Inductive Events in Oral Tolerance in the TCR Transgenic Adoptive Transfer Model

CELLULAR IMMUNOLOGY ARTICLE NO.

178, 62–68 (1997)

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Inductive Events in Oral Tolerance in the TCR Transgenic Adoptive Transfer Model1 Youhai Chen,2,3 Jun-ichi Inobe, and Howard L. Weiner Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115 Received January 8, 1997; accepted March 19, 1997

INTRODUCTION Oral administration of antigen induces a systemic hyporesponsiveness termed oral tolerance. High doses of oral antigen lead to deletion or anergy of T-cells whereas low doses induce regulatory T-cells that secrete Th2 cytokines (IL-4/IL-10) and TGF-b. The initiating events associated with oral tolerance have not been well characterized. We investigated the induction phase of oral tolerance by adoptively transferring ovalbulumin (OVA) p (323-339) TcR specific transgenic (Tg/) T-cells into BALB/c recipients that were then fed either a high (5 mg 1 5) or a low (0.1 mg 1 5) dose of OVA 323-329 peptide. The frequency of Tg/ T-cells in lymphoid tissues was determined by flow cytometry using an anti-clonotypic monoclonal antibody. In highdose-fed animals, Tg/ cells increased six- to eightfold in Peyer’s patches after one feeding and then progressively decreased to 44% of those in the control by Day 20. In contrast, a biphasic-type response was observed in lymph node and spleen where Tg/ cells decreased after the first feeding, returned to the control level, and then decreased to 36–63% of the control level by Day 20. In low-dose-fed animals, changes in Tg/ T cells were only observed in Peyer’s patches after five feedings, where cells increased approximately twofold. Tcell activation as measured by proliferation and IFNg secretion occurred in both low- and high-dose-fed animals after only one feeding and then declined whereas secretion of Th2 cytokines and TGF-b remained high even 10 days after the last feeding in low-dose-fed animals. Immunization with OVA/CFA demonstrated peripheral tolerance as measured by decreased proliferation and IFN-g secretion and was associated with increased production of TGF-b and IL10. These results suggest that the inductive phase of oral tolerance is characterized by an activation of antigen-specific T-cells that involves the initial secretion of IFN-g followed by prolonged secretion of Th2 cytokines and TGF-b. q 1997 Academic Press

Oral administration of antigen (oral tolerance) is a long-recognized method for inducing peripheral immunologic tolerance (1, 2). In addition to its physiologic role of preventing adverse immune responses to ingested proteins (3), oral tolerance has been successfully employed for the treatment of autoimmune diseases in animals (reviewed in 4) and is being tested in humans (5–9). The induction of tolerance following the oral administration of antigen may occur by a variety of mechanisms and is primarily dependent on the dose of antigen fed. Higher doses favor clonal deletion (10) and clonal anergy (11, 12), whereas lower doses favor the induction of regulatory cells which mediate suppression by the secretion of antiinflammatory cytokines such as TGF-b4 (13, 14). How these distinct mechanisms are induced after oral administration of antigen remains to be elucidated. We have recently investigated the immunologic effects of oral antigen in OVA TcR transgenic mice (10) and in MBP TcR transgenic (Tg/) mice (15) and found evidence of both deletion and immune deviation following oral antigen. In the present study, we investigated the induction phase of oral tolerance using an approach described by Kearney et al. (16) in which small numbers of OVA-specific cells from OVA TcR transgenic animals are transferred into conventional BALB/c animals. This allows investigation of the in vivo distribution of OVA-specific T-cells by flow cytometry using an anti-clonotypic mAb. Furthermore, the T-cell responses to fed antigen could be investigated ex vivo after oral administration of antigen without the need for immunization with adjuvant. MATERIALS AND METHODS Mice Female BALB/c mice, 6–8 weeks of age, were purchased from Jackson Laboratory (Bar Harbor, ME).

1 This work was supported by NIH Grants NS29352 and PO1AR/ AI43220. 2 Youhai Chen is the recipient of a fellowship from the Medical Research Council of Canada. 3 Current address: Institute for Human Gene Therapy, University of Pennsylvania, Philadelphia, PA 19104.

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0008-8749/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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4 Abbreviations used: IFN, interferon; OVA, ovalbumin; TGF-b, transforming growth factor beta.

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OVA-specific TcR transgenic mice (DO11.10) were provided by Dr. Dennis Y. Loh (Washington University, St. Louis, MO) (17) and were extensively backcrossed (ú15 generations) onto the BALB/c background. Mice were screened for the expression of OVA-specific TcR by flow cytometry with anti-clonotypic mAb KJ1-26. All mice were housed in the animal resource center of Children’s Hospital, Boston, under pathogen-free conditions. Adoptive Transfer This was performed essentially as described by Kearney et al. (16) except that enriched splenic T-cells instead of total splenocytes were used. A single cell suspension was prepared from the spleen of female OVA-specific TcR transgenic mice, 6 –8 weeks of age. Erythrocytes were removed by hemolysis and T-cells enriched by passage over a nylon wool column, after which they were washed and injected intraperitoneally into recipient female BALB/c mice. Each recipient mouse received 5 1 106 nylon wool enriched splenic T-cells. Feeding was initiated 2 days after adoptive transfer. Induction of Oral Tolerance Each mouse was fed with 0.1 or 5 mg of OVA peptide 323-339 in 0.5 ml of PBS by gastric intubation with an 18-gauge stainless-steel feeding needle (Thomas Scientific, Swedesboro, NJ). Feeding was repeated every other day for a total of five times.

ELISA for Cytokines Quantitative ELISA assays for IL-2, IL-4, IL-10, and IFN-g were performed using paired mAbs specific for corresponding cytokines per the manufacturer’s recommendations. TGF-b was determined as follows: 96-well microtiter plates (Dynatech, Chantilly, VA) were coated overnight at 47C with 5 mg/ml of chicken antiTGF-b1 polyclonal antibodies in 100 ml of carbonate buffer, pH 8.0. The plates were then washed three times with PBS containing 0.5% Tween 20, blocked with 1% BSA in PBS, washed, and incubated with culture supernatants or TGF-b1 standard overnight at 47C. The plates were washed again and incubated with mouse anti-TGF-b mAb (clone 1D11, 1 mg/ml) for an hour followed by peroxidase-labeled goat anti-mouse IgG (h / L) (KPL, Gaithersberg, MD) for 1 hr at room temperature. Color was developed with a one-component TMB reagent (KPL, Gaithersberg, MD). The sensitivity of this ELISA assay is approximately 50 pg/ml. Statistical Analysis Frequencies of transgenic T-cells were compared by x2 analysis. Cytokine concentration and stimulation index were analyzed by ANOVA. RESULTS Oral Administration of Antigen Results in an Increase of Tg/ T-Cells in Mucosal Lymphoid Tissue

OVA 323-339 peptide (ISQAVHAAHAEINEAGR) and myelin basic protein (MBP) 1-11 peptide (Ac-ASQKRPSQRHG) were synthesized and purified by HPLC. Polyclonal chicken anti-TGF-b1 antibody was purchased from R&D Systems (Minneapolis, MN); purified bovine TGF-b1 and monoclonal mouse anti-TGF-b antibody (clone 1D11.16) were kindly provided by Celtrix Pharmaceuticals (Santa Clara, CA). The following reagents were purchased from Pharmingen (San Diego, CA): purified rat anti-mouse IL-2 (clone JES-1A12), IL4 (clone BVD4-1D11), IL-10 (clone JES5-2A5), and IFN-g (clone R4-6A2) mAb; Biotinylated rat antimouse IL-2 (clone JES6-5H4), IL-4 (clone BVD6-24G2), IL-10 (clone SXC-1), and IFN-g (clone XMG1.2) mAb; and recombinant mouse IL-2, IL-4, IL-10, and IFN-g.

To investigate the effect of mucosal exposure of antigen on T-cells in vivo, we utilized an adoptive transfer model as described by Kearney et al. (16). OVA-specific Tg/ T-cells were injected intraperitoneally into syngeneic BALB/c mice and their distribution in vivo was monitored by flow cytometry using FITC-labeled anticlonotypic mAb KJ1-26. Figure 1 illustrates the FACS profile of Peyer’s patches stained for CD4 and the transgenic TcR. As shown in Fig. 1A, virtually no OVAspecific transgenic T-cells were detected in Peyer’s patches of normal BALB/c mice fed 0.1 mg OVA peptide only (0.04%). However, OVA-specific transgenic T-cells were detected in Peyer’s patches of animals that received Tg/ T-cells and were fed PBS (0.6%, Fig. 1B), and an increased number of Tg/ T-cells (1.3%) was observed in animals fed low doses (0.1 mg) of OVA peptide five times following the transfer of Tg/ T-cells (Fig. 1C). To further investigate the distribution of the OVAspecific transgenic T-cells in vivo, we performed the same flow cytometry analysis for Peyer’s patches, spleen, and lymph nodes at different time points after oral administration of both low (0.1 mg)- and high (5 mg)-dose OVA peptide. As shown in Fig. 2A, the most marked change in Tg/ T-cell frequency occurred in Peyer’s patches after high-dose feeding. After only one

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Flow Cytometry Cells were stained for CD4 with PE-conjugated YTS 191.1 mAb (Caltag, San Francisco, CA) and for TcR with FITC-conjugated KJ1-26 mAb. Fluorescence intensity was analyzed on a Becton–Dickinson FACSort using Lysis II software. Data collection was gated on live cells through propidium iodide exclusion. Antigens, Antibodies, and Recombinant Cytokines

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T-Cell Activation Following Oral Antigen Administration

FIG. 1. Detection of OVA-specific TcR transgenic T-cells in Peyer’s patches of BALB/c mice. Six- to eight-week-old female BALB/c mice were divided into 3 groups with 3 mice per group. Group A received no injection of transgenic T-cells; groups B and C received an intraperitoneal injection of OVA-specific TcR transgenic T-cells (5 1 106 cells/mouse). Two days later, animals were fed either 0.1 mg OVA 323-339 peptide in 0.5 ml PBS (A and C) or PBS (B) on alternate days for a total of 5 times. Two days after the last feeding, animals were sacrificed and Peyer’s patches removed. Peyer’s patch cell suspensions were then prepared (28) and stained for CD4 with PE-conjugated YTS 191.1 mAb and for transgenic TcR with FITCconjugated KJ1-26 mAb. Data presented represent 10,000 events of live Peyer’s patch cells.

Since tolerance induced by oral antigen is antigenspecific, interaction between the fed-antigen and antigen-specific T-cells must occur during tolerance induction. However, due to the low frequency of specific Tcells in conventional animals, investigation of immune responses to the fed antigen has been difficult to demonstrate without immunization. We thus had the opportunity to examine T-cell activation in the absence of immunization as measured by proliferation and cytokine secretion in animals that had received Tg/ T-cells. As shown in Fig. 3, there were increased proliferative responses to OVA following one feeding of either 0.1 or 5 mg OVA peptide (52,382 { 6997 and 35,828 { 2300 cpm vs 13,708 { 1821 cpm in PBS fed; P õ 0.001 and 0.001 vs control). This increase was also seen at Day 10 in 5-mg-fed animals (P õ 0.01). By Day 20, T cell reactivity was below that observed in PBS-fed animals for both doses fed. We then examined cytokine production following oral antigen administration. As shown in Fig. 4, there was no measurable cytokine production in PBS-fed animals. IFN-g production was induced in both high (165 { 22 pg/ml)- and low-dose-fed (140 { 19 pg/ml) animals but disappeared over time. In contrast, low-dose feeding induced prolonged secretion of IL-4, IL-10, and TGF-b (102 { 26, 502 { 67, and 201 { 22 pg/ml at 20 days). In high-dose-fed groups there was induction of TGF-b but only minimal induction of IL-4 and no induction of IL-10. IL-2 was not induced by either feeding regimen.

feeding of 5 mg OVA peptide, the frequency of Tg/ Tcells increased six- to eightfold (49 { 6/103 cells) compared to that with PBS (6 { 1.5/103 cells) of 0.1-mg-fed (8 { 2.1/103 cells) animals (P õ 0.001). This increase was followed by a decrease to control levels by Day 10 and to 44% of the control level (2 { 0.4/103 cells) by Day 20 (P õ 0.01 vs control). In low-dose-fed animals a small but significant increase relative to the control level was observed on Day 10 (11.9 { 1.7 vs 5.6 { 0.9/ 103 cells; P õ 0.01) with the frequency of Tg/ T-cells returning to control levels by Day 20. In contrast to Peyer’s patches, after a single feeding of high-dose (5 mg) OVA peptide there was a decrease in the Tg/ T-cell frequency in the spleen (10 { 0.4 vs 14.5 { 0.3/103 cells; P õ 0.01 vs control; Fig. 2B) and lymph nodes (4 { 0.1 vs 9 { 0.7/103 cells; P õ 0.05 vs control; Fig. 2C). This decrease coincided with the peak increase of Tg/ T-cells in the Peyer’s patches (Fig. 2A). Also in contrast to Peyer’s patches, after subsequent feedings of 5 mg, Tg/ T-cells in the spleen and lymph nodes were increased at Day 10 compared to the levels after one feeding. At Day 20, the Tg/ T-cell frequency was 63% of the control level in spleen and 36% of the control level in lymph node. No significant changes were observed in spleen or lymph nodes of mice fed low-dose OVA peptide (0.1 mg). In initial experiments, we analyzed lymph nodes individually from cervical, axillary, inguinal, popliteal, periaortic, and mesenteric sites after a single feeding of 5 mg OVA peptide and found less than 40% differences among different lymph nodes. Thus, we pooled lymph nodes for most experiments to ensure an adequate number of cells for multiple cytokine assays and flow cytometry.

To confirm that oral tolerance was induced in animals that had received Tg/ T-cells, we immunized animals after oral administration of OVA peptide. As shown in Fig. 5, T-cell proliferative responses were suppressed in both low- and high-dose-fed animals. We also tested the cytokine production by these cells. Tcells from nonfed animals produced 813 { 216 pg/ml IFN-g and 13 { 4 pg/ml IL-2 with no detectable IL-4, IL-10, and TGF-b. Feeding either low- or high-dose OVA peptide completely abrogated IL-2 production. The production of IFN-g was reduced to 171 { 23 and 50 { 17 pg/ml in low- and high-dose-fed mice, respectively. In mice fed low-dose OVA peptide, IL-10 (628 { 64 pg/ml) and TGF-b (994 { 213 pg/ml) were detected. TGF-b but not IL-10 was detected in high-dose-fed and immunized animals (821 { 187 pg/ml). IL-4 was not detected in any of the cultures. When unfed mice that received no injection of transgenic T-cells were immunized with OVA peptide, the proliferation and cytokine responses to OVA were approximately 10% of that seen in mice that received Tg/ T-cells, demonstrating that the proliferative responses we observed were primarily

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Oral Tolerance Was Induced in Mice Transferred with Tg/ T-Cells

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FIG. 3. Proliferative responses following oral administration of antigen. Mice were fed as in Fig. 2. Splenocytes, 1 1 106 cells/well, were cultured in 0.2 ml of serum-free medium X-vivo 20 (Biowhitacker, Walkersville, MD) containing various concentrations of OVA323-339 peptide. Seventy-two hours later, 1 mCi of [3H]thymidine was added to each culture. Cells were harvested and radioactivity was measured 16 hr later. Data presented represent the stimulation index of cultures with 100 mg/ml of OVA323-339 peptide (using cultures with MBP1-11 peptide as control). Maximum cpm, 52,382 { 6997 (0.1 mg fed), 35,828 { 2300 (5 mg fed), and 13,708 { 1821 (PBS fed). Each data point represents an average from a minimum of 3 mice; the standard deviations are within 25% of the mean. The experiments were repeated twice with similar results. The statistical significance (ANOVA) of the differences in stimulation index between PBS- and OVA-fed groups was as follows: for mice fed 0.1 mg OVA peptide, P õ 0.01 on Days 2, 10, and 20; for mice fed 5 mg OVA peptide, P õ 0.001 on Day 2 and P õ 0.05 on Day 20.

from transferred Tg/ T cells. These data confirm our earlier observation that both high- and low-dose oral antigen tolerize Th1 responses and that Th2 responses are not tolerized in low-dose-fed animals (10, 14). DISCUSSION

FIG. 2. Frequency of transgenic T-cells in Peyer’s patch, lymph node, and spleen following oral administration of antigen. Three groups of BALB/c mice, 14 mice per group, received an intraperitoneal injection of OVA-specific TcR transgenic T-cells (5 1 106/mouse). Two days later (Day 0), animals in each group were fed with 0.1 or 5 mg OVA323-339 peptide in 0.5 ml PBS, or PBS alone. Feeding was repeated on Days 2, 4, 6, and 8. Mice were sacrificed on Day 0 (before the first feeding), Day 2 (before the second feeding), and on Day 10 or Day 20 after the first feeding. Peyer’s patches (A), lymph nodes (B), and spleens (C) were removed and single cell suspensions were prepared. Cells were analyzed by flow cytometry as in Fig. 1. Each data point represents an average of transgenic T-cells from a minimum of 3 mice; the standard error is within 11–25% of the mean. Data presented are representative of two experiments. The statistical significance (x2 analysis) of the differences in transgenic T-cell frequency between PBS- and OVA-fed groups was as follows: for Peyer’s

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Although the gut-associated lymphoid tissue harbors more than half of the peripheral lymphocytes, the activation of antigen-specific cells in response to oral antigen remains uncharacterized. In the present study, using a sensitive adoptive transfer model (16), we investigated the effect of orally administered OVA peptide on adoptively transferred OVA-specific Tg/ T-cells. We found that oral administration of OVA peptide resulted in an increase of OVA-specific Tg/ T-cells to the gut-

patches of mice fed 5 mg OVA peptide, P õ 0.0001 on Day 2 and P õ 0.01 on Day 20; for Peyer’s patches of mice fed 0.1 mg OVA, P õ 0.01 on Day 10; for lymph nodes of mice fed 5 mg OVA peptide, P õ 0.01 on Day 2 and P õ 0.05 on Day 20; and for spleens of mice fed 5 mg OVA peptide, P õ 0.05 on Day 2 and P õ 0.01 on Day 20.

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FIG. 4. Cytokine production following oral administration of antigen. Mice were treated and sacrificed as in Fig 2. Splenocytes were cultured as in Fig. 3. Culture supernatants were collected 40 (for IL-2, IL-4, IL-10, IFN-g) or 72 hr (for TGF-b) later. Cytokine concentration was determined by ELISA. Data presented represent the mean of cultures with 100 mg/ml of OVA323-339 peptide minus the mean of cultures with 100 mg/ml of MBP1-11 peptide. Each data point represents an average from a minimum of 3 mice; the standard deviation is within 20% of the mean. The experiments were repeated twice with similar results. The statistical significance (ANOVA) of the differences in cytokine concentration between PBS- and OVA-fed groups was as follows: IFN-g, P õ 0.0001 on Day 2 for both OVA-fed groups; IL-4, P õ 0.001 for 0.1-mg-fed mice on Days 10 and 20, P õ 0.01 for 5-mg-fed mice on Day 2; IL-10, P õ 0.0001 for 0.1-mg-fed mice on Days 2, 10, and 20; TGF-b, P õ 0.01 on Days 2, 10, and 20 for both OVA-fed groups.

associated lymphoid tissue (Peyer’s patches) in vivo especially in animals fed high-dose (5 mg) antigen. A monophasic response occurred in Peyer’s patches where a marked increase in the number of Tg/ T-cells was observed followed by a decrease in Tg/ T-cells. In contrast, in the spleen and to some extent in lymph nodes, a biphasic response was observed in which there was an initial decrease followed by an increase and then a subsequent decrease. It has been demonstrated by others that intravenous administration of antigen led to sequestration of specific T-cells in the spleen (18). Similarly, it appears that following oral administration of OVA in our system, antigen-specific T-cells increase in mucosal tissue most probably secondary to sequestration, especially in highdose-fed animals, although a component of local proliferation of cells may contribute. This presumably allows the immune system to focus on the site where the antigen is processed. However, this increase in antigenspecific T-cells is transient as by Day 10 there was a decrease of antigen-specific cells in Peyer’s patches. Since tolerance induced by oral antigen is antigenspecific, it must follow that interaction of T-cells with its specific antigen occurs during the induction phase

of oral tolerance. The immunologic events associated with this interaction have been difficult to study as immunization is required to detect T-cell responses in conventional animals. The model used here allowed investigation of this process. Activation of specific T-cells as measured by proliferation and IFN-g secretion was observed after a single feeding of either a low or a high dose of OVA. The activated T-cells were detected in the spleen and lymph nodes. These results confirm our earlier observations in intact transgenic mice in which activation of Th1 and Th2 cells was observed following oral administration of antigen (10). In this regard, Gautam et al. (19) reported activation of Th1 cells by oral feeding of the hapten trinitrochlorobenzene and found that it preceded oral tolerance. Marth et al. (20) found an initial increase in IFN-g secretion when high-dose antigen was fed to OVA TCR transgenic animals and we have observed an initial increase of IFN-g secretion in Peyer’s patches followed by TGF-b secretion when multiple low-dose antigen was fed to MBP TCR transgenic mice (J.-i. Inobe and H. L. Weiner, unpublished observations). Thus, oral antigen may be associated with initial priming of Th1-type responses, especially after the initial feeding. Consistent with this, in the

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FIG. 5. Transgenic T-cells were tolerized by both low- and highdose feeding regimens. Three groups of BABL/c mice, 4 mice per group, received an intraperitoneal injection of OVA-specific TcR transgenic T-cells (5 1 106/mouse). Two days later, mice in each group were fed with 0.1 or 5 mg OVA323-339 peptide in 0.5 ml PBS, or PBS, on alternate days for a total of 5 times. Two days after the last feeding, mice were immunized subcutaneously on the flank with 100 mg OVA323-339 peptide in 0.1 ml of PBS emulsified in an equal volume of complete Freund’s adjuvant containing 1 mg/ml of mycobacterium tuberculosis H37 RA. Mice were sacrificed 14 days later and splenocytes were cultured and tested as in Figs. 3 and 4. Data presented are (A) the stimulation index of cultures with 100 mg/ml of OVA323-339 peptide (using cultures with MBP1-11 peptide as control whose cpm values are between 4000 and 18,000) and (B) the cytokine concentration of cultures with OVA peptide minus that of cultures with MBP peptide. Data are representative of two experiments. The statistical significance (ANOVA) of the differences is as follows: (A) For the stimulation index between PBS- and OVA-fed groups: P õ 0.01 for mice fed 0.1 mg OVA; P õ 0.001 for mice fed 5 mg OVA. (B) For all cytokines, P õ 0.01 for PBS- and OVA-fed groups. No IL-4 secretion was observed.

sistent with the possibility that TGF-b-secreting cells may represent a distinct lineage of T-cells. Recent studies in which investigators also examined oral administration of antigen following adoptive transfer of Tg/ T-cells found anergy as a primary mechanism (25). Because higher doses (25 and 100 mg) of antigen were fed than those in the current study, a direct comparison to our results cannot be made. Nonetheless, the authors found no increase of Tg/ T-cells in the lamina propria or intraepithelial compartments. Peyer’s patches were not examined and cytokine secretion was not measured. Their finding of anergy is consistent with previous studies by a number of investigators that angery is observed when high doses of oral antigen are administered (11, 13, 14). Although T-cell activation after oral antigen may primarily occur in the mucosal tissue, some antigen is detectable in the blood circulation a few hours after feeding, which raises the possibility that interaction between T-cells and oral antigen may also occur in other tissues (26, 27). Indeed, we detected activated T-cells in the spleen 48 hr after feeding even though the frequency of transgenic T-cells was lower relative to other time points examined. Thus, it is possible that activation of antigen-specific T-cells may occur in the systemic as well as mucosal lymphoid tissues following oral administration of antigen, even though there appears to be preferential stimulation of cells secreting cytokines such as TGF-b in the gut-associated lymphoid tissue (28). ACKNOWLEDGMENTS

EAE model, Meyer et al. (21) have found a worsening of disease when one low-dose feeding was given prior to immunization. In another system, Blanas et al. (22) reported activation of OVA-specific CTL after oral administration of large amounts of OVA. With intravenous antigen, Vidard et al. (23) observed evidence of preactivation in an intravenous tolerance model using immunized animals. Using the same model as that reported here, Kearney et al. (16) detected T-cell activation of OVA transgenic T-cells after intravenous administration of OVA. Following activation, we observed immune deviation with the induction of IL-4, IL-10, and TGF-b. Thus, a bias against Th1 responses occurs quickly and appears to downregulate Th1 priming. As with our previous studies using soluble OVA protein, we found more Th2 responses with low- than with high-dose OVA peptide, which is consistent with the observation that immune deviation is favored by low doses of antigen whereas anergy and deletion are preferentially observed at higher doses. By contrast, Degermann et al. (24) recently reported that intravenous OVA protein, but not peptide, activates Th2 cells. TGF-b secretion was observed at both doses in the current study, which was also observed in intact transgenic animals and is con-

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We thank D. Y. Loh for providing OVA-specific TcR transgenic mice, V. Perez and A. Abbas for providing mAb KJ1-26, and P. Hyde for technical assistance.

REFERENCES 1. Wells, H. G., J. Infect. Dis. 8, 147, 1911. 2. Chase, M., Proc. Soc. Exp. Biol. Med. 61, 257, 1946. 3. Mowat, A. M., Immunol. Today 8, 93, 1987. 4. Weiner, H. L., Friedman, A., Miller, A., Khoury, S. J., Al-Sabbagh, A., Santos, L. M. B., Sayegh, M., Nussenblatt, R. B., Trentham, D. E., and Hafler, D. A., Annu. Rev. Immunol. 12, 809, 1994. 5. Weiner, H. L., Mackin, G. A., Matsui, M., Orav, E. J., Khoury, S. J., Dawson, D. M., and Hafler, D. A., Science 259, 1321, 1993. 6. Trentham, D. E., Dynesius-Trentham, R. A., Orav, E. J., Combitchi, D., Lorenzo, C., Sewell, K. L., Hafler, D. A., and Weiner, H. L., Science 261, 1727, 1993. 7. Barnett, M. L., Combitchi, D., and Trentham, D. E., Arthritis Rheum. 39, 623, 1996. 8. Sieper, J., Kary, S., So¨rensen, H., Alten, R., Eggens, U., Hu¨ge, W., Hiepe, F., Ku¨hne, A., Listing, J., Ulbrich, N., Braun, J., Zink, A., and Mitchison, N. A., Arthritis Rheum. 39, 41, 1996. 9. Nussenblatt, R. B., Gery, I., Weiner, H. L., Ferris, F., Shiloach, J., Ramaley, N., Perry, C., Caspi, R. R., Hafler, D., Foster, S., and Whitcup, S. M., Am. J. Opthalmol., in press.

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10. Chen, Y., Inobe, J.-i., Marks, R., Gonnella, P., Kuchroo, V. K., and Weiner, H. L., Nature 376, 177, 1995. 11. Whitacre, C. C., Gienapp, I. E., Orosz, C. G., and Bitar, D., J. Immunol. 147, 2155, 1991. 12. Melamed, D., and Friedman, A., Eur. J. Immunol. 23, 935, 1993. 13. Gregerson, D. S., Obritsch, W. F., and Donoso, L. A., J. Immunol. 151, 5751, 1993. 14. Friedman, A., and Weiner, H. L., Proc. Natl. Acad. Sci. USA 91,6688, 1994. 15. Chen, Y., Inobe, J.-i., Kuchroo, V. K., Baron, J. L., Janeway, C. A., and Weiner, H. L., Proc. Natl. Acad. Sci. USA 93, 388, 1996. 16. Kearney, E., Pape, K., Loh, D., and Jenkins, M., Immunity 1, 327, 1994. 17. Murphy, K., Heimberger, A., and Loh, D., Science 250, 1720, 1990. 18. Sprent, J., Miller, J., and Mitchell, G., Cell. Immunol. 2, 171, 1971.

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19. Gautam, S. C., Chikkala, N. F., and Battisto, J. R., Cell. Immunol. 125, 437, 1990. 20. Marth, T., Strober, W., and Kelsall, B. L., J. Immunol. 157, 2348, 1996. 21. Meyer, A. L., Benson, J. M., Gienapp, I. E., Cox, K. L., and Whitacre, C. C., J. Immunol. 157, 4230, 1996. 22. Blanas, E., Carbone, F. R., Allison, J., Miller, J. F. A. P., and Heath, W. R., Science 274, 1707, 1996. 23. Vidard, L., J., C. L., and Benacerraf, B., Proc. Natl. Acad. Sci. USA 91, 5627, 1994. 24. Degermann, S., Pria, E., and Adorini, L., J. Immunol. 157, 3260, 1996. 25. Van Houten, N., and Blake, S. F., J. Immunol. 157, 1337, 1996. 26. Peng, H. J., Turner, M. W., and Strobel, S., Clin. Exp. Immunol. 81, 510, 1990. 27. Mowat, A. M., Lamont, A. G., Strobel, S., and Mackenzie, S., Adv. Exp. Med. Biol. 216A, 709, 1987. 28. Santos, L. M. B., Al-Sabbagh, A., Londono, A., and Weiner, H. L., Cell. Immunol. 157, 439, 1994.

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