IFN-α and IL-10 Induce the Differentiation of Human Type 1 T Regulatory Cells

Megan K. Levings, Romina Sangregorio, Francesca Galbiati, Stefania Squadrone, Rene de Waal Malefyt and Maria-Grazia Roncarolo
J Immunol May 1, 2001, 166 (9) 5530-5539; DOI: https://doi.org/10.4049/jimmunol.166.9.5530

Abstract

CD4+ T regulatory type 1 (Tr1) cells suppress Ag-specific immune responses in vitro and in vivo. Although IL-10 is critical for the differentiation of Tr1 cells, the effects of other cytokines on differentiation of naive T cells into Tr1 cells have not been investigated. Here we demonstrate that endogenous or exogenous IL-10 in combination with IFN-α, but not TGF-β, induces naive CD4+ T cells derived from cord blood to differentiate into Tr1 cells: IL-10+IFN-γ+IL-2−/lowIL-4−. Naive CD4+ T cells derived from peripheral blood require both exogenous IL-10 and IFN-α for Tr1 cell differentiation. The proliferative responses of the Tr1-containing lymphocyte populations, following activation with anti-CD3 and anti-CD28 mAbs, were reduced. Similarly, cultures containing Tr1 cells displayed reduced responses to alloantigens via a mechanism that was partially mediated by IL-10 and TGF-β. More importantly, Tr1-containing populations strongly suppressed responses of naive T cells to alloantigens. Collectively, these results show that IFN-α strongly enhances IL-10-induced differentiation of functional Tr1 cells, which represents a first major step in establishing specific culture conditions to generate T regulatory cells for biological and biochemical analysis, and for cellular therapy to induce peripheral tolerance in humans.

The qualitative characteristics of immune responses are regulated by T cell subsets through their production of distinctive cytokines. Two well-characterized T cell subsets are Th1 cells which, via production of IFN-γ, promote cell-mediated responses against bacteria, and Th2 cells which, by producing IL-4, IL-5, and IL-13, induce Ab synthesis and the anti-parasite mast cell and eosinophil responses (1). Both subsets originate from a naive T cell precursor, whose differentiation is influenced by both the mode of activation and the environment in which it is initially stimulated. Variables known to influence the development of T cell subsets include the affinity of the TCR for Ag (2), the duration of the interaction between the TCR and Ag (3), and differential costimulation by APCs (4). In addition, it has been shown that cytokines present upon T cell activation define T cell differentiation. It is well established that priming of naive T cells in the presence of IL-12 favors the development of Th1 cells, whereas IL-4 favors the development of Th2 cells (5, 6).

Activation, in vitro or in vivo, of human or mouse CD4+ T cells in the presence of IL-10 has been shown to result in the generation of T cell clones with a cytokine production profile different from that of Th1 or Th2 cells. These T cell clones produce significant amounts of IL-10, IFN-γ, TGF-β, and IL-5, but low amounts of IL-2 and no IL-4 (7, 8). Functionally, these T cell clones have inhibitory effects on Ag-specific activation of naive autologous T cells, which are partially mediated by IL-10 and TGF-β. This novel CD4+ T cell subset was termed T regulatory type 1 (Tr1)3 cells (7). In addition, in a murine model of inflammatory bowel disease in SCID mice, cotransfer of Tr1 cell clones together with pathogenic CD4+ CD45RBhigh T cells prevented the induction of disease (7). Prevention of inflammatory bowel disease was only observed in mice that were administered the Ag recognized by Tr1 cells, demonstrating that Tr1 cells must be activated in vivo via the TCR to exert their regulatory effects. Donor-derived T cells specific for host alloantigens that possessed a Tr1 profile of cytokine production were also isolated from tolerant SCID patients who had been reconstituted with HLA-mismatched stem cells (8, 9, 10). Together, these data indicate that Tr1 cells can function as regulatory cells in vivo.

CD4+ T cells that, in addition to IL-10, secrete high levels of TGF-β and/or IL-4 and that suppress responses to self peptides, have been described in several experimental models of autoimmune diseases (11, 12, 13, 14, 15). Notably, in most cases, these T regulatory cells appear to arise following repeated Ag stimulation either in vitro or in vivo. Buer et al. (16) reported that IL-10-producing T cells generated following repeated stimulation with influenza hemagglutinin could regulate immune responses in vivo. Similarly, repetitive in vitro stimulation with Ag-loaded APCs (17) or in vivo stimulation with superantigen (18, 19) led to the emergence of CD4+ T cells that suppressed naive T cell responses via an IL-10-dependent mechanism. A number of investigators have also documented the presence of Ag-specific regulatory CD4+ T cells that, via a TGF-β-dependent mechanism, can prevent T cell-mediated diseases (13, 14, 15, 20, 21). These observations support the notion that in addition to clonal deletion and clonal anergy, clonal suppression mediated by regulatory T cells that produce suppressive cytokines is an important mechanism of peripheral tolerance.

Due to their low proliferative capacity, both human and mouse Tr1 cells are difficult to isolate under standard culture conditions. To better understand the requirements for Tr1 cell differentiation, we determined which immunoregulatory cytokines, in addition to IL-10, could drive the differentiation of this unique T cell subset in vitro. Using culture conditions in which highly purified naive CD4+ T cells were stimulated via their TCR in the absence of professional APCs, it was demonstrated that IFN-α, but not TGF-β, acts synergistically with IL-10 to induce the generation of CD4+ immunosuppressive Tr1 cells.

Materials and Methods

Cell purification

Human blood was obtained from healthy anonymous donors in accordance with local ethical committee approval. The neonatal leukocytes from umbilical cord blood were prepared by centrifugation over Ficoll-Hypaque gradients (Nycomed Amersham, Uppsala, Sweden), and CD4+ T cells were purified by positive or negative selection. For negative selection, purification was performed by the addition of Dynabeads (Dynal, Oxoid, Italy) coupled to mAbs against CD8, CD14, CD19, and CD56. Beads were added at a 4:1 bead-target cell ratio and incubated for 1 h at 4°C. Beads and contaminating cells were removed by magnetic field. For positive selection, cells were purified using the Miltenyi CD4+ T cell isolation kit according to the manufacturer’s instructions (Miltenyi Biotech, Bergische Gladbach, Germany). Results obtained with CD4+ T cells isolated by negative or positive selection were identical. Human PBMCs were centrifuged over Ficoll-Hypaque gradients, adherent cells were removed by two rounds of incubation on tissue-culture treated flasks for 1 h at 37°C, and CD4+ CD45RO− cells were purified by negative selection. Nonadherent cells were incubated with anti-CD45RO mAbs (5 μg/106 target cells) (UCHL1; Valter Occhiena, Torino, Italy) for 45 min at 4°C. Following washing, Dynabeads coupled to mAbs directed against CD8, CD14, CD19, CD56, and mouse IgG were added, and CD4+CD45RO− cells were isolated as described above.

T cell differentiation

Murine L cell transfectants expressing hCD32 (FCγRII), hCD58 (LFA-3), and hCD80 (22) were cultured in RPMI 1640 (BioWhittaker, Bergamo, Italy) supplemented with 10% FCS (Mascia Brunelli, Milan, Italy), 100 U/ml penicillin/streptomycin (Bristol-Myers Squibb, Sermoneta, Italy), and 2 mM glutamine (Life Technologies, Milan, Italy). L cells were detached by incubation with trypsin-EDTA (Life Technologies) and irradiated (7000 rad) by an x-ray source. Following washing, cells were plated in 24-well plates (Corning, Cambridge, MA) at an initial density of 4 × 105 cells/ml in a 500 μl volume of YSSEL medium (Diaclone, Besançon, France) supplemented with 10% FCS, 1% pooled human serum, and 100 U/ml penicillin/streptomycin (hereafter referred to as complete medium) and 100 ng/ml of anti-CD3 (OKT3; Orthoclone, Jansen Cilag, Italy). After the L cells had adhered, 500 μl of CD4+ cord blood or CD4+CD45RO− peripheral blood T cells were added at an initial density of 4 × 105 cells/ml in complete medium. For differentiation by immobilized anti-CD3, the mAb (10 μg/ml) was immobilized by overnight incubation in 24-well plates in 0.1 M Tris buffer, pH 9.5. Wells were washed three times with PBS before the addition of 106 CD4+ cord blood T cells in complete medium.

All experiments were conducted in the presence of recombinant human (rh) IL-2 (100 U/ml; Chiron Italia, Milan, Italy) and rhIL-15 (1 ng/ml; R&D Systems, Minneapolis, MN). In addition, the following polarizing cytokines were added as indicated: rhIL-10 (100 U/ml; Schering-Plough Research Institute, Kenilworth, NJ), rhIFN-α 2b (5 ng/ml; Schering-Plough Research Institute or PeproTech, Rocky Hill, NJ), rhIL-4 (200 U/ml; Schering-Plough Research Institute), rhIL-12 (5 ng/ml; R&D Systems), TGF-β2 (1 ng/ml; R&D Systems). Anti-human IL-4 mAb (200 ng/ml; PharMingen, San Diego, CA) and anti-human IL-12 mAb (10 μg/ml; PharMingen) were added to the Th1 and Th2 polarizing conditions, respectively. T cells were split as necessary, IL-2 and IL-15 were replenished in all cultures, and IL-4 was replenished only in cultures of Th2 cells. At day 7, T cells were collected, washed, counted, and restimulated under identical conditions for an additional 7 days. At day 14 of in vitro culture, cells were collected, washed, counted, and analyzed for their profile of cytokine production and proliferative capacity. All cells were cultured in humidified incubators at 37°C with 5% CO2.

Analysis of cytokine production

Intracellular cytokines were detected by flow cytometry as described in Sornasse et al. (23), with slight modifications. T cells (1 × 106/ml) were stimulated with immobilized anti-CD3 (10 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs (PharMingen) in complete medium. Upon initiation of the culture, the plates were centrifuged for 5 min at 800 × g. After 3 h of activation, brefeldin A (10 μg/ml; Sigma, Milan, Italy) was added. After a total of 6 h of activation, T cells were collected, washed in PBS, and fixed with 2% formaldehyde. After fixation, T cells were permeabilized by incubation in PBS supplemented with 2% FCS and 0.5% saponin (Sigma). Permeabilized T cells were incubated with PE-labeled anti-hIL-4, anti-hIL-2, or anti-hIL-10, and FITC-coupled anti-hIFN-γ or anti-hIL-4 mAbs. All mAbs were obtained from PharMingen. After washing, cells were analyzed using a FACScan flow cytometer (BD Biosciences, Mountain View, CA), and data were analyzed with CellQuest software (BD Biosciences). Quadrant markers were positioned to include 95% of stained, unstimulated cells in the lower left square.

For detection of IL-5 and TGF-β, capture ELISAs were performed on supernatants of cells that had been stimulated (1 × 106 T cells/ml) with immobilized anti-CD3 (10 μg/ml) and anti-CD28 (1 μg/ml) for 72 h. ELISAs were performed as described (24), and capture and detection mAbs were purchased from PharMingen.

Proliferation of polarized T cells

Polarized T cell subsets were compared for their proliferative capacity following polyclonal or Ag-specific activation. To analyze proliferation in response to polyclonal activation, 96-well flat-bottom plates (Costar) were coated overnight with anti-CD3 mAbs (10 μg/ml) and washed three times with PBS. T cells were plated at an initial density of 2.5 × 105 cells/ml in a final volume of 200 μl of complete medium and soluble anti-CD28 mAbs (1 μg/ml). Control cultures consisted of T cells cultured in the absence of anti-CD3 and anti-CD28 mAbs. To determine proliferation induced by Ag-dependent activation, MLRs were performed. T cells (5 × 105 cells/ml) were stimulated with irradiated (6000 rad) allogenic PBMCs (5 × 105 cells/ml) that had been depleted of CD3+ cells by negative selection. Cells were cocultured in a final volume of 200 μl of complete medium in 96-well round-bottom plates (Costar). Neutralizing anti-IL-10R (20 μg/ml) (3F9, a gift of Kevin Moore, DNAX Research Institute) and/or anti-TGF-β1,2,3 (20 μg/ml) (R&D Systems) mAbs were added as indicated. After 48 h, wells were pulsed for 16 h with 1 μCi/well [3H]thymidine (Amersham, Uppsala, Sweden). Cells were harvested, and counted in a scintillation counter.

To asses the effects of IFN-α on proliferation, cord blood T cells were activated with L cells as described above in the presence of IL-4, IL-12, or IL-10, with or without IFN-α, for the primary stimulation. After 7 days, the cells were washed and restimulated in the presence of IL-4, IL-12, or IL-10, with or without IFN-α, and incorporation of [3H]thymidine was measured 3 days after initiation of the secondary stimulation.

Suppression of naive T cells

Polarized T cell subsets were tested for their ability to suppress the proliferation of naive T cells to alloantigens. Naive autologous CD4+ cord blood T cells were purified as described above and cocultured (2.5 × 105 cells/ml) together with irradiated, allogenic CD3-depleted PBMCs (5 × 105 cells/ml), in the absence or presence of polarized T cells (2.5 × 105 or 5 × 105 cells/ml) in a final volume of 200 μl in complete medium. Control cultures consisted of naive and polarized T cells in the absence of allogenic PBMCs, and polarized T cells plus allogenic PBMCs in the absence of naive T cells. After 4 days, wells were pulsed for 16 h with 1 μCi/well [3H]thymidine.

Effects of IFN-α and IL-10 on TCR-mediated signal transduction

CD4+ T cells were purified from PBMCs by positive selection. Immediately after purification, cells were resuspended at 8 × 106 cells/ml in serum-free medium (X-vivo 15; BioWhittaker) and incubated at 37°C for 16 h in the absence or presence of IL-10 (1000 U/ml) and/or IFN-α (50 ng/ml). After 16 h, the cells were stimulated for 10 min at 37°C with 5-μm latex beads (Interfacial Dynamics, Portland, OR) coated with 1 μg/ml anti-CD3 and 10 μg/ml anti-CD28 mAbs or with 10 μg/ml mouse IgG (Sigma) as a control, at a ratio of 2.5 beads per cell. For samples incubated for 16 h in medium, IL-10 and/or IFN-α were added together with the latex beads for 10 min. Cells were lysed, and whole cell lysates were subjected to SDS-PAGE and immunoblotting (25). Membranes were immunoblotted with 4G10 (Upstate Biotechnology, Lake Placid, NY) to assess whole-cell tyrosine phosphorylation, anti-phospho-extracellular signal-related kinase 1/2 (Cell Signaling Technology, Beverly, MA), anti-phospho-p38 (Cell Signaling Technology), or anti-phospho-c-Jun N-terminal kinase (Santa Cruz Biotechnology, Santa Cruz, CA).

Statistical analysis

All analyses for statistically significant differences were performed with Student’s paired t test. Values of p < 0.05 were considered significant.

Results

IL-10 does not synergize with TGF-β to induce the differentiation of Tr1 cells

We have shown that IL-10 is a critical factor for the generation of Tr1 cells in primary MLRs and in OVA-stimulated cultures of naive T cells and autologous APCs (7). To determine whether IL-10 alone could induce the differentiation of naive CD4+ T cells into Tr1 cells, we established in vitro culture conditions in the absence of professional APCs. In these cultures, highly purified naive CD4+ cord blood lymphocytes were stimulated with anti-CD3 mAbs cross-linked onto CD32+ mouse L cells in the presence or absence of exogenous IL-10. Similar cultures performed in the presence of IL-12 or IL-4 resulted in highly polarized populations of Th1 and Th2 cells, respectively (23). Although activation in the presence of IL-10 reproducibly increased the total number of IL-10-producing cells (Fig. 1⇓, 8.4% compared with 1.6% in the absence of IL-10), only a small population (on average 1.0%, range 0.4–2.3%, n = 12) of cells displayed the typical Tr1 phenotype, as judged by their characteristic cytokine production profile: IL-10+IFN-γ+IL-2−/lowIL-4− (Fig. 1⇓).

FIGURE 1.
FIGURE 1.

IL-10 does not synergize with TGF-β to induce the in vitro differentiation of Tr1 cells. CD4+ cord blood T cells were activated by anti-CD3 mAbs cross-linked on CD32+CD80+CD58+ L cells in the presence or absence of IL-10 and/or TGF-β2. Following two rounds of identical stimulation, T cells were restimulated with immobilized anti-CD3 (10 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs, and cytokine production was determined by intracytoplasmic staining and cytofluorometric analysis, as described in Materials and Methods. One representative experiment of three is shown.

Tr1 cells also produce TGF-β, and TGF-β has previously been reported to positively regulate its own production (26). In addition, both exogenous IL-10 and TGF-β are required for induction of Ag-specific anergy of murine T cells in vitro (27). Therefore, we analyzed the effect of TGF-β on IL-10-induced Tr1 cell differentiation. Cord blood T cells activated in the presence of TGF-β, with or without IL-10, did not differentiate into a population of Tr1-polarized cells. Rather, addition of TGF-β resulted in a general suppression of cytokine production, including that of IL-10. These results are consistent with previous reports indicating that TGF-β strongly inhibits the production of IL-4 and to a lesser extent inhibits that of IL-2 and IFN-γ (28, 29).

CD4+ T cells primed in the presence of IL-10 and IFN-α differentiate into Tr1 cells

It has been shown previously that CD4+ T cells activated by anti-CD3 cross-linked to CD32+ L cells or in vitro-differentiated dendritic cells, in the presence of IFN-α, produced high levels of IL-10 (29, 30). Therefore, we investigated the effects of IL-10 + IFN-α on the differentiation of CD4+ cord blood T cells. As shown in Fig. 2⇓A, activation of CD4+ cord blood T cells in the presence of IL-10 + IFN-α resulted in the differentiation of a significant population of T cells with a Tr1 cytokine production profile. Under these culture conditions a significant subset of T cells produced IL-10 (average: 11.5%, range: 7.5–18.8%, n = 12), whereas a significantly lower proportion of T cells produced IL-2 (average: 4.0%, range: 2.2–11%) or IL-4 (average: 2.1%, range: 0.9–4.2%) in comparison to the Th1 and Th2 cultures, respectively (Fig. 2⇓C). Cultures differentiated in IL-10 + IFN-α contained more IFN-γ-producing cells (average 15%, range: 7.3–28%) than Th2 cultures, but the proportion of IFN-γ-producing cells was lower than in Th1 cultures. As expected, addition of IL-4 or IL-12 resulted in the differentiation of Th2 (IL-4+ IL-10+) and Th1 cells (IFN-γ+ IL-2+), respectively. It is important to note that although in all three culture conditions a similar proportion of IL-10+ T cells was induced, only in cultures with IL-10 + IFN-α did ∼50% of IL-10+ T cells display the expected Tr1 phenotype and were positive for IFN-γ (average 4.3%, range: 1.5–7.7%) but negative or low for IL-4 and IL-2. Addition of neutralizing anti-IL-4 and/or anti-IL-12 mAbs (which inhibited differentiation of Th1 or Th2 cells, respectively) during priming in the presence of IL-10 + IFN-α did not significantly alter this cytokine production profile (data not shown). T cells primed with IL-10 + IFN-α also produced IL-5 and TGF-β (average TGF-β levels detected in supernatants 1.7 ng/ml, range: 0.8–2.8 ng/ml, and average IL-5 levels 0.6 ng/ml, range: 0.4–0.9 ng/ml, n = 3) as previously described for Tr1 cells (7).

FIGURE 2.
FIGURE 2.

CD4+ T cells primed in the presence of IL-10 and IFN-α differentiate into Tr1 cells. Naive CD4+ cord blood T cells (A) or peripheral blood T cells (B) were activated by anti-CD3 mAbs cross-linked on CD32+CD80+CD58+ L cells in the presence of IL-4, IL-12, or IL-10 + IFN-α. Following two rounds of identical stimulation, T cells were restimulated with immobilized anti-CD3 (10 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs, and cytokine production was determined by intracytoplasmic staining and cytofluorometric analysis, as described in Materials and Methods. In C, the average percent positive cells from 12 experiments with cord blood and nine with peripheral blood is shown. ∗, Statistically significant difference in the percentage of cytokine-producing cells in cultures with IL-10 + IFN-α compared with cultures with IL-4 for %IL-4+ cells, to cultures with IL-12 for %IL-2+ cells or IFN-γ+ cells and to cultures with IL-4 or IL-12 for %IL-10+ cells. Error bars represent the SEM.

To establish that the cytokine effects were direct, and not mediated by unknown cell-surface or soluble proteins expressed by the murine L cells, we determined whether IL-10 + IFN-α could promote the differentiation of Tr1 cells from CD4+ cord blood T cells activated by anti-CD3 mAbs immobilized on plastic. Following two rounds of stimulation with immobilized anti-CD3 mAbs, polarized T cell populations, which displayed profiles of cytokine production similar to those obtained with the cells activated by anti-CD3 cross-linked to CD32+CD80+CD58+ L cells, were generated (data not shown). However, the overall percentages of Tr1 cells were lower. These data demonstrate that IL-10 and IFN-α act directly on T cells to induce differentiation of Tr1 cells.

We next determined whether priming of naive CD4+ T cells from peripheral blood in the presence of IL-10 + IFN-α also led to the differentiation of Tr1 cells. Similar to cord blood cells, activation of peripheral blood T cells in the presence of IL-10 + IFN-α reproducibly resulted in a population that contained IL-10+ T cells (average 7.5%, range 3.8–11.5%) and a small but statistically significant percentage of IL-10+IFN-γ+ cells (average 4.0%, range 1.4–7.2%, n = 9; Fig. 2⇑B). Peripheral blood T cells activated in the presence of IL-10 + IFN-α produced less IL-4 (average 3.1%, range: 0.6–4.8%) and IL-2 (average 7.4%, range 1.2–14%) than cultures differentiated in the presence of IL-4 or IL-12, respectively (Fig. 2⇑C). Significant levels of TGF-β (average 3.5 ng/ml, range: 0.8–7.6 ng/ml, n = 3) and IL-5 (average 1.5 ng/ml, range: 1.2–1.6 ng/ml, n = 3) were detected in the culture supernatants of cells primed with IL-10 + IFN-α and restimulated for 72 h with anti-CD3 and anti-CD28 mAbs (data not shown). Thus, priming of CD4+ T cells from cord blood or peripheral blood in the presence of IL-10 + IFN-α results in a population of cells that display a Tr1 phenotype of cytokine production: IL-10+IFN-γ+IL-2−/lowIL-4−.

Comparison of the effects of IL-10 and IFN-α on cord blood and peripheral blood CD4+ T cells

Analysis of the relative roles of exogenous IL-10 and IFN-α in the differentiation of IL-10-producing T cells from cord blood revealed that addition of IFN-α alone was sufficient to induce the differentiation of a population of cells with a Tr1 profile of cytokine production (Fig. 3⇓A). Addition of either IL-10 or IFN-α resulted in the presence of IL-10+ IL-4− T cells, and addition of both IL-10 and IFN-α induced a variable enhancement of IL-10+ IL-4− T cells compared with the numbers induced by IFN-α alone. However, IFN-α alone reproducibly induced a higher proportion of IL-10+ IFN-γ+ T cells compared with IL-10 alone. Although the IL-10+ IFN-γ+ cells were clearly IL-4−, it cannot be excluded that they produced low levels of IL-2. Addition of IL-10 to IFN-α resulted in a variable increase in the proportion of IL-10+IFN-γ+ cells, but upon comparison of data from 12 donors, this increase was not statistically significant (Fig. 3⇓C). As demonstrated previously, cord blood T cells have an intrinsic ability to produce high levels of endogenous IL-10 in comparison to peripheral blood T cells (31, 32). Unpolarized cord blood T cells produced 7- to 13-fold higher levels of IL-10 (range: 40–130 ng/ml) in comparison to unpolarized peripheral blood T cells (range: 6–10 ng/ml; data not shown). Therefore, the variable effects of exogenous IL-10 in enhancing the proportion of IL-10+IFN-γ+ T cells may be due to the variable production of endogenous IL-10.

FIGURE 3.
FIGURE 3.

Comparison of the relative roles of IL-10 and IFN-α in the differentiation of naive CD4+ T cells derived from cord blood or peripheral blood. CD4+ T cells from cord blood (A) or peripheral blood (B) were activated by anti-CD3 mAbs cross-linked on CD32+CD80+CD58+ L cells in the absence (−) or presence of IL-10 and/or IFN-α. Following two rounds of identical stimulation, T cells were restimulated with immobilized anti-CD3 (10 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs, and cytokine production was determined by intracytoplasmic staining and cytofluorometric analysis, as described in Materials and Methods. For C, the average percentage of IL-10+IFN-γ+ cells from 12 experiments with cord blood and nine with peripheral blood is shown.∗, Statistically significant difference in the percentage of IL-10+IFN-γ+ cells in cultures with IFN-α or IL-10 + IFN-α compared with cells cultured in IL-10 alone.Error bars represent the SEM.

To further investigate the role of IL-10 vs IFN-α in inducing the differentiation of Tr1 cells, we tested naive T cells derived from peripheral blood. In Fig. 3⇑B it is shown that, in contrast to cord blood T cells, naive CD4+ peripheral blood T cells reproducibly required both exogenous IL-10 and IFN-α to differentiate into cells with a Tr1 cytokine production pattern. Cultures with IFN-α alone did not contain statistically significant higher proportions of IL-10+IFN-γ+ cells than cultures differentiated in the presence of IL-10 alone (average 0.5% with IL-10 and 1.0% with IFN-α, n = 9). In contrast, addition of both IL-10 and IFN-α resulted in a statistically significant increase in both the total proportion of IL-10-producing T cells (average 7.5, range 3.8–11.5%) and the IL-10+IFN-γ+ cells (average 4.0%, range 1.4–7.2%) in comparison to IFN-α alone (Fig. 3⇑C).

CD4+ T cells activated in the presence of IL-10 and IFN-α are impaired in their responses to polyclonal activation and alloantigens

In addition to their cytokine production profile, Tr1 cells are functionally characterized by their intrinsic low proliferative capacity (7, 8). Therefore, we investigated whether the presence of a population of cells with a Tr1 cytokine production profile correlated with an impaired ability of the cultures to proliferate in response to polyclonal activation or alloantigens in primary MLRs.

In comparison to Th1 and Th2 control cultures, cultures of cord blood containing Tr1 cells had a reduced proliferative responses to activation by anti-CD3 and anti-CD28 mAbs (Fig. 4⇓A). In addition, the cells differentiated in IL-10 + IFN-α reproducibly failed to proliferate significantly in response to alloantigens in comparison to Th2 and Th1 cultures (Fig. 4⇓B). Importantly, addition of neutralizing anti-IL-10R and anti-TGF-β mAbs resulted in partial (average 5-fold increase) restoration of the proliferation in response to alloantigens (Fig. 4⇓B), indicating that the reduced proliferative responses were due, in part, to the production of IL-10 and TGF-β in the cultures. Thus, IL-10 + IFN-α induced the differentiation of a population of T cells that not only displayed a Tr1-like profile of cytokine production, but also had the intrinsically low proliferative potential of regulatory T cells.

FIGURE 4.
FIGURE 4.

CD4+ cord blood T cells activated in the presence of IL-10 and IFN-α display a reduced proliferative capacity following polyclonal or Ag-specific activation. CD4+ cord blood T cells were activated by anti-CD3 mAbs cross-linked to CD32+CD80+CD58+ L cells in the presence of IL-4, IL-12, or IL-10 and IFN-α. Following two rounds of stimulation, T cells were tested for their ability to proliferate in response to immobilized anti-CD3 (10 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs (A) or CD3-depleted allogeneic PBMCs (MLR) (B). In B, neutralizing anti-IL-10R (20 μg/ml) and/or anti-TGF-β (20 μg/ml) mAbs were added as indicated. Proliferation was measured by incorporation of [3H]thymidine into de novo synthesized DNA 3 days after initiation of the culture. All cultures were performed in triplicate, and error bars represent the SEM. ∗, Statistically significant reduction compared with cultures with IL-4 or IL-12. One representative experiment of 12 (A) and three (B) is shown.

IFN-α alone was sufficient to differentiate cord blood T cells into cells with a reduced capacity to proliferate following polyclonal activation (Fig. 5⇓A) or stimulation with alloantigens (Fig. 5⇓B). In contrast, addition of IFN-α alone had a minimal effect on the proliferative responses of peripheral blood T cells, and only T cells cultured in both IL-10 and IFN-α showed the characteristic reduction in proliferation to polyclonal activation (Fig. 5⇓C) and a strongly reduced proliferative response to alloantigens (Fig. 5⇓D).

FIGURE 5.
FIGURE 5.

Comparison of the relative roles of IL-10 and IFN-α in the generation of cells with reduced proliferative capacities from cord blood or peripheral blood. CD4+ cord blood (A and B) or CD4+CD45RO− peripheral blood (C and D) T cells were activated by anti-CD3 mAbs cross-linked on CD32+CD80+CD58+ L cells in the absence (−) or presence of IL-10 and/or IFN-α. Following two rounds of identical stimulation, T cells were tested for their ability to proliferate in response to polyclonal activation by immobilized anti-CD3 (10 μg/ml) and soluble anti-CD28 (1 μg/ml) mAbs (A and C) or CD3-depleted allogeneic PBMCs (MLR) (B and D). Proliferation was measured by incorporation of [3H]thymidine into de novo synthesized DNA 3 days after initiation of the culture. All cultures were performed in triplicate, and error bars represent the SEM. ∗, Statistically significant reduction compared with cultures with IL-10. One representative experiment of 12 (A and B) and six (C and D) is shown.

CD4+ cord blood T cells activated in the presence of IL-10 and IFN-α suppress the proliferative response of naive autologous T cells to alloantigens

To establish the functional capacity of in vitro-differentiated Tr1 cells to act as regulatory/suppressor cells (7), we tested whether IL-10+IFN-γ+IL-2−/lowIL-4− T cells differentiated in the presence of IFN-α and/or IL-10 could suppress the response of autologous CD4+ T cells. As shown in Fig. 6⇓, addition of T cells primed in the absence or presence of IL-10 had a minimal effect on the proliferation of naive autologous cells. In contrast, T cells cultured in the presence of IFN-α, or IL-10 + IFN-α, strongly suppressed the proliferation of naive autologous T cells. On average, T cells from cultures with IFN-α + IL-10 inhibited the proliferation of naive cells by 54.8 ± 11.4% (n = 3) when cocultured at a ratio of 1:1 (naive:polarized) and by 58.1 ± 12.6% at a ratio of 1:2.

FIGURE 6.
FIGURE 6.

CD4+ cord blood T cells differentiated in the presence of IFN-α and/or IL-10 actively suppress the proliferation of naive autologous CD4+ T cells to alloantigens. Responses of naive CD4+ cord blood T cells to CD3-depleted allogenic PBMCs were tested in a primary MLR (black column). CD4+ cord blood T cells were activated by anti-CD3 mAbs cross-linked on CD32+CD80+CD58+ L cells in the absence (−) or presence of IL-10 and/or IFN-α. Following two rounds of identical stimulation, T cells were tested for their ability to suppress the proliferation of naive autologous CD4+ T cells to alloantigens at a ratio of 1:1 (naive:polarized, gray columns) or 1:2 (open columns). Proliferation was measured by incorporation of [3H]thymidine into de novo synthesized DNA 5 days after initiation of the culture. Proliferation of polarized cord blood T cells to alloantigens in the absence of naive T cells was similar to that shown in Fig. 5⇑B. Proliferation of naive and polarized T cells together, in the absence of allogenic PBMCs, was <2000 cpm. All cultures were performed in triplicate, and error bars represent the SEM. ∗, Statistically significant reduction in proliferation of naive T cells compared with control (black columns). One representative experiment of three is shown.

IL-10 and IFN-α do not affect TCR-mediated intracellular signaling or proliferation of CD4+ T cells

It has been described that IL-10 prevents T cell activation (33) and that IFN-α may mediate anti-proliferative effects by interfering with TCR signaling (34). Therefore, we determined whether IL-10 and/or IFN-α act by altering TCR-mediated intracellular signals in our system. As shown in Fig. 7⇓A, addition of IFN-α and/or IL-10 either 16 h before, or at the time of, TCR activation affected neither whole-cell tyrosine phosphorylation nor activation of the mitogen-activated protein (MAP) family kinases, extracellular signal-related kinase 1/2, c-Jun N-terminal kinase 1/2 or p38. However, both IL-10 and IFN-α were biologically active as judged by their ability to induce tyrosine phosphorylation of STAT-3 and STAT-1, respectively (data not shown).

FIGURE 7.
FIGURE 7.

Effects of IL-10 and IFN-α on TCR-mediated intracellular signaling and CD4+ T cell proliferation. A, CD4+ T cells were purified from peripheral blood and incubated overnight in the absence or presence of IFN-α (50 ng/ml) and/or IL-10 (1000 U/ml). The following day, T cells were stimulated with latex beads coated with anti-CD3 (1 μg/ml) and anti-CD28 (10 μg/ml) mAbs for 10 min at 37°C. In one set of samples, cytokines were added only upon addition of the latex beads. Cell lysates were subjected to SDS-PAGE and were immunoblotted with Abs recognizing anti-phospho (p)-tyrosine or anti-p-MAP family kinases. B, CD4+ cord blood T cells were activated by anti-CD3 mAbs cross-linked on CD32+CD80+CD58+ L cells in the presence of IL-4, IL-12, or IL-10, in the absence or presence of IFN-α in the primary (I), secondary (II), or primary and secondary stimulations (I+II). Incorporation of [3H]thymidine was assessed 3 days after initiation of the secondary stimulation. All cultures were performed in triplicate, and error bars represent the SEM. One representative experiment of three is shown. C, T cells were activated as in B in the presence of IL-4, IL-12, or IL-10 + IFN-α. At the end of the primary (day 7) and secondary (day 14) stimulations cells were counted and the fold increase in total cell number was determined. C, Average of seven independent experiments; ∗, statistically significant reduction in fold increase of cell number compared with the controls in IL-4 or IL-12.

In addition, IFN-α did not have significant anti-proliferative effects when we activated cord blood T cells at day 0 and/or at day 7 in the presence of IFN-α and the indicated polarizing cytokines (IL-4, IL-12, or IL-10) and measured the proliferative responses after 3 days. Addition of IFN-α during the primary, secondary, or both the primary and secondary stimulation periods had only a modest inhibitory effect (∼30% reduction) on proliferation of cells cultured with IL-12 or IL-10, whereas no significant effect was observed in cells cultured with IL-4 (Fig. 7⇑B). Moreover, results in Fig. 7⇑C show that T cells cultured in IL-4, IL-12, or IL-10 + IFN-α had similar increases in numbers during the primary (7-day) stimulation period. In contrast, at the end of the secondary stimulation, during which significant differentiation of Tr1, Th1, and Th2 cells was observed, the recovery of T cells cultured in the presence of IL-10 + IFN-α was significantly lower compared with that of cultures with IL-4 or IL-12.

Together, these data indicate that IFN-α does not act as a general anti-proliferative agent, but that it drives the differentiation of Tr1 cells which, upon reactivation, suppress the proliferation of bystander non-Tr1 cells. It should be noted that despite the low cell recovery at day 14 in cultures with IL-10 + IFN-α, a significant number of Tr1 cells could be recovered. Based on the average percentage of IL-10+IFN-γ+ cells (Fig. 3⇑C) and the average fold increases, culture of 106 naive CD4+ T cells in IL-10 + IFN-α would result in ∼2 × 106 Tr1 cells at day 14 of culture.

Discussion

Naive CD4+ T cells from cord blood or peripheral blood can be differentiated in vitro not only into Th1 or Th2 cells, but also into Tr1 cells. Similar to Th1 and Th2 cells, the differentiation of Tr1 cells is regulated by cytokines present during T cell activation. We demonstrate that IL-10 and IFN-α have additive effects on the differentiation of a population of cells that displays a Tr1 cytokine production profile: IL-10+IFN-γ+IL-2−/lowIL-4−. Furthermore, a direct differentiation from naive T cells toward a Tr1 phenotype was observed, suggesting that Tr1 cells arise directly from a CD4+ T cell precursor. Analysis of the proliferative responses of populations containing Tr1 cells revealed that priming in the presence of IL-10 + IFN-α results in a synergistic reduction in the proliferative capacity following polyclonal activation or stimulation with alloantigens. Proliferation to alloantigens was partially restored by addition of neutralizing anti-IL-10R and anti-TGF-β mAbs. These data correlate with our previous observations that Tr1 cells have an intrinsically low proliferative capacity (8) and that they have immunoregulatory functions that are partially mediated by IL-10 and TGF-β (7). Furthermore, our results suggest that, in addition to IL-10 and TGF-β, other soluble or cell-surface proteins are involved in regulation of immune responses by Tr1 cells. Importantly, cultures containing Tr1 cells suppressed the proliferation of naive autologous T cells, demonstrating that in vitro-differentiated Tr1 possess the key functional property of T regulatory cells and can mediate active suppression (35). The suppressive effects of in vitro-differentiated Tr1 cells are remarkably potent considering that only 1.5–7.7% of the cells in cultures with IL-10 + IFN-α are Tr1 cells (i.e., IL-10+IFN-γ+). Thus fewer than 4000 Tr1 cells are capable of suppressing the proliferation of 50,000 naive CD4+ T cells by ∼50%.

Cord blood and peripheral blood CD4+ T cells differ in their requirements for exogenous cytokines for the differentiation of Tr1 cells. Addition of IFN-α alone to cord blood T cells was, in some cases, sufficient to induce a population of T cells with a Tr1-cytokine production profile and immunosuppressive properties. Cord blood T cells have the intrinsic ability to produce high levels of IL-10 (31, 32), which could be further enhanced by the presence of IFN-α (29, 30). Therefore, autocrine IL-10 production by cord blood T cells may alleviate the requirement for exogenous IL-10. In contrast, peripheral blood T cells, which produced 7- to 13-fold less endogenous IL-10 than cord blood T cells, required both exogenous IL-10 and IFN-α to induce the differentiation of functional Tr1 cells.

It has previously been shown that IL-12 can also induce the differentiation of IL-10+IFN-γ+ cells from PBMC (36, 37). In our system using purified CD4+ T cells, we observed that differentiation of cord blood, but not peripheral blood, in the presence of IL-12 resulted in a significant population of IL-10+IFN-γ+ T cells. However, the presence of these IL-10+IFN-γ+ T cells was also associated with a high percentage of IL-2-positive cells, and no reduction in proliferation or suppressive activity. These data suggest that if Tr1 cells were present, their regulatory effects were largely reversed in the presence of “classical” Th1 cells that clearly represent the majority of T cells following culture with IL-12.

IL-10 and IFN-α have been shown to prevent activation of T cells and can affect the growth of multiple cell types (33, 34). In addition, both cytokines can promote cell-cycle arrest in CD4+ T cells (38, 39). However, there are also reports documenting enhancing effects of both IL-10 (40) and type I IFNs (41, 42) on survival and proliferation of T cells. Our experiments showed that neither IFN-α nor IL-10 had an effect on TCR-mediated activation of MAP family kinases, indicating that these cytokines did not act by preventing activation of early events in TCR-mediated signal transduction. In addition, we did not observe any significant anti-proliferative effects of IFN-α and/or IL-10, as measured by thymidine incorporation 3 days after activation, whereas there was a striking decrease in the total number of cells recovered at the end of the culture in the presence of these cytokines. These data suggest that IFN-α and IL-10 do not act as general anti-proliferative agents, but rather as factors that induce the differentiation of Tr1 cells, which themselves have a low proliferative capacity, and in addition inhibit the growth of non-Tr1 cells present in the culture.

Our data indicate that there is a difference between the cytokines required for the differentiation and the effector functions of Tr1 cells. Although TGF-β in part mediates the immunosuppressive functions of Tr1 cells, and has immunosuppressive effects on T cell-mediated pathology (13, 43), we found that TGF-β had no effect on the differentiation of these cells. Rather, a general suppression of cytokine production (IL-2, IL-4, IL-10, and IFN-γ) by T cells primed in the presence of TGF-β was observed. These data are in agreement with recent reports describing that TGF-β inhibits production of IFN-γ and IL-4, and differentiation of murine Th1 and Th2 cells (44, 45).

In contrast to TGF-β, IL-10 was important for both the differentiation and effector functions of Tr1 cells. IL-10 was required for the differentiation of Tr1 cells derived from peripheral blood, and despite the fact that cord blood cells produced considerable amounts of endogenous IL-10, in several independent experiments, exogenous IL-10 further increased the number of IL-10+ cord blood T cells. IL-10 is well characterized as an effector cytokine involved in regulation of T cell-mediated responses via both direct and indirect mechanisms (reviewed in Ref. 33), but its role in differentiation of Th cells has not been extensively addressed. The present data support previous studies that demonstrated that T cell priming in the presence of APCs and IL-10 results in cells that produce IL-10 and TGF-β and have immunoregulatory properties (7).

In this study, addition of IFN-α resulted not only in enhanced IL-10 and IFN-γ production compared with cultures in IL-10 alone, but also in down-regulation of IL-4 and IL-2 synthesis, consistent with previous reports (29, 30, 46, 47, 48). Through its ability to inhibit production of IL-4, IFN-α has previously been implicated in the differentiation of human Th1 cells (49). However, our data show that IFN-α is not involved in the differentiation of Th1 cells, but rather, together with IL-10, is a key factor in the differentiation of Tr1 cells.

The receptors for IL-10 and IFN-α are structurally similar: both are members of the class II cytokine receptor family (50) and they activate similar pathways of intracellular signal transduction. However, the molecular mechanism(s) by which IL-10 and IFN-α induce the differentiation of Tr1 cells are unknown. It is well documented that the Jak/STAT pathway is the key intracellular pathway involved in differentiation of Th1 and Th2 cells (51). IL-10 activates STAT-1 and STAT-3 (52, 53), whereas IFN-α activates STAT-1, -2, and -3 in most cells (54), and in human lymphocytes can also activate STAT-4 and -5 (42, 49). It remains to be determined which members of the Jak/STAT pathway are required for the differentiation of Tr1 cells.

In the human, priming of naive T cells with type 2 dendritic cells (DC2) cultured in the presence of IL-3 and activated by CD40 ligand results in polarization toward Th2 cells (55). However, viral-infected DC2 cells produce high levels of IFN-α (56, 57); therefore, it is tempting to speculate that DC2 cells may also be involved in the differentiation of Tr1 cells.

The hypothesis that type I IFNs may not only be involved in innate immunity but also in the generation of immunoregulatory T cells in vivo is supported by the observation that in the absence of functional IFN responses, viral infection results in a profound Th2-mediated inflammation (58). Furthermore, treatment with IFN-β of patients suffering from relapsing-remitting multiple sclerosis results in increased levels of IL-10 production by mononuclear cells and favorably alters the disease course (59, 60, 61).

A number of T regulatory cell subsets have been identified, and there is growing evidence that these cells are essential for controlling immune responses (reviewed in Refs. 35, 62, 63). The relationship between Tr1 cells and other types of CD4+ T regulatory cells, such as those that are CD25+, CD45RBlow, or TGF-β+ (Th3) is unclear. Although we have much to learn about the biology of T regulatory cells, it is clear that via expression of inhibitory cell-surface molecules and production of cytokines such as IL-10 and TGF-β, T regulatory cells maintain peripheral tolerance to self and foreign Ags, and that in their absence the host may succumb to a variety of autoimmune and chronic inflammatory diseases (11, 13, 35, 63, 64). We have shown that IL-10 and IFN-α promote the in vitro differentiation of type 1 T regulatory cells. The ability to isolate and culture Tr1 cells in vitro is a crucial step toward learning more about their basic biology and exploring the use of these cells as a “cellular therapy” to regulate immune responses to self- or allo-Ags in vivo.

Acknowledgments

We thank Satwant Narula, Claudio Bordignon, Rosa Bacchetta, and Paul Orban for many helpful scientific discussions and advice.

Footnotes

  • ↵1 This work was partially supported by a grant from the Italian Telethon Foundation. M.K.L. is the recipient of a postdoctoral fellowship from the Canadian Institutes of Health Research.

  • ↵2 Address correspondence and reprint requests to Dr. Maria-Grazia Roncarolo, San Raffaele Telethon Institute for Gene Therapy, Via Olgettina 58, Milan, Italy 20132. E-mail address: m.roncarolo{at}hsr.it

  • 3 Abbreviations used in this paper: Tr1, T regulatory type 1; MAP, mitogen-activated protein; DC2, type 2 dendritic cell; rh, recombinant human.

  • Received December 5, 2000.
  • Accepted March 1, 2001.

References

  1. ↵
    Abbas, A. K., K. M. Murphy, A. Sher. 1996. Functional diversity of helper T lymphocytes. Nature 383: 787
    OpenUrlCrossRefPubMed
  2. ↵
    Constant, S. L., K. Bottomly. 1997. Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches. Annu. Rev. Immunol. 15: 297
    OpenUrlCrossRefPubMed
  3. ↵
    Iezzi, G., E. Scotet, D. Scheidegger, A. Lanzavecchia. 1999. The interplay between the duration of TCR and cytokine signaling determines T cell polarization. Eur. J. Immunol. 29: 4092
    OpenUrlCrossRefPubMed
  4. ↵
    McAdam, A. J., A. N. Schweitzer, A. H. Sharpe. 1998. The role of B7 co-stimulation in activation and differentiation of CD4+ and CD8+ T cells. Immunol. Rev. 165: 231
    OpenUrlCrossRefPubMed
  5. ↵
    O’Garra, A.. 1998. Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity 8: 275
    OpenUrlCrossRefPubMed
  6. ↵
    Romagnani, S.. 1997. The Th1/Th2 paradigm. Immunol. Today 18: 263
    OpenUrlCrossRefPubMed
  7. ↵
    Groux, H., A. O’Garra, M. Bigler, M. Rouleau, S. Antonenko, J. E. de Vries, M. G. Roncarolo. 1997. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389: 737
    OpenUrlCrossRefPubMed
  8. ↵
    Bacchetta, R., M. Bigler, J. L. Touraine, R. Parkman, P. A. Tovo, J. Abrams, R. de Waal Malefyt, J. E. de Vries, M. G. Roncarolo. 1994. High levels of interleukin 10 production in vivo are associated with tolerance in SCID patients transplanted with HLA mismatched hematopoietic stem cells. J. Exp. Med. 179: 493
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Bacchetta, R., B. A. Vandekerckhove, J. L. Touraine, M. Bigler, S. Martino, L. Gebuhrer, J. E. de Vries, H. Spits, M. G. Roncarolo. 1993. Chimerism and tolerance to host and donor in severe combined immunodeficiencies transplanted with fetal liver stem cells. J. Clin. Invest. 91: 1067
    OpenUrlCrossRefPubMed
  10. ↵
    Bacchetta, R., R. de Waal Malefijt, H. Yssel, J. Abrams, J. E. de Vries, H. Spits, M. G. Roncarolo. 1990. Host-reactive CD4+ and CD8+ T cell clones isolated from a human chimera produce IL-5, IL-2, IFN-γ and granulocyte/macrophage-colony-stimulating factor but not IL-4. J. Immunol. 144: 902
    OpenUrlAbstract
  11. ↵
    Asseman, C., S. Mauze, M. W. Leach, R. L. Coffman, F. Powrie. 1999. An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. J. Exp. Med. 190: 995
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Kitani, A., K. Chua, K. Nakamura, W. Strober. 2000. Activated self-MHC-reactive T cells have the cytokine phenotype of Th3/T regulatory cell 1 T cells. J. Immunol. 165: 691
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Powrie, F., J. Carlino, M. W. Leach, S. Mauze, R. L. Coffman. 1996. A critical role for transforming growth factor-β but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RBlow CD4+ T cells. J. Exp. Med. 183: 2669
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Han, H. S., H. S. Jun, T. Utsugi, J. W. Yoon. 1996. A new type of CD4+ suppressor T cell completely prevents spontaneous autoimmune diabetes and recurrent diabetes in syngeneic islet-transplanted NOD mice. J. Autoimmun. 9: 331
    OpenUrlCrossRefPubMed
  15. ↵
    Chen, Y., V. K. Kuchroo, J. Inobe, D. A. Hafler, H. L. Weiner. 1994. Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science 265: 1237
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Buer, J., A. Lanoue, A. Franzke, C. Garcia, H. von Boehmer, A. Sarukhan. 1998. Interleukin 10 secretion and impaired effector function of major histocompatibility complex class II-restricted T cells anergized in vivo. J. Exp. Med. 187: 177
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Chakraborty, N. G., L. Li, J. R. Sporn, S. H. Kurtzman, M. T. Ergin, B. Mukherji. 1999. Emergence of regulatory CD4+ T cell response to repetitive stimulation with antigen-presenting cells in vitro: implications in designing antigen-presenting cell-based tumor vaccines. J. Immunol. 162: 5576
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Miller, C., J. A. Ragheb, R. H. Schwartz. 1999. Anergy and cytokine-mediated suppression as distinct superantigen-induced tolerance mechanisms in vivo. J. Exp. Med. 190: 53
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Sundstedt, A., I. Hoiden, A. Rosendahl, T. Kalland, N. van Rooijen, M. Dohlsten. 1997. Immunoregulatory role of IL-10 during superantigen-induced hyporesponsiveness in vivo. J. Immunol. 158: 180
    OpenUrlAbstract
  20. ↵
    Khoo, U. Y., I. E. Proctor, A. J. Macpherson. 1997. CD4+ T cell down-regulation in human intestinal mucosa: evidence for intestinal tolerance to luminal bacterial antigens. J. Immunol. 158: 3626
    OpenUrlAbstract
  21. ↵
    Bridoux, F., A. Badou, A. Saoudi, I. Bernard, E. Druet, R. Pasquier, P. Druet, L. Pelletier. 1997. Transforming growth factor β (TGFβ)-dependent inhibition of T helper cell 2 (TH2)-induced autoimmunity by self-major histocompatability complex (MHC) class II, regulatory CD4+ T cell lines. J. Exp. Med. 185: 1769
    OpenUrlAbstract/FREE Full Text
  22. ↵
    de Waal Malefyt, R., S. Verma, M. T. Bejarano, M. Ranes-Goldberg, M. Hill, H. Spits. 1993. CD2/LFA-3 or LFA-1/ICAM-1 but not CD28/B7 interactions can augment cytotoxicity by virus-specific CD8+ cytotoxic T lymphocytes. Eur. J. Immunol. 23: 418
    OpenUrlCrossRefPubMed
  23. ↵
    Sornasse, T., P. V. Larenas, K. A. Davis, J. E. de Vries, H. Yssel. 1996. Differentiation and stability of T helper 1 and 2 cells derived from naive human neonatal CD4+ T cells, analyzed at the single-cell level. J. Exp. Med. 184: 473
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Levings, M. K., J. W. Schrader. 1999. IL-4 Inhibits the production of TNF-α and IL-12 by STAT6-dependent and -independent mechanisms. J. Immunol. 162: 5224
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Orban, P. C., M. K. Levings, J. W. Schrader. 1999. Heterodimerization of the α and β chains of the interleukin-3 (IL-3) receptor is necessary and sufficient for IL-3-induced mitogenesis. Blood 94: 1614
    OpenUrlAbstract/FREE Full Text
  26. ↵
    Seder, R. A., T. Marth, M. C. Sieve, W. Strober, J. J. Letterio, A. B. Roberts, B. Kelsall. 1998. Factors involved in the differentiation of TGF-β-producing cells from naive CD4+ T cells: IL-4 and IFN-γ have opposing effects, while TGF-β positively regulates its own production. J. Immunol. 160: 5719
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Zeller, J. C., A. Panoskaltsis-Mortari, W. J. Murphy, F. W. Ruscetti, S. Narula, M. G. Roncarolo, B. R. Blazar. 1999. Induction of CD4+ T cell alloantigen-specific hyporesponsiveness by IL-10 and TGF-β. J. Immunol. 163: 3684
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Swain, S. L., G. Huston, S. Tonkonogy, A. Weinberg. 1991. Transforming growth factor-β and IL-4 cause helper T cell precursors to develop into distinct effector helper cells that differ in lymphokine secretion pattern and cell surface phenotype. J. Immunol. 147: 2991
    OpenUrlAbstract
  29. ↵
    Demeure, C. E., C. Y. Wu, U. Shu, P. V. Schneider, C. Heusser, H. Yssel, G. Delespesse. 1994. In vitro maturation of human neonatal CD4 T lymphocytes. II. Cytokines present at priming modulate the development of lymphokine production. J. Immunol. 152: 4775
    OpenUrlAbstract
  30. ↵
    McRae, B. L., R. T. Semnani, M. P. Hayes, G. A. van Seventer. 1998. Type I IFNs inhibit human dendritic cell IL-12 production and Th1 cell development. J. Immunol. 160: 4298
    OpenUrlAbstract/FREE Full Text
  31. ↵
    de La Selle, V., E. Gluckman, M. Bruley-Rosset. 1998. Graft-versus-host disease and graft-versus-leukemia effect in mice grafted with peripheral newborn blood. Blood 92: 3968
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Roncarolo, M. G., E. Vaccarino, P. Saracco, R. Miniero, E. Madon, H. Yssel. 1998. Immunologic properties of cord blood. H. E. Broxmeyer, ed. Cellular Characteristics of Cord Blood and Cord Blood Transplantation 67 AABB Press, Bethesda, MD.
  33. ↵
    Moore, K. W., A. O’Garra, R. de Waal Malefyt, P. Vieira, T. R. Mosmann. 1993. Interleukin-10. Annu. Rev. Immunol. 11: 165
    OpenUrlCrossRefPubMed
  34. ↵
    Petricoin, E. F. 3rd, S. Ito, B. L. Williams, S. Audet, L. F. Stancato, A. Gamero, K. Clouse, P. Grimley, A. Weiss, J. Beeler, et al 1997. Antiproliferative action of interferon-α requires components of T-cell-receptor signalling. Nature 390: 629
    OpenUrlCrossRefPubMed
  35. ↵
    Shevach, E. M.. 2000. Regulatory T cells in autoimmmunity. Annu. Rev. Immunol. 18: 423
    OpenUrlCrossRefPubMed
  36. ↵
    Gerosa, F., C. Paganin, D. Peritt, F. Paiola, M. T. Scupoli, M. Aste-Amezaga, I. Frank, G. Trinchieri. 1996. Interleukin-12 primes human CD4 and CD8 T cells clones for high production of both interferon-γ and interleukin-10. J. Exp. Med. 183: 2559
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Windhagen, A., D. E. Anderson, A. Carrizosa, R. E. Williams, D. A. Hafler. 1996. IL-12 induced human T cells secreting IL-10 with IFN-γ. J. Immunol. 157: 1127
    OpenUrlAbstract
  38. ↵
    Essayan, D. M., G. Krishnaswamy, A. Oriente, L. M. Lichtenstein, S. K. Huang. 1999. Differential regulation of antigen-induced IL-4 and IL-13 generation from T lymphocytes by IFN-α. J. Allergy Clin. Immunol. 103: 451
    OpenUrlCrossRefPubMed
  39. ↵
    Perrin, G. Q., H. M. Johnson, P. S. Subramaniam. 1999. Mechanism of interleukin-10 inhibition of T-helper cell activation by superantigen at the level of the cell cycle. Blood 93: 208
    OpenUrlAbstract/FREE Full Text
  40. ↵
    Groux, H., M. Bigler, J. E. de Vries, M. G. Roncarolo. 1998. Inhibitory and stimulatory effects of IL-10 on human CD8+ T cells. J. Immunol. 160: 3188
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Marrack, P., J. Kappler, T. Mitchell. 1999. Type I interferons keep activated T cells alive. J. Exp. Med. 189: 521
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Matikainen, S., T. Sareneva, T. Ronni, A. Lehtonen, P. J. Koskinen, I. Julkunen. 1999. Interferon-α activates multiple STAT proteins and upregulates proliferation-associated IL-2Rα, c-myc, and pim-1 genes in human T cells. Blood 93: 1980
    OpenUrlAbstract/FREE Full Text
  43. ↵
    Gorelik, L., R. A. Flavell. 2000. Abrogation of TGFβ signaling in T cells leads to spontaneous T cell differentiation and autoimmune disease. Immunity 12: 171
    OpenUrlCrossRefPubMed
  44. ↵
    Ludviksson, B. R., D. Seegers, A. S. Resnick, W. Strober. 2000. The effect of TGF-β1 on immune responses of naive versus memory CD4+ Th1/Th2 cells. Eur. J. Immunol. 30: 2102
    OpenUrl
  45. ↵
    Heath, V. L., E. E. Murphy, C. Crain, M. G. Tomlinson, A. O’Garra. 2000. TGF-β1 down-regulates Th2 development and results in decreased IL-4-induced STAT6 activation and GATA-3 expression. Eur. J. Immunol. 30: 2639
    OpenUrlCrossRefPubMed
  46. ↵
    Schandene, L., G. F. Del Prete, E. Cogan, P. Stordeur, A. Crusiaux, B. Kennes, S. Romagnani, M. Goldman. 1996. Recombinant interferon-α selectively inhibits the production of interleukin-5 by human CD4+ T cells. J. Clin. Invest. 97: 309
    OpenUrlCrossRefPubMed
  47. ↵
    Erickson, S., O. Sangfelt, J. Castro, M. Heyman, S. Einhorn, D. Grander. 1999. Interferon-α inhibits proliferation in human T lymphocytes by abrogation of interleukin 2-induced changes in cell cycle-regulatory proteins. Cell Growth Differ. 10: 575
    OpenUrlAbstract/FREE Full Text
  48. ↵
    Zella, D., F. Romerio, S. Curreli, P. Secchiero, C. Cicala, D. Zagury, R. C. Gallo. 2000. IFN-α 2b reduces IL-2 production and IL-2 receptor function in primary CD4+ T cells. J. Immunol. 164: 2296
    OpenUrlAbstract/FREE Full Text
  49. ↵
    Rogge, L., D. D’Ambrosio, M. Biffi, G. Penna, L. J. Minetti, D. H. Presky, L. Adorini, F. Sinigaglia. 1998. The role of Stat4 in species-specific regulation of Th cell development by type I IFNs. J. Immunol. 161: 6567
    OpenUrlAbstract/FREE Full Text
  50. ↵
    Ho, A. S., Y. Liu, T. A. Khan, D. H. Hsu, J. F. Bazan, K. W. Moore. 1993. A receptor for interleukin 10 is related to interferon receptors. Proc. Natl. Acad. Sci. USA 90: 11267
    OpenUrlAbstract/FREE Full Text
  51. ↵
    Murphy, K. M., W. Ouyang, J. D. Farrar, J. Yang, S. Ranganath, H. Asnagli, M. Afkarian, T. L. Murphy. 2000. Signaling and transcription in T helper development. Annu. Rev. Immunol. 18: 495
    OpenUrlCrossRefPubMed
  52. ↵
    Finbloom, D. S., K. D. Winestock. 1995. IL-10 induces the tyrosine phosphorylation of tyk2 and Jak1 and the differential assembly of STAT1 α and STAT3 complexes in human T cells and monocytes. J. Immunol. 155: 1079
    OpenUrlAbstract/FREE Full Text
  53. ↵
    Ho, A. S., S. H. Wei, A. L. Mui, A. Miyajima, K. W. Moore. 1995. Functional regions of the mouse interleukin-10 receptor cytoplasmic domain. Mol. Cell. Biol. 15: 5043
    OpenUrlAbstract/FREE Full Text
  54. ↵
    Leonard, W. J., J. J. O’Shea. 1998. Jaks and STATs: biological implications. Annu. Rev. Immunol. 16: 293
    OpenUrlCrossRefPubMed
  55. ↵
    Rissoan, M. C., V. Soumelis, N. Kadowaki, G. Grouard, F. Briere, R. de Waal Malefyt, Y. J. Liu. 1999. Reciprocal control of T helper cell and dendritic cell differentiation. Science 283: 1183
    OpenUrlAbstract/FREE Full Text
  56. ↵
    Cella, M., D. Jarrossay, F. Facchetti, O. Alebardi, H. Nakajima, A. Lanzavecchia, M. Colonna. 1999. Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon. Nat. Med. 5: 919
    OpenUrlCrossRefPubMed
  57. ↵
    Kadowaki, N., S. Antonenko, J. Y. Lau, Y. J. Liu. 2000. Natural interferon α/β-producing cells link innate and adaptive immunity. J. Exp. Med. 192: 219
    OpenUrlAbstract/FREE Full Text
  58. ↵
    Durbin, J. E., A. Fernandez-Sesma, C. K. Lee, T. D. Rao, A. B. Frey, T. M. Moran, S. Vukmanovic, A. Garcia-Sastre, D. E. Levy. 2000. Type I IFN modulates innate and specific antiviral immunity. J. Immunol. 164: 4220
    OpenUrlAbstract/FREE Full Text
  59. ↵
    Rep, M. H., H. M. Schrijver, T. van Lopik, R. Q. Hintzen, M. T. Roos, H. J. Ader, C. H. Polman, R. A. van Lier. 1999. Interferon (IFN)-β treatment enhances CD95 and interleukin 10 expression but reduces interferon-γ producing T cells in MS patients. J. Neuroimmunol. 96: 92
    OpenUrlCrossRefPubMed
  60. ↵
    Ozenci, V., M. Kouwenhoven, Y. M. Huang, B. Xiao, P. Kivisakk, S. Fredrikson, H. Link. 1999. Multiple sclerosis: levels of interleukin-10-secreting blood mononuclear cells are low in untreated patients but augmented during interferon-β-1b treatment. Scand. J. Immunol. 49: 554
    OpenUrlCrossRefPubMed
  61. ↵
    Ozenci, V., M. Kouwenhoven, Y. M. Huang, P. Kivisakk, H. Link. 2000. Multiple sclerosis is associated with an imbalance between tumour necrosis factor-α (TNF-α)- and IL-10-secreting blood cells that is corrected by interferon-β (IFN-β) treatment. Clin. Exp. Immunol. 120: 147
    OpenUrlCrossRefPubMed
  62. ↵
    Roncarolo, M. G., M. K. Levings. 2000. The role of different subsets of T regulatory cells in controlling autoimmunity. Curr. Opin. Immunol. 12: 676
    OpenUrlCrossRefPubMed
  63. ↵
    Sakaguchi, S.. 2000. Regulatory T cells: key controllers of immunologic self-tolerance. Cell 101: 455
    OpenUrlCrossRefPubMed
  64. ↵
    Salomon, B., D. J. Lenschow, L. Rhee, N. Ashourian, B. Singh, A. Sharpe, J. A. Bluestone. 2000. B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 12: 431
    OpenUrlCrossRefPubMed
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section