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Originally published online as doi:10.1189/jlb.1006619 on March 27, 2007

Published online before print March 27, 2007
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(Journal of Leukocyte Biology. 2007;81:1455-1465.)
© 2007 by Society for Leukocyte Biology

4-1BB triggers IL-13 production from T cells to limit the polarized, Th1-mediated inflammation

Su M. Shin*, Young H. Kim*, Beom K. Choi*, Patrick M. Kwon*, Hyeon-Woo Lee{dagger} and Byoung S. Kwon*,{ddagger},1

* The Immunomodulation Research Center, University of Ulsan, Ulsan, Korea;
{dagger} Department of Pharmacology, School of Dentistry, Kyung Hee University, Seoul, Korea; and
{ddagger} LSU Eye Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana, USA

1 Correspondence: Immunomodulation Research Center, University of Ulsan, San29, Mukeo-Dong, Nam-Ku, Ulsan, Korea 680-749. E-mail: bskwon{at}mail.ulsan.ac.kr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
4-1BB (CD137) triggering typically induces Th1 response by increasing IFN-{gamma} from T cells upon TCR ligation. We found recently that 4-1BB costimulation increased the expression of IL-13 from CD4+ T cells, as well as CD8+ T cells. The enhanced IL-13 expression by agonistic anti-4-1BB treatment was mediated via MAPK1/2, PI-3K, JNK, mammalian target of rapamycin, NF-AT, and NF-{kappa}B signaling pathways. The signaling for IL-13 induction was similar to that of IFN-{gamma} production by anti-4-1BB treatment in T cells. When the anti-4-1BB-mediated IL-13 expression was tested in an in vivo viral infection model such as HSV-1 and vesicular stomatitis virus, 4-1BB stimulation enhanced IL-13 expression of CD4+ T, rather than CD8+ T cells. Although IL-13 was enhanced by anti-4-1BB treatment, the increased IL-13 did not significantly alter the anti-4-1BB-induced Th1 polarization of T cells—increase of T-bet and decrease of GATA-3. Nevertheless, anti-4-1BB treatment polarized T cells excessively in the absence of IL-13 and even became detrimental to the mice by causing liver inflammation. Therefore, we concluded that IL-13 was coinduced following 4-1BB triggering to maintain the Th1/2 balance of immune response.

Key Words: costimulation • CD8+ T • CD4+ T • cytokine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The activation of a naïve T cell requires two signals: ligation of the TCR with the MHC/peptide complex on the APC and cross-linking of costimulatory receptors on the T cell with the corresponding ligands on the APC [1 ]. Many costimulatory molecules influence T cell activation and differentiation positively or negatively [2 3 4 ]. 4-1BB is a TNF receptor (TNFR) family member, which is known to evoke various T cell responses through TNFR-associated factor-mediated activation of NF-{kappa}B, p38 MAPK1/2, stress-activated protein kinase/JNK, or ERK1/2 [5 6 7 8 ]. Interaction of 4-1BB with 4-1BB ligand or anti-4-1BB mAb can costimulate the CD4+ and CD8+ T cells, which leads to the expansion and cytokine production of T cells, development of CTL, and prevention of activation-induced cell death [7 , 9 10 11 12 13 14 15 ]. In CD8+ T cells, 4-1BB promotes survival by stimulating antiapoptotic gene expression via the I{kappa}B-{alpha}/NF-{kappa}B pathway, whereas 4-1BB-mediated expansion of T cells results from increasing cell cycle-related gene transcription and translation through the combined action of the ERK1/2 and IL-2 receptor (IL-2R)/PI-3K/Akt/mammalian target of rapamycin (mTOR)/p70S6K pathways [16 , 17 ]. As reported in previous studies, 4-1BB stimulation preferentially induces Th1 responses and CD8+ T cell proliferation [12 , 18 ]. However, a recent report showed that 4-1BB-knockout (KO) mice did not generate rapid IL-4 responses after systemic T cell activation or effective antigen-specific Th2 responses in a model of LPS-induced shock syndromes [19 ].

We reported previously that cross-linking of 4-1BB up-regulates IL-13 expression in primary CD8+ T lymphocytes, and 4-1BB-mediated IL-13 expression is partially responsible for the CD8+ T cell expansion in an autocrine or paracrine manner [20 ]. IL-13 was produced predominantly by Th2-polarized CD4+ T cells [21 ] and shown to be a key mediator of allergic inflammation, independent of IL-4 in mouse models [22 , 23 ]. Therefore, we investigated the effect of 4-1BB in the expression of IL-13 and its receptors on CD4+ and CD8+ T cells in vitro and in vivo. We found that in vitro cross-linking of 4-1BB increased the expression of IL-13 mRNA and protein in CD4+ T cells, as well as in CD8+ T cells [20 ]. Also, the enhanced IL-13 expression by agonistic anti-4-1BB was mediated via MAPK1/2, PI-3K, JNK, mTOR, NF-AT, and NF-{kappa}B signaling pathways, which was similar to the signaling pathway by 4-1BB stimulation that led to the IFN-{gamma} production in T cells. In vivo experiments also showed that anti-4-1BB treatment increased the expression of IL-13 and IL-13R{alpha} in CD4+ T and CD8+ T cells of virus-infected mice but decreased IL-13R{alpha}2. Nevertheless, the enhanced IL-13 did not alter the anti-4-1BB-mediated Th1 immune responses but limited the magnitude of immune response to prevent the unnecessary inflammation. Therefore, our data suggest that 4-1BB triggering increases the expression of IL-13 and its functional receptors on T cells, which seems to be a feedback reaction of excessively polarized immune response.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice, reagents, and antibodies
Female C57BL/6 and BALB/c mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA), and IL-13-deficient mice (IL-13 KO) were kindly provided by Dr. Andrew N. J. McKenzie (Medical Research Council Laboratory of Molecular Biology, Cambridge, UK) and maintained under specific pathogen-free conditions. Anti-CD3 mAb (145-2C11 clone) and biotin-labeled CD25 (7D4) were purchased from BD PharMingen (San Diego, CA, USA). CD4-, CD8-, and streptoavidin-conjugated microbeads were purchased from Miltenyi Biotec (Auburn, CA, USA). Agonistic anti-4-1BB mAb (3H3 and 3E1) were kind gifts from Dr. Robert Mittler of Emory University (Atlanta, GA, USA). Rapamycin (mTOR inhibitor), SP600125 (JNK inhibitor), pyrrolidine derivative of dithiocarbamate (PDTC; NF-{kappa}B inhibitor), NF-AT inhibitory peptide VIVIT, PD98059 [MAPK kinase (MEK)1/2 inhibitor], and LY294002 (PI-3K inhibitor) were purchased from Calbiochem-Novabiochem (La Jolla, CA, USA). Antimouse IL-13 mAb (38213), biotinylated antimouse IL-13 antibody, murine (r)IL-13, and IFN-{gamma} ELISA kit were purchased from R&D Systems (Minneapolis, MN, USA).

CD4+ and CD8+ T cell purification
Cell suspensions were prepared from the spleens and lymph nodes (LNs), preincubated with FcR-blocking mAb 2.4G2 for 10 min at 4°C, and then incubated with biotin-labeled, anti-CD25 mAb for 30 min. The cells were incubated further with streptavidin microbeads. CD25+ T cells, including CD4+CD25+ regulatory T cells, were removed by loading the incubated cells on an LS column. CD4+ or CD8+ T cells were isolated from the CD25+ cell-depleted fraction by incubating with CD4 or CD8 microbeads for 30 min at 4°C. The purified CD4+ and CD8+ T cells were >95% pure by flow cytometry.

T cell stimulation and the treatment of inhibitors, antibodies, and cytokine
The purified CD4+ and CD8 + T cells were incubated with 0.2 µg/ml anti-CD3 mAb at 106 cells/well in 96-well, round-bottom microplates. The activated CD4+ or CD8+ T cells were stained with anti-4-1BB-FITC (3E1-FITC) 16 h after the incubation, and >70% were found routinely by flow cytometry to express 4-1BB on their surface. After verifying the 4-1BB expression, we preincubated them with various pharmacological inhibitors or DMSO as a control for 1 h and then added 5 µ/ml agonistic anti-4-1BB mAb (3H3) or rat IgG2a (as an isotype control). In separate experiments, the anti-CD3-treated CD4+ or CD8+ T cells were preincubated with rIL-13 or anti-IL-13 mAb for 1 h before the treatment of anti-4-1BB mAb (3H3) or rat IgG2a. To remove anti-CD3, the anti-CD3-treated T cells were washed intensively with RPMI 1640 and rested in the 96-well plate for 24 h. The cells were stimulated further with anti-4-1BB or rat IgG as described above.

Virus infection and antibody treatment
For the infection of HSV-1 KOS strain, C57BL/6 mice were anesthetized by an i.p. injection of ketamine hydrochloride (1 mg/kg, vetamine, Phoenix Scientific Inc., St. Joseph, MO, USA) and xylazine (0.5 mg/kg, Ben Venue Laboratories, Bedford, OH, USA). Mice were infected with 5 x 105 PFU HSV-1 into each hind footpad or i.v. For the infection of vesicular stomatitis virus (VSV), BALB/c mice were infected i.v. with 1 x 106 PFU VSV. The infected mice were received i.p. 200 µg anti-4-1BB (3H3) mAb or rat IgG on postinfection (PI) Days 0 and 2. CD4+ and CD8+ T cells were isolated from popliteal LNs (PLNs) of HSV-1-infected mice on PI Day 5 or spleens of VSV-infected mice on PI Day 7 and used for the next experiments.

Cytokine measurement
To measure IFN-{gamma} or IL-13 from CD4+ or CD8+ T cells in vitro, the culture supernatants were harvested from CD4+ or CD8+ T cells following anti-CD3 and/or anti-4-1BB treatment at the indicated time. To measure IFN-{gamma} or IL-13 from CD4+ or CD8+ T cells following HSV-1 or VSV infection, CD4+ or CD8+ T cells were isolated from the virus-infected mice and restimulated in vitro with irradiated and T cell-depleted splenocytes pulsed with UV-inactivated HSV [multiplicity of infection (MOI) 10.0 before UV inactivation] for another 24 h. Cytokines in supernatants were detected by conventional, double-sandwich ELISA according to the manufacturer’s instructions (R&D Systems). In a separate experiment, CD4+ or CD8+ T cells of wild-type and IL-13-deficient mice were activated with anti-CD3 and/or anti-4-1BB mAb as described above, and the culture supernatant was collected 48 h after the activation. Th1/2 cytokine profile was determined by using cytometric bead array kit (CBA; BD Biosciences, San Jose, CA, USA) from the culture supernatant according to the manufacturer’s instruction.

RT-PCR
Total RNA was extracted from cultured cells using TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA) following the instructions provided by the manufacturer. Total RNA (1 µ) was reverse-transcribed into cDNA using a SuperScriptTM first-strand synthesis system (Invitrogen Life Technologies). Gene-specific primers used were as follows: IL-2, forward 5'-TCCACTTCAAGCTCTACAG-3', reverse 5'-AGTCAAATCCAGAACATGCC-3'; IFN-{gamma}, forward 5'-AACGCTACACACTGCATCTTGG-3', reverse 5'-GCCGTGGCAGTAACAGCC-3'; IL-13, forward 5'-ATCACACAAGACCAGACTCC-3', reverse 5'-TGTGTAGCTGAGCAGTTTTG-3'; IL-15, 5'-AGGAATACATCCATCTCGTGCTA-3', reverse 5'-GGAGAAAGCAGTTCATTGCAGTA-3'; IL-2R, forward 5'-CACGGGCTCCAGAAAATCTA-3', reverse 5'-AGGGATAAGCACAGCTTCCA-3'; Il-4R, forward 5'-AGTGCCAGCACTGTGTGTGT-3', reverse 5'-TGGAGTGTGAGGTTGTCTGG-3'; IL-13R{alpha}1, forward 5'-GGCCATCCTGCAAAATAGTGTC-3', reverse 5'-GGGTATCATCATTCTGGTCTCC-3'; IL-13R{alpha}2, forward 5'-CGCATTTGTCAGAGCATTGT-3', reverse, 5'-CCAAGCCCTCATACCAGAAA-3'; T-bet, forward 5'-TGCCTGCAGTGCTTCTAACA-3', reverse 5'-TGCCCCGCTTCCTCTCCAACCAA-3'; GATA-3, forward 5'-GAAGGCATCCAGACCCGAAAC-3', reverse 5'-ACCCATGGCGGTGACCATGC-3'; glyceraldehyde-3 phosphate dehydrogenase (GAPDH), forward 5'-GAACGGGAAGCTTGTCATCAA-3', reverse 5'-CTAAGCAGTTGGTGGTGCAG-3'. PCR conditions were as follows: for IL-13, 94ºC for 5 min, followed by 35 cycles of 40 s at 94ºC, 40 s at 55ºC, and 1 min at 72ºC with a final extension at 72ºC for 5 min; for detection of IFN-{gamma}, 94ºC for 5 min, followed by 32 cycles of 30 s at 94ºC, 30 s at 49ºC, and 30 s at 72ºC with a final extension at 72ºC for 5 min; for IL-2, IL-2R, IL-4R{alpha}, IL-13R{alpha}1, and IL-13R{alpha}2, annealing was performed at 58ºC; for IL-15, annealing was performed at 58ºC; for detection of T-bet and GATA-3, annealing was 55ºC. PCR products were visualized by ethidium bromide following electrophoresis on 1% agarose gels.

Immunohistochemistry
Wild-type and IL-13-deficient BALB/c mice were i.v.-infected with 5 x 105 PFU of HSV-1 and i.p.-treated with anti-4-1BB or rat IgG on PI Days 0 and 2. Liver was snap-frozen in isopentane (2-methylbutane, Sigma-Aldrich, St. Louis, MO, USA) and embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA, USA) on liquid nitrogen. Cryostat sections (6 mm) were prepared and stained with hemotoxin and eosin (H&E), mounted in the mounting solution (Zymed, San Francisco, CA, USA), and photographed at 100x under light microscope.

Statistical analysis
All data were analyzed using a statistical program, Prism 4.0 (GraphPad, San Diego, CA, USA). Student’s t-test was used to determine statistical significance between groups.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
4-1BB stimulation enhances the secretion of IL-13 along with IFN-{gamma} from CD4+ and CD8+ T cells
We reported previously that cross-linking of 4-1BB up-regulates IL-13 expression in primary CD8+ T lymphocytes, and anti-4-1BB-mediated IL-13 expression is partially responsible for the CD8+ T cell expansion in an autocrine and paracrine manner [20 ]. In addition to this finding, we tested whether 4-1BB stimulation could increase the IL-13 expression from CD4+ T cells as well as CD8+ T cells. CD4+ and CD8+ T cells were purified from LNs and spleens of C57BL/6 mice by using a magnetic bead and stimulated with anti-CD3 and/or anti-4-1BB. Cross-linking of 4-1BB with agonistic anti-4-1BB mAb increased the IL-13 production from CD4+ and CD8+ T cells (Fig. 1A ), as well as IFN-{gamma} (Fig. 1B) . Anti-4-1BB-mediated IL-13 production was higher in CD4+ T cells than CD8+ T cells, and IFN-{gamma} production was higher in CD8+ T than in CD4+ T cells. To exclude the possibility that the positive selection of CD4+ or CD8+ T cells influences the subsequent results as a result of the binding of anti-CD4 or -CD8 mAb, CD4+ or CD8+ T cells were negatively selected by using an enrichment column (R&D Systems) and treated as described above. IL-13 was produced in the negatively selected CD4+ or CD8+ T cells at the levels similar to positively selected T cells (data not shown).


Figure 1
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  		Figure 1. IL-13 is partially responsible for proliferation of CD4+ T cells in a 4-1BB-independent manner. CD4+ T cells were purified from spleen and LNs of naive mice and stimulated with 0.1 µg/ml anti-CD3 mAb for 16 h. Following the confirmation of 4-1BB expression by flow cytometry, the anti-CD3-treated T cells were stimulated further with 5 µg/ml anti-4-1BB mAb. Production of IL-13 (A) and IFN-{gamma} (B) proteins was determined from the culture supernatant of CD8+ and CD4+ T cells at the indicated time by ELISA. To test the effect of IL-13 on CD4+ T cell proliferation, the anti-CD3-treated CD4+ T cells were preincubated with anti-IL-13 antibody or isotype control for 1 h and stimulated further with anti-4-1BB mAb for another 48 h (C). In a separate experiment, rIL-13 was added to anti-CD3-treated or anti-CD3 plus anti-4-1BB-treated CD4+ T cells for 48 h (D). The cells were pulsed with 1.0 µCi/well [3H]-thymidine to assess the proliferation for last 8 h, and the incorporation of thymidine was counted by liquid scintillation spectroscopy. The results are represented as means ± SD of triplicates. Similar results were obtained in three independent experiments. NT, no treatment.

 
To test the effect of IL-13 on the proliferation of CD4+ T cells, the anti-CD3-treated CD4+ T cells were preincubated with a blocking anti-IL-13 and followed by anti-4-1BB treatment. Similar to proliferation of CD8+ T cells [20 ], blockade of IL-13 partially inhibited the proliferation of CD4+ T cells in a dose-dependent manner but did not selectively inhibit anti-4-1BB-mediated CD4+ T cell proliferation (Fig. 1C) . Addition of rIL-13 did not significantly alter the proliferation of activated CD4+ T cells in the presence or absence of anti-4-1BB mAb (Fig. 1D) . These results imply that anti-4-1BB treatment enhances the secretion of IL-13 as well as IFN-{gamma} from CD4+ and CD8+ T cells, and the IL-13 is partially involved in the proliferation of CD4+ T cells.

4-1BB stimulation induces IL-13 from CD4+ T and CD8+ T cells, which are mediated via PI-3K, MEK1/2, TOR, JNK, NF-{kappa}B, and NF-AT
Anti-4-1BB treatment induced the IFN-{gamma} at an early time (12 h) but increased IL-13 production from 48 h after the stimulation. We next investigated signaling pathways for the IL-13 production and whether there were any differences in the signaling to trigger the IL-13 and IFN-{gamma} production following anti-4-1BB treatment. The purified CD4+ or CD8+ T cells were stimulated with anti-CD3 and/or anti-4-1BB in the presence or absence of PD98059 (ERK1/2 inhibitor) or LY294002 (PI-3K inhibitor). Treatments of PD98059 and LY294002 abolished the anti-4-1BB-mediated IL-13 production effectively in CD8+ and CD4+ T cells (Fig. 2A and 2B ). In the case of IFN-{gamma} expression, LY294002 completely inhibited the anti-4-1BB-mediated induction of IFN-{gamma} in CD8+ T cells, whereas PD98059 had a minimal effect on the IFN-{gamma} production (Fig. 2C) . However, both treatments effectively inhibited the IFN-{gamma} production from CD4+ T cells by anti-4-1BB treatment (Fig. 2D) . Consistent with the protein level, the treatment of PD98059 or LY294002 decreased the mRNA expression of IL-13 and IFN-{gamma} from CD8+ and CD4+ T cells, which were induced by anti-4-1BB treatment (Fig. 2E and 2F) .


Figure 2
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  		Figure 2. The signal pathways for IL-13 and IFN-{gamma} production on CD4+ T and CD8+ T cells. Purified, naïve CD4+ T and CD8+ T cells were stimulated with 0.2 µ/ml anti-CD3 mAb. After the incubation for 16 h, the cells were preincubated with 30 µM PD98059 (PD; MEK1/2 inhibitor) or 20 mM LY294002 (LY; PI-3K inhibitor) for 1 h and then treated with 5 µg/ml anti-4-1BB mAb or rat IgG as a control. Production of IL-13 and IFN-{gamma} proteins was determined from the culture supernatant of CD8+ T (A, C) and CD4+ T cells (B, D) by ELISA. RT-PCR for IL-2, IL-13, and IFN-{gamma} was performed from the cultured CD8+ (E) and CD4+ T cells (F) at 24 h after 4-1BB stimulation. PCR products were visualized by ethidium bromide following electrophoresis on 1% agarose gels.

 
In a separate experiment, we treated rapamycin (mTOR inhibitor), SP600125 (JNK inhibitor), PDTC (NF-{kappa}B inhibitor), and VIVIT (NF-AT inhibitor) to determine the signaling pathways that were involved in the anti-4-1BB-mediated IL-13 expression. Inhibition of mTOR, JNK, and NF-{kappa}B reversed the anti-4-1BB-mediated IL-13 and IFN-{gamma} production completely (Fig. 3A 3B 3C ), but NF-AT inhibitor decreased the anti-4-1BB-mediated cytokine production partially (Fig. 3D) .


Figure 3
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  		Figure 3. The signal pathways for IL-13 and IFN-{gamma} production on CD4+ T and CD8+ T cells. Purified, naïve CD4+ T and CD8+ T cells were stimulated with 0.2 µ/ml anti-CD3 mAb. After the incubation for 16 h, the anti-CD3-treated cells were pretreated with rapamycin (RAPA; 200 nM, mTOR inhibitor; A), SP600125 (SP; 20 µM, JNK inhibitor; B), PDTC (1 µM, NF-{kappa}B inhibitor; C), VIVIT (5 µM, NF-AT inhibitor; D), or DMSO as a control for 1 h and then stimulated with 5 µg/ml anti-4-1BB mAb or rat IgG as a control for another 72 h. IL-13 and IFN-{gamma} protein was measured in the culture supernatant of CD4+ T and CD8+ T cells by ELISA. NT, no treatment.

 
These data suggest that in vitro 4-1BB stimulation up-regulates IL-13 and IFN-{gamma} production from CD4+ and CD8+ T cells, and they require similar signaling pathways, including ERK1/2, PI-3K, mTOR, JNK, NF-{kappa}B, and NF-AT.

4-1BB stimulation in vivo enhances IL-13 expression and its functional receptor following HSV-1 infection
As in vitro 4-1BB stimulation up-regulated the expression of IL-13 from CD4+ and CD8+ T cells, we tested whether 4-1BB stimulation could enhance the expression of IL-13 from CD4+ and CD8+ T cells in vivo. We infected C57BL/6 mice with HSV-1 KOS strain via footpads, treated with agonistic anti-4-1BB (3H3) mAb or rat IgG as a control on PI Days 0 and 2 and killed the mice on PI Day 5 for the analysis. CD4+ or CD8+ T cells were purified from draining LNs (PLNs) and restimulated in vitro with irradiated and T cell-depleted splenocytes, which were pulsed with UV-inactivated HSV (MOI, 10.0 before UV inactivation) for another 24 h. IL-13 and IFN-{gamma} were measured in the culture supernatant by ELISA. In CD8+ T cells, 4-1BB stimulation in vivo had only a slight effect on IL-13 production (Fig. 4A ). However, 4-1BB stimulation increased IL-13 in CD4+ T cells (Fig. 4B) significantly. IFN-{gamma} was increased from CD8+ and CD4+ T cells (Fig. 4A and 4B) .


Figure 4
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  		Figure 4. Expression of IL-13 and IFN-{gamma} from T cells by 4-1BB stimulation in HSV-1-infected mice in vivo. C57BL/6 mice were infected s.c. with 5 x 105 PFU HSV-1 KOS into footpads. Rat IgG (200 µg) or anti-4-1BB mAb was injected i.p. on PI Days 0 and 2. Mice were killed on PI Day 5, and CD4+ T and CD8+ T cells were purified from PLNs of infected mice. Purified CD4+ T or CD8+ T cells were restimulated in vitro with irradiated and T cell-depleted splenocytes pulsed with UV-inactivated HSV (MOI, 10.0 before UV inactivation) for another 24 h. IFN-{gamma} and IL-13 proteins were measured in the culture supernatant of CD8+ T (A) and CD4+ T cells (B) from PLNs by ELISA. The mRNA expression for cytokines (C) and cytokine receptors (D) was determined by performing RT-PCR with the freshly isolated, total RNA of CD4+ T and CD8+ T cells on PI Day 5.

 
We also measured the mRNA level of IL-13, IFN-{gamma}, and IL-2 and IL-15 as a positive control, which was known to be induced by anti-4-1BB treatment. CD4+ T and CD8+ T cells were isolated from the PLNs of HSV-1-infected mice on PI Day 5, which received rat IgG or anti-4-1BB mAb, and total RNAs were isolated immediately from the purified T cells. Consistent with the protein level, the transcripts of IL-13 and IFN-{gamma} were up-regulated by anti-4-1BB treatment, which was confirmed by RT-PCR (Fig. 4C) . In particular, IL-13 was notably increased by anti-4-1BB treatment in CD4+ T cells. As we expected, IL-2 and IL-15 were increased by treating anti-4-1BB mAb in CD8+ T and CD4+ T cells.

We subsequently performed RT-PCR to analyze the expression of IL-13-related receptors on CD4+ T and CD8+ T cells. Previous reports indicated that mRNA for the functional receptor of IL-13, IL-4{alpha}/IL-13R{alpha}1, was expressed in CD8+ T cells [20 , 24 ], and in vitro cross-linking of 4-1BB down-regulated transcripts of IL-13R{alpha}2, which was known as a decoy receptor for IL-13. In vivo anti-4-1BB treatment increased transcription of IL-13R{alpha}1 on CD8+ and CD4+ T cells but decreased IL-13R{alpha}2 transcripts (Fig. 4D) . IL-2R{gamma} and IL-4R{alpha} mRNA was expressed constantly in CD8+ and CD4+ T cells.

These data indicate that in vivo 4-1BB stimulation up-regulates the expression of IL-13 and its functional receptor IL-13R{alpha}1 on CD4+ and CD8+ T cells.

4-1BB stimulation in vivo enhances IL-13 expression and its functional receptor following VSV infection
We found that CD4+ and CD8+ T cells from HSV-1-infected mice increased IL-13 and its functional receptor IL-13R{alpha}1 by treating anti-4-1BB mAb, but it was not clear whether the enhanced IL-13 by 4-1BB stimulation was a universal phenomenon. Therefore, we tested whether 4-1BB stimulation could enhance the IL-13 expression following VSV infection. BALB/c mice were i.v.-infected with VSV, treated with agonistic anti-4-1BB mAb or rat IgG on PI Days 0 and 2, and killed for analysis on PI Day 7. Similar with HSV-1 infection, anti-4-1BB treatment up-regulated IL-13 significantly in only CD4+ T cells in the protein and mRNA level, but IFN-{gamma} was up-regulated in CD8+ and CD4+ T cells (Fig. 5A 5B 5C ). Again, anti-4-1BB treatment increased the expression of IL-13R{alpha}1 and decreased IL-13R{alpha}2 on CD8+ and CD4+ T cells (Fig. 5D) .


Figure 5
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  		Figure 5. IL-13 and IFN-{gamma} production from T cells by 4-1BB stimulation in VSV-infected mice in vivo. BALB/c mice were infected i.v. with 1 x 106 PFU VSV. Rat IgG (200 µg) or anti-4-1BB was injected i.p. on PI Days 0 and 2. Mice were killed on PI Day 7, and CD4+ T and CD8+ T cells were purified from spleens of VSV-infected mice. Purified CD4+ T or CD8+ T cells were restimulated with irradiated and T cell-depleted splenocytes pulsed with UV-inactivated VSV (MOI, 10.0 before UV inactivation) for another 24 h. IL-13 and IFN-{gamma} proteins were determined in the culture supernatant of CD8+ T (A) and CD4+ T cells (B) by ELISA. The mRNA expression for cytokines (C) and cytokine receptors (D) was determined by performing RT-PCR with the freshly isolated, total RNA of CD4+ T and CD8+ T cells on PI Day 7.

 
These data suggest that in vivo 4-1BB stimulation is able to up-regulate the expression of IL-13 and its functional receptor, IL-13R{alpha}1, on CD4+ and CD8+ T cells of VSV-infected mice. We, therefore, concluded that 4-1BB stimulation was inducing the expression of IL-13 from T cells, particularly CD4+ T cells, along with IFN-{gamma}, and the increased IL-13 might act on the CD4+ and CD8+ T cells via the autocrine or paracrine manner, as CD4+ and CD8+ T cells induced its functional receptor IL-13R{alpha}1.

Anti-4-1BB treatment directly induces the production of IL-13 from T cells, which does not alter the expression of T-bet and GATA-3 in CD4+ and CD8+ T cells
The purified CD4+ or CD8+ T cells were stimulated with anti-CD3 for 16–32 h, washed to remove TCR signal, and restimulated with anti-4-1BB mAb for another 72 h. As shown in Figure 6A , 4-1BB triggering alone was sufficient to induce the expression of IL-13 and IFN-{gamma} from preactiveated CD4+ and CD8+ T cells, and expression level of IL-13 was comparable between washed and unwashed T cells in the presence of anti-4-1BB.


Figure 6
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  		Figure 6. Direct induction of IL-13 by 4-1BB stimulation and the effects of IL-13 on T-bet and GATA-3 in T cells. Purified, naïve CD4+ and CD8+ T cells were stimulated with 0.2 µ/ml anti-CD3. After the incubation for 16 h, some of cells were washed intensively with RPMI-1640 medium to remove anti-CD3 mAb and rested for 24 h. The cells were treated with 5 µg/ml anti-4-1BB mAb or rat IgG as a control (A) or pretreated with 100 and 1000 ng/ml blocking anti-IL-2 mAb (BD Biosciences) for 1 h before the anti-4-1BB treatment (B). IL-13 was determined from the culture supernatant 72 h after 4-1BB stimulation by ELISA. (C) CD8+ or CD4+ T cells were activated as described above, preincubated with 20 ng/ml rIL-13, and then stimulated with 5 µg/ml anti-4-1BB mAb or rat IgG as a control. Total RNA was extracted from the cells 48 h after anti-4-1BB treatment, and first-strand cDNA was synthesized with 0.5 µg total RNA. SYBR Green I-based, real-time quantitative PCR was carried out on a continuous fluorescence detection system (Opticon DNA engine, MJ Research Inc., Waltham, MA, USA). The relative gene expression was determined using the comparative threshold cycle method and normalized to the housekeeping gene GAPDH. To simplify the representation of T-bet and GATA-3 expression, the gene expression data normalized for GAPDH are shown as fold increase compared with levels in the rat IgG group and are means ± SD of triplicate for T-bet and sixplicate for GATA-3.

 
Anti-4-1BB treatment enhanced the expression of IFN-{gamma} and IL-13 via similar signal pathways, but their expression time was different (Fig. 2A 2B 2C 2D) . Therefore, it was possible that anti-4-1BB-mediated IL-13 expression might be a result of a series of action of cytokines, which were increased by anti-4-1BB treatment such as IL-2—the early consequence of anti-CD3 plus 4-1BB stimulation. Therefore, we neutralized IL-2 and determined the mRNA level of IL-13 and IFN-{gamma} 48 h after anti-4-1BB treatment. As shown in Figure 6B , IL-13 expression by anti-4-1BB treatment was not affected by IL-2 neutralization. Therefore, it seemed that anti-4-1BB-mediated IL-13 expression was not merely a result of the cascade actions of cytokines, which were enhanced by 4-1BB stimulation.

4-1BB signaling preferentially induces the Th1 response of T cells by increasing IFN-{gamma} [12 , 18 ], whereas IL-13 is a typical Th2 cytokine [21 , 25 , 26 ]. Thus, we examined whether the enhanced IL-13 expression altered the anti-4-1BB-mediated differentiation of CD4+ and CD8+ T cells into Th1 cells. T-bet and GATA-3 expression was tested in CD4+ and CD8+ T cells, following the treatment of anti-4-1BB mAb and/or IL-13. Activated CD4+ or CD8+ T cells stimulated with anti-4-1BB mAb in the presence/absence of rIL-13 for 48 h. T-bet and GATA-3 are responsible for the shift of CD4+ T cells into the Th1 or Th2 phenotype—T-bet for Th1 and GATA-3 for Th2 [27 , 28 ]. 4-1BB stimulation clearly polarized CD8+ and CD4+ T cells into Th1 cells by increasing T-bet and decreasing GATA-3 (Fig. 6C) . The treatment of rIL-13 alone decreased T-bet expression slightly in CD8+ T cells and increased GATA-3 expression in CD4+ and CD8+ T cells. The increased T-bet by anti-4-1BB treatment was reversed partially by adding IL-13 in only CD8+ T cells, and the decreased GATA-3 by anti-4-1BB treatment was not reversed significantly by adding IL-13 in CD4+ and CD8+ T cells (Fig. 6C) . Although there were slight differences in the effects of IL-13 on the expression of T-bet and GATA-3 between CD4+ T and CD8+ T cells, Th1 polarization of T cells by anti-4-1BB treatment was not altered significantly by adding IL-13.

Taken together, these data demonstrated that 4-1BB stimulation alone could up-regulate IL-13 directly from CD4+ and CD8+ T cells but did not alter the anti-4-1BB-mediated Th1 polarization of CD4+ or CD8+ T cells, even if IL-13 enhanced the proliferation of CD4+ or CD8+ T cells.

Anti-4-1BB treatment leads to liver injury in the absence of IL-13
To test the contribution of IL-13 directly on Th1/2 polarization of CD4+ and CD8+ T cells, we compared the production of Th1/2 cytokines from CD4+ and CD8+ T cells of wild-type and IL-13-deficient BALB/c mice following the polyclonal activation. CD4+ and CD8+ T cells from wild-type and IL-13-deficient BALB/c mice were stimulated with anti-CD3 and/or anti-4-1BB mAb, and Th1/2 cytokines were determined from the culture supernatants. 4-1BB stimulation preferentially enhanced the secretion of IL-2, IFN-{gamma}, and TNF-{alpha} from CD8+ T cells but not IL-4 and IL-5 (Fig. 7A ). In particular, IFN-{gamma} and TNF-{alpha} were enhanced massively in IL-13-deficient CD8+ T cells. However, consistent with a previous report [15 ], in wild-type CD4+ T cells, anti-4-1BB mAb produced only a marginal increase in IL-4, IL-5, IL-2, IFN-{gamma}, and TNF-{alpha} (Fig. 7A) . In IL-13-deficient CD4+ T cells, anti-4-1BB treatment produced a reduced level of IL-4 and IL-5 but no IL-2, IFN-{gamma}, and TNF-{alpha} (Fig. 7A) .


Figure 7
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  		Figure 7. Enhanced Th1 polarization of IL-13-deficient CD8+ T cells. (A) CD4+ or CD8+ T cells were purified from wild-type (WT) and IL-13-deficient BALB/c mice (KO) and stimulated with anti-CD3 and/or anti-4-1BB mAb. Culture supernatants were collected 48 h after the anti-4-1BB treatment, and Th1/2 cytokines were determined from the culture supernatant by using CBA (BD Biosciences), according to the manufacturer’s instruction. (B) Wild-type and IL-13-deficient mice were i.v.-infected with 5 x 105 PFU HSV-1 KOS and treated with 200 µg anti-4-1BB mAb or rat IgG as control on PI Days 0 and 2. Sections (6 µm) were prepared from each group of mice on PI Day 5, stained with H&E, and mounted in GVA mounting solution (Zymed).

 
Several studies have demonstrated that highly polarized, immune responses may become detrimental or even lethal to the host if they are too vigorous or become chronic [29 30 31 ]. As we found an enhanced Th1 response in IL-13-deficient T cells, we examined whether anti-4-1BB treatment induced an inflammation in IL-13-deficient mice. We infected mice with HSV-1 KOS i.v. and treated with anti-4-1BB mAb on PI Days 0 and 2. Sections were prepared from the liver of each group of mice on PI Day 5 and stained with H&E. As shown in Figure 7B , HSV-1 infection itself did not induce the inflammation in liver of wild-type and IL-13-deficient mice. Anti-4-1BB treatment induced a mild inflammation only around the blood vessel in wild-type but produced a severe inflammation and injury in the liver of IL-13-deficient mice. These results suggest that IL-13 plays a regulatory role in limiting an excess level of Th1 polarization in CD8+ T cells and in increasing the secretion of Th2 cytokines in CD4+ T cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown that the signaling through 4-1BB receptors on the activated CD4+ and CD8+ T cells promoted their expansion, survival, differentiation, and cytokine expression [7 , 9 10 11 12 13 14 15 ]. Although we reported previously that 4-1BB triggering increased the expression of IL-13 mRNA and protein from CD8+ T cells along with IFN-{gamma}, which caused their proliferation in a paracrine and autocrine manner [20 ], it was still not clear why 4-1BB stimulation increased IL-13 and IFN-{gamma}, which had a contradictory effect on the immune response, and what the effect was of IL-13 on the anti-4-1BB-mediated, immune response.

In the present study, we have elucidated the signaling pathway related to the anti-4-1BB-mediated IL-13 expression from CD4+ and CD8+ T cells and that the enhanced IL-13 expression might not alter the anti-4-1BB-mediated Th1 differentiation of CD4+ and CD8+ T cells. First, there were no significant differences in the signaling pathway, which was required for the expression of IFN-{gamma} and IL-13 by triggering 4-1BB. The treatment of PI-3K, mTOR, JNK, NF-{kappa}B, and NF-AT inhibitors blocked the anti-4-1BB-mediated IFN-{gamma} and IL-13 expression completely (Figs. 2 and 3) . However, the anti-4-1BB-mediated IFN-{gamma} expression was not affected significantly by treating the ERK1/2 inhibitor (PD98059; Fig. 2C ), which was consistent with the previous reports that JNK2 was involved in the IFN-{gamma} production and CD4+ Th1 differentiation and that the ERK signaling pathway was required for Th2 differentiation [32 33 34 35 ]. IL-13 production from CD4+ and CD8+ T cells required at least 48 h after the 4-1BB stimulation, although the antibody treatment evoked the similar signaling pathway to induce IFN-{gamma} and IL-13 (Figs. 2 and 3) . Possible explanations of the delayed IL-13 production are likely to be the requirement of additional transcriptional factors or full activation of CD4+ and CD8+ T cells.

IL-13 cytokine has been shown to have many different activities on numerous cell types including T, B, and endothelial cells, as well as in monocytic, basophilic, and mast cell lines [36 ]. Several studies have demonstrated that IL-13 increases IgE production from human B cells in vitro [37 ], and administration of rIL-13 in in vivo or transgenic overexpression of IL-13 affords protection to LPS-induced endotoxemia [38 , 39 ]. However, the 4-1BB stimulation in vivo inhibits the allergic asthma by decreasing IgE production [40 ], suppresses B cell response [41 , 42 ], and exacerbates the LPS-induced endotoxemia [19 ]. Therefore, it seems that the increased IL-13 by 4-1BB stimulation has a limited role on the immune response as a result of the overwhelmed coexpression of IFN-{gamma}.

Several studies have demonstrated that highly polarized immune responses may become detrimental or even lethal to the host if they are too vigorous or become chronic, regardless of whether Type 1 or 2 cytokines dominate the response [29 30 31 ]. Indeed, anti-4-1BB treatment strongly enhances Th1 polarization of T cells in vitro and eventually resulted in a liver inflammation in vivo in the absence of IL-13 (Fig. 7A and 7B) . Anti-4-1BB treatment not only enhanced the IFN-{gamma} and TNF-{alpha} production from CD8+ T cells but also reduced the IL-4 and IL-5 expression from CD4+ T cells in the absence of IL-13 (Fig. 7A) . Therefore, we concluded that IL-13 has a protective role to prevent the unnecessary inflammation caused by an excessively polarized Th1 immune response and seems to compose one of feedback or compensatory mechanisms to maintain the Th1/2 balance of immune response. As such, our findings illustrate a novel and previously unappreciated mechanism for 4-1BB-mediated immune regulation.


   ACKNOWLEDGEMENTS
 
This study was supported in part by National Institutes of Health grant R01EY013325 (B. S. K.), KRF-2005-201-E00008, KRF-2005-084-E00001, Korean Health R&D21 #A050260, and the Science Research Center Fund to the Immunomodulation Research Center at the University of Ulsan from KOSEF and the Korean Ministry of Science and Technology.

Received October 8, 2006; revised January 25, 2007; accepted February 27, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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