A co-stimulatory molecule on activated T cells, H4/ICOS, delivers specific signals in Th cells and regulates their responses
Yutaka Arimura1,
Hidehito Kato1,
Umberto Dianzani4,
Toshihiro Okamoto2,
Soichiro Kamekura1,
Donatella Buonfiglio4,
Tohru Miyoshi-Akiyama1,
Takehiko Uchiyama1,3 and
Junji Yagi1
Departments of 1 Microbiology and Immunology, and 2 Oral and Maxillofacial Surgery, and 3 Institute of Laboratory Animals, Tokyo Womens Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan 4 Laboratory of Immunology, Department of Medical Science, A. Avogadro University of Eastern Piedmont, 28100 Novara, Italy
Correspondence to: J. Yagi; E-mail: jyagi1{at}research.twmu.ac.jp
Transmitting editor: S. Koyasu
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Abstract
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We examined the co-stimulatory activity of H4/ICOS on murine activated CD4+ T cells and found that the cross-linking of H4/ICOS enhanced their proliferation, in addition to raising IFN-
, IL-4 and IL-10 production to levels comparable to those induced by CD28. However, IL-2 production was only marginally co-stimulated by H4/ICOS. This distinct pattern of lymphokine production appears to be induced by a specific intracellular signaling event. Compared with CD28, H4/ICOS dominantly elicited the Akt pathway via phosphatidylinositol 3-kinase. In addition, mitogen-activated protein kinase family kinases were activated in different ways by CD28 and H4/ICOS. The strong phosphorylation of p46 c-Jun N-terminal kinase was observed upon CD28 co-stimulation, but was less potently induced by H4/ICOS. The strain diversity in the induction of H4/ICOS was recognized. The expression of H4/ICOS on BALB/c activated CD4+ T cells was >6-fold higher compared with C57BL/6 activated CD4+ T cells. Furthermore, BALB/c activated CD4+ T cells exhibited more Th2-deviated lymphokine production as compared with C57BL/6 activated CD4+ T cells and signaling through H4/ICOS during the primary stimulation of naive CD4+ T cells promoted the generation of Th2 cells. Thus, the difference in H4/ICOS expression on activated CD4+ T cells, which is regulated among the mouse strains, may also regulate the polarization of Th cells.
Keywords: immunoregulation, inducible co-stimulator, signal transduction, Th2 cells
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Introduction
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Activation of CD4+ Th cells is achieved by at least two signals (14). One signal, determining the specificity of the immune response, is delivered through the interaction of TCR with the antigenic peptideMHC complex on antigen-presenting cells (APC). A second, co-stimulatory signal is provided through engagement of CD28 on the T cells by its ligands, B7-1 and B7-2, on APC, and promotes the proliferation and effector function of T cells. A structurally related molecule of CD28, CTLA-4, appears to deliver negative signal in activated T cells, thereby negatively regulating the immune responses (57). Recently, a third CD28-related molecule has been identified. The molecule is induced during the activation of T cells, is thus referred to as an inducible co-stimulator (ICOS), and enhances T cell proliferation and lymphokine production (8,9). The ligand of ICOS, B7H/B7RP-1, was identified as a close homolog of B7-1/2 (911). It has been shown that the ICOS/B7H pathway controls T cell-dependent immune responses (1214). The contribution of ICOS to the development of both Th1 and Th2 cells (12) or the predominant expression of ICOS on Th2 cells and its possible effect on the development of Th2 cells have been reported (13,14). Consistent with these findings, ICOS-deficient mice revealed a remarkable feature of immunodeficiency in which the production of lymphokines such as IL-4 and IL-13, and antibody class switching were greatly impaired (1517). ICOS co-stimulation appears to be under the stringent control. It synergizes with IL-2 and can be prevented by CTLA-4 ligation (18).
In 1996, two of the co-authors (U. D. and D. B.) and colleagues reported a T cell activation marker, H4, which co-stimulates murine T cells and physically associates with TCR (19). Subsequent reports in humans indicated that H4 is expressed on activated and germinal center T cells, and on a large proportion of peripheral blood T cells in acute viral infections (20). These results together with the similar molecular structure (8,19) of the two molecules suggested a resemblance between H4 and ICOS, and it was indeed shown recently that these molecules are identical (21). On the other hand, we found that TCR
ß+ CD4 single-positive (SP) thymic T cells in mice are divided into three distinct subpopulations based upon their H4 levels (22). Thymic T cells with none to a low amount of H4 branch off the mainstream differentiation pathway. Thymic T cells with an intermediate amount of H4 are ordinary NKT cells, whereas those with a high amount of H4 constitute a new type of invariant V
14+ T cell, whose phenotype is totally different from ordinary NKT cells (22). Strain diversity was recognized in the frequency of a new type of invariant V
14+ T cell in TCR
ß+ CD4 SP and CD4CD8 double-negative (DN) thymocytes: 0.7 and 3.0% in C57BL/6 and BALB/c mice respectively (22). The question arises as to whether the expression of H4/ICOS is regulated in specific ways in different strains of mice.
Although accumulating evidence has indicated the important role of H4/ICOS in T cell activation and function, H4/ICOS-dependent signaling pathways remain to be elucidated. Only Coyle et al. have recently reported that ICOS associates with phosphatidylinositol 3-kinase (PI3-K) and shows lipid kinase activity, suggesting the sharing of a common signaling pathway between CD28 and H4/ICOS (13). However, as inferred by the fact that the asparagine of the pYMNM motif in CD28, which is necessary for the binding of Grb2 (23), is not conserved in H4/ICOS (8,9), H4/ICOS may engage multiple signaling pathways in T cells in different ways. In the present study, in an attempt to clarify this possibility, we compared the extent of activation of key signaling proteins between CD28 and H4/ICOS engagement. In addition, strain diversity in the induction of H4/ICOS was examined between C57BL/6 and BALB/c activated CD4+ T cells. Our results indicate that different signaling is delivered upon cross-linking of H4/ICOS molecules as compared with CD28, resulting in specific production of lymphokines for each of the molecular types. The amount of H4/ICOS induced on BALB/c activated CD4+ T cells was >6-fold higher than on C57BL/6 activated CD4+ T cells. Furthermore, BALB/c activated CD4+ T cells exhibited Th2-deviated lymphokine production, and H4/ICOS-dependent signaling promoted the generation of Th2 cells. Thus, it is most likely that the expression of H4/ICOS, regulated differently depending on the murine strain, is an important determinant of the development of Th1 or Th2 cells.
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Methods
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Animals
BALB/c and C57BL/6 mice were bred in our own colony at the Department of Microbiology and Immunology, Tokyo Womens Medical University. Female mice (810 weeks old) were used in this study.
Superantigen (SAg) and antibodies
Yersinia pseudotuberculosis-derived mitogen (YPM) was purified from an extract of Escherichia coli XL1-Blue carrying pQE30-6xH·ypm (24) using Ni-NTAAgarose (Qiagen Chatsworth, CA) followed by Sepharose Fast Flow (Pharmacia LKB, Tokyo, Japan). The YPM SAg stimulates murine Vß7- and Vß8-bearing T cells (25). The C398.4A mAb specific for H4/ICOS was produced in Armenian hamsters by immunization with the murine T cell clone D10.G4.1 as described previously (19). mAb to I-Ab,d (28-16-8S), CD3 (145-2C11), CD8 (83.12.5) and Thy1.2 (HO13) were described previously (26,27). mAb to CD28 (37.51), CD44 (IM7), NK1.1 (PK136) and TCR
(GL3), FITCanti-CD4 (RM4-5), PerCPanti-CD4 (RM4-5), biotinylated anti-CD28 (37.51) and anti-CD44 (IM7), and phycoerythrin (PE)anti-CD44 (IM7) were purchased from PharMingen (San Diego, CA). mAb to CD28 (37.51) was also kindly provided by Dr J. Allison (University of California at Berkeley, Berkeley, CA). Goat anti-hamster IgG (anti-HIgG) was obtained from ICN (Aurora, OH). PEstreptavidin was obtained from Becton Dickinson (Mountain View, CA). The following rabbit polyclonal antibodies and reagents were used for the analysis of intracellular signaling: anti-Akt and anti-phospho-Akt (Cell Signaling Technology, Beverly, MA), anti-phospho-extracellular signal regulated kinase (anti-phospho-ERK) (New England Biolabs, Beverly, MA), anti-phospho-c-Jun N-terminal kinase (anti-phospho-JNK) and anti-phospho-p38 (Promega, Madison, WI), anti-ERK, anti-JNK and anti-p38 (Santa Cruz Biotechnology, Santa Cruz, CA), and PI3-K inhibitors, LY294002 and Wortmannin (Sigma, St Louis, MO). Anti-CD3 mAb was used as dialyzed ammonium sulfate precipitates of ascitic fluid. HIgG and anti-H4/ICOS mAb were purified from serum by Protein ASepharose (Pharmacia Fine Chemicals, Uppsala, Sweden) and from culture supernatant by Protein GSepharose (Pharmacia Fine Chemicals) respectively. Purified HIgG and anti-H4/ICOS mAb were conjugated with biotin in our laboratory.
Culture medium
RPMI 1640 supplemented with 100 µg streptomycin/ml, 100 U penicillin/ml, 10% FBS and 5 x 105 M 2-mercaptoethanol was used for the culture.
Preparation of cells
Single spleen cell suspensions were prepared in HBBS (Nissui Pharmaceutical, Tokyo, Japan) with 2% FCS. CD4+ T cells were obtained by two rounds of treatment of spleen cells with 28-16-8S and 83.12.5, and guinea pig complement. Naive CD4+ TCR
ß+ T cells were then enriched by treatment with a cocktail of mAb IM7, PK136 and GL3, and by depletion of mAb-coated cells using sheep anti-rat and sheep anti-mouse IgG-bound magnetic beads (Dynabeads; Dynal, Great Neck, NY) followed by taking non-adherent cells from goat anti-HIgG (30 µg/ml)-coated dishes (25020; Corning, Corning, NY). PK136 and anti-mouse IgG beads were omitted in the treatment of BALB/c spleen cells. The cells contained <2% CD44highCD4+ T cells. T-depleted spleen cells were obtained by treatment of spleen cells with HO13 and complement followed by inactivation with mitomycin C (Kyowa Hakko Kogyo, Tokyo, Japan), and used as APC.
To obtain CD4+ T cell blasts, an indicated number of naive CD4+ T cells was cultured with 2 µg/ml of anti-CD3 mAb or 3 µg/ml of YPM in the presence of indicated numbers of syngenic APC in a 24-well culture plate (3047 Falcon; Becton Dickinson Labware, Franklin Lakes, NJ) or a 48-well culture plate (3078 Falcon; Becton Dickinson Labware). After 2 days of culture for the stimulation with anti-CD3 mAb or 3 days of culture for the stimulation with YPM, blasts were collected by applying the cell suspension to a Percoll gradient (density 1.075) and expanded with 100 U/ml of human rIL-2 (a gift from Shionogi, Osaka, Japan) for an additional 2 days. The blasts finally obtained were >95% CD4+, <1.0% NK1.1+ and <0.2% TCR
+.
Assay for the co-stimulatory function of H4/ICOS
For the primary proliferative response, 2 x 105 naive CD4+ T cells were cultured for 2.5 days in a 96-well flat-bottomed culture plate (3072 Falcon; Becton Dickinson Labware) coated with antibodies as follows. The plate was first coated with anti-HIgG (10 µg/ml) at 37°C for 2 h. After being intensively washed, it was serially bound with titrated amounts of anti-CD3 mAb at 37°C for 1 h and, after being washed, with a fixed amount (3 µg/ml) of anti-H4/ICOS mAb, anti-CD28 mAb or control HIgG at 37°C for 2 h. Cells were pulsed with 0.5 µCi of [3H]thymidine for the last 16 h and the amount of [3H]thymidine incorporated was measured. Results were expressed as mean c.p.m ± SD of triplicate cultures. For the secondary proliferative response, 1 x 105 CD4+ T cell blasts were stimulated and their responses were assessed as described above. To assay lymphokine production, supernatants were collected at indicated times from the secondary culture of 2.5 x 105 CD4+ T cell blasts in a 48-well culture plate coated with antibodies as described above.
Determination of lymphokine concentrations
The concentration of IL-2 in culture supernatants was determined in a bioassay as the proliferation of IL-2-dependent CTLL-2 cells as described previously (28). IL-4, IL-5, IL-10 and IFN-
in culture supernatants were quantitated by sandwich ELISA according to the manufacturers instructions (PharMingen). Results were expressed as mean concentration ± SD of triplicate cultures.
Assay for the intracellular signaling through H4/ICOS
For analysis of the intracellular signaling, 4 x 106 anti-CD3-induced CD4+ T cell blasts were reacted with 1 µg/ml of anti-CD3 mAb and/or various concentrations of anti-CD28 or anti-H4/ICOS mAb for 15 min in a microcentrifuge tube on ice. These cells were then spun down, resuspended with 200 µl of cold RPMI medium, cross-linked with anti-HIgG at a final concentration of 20 µg/ml and stimulated by incubation at 37°C for the indicated times. The reactions were stopped by the addition of cold PBS with 1 mM EDTA and 1 mM Na3VO4. After centrifugation, the cells were lysed by incubation for 20 min on ice with TNE lysis buffer (10 mM Tris, pH 7.5, 0.15 M NaCl, 2 mM EDTA and 10% glycerol) containing protease inhibitors. The lysates were cleared by centrifugation to remove insoluble materials, boiled for 3 min in sample buffer and stored at 80°C until needed. The lysates from stimulated cells were applied to SDSPAGE, transferred to a nitrocellulose membrane and blocked with 13% skim milk in TBST (10 mM Tris pH 7.5, 0.15 M NaCl and 0.1% Tween) for 1 h to reduce the background. The membranes were incubated overnight with specific antibodies of appropriate concentration in the TBST containing 1% BSA. After washing 4 times with TBST, they were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h and developed to film using an ECL substrate (Santa Cruz Biotechnology) according to the manufacturers information.
Flow cytometry analysis
The entire procedure was carried out on ice. To detect H4/ICOS expression on naive CD4+ T cells, spleen cells were treated with 83.12.5 and guinea pig complement, and cells were stained by incubation with a combination of HIgG or anti-H4/ICOS mAb and FITCanti-HIgG, and were incubated with an excess amount of normal rat IgG to block unbound binding sites for rat IgG on FITCanti-HIgG. After washing, FITCstained cells were incubated with a combination of PEanti-CD44 and PerCPanti-CD4 mAb. Samples were analyzed for H4/ICOS and CD44 expression on 20,000 gated CD4+ T cells by an Epics XL flow cytometer (Coulter Immunology, Hialeah, FL), using System II software. The percentage of H4/ICOS+ cells was calculated by subtracting background staining with HIgG. To detect expression of surface molecules on CD4+ T cell blasts, cells were stained by incubation with biotinylated anti-CD28, anti-H4/ICOS or anti-CD44 mAb followed by a cocktail of FITCanti-CD4 mAb and PEstreptavidin. Samples of 10,000 viable cells were analyzed as above. Expression of CD28, H4/ICOS and CD44 on CD4+ T cell blasts was calculated by the subtraction of mean log fluorescence intensity with background biotinylated HIgG staining in the primary antibodies from mean log fluorescence with corresponding biotinylated mAb.
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Results
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Co-stimulatory activity of H4/ICOS in CD4+ T cell proliferation
We first examined the co-stimulatory activity of H4/ICOS in naive CD4+ T cell proliferation. It has been found that H4/ICOS expression differs depending on the strain of the mouse. As reported previously, the percentage of H4/ICOShigh thymic T cells in TCR
ß+ CD4 SP + DN thymocytes is quite different between C57BL/6 and BALB/c mice, 0.7 versus 3.0% respectively (22). Because these results suggest that H4/ICOS expression may be differently regulated in different mouse strains, we used both C57BL/6 and BALB/c mice in the present study. Naive CD4+ T cells were stimulated in wells coated with titrated amounts of anti-CD3 mAb and a fixed amount of anti-CD28, anti-H4/ICOS or control HIgG. As shown in Fig. 1(Aa and Ba), signaling through CD28 markedly co-stimulated C57BL/6 and BALB/c naive CD4+ T cells to proliferate under suboptimal doses of anti-CD3 mAb. In contrast, signaling through H4/ICOS promoted only a weak proliferation of both naive CD4+ T cells.

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Fig. 1. Co-stimulatory effect of H4/ICOS in naive and activated CD4+ T cell proliferation. Naive CD4+ T cells (2 x 105) obtained from C57BL/6 (A, a) or BALB/c (B, a) mice were stimulated in a 96-well plate coated with titrated amounts of anti-CD3 mAb and a fixed amount of HIgG, anti-H4/ICOS or anti-CD28 mAb (3 µg/ml). The plate was precoated with anti-HIgG antibodies (10 µg/ml). After 2.5 days of culture, uptake of [3H]thymidine was examined. Naive CD4+ T cells (1.5 x 106) T cells from C57BL/6 or BALB/c mice were stimulated with anti-CD3 mAb (2 µg/ml) in the presence of 1.5 x 106 APC in a 24-well plate. After 2 days of culture, blasts were collected and expanded with 100 U/ml of rIL-2 for 2 more days. For secondary proliferative responses, 1 x 105 CD4+ T cell blasts from C57BL/6 (A, b) or BALB/c (B, b) mice were examined as in primary cultures. Results are expressed as mean ± SD of triplicate cultures. Data shown are typical results from one of three experiments.
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We next prepared CD4+ T cell blasts, nearly all of which express H4/ICOS (see the profiles in Fig. 5B), by stimulating naive CD4+ T cells with soluble anti-CD3 mAb and syngenic APC, and examined them for co-stimulatory activity of CD28 and H4/ICOS. The same experimental conditions gave rise to an equal enhancement of the proliferative response of both CD4+ T cell blasts by anti-CD28 mAb under a slightly lower dose of anti-CD3 mAb (>0.08 µg/ml) than that used in the primary stimulation (>0.19 µg/ml) (Fig. 1Ab and Bb). As compared to CD28, the ability of H4/ICOS in co-stimulation was almost equivalent in both strains or a little weak, especially in C57BL/6 CD4+ T cell blasts (Fig. 1Ab and Bb). Since the results suggest that H4/ICOS has a particular role in activated T cells, in the present study we used the CD4+ T cell blasts to further analyze the function of H4/ICOS.
Co-stimulatory activity of H4/ICOS in lymphokine secretion
In a previous report, ICOS co-stimulated human CD4+ T cells to markedly secrete IL-10, but not IL-2 (8). Therefore, the ability of H4/ICOS to co-stimulate CD4+ T cell blasts to secrete various lymphokines was next examined. The concentration of lymphokines in culture supernatants was assayed at times when a peak concentration is observed; 24 h for IL-2 and IL-4, and 72 h for IFN-
and IL-10 after the simulation of CD4+ T cell blasts as described above. In agreement with previous reports, H4/ICOS co-stimulated both C57BL/6 and BALB/c CD4+ T cell blasts to secrete IL-2 very weakly, compared with the high level of IL-2 secretion by CD28 co-stimulation (Fig. 2Ab and Bb). The levels of IFN-
, IL-4 and IL-10 secretion from both CD4+ T cell blasts co-stimulated by H4/ICOS were markedly increased relative to control HIgG under suboptimal doses of anti-CD3 mAb and were comparable to or slightly lower than those co-stimulated by CD28 (Fig. 2Aa, c, d and Ba, c, d). Thus, signaling through H4/ICOS can provide a co-stimulatory effect in various lymphokines, IFN-
, IL-4 and IL-10 production from activated CD4+ T cells. In addition, it is noteworthy that these experimental conditions allow C57BL/6 and BALB/c CD4+ T cell blasts to exhibit a distinct pattern of lymphokine production. IFN-
and IL-2 production from C57BL/6 CD4+ T cell blasts was higher than BALB/c CD4+ T cell blasts. Conversely, IL-4 was produced in much greater quantities by BALB/c CD4+ T cell blasts than C57BL/6 CD4+ T cell blasts. Given a distinct pattern of lymphokine production by H4/ICOS co-stimulation, it is conceivable that differential signaling is delivered by CD28 and H4/ICOS. The next series of experiments was performed in order to clarify this possibility.

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Fig. 2. Co-stimulatory effect of H4/ICOS in lymphokine secretion from activated CD4+ T cells. C57BL/6 (A) or BALB/c (B) CD4+ T cell blasts (2.5 x 105) obtained as in Fig. 1 were stimulated in a 48-well plate coated as in Fig. 1. Culture supernatants were collected at 24 and 72 h of the secondary cultures for the assay of IL-2/IL-4 and IFN- /IL-10 respectively. Concentrations of IL-2 were determined in a bioassay using CTLL-2 cells and those of other lymphokines were quantitated by sandwich ELISA. Results are expressed as mean ± SD of triplicate cultures. Data shown are typical results from one of three experiments.
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H4/ICOS dominantly elicits the Akt pathway via PI3-K
The activated CD4+ T cell blasts express both CD28 and H4/ICOS which have the PI3-K binding motif in their cytoplasmic domain, and the recruitment of PI3-K to these receptors has in fact been demonstrated (13). To further elucidate the intracellular events in activated CD4+ T cell blasts following H4/ICOS co-stimulation, we examined the activation of serine/threonine protein kinase Akt, one of the downstream molecules for PI3-K. As shown in Fig. 3, H4/ICOS cross-linking indeed elicited the phosphorylation at Ser473 of Akt, which indicates the up-regulation of kinase activity (29). Furthermore, H4/ICOS induced phosphorylation much more strongly than CD28 at all time points examined (Fig. 3A) and even at maximal antibody dose (Fig. 3B). The activation of Akt induced by CD28 co-stimulation was stronger than that induced by anti-CD3 alone, indicating the co-stimulatory effect of CD28. To confirm that upon H4/ICOS co-stimulation Akt is activated downstream of PI3-K, we used PI3-K-specific inhibitors, Wortmannin and LY294002. These inhibitors indeed blocked the Akt phosphorylation upon H4/ICOS co-stimulation in a dose-dependent manner (Fig. 3C). These results suggest that the Akt activation via H4/ICOS is under the control of PI3-K activity.

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Fig. 3. H4/ICOS co-stimulation dominantly elicits the activation of Akt via PI3-K. (A) C57BL/6 anti-CD3-induced CD4+ T cell blasts were incubated with 1 µg/ml of anti-CD3 mAb and 10 µg/ml of anti-CD28 mAb or anti-H4/ICOS mAb for 15 min on ice and spun down, and then cross-linked with 20 µg/ml of goat anti-HIgG antibodies at 37°C for the times indicated. The cell lysates were subjected to SDSPAGE followed by blotting with anti-phospho-serine at 473 of Akt and anti-Akt antibodies. (B) The CD4+ T cell blasts were stimulated with various concentrations of antibodies indicated above for 2 min at 37°C. (C) The cells were stimulated as in (A) in the presence of PI3-K inhibitors, LY294002 and Wortmannin, and DMSO (mock). These figures are representative of at least four experiments with similar results.
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Differential activation of mitogen-activated protein kinase (MAPK) family kinases by CD28 and H4/ICOS
As the downstream signaling molecules, we next tried to examine the MAPK family members. No difference in activation of ERK and p38 MAPK was observed between CD28 and H4/ICOS co-stimulation in repeated experiments, although the variation in experiments was recognized as shown in Fig. 4(A and B). However, p46 JNK activation was markedly different. The phosphorylation of p46 JNK was observed at 520 min upon CD28 co-stimulation, whereas H4/ICOS consistently induced much weaker p46 JNK phosphorylation than CD28 (Fig. 4A), even at the maximal antibody concentration (Fig. 4B). p54 JNK was constitutively phosphorylated in the CD4+ T cell blasts and in our experiments changes in phosphorylation level following stimulation could not be detected. Taken collectively, CD28 and H4/ICOS provide qualitatively different signals as well as overlapping ones.


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Fig. 4. MAPK family kinases are differentially activated by CD28 and H4/ICOS. (A) The total lysates from the cells stimulated as in Fig. 3 were subjected to SDSPAGE followed by blotting with anti-phospho-MAPK family members, ERK, JNK and p38. (B) The dose responses were assessed at 2 min for ERK and p38, and at 10 min for JNK, after the stimulation. These figures are representative of at least five experiments with similar results.
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Higher expression of H4/ICOS and stronger activation of Akt in BALB/c activated CD4+ T cells than C57BL/6 activated CD4+ T cells
As mentioned earlier in this section, H4/ICOS expression may be regulated differently depending on the mouse strain. If the difference of H4/ICOS expression is genetically controlled, the expression of H4/ICOS on naive CD4+ T cells might be different among the mouse strains. To examine this possibility, H4/ICOS expression on CD4+ T cells obtained from BALB/c and C57BL/6 mice was examined in relation to their CD44 expression (Fig. 5A). The expression of CD44 is low on naive T cells, and high on activated and memory T cells (30). As shown in Fig. 5,
7% of splenic CD4+ T cells express H4/ICOS in C57BL/6 mice, whereas splenic H4/ICOS+ CD4+ T cells are seen at much higher levels (
15%) in BALB/c mice. The majority of H4+ CD4+ T cells in both strains of mouse were found in CD44high cells. The percentage of H4+ CD4+ T cells in CD44lowCD4+ T cells was very small, and at similar levels in BALB/c and C57BL/6 mice, 0.8 and 0.5% respectively, thus ruling out the above possibility.
Next, to clarify whether the induction of H4/ICOS expression is different among the mouse strain, we compared H4/ICOS expression on activated CD4+ T cells between C57BL/6 and BALB/c mice. CD4+ T cell blasts were obtained from C57BL/6 and BALB/c naive CD4+ T cells by stimulating them with soluble anti-CD3 mAb or SAg, YPM in the presence of syngenic APC. As shown in Fig. 5(B and C), the levels of H4/ICOS expression on anti-CD3-induced and YPM-induced BALB/c CD4+ T cell blasts were 6.2- and 7.3-fold higher than C57BL/6 CD4+ T cell blasts respectively. In contrast, CD28 expression on anti-CD3-induced and YPM-induced BALB/c CD4+ T cell blasts was only 1.3- and 1.2-fold higher respectively as compared to C57BL/6 CD4+ T cell blasts (Fig. 5C). Since differences in the number of blast cell recovery and cell size of blast cells (data not shown) and in CD44 expression (Fig. 5C), were not recognized between the corresponding two blasts induced by anti-CD3 mAb or YPM, it is unlikely that lower H4/ICOS expression on C57BL/6 CD4+ T cell blasts is due to insufficiency of cell activation. Thus, H4/ICOS is predominantly induced on BALB/c CD4+ T cells over C57BL/6 CD4+ T cells after being stimulated through TCRCD3 molecules.
Next, experiments were performed to assess the difference of intracellular events in BALB/c and C57BL/6 CD4+ T cell blasts following H4/ICOS co-stimulation. As shown in Fig. 5(D), H4/ICOS cross-linking induced much stronger phosphorylation of Akt in BALB/c CD4+ T cell blasts than that in C57BL/6 CD4+ T cell blasts, although the basal level of, and CD28 co-stimulation-induced phosphorylation of Akt were also stronger in BALB/c cells than in C57BL/6 cells. The results indicate that the downstream Akt pathway for PI3-K is highly activated following H4/ICOS co-stimulation in BALB/c CD4+ T cell blasts as compared to C57BL/6 CD4+ T cell blasts.
Signaling through H4/ICOS during Th cell differentiation promotes the generation of Th2 cells
Recent reports using cloned CD4+ T cell lines or CD4+ T cells from TCR-transgenic mice, but lacking the evidence for normal CD4+ T cells have indicated that the expression of ICOS in Th2 cells is higher than in Th1 cells, and has a preferential role in the development and function of Th2 cells (13,14). Therefore, the pattern of lymphokine production of the activated CD4+ T cells was examined by stimulating them with immobilized anti-CD3 and anti-CD28 mAb. To assess the skewness of CD4+ T cell differentiation, the concentration of lymphokines was uniformly analyzed at 24 h of secondary culture. As shown in Fig. 6(Aa), anti-CD3-induced BALB/c CD4+ T cell blasts revealed more Th2-deviated lymphokine production, as compared with anti-CD3-induced C57BL/6 CD4+ T cell blasts. Accordingly, BALB/c CD4+ T cell blasts expressed Th2-associated GATA-3 (31) at much higher levels than did C57BL/6 CD4+ T cell blasts (data not shown). Even stronger deviation toward Th2 cells in BALB/c CD4+ T cell blasts was observed by a different primary stimulation using YPM and APC (Fig. 6Ab). Taken collectively, the results suggest the high level of H4/ICOS expression on activated Th2 type CD4+ T cells and a possible effect of H4/ICOS signaling on Th2 differentiation in normal CD4+ T cells.

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Fig. 6. BALB/c activated CD4+ T cells are deviated more into Th2 cells than C57BL/6 activated CD4+ T cells and H4/ICOS signaling promotes the generation of Th2 cells. (A) Anti-CD3-induced (a) and YPM-induced (b) CD4+ T cell blasts from C57BL/6 and BALB/c mice were obtained as in Fig. 5, and were stimulated in a 48-well plate serially coated with anti-CD3 mAb (0.1 µg/ml) and anti-CD28 mAb (3 µg/ml). The plate was precoated with anti-HIgG antibodies (10 µg/ml). Culture supernatants were collected at 24 h of the secondary culture, and concentrations of IFN- , IL-2 and IL-4 were determined as in Fig. 2, and that of IL-5 was quantitated by sandwich ELISA. Data shown are typical results from one of three experiments. (B) CD4+ T cell blasts from BALB/c mice were obtained by the stimulation of 5 x 105 naive CD4+ T cells in a 48-well plate serially coated with anti-CD3 mAb (1 µg/ml) for 1 h, and a mixture of a fixed amount of anti-CD28 mAb (3 µg/ml) and HIgG or anti-H4/ICOS mAb (0.3 µg/ml) for 2 h. The plate was precoated with anti-HIgG antibodies (10 µg/ml). CD4+ T cell blasts were then examined for the pattern of lymphokine production as in (A). Data shown are typical results from one of three experiments.
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To examine the role of H4/ICOS on Th cell differentiation more directly, signaling through H4/ICOS by cross-linking with anti-H4/ICOS mAb was added to the stimulation of naive CD4+ T cells with anti-CD3 and anti-CD28 mAb. Optimal concentrations of anti-CD3, anti-H4/ICOS and anti-CD28 mAb were determined according to the results from preliminary experiments. The pattern of lymphokine production from CD4+ T cell blasts thus obtained was analyzed after the stimulation with a fixed amount of anti-CD3 and anti-CD28 mAb (Fig. 6B). H4/ICOS cross-linking during primary stimulation gave rise to
2-fold increase in IL-4 and IL-5 production, and
2-fold decrease in IFN-
and IL-2 production from CD4+ T cell blasts, compared with the control antibodies. Thus, the results indicate that signaling through H4/ICOS during Th cell differentiation preferentially induces differentiation into Th2 cells.
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Discussion
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In the present study, when comparing the primary and secondary stimulation of splenic CD4+ T cells, we observed that H4/ICOS functions as a co-stimulator comparable to CD28 in the proliferation of activated CD4+ T cells. The results further indicated that cross-linking of H4/ICOS molecules elicited multiple effects including co-stimulation of the production of various lymphokines in activated CD4+ T cells and skewing Th cell differentiation into Th2 cells.
Signaling through H4/ICOS in activated CD4+ T cells in the presence of suboptimal cross-linking of CD3 remarkably promoted the production of IL-4, IL-10 and IFN-
, which was comparable to the levels induced by CD28 (Fig. 2). This result supports a recent report indicating that ICOS is an important co-stimulator in both Th1 and Th2 responses (12). However, in agreement with other reports (8,9), IL-2 production was only marginally co-stimulated by the cross-linking of H4/ICOS in contrast to high co-stimulatory activity of CD28 (Fig. 2). Analysis for intracellular signaling revealed that differential signaling is induced by H4/ICOS co-stimulation, thereby leading to a distinct pattern of lymphokine production. The cross-linking of H4/ICOS induced much less phosphorylation of p46 JNK than did the cross-linking of CD28, whereas there was no difference in the phosphorylation of p38 and ERK by the signaling through H4/ICOS and CD28 (Fig. 4). Whereas TCR-mediated signaling alone can induce the activation of ERK, co-stimulation through CD28 in addition to TCR-mediated signaling is required for JNK activation, which leads to the activation of AP-1 and the transcription of IL-2 (32). Therefore, the much weaker phosphorylation of JNK induced by H4/ICOS, as compared to that by CD28, may be ascribed to a marginal co-stimulatory effect of H4/ICOS in IL-2 production. However, a recent report by Dong et al. has shown that JNK is not necessarily required for activation and IL-2 production in naive CD4+ T cells (33). The mode of stimulation and the status of CD4+ T cells are different between the above report and ours, and IL-2 production of activated CD4+ T cells may be more dependent on JNK than is the case in naive CD4+ T cells.
Alternatively, another signaling pathway could be a key to the low IL-2 production induced by H4/ICOS. As reported previously, PI3-K has a role in the co-stimulation of IL-2 production by CD28 (34). Different signaling via PI3-K could result in the low production of IL-2. In support of this possibility, a previous and a recent report indicated the negative regulation of PI3-K in TCR-dependent activation of NF-AT responses (35) and in CD28-mediated IL-2 promotor activity (36) respectively. The asparagine of the pYMNM motif in CD28 which is necessary for the binding of the Grb2 SH2 domain is not conserved in H4/ICOS (8,9). This suggests that multiple molecules including Grb2 associated with CD28 associate in a different way with H4/ICOS and specific signaling molecules may bind to H4/ICOS, leading to a distinct activation status of PI3-K and its downstream targets. In support of this possibility, signaling through H4/ICOS in the present study implicates the much stronger phosphorylation of Akt, one of the downstream molecules for PI3-K, as compared to signaling through CD28 (Fig. 3).
Kane et al. have recently shown that Akt can provide a co-stimulatory signal that is indistinguishable from the CD28 signal for up-regulation of IL-2 and IFN-
, but not IL-4 and IL-5 (37). This suggests that the strong phosphorylation of Akt, as induced by H4/ICOS co-stimulation, may lead to high, but not low, production of IL-2. The activation of T cells triggered by TCR and CD28 is complicatedly regulated. It has been shown that protein kinase C (PKC)
is required in a Vav signaling pathway that mediates the TCRCD28-induced activation of JNK and the IL-2 gene (38). The marginal phosphorylation of p46 JNK induced by H4/ICOS thus suggests that the PKC
and Vav signaling pathway may be poorly activated by H4/ICOS as compared with CD28. Akt was shown to synergize with PKC
for activation of IL-2 promoter (37). Given such a synergism, it is conceivable that the poor activation of PKC
and Vav signaling pathway causes the low production of IL-2 even when Akt is highly phosphorylated. Furthermore, molecules such as the adaptor protein Cbl-b (39) and the serine/threonine phosphatase PP2A (40) act as negative regulators of T cell activation. Therefore, it should be elucidated whether these positive or negative regulators in IL-2 production are differently activated by H4/ICOS co-stimulation as compared with CD28. Obviously, further study is needed to clarify the mechanism underlying H4/ICOS-specific low IL-2 production.
Another feature of H4/ICOS in the present study is the strain diversity in the expression on activated CD4+ T cells. BALB/c CD4+ T cell blasts obtained by stimulating naive CD4+ T cells with soluble anti-CD3 mAb or bacterial SAg, YPM, with APC exhibited expression of H4/ICOS on their surfaces >6-fold higher than corresponding C57BL/6 CD4+ T cell blasts (Fig. 5B and C). More importantly, an association was observed between the Th2-skewed response of BALB/c CD4+ T cell blasts and their high expression of H4/ICOS, and between Th1-skewed C57BL/6 CD4+ T cell blasts and their low expression of H4/ICOS (Figs 5B and C and 6A). Consistent with this result, recent reports have shown that cloned Th2 cell lines and Th2 cells derived from TCR-transgenic mice predominantly express ICOS compared with Th1 cells (13,14). To clarify that the above association is a general phenomenon, further study using other strains of mouse should be required and is now under way. The present result, however, first indicates that the induction of H4/ICOS molecules is regulated differently depending on the strain of mouse, suggesting that the genetic background, which is not assigned, determines the induction of H4/ICOS in activated CD4+ T cells. It has not been elucidated whether the high induction of H4/ICOS in BALB/c CD4+ T cells is due to an intrinsic property of T cells or due to an environmental factor like lymphokines produced by APC. However, the above notion promptly suggests an interesting mechanism underlying Th2-mediated diseases such as asthma and atopy. CD4+ T cells from patients suffering from these diseases would show a high induction of H4/ICOS, thereby leading to a promoted Th2 effector function. In the mouse model, involvement of ICOS in Th2-meidated diseases has already been suggested, since the treatment with ICOS-IgG or ICOS-deficient mice reduced the Th2 cell-mediated airway hyper-responsiveness (13,15).
Signaling through H4/ICOS affected the Th cell differentiation of naive CD4+ T cells. Anti-H4/ICOS mAb to cross-link the molecule were added during the primary stimulation of naive CD4+ T cells with immobilized anti-CD3 and anti-CD28 mAb, and promoted Th cell differentiation toward Th2 cells (Fig. 6B). However, Th2 polarization was not remarkable in repeated experiments. Induced CD4+ T cell blasts in the presence of H4/ICOS cross-linking revealed only at most a 2-fold increase of Th2 type lymphokine production with
2-fold decrease in, but still a substantial level of, Th1-type lymphokine production, as compared with the control levels. From these results, we favor the assumption that H4/ICOS signaling is not decisive for Th2 cell differentiation. Rather, the predominant expression of H4/ICOS on Th2 cells compared with Th1 cells could facilitate further generation of Th2 cells and their IL-4 produced advances the skewness of this type of Th cell. Much stronger activation of the Akt pathway following H4/ICOS co-stimulation in BALB/c activated CD4+ T cell blasts than in C57BL/6 activated CD4+ T cell blasts was observed in the present study (Fig. 5D) and may support this assumption. It is also possible, however, that unidentified factors related to the activation of CD4+ T cells rather than the difference of the level of H4/ICOS expression affects the activation of Akt following H4/ICOS co-stimulation since the basal level of, and CD28 co-stimulation-induced phosphorylation of, Akt were also stronger in BALB/c activated CD4+ T cell blasts than in C57BL/6 activated CD4+ T cell blasts. The cross-linking with anti-H4/ICOS mAb is likely to trigger Th1 cells as well and to a lesser extent promotes the generation of Th1 cells. Collectively, it is conceivable that the difference in H4/ICOS expression on Th1 and Th2 cells at least partially determines the extent of polarization of the Th cells. However, an alternative possibility that H4/ICOS signaling specifically promotes Th2 differentiation is not excluded. Flavell et al. have recently indicated that JNK is required for the polarization of naive CD4+ T cells toward Th1 cells (33,41,42). Hence, the weak phosphorylation of JNK induced by H4/ICOS co-stimulation may make the differentiation into Th2 cells easier than Th1 cells.
Another function of H4/ICOS has been suggested. We recently reported that ordinary NKT cells and a new type of invariant V
14+ T cell, which are NK1.1, express an intermediate and a high amount of H4/ICOS respectively in the mouse thymus (22). Thus, expression and function of H4/ICOS in regulatory cells such as NKT cells remain to be investigated. Two of the co-authors (D. B. and U. D.) and colleagues also reported that H4/ICOS signaling by mAb protected human CD4+ T cells from cell death induced by HIV gp120 or anti-Fas mAb (43). One of the downstream targets of Akt is Bad and phosphorylated Bad is thought to promote cell survival by releasing Bcl-xL (44). Therefore, further studies should be performed to explore whether the hyperactivation of Akt as shown in this study leads to the cell survival through Bad and Bcl-xL. It is noteworthy that similarly to Bcl-2 expression in thymocytes (45), CD4 SP and CD8 SP thymic conventional T cells express a low but substantial amount of H4/ICOS (25 and 55% respectively), whereas CD4/CD8 double-positive cells do not express H4/ICOS at all (22).
In conclusion, H4/ICOS has been suggested to have multiple roles in the immune responses of activated T cellsco-stimulation, promotion for Th2 cell differentiation and cell survival. The induction of H4/ICOS in activated CD4+ T cells exhibited strain diversity, thus suggesting the different regulation of H4/ICOS expression among the mouse strains. These multiple functions are likely to be elicited by a specific signaling triggered by H4/ICOS molecules. Obviously, further elucidation of the H4/ICOS signaling pathway and the regulation of H4/ICOS expression is required before these can be used therapeutically, in that their inhibition could be effective in the alleviation of immune-mediated diseases.
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Acknowledgements
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We are grateful to Drs G. Suzuki and J. Allison for providing the mAb. We would also like to thank Hisako Yagi for her technical help. This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan, the Ministry of Public Welfare of Japan, and the Associazione Italiana Ricerca sul Cancro (Milan).
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Abbreviations
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APCantigen-presenting cell
DNdouble negative
ERKextracellular signal regulated kinase
HIgGhamster IgG
ICOSinducible co-stimulator
JNKc-Jun N-terminal kinase
MAPKmitogen-activated protein kinase
PEphycoerythrin
PI3-Kphosphatidylinositol 3-kinase
PKCprotein kinase C
SAgsuperantigen
SPsingle positive
YPMYersinia pseudotuberculosis-derived mitogen
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