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The Journal of Immunology, 2002, 169: 4723-4731.
Copyright © 2002 by The American Association of Immunologists

T Cell Hyporesponsiveness Induced by Oral Administration of Ovalbumin Is Associated with Impaired NFAT Nuclear Translocation and p27kip1 Degradation

Kazumi Asai1,*, Satoshi Hachimura2,*, Motoko Kimura{dagger}, Terumasa Toraya*, Masakatsu Yamashita{dagger}, Toshinori Nakayama{dagger} and Shuichi Kaminogawa*

* Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan; and {dagger} Departments of Medical Immunology and Molecular Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Oral tolerance is an important physiological component of the immune system whereby the organism avoids dangerous reactions such as hypersensitivity to ingested food proteins and other luminal Ags which may cause tissue damage and inflammation. In addition, it has been shown in animal models and in humans that oral tolerance can be applied to controlling undesired immune responses, including autoimmune diseases, allergies, and organ transplant rejections. However, the molecular mechanisms of oral tolerance have been poorly defined. In this study, we investigated the molecular basis underlying the hyporesponsiveness of orally tolerant CD4 T cells using a TCR transgenic mouse system in which oral tolerance was induced by long-term feeding with high dose Ag. We demonstrate that the hyporesponsive state of the CD4 T cells was maintained by a selective impairment in the TCR-induced calcium/NFAT signaling pathway and in the IL-2R-induced degradation of p27kip1 and cell cycle progression. Thus, physiological mucosal tolerance is revealed to be associated with a unique type of T cell hyporesponsiveness which differs from previously described anergic T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Oral tolerance is the state of systemic Ag-specific hyporesponsiveness induced by prior oral administration of Ag under certain conditions (1, 2). Soluble nonreplicating proteins delivered through intact mucosal surfaces do not normally provoke strong immune reactions, but instead induce a state of Ag-specific hyporesponsiveness. This phenomenon was first described in 1911 by Wells (3) as a state in which systemic anaphylaxis in guinea pigs was prevented by previous feeding of hen egg protein. Since then, various experimental models have been reported. Systemic hyporesponsiveness occurs after oral administration of various soluble thymus-dependent Ags, including contact-sensitizing agents (4) and heterologous red cells (5, 6). Once oral tolerance has been induced, various aspects of Ag-specific immune responses including in vitro lymphocyte proliferation (7, 8), cytokine production (9, 10), in vivo delayed-type hypersensitivity (11, 12), and Ab production (7, 13) are suppressed. Thus, oral tolerance appears to be a complex phenomenon, which may involve various molecular mechanisms.

In our laboratory, we have shown that feeding mice with protein Ag as a constituent of the diet induces profound oral tolerance (10, 14, 15). Using this system, we have shown that oral tolerance induced by long-term feeding of the milk protein casein to BALB/c mice was mediated by CD4 T cells (15). However, the mechanisms that induce oral tolerance have not been clearly elucidated. From other reports, oral tolerance appears to be established and maintained by at least two biological mechanisms, i. e., apoptotic cell death (clonal deletion; Ref. 16) and functional unresponsiveness induced (9, 17) in peripheral T cells (anergy) that are characterized by an inability to proliferate and a reduced IL-2 production. In addition, regulatory T cells which secrete suppressive cytokines such as TGF-{beta} have been shown to mediate oral tolerance (2, 18).

One of the most extensively studied models of oral tolerance uses OVA administration to mice, in which systemic immune responses can be suppressed dramatically after feeding relatively high doses of OVA. In addition, OVA-specific TCR transgenic (Tg)3 mice were established where most T cells expressed OVA-reactive TCR (19). Although intracellular signaling of tolerant T cells has been examined in previous studies (20), little is known about the molecular basis responsible for the state of peripheral T cell tolerance, particularly those induced by exogenous Ag. Thus, we decided to use OVA and OVA-specific TCR Tg mice for investigating the molecular mechanisms underlying the induction and maintenance of the state of OVA-induced oral tolerance in vivo. Adoptive transfer systems in which T cells from TCR Tg mice are transferred to syngeneic recipients have been recently used to study peripheral tolerance (17, 21, 22). However, in such systems, the frequency of Ag-specific CD4 T cells is too low to evaluate differences in signaling events in these cells by biochemical analysis (Refs. 17 , 21 , and 22 , and our unpublished observations). Therefore, we used intact TCR Tg mice.

Oral tolerance avoids dangerous hypersensitive reactions to ingested food proteins and other luminal Ags. Indeed food allergy is thought to be one of the consequences of a break down of the state of oral tolerance. Because this state of immunological tolerance is specific to the initially ingested Ag, it is an alternative to drug therapy and can be applied to controlling undesired immune responses. The strategy of oral tolerance induction has been used successfully to treat allergy (23) and autoimmune diseases in animal models and in humans (24, 25, 26). Given that we understand the details of the molecular mechanisms, we could establish a new approach for treating food allergy and other immune diseases.

In this study, we have analyzed the TCR and IL-2R-mediated signal transduction pathways in the hyporesponsive T cells induced by oral administration of OVA. The following results are demonstrated: 1) impaired phosphorylation of TCR-{zeta}, ZAP-70, linker for activation of T cells (LAT), and phospholipase C (PLC){gamma}-1; 2) impaired calcium responses and decreased NFAT nuclear translocation; 3) normal activation of extracellular signal-related kinase (ERK) and stress-activated protein kinase (SAPK) (mitogen-activated protein kinase (MAPK) pathway); and 4) impaired degradation of p27kip1 induced by IL-2 stimulation accompanying cell cycle arrest. Thus, hyporesponsiveness in the orally tolerant CD4 T cell appears to be associated with two defects, i.e., an impaired calcium/NFAT pathway from TCR and impaired p27kip1 degradation induced by stimulation through the IL-2R.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

BALB/c mice were purchased from CLEA Japan (Tokyo, Japan). OVA 323–339-specific TCR Tg (OVA 23-3 TCR Tg) mice on a BALB/c genetic background were produced as described previously (19). The Tg TCR uses the V{alpha}3 and the V{beta}15 chains. After back-crossing with BALB/c mice (mice expressing the Tg TCR selected by PCR), homozygotes were produced and mated with BALB/c mice to produce heterozygotes which were used for experiments. Recombination-activating gene (Rag)-2-/-/OVA 23-3 TCR Tg mice were produced by crossing the OVA 23-3 TCR Tg strain with Rag-2-/- mice (BALB/c background; Ref. 27). All mice used in this study were maintained in our animal facilities at the University of Tokyo (Tokyo, Japan), and used in accordance with the guide lines of University of Tokyo.

OVA-specific oral tolerance induced in OVA 23-3 TCR Tg mice

Eight- to 10-wk-old OVA 23-3 TCR Tg mice and Rag-2-/-/OVA 23-3 TCR Tg mice were orally administrated with a diet containing 8% OVA (egg white diet; Funabashi Farm, Funabashi, Japan) or a control diet for >28 days. The daily intake of OVA was estimated and was ~250 mg/mouse.

Cell preparation and culture

The purification of splenic CD4 T cells was performed using a MACS (Miltenyi Biotec, Bergisch Gladbach, Germany). CD4-positive splenic T cells from OVA 23-3 TCR Tg mice and Rag-2-/-/OVA 23-3 TCR Tg mice were positively selected with anti-CD4 mAb (GK1.5). The purity was checked by flow cytometry (FACSort; BD Biosciences, Franklin Lakes, NJ) following staining with PE-conjugated anti-CD4 Ab and the percentage of CD4 T cells in the separated cells was >95%. CellQuest software program (BD Biosciences) was used for collecting and analyzing data. The numbers of CD4+ T cells were appropriately adjusted before adding to each stimulation culture.

Antibodies

The anti-IL-2 mAbs (JES6-1A12 and JES6-5H4) and anti-IL-4 mAb (BVD4-1D11 and BVD6-24G2) were purchased from BD PharMingen (San Diego, CA), and were used for ELISA. The anti-IFN-{gamma} mAbs (R4-6A2 and XMG1.2) were prepared in our laboratory and were used for ELISA. Anti-PLC{gamma}-1 antiserum (catalog no. 06-152; Upstate Biotechnology, Lake Placid, NY) was used for immunoprecipitation, and anti-PLC{gamma}-1 Ab (catalog no. 06-159; Upstate Biotechnology) was used for immunoblotting. Anti-TCR-{zeta} antiserum (no. 551; a gift from Dr. A. Singer, National Institutes of Health, Bethesda, MD), anti-ZAP70 (2F3; a gift from Dr. M. Iwashima, Medical College of Georgia, Augusta, GA), anti-LAT polyclonal Ab (Upstate Biotechnology), and anti-STAT5a antiserum (Genzyme, Cambridge, MA) were used for immunoprecipitaion or immunoblotting. Anti-cytokine-inducible Src homology 2 protein (CIS) (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-p27kip1 (Transduction Laboratories, Lexington, KY) were used for immunoblotting. RC20 (Transduction Laboratories) was used for anti-phosphotyrosine immunoblotting.

Proliferation assay and ELISA for measuring cytokine production

Purified splenic CD4 T cells (1 x 105/well) were stimulated with 0.1 or 1 mg/ml of OVA and mitomycin C-treated BALB/c splenic cells (used as APCs) for 2 days. Where indicated, a final concentration of 100 U/ml of rIL-2 was added at the beginning of the stimulation culture. [3H]thymidine (1 µCi/well) was added for a further 24 h. Cells were collected on glass fiber and the incorporated radioactivity was measured by scintillation counting. The concentrations of IL-2, IL-4, and IFN-{gamma} in the culture supernatant were measured by ELISA using commercial mAb pairs (BD PharMingen) and standard methods as described (10). Results were analyzed by the Microplate Manager system (Bio-Rad, Hercules, CA).

Measurement of intracellular free calcium ion concentration ([Ca2+]i)

Purified splenic T cells were loaded with Indo-1 (Indo-1 AM; Molecular Probes, Eugene, OR) in the presence of F127 (28, 29). After washing, the cells were incubated with anti-CD4-PE (RM4-5-PE; BD PharMingen), FITC-conjugated anti-CD8 Ab (53.6-72; BD PharMingen), anti-TCR-biotin (H57-597-biotin), and anti-CD4-biotin (GK1.5-biotin) at 4°C. The stained cells were washed and subjected to calcium analysis on FACSVantage (BD Biosciences). TCR and CD4 were co-cross-linked with avidin, and [Ca2+]i was monitored for 512 s. Results were analyzed by CellQuest software.

Immunoprecipitation and immunoblotting

Purified splenic CD4 T cells were treated with anti-TCR mAb (H57-579) and anti-CD4 mAb (GK1.5) at 4°C, washed, and the TCR and CD4 molecules co-cross-linked with goat anti-hamster IgG (100 µg/ml; ICN Pharmaceuticals, Costa Mesa, CA) at 37°C. For the induction of STAT5a phosphorylation, CIS1 expression, and p27kip1 degradation, purified splenic CD4 T cells were stimulated with anti-TCR mAb (3 µg/ml; plate coated for 1 h at room temperature) and rIL-2 (final 100 U/ml) for 10, 20, or 30 h at 37°C. The stimulated cells were lysed in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA containing 1% Nonidet P-40, 1 mM Na3VO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM NaF, and clarified by centrifugation. The clarified lysates were incubated with indicated Abs for 1 h, followed by protein G-Sepharose beads for 1 h at 4°C. Immune complexes were washed three times with lysis buffer and suspended in SDS-sample buffer. Boiled samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes for immunoblotting. Immunoblotting was performed with the indicated Abs followed by peroxidase-conjugated anti-mouse IgG (ICN Pharmaceuticals), anti-rabbit IgG (New England Biology, Boston, MA), or anti-goat IgG (Santa Cruz Biotechnology). The immunoblots were developed by the ECL technique (Amersham Pharmacia Biotech, Arlington Heights, IL). Band intensities were measured by a densitometer (Bio-Rad), and arbitrary densitometric units are shown.

Lck kinase assay

Immunoprecipitation of the Lck molecule and immunoblotting with anti-Lck antiserum (no. 688, specific for a part of the unique sequence of Lck; RNGSEVRDPLVTYEGSLPPASPLQDN; a gift from Dr. A. Singer) were performed as previously described (30). In brief, splenic CD4 T cells were solubilized in lysis buffer (50 mM Tris (pH 7.4), 0.15 M NaCl, 1 mM sodium vanadate) containing Nonidet P-40 at 1%. Lck molecules were immunoprecipitated with no. 688 preabsorbed to protein G-Sepharose. After washing the beads with lysis buffer lacking EDTA and sodium vanadate, the precipitates were incubated with 15 µCi of [{gamma}-32P]ATP (5000 Ci/mmol; Amersham Pharmacia Biotech) for 15 min on ice in a kinase buffer (20 mM HEPES (pH 7.5), 100 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 1 µM NaATP). Enolase (2 µg) was added in each reaction tube. Kinase reactions were quenched with 50 µl of 2 Laemmli sample buffer with 4 mM EDTA and resolved on 7.5% gels.

MAPK assay

Purified splenic CD4 T cells were treated with anti-TCR and anti-CD4, and the TCR and CD4 molecules were co-cross-linked with anti-hamster IgG (100 µg/ml) at 37°C. The cells were lysed and cell lysates were applied to SDS-PAGE. Proteins were transferred to a polyvinylidene difluoride membrane and subjected to immunoblotting using a phospho-MAPK detection kit (New England Biology) and a phospho-c-Jun N-terminal kinase/SAPK detection kit (New England Biology).

Detection of nuclear translocation of NFAT

Purified splenic CD4 T cells were stimulated with immobilized anti-TCR mAb (3 µg/ml) and anti-CD4 mAb (1 µg/ml) for 24 h and nuclear extracts were prepared with a NE-PER Nuclear and Cytoplasmic Extraction reagent (Pierce, Rockford, IL) according to the manufacturer’s protocol. The amounts of NFATc and NFATp in the cytoplasmic and nuclear extracts were assessed by immunoblotting with anti-NFATc and anti-NFATp mAb (BD PharMingen).

Immunofluorescent staining

For analysis of cell surface TCR, CD3, and CD4 expression on splenic CD4 T cells, freshly isolated spleen cells were incubated with FITC-conjugated anti-TCR Ab (H57-597; BD PharMingen) or FITC-conjugated anti-CD3 Ab (145-2C11; BD PharMingen) and PE-conjugated anti-CD4 Ab (H129.19; BD PharMingen). Analysis of intracellular TCR expression was performed by method of Benson et al. (31) with minor modifications. Briefly, splenic CD4 T cells were incubated with FITC-conjugated anti-TCR Ab. After washing, cells were incubated with excess nonlabeled Ab to saturate extracellular TCR sites. Cells were then washed, fixed with 4% parafolmaldehyde, and permeabilized with permeabilization buffer. Cells were then incubated with PE-conjugated anti-TCR Ab (H57-597; BD PharMingen) for intracellular TCR protein detection, then washed and then analyzed by flow cytometry.

For detection of IL-2R {alpha}-chain expression, freshly prepared and stimulated splenic cells were incubated with anti-TCR mAb (3 µg/ml) and anti-IL-2R {alpha}-chain Ab, and stained with FITC-conjugated goat anti-rat IgG Ab (Caltag Laboratories, Burlingame, CA). After washing, the cells were incubated with anti-FcR Ab (2.4G2; BD PharMingen) to block residual binding sites of the second Ab, and then stained with anti-CD4-PE. Cell cycle progression of CD4 T cells was analyzed after propidium iodide staining as described (32). A FACSort flow cytometer and CellQuest software program were used for collecting and analyzing data.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of oral tolerance: diminished anti-OVA responses of splenic CD4 T cells from OVA 23-3 TCR Tg mice with long-term oral administration of OVA

This study aims to clarify the molecular basis underlying the OVA-specific hyporesponsiveness of CD4 T cells induced by long-term oral administration of OVA. Splenic CD4 T cells from OVA 23-3 TCR Tg mice with long-term egg white feeding were purified by MACS, and stimulated in vitro with OVA and BALB/c (H-2d) APCs. As shown in Fig. 1Go, the OVA-induced proliferative responses of the CD4 T cells, as well as the production of cytokines including IL-2, IL-4, and IFN-{gamma} were significantly decreased. OVA-induced IL-10 production and mRNA expression of TGF-{beta} were not up-regulated (data not shown). Furthermore, to address the possibility that the hyporesponsiveness was due to TCR down-modulation, we examined surface TCR and CD3 expression on splenic CD4 T cells (Table IGo). As shown in Table IGo, neither the percentage of splenic CD4+ cells expressing TCR, nor the intensity of TCR expression on these cells decreased after long-term egg white feeding. Similar results were obtained in CD3 staining, and the expression level of CD4 was unaltered after feeding the egg white diet (Table IGo). We also examined the levels of intracellular TCR which increases when surface TCR is down-regulated; however, an increase was not detected (data not shown). Thus, TCR expression was not down-regulated in orally tolerant CD4 T cells induced in this system. T cell hyporesponsiveness persisted for at least 1 mo, even if oral administration of OVA was terminated (data not shown). Thus, we conclude that oral tolerance can be experimentally induced in OVA 23-3 TCR Tg mice by the long-term feeding of egg white. The orally tolerant CD4 T cells were in a hyporesponsive state in the absence of regulatory T cells, which produced suppressive factors of TGF-{beta} and/or IL-10. Consequently, we used this system to clarify the molecular basis underlying the hyporesponsive state of CD4 T cells induced by oral administration of OVA.



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  		FIGURE 1. Induction of oral tolerance; proliferative responses and cytokine production of splenic CD4 T cells from OVA 23-3 TCR Tg mice with long-term oral administration of OVA. Mean [3H]thymidine incorporation and cytokine concentration of each culture is shown with SD. Data represent the results from at least five independent experiments.

 

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  		Table I. Expression of TCR, CD3, and CD4 on splenic CD4 T cells from OVA 23-3 TCR Tg mice with long-term administration of OVAa

 
Decreased tyrosine phosphorylation of TCR-{zeta}, ZAP-70, and LAT in the orally tolerant CD4 T cells

The phosphorylation of TCR-{zeta}, ZAP-70, and LAT are known to be early signaling events in TCR-mediated signal transduction (33); therefore, we examined the levels of anti-TCR-induced phosphorylation on these signaling molecules in orally tolerant CD4 T cells. The phosphorylation of TCR-{zeta} in the stimulated T cells was examined by immunoprecipitation with anti-TCR-{zeta} serum (no. 551) and subsequent immunoblotting with anti-phosphotyrosine mAb (Fig. 2GoA). The total amount of TCR-{zeta} protein was also assessed by immunoblotting the precipitates with anti-TCR-{zeta} antiserum. As can be seen in Fig. 2GoA, the 21-kDa phosphorylated form of TCR-{zeta} was detected even in freshly prepared CD4 T cells. Endogenously phosphorylated TCR-{zeta} has been previously reported in splenic T cells (34). In contrast, the hyperphosphorylated 23-kDa TCR-{zeta} was detectable within 2 min after stimulation and the band intensities were significantly lower in the egg white diet group. The protein expression levels of the 16-kDa TCR-{zeta} chain were not altered (Fig. 2GoA, bottom panel).



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  		FIGURE 2. Tyrosine phosphorylation of TCR-{zeta}, ZAP-70, and LAT and Lck kinase activity in the orally tolerant CD4 T cells of OVA 23-3 TCR Tg mice. Purified splenic CD4 T cells were treated with anti-TCR mAb (H57-579) and anti-CD4 mAb (GK1.5). Phosphorylation status and the amount of protein of TCR-{zeta} (A), ZAP-70 (B), and LAT (C) were assessed after co-cross-linking of TCR and CD4 molecules with anti-hamster IgG. Arbitrary densitometric units of each band (p21 and p23 for TCR-{zeta}, p70 for ZAP-70, p36 for LAT) are indicated. Similar results were obtained from three independent experiments. D, Lck tyrosine kinase activity was determined by in vitro kinase assay. In addition to autophosphorylation of Lck, enolase was used for an indicator of transphosphorylation activity. The phosphorylated proteins migrating at the expected size of Lck (p56) and enolase, and arbitrary densitometric units of these bands are indicated. Similar results were obtained from two independent experiments.

 
The phosphorylation of ZAP-70 was also assessed in the cells stimulated as in Fig. 2GoA (Fig. 2GoB). As expected, the levels of tyrosine-phosphorylated ZAP-70 were also reduced in the egg white diet group. The protein expression levels of ZAP-70 were not decreased (Fig. 2GoB, bottom panel). Fig. 2GoC shows the level of tyrosine-phosphorylated LAT, which is known to be phosphorylated by activated ZAP-70 (35). The tyrosine phosphorylation of LAT was induced within a few minutes after TCR stimulation in both control or egg white diet groups, and the magnitude was significantly reduced in the egg white diet group. Again, the expression levels of LAT protein were equivalent (Fig. 2GoC, bottom panel). Taken as a whole, the activation of proximal upstream signaling events in TCR-mediated signal transduction, i.e., the phosphorylation of TCR-{zeta}, ZAP-70, and LAT, were revealed to be significantly impaired in the orally tolerant CD4 T cells. However, Lck tyrosine kinase activity assessed by in vitro kinase assay was not reduced in the orally tolerant CD4 T cells when measured by autophosphorylation and by transphosphorylation of an exogenous substrate, enolase (Fig. 2GoD).

Decreased tyrosine phosphorylation of PLC{gamma}-1, impaired calcium responses and decreased NFAT nuclear translocation in the orally tolerant CD4 T cells

It is known that there are two major downstream signal transduction pathways from the TCR, the calcium/calcineurin pathway and the MAPK signaling pathway (36, 37, 38). Phosphorylation of LAT is required for the phosphorylation of PLC{gamma}-1, calcium mobilization, and NFAT-mediated gene expression, and is also required for the activation of the ERK/MAPK cascade (39). Consequently, we assessed the activation of PLC{gamma}-1 in the orally tolerant CD4 T cells by measuring the phosphorylation levels of PLC{gamma}-1 after TCR stimulation. As shown in Fig. 3GoA, the increase in PLC{gamma}-1 phosphorylation after co-cross-linking of TCR and CD4 molecules was significantly impaired in the egg white diet group. The PLC{gamma}-1 protein levels were unaltered (Fig. 3GoA, bottom panel).



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  		FIGURE 3. Tyrosine phosphorylation of PLC{gamma}-1, calcium responses, and NFAT nuclear translocation in the orally tolerant CD4 T cells of OVA 23-3 TCR Tg mice, and induction of oral tolerance in Rag-2-/-/OVA 23-3 TCR Tg mice. A, The phosphorylation status and amount of PLC{gamma}-1 protein in CD4 T cells which were treated as in Fig. 2GoC. Anti-PLC{gamma}-1 antiserum was used for immunoprecipitation. Arbitrary densitometric units of bands corresponding to phosphorylated PLC{gamma}-1 (p135) and PLC{gamma}-1 protein are indicated. B, Splenic T cells from OVA 23-3 TCR Tg mice with control and egg white diet were loaded with calcium-sensitive dye Indo-1 and were incubated with anti-CD4 (GK1.5)-biotin and anti-TCR (H57-597)-biotin. TCR and CD4 molecules were co-cross-linked by the addition of streptavidin at the 30-s time point (white gap) during the monitoring of [Ca2+]i for 512 s. Percentage of responding cells and mean Indo-1 ratio of each reaction are indicated in each panel. Splenic CD4 T cells (1 x 107/lane) were stimulated with anti-TCR (H57-597) and anti-CD4 (GK1.5) mAbs for 24 h. The extracts were subjected to immunoblotting with anti-NFATc (C) and NFATp (D) mAbs. Arbitrary densitometric units of NFAT bands are indicated. Representative results of three independent experiments are shown. E, Proliferative responses and IL-2 production of splenic CD4 T cells from Rag-2-/-/OVA 23-3 TCR Tg mice with long-term oral administration of OVA. Splenic CD4 T cells were stimulated with OVA and BALB/c splenic APCs. Mean [3H]thymidine incorporation and IL-2 concentration of each culture is shown with SD. Data represent the results from two independent experiments. F, The phosphorylation status of PLC{gamma}-1 in orally tolerant CD4 T cells from Rag-2-/-/OVA 23-3 TCR Tg mice. CD4 T cells were treated as in Fig. 3GoA. Arbitrary densitometric units of bands corresponding to phosphorylated PLC{gamma}-1 (p135) are indicated.

 
Next, the ability to mobilize Ca2+ was examined in the orally tolerant CD4 T cells. Splenic T cells from OVA 23-3 TCR Tg mice with the control or egg white diet were loaded with the calcium-sensitive dye Indo-1, and then incubated with anti-CD4-PE (Rm4-5), anti-CD8-FITC, anti-TCR-biotin, and anti-CD4-biotin (GK1.5). The TCR and CD4 molecules were co-cross-linked by the addition of streptavidin at the 30-s time point during the monitoring of [Ca2+]i. The [Ca2+]i of electronically gated CD4 T cells are shown in Fig. 3GoB. The orally tolerant CD4 T cells mobilized intracellular calcium poorly, suggesting a reduced PLC{gamma}-1 activation resulting in impaired calcium responses.

Nuclear translocation of NFATc and NFATp, which are thought to be a result of calcium-dependent calcineurin activation, was assessed in the orally tolerant CD4 T cells. As can be seen in Fig. 3GoC, NFATc was not significantly detected in either nuclear or cytoplasmic extracts before stimulation. After stimulation for 24 h, NFATc protein could be detected in the cytoplasmic extract, and a substantial amount was found in the nuclear extract of the control CD4 T cells. As expected, a significant decrease in the amount of NFATc in nuclear extracts was detected in the egg white diet group (Fig. 3GoC, right panel; 1.0 vs 3.0). This result shows that in the orally tolerant CD4 T cells, NFATc could not be induced in the cytoplasm and translocate to the nucleus. Multiple bands in the NFATc immunoblots may represent differentially glycosylated NFATc molecules (40). Furthermore, the same results were obtained with NFATp (Fig. 3GoD). NFATp protein was detected in cytoplasmic fraction after stimulation of both groups (Fig. 3GoD, right upper panel); however, nuclear translocation was not induced after stimulation in the egg white diet group (Fig. 3GoD, right bottom panel). These results suggest that the levels in the nuclear translocation of NFATc and NFATp are significantly reduced in the orally tolerant CD4 T cells.

Furthermore, we confirmed that oral tolerance was induced in Rag-2-/-/OVA 23-3 TCR Tg mice in which all T cells express the Tg TCR. A significant decrease in OVA-induced proliferative responses and OVA-induced IL-2 production of the splenic CD4 T cells was detected (Fig. 3GoE). Impaired phosphorylation of PLC-{gamma} after TCR stimulation was also confirmed in these mice (Fig. 3GoF). Thus, it is most conceivable that the alterations observed in the signaling experiments were due to changes in the Ag-specific CD4 T cell population.

ERK/MAPK and SAPK/MAPK cascades are unaffected in the orally tolerant CD4 T cells

It is reported that some anergic T cell clones (41, 42) and super Ag-induced anergic CD4 T cells show a certain level of impaired activation in MAPK cascades. In our orally tolerant CD4 T cells, impaired phosphorylation of LAT was detected (Fig. 2GoC); thus, we wanted to assess the TCR-induced activation of two MAPK pathways (ERK and SAPK). Orally tolerant CD4 T cells were stimulated by co-cross-linking of TCR and CD4 molecules, and the phosphorylation status of MAPK (ERK1, ERK2, and SAPK) was determined. As can be seen in Fig. 4Go, no impairment in MAPK phosphorylation was detected in the egg white diet groups. Indeed, at some time points increased levels in the phosphorylation on ERK1, ERK2, and SAPK were detected. Thus, the activation of ERK/MAPK and SAPK/MAPK cascade appears not to be compromised in the orally tolerant CD4 T cells. These results differ from those of anergic T cells induced in vitro as well as other forms of in vivo tolerance.



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  		FIGURE 4. Activation of ERK/MAPK and SAPK/MAPK cascades in the orally tolerant CD4 T cells of OVA 23-3 TCR Tg mice. Splenic CD4 T cells (1 x 107/lane) were stimulated and lysed as in Fig. 2Go. The lysates were subjected to immunoblotting with anti-phospho-ERK (A) or anti-phospho-SAPK (B) Ab. Arbitrary densitometric units of phospholyrated bands (p42 and p44 for ERK, p56 for SAPK) are shown under each band. The amount of protein was determined by reblotting the same membrane with specific anti-MAPK mAbs. Representative results of three independent experiments are shown.

 
The hyporesponsiveness of the orally tolerant CD4 T cells was not rescued by the addition of calcium ionophore or excess amounts of exogenous IL-2

These results suggest that the orally tolerant CD4 T cells have an impairment in calcium/calcineurin/NFAT pathways from the TCR. Thus, the hyporesponsiveness of the orally tolerant CD4 T cells might be rescued by the addition of ionomycin, which induces an increase of Ca2+ to activate calcineurin. As shown in Fig. 5Go, even in the presence of ionomycin (250 ng/ml), the hyporesponsive state in the egg white diet group was not rescued, suggesting that other signaling pathways are also impaired. The hyporesponsive state of various anergic T cells was reported to be rescued by the addition of exogenous IL-2 during anti-TCR stimulation in vitro (20). Therefore, we tested whether the hyporesponsiveness of the orally tolerant CD4 T cells was rescued by exogenous IL-2 or not. As shown in Fig. 5Go, even in the presence of excess rIL-2 (100 U/ml), the decreased proliferative responses in the orally tolerant CD4 T cells were not rescued significantly. Thus, the hyporesponsiveness of the orally tolerant CD4 T cells is not simply due to impaired IL-2 production or calcium/calcineurin/NFAT pathways from the activated TCR.



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  		FIGURE 5. The hyporesponsiveness of the orally tolerant CD4 T cells was not rescued by the addition of calcium ionophore or excess amounts of exogenous IL-2. Splenic CD4 T cells (1 x 105 cells/well) were stimulated with OVA as in Fig. 1Go in the presence of ionomycin (250 ng/ml) or rIL-2 (100 U/ml). Similar results were obtained from three independent experiments.

 
Normal expression of the IL-2R {alpha}-chain, IL-2-induced tyrosine phosphorylation of STAT5 and CIS1 induction in the orally tolerant CD4 T cells

The failure to rescue the impaired proliferative responses by exogenous IL-2 prompted us to examine IL-2R expression and STAT5 activation, a downstream molecule of the IL-2R signaling pathway. We assessed the induction of the IL-2R {alpha}-chain on the orally tolerant CD4 T cells after stimulation with anti-TCR and rIL-2 (100 U/ml). First, purified CD4 T cells were stimulated by rIL-2 alone; however, IL-2R expression and IL-2R signaling were not detected. Therefore, we examined the cells after stimulation through TCR and IL-2R. As shown in Fig. 6GoA, 24 h after the stimulation, the percentages of T cells expressing the IL-2R {alpha}-chain were equivalent in the control and egg white diet groups. These results suggest that the failure to rescue T cell responsiveness by the addition of exogenous IL-2 is not due to the impaired up-regulation of the IL-2R {alpha}-chain.



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  		FIGURE 6. Expression of the IL-2R {alpha}-chain, IL-2-induced tyrosine phosphorylation of STAT5a and CIS1 induction in the orally tolerant CD4 T cells of OVA 23-3 TCR Tg mice. A, IL-2R {alpha}-chain expression on electronically gated CD4 T cells are shown. The percentages of the positive cells are shown. Background staining is shown by dotted lines. B, Phosphorylation status and protein levels of STAT5a in CD4 T cells after IL-2 stimulation. Splenic CD4 T cells (1 x 107/lane) were stimulated with anti-TCR (H57-597) mAb and rIL-2 (100 U/ml) for 24 h. The lysates of the stimulated cells were subjected to immunoprecipitation with anti-STAT5a antiserum, and subsequent anti-phosphotyrosine immunoblotting. Arbitrary densitometric units of phosphorylated STAT5a (p92 band) are shown under each band. The amount of protein was determined by reblotting the same membrane with anti-STAT5a mAb. C, Expression level of CIS1 protein in the CD4 T cells. Splenic CD4 T cells (1 x 107 cells/well) were treated as in Fig. 6GoB. The lysates were subjected to immunoblotting with anti-CIS1 mAb. Arbitrary densitometric units of CIS1 (p36 band) are shown under each band.

 
Next, the phosphorylation status of STAT5 was assessed in the orally tolerant CD4 T cells after stimulation with anti-TCR and rIL-2 (100 U/ml). As can be seen in Fig. 6GoB, the levels of STAT5a phosphorylation were not reduced but rather slightly enhanced. The levels of STAT5a protein were equivalent among these stimulated cells. Thus, the IL-2R expressed on the orally tolerant CD4 T cells appeared to be functionally normal.

Recently, a new adaptor protein family has been identified, which has a motif designated as the suppressors of cytokine signaling box (43). CIS1 is a member of this family and was found to negatively regulate cytokine signal transduction via the interaction with phospholyrated cytoplasmic domains of cytokine receptors (44), with activated Janus kinase (45), or with STAT5 (46). CIS1 is reported to regulate the activation of STAT5 negatively in the IL-2R signaling complex (47). Therefore, the expression levels of CIS1 protein were examined in the egg white diet group after stimulation with TCR and IL-2R and were revealed to be normal (Fig. 6GoC). These results suggest that the failure to rescue T cell unresponsiveness by the addition of IL-2 is not due to changes in the CIS1-mediated negative regulation of IL-2R signaling.

p27kip1 degradation and cell cycle progression is impaired in the orally tolerant CD4 T cells

Finally, we examined one of the downstream signaling events of IL-2R-mediated signaling. p27kip1 is an inhibitor of cyclin-dependent kinase (48) in normal T cells, p27kip1 is degraded by IL-2R-mediated signaling (49), and its activity is decreased gradually in cells reaching S phase. Therefore, we examined IL-2-induced p27kip1 degradation in the orally tolerant CD4 T cells. As shown in Fig. 7GoA, the levels in p27kip1 degradation were significantly reduced in the egg white diet group. IL-2-mediated degradation of p27kip1 protein allows progression of the cell cycle. Thus, cell cycle analysis was performed with propidium iodide staining. As shown in Fig. 7GoB, the ratio of CD4 T cells which were in the G2/M phase was significantly reduced in the group of egg white diet. In the control diet group, 46.6% of cells were in S plus G2/M phases compared with 21.7% in the egg white diet group. The results suggest that the decreased proliferative responses in the orally tolerant CD4 T cells are due at least in part to the impaired IL-2-mediated degradation of p27kip1.



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  		FIGURE 7. IL-2-induced p27kip1 degradation and cell cycle progression in the orally tolerant CD4 T cells of OVA 23-3 TCR Tg mice. A, Splenic CD4 T cells (1 x 107 cells/well) were treated as in Fig. 6GoB. Arbitrary densitometric units of p27kip1 (p27 band) are shown under each band. B, Splenic CD4 T cells (1 x 105 cells/lane) were treated as in Fig. 5Go in the presence of rIL-2 and propidium iodide positive staining cells on electronically gated CD4 T cells are shown. The same findings were obtained from four independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we have provided biochemical evidence for the hyporesponsiveness of in vivo orally tolerant CD4 T cells. The behavior of the orally tolerant CD4 T cells was similar to that of anergic T cells induced in vitro. Anergic T lymphocytes are viable but fail to display certain functional responses, such as IL-2 production and proliferation (50). It has been established that the major defect in the in vitro-induced anergic T cells is impaired IL-2 production resulting from a failure to activate p21ras (Ras) after TCR ligation (41, 51). However, the intracellular biochemical basis underlying the T cell hyporesponsive state maintained in vivo is poorly defined.

Our study indicated that a novel type of hyporesponsive T cells was induced in oral tolerance and was maintained by two intracellular defects. The first was the impaired activation of the calcium signaling pathway. This was reported recently in several experimental systems of in vivo tolerance including super Ag-induced anergy (52), self Ag-induced anergy (53), and CD8 T cell tolerance (54). However, the hyporesponsive state of these cells was reversed by the addition of IL-2, which differed from the results obtained in the present study. The second defect in our orally tolerant CD4 T cells appeared to be a reduced IL-2-induced degradation of p27kip1. The elevation of p27kip1 has been observed in other hyporesponsive T cells induced in vitro by TCR stimulation in the absence of costimulation (55), or by blocking cell division (56, 57). A recent study demonstrated that p27kip1 is up-regulated in tolerant T cells induced in vivo by the use of Ab against CD40 ligand (58). However, impairment in the calcium pathway was not observed in these studies. Thus, the orally tolerant CD4 T cells in our system appear to be maintained in an unresponsive state by a unique type of signaling defect, which is distinct from previously reported hyporesponsive T cells.

LAT has been shown to link proximal TCR signaling events to calcium and MAPK signaling pathways (39). Thus, impairment in TCR-{zeta}/ZAP-70/LAT phosphorylation should affect both pathways. However, in our experiments MAPK activation appeared to be normal (Fig. 4Go). Interestingly, the same finding has been reported in in vivo self Ag-induced anergy (53). Taken together these observations suggest that the activation of MAPK is normal in some hyporesponsive T cells. The reason for this is not clear, but it should be noted that the phosphorylation levels of TCR-{zeta}/ZAP-70/LAT are reduced but not totally absent (Fig. 2Go). Thus, it is conceivable that the lower levels of phosphorylation of TCR-{zeta}/ZAP-70/LAT are sufficient for the activation of MAPK pathways in normal CD4 T cells. Alternatively, it is possible that the activation of MAPK pathways can be induced by different signaling events. Indeed, it has been reported that the Ras/MAPK pathway can be activated by an alternative mechanism which is independent of ZAP-70 kinase activity (59) and LAT phosphorylation (60) when T cells were stimulated with TCR partial agonist ligands.

In general, Lck through association with the intracellular domains of the CD4 and/or CD8 is believed to be the kinase that primarily phosphorylates the TCR-{zeta} dimer following activation (61, 62). However, in this study, despite the reduction of TCR-{zeta} phosphorylation, Lck kinase activity appeared to be normal. This was not expected, and the reason why tyrosine phosphorylation of TCR-{zeta} was impaired despite intact Lck activity is currently unclear, but we consider that the nature of the functional TCR/CD3 signaling complex is different in orally tolerant CD4 T cells. The results in Fig. 2Go, A–C, show that the decrease in phophorylation of signaling molecules become more evident in the downstream molecules, suggesting that the difference in the structure of the TCR/CD3 signaling complex is subtle but significant.

The hyporesponsiveness of the orally tolerant CD4 T cells was not rescued by the addition of rIL-2 (Fig. 5Go), suggesting another defect in the IL-2-induced signaling cascade. Two major IL-2R-coupled signaling pathways have been reported for the transduction of IL-2-mediated proliferative signals in T cells. One pathway, which is specifically coupled to the common {gamma}-chain of the IL-2R, involves Janus kinase 3-mediated activation of the transcription factor STAT5 (63), while the other pathway is coupled to the IL-2R {beta}-chain and involves phosphatidylinositol 3-kinase-mediated activation of PKB/Akt (64).

Several studies have characterized hyporesponsive T cells which are not rescued by IL-2 addition. Some T cells are found to be unable to express sufficient levels of the IL-2R {alpha}-chain on the T cell surface (65). In another case, IL-2 unresponsiveness appeared to be due to a defect in signaling through the common {gamma}-chain of the IL-2R (66). However, in our studies, the orally tolerant CD4 T cells expressed normal levels of the IL-2R {alpha}-chain (Fig. 6GoA). Furthermore, the activation of STAT5 and also CIS1 expression was unaltered in orally tolerant CD4 T cells (Fig. 6Go, B and C). Our results suggest that the IL-2 unresponsiveness may be due to the abnormal degradation of p27kip1, one of the cyclin-dependent kinase inhibitors. The PKB/Akt kinase has been shown to be required for the down-regulation of p27kip1 (67); however, we did not detect PKB/Akt activity in either the control or orally tolerant CD4 T cells (data not shown). Although the precise molecular mechanism governing the impaired p27kip degradation in the orally tolerant CD4 T cells is not clear at this time, it is clear that they show a significant impaired down-regulation of p27kip1. Further investigation into the mechanism of p27kip degradation is required to address this issue.

At the cellular level, OVA-induced oral tolerance appears to be established and maintained by several mechanisms, which include T cell clonal deletion (16), T cell anergy (9, 17), and the induction of regulatory T cells (18, 24). In the case of Rag-2-/-/OVA 23-3 TCR Tg mice, in which all T cells express the OVA-specific TCR, the average number of CD4 T cells in the spleens of control mice was 7.0 x 106 ± 0.5/mouse, while that of orally tolerant mice was 2.0 x 106 ± 0.2/mouse. Thus, it appeared that considerable numbers of Ag-specific T cells were deleted probably by apoptosis. Nevertheless, the remaining CD4 T cells in the OVA-fed mice were in a hyporesponsive state. Another explanation for the hyporesponsiveness may be due to TCR down-regulation (31). However, in the present study, we did not observe any decrease in TCR expression in CD4 T cells of OVA-fed mice. The regulatory T cells mediate active cellular suppression by the secretion of suppressive cytokines such as TGF-{beta}, IL-4, and IL-10 following Ag-specific triggering (18), especially in experiments with low-dose feeding. However, real-time PCR analysis did not reveal any increase in the TGF-{beta} mRNA levels of the orally tolerant T cells upon TCR restimulation (data not shown), and the production of IL-10 was significantly decreased (data not shown). Furthermore, OVA-induced IL-4 production in vitro was significantly decreased in the orally tolerant CD4 T cells (Fig. 1Go). Thus, our OVA-induced oral tolerance did not appear to be maintained by regulatory T cells that secrete these cytokines.

Recently, the suppressive function of CD25+CD4+ T cells (68, 69) has been demonstrated in oral tolerance (70). These immunoregulatory CD25+CD4+ T cells are naturally unresponsive (anergic) in vitro to TCR stimulation, and upon stimulation, are able to suppress the proliferation of CD25-CD4+ T cells (68). However, depletion of the CD25+CD4+ T cells did not affect the responses of CD25-CD4+ T cells from the egg white diet group (data not shown). These results suggest that the down-regulation of CD4 T cell responsiveness in our system was not mediated by the CD25+CD4+ regulatory T cells, but was a result of the hyporesponsive state of effector T cells.

In conclusion, we investigated the molecular mechanisms governing the hyporesponsive state of orally tolerant CD4 T cells. The results suggest that the hyporesponsive state of these T cells is maintained by at least two distinct defects, i.e., impaired NFAT translocation to the nucleus upon TCR recognition of Ag and reduced IL-2-induced degradation of p27kip1 molecules. These defects may be characteristic to hyporesponsive CD4 T cells induced and maintained by oral administration of Ag.


   Acknowledgments
 
We thank Drs. Sonoko Habu and Takehito Sato of Tokai University (Isehara, Kanagawa, Japan) for providing the OVA 23-3 TCR Tg mice. We also thank Drs. Alfred Singer and Makio Iwashima for providing anti-TCR-{zeta} serum, anti-Lck serum, and anti-ZAP-70 Ab. We thank Dr. Wataru Ise for helpful suggestions. We also thank Kaoru Sugaya for excellent technical assistance.


   Footnotes
 
1 Current address: National Institute of Vegetable and Tea Science, National Agricultural Research Organization, Kanaya, Shizuoka 428-8501, Japan. Back

2 Address correspondence and reprint requests to Dr. Satoshi Hachimura, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. E-mail address: ahachi{at}mail.ecc.u-tokyo.ac.jp Back

3 Abbreviations used in this paper: Tg, transgenic; LAT, linker for activation of T cells; PLC, phospholipase C; ERK, extracellular signal-related kinase; SAPK, stress-activated protein kinase; MAPK, mitogen-activated protein kinase; Rag, recombination-activating gene; [Ca2+]i, intracellular free calcium ion concentration; CIS, cytokine-inducible Src homology 2 protein. Back

Received for publication October 26, 2001. Accepted for publication August 22, 2002.


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 Introduction
 Materials and Methods
 Results
 Discussion
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