose ([3H]TdR) (1 μCi/well) incorporation by proliferating lymphocytes was measured after the last 5 h of culture. [3H]TdR incorporation was mesured by Matrix 96 Direct Beta Counter Packard. The degree of T cell proliferation was defined as 100 × (cpm of the mixed CD4+CD25− Th and CD4+CD25+ Treg cell populations/cpm of CD4+CD25− Th cells). Where indicated, CD4+CD25− Th or CD4+CD25+ Treg cells were cultured in 24-well plates precoated with anti-CD3 mAb (10 μg/ml) for 2 h at 37°C. Anti-CD28 mAb (1 μg/ml) and IL-2 (10 ng/ml) were used in soluble form.
Cytokine titration
The following cytokines were titrated in culture supernatants by ELISA: IL-2 (Endogen mini-kit; Endogen, Thermo Scientific, Waltham, MA) and IL-6 (R&D Systems kit; R&D Systems, Minneapolis, MN). Avidin-peroxidase (A3151, 1:1000; Sigma-Aldrich) was then added. Thereafter, ABTS substrate (506400; Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added, and reactions were blocked by addition of 0.2 M citric acid. Absorbance was measured at 405 nm.
Flow cytometry analysis
Where indicated, cells were labeled with 5 μM CFSE (Molecular Probes, Eugene, OR) for 5 min at room temperature. Cells were washed and set up in culture in round bottom 96-well plates in quadruplicate. After 3 d, cells were pooled and analyzed by flow cytometry.
In some experiments, after washing, cells were incubated with labeled anti-CD3, anti-CD4, anti-CD25, and anti-Ly6C mAbs. IL-2 production was also assessed by intracellular staining. 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) for the last 4 h. After staining for CD4, CD3, and CD25, cells were 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 APC-conjugated anti–IL-2 mAb. Isotype-matched mAbs were used as controls.
Samples of viable 2.5 × 105 cells were analyzed using a FACSCalibur (BD Biosciences), using CellQuest research (BD Biosciences) and FlowJo softwares. The proliferation index was evaluated using FlowJo software.
Western blot analysis
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 × 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 indicated Abs for 60 min at room temperature. Membranes were washed and probed with phosphatase-alkaline-conjugated anti-rabbit, anti-mouse, or anti-goat/sheep-peroxidase conjugate Abs for 60 min at room temperature. Blots were visualized by electron-chemio fluorescence or ECL (Amersham Biosciences), according to the manufacturer’s protocol, and acquired by the phosphor/fluorescence imager STORM 860 (Molecular Dynamics, Sunnyvale, CA). The intensity of the bands was directly quantified by Image QuaNT 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 β-actin (16). Abs were subsequently stripped off from membranes for reprobing as described above.
Statistical analysis
SE of the ratio between means (SE ratio mean) was calculated as described elsewhere (16). Two-way ANOVA and Student t tests were performed where indicated. The following symbols were used: *p < 0.05; **p < 0.01; ***p < 0.001; and n.s., not significant.
Results
Analysis of IFNAR expression on CD4+CD25− Th and CD4+CD25+ Treg cell subsets
IFNAR is composed of two distinct subunits, respectively, IFNAR1 and IFNAR2 chains (25). As we intended to characterize the effects of IFN-α on CD4+ T cell activation, we first analyzed IFNAR1 and IFNAR2 chain expression on murine APCs, CD4+CD25− Th and CD4+CD25+ Treg cells, freshly isolated (resting) or following polyclonal stimulation for 48 h. The cells were lysed, and subsequently IFNAR1 and IFNAR2 chain expression was analyzed by Western blot. The results in Fig.1 indicate that the level of expression of IFNAR1 was higher in resting CD4+CD25− Th cells as well as APCs as compared with resting CD4+CD25+ Treg cells. Conversely, IFNAR2 was more expressed on purified CD4+CD25+ Treg cells. Upon activation, IFNAR1 expression decreased in CD4+CD25− Th cells to similar level to CD4+CD25+ Treg cells. The expression of IFNAR2 also decreased on activated CD4+CD25+ Treg cells. These results show that all the analyzed cellular subsets express IFNAR, but the pattern of expression of the two IFNAR subunits is different between the resting subsets, and it ends up to lower level following T cell activation.
Effects of IFN-α on CD4+ CD25- Th cell activation
After analyzing the IFNAR chain expression, we evaluated the effects of IFN-α on CD4+CD25− Th cell activation. First, purified CD4+CD25− Th cells were activated with coated anti-CD3 and soluble anti-CD28 mAbs in medium supplemented or not with increasing concentration of IFN-α for 3 d. In agreement with previous observations (11–14), the addition of IFN-α decreased CD4+ T cell proliferation (Fig. 2A, left panel) and IL-2 production (Fig. 2A, right panel). Conversely, the addition of IFN-α affected neither IFNAR−/−CD4+CD25− Th cell proliferation nor IL-2 production (Fig. 2A).
As IFN-α can act on T cell priming also indirectly through APC activation (26), we decided to perform a similar experiment activating CD4+CD25− Th cells for 3 d with anti-CD3 mAb but in the presence of splenic APCs and IFN-α (Fig. 2B). Notably, in these culture condition, the addition of increasing concentrations of IFN-α did not affect CD4+CD25− Th cell growth (Fig. 2B, left panel), but induced a dose-dependent increase of IL-2 release in the culture supernatants (Fig. 2B, right panel). These data demonstrate the ability of IFN-α to promote CD4+CD25− Th cell activation in the presence of APCs. Even though in this culture setting the supplement of IFN-α did not affect CD4+CD25− Th cell proliferation, these cells produced large amounts of IL-2. However, no autocrine proliferative effect exerted by IL-2 was observed under these conditions.
IFN-α enhances the early production of IL-2 without affecting CD4+CD25− Th cell proliferation
The results shown in Fig. 2B suggested that CD4+ Th cells were unresponsive to the autocrine mitogenic effect exerted by IL-2, even if IL-2 was produced in large amounts following IFN-α administration. To verify this hypothesis, purified CD4+CD25− Th cells were polyclonally activated with anti-CD3 mAb and APCs in the presence of exogenous IL-2, IFN-α, or their combination. The time course of Th cell proliferation was then analyzed.
The addition of IFN-α delayed but did not inhibit CD4+CD25− Th cell proliferation (Fig. 3A), whereas, as expected, exogenous IL-2 exerted a significant proliferative effect on these cells. Of note, CD4+ Th cell proliferation induced by the supplement of IL-2 was inhibited in the presence of IFN-α. The results obtained by ELISA on the culture supernatants confirmed that the addition of IFN-α induced a significant increase of IL-2 levels, but this effect was not associated with an enhancement of CD4+ Th cell proliferation (Fig. 3A, 3B).
IL-2 production by CD4+ Th cells was also assessed by intracellular cytokine staining. The results demonstrated that since the earliest time point (15 h), IFN-α promoted IL-2 production, as shown by the percentage and mean fluorescence intensity (MFI) of recovered IL-2+ cells (Fig. 3C, 3D). Interestingly, IFN-α also enhanced IL-2 production in medium supplemented with IL-2 (Fig. 3C, 3D). These results confirm the prediction that IFN-α promotes IL-2 production by CD4+ Th cells, but at the same time, the IL-2–mediated mitogenic effect is reduced.
In the same experiment, we also performed the analysis the IL-2Rα–chain (CD25) expression. The results showed that IFN-α enhances and sustains CD25 expression (Fig. 3C).
IFN-α inhibits IL-2–induced Th and Treg cell proliferation
Although the previous data demonstrated the ability of IFN-α to promote IL-2 production by CD4+CD25− Th cells, they did not allow distinguishing if this result was a direct effect of IFN-α signal on CD4+CD25− Th cells or on APCs, which were also present in the culture. To establish the relative contribution of APCs and CD4+CD25− Th cells to this effect, we decided to combine APCs and CD4+CD25− Th cells isolated from IFNAR−/− or littermate control mice with or without IFN-α. The results in Fig. 4A demonstrated that IL-2 production was increased by the supplement of IFN-α only in the presence of IFNAR+/+APCs. This effect was still evident when APCs from IFNAR+/+ mice were cocultured with IFNAR−/−CD4+CD25− Th cells. Interestingly, in this culture setting, IFNAR−/−CD4+CD25− Th cell proliferation was also increased (Fig. 4A, upper panel). Thus, by acting through APCs, IFN-α promotes IL-2 production by CD4+CD25− Th cells.
IL-2 is a well-known T cell growth factor, principally involved in the generation and maintenance of CD4+CD25+ Treg cell subset. In vitro CD4+CD25+ Treg cells are anergic, but their proliferative capacity can be restored following the addition of IL-2 (22). On the basis of these findings, we subsequently analyzed the effect of IFN-α on IL-2–induced CD4+CD25+ Treg cell proliferation. As illustrated in Fig. 4B, IFN-α affected neither the proliferation of CD4+CD25− Th and CD4+CD25+ Treg cell subsets nor the anergic profile of CD4+CD25+ Treg cells. The addition of IL-2 promoted both CD4+CD25− Th and CD4+CD25+ Treg cell proliferation. Conversely, when IL-2 was combined with IFN-α, the proliferation was decreased.
Although in vivo observations have sometimes indicated a protective effect exerted by type I IFNs on activated CD4+ Th cells (4), IFN-α has often been associated with a proapoptotic effect on T cells (27). To better understand the results shown above, we performed additional experiments to eliminate the potentially confounding proapoptotic effects of IFN-α on CD4+CD25− Th and CD4+CD25+ Treg cell survival. IFN-α did not significantly affect the survival of both CD4+ Th and CD4+CD25+ Treg cells (data not shown).
IFN-α promotes CD25 and Ly6C expression on CD4+CD25− Th cells
To further understand the mechanism involved in the inhibition of IL-2–induced proliferation in the presence of IFN-α, we decided to analyze, in more detail, the expression of CD25, a component of the high-affinity IL-2R. To this purpose, purified CD4+CD25− Th or CD4+CD25+ Treg cells were polyclonally activated with APCs in medium supplemented or not with IFN-α. The time course of CD25 expression on both CD4+CD25− Th (i.e., CD25− at the beginning of the culture) and CD4+CD25+ Treg cell subsets was subsequently analyzed. As shown in Fig. 5A and 5B, the addition of IFN-α promoted and sustained the expression of CD25 on activated CD4+ Th cells in terms of both percentage and MFI of CD25+ cells. Conversely, IFN-α did not influence CD25 expression on Treg cells. These results indicate that the reduced ability of CD4+ Th cells and Treg cells to respond to IL-2 in the presence of IFN-α cannot be ascribed to the inhibition of CD25 expression, thus suggesting that other mechanisms are involved.
A similar experiment was performed using serial dilution of IFN-α. The results showed a dose-response effect of IFN-α on CD25 expression on CD4+ Th cells. As control, we also measured the induction of the activation, differentiation and memory Ag Ly6C (28–31), whose expression was previously shown to be specifically induced by type I IFNs on bulk T cell population (Supplemental Fig. 1).
To functionally exclude the role of APCs in the induction of both markers on CD4+ Th cells, we compared the effect of IFN-α on CD4+CD25− Th cells when the cells were activated with coated anti-CD3 plus soluble anti-CD28 mAbs or otherwise stimulated with APCs combined with soluble anti-CD3 mAb. The results in Fig. 5C and Supplemental Fig. 1 show that IFN-α was able to enhance CD25 and LyC6 expression on CD4+ Th cells both in the presence or absence of APCs. Thus, these findings clearly indicate a direct effect of IFN-α on CD25 and LyC6 expression on CD4+ Th cells.
As the previous experiments indicated that CD25 chain expression could be specifically upregulated on CD4+ Th cells following the IFN-α treatment, we decided to compare the functional properties of IFN-α on naive versus activated Th cells by analyzing CD25 expression. Thus, in the subsequent experiments, naive CD4+CD25− Th cells or activated CD4+ Th cells preactivated 36 h before (blasts) were both stimulated in the presence of APCs in medium supplemented or not with IFN-α. After 36 h, the cells were harvested and analyzed for CD25 and Ly6C expression (Fig. 5D, 5E). The results demonstrate that the addition of IFN-α induced the upregulation of CD25 as well as LyC6 expression on both naive and blast CD4+ Th cells (Fig. 5D, 5E, right panel). Of note, we observed a double increase in the percentage of Ly6Chigh CD4+ Th cell subset in the presence of IFN-α (Fig. 5E). However, the effect of IFN-α on CD25 expression on blast Th cells was less evident than on naive CD4+ Th cells (Fig. 5D). A control group of CD4+ blasts cells was preactivated with IFN-α and afterward stimulated in the presence or absence of IFN-α. We found that blast CD4+ Th cells, preactivated with IFN-α and restimulated in the absence of this cytokine, showed a decrease in CD25 and LyC6 levels. Conversely, when CD4+ Th cells were activated and then cultured again with IFN-α, these cells displayed the highest level of expression for both markers. Moreover, in all culture conditions examined, IFN-α induced an increase in the percentage of Ly6Chigh CD4+ Th cell subset. These data suggest that both CD25 and LyC6 expression preferentially required the presence of IFN-α to remain upregulated. When similar experiments were performed using CD4+CD25+ Treg cells, no increase in CD25 expression was observed (Fig. 5A and Supplemental Fig. 1). Thus, IFN-α was able to induce the expression of both CD25 and Ly6C markers on naive and activated CD4+ Th cells. However, the effect of IFN-α on CD25 expression was more evident on naive CD4+ Th cells. Notably, the observed upregulation for both CD25 and Ly6C markers required a prolonged exposure to this cytokine.
Effects of IFN-α on CD4+CD25+ Treg cell-mediated suppression
The results in Fig. 4B showed a critical involvement of IFN-α on the expansion of CD4+CD25+ Treg cell subset. To determine whether IFN-α could also affect CD4+CD25+ Treg cell suppressive activity, the cytokine was then added to the in vitro coculture suppression assay. The CD4+CD25+ Treg cell suppression assay was set up using CD4+CD25− Th as target cells cultured in the presence or absence of CD4+CD25+ Treg cells. Suppression was evaluated in terms of inhibition of both CD4+CD25− Th proliferation and IL-2 production. When CD4+CD25− Th cells where incubated with CD4+CD25+ Treg cells, Th cell proliferation was strongly inhibited. The addition of IFN-α promoted a 2-fold increase in total T cell proliferation as compared with the control cultures (Fig. 6A, 6B). As previously described (18, 19), the addition of IL-2 increased total T cell proliferation. The combined supplement of IL-2 and IFN-α did not induce a further proliferation increase.
CD4+CD25+ Treg cell suppressive activity is usually evaluated by measuring the inhibition of cpm in coculture suppression assay. However, this analysis cannot distinguish between the effective contribution of CD4+CD25− Th and CD4+CD25+ Treg cells to total cell proliferation, especially in the presence of exogenous factors. Thus, we attempted to define in more detail the relative contribution of CD4+CD25− Th and CD4+CD25+ Treg cells to total cell proliferation in the coculture assay. In the subsequent experiments, CD4+CD25− Th cells were stained with the cell division marker CFSE before cocolture to evaluate their proliferation after coincubation with unstained CD4+CD25+ Treg cells. As shown in Fig. 6C, CD4+ Th cell proliferation was inhibited in the presence of CD4+CD25+ Treg cells. Conversely, the addition of IL-2 prevented this suppression. Of note, in medium supplemented with IFN-α, the proliferation of CFSE+CD4+ Th cells coincubated with CD4+CD25+ Treg cells was much higher as compared with the control cultures. Thus, according to the results obtained by total T cell proliferation evaluated by cpm, CFSE analysis revealed an inhibition of CD4+CD25+ Treg cell-mediated suppression in the presence of IFN-α (Fig. 6C, 6E). These results confirmed that when IFN-α was combined with IL-2, IFN-α did not interfere with IL-2–mediated block of suppression.
We also analyzed the proliferative contribution of CD4+CD25+ Treg cells in these coculture (Fig. 6D, 6F). In these experiments, CFSE+CD4+CD25+ Treg cells were coincubated with unstained CD25− CD4+ Th cells and APCs in medium supplemented or not with IFN-α, with or without the addition of IL-2. Of note, CFSE+ Treg cell proliferation was not affected in the presence IFN-α treatment as compared with control culture.
A further analysis was performed to assess IL-2 production in the same culture supernatants. As shown in Fig. 6G, upper panel, the addition of IFN-α strongly increased IL-2 production. Although in the presence of CD4+CD25+ Treg cells the IFN-α–induced IL-2 production was partially inhibited, the percentage of inhibition of IL-2 production was reduced as compared with control coculture stimulated with anti-CD3 mAb alone (Fig. 6H, upper panel). Thus, these results demonstrate that IFN-α is able to inhibit CD4+CD25+ Treg cell suppressive function in terms of inhibition of both Th cell proliferation and IL-2 production.
IL-6 is not involved in the block of CD4+CD25+ Treg cell suppression induced by IFN-α
To definitively exclude the role of IL-6 in the block of CD4+CD25+ Treg cell-mediated suppression observed in the presence of IFN-α, we decided to perform a suppression coculture assay by adding a blocking anti–IL-6 mAb. The results shown in Fig. 7 indicate that the supplement of anti–IL-6 mAb to the coculture of CD4+CD25− Th and CD4+CD25+ Treg cells did not affect the CD4+CD25+ Treg cell suppressive activity. Moreover, the addition of anti–IL-6 mAb did not inhibit the block of CD4+CD25+ Treg cell-mediated suppression when the culture was supplemented with IFN-α (Fig. 7A, right panel). Fig. 7C shows the analysis of IL-2 production in the supernatants. The addition of the anti–IL-6 mAb induced a strong decrease in IL-2 production in all the culture conditions examined. Of note, the increased IL-2 production by CD4+CD25− Th cells stimulated with APCs and IFN-α was inhibited in the presence of anti–IL-6 mAb. Thus, the ensemble of these results indicates that 1) IL-6 is not involved in the IFN-α–mediated block of CD4+CD25+ Treg cell suppression, and 2) the enhancement of IL-2 production triggered by IFN-α is strictly IL-6 dependent.
IFN-α–mediated inhibition of CD4+CD25+ Treg cell suppressive activity requires IFNAR expression on both APCs and CD4+CD25+ Treg cells
The previous experiments demonstrated that IFN-α inhibited the CD4+CD25+ Treg cell suppressive activity on CD4+ Th cell activation. However, as all the cell subsets used in the suppressive coculture test expressed the IFNAR chains (Fig. 1), it was not feasible to identify the real cellular target of IFN-α in the suppressive coculture experiments. To better understand the effect of IFN-α in this coculture setting, the mechanism of inhibition of suppression by IFN-α was revealed using APCs and CD4+CD25− Th and CD4+CD25+ Treg cells from IFNAR−/− or littermate control mice. Thus, these different IFNAR+/+ or IFNAR−/− cell subsets were combined in different ways in mix and match experiments. To easily distinguish the target Th cell proliferation, only the CD4+CD25− Th cell subset was stained with CFSE marker (Fig. 8A, 8B). The results indicated that in the absence of IFN-α, CFSE-labeled IFNAR+/+ Th cells when cocultured with IFNAR+/+ or IFNAR−/− Treg cells in the presence of IFNAR+/+ APCs (Fig. 8A, left panel) showed reduced cell cycle progression. Similar results were obtained when IFNAR−/− Th cells were used as target cells. Conversely, the addition of IFN-α to the coculture of IFNAR+/+ or IFNAR−/− Th cells combined with IFNAR+/+ or IFNAR−/−CD4+CD25+ Treg cells led to increased proliferation for both IFNAR+/+ or IFNAR−/− Th cells, because of a block of CD4+CD25+ Treg cell-mediated suppression. This effect was evident when APCs were from IFNAR+/+ mice (Fig. 8A). Conversely, when the same coculture of CD4+CD25− Th and CD4+CD25+ Treg cells isolated from IFNAR+/+ or IFNAR−/− mice was performed in the presence of IFNAR−/− APCs (Fig. 8B), the protective effect of IFN-α was not detected. Thus, as opposed to the CD4+CD25− Th cell proliferation observed in the presence of CD4+CD25+ Treg cells with IFNAR+/+ APCs supplemented with IFN-α, we found that CD4+CD25+ Treg cell exerted their suppressive activity in all the coculture conditions with IFNAR−/−APCs. Thus, the protective effect of IFN-α on CD4+CD25− Th cell proliferation was found to be an indirect effect mediated by IFN-α on APCs. Of note, a slight increase in the suppressive activity was observed in the presence of IFNAR−/− Treg cells in the presence of IFN-α.
To further explore the role of IFN-α in these coculture settings, we decided to analyze the IL-2 concentration in the culture supernatants collected from the cocultures described in Fig. 8A and 8B. The results in Fig. 8D confirmed that the increased IL-2 production observed in the presence of IFN-α was strictly related to the expression of IFNAR on APCs, as this effect was completely lost when IFNAR+/+ APCs were replaced by IFNAR−/− APCs. Moreover, when the coculture was performed in the presence of IFNAR−/− APCs, we observed a broad decrease in IL-2 production either in the presence of IFNAR+/+ and IFNAR−/− Th cells. The results confirmed that, in the presence IFNAR+/+ APCs with IFN-α, IL-2 production was increased, and that the addition of IFNAR+/+CD4+CD25+ Treg cells had only a slight effect on IL-2 production. An interestingly observation was that IFNAR−/−CD4+CD25+ Treg cells were able to inhibit IL-2 production in all the coculture settings examined, in the presence of either IFNAR+/+ or IFNAR−/− Th cells.
Thus, these experiments allowed us to determine that IFN-α was able to block CD4+CD25+ Treg cell suppressive activity on CD4+CD25− Th cell proliferation mainly by acting through APCs. Moreover, by directly affecting CD4+CD25+ Treg cells, IFN-α inhibited the suppression of IL-2 production.
Discussion
Several findings indicate that the type I IFNs can act as immune adjuvant factors, as suggested by their widespread effects on dendritic cell cross-priming, generation of CD8+ T cell response, and Ab production (5–8). However, although great efforts have been addressed to characterize the role of IFN-α on CD8+ T cell activation (5–8), little is known about its immunomodulatory effects on CD4+ T cell subsets. Thus, the current study was aimed at further understanding the role played by IFN-α on CD4+ T cell activation as well as on CD4+CD25+ Treg cell function.
An ensemble of past and more recent data indicate that multiple and opposing signaling pathways can be activated by type I IFNs and the integration of these signaling cascades can lead to the generation of the different and sometimes opposite biological properties ascribed to these cytokines. The IFN-α effect on CD4+ T cell proliferation can represent one of the examples of the complexity of the type I IFN-mediated regulatory effects. Thus, even though IFN-α inhibits CD4+ T cell proliferation (11–14), the supplement of APCs is able to prevent this effect. In particular, in this study, we show that, in the presence of APCs, the addition of IFN-α turns on CD4+ Th cell activity by inducing IL-6 and IL-2 production and by increasing the level of CD25 and Ly6C expression. Nevertheless, the addition of IFN-α inhibits the signal of cell proliferation induced by IL-2. Thus, although IFN-α enhances through APCs endogenous IL-2 production by CD4+ Th cells, at the same time the sensitivity to its mitogenic effect is reduced. The inhibitory effect exerted by IFN-α on IL-2–induced T cell proliferation gains a more relevant significance in terms of CD4+CD25+ Treg cell proliferation, as CD4+CD25+ Treg cell proliferation and homeostasis are strictly dependent on the signal delivered by IL-2 (32).
Although IFN-α inhibits the signal of cell proliferation induced by IL-2, this cytokine upregulates the expression of CD25 on CD4+ Th cells. This evidence indicates that although the magnitude of clonal expansion is reduced, at the same time, the signal delivered by IL-2 can be reinforced as virtually suggested by the highest level of CD25 expression on CD4+ Th cells. Consequently, it is conceivable that, other signaling pathways could be activated by IL-2 on CD4+ Th cells, not strictly related with T cell proliferation. It should be pointed that the major role of IL-2 produced by CD4+ Th cells is not limited to enhance their own proliferation. In fact, IL-2 can also be involved in other important functions, including dendritic cell properties and T cell and NK cell effector activities and the suppressive activity exerted by CD4+CD25+ Treg cells (33). On the basis of these findings, it follows that the marked IFN-α–induced IL-2 production might contribute to other important activities on other cellular subsets, and not to be exclusively directed to affect CD4+ Th cell proliferation. As an example, IL-2 produced by CD4+ T cells has now become clearly relevant for the generation of functional memory CD8+ T cell responses (34, 35). Thus, the effect of IFN-α on IL-2 production by CD4+ Th cells might be crucial for the outcome of the overall immune response following secondary challenge. In accordance, our previous observations have already shown a close relationship between IFN-α and long-lived memory T cells (36). Of note, our results indicate that IFN-α induces and sustains the expression of the memory marker Ly6C on CD4+ T cells (31). However, further investigation is required to clarify the link between the effects of type I IFNs on the early activation of immune response and the subsequent development of memory CD4+ and CD8+ T cells.
The present study reveals a dual effect of IFN-α on CD4+ T cells, the first as inducer of IL-2 production and the latter as antagonist of CD4+CD25+ Treg cell suppressive function. When IFN-α was added in CD4+CD25+ Treg cell suppression assay, the results showed a decrease of the Treg cell suppressive activity exerted on Th cell proliferation, which was also associated with a persistent IL-2 production. Moreover, our results show that this effect was due to an indirect effect of IFN-α on APCs, as the protective effect was completely lost when APCs were derived from IFNAR−/− mice. Although we have identified the cellular target of IFN-α in the block of CD4+CD25+ Treg cell suppression, the molecular mechanism by which IFN-α is able to promote the protective effect exerted by APCs still remains to be clarified.
Thus, in addition to the well described role of IFN-α as activator of innate and CD8+ T cell-mediated adaptive immune responses, the present results shed light on a new function exerted by this cytokine on CD4+CD25+ Treg cell activity, thus unraveling an important role of IFN-α in the control of the CD4+ T cell-mediated adaptive immune responses. Of note, a previous work had already indicated an inhibitory activity of IFN-α on the differentiation of active suppressor cells after allogenic stimulation (37).
Of interest, the current study indicates that IL-2 production is strictly IL-6 dependent. Notably, the relationship between IL-6 and IL-2 has been already observed in the IL-6−/− mice (38). In addition, the results are also in line with and strengthen some recent evidences on the role of IFN-α on IL-6 production by dendritic cells (39). IL-6, together with other TLR-induced cytokines, has been described to render CD4+ T cell resistant to the suppressive activity exerted by CD4+CD25+ Treg cells (21). In our culture conditions, IFN-α was able to induce IL-6 production; nevertheless, this effect was prevented in the presence of CD4+ CD25+ Treg cells. This evidence and the use of a blocking anti–IL-6 mAb rule out a potential role of IL-6 on the block of suppression mediated by IFN-α.
The results demonstrate a direct effect of IFN-α on both CD4+CD25+ Treg and CD4+CD25− Th cell functions. With regard to these effects, the expression of IFNAR chains might play a key role to tuning Th as well as CD4+CD25+ Treg cell activities by type I IFNs. Accordingly, the present research indicates that the IFNAR chain expression can be modulated following activation. The understanding of the mechanisms involved in the modulation of IFNAR chains on both CD4+ Th cells and CD4+CD25+ Treg cells as well as in the extent of responsiveness to this cytokine might have significant implications for the development of new immunotherapeutical approaches.
The inhibitory activity of IFN-α on CD4+CD25+ Treg cell suppression is consistent with the role of IFN-α as general activator of the immune response. The type I IFNs are largely used in clinical practice to treat several malignances, and some viral infections (1). However, several observations indicate that IFN-α can contribute to the breaking of peripheral tolerance through activation and differentiation of naive autoreactive T lymphocytes. Some results have suggested a link between IFN-α/β and the pathogenesis of SLE and type I diabetes mellitus in both rodent models and humans (40). Although other cytokines are also increased in sera from SLE patients, enhanced IFN-α levels often coincide with the disease exacerbation (9, 40, 41). Even if type I IFNs have been indicated to be the master switch participating in the main pathways involved in these autoimmune disorders, to date the mechanism by which this switch is turned on remains unknown. In particular, very little is known about the relationship between CD4+CD25+ Treg cell activity and type I IFNs in those autoimmune diseases where a pathogenetic role of IFN-α has been claimed. In this regard, it is of interest to mention that a recent work showed a correlation between dysfunctional CD4+CD25+ Treg cell activity and highest level of IFN-α in SLE patients (41).
In conclusion, the current study extends our understanding of the complex role of IFN-α in the orchestration of the innate and adaptive immunity by providing new insights into the IFN-α–mediated regulation of the CD4+ T cell responses. The identification of the dual effect of IFN-α on Th and CD4+CD25+ Treg cell activities can inspire new investigation efforts relevant for defining novel intervention strategies in patients with certain types of cancer and autoimmune diseases.
Acknowledgements
We thank Vittorio Colizzi for support, Massimo Spada for technical assistance, and Monica Boirivant for critical review of the manuscript. We thank Ezio Giorda and Rita Carsetti for cell sorting.
Disclosures The authors have no financial conflicts of interest.
Footnotes
This work was supported by Ministero dell’Università e Ricerca, Istituto Superiore di Sanità, Alleanza Contro il Cancro prog.3, and Associazione Italiana per la Ricerca sul Cancro (Italy).
The online version of this article contains supplemental material.