An IL-4-independent and CD25-mediated function of c-maf in promoting the production of Th2 cytokines

Eun Sook Hwang; Ian Alexander White; I-Cheng Ho

doi:10.1073/pnas.202474499

. 2002 Sep 23;99(20):13026–13030. doi: 10.1073/pnas.202474499

An IL-4-independent and CD25-mediated function of c-maf in promoting the production of Th2 cytokines

Eun Sook Hwang ^*, Ian Alexander White ^†,‡, I-Cheng Ho ^*,†,‡,^§

PMCID: PMC130580 PMID: 12271139

Abstract

c-maf is a T helper (Th)2 cell-specific transcription factor, which promotes the differentiation of Th2 cells mainly by an IL-4-dependent mechanism. It remains unclear whether c-maf possesses any IL-4-independent function in regulating the production of Th2 cytokines. Here, we provide evidence demonstrating that c-maf, independent of IL-4, is essential for normal induction of CD25 in developing Th2 cells. The levels of CD25 are significantly higher in developing Th2 cells than in developing Th1 cells during in vitro differentiation. In addition, timely blockade of IL-2 receptor signaling selectively inhibits the production of Th2 cytokines, but not IFN-γ or IL-2. Taken together, our results uncover an IL-4-independent and CD25-mediated function of c-maf in promoting the production of Th2 cytokines.

c-maf was the first T helper (Th)2 cell-specific transcription factor to be cloned (1, 2). Naïve Th cells express negligible levels of c-maf, which are substantially induced in developing Th2 cells as early as 72 h after initial stimulation. In in vitro systems, c-maf can bind to a half consensus MARE site located in the IL-4 promoter and potently transactivate the IL-4 promoter (1). In agreement with these observations, c-maf-transgenic (mafTg) mice produced higher serum levels of IgE and IgG1, and their Th cells spontaneously developed into Th2 cells in vitro (2). The Th2-promoting effects of c-maf were nullified by the absence of IL-4. In contrast, c-maf-deficient (c-maf^−/−) Th cells were unable to differentiate into Th2 cells in the absence of exogenous IL-4. Although c-maf^−/− Th2 cells, differentiated in the presence of exogenous IL-4, produced normal levels of IL-5, IL-10, and IL-13, the production of IL-4 was severely impaired (3). Taken together, these results suggest that c-maf is a specific and potent transcription factor for the IL-4 gene and promotes the differentiation of Th2 cells mainly by an IL-4-dependent mechanism.

IL-2 is a critical cytokine that regulates the activation, proliferation, and homeostasis of T cells (4, 5). The high affinity receptor for IL-2 is composed of the IL-2 receptor (IL-2R) α (CD25), which functions solely in IL-2 binding, and IL-2Rβ (CD122) and IL-2Rγ, which contribute to IL-2 binding and mediate signal transduction (6). Although CD25, by itself, is unable to activate the IL-2R downstream signaling pathways, including Janus kinase/signal transducer and activator of transcription 5 (STAT5), phosphatidylinositol 3-kinase, and Ras/mitogen-activated protein kinase, the expression of CD25 in T cells is tightly linked to cell activation, whereas CD122 is constitutively expressed (7, 8). These observations suggest that CD25 might play a critical role in regulating the strength of IL-2R signals. Indeed, CD25-deficient T cells, in which IL-2 can still bind to CD122 and IL-2Rγ, were very resistant to activation-induced cell death, a defect also seen in CD122-deficient T cells (9, 10).

Although the functions and downstream signaling events of IL-2/IL-2R in regulating the proliferation and apoptosis of Th cells are well characterized, it is unclear whether IL-2/IL-2R signals have differential effects on the expression of Th1 and Th2 cytokine genes. In this article, we demonstrate that mafTg Th cells express higher levels of CD25 and are more sensitive to exogenous IL-2. In addition, c-maf^−/− Th cells, on stimulation with anti-CD3, display delayed induction of CD25 that could not be rectified by exogenous IL-4. We also show that developing Th2 cells, as compared with developing Th1 cells, express significantly higher levels of CD25 and perceived stronger STAT5 signals during in vitro differentiation. Furthermore, timely blockade of IL-2R signaling in vitro and in vivo dramatically reduces the production of Th2 cytokines, but not IFN-γ or IL-2. Taken together, our data demonstrate that c-maf, in addition to transactivating the IL-4 promoter, can enhance the production of Th2 cytokines by an IL-4-independent and CD25-mediated mechanism.

Methods

Mice.

The generation of mafTg, mafTg/IL-4-deficient (IL-4^−/−), and c-maf^−/− mice has been described (2, 3). C57BL/6 and DO11.10 α/β-T cell receptor (TCR) transgenic mice were purchased from The Jackson Laboratory, housed in sterilized microisolator cages, fed with autoclaved food and water, and handled in laminar airflow hoods.

Purification and in Vitro Differentiation of Naïve Th Cells.

Single cell suspensions were prepared from lymph nodes and spleens and depleted of RBC and B cells by using RBC lysis buffer (GIBCO/BRL) followed by negative selection with anti-B220 MACS magnetic beads (Miltenyi Biotec, Auburn, CA). Naïve Th cells were then isolated by using the Mouse Naïve T cell CD4⁺/CD62L⁺/CD44^low Column kit according to the manufacturer's instruction (R&D Systems). We routinely achieved approximately 90% purity of CD4⁺CD62L⁺ cells with this protocol. For in vitro differentiation assays, purified naïve Th cells, at 1 × 10⁶ cells/ml, were cultured in RPMI 1640 medium supplemented with 10% FCS and stimulated with 2 μg/ml plate-bound anti-CD3ɛ antibody and 1 μg/ml plate-bound anti-CD28 antibody. On the day of stimulation, IL-12 (1 ng/ml) was added for the Th1-skewing condition, and IL-4 (10 ng/ml) was added for the Th2-skewing condition. Twenty-four hours after stimulation, anti-IL-4 (5 μg/ml) or anti-IL-12 (20 μg/ml) were added to Th1 or Th2 culture, respectively. Unless specified, recombinant human IL-2 (100 units/ml) was added to all cultures, each of which was cultivated on the same plate during the period of observation. A total of 10 μg/ml of each anti-CD25 (PC61) and/or anti-CD122 (TM-β1) was used for anti-IL-2R blockade in vitro. All antibodies and cytokines were purchased from PharMingen.

Th Cell Proliferation Assay.

For Th cell proliferation, 5 × 10⁴ naïve Th cells in 50 μl medium per well of 96-well plates previously coated with anti-CD3ɛ antibody were used. Twenty-four hours after stimulation, human IL-2 was added to cultures as described in the figure legends. The cells were then pulsed with 1 μCi of [³H]thymidine (NEN Life Science) 48 h after stimulation and harvested 24 h later. The uptake of [³H]thymidine was measured by scintillation counters. All assays were conducted in triplicate.

Real-Time PCR.

Total RNA was prepared by using TRIzol reagent (GIBCO/BRL), and 1 μg of total RNA was used for reverse transcription and amplification by using a superscript II RT kit according to the manufacturer's manual (Invitrogen). A master mix of TaqMan reagents was prepared, and 10 ng of each reverse transcription product was used in a TaqMan PCR (Applied Biosystems). The standard curve method was used to measure the amounts of each species of transcripts relative to that of β-actin in each reaction. Reactions were carried out in 96-well plates by using the Applied Biosystems PRISM 7700 Sequence Detection System. The sequences of primers and probes used for each gene were: mIL-2 primers, 5′-CCTGAGCAGGATGGAGAATTACA-3′, 5′-TCCAGAACATGCCGCAGAG-3′, probe, 6FAM-CCCAAGCAGGCCACAGAATTGAAAG-TAMRA; and mCD25 primers, 5′-CGTTGCTTAGGAAACTCCTGGA-3′, 5′-GCTTTCTCGATTTGTCATGGG-3′, probe, 6FAM-CAGCAACTGCCAGTGCACCAGCA-TAMRA. All primers and probes were designed by using PRIMER EXPRESSION 1.0 (Applied Biosystems).

Generation of c-maf^−/− Fetal Liver Chimeras.

To produce fetal liver chimeras, 10–15 × 10⁶ liver cells (an entire fetal liver) from embryonic day 14.5 c-maf^+/+ or c-maf^−/− embryos were adoptively transferred into sublethally irradiated (600 rad) RAG-2-deficient mice. Chimeras thus generated were analyzed 12 weeks after repopulation.

Immunoblot Analysis.

Whole-cell extracts were prepared from developing Th1 and Th2 cells. Extracts (30 μg) were separated on 4–20% gradient PAGE gels, transferred to nitrocellulose membranes, and probed with indicated antibodies. Antibodies against phospho-STAT5, extracellular signal-regulated kinase (ERK), phospho-ERK, phospho-AKT, and AKT were purchased from Cell Signaling Technology (Beverly, MA), and STAT5 antibody was from Santa Cruz Biotechnology.

In Vivo Immunization and Administration of Antibodies.

Each female DO11.10 α/β TCR transgenic mouse (4–6 wk of age) received a s.c. injection of 100 μg of ovalbumin mixed with complete Freund's adjuvant. Each experimental group (eight animals per group) received either 400 μg/animal of anti-CD25 (PC61)/anti-CD122 (TM-β1) antibodies (200 μg of each per animal) or control IgG (PharMingen) via single i.p. injection 48 h after immunization. Three days after immunization, single cell suspensions were collected from draining lymph nodes and restimulated with 0.3 μM ovalbumin peptide_323–339 [OVA (323–339)] for 24 h, and the supernatant was assayed for cytokine production by ELISA.

Results

mafTg Th Cells Are More Sensitive to IL-2.

To characterize IL-4-independent functions of c-maf in regulating the differentiation and activation of Th cells, we generated and studied mafTgIL-4^−/− mice. We found that naïve mafTgIL-4^−/− Th cells, on stimulation with anti-CD3, proliferated significantly better than naïve IL-4^−/− Th cells did, but only in the presence of exogenous human IL-2 (hIL-2) (Fig. 1A). This observation suggests that the enhanced proliferation is caused by hypersensitivity of mafTgIL-4^−/− Th cells to IL-2, but not to anti-CD3 stimulation. Indeed, the effects of hIL-2 on the anti-CD3-induced proliferation of mafTgIL-4^−/− Th cells were nearly maximized at 50 units/ml, whereas 200 units/ml of hIL-2 was required to reach maximal effects on IL-4^−/− Th cells (Fig. 1B). In contrast, exogenous mouse IL-4 comparably enhanced the anti-CD3-induced proliferation of both mafTgIL-4^−/− and IL-4^−/− Th cells (data not shown). These results suggest that overexpression of c-maf heightens the sensitivity of Th cells specifically to IL-2 in an IL-4-independent fashion.

Hypersensitivity to IL-2 and enhanced expression of CD25 of mafTg Th cells. Naïve Th cells derived from IL-4^−/− (non-Tg) or mafTgIL-4^−/− (Tg) mice were stimulated *in vitro* with indicated concentrations of anti-CD3 in the absence or presence of hIL-2 (100 units/ml) (A) or with anti-CD3 (0.1 μg/ml) in the presence of indicated concentrations of hIL-2 (B). [³H]thymidine was added 48 h after stimulation, and the uptake of [³H]thymidine was determined 24 h later. [³H]thymidine uptake of each sample in B was normalized against that of Th cells stimulated in the absence of exogenous hIL-2, which was arbitrarily set as 100%. In parallel experiments, naïve Th cells were purified from IL-4^−/− (non-Tg) or mafTgIL-4^−/− (Tg) mice and stimulated *in vitro* with 1 μg/ml of anti-CD3. The expression of IL-2 and/or IFN-γ was determined by ELISA (C) and real-time PCR (D) 48 h after stimulation, and the expression of CD25, CD122, and CD44 was examined by fluorescence-activated cell sorting (E) and real-time PCR (F).

mafTg Th Cells Express Higher Levels of CD25.

To understand the mechanisms that cause the IL-2-induced hyperproliferation, we first examined the endogenous level of mouse IL-2 produced by mafTgIL-4^−/− Th cells during in vitro differentiation. Surprisingly, very little IL-2 was detected by ELISA in supernatant of mafTgIL-4^−/− Th cells, when compared with IL-4^−/− Th cells, cultured for 2 days after anti-CD3 stimulation (Fig. 1C). This finding is in sharp contrast to IFN-γ, which was comparably detected in the supernatants of all cultured Th cells. Interestingly, comparable levels of intracellular IL-2 and IL-2 transcripts were detected between mafTgIL-4^−/− and IL-4^−/− Th cells at the same time points (Fig. 1D and data not shown). These results suggest that the near absence of IL-2 in the supernatant of mafTgIL-4^−/− Th cells most likely is caused by increased consumption. In agreement with the notion, we found that mafTgIL-4^−/− Th cells, when compared with IL-4^−/− Th cells, expressed substantially higher levels of surface CD25, the α subunit of IL-2R, on stimulation with anti-CD3 (Fig. 1E). In contrast, CD122, the β subunit of IL-2R, and CD44, an activation marker, were expressed comparably in mafTgIL-4^−/− and IL-4^−/− Th cells. The enhanced level of CD25 in mafTgIL-4^−/− Th cells was at least partly regulated at the level of transcription because the level of CD25 transcripts, as determined by real-time PCR, was also substantially higher in mafTgIL-4^−/− Th cells (Fig. 1F).

Deficiency of c-maf Results in Aberrant CD25 Expression in Th Cells.

To determine whether c-maf is essential for normal up-regulation of CD25 in Th cells on stimulation, we chose to examine the kinetics of CD25 expression during in vitro differentiation of c-maf^−/− Th cells. Because c-maf deficiency resulted in nearly 100% perinatal fatality in our colony, we generated c-maf^−/− Th cells by reconstituting RAG-2-deficient mice with c-maf^−/− fetal liver cells. Both c-maf^+/+ and c-maf^−/− fetal liver cells efficiently reconstituted lymphoid compartments of RAG-2-deficient mice. The total numbers of peripheral T cells and the ratio between CD4 and CD8 T cells were comparable among reconstituted mice, and naïve c-maf^−/− Th cells produced very low levels of IL-4 on stimulation with anti-CD3 (Fig. 6, which is published as supporting information on the PNAS web site, www.pnas.org). To avoid any confounding effect on the expression of CD25 caused by the differences in IL-4 levels between wild-type and c-maf^−/− Th cell culture, naïve Th cells thus obtained were stimulated in vitro under Th2-skewing conditions (with a saturating dose of exogenous IL-4), and the expression of CD25 was examined. Although the levels of CD25 were comparable between developing c-maf^−/− and c-maf^+/+ Th2 cells during the first 2 days after stimulation, the up-regulation of CD25 from the third day on was significantly delayed in developing c-maf^−/− Th cells as compared with that of developing c-maf^+/+ Th cells (Fig. 2A). The delayed up-regulation of CD25 was not caused by low levels of IL-4, because equivalent concentrations of IL-4 in cultures of both c-maf^−/− and c-maf^+/+ Th cells on the third day after initial stimulation were detected under Th2 polarizing conditions (Fig. 2B). In addition, the delayed up-regulation of CD25 was not caused by insufficient or weaker activating signals perceived by developing c-maf^−/− Th cells, because the expression of CD44 and CD122 was undisturbed by the deficiency of c-maf (Fig. 2A). Taken together, these results suggest that c-maf, independent of IL-4, is essential for normal expression kinetics of CD25, but not CD122, during in vitro differentiation of Th cells.

Aberrant CD25 expression of c-maf^−/− Th2 cells. Naïve Th cells purified from RAG-2-deficient mice reconstituted with c-maf^+/+ (WT) or c-maf^−/− (KO) fetal liver cells were stimulated *in vitro* under Th2-skewing conditions. The expression of CD25, CD122, and CD44 was examined by fluorescence-activated cell sorting at indicated time points (A), and the concentrations of IL-4 and IFN-γ in supernatant were measured by ELISA on day 3 (B).

The Expression Kinetics of CD25 Is Different Between Th1 and Th2 Cells in Vitro.

The fact that c-maf, a Th2 cell-specific transcription factor, is essential for normal induction of CD25 in developing Th2 cells, prompted us to examine the expression kinetics of CD25 in both Th1 and Th2 cells. Naïve Th cells were isolated from wild-type C57BL/6 mice and stimulated in vitro under Th1- or Th2-skewing conditions. At different time points, surface CD25 levels were analyzed by flow cytometry. We found that surface levels of CD25 started to increase in both developing Th1 and Th2 cells approximately 24 h after initial stimulation and continued to do so over the next 2–3 days. The levels of CD25, however, were substantially higher in developing Th2 cells than in developing Th1 cells on the fourth day after stimulation (Fig. 3A). Similar results were obtained when naïve Th cells derived from DO11.10 TCR transgenic mice were stimulated in vitro with an ovalbumin peptide, OVA (323–339), and antigen-presenting cells (Fig. 3B), suggesting that the differences in the expression kinetics of CD25 between Th1 and Th2 cells are independent of genetic background or mode of stimulation. In contrast, the expression kinetics of CD122 and CD44 was similar between Th1 and Th2 cells. Of note, the onset of the discordant CD25 expression between developing Th1 and Th2 cells, approximately 72 h after the initial stimulation, correlates very well with the induction of c-maf during in vitro differentiation of Th2 cells (2).

Expression kinetics of IL-2R in developing Th1 and Th2 cells. Naïve wild-type C57BL/6 (A) or DO11.10 TCR transgenic (B) Th cells were stimulated with anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) (A) or 0.3 μM OVA (323–339)/antigen-presenting cells (B) under Th1- or Th2-skewing conditions. The expression of CD25, CD122, and CD44 was examined at indicated time points by fluorescence-activated cell sorting.

Developing Th2 Cells Perceive Stronger STAT5 Signals.

To determine whether the higher levels of CD25 result in stronger IL-2R signals in developing Th2 cells, we examined the levels of IL-2R downstream signal molecules, such as STAT5, ERK, and AKT, during in vitro differentiation. Naïve Th cells were stimulated in vitro under Th1- or Th2-skewing conditions. On the fourth day after initial stimulation, the levels of IL-2R downstream signal molecules were examined by immunoblot analysis. As shown in Fig. 4A, considerably more total phospho-STAT5 was readily detected in developing Th2 cells than in developing Th1 cells, indicating enhanced IL-2R signaling in developing Th2 cells. The difference in the level of phospho-STAT5 is not caused by higher levels of IL-2 in the supernatant of developing Th2 cells, because comparable, albeit low, concentrations of IL-2 were detected by ELISA (data not shown). In contrast to phospho-STAT5, we found no significant difference in the levels of phospho-ERK and phospho-AKT between developing Th1 and Th2 cells (Fig. 4A Lower). In parallel experiments, we also examined the levels of phospho-STAT5 in developing mafTg and c-maf^−/− Th cells, which respectively express higher and lower levels of CD25 as compared with those of wild-type cells. We found that developing mafTg Th cells contained considerably more phospho-STAT5 on the fourth day after initial stimulation (Fig. 4B). Conversely, very little, if none at all, phospho-STAT5 was detected in developing c-maf^−/− Th cells (Fig. 4C). Taken together, these results indicate that the differences in the levels of CD25 seen among developing mafTg, c-maf^−/−, and wild-type Th, and between developing Th1 and Th2 cells can, indeed, yield substantial differences in the strength of IL-2R signals.

Analysis of IL-2R downstream signals. Naïve C57BL/6 Th cells were subjected to *in vitro* differentiation under Th1- or Th2-skewing conditions (A), and naïve mafTg (Tg), c-maf^−/− (KO), and respective control wild-type Th cells (non-Tg or WT) under nonskewing conditions (B and C). On the fourth day after initial stimulation, cells were harvested, and whole-cell extracts were subjected to immunoblot analysis by using the indicated antibodies.

Sustained IL-2R Signaling During in Vitro Differentiation Is Essential for Optimal Production of Th2, But Not Th1, Cytokines by Developing Th Cells.

The fact that developing Th2 cells express higher levels of CD25 implies that signaling through the IL-2R might be especially important in regulating the production of Th2 cytokines. To address this question, we examined the effects of IL-2R blockade on the production of cytokines by Th cells. Naïve Th cells were stimulated under either Th1- or Th2-skewing conditions, and anti-CD25 (PC61) and/or anti-CD122 (TM-β1) antibodies were added 2 days after the initial stimulation. The production of cytokines was then measured by ELISA 24 h later. Anti-CD25 (PC61) and anti-CD122 (TM-β1) antibodies were chosen because both antibodies have been independently shown to block the binding of IL-2 to IL-2R in vivo and in vitro (7, 8, 11). We found that IL-2R blockade exclusively reduced the production of Th2 cytokines, such as IL-4 and IL-10, by 75–90% (Fig. 5A), whereas the production of IL-2 and IFN-γ by developing Th1 cells was not affected (Fig. 5B). In addition, the inhibition of Th2 cytokine production by IL-2R blockade was not caused by a “global” defect in the expression of cytokine genes, because the anti-IL-2R antibodies-treated Th2 cells were capable of producing normal levels of IL-2 (Fig. 5A). Of note, the inhibition of Th2 cytokine production was achieved only with a combination of anti-CD25 and anti-CD122 antibodies.

Inhibition of Th2 cytokine production by IL-2R blockade. Naïve wild-type C57BL/6 Th cells were subjected to *in vitro* differentiation under Th2- skewing (A) or Th1-skewing (B) conditions. On the second day after initial stimulation, cells were incubated with anti-CD25 and/or anti-CD122 antibodies for 24 h, and the production of cytokines was determined by ELISA. (C) DO11.10 TCR transgenic mice were immunized with ovalbumin (100 μg/mouse, mixed with complete Freund's adjuvant) s.c., and injected i.p. with anti-IL-2R antibodies (+) or control IgG (−) 2 days after immunization. Single cell suspensions were prepared from draining lymph nodes 1 day later and restimulated *in vitro* with OVA (323–339) for 24 h, and the production of cytokines was measured by ELISA.

In Vivo IL-2R Blockade also Selectively Inhibits the Production of Th2 Cytokines.

To determine whether in vivo IL-2R blockade can also inhibit the production of Th2 cytokines, DO11.10 TCR transgenic mice were immunized with ovalbumin s.c. Two days after immunization, anti-CD25/anti-CD122 (anti-IL-2R) antibodies or control antibodies were administered via i.p. injections. One day after administration of anti-IL-2R antibodies, single cell suspensions were prepared from draining lymph nodes of immunized mice. The cells, thus prepared, were stimulated in vitro with OVA (323–339) for 24 h, and the production of cytokines was measured by ELISA. As shown in Fig. 5C, in vivo administration of IL-2R antibodies, when compared with control antibodies, also significantly suppressed the production of Th2 cytokines. In contrast, the production of IFN-γ and IL-2 was not affected by in vivo administration of IL-2R antibodies.

Discussion

Until this report, the only proven function of c-maf in T cells was serving as a specific transcription factor for the IL-4 gene (1, 2). The data described above strongly indicate that c-maf can also promote the production of Th2 cytokines and anti-CD3-induced proliferative responses by an IL-4-independent and CD25-mediated mechanism.

It was demonstrated that the expression of CD25 was regulated by nuclear factor of activated T cells (NF-AT) and STAT5 (12, 13). T cells derived from NF-ATc2-deficient or STAT5a-deficient mice expressed subnormal levels of CD25 on stimulation. Several NF-AT and STAT5 binding sites have been identified in the promoter and introns of the CD25 gene, and overexpression of NF-AT or addition of exogenous IL-2 substantially enhanced the activity of exogenous CD25 promoters in vitro (12, 14–16). However, neither STAT5a nor NF-AT is preferentially expressed in Th2 cells and can explain the higher levels of CD25 in developing Th2 cells. Our data highly suggest that the presence of c-maf in Th2 cells is an attractive explanation for the differences in the expression kinetics of CD25 between Th1 and Th2 cells. It is still unclear whether c-maf directly regulates the expression of CD25. We found that overexpression of c-maf only modestly transactivated reporter constructs containing the murine IL-2 promoter and/or the first intron and that there was no synergistic effect between c-maf and NF-AT (Fig. 7, which is published as supporting information on the PNAS web site, and data not shown). C-maf, therefore, very likely enhances the expression of CD25 by an indirect mechanism, such as by augmenting the STAT5 signals. Alternatively, c-maf could still directly transactivate the CD25 promoter only in developing Th2 cells but not in mature Th clones or tumor cells, which were used in our in vitro transfection assays.

Our observation that sustained IL-2R signaling is essential for optimal production of Th2 cytokines is in agreement with a recent report showing that the deficiency of STAT5a led to significant defects in Th2 cytokine production and Th2 cell differentiation (17). More recently, it was also reported that IL-2 was essential for the production of Th2 cytokines in an in vitro Th cell differentiation protocol (18). Splenocytes preactivated with soluble anti-CD3 for 48 h produced more Th2 cytokine on restimulated with plate-bound anti-CD3 in the presence of exogenous IL-2. Interestingly, the production of Th2 cytokines by the preactivated splenocytes was nearly completely inhibited by anti-IL-2. In contrast, no such IL-2 dependence was noticed for the production of IFN-γ and tumor necrosis factor α.

How does IL-2R blockade inhibit the production of Th2 cytokines? The answer to this question remains unclear. We found that IL-2R blockade did not affect the levels of c-maf or GATA-3 transcripts, two subset-specific transcription factors that are critical for the expression of the Th2 cytokine genes (data not shown). It was reported that developing Th cells had to undergo at least three or four cell divisions before the production of Th2 cytokines but only one or no cell division is required for the production of IFN-γ and IL-2, respectively, by developing Th cells (19). Thus, IL-2R blockade might prevent developing Th cells from traveling through three cell divisions and subsequently inhibit the expression of Th2, but not Th1, cytokines. In agreement with this hypothesis, we found that IL-2R blockade inhibited the proliferation of both developing Th1 and Th2 cells (Fig. 8, which is published as supporting information on the PNAS web site). Although anit-CD25 or anti-CD122 antibody alone has been shown to attenuate the effects of IL-2, synergy between anti-CD25 and anti-CD122 antibodies has been reported in both in vivo and in vitro experiments (20). The synergistic effect might be partly explained by the fact that IL-2 can still bind to and signal through the “intermediate affinity” IL-2R, which contains CD122 and IL-2Rγ. Therefore, it is not surprising that both anti-CD25 and anti-CD122 antibodies were required to efficiently inhibit the production of Th2 cytokines and the proliferation of developing Th.

Anti-IL-2R antibodies, because of their antiproliferation effect on T cells, have been used in several clinical trials for prevention of graft rejection (21). Our data demonstrate that IL-2R blockade, when applied in a timely fashion during Th immune responses, can strongly inhibit the production of Th2 cytokines. This observation points out a potential therapeutic application for anti-IL-2R antibodies in the treatment of Th2 cell-mediated diseases, such as allergic asthma.

Supplementary Material

Supporting Figures

pnas_202474499_index.html^{(1.1KB, html)}

Acknowledgments

We thank Drs. Laurie H. Glimcher, Sung-Yun Pai, and Andrea Wurster for critical review of this manuscript and Dr. Neal Iwakoshi for helping with the generation of c-maf^−/− fetal liver chimeric mice. We also thank Dr. Warren Leonard for providing the CD25 promoter/reporter constructs. This work was supported by a junior award from the Sandler Program for Asthma Research (to I-C.H.), a grant from the Juvenile Diabetic Research Foundation (to I-C.H.), and postdoctoral fellowships from the Arthritis Foundation (to E.S.H.) and the Korea Science and Engineering Foundation (to E.S.H.).

Abbreviations

mafTg: c-maf-transgenic
c-maf^−/−: c-maf-deficient
IL-2R: IL-2 receptor
hIL-2: human IL-2
TCR: T cell receptor
IL-4^−/−: IL-4-deficient
NF-AT: nuclear factor of activated T cells
OVA (323–339): ovalbumin peptide_323–339
ERK: extracellular signal-regulated kinase
STAT5: signal transducer and activator of transcription 5

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Figures

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PERMALINK

An IL-4-independent and CD25-mediated function of c-maf in promoting the production of Th2 cytokines

Eun Sook Hwang

Ian Alexander White

I-Cheng Ho

Abstract