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,§
*
Mucosal Immunology Laboratory, Massachusetts General Hospital, Charlestown, MA 02129;
Department of Immunology and Infectious Diseases, Harvard School of Public Health, and Departments of
Pediatrics and
§
Medicine, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generalizability of the initial observation that experimental
models of autoimmune disease can be suppressed by oral administration
of the corresponding autoantigen (8, 9) has suggested that the
induction of peripheral tolerance to orally administered Ags might
provide a safe, specific, and effective treatment for autoimmune
disease. Inspired by encouraging trends in clinical trials in patients
with multiple sclerosis and rheumatoid arthritis, much of current
effort is focused on potentiating orally induced nonresponsiveness,
particularly through the use of cytokines (7). Recent efforts at
modulation of oral tolerance have led to the suggestion that,
regardless of dose administered, cytokines or other immune-modulating
agents that favor Th1 (vs Th2 responses) diminish oral tolerance while
those that favor Th2 responses enhance tolerance (7). Thus, treatment
with anti-IL-12 (2) or rIL4/IL-10 (7, 10) have been reported to
augment oral tolerance while treatment with IFN-
(11) suppresses its
induction. To gain insight into cytokine regulation of oral tolerance,
we have examined responses to orally administered Ag in both the
peripheral and mucosal tissues of mice deficient in Th1 or Th2 cells.
STATs are a family of DNA-binding proteins integrally involved in the
responses of lymphocytes to signaling via cytokines. Although some Stat
family members are activated by a variety of cytokines, Stat 4 is
activated only in response to the Th1 inducer IL-12 while Stat 6 is
activated in response to the Th2 inducer IL-4 (reviewed in 12).
Inactivation of the Stat 4 and Stat 6 genes, through gene-targeting
technology, has resulted in mice deficient in the functional
differentiation of Th1 (13, 14) and Th2 cells (15, 16, 17). Stat
4-/- and Stat 6-/- mice therefore provide
an ideal in vivo model system in which to explore the role of cytokine
regulation in oral tolerance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stat 4 (13)-deficient and Stat 6 (15)-deficient mice were bred at the Harvard School of Public Health and maintained under specific pathogen-free conditions at Massachusetts General Hospital. All of these experiments involved female mice, 9–16 wk of age. The experiments shown were performed using Stat 4- and Stat 6-deficient mice at between six and ten generations of backcross to BALB/c and normal BALB/c (Taconic, Germantown, NY) mice as controls.
Induction of tolerance
Either PBS or OVA (25 mg in PBS; Sigma, St. Louis, MO) was
administered intragastrically in 0.5 ml using a ball-tipped feeding
needle to groups of three to five mice. Preliminary experiments
established that the response of mice fed 25 mg of human serum albumin
to immunization with OVA was equivalent to that of mice fed PBS.
Control mice were therefore fed PBS in subsequent experiments. Five
days after feeding, mice were immunized either in the hind footpads
(see
Figs. 1–3
) or i.p. (see Figs. 4 and 5) with 100 µg of OVA in
CFA. Two weeks after immunization, cells from the draining popliteal
lymph nodes (PLN)3 or Peyer’s
patches (PP) were harvested for restimulation in vitro, and serum and
feces were collected for determination of OVA-specific Ab responses.
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PLN or PP were pooled from each group of three to five mice and pressed through a 70-µM nylon cell strainer (Falcon, BD Labware, Franklin Lakes, NJ) to create a single cell suspension. Each cell suspension was washed three times before culture at 1 x 106 cells/ml (PLN) or 2 x 106/ml (PP), in triplicate, in round-bottom microtiter plates (Costar, Cambridge, MA) in complete DMEM (Life Technologies, Grand Island, NY; cDMEM contains 10% FCS (HyClone, Logan, UT), 10 mM HEPES, 2 mM L-glutamine, 100 U penicillin/ml, 100 µg of streptomycin/ml, 50 µM 2-ME, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate) in 200 µl with or without varying concentrations of OVA (1–1000 µg/ml). Some wells were coated with purified anti-CD3 (PharMingen, San Diego, CA; 10 µg/ml) as a positive control. Some of the cultures also contained a neutralizing, polyclonal chicken Ab to TGF-ß (R&D Systems, Minneapolis, MN) or chicken Ig (R&D Systems) at a final concentration of 10 µg/ml. To measure cellular proliferation, plates were pulsed with 1 µCi/well of [3H]TdR (New England Nuclear, Boston, MA) for 16–18 h and harvested at the indicated time points. [3H]Thymidine incorporation was determined by liquid scintillation counting.
Measurement of cytokine production
Pooled PLN (4 x 106 cells/ml) from each group
of mice were stimulated with 1, 10, or 100 µg/ml of OVA or
plate-bound anti-CD3 (10 µg/ml), and cytokines were assayed as
previously described (18). Briefly, 24 h supernatants were assayed
for IL-2 production using the indicator cell line HT-2 in the presence
of neutralizing concentrations of the IL-4-specific Ab 11B11. IL-4,
IFN-, IL-5, and IL-10 production was assayed in 72-h culture
supernatants by ELISA. ELISA capture (BVD4-1D11, IL-4; R4-6A2, IFN-;
TRFK-5, IL-5; and JESS-2A5, IL-10) and biotinylated second Abs
(BVD6-24G2, IL-4; XMG1.2, IFN-; TRFK4, IL-5; and SXC-1, IL-10) were
purchased from PharMingen. Standard curves were obtained using
recombinant murine IFN-, IL-4 (Genzyme, Cambridge, MA), IL-10
(R&D Systems), and IL-5 (PharMingen) and are expressed in pg/ml ±
SEM. OD values were converted to pg/ml for each cytokine by linear
regression with Delta Soft II (Biometallics, Princeton, NJ). The limits
of detection of the ELISA assays are 5 pg/ml for IFN- and IL-4, 40
pg/ml for IL-10, and 8 pg/ml for IL-5.
Ab assays
Each mouse was bled at sacrifice, and individual sera were assayed for OVA-specific IgG1 and IgG2a by ELISA on OVA-coated Immulon 2 plates as previously described (18). OD values were converted to µg/ml of OVA-specific IgG1 or IgG2a by comparison to a standard curve of purified OVA-specific Ig (developed with either HRP-conjugated goat anti-mouse IgG1 or goat anti-mouse IgG2a (Southern Biotechnology, Birmingham, AL) with Delta Soft linear regression analysis and are expressed as the mean concentration for each group of mice ± SEM. OVA-specific Ig was purified from the pooled serum of OVA-immunized mice by affinity chromatography on an OVA-Sepharose 4B column (Pharmacia, Piscataway, NJ) prepared according to the manufacturer’s instructions.
To examine total and OVA-specific IgA secretion, fecal pellets were collected at sacrifice, 14 days after immunization. Four to five fecal pellets from each mouse were collected into preweighed Eppendorf tubes containing PBS and protease inhibitors, as described (19). Fecal weight was calculated by subtracting the preweigh values. The fecal samples were vortexed, incubated at 4°C for 1 h, and spun for 10–15 min at 13,000 rpm to remove insoluble material. The supernatant was then removed and frozen at -20°C until assay. Before assay, all extracts were adjusted to the same concentration (between 0.06 and 0.1 g/ml) based on fecal weight. OVA-specific IgA secretion into fecal extracts was assayed by ELISA on OVA-coated Immulon 2 plates essentially as previously described (20). The plates were blocked with 10% normal goat serum in PBS-1% BSA before an overnight incubation at 4°C with fecal extracts from individual mice, assayed in triplicate. The assay was then developed by the addition of HRP-conjugated goat anti-mouse IgA (Southern Biotechnology) with O-phenylenediamine (Zymed Laboratories, San Francisco, CA) as the substrate and read at 492 nm. Total IgA in fecal extracts was also detected by ELISA on plates coated with goat anti-mouse Ab to IgA (Southern Biotechnology). Blocked, washed plates were incubated with fecal extracts, in triplicate, overnight at 4°C and detected with HRP-conjugated goat anti-mouse IgA. The reaction was developed with O-phenylenediamine and read at 492 nm. OD values were converted to µg/ml by comparison with a titration of a monoclonal IgA hybridoma (Sal4; 21) to generate a standard curve and are expressed as micrograms per gram of fecal weight.
Statistical differences in serum and fecal Ab levels were determined using a two-tailed Student’s t test with StatView software (Abacus Concepts, Berkeley, CA). A p value of < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Groups of Stat 4-/- (Th1-deficient), Stat
6-/- (Th2-deficient), or BALB/c mice were fed a single
high (25-mg) dose of OVA or PBS and immunized in the hind footpads 5
days later with OVA/CFA. Two weeks after immunization, cells from the
draining PLN were restimulated in vitro with varying doses of OVA or
with plate-bound anti-CD3.
Fig. 1
A shows that OVA feeding
greatly reduced the dose-dependent proliferative response to OVA
restimulation in vitro in Stat 4-/- and Stat
6-/- mice as well as in BALB/c mice. Each of the cultures
responded similarly to plate-bound anti-CD3 at 72 h after the
initiation of the culture (Fig. 1B). To examine the role of
TGF-ß, a neutralizing Ab to TGF-ß previously shown to be effective
in abrogating orally induced peripheral nonresponsiveness to in vitro
restimulation was included in each of the cultures assayed (2). Fig. 1
shows that anti-TGF-ß had no effect on the nonresponsiveness of
cells from the PLN of any of the mice tested to OVA or their
proliferation in response to anti-CD3 restimulation in vitro.
Control cultures containing chicken Ig gave results similar to those
obtained with media alone.
We next examined the production of both Th1 and Th2 cytokines by OVA-
and PBS-fed mice in response to OVA restimulation in vitro. We (18) and
others (22) have previously shown that both Th1 and Th2 responses are
suppressed by OVA feeding in BALB/c mice. Fig. 2 shows that each of the Th1 (IL-2 and
IFN-) and Th2 (IL-4, IL-5, and IL-10) cytokines tested is suppressed
by OVA feeding in BALB/c mice. Stat 4-/- mice are Th1
deficient and, in the absence of Th1 counter-regulation, Th2 enhanced.
As shown in Fig. 2, they produce much lower levels of IFN- but
higher levels of IL-4, IL-5, and IL-10 than BALB/c mice in response to
OVA restimulation in vitro. In spite of the enhanced Th2 cytokine
production by PLN cells from PBS-fed mice, OVA feeding reduced each of
these responses to near background levels. Stat 6-/-mice,
on the other hand, are Th2 deficient and Th1 enhanced and make elevated
levels of IFN- upon restimulation in vitro. PLN cells from Stat
6-/- mice did not produce detectable levels of IL-4 and
produced only low levels of IL-5 and IL-10 upon restimulation with OVA
in vitro. Each of the cytokines produced was suppressed by OVA feeding
in vivo. When we examined the responses of each of these cultures to
stimulation with immobilized anti-CD3, we found that, while IFN-
and IL-10 production was similar for each group of mice, Stat
6-/- mice produced less IL-5 and that Stat
4-/- mice produced more IL-4 than the BALB/c controls or
their reciprocal Stat-deficient counterpart (data not shown). Cytokine
secretion was not altered by the inclusion of anti-TGF-ß in the
culture medium.
Analysis of serum OVA-specific Ab responses provides an assessment of
peripheral nonresponsiveness to orally administered OVA in the absence
of restimulation in vitro. Serum was collected from individual mice at
the time of sacrifice and analyzed for OVA-specific Ab responses of
both Th1-dependent (IgG2a) and Th2-dependent (IgG1) isotypes of IgG.
Fig. 3 shows that OVA-specific IgG2a
(Fig. 3A) and IgG1 (Fig. 3B) were suppressed by
intragastric administration of OVA in each strain of mouse tested. As
predicted, the Stat 6-/- mice exhibit an enhanced
OVA-specific IgG2a response and an impaired IgG1 response. The
OVA-specific IgG2a response is reduced in Th1-deficient Stat
4-/- mice without an obvious enhancement of the IgG1
response.
Mucosal responsiveness to OVA is also suppressed by intragastric administration of OVA to Stat4-/- and Stat 6-/- mice
To compare mucosal immune responses to OVA to those seen in the
periphery, we examined the proliferative responses of PP cells from
OVA-fed Stat 4-/- and Stat 6-/- mice to
restimulation with OVA in vitro. For the experiments shown in Figs. 4 and 5,
groups of mice were immunized i.p. with OVA/CFA after OVA feeding
instead of in the footpads. Systemic immunization does not drain well
to the gut-associated lymphoid tissue; when we compared mucosal
immune responses after footpad and i.p. immunization, we found that the
response elicited by i.p. immunization was substantially greater. Fig. 4 shows the proliferative responses of PP cells from BALB/c, Stat
4-/-, and Stat 6-/- mice to OVA (Fig. 4A) and plate-bound anti-CD3 (Fig. 4B). After
i.p. immunization, the PP were enlarged in OVA-fed compared with
PBS-fed mice (in the experiment shown, the cell counts were increased
3-, 1.25-, and 2.25-fold in OVA-fed BALB/c, Stat 4-/-,
and Stat 6-/- mice, respectively). This PP enlargement
parallels the increased response to anti-CD3 seen in Fig. 4B for OVA-fed Stat 4-/- and Stat
6-/- mice and apparently reflects the recruitment or
expansion of T cells in the PP in mice fed OVA and immunized with
OVA/CFA i.p. Strikingly, although T cells are present in increased
numbers in the PP of OVA-fed mice and respond well to anti-CD3,
their Ag-specific response to restimulation with OVA is suppressed in
both Stat 4-/- and Stat 6-/- mice. This
nonresponsiveness is abrogated by the inclusion of a neutralizing Ab to
TGF-ß in the culture medium. The fecal OVA-specific IgA response,
which, like the serum Ab response, provides a direct ex vivo
measurement, is also significantly reduced by OVA feeding in both Stat
4-/- and Stat 6-/- mice (Fig. 5). Although
in Fig. 4A, the response to restimulation with OVA is
increased in BALB/c mice, it parallels the increase in the response to
anti-CD3 (Fig 4B) seen in the PP of these mice (both are
increased 
4-fold) and, in agreement with the fecal OVA-specific IgA
response (Fig. 5), suggests that neither mucosal tolerance nor immunity
is induced by Ag feeding in BALB/c mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R-/- mice (24) while peripheral nonresponsiveness
to orally administered Ags is intact (22, 24). A recent study, however,
suggests that peripheral tolerance to orally administered Ag is not
induced in IFN--/- mice (25). To clarify the role of
Th1 and Th2 cytokines in tolerance to orally administered Ag, we have
directly compared the induction of nonresponsiveness in Th1- (Stat
4-/-) and Th2 (Stat 6-/-)-deficient mice to
that obtained in normal BALB/c mice.
Clonal deletion/anergy and the induction of TGF-ß-secreting
regulatory cells have been proposed as the primary mechanisms for oral
tolerance induction, the relative importance of each varying with the
dose of Ag fed (7). Mounting evidence also indicates that the induction
of tolerance to oral Ag, like other forms of peripheral tolerance, is
preceded by transient T cell activation and clonal expansion (26, 27, 28).
Studies in OVA TCR-transgenic mice have suggested that the induction of
TGF-ß-secreting Th3 cells by Ag feeding occurs in conjunction with
mucosal production of Th2 cytokines (1, 7) and is negatively regulated
by IL-12 (2, 29), implying that Th1 and Th2 cytokines play key roles in
regulating the response to oral Ag. The Stat mutant mice provide an
ideal in vivo model system in which to explore this hypothesis and to
examine, in parallel, the interplay of TGF-ß and Th1 and Th2 cytokine
regulation in both mucosal and peripheral tissues in response to high
doses of oral Ag in non-TCR-transgenic mice. The proliferative (Fig. 1)
and cytokine (Fig. 2) responses of draining PLN to restimulation with
OVA in vitro were tolerized in Th1-deficient (Stat 4-/-),
Th2-deficient (Stat 6-/-), and BALB/c mice, and this
suppression was not affected by inclusion of a neutralizing Ab to
TGF-ß in the culture medium. Both Th1- and Th2-dependent serum
OVA-specific Ab responses were tolerized by OVA feeding in each strain
of mouse (Fig. 3). Neither functional Th1 or Th2 cells are therefore
required for the induction of peripheral cell-mediated or humoral
tolerance to orally administered Ag.
Whether or not the mucosal immune response is tolerized by oral Ag has
remained controversial, due in large part to the difficulty of
detecting mucosal immune responses in the context of the systemic
immunization used to demonstrate peripheral tolerance. Indeed PP
lymphocytes from mice immunized in the footpads with OVA in CFA
proliferate very poorly to restimulation with OVA, even when their
ability to respond to anti-CD3 is comparable to that in the PLN. We
were able to enhance the response of PP lymphocytes to in vitro
restimulation by immunizing with OVA in CFA i.p., rather than in the
footpads. We confirmed that the mice were tolerized by oral Ag by
examining the serum OVA-specific Ab response. As shown in Fig. 4, the
responses of PP cells from Stat 4-/- and, to a lesser
extent, Stat 6-/- mice to restimulation with OVA in vitro
were suppressed by OVA feeding, and this nonresponsiveness was
abrogated by anti-TGF-ß. Nonresponsiveness was not induced in the
PP of BALB/c mice. In each strain of mouse, OVA feeding, following by
i.p. immunization with OVA in CFA, resulted in a marked enlargement of
the PP and an apparent increase in the numbers of CD3+ T
cells (note that we did not see this PP enlargement when the mice were
immunized in the footpads). In spite of this enhanced responsiveness to
anti-CD3, PP cells from Stat 4-/- and Stat
6-/- mice fed OVA responded poorly to restimulation with
OVA in vitro. In the experiment shown, the response of PP cells from
normal BALB/c mice to restimulation with OVA in vitro was greater for
OVA-fed than PBS-fed mice but was proportional to the increased
responsiveness to anti-CD3 (both responses were increased about
4-fold). Since the number of cells in the PP was 3-fold greater in the
OVA-fed BALB/c mice immunized with OVA/CFA in this experiment, this
result suggests that OVA feeding did not induce either priming or
nonresponsiveness in BALB/c mice. Indeed in two additional experiments
in which the enlargement of the PP induced by OVA feeding was less
pronounced, PP from OVA- and PBS-fed BALB/c mice responded similarly to
restimulation with OVA in vitro. In agreement with this observation,
fecal OVA-specific IgA responses were significantly reduced by OVA
feeding in Stat 4-/- and Stat 6-/- mice but
only marginally suppressed in normal BALB/c mice (Fig. 5).
Detection of TGF-ß secreting cells in the PP but not in the PLN is not surprising since several reports suggest that the PP is the major inductive site for TGF-ß-secreting Th3 cells, which are purported to play a role in both tolerance to oral Ags and regulation of IgA (7). An explanation for our findings may be suggested by a recent report that showed that feeding of a single, relatively high dose of Ag, followed by immunization with Ag in CFA, led to up-regulation of mucosal expression of MCP-1, the monocyte chemotactic protein (30). This suggests a mechanism by which Ag feeding could induce migration of T cells into the PP. Interestingly, this report also showed that up-regulation of mucosal MCP-1 was associated with an increase in IL-4 and down-regulation of IL-12 expression in the mucosa but not in the periphery. Administration of a neutralizing Ab to MCP-1 blocked mucosal down-regulation of IL-12 and abrogated oral tolerance. Since the induction of TGF-ß-secreting regulatory cells by oral Ag has been shown to be counter-regulated by IL-12 (2, 29), the up-regulated expression of mucosal MCP-1 induced by oral Ag may exert its effect by potentiating the effectiveness, or increasing the numbers, of TGF-ß-secreting regulatory cells. IL-12 responses are impaired in the Th1-deficient Stat 4-/- mice, resulting, in our model, in mucosal tolerance to oral Ag that is, in part, mediated by TGF-ß. Detection of TGF-ß-secreting cells in the PP of Stat 6-/- mice may suggest that chemokine (i.e., MCP-1)-mediated down-regulation of IL-12 is unimpeded by the absence of Th2 cells. Although it has been suggested that IL-4 is a differentiation factor for Th3 cells (10), we and others have now shown that peripheral tolerance to oral Ag does not require Th2 cells (22). Moreover, recent studies have also shown that, in vitro, CD4+ T cells can be induced to secrete TGF-ß in the absence of both IL-4 and IL-13 (albeit suboptimally), paralleling the conditions present in the Stat 6-/- mice (31). The apparent enhancement of mucosal tolerance in Stat 6-/- mice when compared with that observed in BALB/c mice may indicate that mucosal Th2 cells are more important for regulating, than actually mediating, the response to oral Ag. The responses seen in Stat 4-/- and Stat 6-/- mice may therefore represent an exaggeration of the response in normal mice, i.e., that the induction of mucosal TGF-ß-secreting cells by oral Ag is regulated by both Th1 and Th2 cytokines without inducing either an apparent mucosal nonresponsiveness or immunity to the fed Ag.
Our results also provide some interesting insights into cytokine regulation of IgA. Although early reports suggested that oral tolerance is associated with concomitant local (IgA) immunity (32, 33), there is actually very little experimental data to support this contention, which remains controversial (reviewed in 34). Our data show that total and OVA-specific IgA responses are equivalent in BALB/c, Stat 4-/-, and Stat 6-/- mice fed PBS. The impairment of Th2 development in Stat 6-/- mice is more pronounced than in IL4-/- mice, but serum IgA levels in mutant mice are equivalent to those in controls (16, 17), in spite of a proposed role for Th2 cytokines in IgA switching. The OVA-specific IgA response, induced by i.p. immunization with OVA in CFA, is significantly suppressed by OVA feeding in both Stat 4-/- and Stat 6-/- mice and marginally suppressed in BALB/c mice, arguing against the induction of IgA immunity by oral Ag and supporting a role for both Th1 and Th2 cytokines in regulating this response. This is in marked contrast to the result seen when oral Ag is administered with the mucosal adjuvant cholera toxin, which induces both peripheral immunity and a mucosal Ag-specific IgA response (which may also be regulated by TGF-ß) and is dependent upon Th2 cells (20, 23, 35, 36).
In summary, both cell-mediated and humoral peripheral tolerance to oral Ag is maintained in the absence of Th1 or Th2 cells. Mucosal, TGF-ß-mediated nonresponsiveness to restimulation with OVA in vitro is detectable in PP lymphocytes from mice deficient in Th1 or Th2 cells and correlates with the suppression of OVA-specific IgA responses by fed Ag in these mice. OVA feeding does not significantly alter either the PP proliferative responsiveness or Ag-specific fecal IgA response in normal BALB/c mice. Moreover, we did not obtain evidence for the induction of local immunity by oral Ag in normal, Th1-deficient, or Th2-deficient mice.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Cathryn Nagler-Anderson, Mucosal Immunology Laboratory, Room 3308, Massachusetts General Hospital East, 3 West, Building 149, 13th Street, Charlestown, MA 02129. E-mail address:
3 Abbreviations used in this paper: PLN, popliteal lymph node; PP, Peyer’s patch; HRP, horseradish peroxidase; MCP, monocyte chemotactic protein.
Received for publication June 12, 1998. Accepted for publication February 8, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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treatment. J. Immunol. 144:4163.[Abstract]
receptor deficient mice exhibit impaired gut mucosal immune responses but intact oral tolerance. Immunology 92:60.[Medline]
knockout mice. J. Immunol. 160:1687. have opposing effects, while TGF-ß positively regulates its own production. J. Immunol. 160:5719.This article has been cited by other articles:
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  C. R. Cardoso, P. R. Provinciatto, D. F. Godoi, B. R. Ferreira, G. Teixeira, M. A. Rossi, F. Q. Cunha, and J. S. Silva IL-4 regulates susceptibility to intestinal inflammation in murine food allergy Am J Physiol Gastrointest Liver Physiol, March 1, 2009; 296(3): G593 - G600. [Abstract] [Full Text] [PDF]
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 J. Mestecky, M. W. Russell, and C. O. Elson Perspectives on Mucosal Vaccines: Is Mucosal Tolerance a Barrier? J. Immunol., November 1, 2007; 179(9): 5633 - 5638. [Abstract] [Full Text] [PDF]
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R. Safadi, C. E. Alvarez, M. Ohta, J. Brimnes, T. Kraus, W. Mehal, J. Bromberg, L. Mayer, and S. L. Friedman Enhanced Oral Tolerance in Transgenic Mice with Hepatocyte Secretion of IL-10 J. Immunol., September 15, 2005; 175(6): 3577 - 3583. [Abstract] [Full Text] [PDF]
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 Y. Chung, S.-H. Lee, D.-H. Kim, and C.-Y. Kang Complementary role of CD4+CD25+ regulatory T cells and TGF-{beta} in oral tolerance J. Leukoc. Biol., June 1, 2005; 77(6): 906 - 913. [Abstract] [Full Text] [PDF]
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H.-C. Chang, S. Zhang, and M. H. Kaplan Neonatal Tolerance in the Absence of Stat4- and Stat6- Dependent Th Cell Differentiation J. Immunol., October 15, 2002; 169(8): 4124 - 4128. [Abstract] [Full Text] [PDF]
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N. S. Ostlie, P. I. Karachunski, W. Wang, C. Monfardini, M. Kronenberg, and B. M. Conti-Fine Transgenic Expression of IL-10 in T Cells Facilitates Development of Experimental Myasthenia Gravis J. Immunol., April 15, 2001; 166(8): 4853 - 4862. [Abstract] [Full Text] [PDF]
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H. Kato, K. Fujihashi, R. Kato, Y. Yuki, and J. R. McGhee Oral Tolerance Revisited: Prior Oral Tolerization Abrogates Cholera Toxin-Induced Mucosal IgA Responses J. Immunol., March 1, 2001; 166(5): 3114 - 3121. [Abstract] [Full Text] [PDF]
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H. N. Shi, H. Y. Liu, and C. Nagler-Anderson Enteric Infection Acts as an Adjuvant for the Response to a Model Food Antigen J. Immunol., December 1, 2000; 165(11): 6174 - 6182. [Abstract] [Full Text] [PDF]
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 K. M. SMITH, A. D. EATON, L. M. FINLAYSON, and P. GARSIDE Oral Tolerance Am. J. Respir. Crit. Care Med., October 1, 2000; 162(4): S175 - 178. [Abstract] [Full Text] [PDF]
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A. McI. Mowat, M. Steel, A. J. Leishman, and P. Garside Normal Induction of Oral Tolerance in the Absence of a Functional IL-12-Dependent IFN-{gamma} Signaling Pathway J. Immunol., November 1, 1999; 163(9): 4728 - 4736. [Abstract] [Full Text] [PDF]
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and 
,
PBS-fed mice; 
and •, OVA-fed mice. The plates were pulsed at
48 h with [3H]TdR and harvested 16–18 h later. The
results are displayed as the mean ± SEM of triplicate wells and
are representative of four independent experiments. The data in
Figs. 1–3
and 
, BALB/c mice; 
