Development of Th1 and not Th2 immune responses in mice lacking IFN-regulatory factor-4
Norio Tominaga1,2,
Kozo Ohkusu-Tsukada1,
Heiichiro Udono1,
Ryo Abe4,
Toshifumi Matsuyama3 and
Katsuyuki Yui1
1 Division of Immunology, Department of Translational Medical Sciences, 2 Division of Medical Virology, and 3 Division of Cytokine Signaling, Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki University, Nagasaki 852-8523, Japan 4 Research Institute for Biological Sciences, Science University of Tokyo, 2669 Yamazaki, Noda, Chiba 278-0022, Japan
Correspondence to: K. Yui; E-mail: katsu{at}net.nagasaki-u.ac.jp
Transmitting editor: T. Saito
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Abstract
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IFN-regulatory factor (IRF)-4 is a member of the IRF family of transcription factors expressed in lymphocytes and macrophages. The previous studies using mice deficient in the IRF-4 gene showed profound defects in function of both B and T cells. To further investigate the role of IRF-4 in CD4+ T cell function, IRF-4/ mice were challenged with the intracellular pathogen Leishmania major. The mice were protected against L. major during the early phase of the infection and CD4+ T cells of the infected mice produced IFN-
in response to L. major antigen. However, during the late phase of infection, lymphocyte numbers were dramatically reduced in the draining lymph nodes, resulting in the deterioration of the lesion, indicating that IRF-4 was required for sustained immune responses against L. major infection. The function of CD4+ T cells was further investigated using TCR transgenic mice lacking the IRF-4 gene. CD4+ T cells from IRF-4/ mice produced IFN-
and expressed T-bet after culture under Th1-skewing conditions in vitro. However, Th2 cell development was not observed after culture under Th2-polarizing conditions. Proliferation of CD4+ T cells to IL-4 was reduced in IRF-4/ mice, suggesting the defects in the responsiveness to IL-4. Furthermore, stimulation of the IRF-4/ CD4+ T cells with IL-4-induced activation of signal transducer and activator of transcription 6, but not expression of growth factor independent-1. Thus, development of CD4+ T cell subsets differentially depends on IRF-4; induction of Th1 response does not depend on IRF-4, while Th2 response depends entirely on IRF-4.
Keywords: IFN-
, IL-4, knockout mouse, Leishmania major
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Introduction
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IFN-regulatory factors (IRF) constitute a family of transcription factors that commonly possess a novel helix-turn-helix DNA-binding motif and bind to the IFN-stimulated response element (ISRE) (1). Members of the IRF family are involved in diverse immune processes that include pathogen response, cytokine signaling, apoptosis, control of cell proliferation, and regulation of the development and function of the immune response (26). In particular, IRF-1-deficient mice fail to mount Th1 responses and instead undergo accelerated Th2 differentiation, suggesting that IRF-1 is required for the development of a Th1-type immune response (5,6).
IRF-4 is a member of the IRF family of transcription factors. It was originally discovered independently as a binding factor to the Ig light chain enhancer in association with PU.1 (7) and as a new IRF family member (8). It is expressed in B cells, mature T cells and macrophages (8,9), and its expression is up-regulated by IgM or TCR cross-linking as well as co-stimulation of B cells with CD40 and IL-4 (8,10,11). Mice deficient in the IRF-4 gene exhibited an age-dependent increase of T and B lymphocytes in spleen and lymph nodes (12). These mice showed profound reduction in serum Ig concentrations, and impaired B and T lymphocyte function. They could not generate cytotoxic or anti-tumor responses, indicating a profound defect in CD8+ T cell function. However, the function of CD4+ T cells was not extensively studied in these mice. IRF-4 has been shown to have dual roles in the regulation of cytokine-regulated gene function. It is repressive over the gene activation induced by IFN stimulation (13), while it interacts with the signal transducer and activator of transcription (Stat)6 and drives the expression of IL-4 inducible genes (11). These features reminded us of the possible link between IRF-4 function and the development of CD4+ T cells to Th1 and Th2 subsets.
In this study we determined whether IRF-4/ mice were able to mount protective immune responses against infection with the intracellular pathogen Leishmania major, one of the best-studied models of CD4+ T cell function in vivo (14). We found that IRF-4/ mice were protected against L. major infection during the early phase of infection and T cells of the infected mice produced IFN-
in response to L. major antigen. However, the protective immunity deteriorated during the late phase of infection, which was due to the striking reduction of lymphocytes in the draining lymph nodes. We also examined whether IRF-4/ CD4+ T cells can differentiate into Th1 or Th2 cells in response to peptide antigen in vitro. The results indicated that Th1 but not Th2 cells can develop in the absence of IRF-4. IRF-4/ CD4+ T cells showed reduced proliferative response to IL-4 due in part to the defective expression of growth factor independent (Gfi)-1.
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Methods
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Animals
IRF-4/ mice were described previously (12) and were maintained by intercrossing. OT-II transgenic mice expressing the TCR specific for OVA323339 and I-Ab (15) were provided by Dr W. R. Heath (The Walter and Eliza Hall Institute of Medical Research, Victoria, Australia). These mice were maintained in the Laboratory Animal Center for Animal Research at Nagasaki University, School of Medicine. Intercross (F2) matings were performed between OT-II and IRF-4/ mice (both C57BL/6 background) to generate TCR transgenic IRF-4/ (OT-II/IRF-4/) mice. Mice were analyzed for transgenic TCR expression by cell surface staining of the TCR using phycoerythrin (PE)anti-CD4 mAb and biotinanti-TCR V
2 mAb (PharMingen, San Diego, CA). IRF-4 genotype was determined by PCR using two pairs of oligonucleotide primers; Neo, 5'-ATTCGGCTATGACTGGGCACAACA-3' and 5'-GAGCAAGGTGAGATGACAGGAGAT-3'; IRF-4, 5'-GCA ATGGGAAACTCCGACAGT-3' and 5'-CAGCGTCCTCCTCA CGATTGT-3'. These PCR products were distinguished by electrophoresis on a 3% agarose gel. Mice that carry the Neo gene and not the IRF-4 gene were determined as IRF-4/, and maintained by interbreeding thereafter. Transgenic mice were used at the age of
10 weeks. Female C57BL/6 and BALB/c mice were purchased from SLC (Hamamatsu, Japan). The animal experiments reported herein were conducted according to the Guidelines of the Laboratory Animal Center for Biomedical Research, Nagasaki University School of Medicine.
Infection of mice with L. major and monitoring of the disease
L. major (MHOM/S U/73-5-ASKH strain) was provided by Dr K. Himeno (Kyushu University, Fukuoka, Japan). Mice were injected in the left hind footpad with 1 x 106 promastigotes of the L. major strain. The thickness of the infected and the contralateral uninfected footpad was measured once per week using a vernier caliper as described (6). The increase in footpad thickness was calculated as: [(thickness of infected footpad) (thickness of uninfected footpad)/(thickness of uninfected footpad)] x 100. Parasite burden was determined using popliteal lymph nodes. Serial dilutions of single-cell suspensions obtained from infected mice were placed in each well of 96-well culture plates in culture medium as previously described (6). After 10 days, growth of parasites was determined and the cell dilution of the wells in which 37% of the wells were negative for parasite growth was taken to represent the original plating of one single parasite according to Poisson statistics. To determine L. major-specific IFN-
production, the draining popliteal lymph node cells (1 x 105/well) were cultured in vitro with mitomycin C-treated syngeneic spleen cells (4 x 105/well) with or without L. major antigen (freezethawed lysates of promastigotes) for 48 h. Culture supernatants were harvested and tested for IFN-
by ELISA. The number of lymphocytes in each lymph node was determined by counting viable cells under a microscope using Trypan blue. The ratio of lymphocyte subsets was determined by flow cytometry analysis of lymph node cells stained with PEanti-CD4 and FITCanti-CD8 antibody or with FITCanti-B220 antibody. The number of each lymphocyte subset was determined by multiplying the total cell number of each lymph node with the ratio of each subset.
Cell culture
To isolate CD4+ T cells, lymph node cells were treated with anti-CD8 mAb (3.155), anti-MHC class II mAb (M5/114.15.2) and complement followed by nylon wool column enrichment. This population consisted of >95% CD4+ T cells. CD4+ T cells (1 x 105) were cultured in RPMI 1640 supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 5 x 105 M 2-mercaptoethanol, penicillin/streptomycin and 10% heat-inactivated FCS in each well of a microtiter plate. Cells were cultured in each well of a flat-bottom 96-well plate in the presence of mitomycin C-treated C57BL/6 spleen cells and varying dose of OVA323339 peptide or with IL-4 (250 U/ml), phorbol myristate acetate (PMA; 20 ng/ml) and ionomycin (1 µM). After culture for 43 h, proliferation was assessed by a terminal pulse of 18.5 kBq [3H]thymidine (NEN Life Science Products, Boston, MA) for 5 h. Alternatively, supernatant was collected from each well after culture for 48 h to determine the level of cytokines. The OVA323339 peptide was purchased from Sawady Technology (Tokyo, Japan). In some experiments, CD4+ T cells were cultured in each well of a flat-bottom 96-well plate coated with anti-TCRß mAb (H57-597, 10 µg/ml) in the presence of anti-CD28 mAb (PV-1, 2 µg/ml) (16).
Cytokine assays
The levels of cytokines in the supernatants of T cell cultures were determined by a sandwich ELISA. IFN-
was measured using R4-6A2 as the capture antibody and biotinylated XMG1.2 as the detecting antibody according to the manufacturers directions (all mAb for ELISA were from BD PharMingen, San Diego, CA). IL-4 was measured using 11B11 as capture antibody and BVD6-24G2 as the detecting antibody. Recombinant mouse IFN-
(PeproTech, London, UK) and IL-4 (Genzyme, Cambridge, MA) were used as standards. The ELISPOT assay was performed using nitrocellulose-lined 96-well microtiter plates (MAHAS45; Millipore, Bedford, MA). Plates were coated with 10 µg/ml of anti-mouse IFN-
antibody (clone R4-6A2; PharMingen). After washing with PBS containing 0.25% Tween 20, plates were overlaid with PBS containing 5% BSA at 37°C for 30 min. CD4+ T cells were prepared from popliteal lymph nodes of mice, and placed in each well of a treated microtiter plate (1 x 105/well) in the presence of various doses of L. major antigen and mitomycin C-treated T cell-depleted syngeneic spleen cells (4 x 105/well). After culture for 24 h, plates were washed, incubated with biotinylated anti-IFN-
mAb, washed, and incubated with streptavidin-conjugated alkaline phosphatase and then with BCIP/NBT. Spots were counted using a stereomicroscope with a magnification of x20.
To induce Th1 and Th2 development in vitro, purified CD4+ T cells of OT-II and OT-II/IRF-4/ mice (105/ml) were cultured in complete medium containing mitomycin C-treated C57BL/6 spleen cells (4 x 105/ml) and OVA323339 peptide (10 µM). Th1 conditions contained IL-2 (20 U/ml), IL-12 (20 ng/ml) and anti-IL-4 mAb (11B11; 10 µg/ml). Th2 conditions contained IL-2 (20 U/ml), IL-4 (100 U/ml), anti-IFN-
mAb (R4-6A2; 15 µg/ml) and anti-IL-12 mAb (C17.8; 10 µg/ml). After culture for 7 days, cells (1 x 105/well) were re-stimulated in the presence of mitomycin C-treated C57BL/6 (4 x 105/well) spleen cells and OVA323339 peptide for 2 days. The levels of cytokines in the culture supernatant were determined by ELISA. For intracellular cytokine staining, polarized CD4+ T cells were stimulated for 5 h in GolgiStop (BD PharMingen, Beverly, MA) containing PMA (50 ng/ml) and ionomycin (1 µM). Intracellular staining was performed according to the protocol of the manufacturer using CytoStain kits (PharMingen). After fixation and permeabilization, cells were stained with FITCanti-IFN-
mAb (R4-6A2) and PEanti-IL-4 mAb (BVD4-1D11), and analyzed using a FACScan (Becton Dickinson, San Jose, CA). The expression of IL-4 receptor and IL-2 receptor was determined by staining cells with anti-IL-4 receptor mAb (M1; BD PharMingen) and FITCanti-rat IgG (MBL, Nagoya, Japan) or with FITCanti-CD25 mAb (BD PharMingen).
RT-PCR
Total RNA was extracted from CD4+ T cells using Isogen (Nippon Gene, Tokyo, Japan). cDNA was generated from 1 µg RNA using random hexamers and MMLV reverse transcriptase (Sawady Technology). Each aliquot of the sample was amplified in a volume of 50 µl for 27 cycles to detect IFN-
and IL-4 (94°C for 30 s, 55°C for 50 s, 72°C for 80 s), and for 35 cycles to detect G3PDH (94°C for 60 s, 60°C for 60 s, 72°C for 60 s) using LA Taq DNA polymerase (Takara, Tokyo, Japan). The primers used for IFN-
were 5'-TGAACGCTACACA CTGCATCTTGG-3' and 5'-CGACTCCTTTTCCGCTTCCTG AG-3', for IL-4 were 5'-TAGTTGTCATCCTGCTCTT-3' and 5'-CTACGAGTAATCCATTTGC-3', and for G3PDH were 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3' and 5'-CATGT AGGCCATGAGGTCCACCAC-3'. PCR analysis of T-bet, GATA-3 and Gfi-1 expression was performed as described (17,18), except that cDNA was amplified 27 cycles for T-bet and GATA-3, and 30 cycles for Gfi-1. The PCR products were size fractionated using a 3% agarose gel and visualized by ethidium bromide staining.
Western blot analysis
CD4+ T cells were resuspended in 50 µl lysis buffer (1% Triton X-100, 10 mM TrisHCl, pH 7.4, 150 mM NaCl, 1 mM EDTA and 1 mM PMSF). After centrifugation, the lysate was mixed with 2 x SDSPAGE sample buffer and size fractionated on 8% SDSPAGE. After transfer to a PVDF membrane, the blot was incubated in blocking buffer (TBS containing 5% skim milk powder and 0.1% Tween 20) and probed with anti-phospho Stat6 or anti-Stat6 antibody (Cell Signaling Tech, Beverly, MA). After washing, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit Ig antibody (1:1000), washed and analyzed using ECL reagents (Amersham Pharmacia Biotech, Piscataway, NJ).
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Results
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Protection of IRF-4/ mice against infection with L. major
One of the best-studied models of CD4+ T cell function in vivo is murine cutaneous Leishmaniasis, induced by inoculation of L. major (14). In the resistant mice, parasite-specific Th1 cells activate parasite-killing macrophages to contain the infection, while in susceptible mice T cells shift to Th2 development resulting in a high parasite burden. To study whether a protective Th1 response can develop in the absence of IRF-4 against L. major in vivo, BALB/c (susceptible strain), C57BL/6 (resistant strain) and IRF-4/ mice were infected s.c. with L. major, and the course of disease was monitored (Fig. 1A). BALB/c mice developed a strong continuous increase in footpad thickness, while C57BL/6 mice showed only a mild and transient increase. The footpad thickness of IRF-4/ mice was lower than that of C57BL/6 mice up to 5 weeks after infection, but did not diminish thereafter. Instead, it increased gradually. The parasite burden was determined 7 weeks after infection (Fig. 1B). The number of L. major in the draining lymph nodes of IRF-4/ mice was much lower than that of BALB/c, but higher than that of C57BL/6 mice, which was consistent to the increased footpad swelling in IRF-4/ mice during the late stage of infection.

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Fig. 1. Increase in lesion size and parasite burden in L. major-infected mice. (A) BALB/c (squares), C57BL/6 (open circles) and IRF-4/ (solid circles) mice were infected with L. major promastigotes, the thicknesses of infected (left) and uninfected (right) footpads measured, and the increase in footpad thickness (%) calculated. The data represent means ± SD of five mice in each group. The data represent three separate experiments with similar results. (B) Parasite burden of the draining popliteal lymph nodes was assessed 7 weeks after L. major infection. Serial dilutions of single-cell suspensions were cultured in 96-well plates for 10 days and the growth of parasites was determined. The dilution containing 37% negative wells was taken as the original plating of a single parasite per well. Results are given as the log of the cell number plated per well in this dilution.
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The number of lymphocytes in the draining lymph nodes of the infected mice was determined during the course of infection (Fig. 2). In both BALB/c and C57BL/6 mice, the number of CD4+ T cells, CD8+ T cells and B cells dramatically increased during infection, while those in the non-draining lymph nodes were constant. In IRF-4/ mice, however, the number of lymphocytes in the draining lymph nodes increased during the first 3 weeks of infection and dramatically declined during the later period of infection. This reduction was specific for the draining lymph nodes, since the number of each lymphocyte subset in the non-draining lymph nodes was much higher than normal mice and generally increased during the period of infection corresponding to aging as reported previously (12). To determine whether specific Th1 cells develop in L. major-infected mice, T cells from the draining lymph nodes were isolated 3 or 7 weeks after infection and cultured for 2 days in the presence or absence of L. major antigen. Culture supernatants of the lymph node cells were tested for IFN-
production by ELISA (Fig. 3A). CD4+ T cells from both C57BL/6 and IRF-4/ mice but not BALB/c mice showed specific IFN-
production in response to L. major antigen. The level of IFN-
produced by T cells of IRF-4/ mice was equivalent to that of C57BL/6 mice 3 weeks after infection and reduced to approximately half of that of C57BL/6 mice 7 weeks after infection, consistent with the level of footpad swelling. Next, we determined the numbers of IFN-
-producing cells in the draining lymph nodes of each mouse by the ELISPOT assay (Fig. 3B). The proportion of CD4+ T cells producing IFN-
in response to L. major-antigen in IRF-4/ mice was approximately half of the C57BL/6 mice, consistent with the result of ELISA. Thus, when the production of IFN-
was monitored on a per cell basis, each CD4+ T cell in IRF-4/ mice produced IFN-
at levels similar to C57BL/6 mice. However, since the number of CD4+ T cells in the draining lymph node of IRF-4/ mice (2.5 x 106/lymph node) was much lower than C57BL/6 mice (6.7 x 107/lymph node) at 7 weeks after infection (Fig. 2), the total number of IFN-
-producing CD4+ T cells in each draining lymph node of IRF-4/ mice (
1.2 x 103) was much lower than C57BL/6 mice (
8.1 x 104). Taken together, the results indicated that IRF-4 is not required for the induction of the protective Th1 immune response against cutaneous infection of L. major, but is required to sustain the protective Th1 response.

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Fig. 2. The kinetic change of the lymphocyte numbers in draining lymph nodes during L. major infection. The number of lymphocytes in the draining (left popliteal) and non-draining lymph nodes (mixture of right axillary and right inguinal, and mesenteric) of BALB/c (squares), C57BL/6 (open circles) and IRF-4/ (solid circles) mice were counted 3 and 7 weeks after inoculation of L. major in the left footpad. The numbers of lymphocytes in each lymph node in naive mice (day 0) were also counted. The lymph node cells were stained with anti-CD4, anti-CD8 and anti-B220 mAb, and the proportion of each subset was determined using flow cytometry. The number of each lymphocyte subset was calculated by multiplying the total cell number with the proportion of each subset. The data shown are means ± SD obtained from three mice at each time point.
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Response of T cells from IRF-4/ mice to TCR engagement in vitro
To monitor the response of CD4+ T cells of the IRF-4/ genotype in response to peptideMHC, OT-II TCR transgenic mice expressing a TCR specific for OVA323339 and I-Ab were bred to IRF-4/ mice (OT-II/IRF-4/ mice). The flow cytometric analysis of T cell populations in the thymus and peripheral lymphoid tissue indicated that the distribution of T cell subpopulations in OT-II/IRF-4/ mice was not significantly different from that of OT-II mice with a marked shift to CD4+CD8 T cells as described (15). The proportions of CD4+CD8 T cells in total lymph node cells were
42% in OT-II and
52% in OT-II/IRF-4/ mice, and CD4CD8+ T cells were
6% in OT-II and
7% in OT-II/IRF-4/ mice. Purified CD4+ T cells from OT-II and OT-II/IRF-4/ mice were cultured in the presence of mitomycin C-treated C57BL/6 spleen cells and a range of antigenic peptide (OVA323339 010 µM) (Fig. 4A). OT-II T cells showed an antigen-specific proliferative response in a dose-dependent manner. OT-II/IRF-4/ T cells also showed a similar antigen dose-dependent proliferation, although the level of the response was reduced when compared with OT-II T cells. We also used ELISA to examine the production of IFN-
in response to the antigenic pep tide (Fig. 4B). Both OT-II and OT-II/IRF-4/ T cells exhibited comparable levels of IFN-
secretion in response to OVA323339.
Differentiation of IRF-4/ T cells to Th1/Th2 in vitro
Since CD4+ T cells from IRF-4/ mice were able to respond to a specific antigen, we investigated whether they could differentiate into Th1 and Th2 cells in vitro. Purified CD4+ T cells from OT-II and OT-II/IRF-4/ mice were cultured in vitro with antigenic peptide and C57BL/6 spleen cells in the presence of IL-2, IL-12 and anti-IL-4 mAb to generate Th1 cells or in the presence of IL-2, IL-4, anti-IL12 mAb and anti-IFN-
mAb to generate Th2 cells. After 1 week in culture, cells were re-stimulated with OVA323339 peptide and C57BL/6 spleen cells, and the levels of IFN-
and IL-4 in the culture supernatant of each T cell subset were determined by ELISA (Fig. 5A). CD4+ T cells from OT-II and OT-II/IRF-4/ mice produced similar levels of IFN-
after they were cultured under Th1 conditions. No significant IL-4 production was detected in these cultures. When CD4+ T cells were cultured in Th2 skewing conditions, cells from OT-II mice produced IL-4 and not IFN-
, whereas those from OT-II/IRF-4/ mice produced neither IL-4 nor IFN-
. Next, we determined cytokine production by individual T cells by intracellular cytokine staining of IFN-
and IL-4 after 1 week of polarizing culture (Fig. 5B). CD4+ T cells from OT-II mice showed significant development to both Th1 and Th2 cells after culture in each condition. About 49.3% became IFN-
producers under Th1 conditions and
18.1% became IL-4 producers under Th2 conditions. A similar proportion of CD4+ T cells from OT-II/IRF-4/ mice (
45.8%) exhibited IFN-
production under Th1 conditions, and the levels of IFN-
in each cell were equivalent to those of OT-II cells. Therefore, Th1 development of CD4+ T cells in vitro was not impaired in IRF-4/ mice. In contrast, very little development of IL-4 producers (
2.9%) was observed under Th2 conditions, suggesting that Th2 development in vitro was impaired in the mice lacking the IRF-4 gene.
The transcription factors T-bet and GATA-3 are putative master regulators of Th1 and Th2 development respectively (19,20). The expression of T-bet and GATA-3 is induced under Th1 and Th2 conditions respectively, and correlates to their production of IFN-
and Th2 cytokines. When expressed ectopically, T-bet and GATA-3 are sufficient to induce expression of IFN-
and IL-4 synthesis respectively. Thus, we determined whether these transcription factors are expressed in CD4+ T cells of IRF-4/ mice after culture in vitro (Fig. 6). RT-PCR analysis was performed using RNA extracted from non-stimulated CD4+ T cells, CD4+ T cells cultured under Th1 conditions and those cultured under Th2 conditions. IFN-
mRNA was detected in CD4+ T cells from both OT-II and OT-II/IRF-4/ mice, while IL-4 mRNA was detected in CD4+ T cells from OT-II and not OT-II/IRF-4/ mice. The expression of T-bet and GATA-3 mRNA in each T cell population was consistent with their production of IFN-
and IL-4. T-bet expression was induced under Th1 conditions in CD4+ T cells from both OT-II and OT-II/IRF-4/ mice, while GATA-3 mRNA was detected under Th2 conditions in CD4+ T cells from OT-II mice and not OT-II/IRF-4/ mice. Taken together, CD4+ T cells from IRF-4/ mice are able to differentiate into Th1 cells, but are impaired in their ability to differentiate into Th2 cells.
The proliferative response to IL-4 of CD4+ T cells from IRF-4/ mice
Since IL-4 is critical for the development of Th2 cells, we investigated the possibility that IL-4-induced CD4+ T cell activation is defective in IRF-4/ mice. CD4+ T cells from IRF-4/ and control C57BL/6 mice were stimulated in the presence of IL-4, and their proliferative response was assessed by [3H]thymidine incorporation (Fig. 7). The proliferative response of CD4+ T cells from IRF-4/ mice in response to IL-4 was reduced when compared with C57BL/6 T cells. These cells, however, proliferated at equivalent levels in response to PMA and ionomycin. These results suggested that CD4+ T cells from IRF-4/ mice have defects in the responsiveness to IL-4. This can be caused by the reduced expression of IL-4 receptor or defects in the subsequent signaling events. Thus, the expression of IL-4 receptor on naive and activated CD4+ T cells was examined by flow cytometry (Fig. 8A). IL-4 receptor
chain was expressed on CD4+ T cells from IRF-4/ mice at levels similar to control mice. After the activation of CD4+ T cells with anti-TCR and anti-CD28 mAb for 40 h, the level of IL-4 receptor expression was similar to naive T cells in both C57BL/6 and IRF-4/ and mice, while the expression of IL-2 receptor was strongly induced. Stat6 is rapidly tyrosine phosphorylated following stimulation of T cells with IL-4 (21), which is essential for signal transduction of IL-4 receptor (2224). Also, it was previously reported that IRF-4 interacts with Stat6 and drives the expression of IL-4-inducible genes in B cells (11). Therefore, we next examined whether Stat6 can be activated by IL-4 in the absence of IRF-4. Naive and activated CD4+ T cells were stimulated with IL-4, and the activation of Stat6 was examined by Western blotting using anti-phophoStat6 antibody (Fig. 8B). Tyrosine phosphorylation of Stat 6 was induced by IL-4 in activated CD4+ T cells of both C57BL/6 and IRF-4/ mice, indicating that activation of Stat6 is not impaired in IRF-4/ mice. It was recently reported that growth factor independent (Gfi)-1, a transcriptional repressor, is induced by IL-4 in a Stat6-dependent manner, and strikingly promotes proliferation of Th2 and ThN cells (18). Thus, we examined the expression of Gfi-1 in CD4+ T cells of both C57BL/6 and IRF-4/ mice by RT-PCR (Fig. 8C). Gfi-1 was expressed in activated CD4+ T cells of C57BL/6 mice and its expression was up-regulated by stimulation with IL-4. However, the expression was not detected in activated CD4+ T cells from IRF-4/ mice even after stimulation with IL-4. The result suggested that the reduced proliferation of IRF-4/ CD4+ T cells in response to IL-4 was at least in part due to the defective expression of Gfi-1.

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Fig. 7. Proliferation of CD4+ T cells from C57BL/6 and IRF-4/ mice in response to IL-4. CD4+ T cells were cultured in medium, IL-4 (250 U/ml), PMA (20 ng/ml), ionomycin (1 µM) and their combinations. After 48 h, proliferation was assessed by [3H]thymidine incorporation. Values represent means ± SD of triplicate wells. The data represent three separate experiments with similar results.
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Discussion
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Mice deficient in IRF-4 gene are severely defective in antibody production, cytotoxic T cell development and cytokine production by T cells, indicating the critical role of IRF-4 in B and T lymphocyte function (12). In this study, we showed that CD4+ T cells from IRF-4/ mice were able to mount protective Th1 immune responses against L. major infection. The protective immunity was particularly effective during early stages of the infection. The level of footpad swelling in IRF-4/ mice was even less than control C57BL/6 mice and CD4+ T cells from IRF-4/ mice produced IFN-
in response to the parasite antigen at levels similar to C57BL/6 mice 3 weeks after infection. The footpad swelling of the infected IRF-4/ mice, however, began to increase 6 weeks after infection when it began to reduce in C57BL/6 mice. In parallel, the number of lymphocytes in the draining lymph nodes was dramatically reduced. Thus, the number of IFN-
-producing T cells of IRF-4/ mice was dramatically reduced when measured at 7 weeks after the infection, although each CD4+ T cell produced IFN-
at levels similar to control C57BL/6 mice. The striking reduction in the lymphocyte number occurred only in the draining lymph nodes, and was observed in all lymphocyte types including CD4+ T cells, CD8+ T cells and B cells. Therefore, IRF-4 is required for sustained protective immune responses against L. major infection. The reason for this reduction of the lymphocytes in the draining lymph nodes is not clear. One possibility is the migration of the lymphocytes out of the draining lymph nodes. Alternatively, these lymphocytes may undergo apoptosis in the draining lymph nodes. These possibilities require further investigation.
CD4+ T cells from OT-II/IRF-4/ mice produced IFN-
at levels similar to control CD4+ T cells in vitro, suggesting that IFN-
production is not impaired in CD4+ T cells of IRF-4/ mice (Figs 4,5). This seemed contradictory to the previous report showing that cytokine secretion by T cells from IRF-4/ mice was defective when they were stimulated with anti-CD3 mAb. It may be due to the differences in the T cell subsets that secrete IFN-
: CD8+ T cells were the main source of IFN-
-secreting cells when T cells were stimulated with anti-CD3 mAb (data not shown), while CD4+ T cells of OT-II mice produced IFN-
in response to OVA323339. Alternatively, the difference might be due to the nature of antigenic stimulation used in both studies: T cells were stimulated with peptideMHC on antigen-presenting cells in this study, while they were stimulated with anti-CD3 antibody alone in the previous study (12). After culture under Th1 conditions, CD4+ T cells from IRF-4/ mice expressed Th1-specific transcription factor, T-bet, and were able to produce IFN-
at levels similar to control T cells, indicating that they differentiated into Th1 cells in vitro. Intracellular staining of IFN-
confirmed that individual T cells produce IFN-
at levels comparable to control T cells. Taken together, these studies indicated that Th1 development and function was not impaired in CD4+ T cells of IRF-4/ mice.
Th2 development was impaired in CD4+ T cells from IRF-4/ mice. The defect in Th2 development was observed by both the defects in IL-4 production and the expression of GATA-3 after culture of CD4+ T cells under Th2 conditions. This observation is consistent with the recent report by Rengarajan et al. (25) showing similar defects in Th2 cytokine production by CD4+ T cells of IRF-4/ mice after culture under Th2 conditions. They also showed that IRF-4 interacted with NFATc2 to enhance NFATc2-driven transcriptional activation of the IL-4 promoter, suggesting that IRF-4 was important for the production of IL-4. Our study of the Th2 defect in these mice is distinct from theirs in two points. First, we stimulated T cells with the natural ligand, peptideMHC, while they used anti-CD3 and anti-CD28 mAb to induce Th cell differentiation. Second, our study suggested that the defect in Th2 development is due to the defective response of IRF-4/ CD4+ T cells to IL-4. Since the Th2-skewing condition of CD4+ T cell culture contains exogenous IL-4, the defect in Th2 development could not be solely explained by the defect in the IL-4 production. In fact, we found that the response to exogenous IL-4 was defective in CD4+ T cells from IRF-4/ mice (Fig. 7). The expression of IL-4 receptor and the phosphorylation of Stat6 by IL-4 were not impaired, but Gfi-1 expression was defective in CD4+ T cells from IRF-4/ mice after activation. Gfi-1 is induced by T cell activation and IL-4. Gfi-1 promotes proliferation and diminishes apoptosis of Th2 cells in cooperation with Gata-3. Thus, the reduced growth of CD4+ T cells from IRF-4/ mice is at least in part mediated by the impaired induction of Gfi-1. Taken together, these results suggest that IRF-4 is involved in both transcriptional activation of IL-4 production (25) and the responsiveness of CD4+ T cells to IL-4.
The critical role of IRF-1 in the development of Th1 immune responses is well established. Mice lacking IRF-1 failed to mount Th1 responses and instead underwent accelerated Th2 differentiation (5,6). On the other hand, we showed here that Th1 but not Th2 cells can develop in mice lacking IRF-4. Therefore, IRF-1 and IRF-4 appear to have crucial functions in the development of two opposing subsets of CD4+ T cells, Th1 and Th2 respectively. These two members of an IRF family might coordinately regulate the development of CD4+ T cells by acting on the production and responsiveness to key cytokines. IRF-1 is important for IL-12 production by macrophages as well as responsiveness of CD4+ T cells to IL-12 (5), while IRF-4 is required for the production and responsiveness to IL-4. Alternatively, it is also possible that these members of the IRF family, which bind to a similar DNA motif, compete for the same DNA binding sequence of a key regulatory gene that determines Th1/Th2 development.
Finally, this and previous studies using mice lacking IRF-4 gene showed that IRF-4 is critical for the development and function of many lymphocyte types including B cells, CD8+ T cells and Th2 cells. There might be a key regulatory gene that is commonly required for the development of these cell types and whose expression is regulated by IRF-4. Th1 appears to be an exception of this regulation, although IRF-4 is required for sustained Th1 response. It is intriguing to find such regulatory mechanisms of lymphocyte development and homeostasis by IRF-4.
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Acknowledgements
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We acknowledge Dr W. Heath and Dr H. Kosaka for OT-II transgenic mice, and Dr K. Himeno and Dr S. Hamano for L. major. We also thank Dr G. Massey for editorial support and Mr M. Ueda for his technical assistance. This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan.
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Abbreviations
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Gfigrowth factor independent
IRFIFN-regulatory factor
ISREIFN-stimulated response element
PEphycoerythrin
PMAphorbol myristate acetate
Statsignal transduction and activation of transcription
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Note added in proof
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After submission of the manuscript, another group reported similar study on the differentiation of helper T cells after infection of IRF-4-deficient mice with >L. major. Lohoff, M., Mittrucker, H.-W., Prechtl, S., Bischof, S., Sommer, F., Kock, S., Ferrick, D.A., Duncan, G.S., Gressner, A. and Mak, T.W. 2002. Dysregulated T helper cell differentiation in the absence of interferon regulatory factor 4. Proc. Natl Acad. Sci. USA 99:11808.
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References
|
---|
- Taniguchi, T., Ogasawara, K., Takaoka, A. and Tanaka, N. 2001. IRF family of transcription factors as regulators of host defense. Annu. Rev. Immunol. 19:623.[CrossRef][ISI][Medline]
- Harada, H., Fujita, T., Miyamoto, M., Kimura, Y., Maruyama, M., Furia, A., Miyata, T. and Taniguchi, T. 1989. Structurally similar but functionally distinct factors, IRF-1 and IRF-2, bind to the same regulatory elements of IFN and IFN-inducible genes. Cell 58:729.[ISI][Medline]
- Tanaka, N., Ishihara, M., Kitagawa, M., Harada, H., Kimura, T., Matsuyama, T., Lamphier, M. S., Aizawa, S., Mak, T. W. and Taniguchi, T. 1994. Cellular commitment to oncogene-induced transformation or apoptosis is dependent on the transcription factor IRF-1. Cell 77:829.[ISI][Medline]
- Matsuyama, T., Kimura, T., Kitagawa, M., Pfeffer, K., Kawakami, T., Watanabe, N., Kundig, T. M., Amakawa, R., Kishihara, K., Wakeham, A., et al. 1993. Targeted disruption of IRF-1 or IRF-2 results in abnormal type I IFN gene induction and aberrant lymphocyte development. Cell 75:83.[ISI][Medline]
- Taki, S., Sato, T., Ogasawara, K., Fukuda, T., Sato, M., Hida, S., Suzuki, G., Matsuyama, M., Shin, E. H., Kojima, S., Taniguchi, T. and Asano, Y. 1997. Multistage regulation of Th1-type immune responses by the transcription factor IRF-1. Immunity 6:673.[ISI][Medline]
- Lohoff, M., Ferrick, D., Mittrucker, H. W., Duncan, G. S., Bischof, S., Rollinghoff, M. and Mak, T. W. 1997. Interferon regulatory factor-1 is required for a T helper 1 immune response in vivo. Immunity 6:681.[ISI][Medline]
- Eisenbeis, C. F., Singh, H. and Storb, U. 1995. Pip, a novel IRF family member, is a lymphoid-specific, PU.1-dependent transcriptional activator. Genes Dev. 9:1377.[Abstract]
- Matsuyama, T., Grossman, A., Mittrucker, H. W., Siderovski, D. P., Kiefer, F., Kawakami, T., Richardson, C. D., Taniguchi, T., Yoshinaga, S. K. and Mak, T. W. 1995. Molecular cloning of LSIRF, a lymphoid-specific member of the interferon regulatory factor family that binds the interferon-stimulated response element (ISRE). Nucleic Acids Res. 23:2127.[Abstract]
- Marecki, S., Atchison, M. L. and Fenton, M. J. 1999. Differential expression and distinct functions of IFN regulatory factor 4 and IFN consensus sequence binding protein in macrophages. J. Immunol. 163:2713.[Abstract/Free Full Text]
- Grumont, R. J. and Gerondakis, S. 2000. Rel induces interferon regulatory factor 4 (IRF-4) expression in lymphocytes: modulation of interferon-regulated gene expression by rel/nuclear factor kappaB. J. Exp. Med. 191:1281.[Abstract/Free Full Text]
- Gupta, S., Jiang, M., Anthony, A. and Pernis, A. B. 1999. Lineage-specific modulation of interleukin 4 signaling by interferon regulatory factor 4. J. Exp. Med. 190:1837.[Abstract/Free Full Text]
- Mittrucker, H. W., Matsuyama, T., Grossman, A., Kundig, T. M., Potter, J., Shahinian, A., Wakeham, A., Patterson, B., Ohashi, P. S. and Mak, T. W. 1997. Requirement for the transcription factor LSIRF/IRF4 for mature B and T lymphocyte function. Science 275:540.[Abstract/Free Full Text]
- Yamagata, T., Nishida, J., Tanaka, S., Sakai, R., Mitani, K., Yoshida, M., Taniguchi, T., Yazaki, Y. and Hirai, H. 1996. A novel interferon regulatory factor family transcription factor, ICSAT/Pip/LSIRF, that negatively regulates the activity of interferon-regulated genes. Mol. Cell. Biol. 16:1283.[Abstract]
- Reiner, S. L. and Loccksley, R. M. 1994. The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13:151.[CrossRef][ISI][Medline]
- Barnden, M. J., Allison, J., Heath, W. R. and Carbone, F. R. 1998. Defective TCR expression in transgenic mice constructed using cDNA-based
- and ß-chain genes under the control of heterogenous regulatory elements. Immunol. Cell Biol. 76:34.
- Abe, R., Vandenberghe, P., Craighead, N., Smoot, D. S., Lee, K. P. and June, C. H. 1995. Distinct signal transduction in mouse CD4+ and CD8+ splenic T cells after CD28 receptor ligation. J. Immunol. 154:985.[Abstract/Free Full Text]
- Grogan, J. L., Mohrs, M., Harmon, B., Lacy, D. A., Sedat, J. W. and Locksley, R. M. 2001. Early transcription and silencing of cytokine genes underlie polarization of T helper cell subsets. Immunity 14:205.[ISI][Medline]
- Zhu, J., Guo, L., Min, B., Watson, C. J., Hu-Li, J., Young, H. A., Tsichlis, P. N. and Paul, W. E. 2002. Growth factor independent-1 induced by IL-4 regulates Th2 cell proliferation. Immunity 16:733.[ISI][Medline]
- Szabo, S. J., Kim, S. T., Costa, G. L., Zhang, X., Fathman, C. G. and Glimcher, l. H. 2000. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 100:655.[ISI][Medline]
- Zhang, W.-P. and Flavell, R. A. 1997. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89:587.[ISI][Medline]
- Hou, J., Schindler, U., Henzel, W. J., Ho, T. C., Brasseur, M. and McKnight, S. L. 1994. An interleukin-4-induced transcription factor: IL-4 Stat. Science 265:1701.[ISI][Medline]
- Takeda, K., Tanaka, T., Shi, W., Matsumoto, M., Minami, M., Kashiwamura, S.-I., Nakanishi, K., Yoshida, N., Kishimoto, T. and Akira, S. 1996. Essential role of Stat6 in IL-4 signalling. Nature 380:627.[CrossRef][ISI][Medline]
- Shimoda, K., van Deursen, J., Sangster, M. Y., Sarawar, S. R., Carson, R. T., Tripp, R. A., Chu, C., Quelle, F. W., Nosaka, T., Vignali, D. A. A., Doherty, P. C., Grosveld, G., Paul, W. E. and Ihle, J. N. 1996. Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted Stat6 gene. Nature 380:630.[CrossRef][ISI][Medline]
- Kaplan, M. H., Schindler, U., Smiley, S. T. and Grusby, M. J. 1996. Stat6 is required for mediating responses to IL-4 and for the development of Th2 cells. Immunity 4:313.[ISI][Medline]
- Rengarajan, J., Mowen, K. A., McBride, K. D., Smith, E. D., Singh, H. and Glimcher, L. H. 2002. Interferon regulatory factor (IRF4) interacts with NFATc2 to modulate interleukin 4 gene expression. J. Exp. Med. 195:1003.[Abstract/Free Full Text]