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The Journal of Immunology, 2001, 166: 5773-5781.
Copyright © 2001 by The American Association of Immunologists

Rapid Depletion of Peripheral Antigen-Specific T Cells in TCR-Transgenic Mice After Oral Administration of Myelin Basic Protein1

Abbie L. Meyer2,*, Jacqueline Benson3,*, Fei Song*, Najma Javed4,*, Ingrid E. Gienapp*, Joan Goverman{dagger}, Thea A. Brabb{dagger}, Leroy Hood{dagger} and Caroline C. Whitacre5,*

* Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University College of Medicine and Public Health, Columbus, OH 43210; and {dagger} University of Washington, Department of Molecular Biotechnology, Seattle, WA 98195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In myelin basic protein (MBP)-specific TCR-transgenic (Tg) mice, peripheral T cells express the V{alpha}2.3/V{beta}8.2-Tg TCR, demonstrate vigorous proliferative responses to MBP in vitro, and can exhibit experimental autoimmune encephalomyelitis (EAE) within 5 days of pertussis toxin injection. We explored the effects of oral administration of MBP on the cellular trafficking of the MBP-specific TCR-Tg cells and the ability of oral MBP to protect Tg mice from EAE. Tg mice were fed MBP, OVA or vehicle and sacrificed at various times after feeding. An immediate and dramatic decrease in V{alpha}2.3/V{beta}8.2+-Tg cells was observed in the periphery within 1 h after feeding. By 3 days after feeding, the percentage of Tg cells increased to near control levels, but decreased again by 10 days. When MBP or vehicle-fed Tg mice were challenged for EAE at this point, disease was severe in the vehicle-fed mice and reduced in the MBP-fed mice over the 40-day observation period. In vitro studies revealed a biphasic pattern of MBP proliferative unresponsiveness and an induction of Th1 cytokines. Immunohistochemical staining showed that the number of Tg cells found in the intestinal lamina propria increased dramatically as the number of Tg cells in the periphery decreased. There was no apparent proliferation of Tg cells in the lamina propria, indicating that Tg cells trafficked there from the periphery. Taken together, these results suggest that T cell trafficking into the site of Ag deposition acts to protect the TCR-Tg mouse from EAE.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Experimental autoimmune encephalomyelitis (EAE)6 is an inflammatory autoimmune disease of the CNS that serves as a useful animal model for testing treatment strategies for multiple sclerosis. EAE is induced in mice and rats by injection of myelin, myelin basic protein (MBP), proteolipid protein, or myelin-derived peptides combined with adjuvant. In susceptible mouse strains such as SJL/J (H-2s), PL/J, and B10.PL (H-2u), EAE can follow a long-term chronic-relapsing course (1, 2, 3, 4, 5, 6) that closely mimics the clinical and histopathologic features of multiple sclerosis. EAE in the mouse has the additional benefit that the efficacy of treatment modalities may be assessed during different stages of disease, such as during a remission period or during a relapse of clinical signs.

EAE is mediated by MHC class II-restricted CD4+ T cells specific for the neuroantigen. After immunization of PL/J mice with the immunodominant epitope of MBP (NAc1-9), 85% of T cell lines generated used the V{beta}8 chain of the TCR (7). The B10.PL mouse also uses the V{beta}8.2 segment together with V{alpha}2.3 in recognition of the NAc1-9 epitope (8, 9). Based on this restricted TCR usage, TCR-transgenic (Tg) mouse lines have been developed that express V{alpha}2.3/V{beta}8.2 and V{alpha}4/V{beta}8.2 as transgenes (10, 11). Fulminant EAE can be induced in these mice by the injection of MBP and pertussis toxin or pertussis toxin alone (12).

Oral tolerance is defined as the Ag-specific suppression of the immune response after the oral administration of a protein Ag. We and others (13, 14, 15) have reported that the oral administration of MBP to Lewis rats and mice before encephalitogenic challenge results in the suppression of acute EAE. The suppression was shown to be highly specific for the fed Ag (16) and was characterized by decreased T cell as well as B cell responses (17, 18, 19). Ag composition has been shown to play an important role in oral tolerance, with suppression of EAE after oral administration of MBP but not myelin (20). Mice were protected from chronic relapsing EAE when a single oral dose of MBP was given before challenge or on the first day of clinical disease. However, multiple oral doses of Ag were required to suppress EAE once relapsing disease was established.

When considering the mechanisms underlying oral Ag-induced unresponsiveness, there are at least five mechanisms that have been put forward: clonal anergy (17, 21), deletion (22, 23), altered trafficking (24), active suppression (25, 26, 27, 28), and immune deviation (Th1-Th2 cytokine shift; Refs. 27, 28, 29). The dose of oral Ag administered appears to play a critical role in determination of the operative mechanism, with low doses favoring active suppression/immune deviation and higher doses favoring induction of anergy and deletion (17, 30, 31). Studies of oral feeding of OVA in OVA-specific TCR-Tg mice have generated data in support of all mechanisms mentioned. In the MBP-specific TCR-Tg mouse, Chen et al. (29) demonstrated regulatory T cells that secrete TGF-{beta} and are capable of transferring protection from EAE. In most of the reports describing Ag feeding in Tg mouse models, a single point in time is chosen for assessment of the mechanism of tolerance. In a cytochrome c-specific TCR-Tg mouse model, the effect of feeding was seen as early as 6 h after oral Ag (32). We reasoned that the response to oral Ag administration is a dynamic and changing process, with some changes evident early on after feeding but resulting in a long-lasting state of tolerance.

Therefore, the present study was designed to monitor the location, phenotype, and function of Tg T lymphocytes in mice over time after oral administration of MBP to MBP-TCR-Tg mice, The results show that the number of Tg cells in the periphery is reduced dramatically as soon as 1 h after oral MBP and that these cells traffic to the lamina propria of the gut. At later time points, functional parameters are decreased and the Tg mice are protected from EAE.


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

Male and female B10.PL mice aged 6–10 wk were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed at Ohio State University (Columbus, OH). Separate V{alpha}2- and V{beta}8-Tg mouse lines were bred with B10.PL mice to generate a colony of breeders made up of a V{alpha}2 male or female plus a V{beta}8 female or male (10). Mice were backcrossed onto the B10.PL line for at least five generations. Progeny were screened by flow cytometry for expression of both V{alpha}2 and V{beta}8 transgenes in PBLs, and V{alpha}2/V{beta}8 double-positive mice were used in experiments at 6–8 wk of age. Single-positive litter mates were used to set up additional breeding pairs, and double-negative litter mates were used as controls.

Antigen

Guinea pig (GP) MBP was extracted from spinal cords (Rockland, Gilbertsville, PA) by the method of Diebler et al. (33) or Swanborg et al. (34). MBP was purified further on a Sephadex G-50 column eluted with 0.01 N HCl. Individual fractions were analyzed by SDS-PAGE, and those fractions containing a single band of the appropriate molecular mass were pooled. The purified MBP was dialyzed against water and lyophilized. Purified protein derivative was obtained from Parke-Davis (Morris Plains, NJ) and ConA was obtained from Sigma (St. Louis, MO).

Induction of oral tolerance

Mice were deprived of food but not water for 5–10 h before oral administration of Ag. Mice then were given 100 mg of GP-MBP or OVA (Sigma) in 0.5 ml of PBS administered by gastric intubation in a single feeding. Alternatively, mice were fed MBP and then challenged with pertussis toxin 10 and 12 days after feeding.

Induction of EAE

EAE was induced in Tg mice in two ways: by two 200-ng i.p. injections of pertussis toxin (List Biological Laboratories, Campbell, CA) 48 h apart or by s.c. injection over four sites on the flank with 200 µg of GP MBP in CFA containing 200 µg of heat-killed Mycobacterium tuberculosis, Jamaica strain. The latter group also received two 200-ng i.p. injections of pertussis toxin 48 h apart as described previously (12). Animals were observed for the onset of clinical disease, which was scored as follows: limp tail or waddling gait with tail tonicity, 1; ataxia or waddling gate with tail limpness, 2; partial hind-limb paralysis, 3; total hind-limb paralysis, 4; death, 5. Additionally a score of 2 was assigned to animals whose paralysis was apparent in forelimbs only. Observations of clinical disease were made for the length of time indicated in figure legends.

Flow cytometry

Cell suspensions containing 0.5 x 106 cells were incubated with 50 µl of direct-labeled mAbs diluted in PBS with azide (S/P Baxter, McGraw Park, IL) containing 2% rat serum in the following Ab combinations: PE-labeled anti-mouse V{beta}8.1/8.2 TCR + FITC-labeled anti-mouse V{alpha}2 TCR; PE-labeled anti-mouse CD4 (L3T4) + FITC-labeled anti-mouse CD8a (Ly-2); or isotype controls PE-labeled mouse IgG2a {kappa} (anti-trinitrophenol) plus FITC-labeled rat IgG2a (all Abs at 1–2 µg/tube; BD PharMingen, San Diego, CA). After a 45-min incubation, cells were washed and resuspended in 1% paraformaldehyde. Analysis was performed on an Epics XL flow cytometer (Coulter, Hialeah, FL). Forward and right-angle light scatter were used to gate the lymphocyte population and to exclude monocytes, granulocytes, and dead cells.

Preparation of lamina propria lymphocytes (LPL)

The entire length of the small bowel was removed and flushed with PBS. The Peyer’s patches were excised from the intestinal wall, and the small intestine was opened longitudinally and cut into pieces measuring 5–10 mm. The intestinal pieces were placed into Medium 199 (Life Technologies, Grand Island, NY) supplemented with 1 mM DTT and shaken at 37°C for 60 min. The tissue fragments then were floated in medium and digested with collagenase type VIII (Sigma) with constant shaking for 60 min at 37°C. Cells in the supernatant were harvested, washed, filtered through a 70-µm cell strainer (Becton Dickinson, Franklin Lake, NJ), washed, and placed on a discontinuous 40–100% Percoll gradient. After centrifugation for 20 min at 600 x g, lymphoid cells were collected from the interface, washed, and resuspended in medium containing 10% FBS. To test for functional activity of LPL, cells (0.5 x 106 LPL) were stimulated with MBP (50 µg/ml) in the presence of peritoneal exudate cells (0.05 x 106) as a source of APCs for 72 h, including a pulse with [3H]thymidine for the final 18 h of culture.

Lymphocyte proliferation

Mice were sacrificed 1, 3, 7, 8, or 10 days after feeding, and peripheral lymph nodes (pooled cervical, axillary, brachial, inguinal, popliteal, and periaortic), mesenteric lymph nodes, spleen, Peyer’s patches, and thymus were harvested and processed into single-cell suspensions. The cells were washed in HBSS (BioWhittaker, Walkersville, MD) and then resuspended in RPMI 1640 medium (BioWhittaker) containing 10% FBS (HyClone Laboratories, Logan, UT), 25 mM HEPES (Life Technologies), 2 mM L-glutamine, 50 U/ml penicillin-50 µg/ml streptomycin (BioWhittaker), and 5 x 10-5 M 2-ME (Bio-Rad Laboratories, Richmond, CA). Cells (4 x 105/well) were distributed into 96-well round-bottom plates (ICN Biomedicals, Costa Mesa, CA) and cultured with MBP (10, 40, 100, or 200 µg/ml), purified protein derivative (40 µg/ml), Con A (2 µg/ml), or medium alone. Cultures were incubated for 72 h in 7% CO2 at 37°C including an 18-h pulse with 1 µCi [3H]thymidine (Amersham, Arlington Heights, IL). The plates were harvested onto glass-fiber filter mats (Skatron, Sterling, VA) and counted on a {beta}-plate scintillation counter (Wallac, Turku, Finland). Data is reported as mean stimulation index, which is calculated by dividing the mean cpm for wells containing cells plus Ag by the mean cpm of wells containing cells in the absence of Ag.

Cytokine determinations

Supernatants were harvested at 24, 48, and 72 h from 24-well plate cultures of cells (4 x 106/ml) stimulated with medium alone, 40 µg/ml MBP, or 5 µg/ml Con A cultured in serum-free medium (X-Vivo; BioWhittaker). Peripheral lymph node cells (LNC), mesenteric LNC, and spleen cells were cultured separately. Capture ELISAs for the detection of IL-2, IFN-{gamma}, and IL-10 were conducted according to manufacturer’s recommendations (BD PharMingen). Capture Abs (2 µg/ml in bicarbonate buffer) were incubated in Immulon II 96-well ELISA plates (Dynatech Laboratories, Chantilly, VA) at 4°C overnight. After washing, the plates were blocked with 3% BSA (Sigma) for 1 h and then washed again. One hundred microliters of each sample or standard dilution (recombinant mouse IL-2, IFN-{gamma}, and IL-10; BD PharMingen) were added to wells in duplicate and incubated by shaking at room temperature for 2 h. Biotinylated detection Abs (anti-IL-2, IFN-{gamma}, and IL-10; BD PharMingen) were diluted in 3% BSA-PBS to 1–2 µg/ml and added to wells for 1 h. For the detection of TGF-{beta}, 2.5 µg/ml chicken anti-TGF-{beta} (R&D Systems, Minneapolis, MN) was used as capture Ab followed by blocking and the addition of 72-h supernatants as described above. Mouse anti-TGF-{beta}1, -TGF-{beta}2, and -TGF-{beta}3 (1 µg/ml; Genzyme, Cambridge, MA) was added as the detection Ab, followed by 1 µg/ml biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA). After extensive washing of all plates, avidin peroxidase (Sigma) was added followed by ABTS substrate (Boehringer Mannheim, Indianapolis, IN). Plates were incubated in the dark for 15–30 min and then read at 405 nm on a Bio-Rad ELISA reader. Cytokine concentrations were determined by comparing the OD of samples to the appropriate standard curve. The lower limits of detection of the cytokine ELISA (as specified by the manufacturer for these assay conditions) were as follows: IFN-{gamma}, 15–30 pg/ml; IL-2, 8–15 pg/ml; IL-10, 15–30 pg/ml; and TGF-{beta}, 10 pg/ml.

Immunohistochemistry

For immunohistochemical detection of cells in the small intestine, animals were sacrificed, and the small intestine was removed immediately. The intestinal lumen was rinsed with chilled HBSS and 1-inch segments were bathed in OCT tissue freezing medium. Tissues then were snap-frozen in liquid nitrogen-cooled isopentane (2-methylbutane) and stored at -70°C. Tissues were cut at a thickness of 4–6 µm with a cryostat, adhered to gelatin-treated glass slides, and fixed in cold acetone for 10 min. Sections were hydrated in TBS for 5 min and then treated with rabbit serum (1:5) for 10 min in a humidified slide chamber at 37°C on a slide warmer. Excess serum was drained and primary unlabeled mAb (anti-V{alpha}2, 1:20 to 1:50; BD PharMingen) was applied for 30 min. Slides were rinsed two or three times with TBS, and biotinylated secondary Ab (1:200; Vector Laboratories) was applied for 10 min at 37°C. Sections were washed, and HRP avidin D (1:500; Vector Laboratories) was applied for 10 min. Sections were washed and incubated in acetate buffer for 5 min. Slides were developed with 3-amino-9-ethylcarbazole (Vector Laboratories) in hydrogen peroxide substrate solution. Sections were examined by light microscopy, and the number of V{alpha}2-staining cells counted in 10-high power fields.

BrdU staining

Two hours before sacrifice, Tg mice were injected i.p. with 1 ml of BrdU (1-bromo-2'deoxyuridine and 5-fluoro-2'deoxyuridine) labeling reagent (Zymed, South San Francisco, CA) per 100 g of body weight. Mesenteric lymph nodes and small intestine sections containing Peyer’s patches were removed from fed mice and immediately fixed in 10% neutral-buffered formalin for 24–36 h. After paraffin embedding, sections measuring 3–5 µm were cut and placed on poly-L-lysine-coated slides. Tissues were stained for detection of BrdU (Zymed). Briefly, endogenous peroxidases were quenched with H2O2 in methanol for 10 min. Tissues were trypsinized with 0.125% trypsin reagent for 3 min, denatured and nonspecific binding blocked. Biotinylated mouse anti-BrdU was applied for 60 min and washed, followed by streptavidin-peroxidase conjugate and diaminobenzidine substrate. Tissues were observed microscopically, and positively stained cells appeared brown to black on a lightly colored background.

Statistical analysis

A two-tailed Student t test was used to determine statistical significance between prefeeding and postfeeding phenotype values. ANOVA with Tukey’s post hoc analysis was used to determine differences among groups over time after feeding Tg mice. All determinations were made with a 95% confidence interval and were considered significant at the p < 0.05 level.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The effects of orally administered MBP on the Tg phenotype

To determine the effect of orally administered MBP on Ag-specific T lymphocytes over time, a single oral dose of MBP was administered to V{alpha}2/V{beta}8.2 TCR-Tg mice. A 100-mg oral dose of MBP in PBS was determined to be the optimal amount in preliminary dose response experiments (data not shown). After feeding, animals were monitored for the presence of Tg lymphocytes 1, 3, 7, and 10 days after gavage. As soon as 1 day after MBP feeding, V{alpha}2/V{beta}8+-Tg cells were dramatically reduced in the blood compared with nonfed and pretreatment controls (Fig. 1Goa). Although the Tg T cells increased on days 3 and 7 after feeding, there was a decrease observed again by 10 days after feeding. Tg+ lymphocytes in the MBP-fed mice were significantly reduced in the blood at all time points examined relative to pretreatment values, whereas the values for the nonfed mice remained nearly the same over a comparable time interval (Fig. 1Goa). We observed that the decrease in double-positive T cells in the blood after feeding MBP is accompanied by an increase in non-double-positive CD4+ T cells, with maximal increases in non-double-positive cells occurring 1 and 10 days after feeding (data not shown). To determine how rapidly the Tg+ cells are reduced after feeding, blood was sampled from Tg mice 1, 6, or 20 h after MBP feeding. Remarkably, V{alpha}2/V{beta}8+ cells were reduced as soon as 1 h after feeding compared with prefeeding values or vehicle-fed controls (Fig. 1Gob). Feeding a similar dose (100 mg) of OVA, an Ag not recognized by MBP TCR-Tg cells, had no effect on the number of V{alpha}2/V{beta}8+ cells in the peripheral blood.



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  		FIGURE 1. V{alpha}2/V{beta}8 double-positive cells are reduced in blood after MBP feeding. Flow cytometric determinations of Tg cells were conducted on the same mice before () and after ({blacksquare}) MBP feeding. a, Tg mice were not fed (control) or fed 100 mg of MBP and sacrificed 1, 3, 7, or 10 days later. A total of 3000 lymphocyte-gated events were analyzed after lysis of anti-V{alpha}2/V{beta}8-stained blood. Data were combined from four separate experiments and represent a total of from three to five mice per group. Control nonfed mice were included with each experiment. b, To examine earlier time points, Tg mice were fed 100 mg of MBP, 100 mg of OVA, or vehicle and sacrificed 1, 6, or 20 h later. Data were combined from two experiments. n = 2 per group. Percentages of double-positive cells are reduced at all time points after feeding MBP when compared with vehicle-fed control or pretreatment values: *, p < 0.05.

 
A variety of lymphoid organs were examined by flow cytometry for the presence or absence of Tg+ lymphocytes after oral MBP administration. A similar pattern was observed in all lymphoid organs, except for the thymus (Fig. 2Go, a–f). Reduced percentages of Tg cells were seen 1 day after MBP feeding followed by increased levels 3 and 7 days after feeding, and a final decrease was observed on day 10. The reduction in Tg+ cells in blood and LNC was highly significant for 1 and 10 days after feeding (Fig. 2Go, a and b). Only day 1 values were significantly reduced in spleen and mesenteric LNC, although the pattern observed was similar to the other lymphoid organs (Fig. 2Go, c and d). In Peyer’s patches, the overall number of double-positive cells increased over nonfed levels 3 and 7 days after feeding (Fig. 2Goe). There was no apparent effect of MBP feeding on V{alpha}2/V{beta}8+-Tg cells found in the thymus (Fig. 2Gof). Examination of lymphoid organs at later time points (days 16–17 after feeding) showed that the reduction in Tg cells was still observed (data not shown), suggestive of long-term reduction in Tg cells.



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  		FIGURE 2. The distribution of Tg lymphocytes in various lymphoid tissues after oral MBP. Mice were fed 100 mg of MBP or not (nonfed control) and were sacrificed on the days indicated after feeding. Cells from (a) blood; (b) LNC; (c) spleen; (d) mesenteric LNC; (e) Peyer’s patch; and (f) thymus were stained and analyzed as in Fig. 1Go. In blood, LNC, spleen, and mesenteric LNC, n = 3–4 per MBP-fed group and n = 10 for nonfed; in Peyer’s patch, n = 1–3 per MBP-fed group and n = 3–5 for nonfed; in thymus, n = 1–2 per MBP-fed group and n = 3 for nonfed. *, Values were significantly different from nonfed controls: *, p < 0.05; **, p < 0.005; ***, p < 0.001.

 
The percentage of CD4-bearing lymphocytes was monitored at various times after feeding to determine whether the decrease in V{alpha}2/V{beta}8+ cells was mirrored in CD4 expression. Like TCR-Tg+ cells, CD4+ cells were observed to be depleted from the blood as quickly as 1 h after feeding, and feeding OVA had no effect on the number of CD4+ cells (data not shown). In LNC, the pattern of CD4 expression over time resembled that of Tg+ cells, with significant decreases at day 1 and day 10.

The effect of orally administered MBP on the course of EAE in the Tg mouse

To assess whether the oral administration of MBP protects mice from EAE, Tg animals were fed 100 mg of MBP or OVA and then were challenged with MBP/CFA/pertussis (Fig. 3Goa) or pertussis toxin alone (Fig. 3Gob) 10 and 12 days after feeding. Both MBP/CFA/pertussis as well as pertussis toxin alone previously have been reported to induce severe EAE in this Tg mouse strain (10, 12). EAE was severe in nonfed or OVA-fed Tg mice challenged with either regimen, with six of seven mice progressing to death by 35 days after challenge (Fig. 3Go). When MBP was given orally before challenge, the severity of disease was markedly suppressed. Only one of six MBP-fed Tg mice developed severe EAE and died, with the remainder displaying only mild signs of EAE. The average clinical score per day (average cumulative score divided by the number of days observed) was significantly reduced in MBP-fed mice compared with controls (0.7 ± 0.7 compared with 2.7 ± 0.9, respectively; p < 0.005). Thus, a single oral administration of MBP protects MBP TCR-Tg mice from severe EAE over the course of 60 days.



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  		FIGURE 3. Orally administered MBP protects MBP TCR-Tg mice from EAE. a, Mice were fed 100 mg of MBP or OVA 10 days before immunization with MBP/CFA/pertussis toxin. Mice were monitored for clinical signs of EAE for 40 days. Data represent two mice per group. b, Mice were fed 100 mg of MBP or not and then received two i.p. injections of pertussis toxin 10 and 12 days later. Mice were observed for 40 days. Data are from two experiments with n = 5 for the nonfed group and n = 4 for the MBP-fed group.

 
The effects of oral MBP on immune function

To determine whether immune function was altered as a result of MBP feeding, proliferative responses and cytokine secretion patterns were analyzed. Fig. 4Go shows that the Ag-specific proliferation for LNC was decreased 1 day after the oral administration of MBP in Tg mice compared with nonfed control and OVA-fed mice. The proliferative response then increased on days 3 and 7, returning to near control levels, and declined by day 10, thus mirroring the pattern that was observed in Tg phenotype (Fig. 2Go). A similar pattern was observed in the spleen and mesenteric LN compartments as well (data not shown). Interestingly, the decreased response observed 1 and 10 days after feeding cannot be explained by normalizing the numbers of double-positive cells placed in each well (data not shown).



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  		FIGURE 4. Proliferative response to MBP in MBP-fed Tg mice. Mice were fed MBP, OVA or nothing (nonfed control) before day of sacrifice. LNC were cultured with MBP or medium and later pulsed with [3H]thymidine. n = 5–6 for the MBP-fed group and n = 7–8 for nonfed. Data is combined from four or five experiments. Background cpm (LNC cultured with medium alone) for the different days of sacrifice are as follows: for day 1, 1800–2700 cpm; day 3, 1100–3000 cpm; day 7, 2500–3200 cpm; and day 10, 200-2700 cpm. LNC response at day 10 is significantly different from control. Responses at days 1, 7, and 10 are significantly different from the OVA-fed group, ***, p < 0.001.

 
Levels of cytokines secreted by spleen cells from Tg mice also were measured. ELISA determinations were performed on MBP-stimulated supernatants to assess IFN-{gamma}, IL-2, IL-10, and TGF-{beta} produced by cells from MBP-fed mice relative to controls. Assessment of the Th1 cytokine IFN-{gamma} revealed a significant increase in IFN-{gamma} 3 days after feeding, followed by a decrease to control levels by day 10 (Fig. 5Goa). A similar pattern was observed for IL-2 (Fig. 5Gob). The Th2 cytokine IL-10 was increased on days 3 and 10, although some variability was noted between fed animals. (Fig. 5Goc). TGF-{beta} was detected in cultures of cells from MBP-fed Tg animals stimulated in vitro with MBP only 7 days after feeding (Fig. 5God). In the majority of instances, TGF{beta} levels did not exceed those demonstrable in cultures from nonfed mice or unstimulated cultures (Fig. 5God).



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  		FIGURE 5. Quantitation of IFN-{gamma}, IL-2, IL-10, and TGF-{beta} in supernatants from MBP-stimulated cultures. Cells (12 x 106) from nonfed or MBP-fed mice were stimulated with 40 µg/ml MBP and supernatants were harvested at 48 h for (a) IFN-{gamma} and (b) IL-2, and 72 h for (c) IL-10 and (d) TGF-{beta} determinations. Values were determined by capture ELISA and average OD of triplicate wells were compared with a standard curve with medium values subtracted (a–c). n = 2–4 per group. Data are from two experiments. **, p < 0.005 relative to nonfed and OVA-fed controls.

 
Trafficking

We examined the possibility that the rapid fluctuations in V{alpha}2/V{beta}8+ cells in the periphery, the changing proliferative responses to MBP, and protection from EAE were attributable to trafficking events. We examined where Ag-specific Tg cells would first encounter MBP, the gut. Tissue sections were treated with Abs specific for V{alpha}2 and visualized by indirect peroxidase staining. Fig. 6Goa illustrates the distribution of Tg+ cells in the gut tissue of a naive, untreated MBP TCR-Tg mouse. A few Tg+ lymphocytes are seen in the lamina propria, but not in the margins of the villi or the epithelium. When gut sections were examined 3 days after MBP feeding (Fig. 6Gob), the number of Tg+ cells was increased compared with controls. Cells were localized largely in the lamina propria (Fig. 6Gob). There was an ~3-fold increase in the number of Tg+ cells in the lamina propria of MBP-fed animals as soon as 1 day after feeding and evident through day 10 (Fig. 6Goc).



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  		FIGURE 6. V{alpha}2+ cells increase in intestinal sections of MBP-fed Tg mice. Frozen sections of gut from (a) nonfed Tg mouse and (b) MBP-fed Tg mouse sacrificed 3 days after MBP feeding were cut and stained with anti-V{alpha}2 Abs. Data is representative of two experiments. c, Average number of V{alpha}2 staining cells per 10-high power fields. Data are combined from two experiments and is significantly different at all time points compared with nonfed controls; p = 0.02.

 
To determine whether the V{alpha}2+ cells in the gut were already in residence there and had proliferated in situ or had trafficked there in response to Ag deposition, BrdU labeling of gut sections was undertaken. In these experiments, mice were fed MBP, vehicle, or OVA and then analyzed 1, 6, or 20 h after feeding. Two hours before sacrifice, animals were given an i.p. injection of BrdU. Paraffin-embedded tissues (mesenteric lymph nodes and small intestine sections containing Peyer’s patches) were prepared. Crypt cells that line the mucosal epithelium normally proliferate to form the mucosal epithelium. Only these cells at the lower margin of the villi stained positive for BrdU. Few other cells were observed as BrdU+ (data not shown), suggesting that Tg+ cells in the lamina propria of the gut were not proliferating in response to MBP-feeding. Because V{alpha}2 cells were observed to increase significantly in the lamina propria by 1 h after feeding, these findings suggest that V{alpha}2/V{beta}8+-Tg cells trafficked to the lamina propria in response to Ag deposition there.

In an effort to determine whether the cells trafficking to the lamina propria exhibited Ag-specific tolerance, LPL were isolated from MBP-fed or vehicle-fed mice and tested for their ability to proliferate in vitro in response to various stimuli. Fig. 7Go shows that LPL from MBP-fed mice did indeed exhibit a significantly reduced proliferative response when restimulated in vitro with the fed Ag, MBP, as well as to the immunodominant MBP peptide, Nac1-11, compared with vehicle-fed controls. However, the T cells responded when stimulated with anti-CD3, demonstrating that they were capable of proliferating. It should be noted that the degree of proliferation was relatively low, as is characteristic of cells isolated from this compartment. It should also be noted that LPL isolated directly from MBP-fed mice and placed in culture without Ag stimulation showed enhanced proliferation, suggestive of a generalized increase in proliferative activity. Thus, these results showed that Tg+ T cells, trafficking into the lamina propria compartment as a result of Ag feeding, exhibited reduced proliferative capacity specific for the fed Ag.



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  		FIGURE 7. LPL exhibit a reduced response to MBP after MBP feeding. Tg+ mice were fed 100 mg of MBP or PBS. One day later, the LPL were isolated from the small intestine and assayed for their ability to proliferate in the presence or absence of MBP (40 µg/ml), the immunodominant peptide of MBP Nac1-11 (10 µg/ml), or anti-CD3 (2 µg/ml) with peritoneal exudate cells from B10.PL mice as a source of APC. LPL were cultured for 48 h and then pulsed with [3H]thymidine for the final 18 h of culture. Results are expressed as cpm ± SEM. *, Significantly different from vehicle-fed control value; p < 0.05

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results reported here show that the oral administration of MBP to MBP TCR-Tg (V{alpha}2/V{beta}8+) mice, wherein the majority of T cells are specific for MBP, results in a rapid removal of Ag-specific cells from the periphery. This depletion occurs as rapidly as 1 h after oral Ag treatment. The initial depletion is followed by a return of Tg+ cells to the periphery lasting ~1 wk, and then a final depletion takes place that results in protection of mice from overwhelming EAE. Although both Th1 and Th2 cytokines are transiently produced, there is no apparent long-term induction of Th2 regulatory cells, nor is there a sustained increase in TGF-{beta} production. There is an influx of Ag-specific cells into the lamina propria soon after Ag feeding, and the cells are observed to be refractory to Ag restimulation.

Ag presentation in the gut is thought to be a critical step in the generation of oral tolerance. Intestinal epithelial cells have been shown to express MHC class II glycoproteins after stimulation and are capable of presenting Ag (35, 36, 37). However, the presentation of Ag may occur in the absence of appropriate costimulatory molecules or in the presence of suppressive costimulatory molecules in situ resulting in unresponsiveness. Sanderson et al. (38) reported that although spleen and intestinal epithelial tissue contained similar amounts of mRNA for MHC class II molecules, the amount of B7, an important costimulatory molecule, was drastically reduced in intestinal epithelium compared with spleen. They found that even as the level of class II message was increased in intestinal epithelial cells after parenteral IFN-{gamma} treatment, the level of B7 remained the same. Kuchroo et al. (39) showed that B7 was implicated in shaping the response to Ag because blocking B7-1 with Ab shifted T cell responses in vitro and in vivo away from the inflammatory Th1 type. Kelsall and Strober (40) identified dendritic cells from the Peyer’s patches that could be loaded with Ag in vivo by feeding mice with OVA. These Ag-loaded cells could stimulate OVA-specific TCR-Tg cell proliferation in vitro, yet animals were unresponsive to OVA challenge after OVA feeding. Further evidence for dendritic cell involvement in Ag presentation in oral tolerance has been shown by Viney et al. (41, 42), using the in vivo administration of the dendritic cell growth factor Flt3 ligand (Flt3L). These investigators showed that administration of Flt3L to mice before administration of OVA resulted in a marked enhancement of oral tolerance. We have made similar observations in EAE, showing enhanced disease suppression after oral feeding of MBP in Flt3L-treated mice. Galliaerde et al. (43) reported that in vivo dinitrochlorobenzene-loaded Peyer’s patch-derived APC were able to activate Ag-specific LNC in vitro, yet class II+ intestinal epithelial cells were unable to do so. When considered together, these results suggest that unresponsiveness after presentation of Ag by gut compartment cells is site-specific and may depend on additional signals or a lack of signals unique to the gut environment.

The fact that peripheral depletion of MBP-specific Tg+ cells occurred within 1 h after Ag feeding was surprising. By using tetramer reagents designed to identify T cells by their Ag specificity, others have shown a peripheral depletion of Ag-specific CD8+-Tg cells as soon as 6 h after feeding (32). Possible tolerizing signals could be the presence of gut-derived soluble Ag in the periphery, Ag carried by APCs, a soluble mediator, or a combination of signals. It has been shown that orally introduced Ag can be detected in the periphery as soon as 1 h after feeding (44). Furrie et al. (45) have reported that serum transferred from BALB/c mice fed OVA an hour earlier confers OVA-specific tolerance in recipient mice. These authors postulated that a fragment of OVA found in the transferred serum is the agent likely responsible for the generation of unresponsiveness. Furthermore, Bruce and Fergueson (46) reported that if serum from OVA-fed animals was absorbed with anti-OVA Ab coupled to Sepharose beads, the tolerogenic effect was abrogated.

Where the MBP-specific Tg T cells traffic after the oral tolerizing signal is a question that can be answered by searching tissues for the expression of the Tg TCR. Our search revealed trafficking of a significant number of Tg+ cells to the lamina propria, which were shown to exhibit decreased proliferative activity. Although naive T cells are observed to recirculate to lymphoid tissues via cell surface homing receptors, most memory and effector lymphocytes can also traffic into extralymphoid immune effector sites, including intestinal lamina propria (47). Once in the lamina propria, Ag-specific cells may be presented with Ag by cells that induce tolerance rather than activation (48). For example, Harper, et al. (49) pulsed lamina propria-derived APC with keyhole limpet hemocyanin (KLH) and injected these cells into the footpads of KLH-primed mice. A decrease in Ag-specific delayed-type hypersensitivity response was observed, whereas injection of similarly prepared spleen-derived KLH-pulsed APC enhanced delayed-type hypersensitivity. Thus, our observation of an increase in Tg cells migrating to the gut could be explained in a number of ways. One possibility is that Ag-specific T cells become activated in the periphery as a result of Ag feeding as shown by Gutegemann et al. (32) and preferentially migrate into multiple tissue sites, including the gut. Another possibility is that in response to Ag feeding, chemokine signals are generated that selectively recruit lymphocytes into the gut ,and it is the presence of MBP on the APC of the gut that retain the lymphocytes at this site. It is possible that the oral Ag-induced signal may also direct Ag-specific T cells to the liver. The liver contains a large number of CD4+ and CD8+ cells, many of which are undergoing apoptosis (50). When Ag is administered via the portal vein, similar to the route eventually taken by oral Ag, systemic tolerance also is induced (51). Furthermore, when the portal vein is ligated or shunted and circulation to the liver is prevented, oral tolerance is prevented (52, 53). Crispe and Mehal (50) argue that once in the liver, T cells encounter NK-like cells that could induce apoptosis via fas/fas ligand or other interactions. Thus, the liver may serve as a site for deletion in oral tolerance. Immunohistochemical studies reveal that there is an increase in the number of Tg cells in the gut (Fig. 6Go) after oral MBP feeding. BrdU studies reveal that the cells are not proliferating in situ in response to Ag found in the gut. Instead, it is envisioned that MBP is presented in the gut to cells circulating through the region. Other signals may induce cells to slow their migration and extravasate into the lamina propria of the gut. Once there, the cells may be induced to traffic to the liver via portal circulation and undergo apoptosis.

Levels of IL-2 and IFN-{gamma} produced after MBP feeding correspond to relative Tg cell numbers as measured by Tg phenotype and the MBP proliferative response over time, with the lowest levels of IFN-{gamma} found in primary cultures of spleen cells 1 and 10 days after MBP feeding. An increase in Th1 cytokine production occurs at the time of cell trafficking to the gut and may reflect the influence of new thymic emigrants. In the cytochrome C-Tg mouse, Gutegemann et al. (32) reported a reduction in IL-2 after feeding with no reciprocal increase in either Th2 cytokines or TGF-{beta}. The precise role of IFN-{gamma} in EAE may vary according to stage of disease (54, 55, 56, 57, 58). Our results suggest that in a Tg mouse with large numbers of MBP-specific cells, a deletion of those cells and, therefore, the ability to produce IFN-{gamma} correlates with protection from EAE. In our hands, the reduction in Th1 cytokines was not accompanied by a lasting reciprocal increase in Th2 cytokines, i.e., IL-10. Rather, the level of IL-10 increased transiently and then decreased. The burst of IL-10 at day 3 after feeding is significant in light of work by Groux, et al. (59) describing a role for IL-10 in the generation of peripheral unresponsiveness. Additionally, like Karpus et al. (60), who fed proteolipid protein peptide to SJL/J mice, we found no evidence of an increase in the amount of TGF-{beta}1, suggesting a mechanism other than the generation of regulatory T cells. We can conclude that the inflammatory response was suppressed, probably due to anergy or deletion of MBP specific cells.

One of the most remarkable findings presented here is the long-term persistence of protection from EAE after MBP feeding, whether the animals are challenged with MBP combined with pertussis or pertussis alone. We have reported previously that B10.PL mice are protected for longer than 100 days when fed before challenge with MBP (15). Similarly, Tg mice, with a vast increase in the Ag-specific cell population, are protected for as long as 40 days after challenge compared with nonfed or irrelevant Ag-fed Tg mice that develop lethal EAE. It is tempting to speculate that the redistribution of Tg cells into the gut is responsible for the lowered susceptibility to EAE. However, although these events are temporally associated, it is also possible that disease protection could be mediated by a response unrelated to T cell trafficking to the gut. For example, a reduction in expression of very late Ag-4 (VLA-4) by T cells would in effect interfere with T cell entry into the CNS. Cohort studies have shown that the incidence of spontaneous disease in these mice can be as high as 43% (12). Moreover, the induction of EAE in the V{alpha}2.3/V{beta}8.2 strain is critically dependent on the ability of Tg T cells to gain access to the CNS. This explains the requirement for pertussis toxin in EAE induction, which acts to increase the permeability of the blood-brain barrier.

These experiments have used direct feeding of one of the currently available MBP-TCR-Tg mouse strains (V{alpha}2.3/V{beta}8.2). Similar approaches have been used in other MBP-TCR-Tg strains (i.e., V{alpha}4/V{beta}8.2) as well as OVA-TCR-Tg mice with comparable but not identical results (22, 23). There are limitations imposed by the direct feeding approach, such as an abnormally skewed T cell repertoire in the Tg mouse that affects mechanisms serving to control autoreactivity. For example, in the V{alpha}4/V{beta}8.2 MBP TCR-Tg mouse, a population of CD4+ TCR {alpha}{beta}-bearing non-Tg regulatory cells has been described that serves to suppress the appearance of spontaneous EAE (61, 62, 63). When the V{alpha}4/V{beta}8.2 strain is crossed onto a Rag-/- background, wherein only Tg cells are present and regulatory cells have been eliminated, spontaneous EAE develops in 100% of the mice (11, 61, 62). The strain used in the studies reported here, expressing V{alpha}2.3/V{beta}8.2, exhibits a much greater degree of spontaneous EAE than the V{alpha}4/V{beta}8.2 strain and thus warrants special attention (10). It will be of interest in future studies to examine the V{alpha}2.3/V{beta}8.2 strain for the presence of such regulatory cells. To circumvent some of the issues surrounding the skewed T cell repertoire and inherent regulatory mechanisms of the Tg mouse, some oral feeding studies have been conducted with the adoptive transfer of Tg T cells to normal wild-type mice (21, 64). It is noteworthy that the same mechanisms, i.e., anergy, have been identified by using both direct feeding and adoptive transfer approaches. Thus, oral administration of Ag is a powerful means to alter the dynamics of experimentally induced or spontaneous autoimmune disease.


   Acknowledgments
 
We thank Drs. K.C. Dowdell and Kim Campbell for helpful discussions and Karen Cox and Scott Stuckman for technical assistance.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants NS23561, AI35960, and AI43376, and National Multiple Sclerosis Society Grant RG2302. Back

2 Current address: Tufts University School of Medicine, Department of Pathology, Boston, MA 02111. Back

3 Current address: Stanford University, Division of Immunology and Rheumatology, Stanford, CA 94305. Back

4 Current address: Ball State University, Department of Physiology and Health Science, Muncie, IN 47306. Back

5 Address correspondence and reprint requests to Dr. Caroline C. Whitacre, Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University College of Medicine, 333 West 10th Avenue, Columbus, OH 43210. Back

6 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; Tg, transgenic; GP, guinea pig; LNC, lymph node cells; BrdU, 1-bromo-2-deoxyuridine; KLH, keyhole limpet hemocyanin; LPL, lamina propria lymphocytes. Back

Received for publication March 21, 2000. Accepted for publication February 16, 2001.


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 Introduction
 Materials and Methods
 Results
 Discussion
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F. Song, Z. Guan, I. E. Gienapp, T. Shawler, J. Benson, and C. C. Whitacre
The Thymus Plays a Role in Oral Tolerance in Experimental Autoimmune Encephalomyelitis
J. Immunol., August 1, 2006; 177(3): 1500 - 1509.
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