Antigen-specific, CD4+CD25+ regulatory T cell clones induced in Peyers patches
Noriko M. Tsuji1,
Koko Mizumachi2 and
Jun-ichi Kurisaki1
1 Department of Molecular Biology and Immunology, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan 2 National Institute of Livestock and Grassland Science, 2 Ikenodai, Tsukuba, Ibaraki 305-0901, Japan
Correspondence to: N. M. Tsuji; E-mail: ten{at}affrc.go.jp
Transmitting editor: T. Saito
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Abstract
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Since intestine is exposed to numerous exogenous antigens such as food and commensal bacteria, the organ bears efficient mechanisms for establishment of tolerance and induction of regulatory T cells (Treg). Intestinal and inducible Treg include Tr1-like and Th3 cells whose major effector molecules are IL-10 and transforming growth factor (TGF)-ß. These antigen-specific Treg are expected to become clinical targets to modify the inflammatory immune response associated with allergy, autoimmune diseases and transplantation. In the present study, we characterized the antigen-specific Treg induced in the intestine by orally administering high-dose ß-lactoglobulin (BLG) to BALB/c mice. Seven days after feeding, only Peyers patch (PP) cells among different organs exerted significant suppressive effect on antibody production upon in vitro BLG stimulation. This suppressive effect was also prominent in six BLG-specific CD4+ T cell clones (OPP16) established from PP from mice orally administered with high doses of BLG and was partially reversed by antibodies to TGF-ß. Intravenous transfer of OPP2 efficiently suppressed BLG-specific IgG1 production in serum following immunization, indicating the role of such Treg in the systemic tolerance after oral administration of antigen (oral tolerance). OPP clones secrete TGF-ß, IFN-
and low levels of IL-10, a cytokine pattern similar to that secreted by anergic T cells. OPP clones bear a CD4+CD25+ phenotype and show significantly lower proliferative response compared to Th0 clones. This lower response is recovered by the addition of IL-2. Thus, antigen-specific CD4+CD25+ Treg, which have characteristics of anergic cells and actively suppress antibody production are induced in PP upon oral administration of protein antigen.
Keywords: active suppression, oral tolerance
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Introduction
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Induction and maintenance of antigen-specific unresponsiveness and/or suppression in peripheral T cells is one of the most effective ways to prevent or cure immune disorders, such as allergy, autoimmune diseases and inflammation induced during transplantation (13). Among multiple mechanisms of immune suppression, recently growing evidence concerning active suppression by regulatory T cells (Treg) especially sheds lights on the possibility of intervention during the immune response (46).
Naturally occurring CD4+CD25+ T cells account for 510% of the normal peripheral CD4+ T cell population, are anergic and have been shown to protect the immune system from autoimmune diseases (4,5,7). Depletion of such a cell population causes detrimental inflammation in various organs. They arise in thymus and circulate throughout the body to exert the suppressive function, and maintain systemic tolerance via cellcell contact using, for example, CTLA-4 and membrane-bound transforming growth factor (TGF)-ß as inhibitory mechanisms (8,9).
In contrast, CD4+ Treg against exogenous foreign antigens are induced following induction of peripheral immunologic tolerance. Inducible Treg subsets include Tr1 and Th3, whose major effector molecules are suppressive cytokines IL-10 and TGF-ß, respectively (1012). Thus, these inducible Treg are unique in the way that they can be generated in the peripheral immune system and use soluble factors, in addition to cellcell contact, as suppressive effector molecules. Interestingly, those Treg are often observed among cell populations associated with the intestinal or mucosal immune system (1215).
Within the gut, immune-competent cells meet a wide variety of exogenous antigens from food and distinguish infectious microorganisms from a large population of intestinal flora. Thus, it is a reasonable supposition that the gut develops tolerance and/or active suppression efficiently. They are efficient ways to deal with numerous exogenous antigens and harmless microorganisms in lieu of raising excess immune responses that may lead to harmful inflammation. Antigen-specific Tr1-like cells and Th3 cells are indeed induced in Peyers patches (PP) and mesenteric lymph nodes (MLN) by feeding exogenous protein to normal and TCR transgenic mice (1214). Moreover, adoptively transferred CD4+ DO11.10 cells (OVA-TCR transgenic) are shown to differentiate to CD25+ Treg after oral administration of ovalbumin (OVA) and exist in the peripheral lymphoid system (16).
It has been reported that the dose of feeding antigen affects the mechanism of tolerance inductionadministration of multiple low doses of antigen elicits a potent induction of Treg that effectively silences inflammatory T cells, while administration of higher doses drives antigen-specific T cells to be anergic or apoptotic (17,18). We have previously shown that low-dose ß-lactoglobulin (BLG) administration to BALB/c mice results in induction of Tr1-like cells that actively suppress Th1-associated immune response (13). Under such conditions, though, antibody suppression (Th2-associated immune response) is incomplete. As is well known, higher antigen dose is required to inhibit Th2 immune response (1922).
In order to characterize inducible and antigen-specific Treg which suppress both Th1- and Th2-associated immune responses in normal mice, we fed high-dose BLG, a potent milk allergen, to BALB/c mice and monitored the active suppressor cells for Th2-associated immune responses. We show that Treg, which suppress antibody production, are present in PP after tolerance induction and are also observed in peripheral LN after parenteral immunization. Further, we established BLG-specific CD4+ T cell clones (OPP clones) from PP of BLG-fed BALB/c mice, and found that all clones share similar suppressive functions and anergic phenotype.
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Methods
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Antigens
Bovine BLG (genetic variant A) was prepared from fresh milk by the method of Aschaffenburg and Drewry, and was purified by ion-exchange chromatography (23). To obtain TNP-BLG and TNP-OVA, BLG and OVA (Sigma, St Louis, MO) were incubated with 2,4,6-trinitrobenzenesulfonic acid (Sigma) in borate-buffered saline for 2 h at 25°C with gentle stirring. After gel filtration through a Sephadex G-25 (Pharmacia, Uppsala, Sweden) column and dialysis in PBS (1 mM phosphate) at 4°C for 2 days, TNP-antigen solutions were stored at 20°C.
Animals
BALB/c mice were purchased from Charles River (Tokyo, Japan) before weaning (2 weeks of age) and fed a commercial diet (MM3; Funabashi Farm, Chiba, Japan) containing no BLG. Mice were bred at the Animal Facility of the National Institute of Agrobiological Sciences and were 68 weeks old at the start of experimentation. Female SD rats were purchased from Charles River at 6 weeks of age. All animals received humane care as outlined in the Guide for the Care and Use of Experimental Animals (National Institute of Agrobiological Sciences Animal Care Committee, and National Institute of Livestock and Grassland Science Animal Care Committee).
Antigen administration and immunization
BLG (100 mg) in saline, or saline alone, was fed to mice 5 times over 2 weeks with 2-day intervals by gastric intubation with a feeding needle. The mice were immunized by i.p. injection with 50 µg/animal of BLG in complete Freunds adjuvant (CFA; Difco, Detroit, MI) 7 days after the last BLG feeding. Two weeks later, the mice were boosted i.p. with 50 µg of BLG in incomplete Freunds adjuvant (IFA; Difco) and then bled 7 days after this injection. The serum was subjected to ELISA for determining the titer of BLG-specific antibodies. The same immunization protocol was employed for the in vivo transfer assay and for obtaining primed spleen (SP) B cells for the in vitro antibody production assay. Two more booster injections were given to obtain the positive control serum for the ELISA for BLG. For some of the in vitro antibody production assay, mice were immunized by s.c. (in the hind footpads and the base of tail) injection with 50 µg/animal and i.p. with 50 µg of BLG in CFA (Difco) 7 days after the last BLG feeding.
Preparation of cells
LN, MLN, SP and PP cells
Seven days after the last feeding, periaortic, popliteal and inguinal LN, MLN, SP, and PP cells were harvested from control mice and mice given 5 x 100 mg BLG without subsequent parenteral immunization. For some experiments, LN and SP cells were harvested from control mice and mice given 5 x 100 mg BLG with immu nization of BLG with CFA, 7 days after the immunization. Single-cell suspensions of cells from periaortic, popliteal and inguinal LN, and MLN cells were prepared by pressing the pooled isolated nodes through a 200 mesh polyester screen with the plunger from a 5-ml polypropylene syringe. Cells were washed 3 times with RPMI 1640 (Sigma). After each wash, cells were centrifuged at 1000 r.p.m. for 10 min at 4°C. Single-cell suspensions of SP cells were prepared in the same manner and erythrocytes were depleted using a red blood cell lysing solution (Sigma) followed by a wash with RPMI 1640 containing 10% FCS (Gibco, Life Technologies, Rockville, MD) and two washes with RPMI 1640 containing 1% FCS. PP were dissected from the small intestine and washed in RPMI 1640 containing 2% FCS. PP were then pressed through a 200 mesh polyester screen as above to make a single-cell suspension. PP cells were washed 4 times with RPMI 1640 containing 2% FCS and kept on ice until use on the same day.
Antigen-presenting cells (APC)
SP cells from non-primed BALB/c mice were treated with mitomycin C (MMC; Wako, Osaka, Japan), washed 3 times with RPMI 1640 (Sigma) and used as APC.
T cell clones
We used the modified method of Kimoto and Fathman (24) to establish BLG-specific T cell clones from PP (OPP clones). Briefly, PP cells were collected from orally tolerized BALB/c mice 7 days after the last feeding of 5 x 100 mg of BLG. Single-cell suspensions were cultured to a density of 5 x 106 cells/well in a 24-well plate in RPMI 1640 (Gibco) culture medium containing 50 µg/ml BLG, and supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES and 50 µM 2-mercaptoethanol, at 37°C in a 5% CO2 atmosphere. After 4 days, the blasts were harvested by density separation on Lympholyte M (Cedarlane, Hornby, Ontario, Canada), and 1 x 105 cells/well were cultured with APC (5 x 106 cells/well) and 10% concanavalin A (Con A) supernatant (see below). After 10 days at rest, viable cells were re-stimulated with the antigen in the presence of feeder cells for 4 days. T cell cloning was achieved by limiting dilution. After 1014 days, the wells showing growth were expanded with antigen, APC and 10% Con A supernatant. To prepare Con A supernatant, a single-cell suspension (5 x 106 cells/ml) of SP cells from SD rats was cultured in 75-cm2 flasks for 20 h in the presence of 5 µg/ml Con A (Type IV, from Canavalia ensiformis; Sigma). Supernatants were harvested, and
-methyl-mannopyranoside (Wako) was added to 20 mM to inactivate Con A before use. BLG-specific, CD4+ T cell clones (H36 and 2.11G) were used as control clones, and were derived from LN from non-tolerized and immunized mice as described above (25). The immunization protocol for control clones was as follows. BALB/c mice were immunized s.c. with 40 µg/animal BLG at the base of the tail and footpads. LN cells were collected from immunized mice after 7 days. Re-stimulation of T cell clones (OPP and control clones) was done after the cells had rested for 2 weeks or longer. Clones that had rested for >2 weeks were harvested, density purified with Lympholyte M, washed 3 times with RPMI 1640 and used for experimentation.
BLG-primed B cells
BALB/c mice were immunized i.p. with 50 µg/animal BLG in CFA and boosted i.p. with 50 µg/animal BLG in IFA after 2 weeks. Whole SP cells were hemolyzed and depleted of adhesive cells by culturing on plastic culture dishes (Falcon; Becton Dickinson, Franklin Lakes, NJ) for 60 min at 37°C. Non-adhesive cells were recovered and B220+ B cells purified by means of magnetic cell sorting (Milteny Biotech, Bergish Gladbach, Germany) with anti-B220 beads and washed 3 times with RPMI 1640.
Flow cytometry
The expression of surface antigens was analyzed by flow cytometry. Sixteen hours after antigen stimulation, T cell clones were incubated with mAb conjugated to fluorescent probes specific for CD4/GK1.5, CD8/53-6.7 and CD25/7D4 (PharMingen, San Diego, CA). CD25 levels were also analyzed at the resting stage. For analysis of the Vß chain, a fluorescent-conjugated mAb specific to Vß8/F23.1 (Phar Mingen) was used after analysis with a panel of antibodies specific to each Vß chain. Cells were washed 3 times after each staining and the fluorescence intensity was analyzed with a flow cytometer (FACSort; Becton Dickinson, San Diego, CA). Calibration of the cytometer was done by FACS Comp program (Becton Dickinson) and further adjusted by eye for mouse lymphocytes. T cell clones shown in the figures were visualized using CellQuest (Becton Dickinson) after gating on forward and side scatter.
ELISA analysis of BLG-specific antibody
For analysis of BLG-specific antibodies, titer plates (MaxiSorp F96; Nunc, Roskilde, Denmark) coated with 100 µg/ml BLG in PBS during overnight incubation at 4°C were washed with PBS and blocked with 1% BSA for 2 h at room temperature. After another wash, cell culture supernatants were added to plate wells for another overnight incubation at 4°C. Wells were washed with PBS/0.05% Tween 20 (PBST) and treated sequentially with biotin-labeled goat anti-mouse IgM, IgG, IgG1 or IgG2a for 1 h, (each in 100 µl/well) followed by avidinperoxidase conjugate (Southern Biotechnology Associates, Birmingham, AL) for 30 min in PBST (100µl/well). A solution of 100 µl 3,3',5,5'-tetramethylbenzidine (Moss, Pasadena, MD) was used as enzyme substrate and 100 µl 0.18 M H2SO4 was used to stop the enzymatic reaction. Absorbance at 450 nm was measured using a microplate reader (VERSA max; Molecular Devices, Sunnyvale, CA). In some experiments, avidinalkaline phosphatase (Zymed, South San Francisco, CA) and disodium p-nitrophenylphosphate (Nacalai Tesque, Kyoto, Japan) were used for the detection of enzymatic activity. In these cases, diethanolamineHCl (1 M, pH 9.8) containing 0.01% MgCl2 was used as buffer and the resulting color was measured at 405 nm. A standard curve was plotted using pooled serum from BALB/c mice hyperimmunized with BLG (control serum). The definition of ELISA units was as follows: for IgM and IgG2a concentration, the resulting absorbancy of control serum at 1000-fold dilution was defined as 100 U. For IgG and IgG1 concentration, the resulting absorbancy of control serum at 1 x 107-fold dilution was defined as 100 U.
In vitro antibody production assay
LN, MLN, SP and PP cells were prepared as described above from mice given 5 x 100 mg BLG without systemic immunization or with one immunization of BLG with CFA. These primary culture cells and T cell clones derived from PP (OPP clones) were analyzed for their suppressive effects on antibody production when co-cultured with an in vitro antibody production system consisting of Th clone cells and primed SP B cells. An in vitro culture system for antibody production was prepared with SP B cells (1 x 106 cells/well) from BALB/c mice pre-immunized i.p. with BLG and a BLG-specific CD4+ T cell clone derived from LN cells from non-tolerized BALB/c mice (H36; 1 x 104 cells/well). The primary culture cells (2 x 106 cells/well) and OPP clones (5 x 104 cells/well) were seeded in culture inserts with 0.45-µm filters (Intercup, Sanko, Tokyo, Japan) for 24-well plates (Falcon) and co-cultured separately with the in vitro antibody production system. In the case of OPP clones tested for their suppressive activity, APC (2 x 106 cells/well) were placed in culture inserts with each OPP clone. After 72 h incubation in the presence of BLG (20 µg/ml), cells were washed and cultured for an additional 3 days in the absence of BLG before supernatants were collected for the detection of secreted BLG-specific antibodies by ELISA. For the neutralization assay, antibodies against regulatory cytokines (anti-IL-10, -TGF-ß and -IFN-
) or control antibodies were added at the initiation of the culture to 30 µg/ml final concentration.
Adoptive transfer assay
PP clones and H36 cells were harvested in their resting stage, purified by density separation (Lympholyte M) and washed 3 times with RPMI 1640. PBS (0.2 ml) containing T cell clones (2 x 106 cells) or PBS alone was injected i.v. into BALB/c mice. All mice were immunized i.p. with 50 µg/animal BLG in CFA after cell transfer. After 2 weeks the mice were given another transfer of T cell clones and boosted i.p. with 50 µg BLG in IFA. One week after the second immunization, the mice were bled and the serum was subjected to ELISA for determination of BLG-specific antibody titer.
Cytokine analysis
Twenty-four-well plates (Falcon) were coated with 10 µg/ml of anti-CD3 antibodies (145-2C11; PharMingen) in PBS by overnight incubation at 4°C. After washing plates twice with PBS, OPP clones (2.5 x 105 cells) in 1 ml of culture medium were placed in each well and cultured for 20 h at 37°C in a 5% CO2 atmosphere and then culture supernatants were collected. The culture medium was RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES and 50 µM 2-mercaptoethanol. Culture medium without FCS was used for the analysis of TGF-ß. To detect cytokines in supernatants of OPP T cell clones, we used commercial kits for TGF-ß (R & D, Minneapolis, MN), and specific antibody sets (PharMingen) for IL-2, IL-4, IL-5, IL-6, IL-10, IFN-
and tumor necrosis factor (TNF)-
. The concentration of TGF-ß was analyzed according to the manufacturers instructions. As a customized analysis for IL-2, IL-4, IL-5, IL-6, IL-10, IFN-
and TNF-
, titer plates (MaxiSorp F96; Nunc) were coated with 3 µg/ml of capture antibodies in 0.1 M sodium carbonate buffer (pH 9.5) for anti-IL-2, -IL-4, -IL-5, -IL-6 and -IFN-
antibodies or 0.2 M sodium phosphate (pH 6.5) for anti-IL-10 and -TNF-
antibodies, and incubated overnight at 4°C. Titer plates were then washed with PBS and blocked with 1% BSA for 2 h at room temperature. After washing with PBST, cell culture supernatants were added to wells for another overnight incubation at 4°C. The wells were washed again and treated sequentially with 1 µg/ml of biotinylated detection antibodies in PBST for 2 h and in avidinperoxidase conjugate (Southern Biotechnology Associates, Birmingham, AL) in PBST for 30 min at room temperature. As above, 3,3',5,5'-tetramethylbenzidine was used as substrate and 0.18 M H2SO4 was used to stop the enzymatic reaction. Absorbance was measured at 450 nm on a microplate reader.
T cell proliferation assay
Resting OPP T cell clones and control clones (1 x 104 cells) were activated in the presence of APC (5 x 105 cells/well, 96 well flat-plate) and BLG (50 µg/ml). Some wells were assayed in the presence of IL-2 (5 ng/ml). Cells were incubated for 48 h at 37°C in a 5% CO2 atmosphere and pulsed with 1 µCi of [3H]thymidine per well. After 16 h further incubation, the cells were harvested and radioactivity was measured in a liquid scintillation counter (Tri-Carb 16-R; Packard, Meriden, CT).
Statistical analysis
Results are expressed as means ± SE. Students t-test was used for determining statistical differences among the various experimental and control groups. P values <0.05 were considered significant.
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Results
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PP cells from mice orally administered with high doses of BLG inhibit antigen-specific antibody production in vitro
Inactivation of the Th2-associated immune response (antibody response) requires high doses of antigen. In BALB/c mice, a multiple feeding regimen consisting of >5 x 10 mg BLG is required to suppress the antibody response efficiently after immunization (data not shown). In contrast, lower doses of BLG (5 x 1 mg) are sufficient to silence the T cell proliferative response (13). Here, we fed BALB/c mice with multiple high doses of BLG (5 x 100 mg) to achieve stable tolerance both in Th1- and Th2-associated immune responses.
To examine whether the low response of antibody production in vivo results from induction of regulatory cells and to identify the location of those regulatory cells in vivo, primary culture cells from PP, SP, inguinal and periaortic LN, and MLN were subjected to co-culture using an in vitro BLG-specific antibody production system. Briefly, single-cell suspensions of each organ from BLG-fed or non-fed mice were co-cultured separately with a BLG-specific Th cell clone (H36) and BLG-primed SP B cells from BALB/c mice, using culture inserts. All co-cultures were carried out in the presence of BLG.
As shown in Fig. 1, in vitro antibody production was suppressed significantly only in the presence of PP cells. Soluble factors are most likely responsible for the suppression given that PP cells were cultured separately in culture inserts with 0.45-µm filters. PP cells from control mice showed moderate, but significant, suppression of IgM production, indicating that inhibitory factors are secreted constitutively by PP cells in normal mice. Still, the effect of BLG on the enhancement of IgM/IgG suppression by PP cells was significant when compared to control mice.

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Fig. 1. Inhibition of BLG-specific antibody production in vitro by primary culture cells from BALB/c mice. PP, SP, periaortic, popliteal and inguinal LN, and MLN cells were collected from the BLG-fed and non-fed mice. Cells (2 x 106 cells/well) were placed in culture inserts (upper well). Primed B cells (1 x 106 cells/well) and a BLG-specific CD4+ Th cell clone (H36; 1 x 104 cells/well) were placed in a 24-well culture plate (bottom well). Primed B cells were prepared as described in Methods. After 72 h of co-culture in the presence of BLG (20 µg/ml), cells were washed and cultured for an additional 3 days in the absence of antigen before collection of supernatants for the detection of secreted BLG-specific antibodies by ELISA. Representative data from three independent experiments are shown. **P < 0.01, ***P < 0.001 (comparison of in vitro antibody levels between groups with or without primary cells in culture inserts).
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Phenotypes and antigen specificity of PP-derived T cell clones (OPP clones)
PP cells from mice fed high doses of BLG contain regulatory cells that are activated by BLG and suppress bystander antibody production in vitro. To further clarify the characteristics and mechanisms of suppression by these regulatory cells, we cloned BLG-specific T cells from PP of mice fed 5 x 100 mg BLG. Six T cell clones (OPP16) with a surface phenotype of Thy1+, CD3+ (data not shown), CD4+, CD8 and Vß8+ (Fig. 2) showed specificity to BLG as measured by antigen-specific cell proliferative response (Fig. 3). The proliferative response of OPP16 clones to BLG was mild relative to Th clones (H36, 2.11G) that we previously established from LN of BLG-immunized mice. However, it was dramatically enhanced in the presence of exogenous IL-2 (Fig. 3). We observed the effect of exogenous IL-2 both in the presence or absence of TCR stimulation (data not shown) and indeed they constitutively express low levels of CD25, a component of IL-2 receptor (Fig. 4).

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Fig. 2. Phenotypes of T cell clones established from PP. The expression of surface antigens on T cell clones established from PP of BALB/c mice fed 5 x 100 mg BLG (OPP16) was analyzed by flow cytometry. Sixteen hours after antigen stimulation, T cell clones were incubated with fluorescence-conjugated mAb specific for CD4, CD8 or Vß8. Unfilled histograms depict unstained controls. Representative data from three independent experiments are shown.
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Fig. 3. Proliferative response of OPP clones against BLG in the presence and absence of IL-2. OPP clones or control T cell clones derived from LN of non-tolerized mice (H36, 2.11G) were activated in the presence of APC (5 x 105/ well, 96 well flat-plate) and BLG (50 µg/ml). Some wells were assayed in the presence of IL-2 (2 ng/ml). Cells were incubated for 48 h at 37°C in a 5% CO2 atmosphere and pulsed with 1 µCi of [3H]thymidine per well. After 16 h, the cells were harvested and radioactivity was measured in a liquid scintillation counter. Representative data from three independent experiments are shown.
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Fig. 4. Flow cytometric analysis of cell-surface CD25 (IL-2 receptor chain) expressed on resting T cell clones. T cell clones were cultured without antigen stimulation over 3 weeks and harvested by density separation. Cells were double stained with the conjugated antibodies phycoerythrinanti-CD4 and FITCanti-CD25. Histograms with light lines depict the unstained control. Representative data from three independent experiments are shown.
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Cytokines produced by OPP clones
A panel of cytokines produced by OPP clones is shown in Table 1. It is characteristic that they produce large amounts of IFN-
. Clones OPP2 and OPP4 produce regulatory cytokines such as IL-10 and TGF-ß, but not helper cytokines such as IL-4 or IL-5. IL-2 production is also minimal in these clones. Together with their efficient production of TNF-
, the pattern of cytokine production by OPP2 and OPP4 especially resembles that of anergic T cells (IL-2 to IL-2/+, IL-4, IL-5, IL-10+, TNF-
+, IFN-
+, TGF-ß+) (26). The rest of the OPP clones differed from OPP2 and OPP4 in that they produced more IL-4 and/or IL-5. With respect to IL-2 production, OPP5 also produced a small amount, associated with the highest production of TNF-
and the least production of TGF-ß.
The pattern of secreted cytokines from OPP clones after stimulating them with APC (MMC-treated SP cells) and BLG (20 µg/ml) was similar to that described in Table 1 (data not shown). Briefly, IL-2 and IL-4 production decreased compared to that from anti-CD3-stimulated culture while regulatory cytokines IL-10, TGF-ß and IFN-
were secreted to a comparable level.
OPP clones inhibit antibody production in vitro
We next examined whether the OPP clones are suppressive in an in vitro antibody production system. Suppression was significant for both IgM and IgG in OPP14, and for IgM in OPP5 and OPP6 (Fig. 5). As stated above, soluble factor(s) were responsible for the suppression since culture inserts were employed. The suppression in antibody production observed for primary culture PP cells (2 x 106 cells) and that observed for BLG-specific OPP clones (5 x 104 cells) with APC (2 x 106) in the present system were comparable (data not shown). We also observed the regulatory functions of OPP clones on antibody production in direct co-culture assay (where cellcell contacts are present), and on T cell proliferation in both separate and direct co-culture assay (data not shown).

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Fig. 5. Inhibition of antibody production by OPP clones in vitro. OPP clones were analyzed for suppressive activity on antibody production in vitro. APC (2 x 106 cells/well) and each OPP clone (5 x 104 cells/well) were placed in culture inserts (upper well), and primed B cells (1 x 106 cells/well) and BLG-specific CD4+ Th cells (H36; 1 x 104 cells/well) were seeded in 24-well culture plates (bottom well). After 72 h incubation in the presence of BLG (20 µg/ml), bottom well cells were harvested, washed 3 times and cultured for an additional 3 days in the absence of BLG before collection of supernatants. BLG-specific antibodies were detected in supernatants by ELISA. Control supernatants were prepared from primed B cells and Th cell cultures in the absence of OPP clones. Representative data from three independent experiments are shown. **P < 0.01, ***P < 0.001 (comparison of in vitro antibody levels between groups with or without individual OPP clones).
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Anti-TGF-ß neutralizes OPP2-induced inhibition of IgM, but not IgG, antibody production in vitro
It should be important to identify the suppressor effector molecule in the regulatory effects observed in the OPP clone system. The in vitro neutralization assay was performed using a panel of antibodies and OPP2 as a representative clone, which mostly produce TGF-ß and best suppress IgG production. Antibodies against IFN-
, IL-10 and TGF-ß were added to the culture media of in vitro antibody assays in the presence of OPP2. Figure 6 shows that anti-TGF-ß antibodies completely neutralized the IgM suppression by OPP2, suggesting that TGF-ß is responsible for OPP2-mediated inhibition of antibody response. However, anti-TGF-ß failed to reverse OPP2-mediated suppression of IgG production. Anti-IL-10 and anti-IFN-
did not influence antibody production in vitro. These results indicate that regulatory cytokines or soluble factors other than the single cytokines IFN-
, IL-10 or TGF-ß are involved in active suppression of IgG.

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Fig. 6. Effects of neutralizing antibodies on OPP2-mediated suppression of IgM and IgG production in vitro. In the presence of APC (2 x 106 cells/well), BLG (20 µg/ml) and neutralizing antibodies (anti-IFN- , anti-IL-10, anti-TGF-ß, or control antibodies), OPP2 cells (5 x 104 cells/well) were seeded in culture inserts and co-cultured using an in vitro antibody production system (primed B cells and H36) separately in 24-well plates. The final concentration of neutralizing antibodies was 30 µg/ml. After 72 h incubation, the cells were washed and cultured for an additional 3 days in the absence of BLG before supernatants were collected for the detection of secreted BLG-specific antibodies by ELISA. Representative data from three independent experiments are shown. *P < 0.05, ** P < 0.01 (comparison of in vitro antibody levels between groups with or without neutralizing antibodies).
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In vivo transfer of OPP2 or OPP4 inhibits IgG1 production in BLG-immunized BALB/c mice
Given that OPP clones 14 exhibited efficient suppressive effects on in vitro antibody production (Fig. 4), we performed adoptive transfers of OPP2, OPP3 and OPP4 to BALB/c mice, immunized with BLG, and measured the induction of antibody response. OPP1 was eliminated from the assay because its proliferation is less efficient than OPP2-4. Figure 7 shows that OPP2 and OPP4 significantly suppressed BLG-specific IgG and IgG1, but enhanced BLG-specific IgG2a production. OPP3 also enhanced IgG2a, but IgG and IgG1 suppression was not significant. Unlike the results from the in vitro antibody production assay, in vivo IgM titers were not significantly suppressed for any of three OPP clones transferred. Control clone H36, a BLG-specific Th clone derived from LN of BLG-immunized mice, enhanced IgM and IgG2a without affecting the IgG and IgG1 titers when transferred (data not shown), suggesting that suppression of IgG and IgG1 in vivo is a characteristic of OPP clones. That these OPP clones substantially suppressed serum titers also suggests that these T cell clones are able to migrate to the sites of antibody formation in vivo and have physiological relevance to the mechanism of antibody suppression upon induction of oral tolerance. With respect to produced cytokines, it may be significant that OPP2 and OPP4 do not produce an effector helper cytokine such as IL-4 (Table 1).

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Fig. 7. BLG-specific antibody titers in the serum of mice following adoptive transfer of OPP clones. Clones OPP2, OPP3 and OPP4 were harvested at their resting stage, purified by density separation (Lympholyte M), and washed 3 times with RPMI 1640. PBS (0.2 ml) containing 2 x 106 T cell clones or PBS alone was injected i.v. into BALB/c mice. All mice were immunized with 50 µg/animal of BLG in CFA 30 min after the cell transfer. Two weeks later, the mice were given another transfer of T cell clones and boosted i.p. with 50 µg BLG in IFA. One week after the second immunization, the mice were bled and sera were subjected to ELISA for determination of the BLG-specific antibody titer. Three to five mice were used for each experimental group. Similar results were obtained in different experiments. Data are shown as relative percents of ELISA units of each group when the mean of ELISA units for non-transferred mice was set to 100% for each isotype. *P < 0.05, **P < 0.01 (comparison of serum antibody levels between groups with or without each OPP clone).
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In accord with the in vitro data, suppression of IgG in vivo was not affected when OPP2 and anti-TGF-ß were injected into mice followed by immunization with BLG (data not shown). Anti-IFN-
reversed the enhancement of serum IgG2a production to normal levels, although it was not effective in reversing the suppression of IgG1 (Fig. 8). Thus, with regard to IgG2a production, IFN-
may be involved in immune deviation when OPP clones are transferred to mice.
After subsequent parenteral immunization, suppressive effects on in vitro antibody production are observed in LN cells of animals fed high-dose BLG
Treg seem to originate in PP after administration of high-dose protein antigen (BLG), and antigen-specific T cell clones derived from such populations elicit suppressive functions both in T and B cell immune responses. Since they not only suppress antibody titer in vitro, but also in vivo (serum titer), when transferred to BALB/c mice followed by immunization, PP-derived regulatory cells may also function in other organs where antigens and adjuvants are provided. To confirm the idea with primary culture cells, we fed BALB/c mice with high-dose BLG, immunized i.p. and s.c. with CFA, and analyzed their LN and SP cells for active suppression using the in vitro antibody production system (Fig. 9). Unlike the results without systemic immunization (Fig. 1), significant suppressive activity on IgG and IgG1 production was observed in LN cells, indicating that Treg migrated to other organs and functionally matured by immunization. When the organs were taken 14 days after the immunization, strong enhancement of antibody production was observed by the presence of LN cells from non-fed and immunized mice (induction of helper-type cells), while LN cells from fed and immunized mice retained their suppressive effect (data not shown). These observation may suggest the present in vitro system reflects the physiological Treg function and may explain part of the mechanisms in high-dose oral tolerance, in which oral administration of protein antigen in advance suppresses the subsequent systemic immune responses to the same protein antigen that are otherwise elicited by a parenteral immunization with adjuvant.

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Fig. 9. Inhibition of BLG-specific antibody production in vitro by primary culture cells from BALB/c mice that were fed BLG and immunized. Periaortic, popliteal and inguinal LN, and SP cells were collected from the BLG-fed and non-fed mice after immunizing them with BLG with CFA. Cells (2 x 106 cells/well) were placed in culture inserts (upper well). Primed B cells (1 x 106 cells/well) and a BLG-specific CD4+ Th cell clone (H36; 1 x 104 cells/well) were placed in a 24-well culture plate (bottom well). Primed B cells were prepared as described in Methods. After 72 h of co-culture in the presence of BLG (20 µg/ml), cells were washed and cultured for an additional 3 days in the absence of antigen before collection of supernatants for the detection of secreted BLG-specific antibodies by ELISA. Representative data from three independent experiments are shown. *P < 0.05, **P < 0.01.
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Discussion
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Based on accumulating evidence for inhibitory effects in functional T cell subsets, CD4+ Treg seem to be divided in two major groups; thymus-derived, CD25+ and anergic naturally occurring Treg that consists of 510% of peripheral mature CD4+ cells, and effector suppressor Treg induced in peripheral immune response, often related to intestinal immunity. They also differ in a way that the former require cellcell contact, while in addition to cellcell contact, soluble factors such as IL-10 and TGF-ß partly describe the suppression for the latter (810,12). It is still to be further addressed, however, if CD25-bearing anergic Treg are also induced via peripheral activation and/or tolerization in normal mice, and if the two groups may share any biological and biochemical characteristics despite of the difference in their origin and activation route.
It was recently reported that transferred TCR transgenic CD4+ T cells from DO11.10 mice differentiate into anergic, CD4+CD25+ anergic and suppressive T cells upon i.v. injection or oral administration of antigen (16). Since recipient mice were irradiated and donor DO11.10 mice were RAG/ in their study, the experimental system was simple and the results were clear. It was also reported that CD4+CD25+ regulatory cells are activated after oral administration of antigen to TCR transgenic mice (27). Such findings suggest that anergic, CD4+CD25+ Treg-like cells are induced among the normal T cell repertoire in non-transgenic mice also.
BLG-specific OPP clones in the present study bear CD4+CD25+ and the anergic phenotype. They were established from PP of normal BALB/c mice after oral administration of high-dose BLG. As the frequency of antigen-specific T cells is so small, cloning was the most reliable way to show such antigen-specific Treg are physiologically induced in normal mice upon oral administration of exogenous foreign antigen. These clones may share some biological and/or biochemical characteristics with thymus-derived, naturally occurring CD25+ Treg or inducible Treg observed in T cells from DO11.10 TCR transgenic mice.
The reason why only PP cells exhibit suppressive activity after BLG feeding in BALB/c mice is not clear yet (Fig. 1). There are several possibilities to answer this question. PP are the physiological route of antigen incorporation and it is the first place where T cells see the antigen, and if T cells are educated in PP, they prefer to migrate back to the mucosal immune site. We observed that OPP clones also affect PP mostly after in vivo transfer (our unpublished observation). In this way, the frequency of inducible Treg may be less in other organs even if there are some, although not enough, to exhibit the antigen-specific activation of the regulatory function. In addition, innate signals provided by commensal bacteria or food components abundant in the mucosal immune site may affect T cell function via the specialized function of APC. Dendritic cells in PP are unique and their cytokine (i.e. IL-10) production, for instance, may affect the differentiation of antigen-specific T cells (28). After parenteral immunization, LN cells also exhibited antigen-specific induction of regulatory effects (Fig. 9). We do not know yet if the functional Treg in LN share common characteristics with inducible Treg in PP as observed in the present study of cloned cells, if Treg in PP migrate to LN and expand or if Treg are newly generated after parenteral immunization. Although these questions are to be answered in future studies, it seems possible that inducible, antigen-specific Treg are responsible for the active suppression and are relevant to the phenomenon of high-dose oral tolerance, where anergy is one of the mechanisms (29,30).
To detect the function of Treg, we employed a system to evaluate the regulation of the Th2-associated immune response and a feeding protocol with a high dose of antigen. Suppression of antibody response often requires a high-dose feeding regimen since the antibody response (Th2-associated response) resists the tolerance-inducing protocol more strongly than the Th1-associated response that is diminished easily (3133). Thus, in order to regulate antibody-associated diseases, such as allergy, it is expected that different types of Treg than that regulate Th1-associated inflammation are induced.
We observed that OPP clones regulate both T and B cell response. They use TGF-ß, IL-10 and IFN-
to inhibit T cell proliferation, as each of the neutralizing antibodies to the cytokine was partially effective in reversing the suppression (data not shown). Moreover, IgM suppression mediated by OPP2 was neutralized with anti-TGF-ß antibodies (Fig. 6). However, IgG suppression was not explained by any single cytokine in the same panel (Fig. 6) and we assume that there is an unknown mechanism involved. The ongoing study on the use of a mouse model for food allergy indicates that OPP clones are also effective in regulating IgE production (our unpublished observation), suggesting the possibility that these CD4+CD25+ Treg clones are able to regulate detrimental humoral responses. It is therefore important for clinical use to identify the suppressor effector molecules, as well as the co-stimulatory factor for their survival and expansion in vivo. Such an understanding about inducible, effector Treg will help clinicians to utilize suitable Treg to design therapies for the treatment of different immune disorders.
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Acknowledgements
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We thank Ms Lyudmila Rachkova for expert technical assistance. This work was partially supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan.
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Abbreviations
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APCantigen-presenting cell
BLGß-lactoglobulin
CFAcomplete Freunds adjuvant
Con Aconcanavalin A
IFAincomplete Freunds adjuvant
LPSlipopolysaccharide
MLNmesenteric lymph node
MMCmitomycin C
PBSTPBS/0.05% Tween 20
PPPeyers patch
SPspleen
TGFtransforming growth factor
TNFtumor necrosis factor
Tregregulatory T cells
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