
T cells regulate mucosally induced tolerance in a dose-dependent fashion
Kohtaro Fujihashi1,
Taeko Dohi2,
Mi-Na Kweon3,
Jerry R. Mcghee2,
Toshiya Koga1,
Max D. Cooper2,4,5,6,
Susumu Tonegawa7 and
Hiroshi Kiyono1,3
1 Departments of Oral Biology,
2 Microbiology,
4 Medicine and
5 Pediatrics, Immunobiology Vaccine Center, and the
6 Howard Hughes Medical Institute, University of Alabama at Birmingham, Medical Center, Birmingham, AL 35294, USA
7 Center for Cancer Research, Department of Biology, Howard Hughes Medical Institute and Massachusetts Institute of Technology, Cambridge, MA 02139, USA
3 Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565, Japan
Correspondence to:
K. Fujihashi, Department of Oral Biology, Immunobiology Vaccine Center, University of Alabama at Birmingham, Medical Center, BBRB 761, Birmingham, AL 35294-2170, USA
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Abstract
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We used 
TCR-deficient (TCR
/) mice to examine the role of 
T cells for induction of mucosal responses and systemic tolerance to high versus low doses of oral antigen. When either TCR
/ or TCR
+/+ mice were immunized orally with a high dose of ovalbumin (OVA) prior to parenteral challenge, systemic IgG and IgE antibody responses were markedly reduced in both types of mice, while mucosal IgA responses were reduced only in the TCR
/ mice. Reduced T cell proliferative responses and delayed-type hypersensitivity were seen in TCR
/ and TCR
+/+ mice given the high dose of OVA. Antigen-induced Th1 and Th2 cytokine production by splenic CD4+ T cells was severely inhibited in orally tolerized TCR
/ and TCR
+/+ mice. In contrast, while oral tolerance associated with increased levels of IL-10 synthesis was induced by a low dose of OVA in TCR
+/+ mice, the TCR
/ mice were not tolerized and failed to produce IL-10. Our findings indicate that 
T cells play a significant immunoregulatory role in IL-10-mediated, low-dose oral tolerance induction, but are not essential participants in the induction of systemic tolerance to orally introduced antigens given in larger doses.
Keywords: CD4+ T cells, knockout mice, 
T cells, oral antigens, tolerance
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Introduction
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Oral administration of proteins or haptens can induce a state of systemic unresponsiveness (1). Sulzberger and Chase established that oral administration of hapten inhibits contact hypersensitivity and this immunologic reaction still bears their name (2). In the early 1980s, this type of immunological response was dubbed oral tolerance and the concept used to refer specifically to immune responses elicited in mucosa-associated as opposed to systemic tissues (3). In general, oral administration of large amounts of antigen resulted in the induction of antigen-specific IgA responses in the mucosal compartment, whereas unresponsiveness to the same antigen was evident in the systemic immune system. Other recent studies have shown that nasal administration of proteins may also induce systemic unresponsiveness (4), leading mucosal immunologists to broaden the earlier term of oral tolerance to the more inclusive `mucosally induced tolerance'.
Current evidence suggests that T lymphocytes are the major cell type involved in the induction of mucosally induced tolerance (1). Oral administration of myelin basic protein (MBP) induced transforming growth factor (TGF)-ß-producing CD8+ T cells that were capable of inducing unresponsiveness and inhibiting experimental autoimmune encephalomyelitis (57). Further, it was shown that experimental tracheal eosinophilia can be down-regulated by TGF-ß-producing CD4+ T cells (8). By contrast, CD4+ T cells anergized by large doses of oral antigen feeding were implicated in the induction of antigen-specific systemic unresponsiveness (9). It has been suggested that Th1-type cells are more sensitive to the induction of tolerance than Th2-type cells (10,11). Thus, oral tolerance is possibly associated with selective down-regulation of Th1 cells which are more sensitive to tolerance induction by Th2 cells via their respective cytokines in the systemic immune compartment. However, recent studies have now shown that feeding high doses of ovalbumin (OVA) inhibited production of both Th1 (IL-2 and IFN-
) and Th2 (IL-4, IL-5 and IL-10) cytokines, and resulted in the reduction of IFN-
- and IL-4-dependent, antigen-specific IgG2a and IgG1 antibody responses respectively (9). These findings indicate that both Th1 and Th2 cell subsets are involved in the induction of oral tolerance.
The mucosal immune system possesses unique anatomical features and is composed of specialized subsets of lymphoid cells. Among the intestinal intraepithelial lymphocyte population, it is the CD8+ T cell subset that is prevalent and 
T cells comprise 3050% of this population (1215). Studies of TCR-deficient mice suggest an important role for the 
T cells in the immune responses to intracellular bacteria and parasites (1618). The 
T cells appear to be necessary for maintenance of mucosal IgA responses in the presence of systemic unresponsiveness induced by oral immunization (19,20). Other studies have provided direct evidence for their influence on both IgA and IgE responses (4,21). These observations suggest that 
T cells are a crucial functional component of the mucosal immune system. Indeed, recent reports indicate that 
T cells are essential for the induction of both low- and high-dose oral tolerance (22,23), although the mechanisms for the immunoregulatory role of 
T cells in the induction of systemic unresponsiveness to low and high antigen doses still remain to be elucidated.
In order to address these issues, we have compared the immune responses in TCR
/ and in control mice given different doses of OVA orally prior to systemic challenge. The results indicate that systemic unresponsiveness to large doses of ingested OVA is unimpaired in TCR
/ mice. In the case of low-dose tolerance, our data demonstrate that increased levels of IL-10 production are associated with the presence of 
T cells for the induction of systemic unresponsiveness. In contrast, low doses of proteins fail to induce IL-10-mediated tolerance in TCR
/ mice.
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Methods
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T cell-deficient mice
The derivation and initial characterization of TCR
/ mice have been described previously (24). TCR
/ mice used in this study were maintained on a C57BL/6 (H-2b) background. The
chain-deficient and normal mice (TCR
+/+) were maintained in Trexler isolators and remained pathogen-free. At 56 weeks of age, the mice were removed from the colony isolator unit, housed in micro-isolator cages in horizontal laminar flow cabinets, and provided sterile food and water ad libitum. The mice were between 7 and 10 weeks of age at the beginning of individual experiments.
Immunization
To establish systemic unresponsiveness to a high dose of oral antigen, mice were given 25 mg of OVA (Fraction V; Sigma, St Louis, MO) dissolved in 0.25 ml of PBS by gastric intubation. Control mice received PBS only. Seven days later, mice were immunized via the i.p. systemic route with 100 µg of OVA in 100 µl of complete Freund's adjuvant (OVA/CFA) (Difco, Detroit, MI). For induction of low-dose oral tolerance, mice were gavaged with 2.5 mg of OVA or hen egg white lysozyme (HEL; Sigma) in 0.25 ml of PBS on days 0, 2 and 5, then immunized s.c. on day 7 with OVA/CFA or 100 µg of HEL in 100 µl of CFA (HEL/CFA) (23). Antigen-specific T and B cell responses were determined 14 days after i.p. or 10 days after s.c. immunization. In some experiments, TCR
+/+ mice were treated by i.p. injection of anti-mouse 
mAb (GL3, 200 µg/mouse) or hamster IgG as a control (Jackson ImmunoResearch, West Grove PA) 3 days before oral administration of OVA or systemic immunization with OVA/CFA (23).
Delayed-type hypersensitivity (DTH) responses
Antigen-specific DTH responses were measured 7 and 14 days after systemic challenge by a standard protocol (25). Briefly, PBS (20 µl) containing 10 µg of antigen (OVA or HEL) was injected into the left ear pinna. Ear swelling was measured 24 h later with a dial thickness gauge (Ozaki, Tokyo, Japan). The DTH response was expressed as the increase of ear swelling after antigen (e.g. OVA or HEL) injection following subtraction of swelling in the control site.
Antigen-specific T cell responses
The spleen was removed aseptically and single cell suspensions prepared in RPMI 1640 (Cellgro Mediatech, Washington, DC) containing HEPES buffer, non-essential amino acids, sodium pyruvate, L-glutamine, penicillin/streptomycin and gentamycin (incomplete medium) (21). CD4+ T cells were purified by the MACS system (Stefen Milteny, Bergish- Gladbach, Germany) as previously described (21). Cells were passed through the magnetized column following incubation with biotin-labeled anti-CD4 mAb (GK 1.5) and streptavidin-conjugated microbeads. The isolated CD4+ T cells were >95% pure and >99% viable. To evaluate OVA or HEL responses, CD4+ T cells were cultured with 1 mg/ml of OVA or HEL in the presence of feeder cells for 4 days. During the last 18 h of incubation, 0.5 µCi of [3H]thymidine was added before the cells were harvested and the amount of incorporation determined by scintillation counting. Control cultures were without antigen stimulation. In other experiments, lymphocytes isolated from spleen or intestinal epithelium of TCR
+/+ mice fed low doses of OVA after 4 days of systemic challenge were stained with FITC-conjugated anti-mouse 
mAb (GL3; PharMingen, San Diego, CA) and then purified by flow cytometry (Becton Dickinson, Oxnard, CA). The purified 
T cells (2.5x1051x106) were cultured with CD4+ T cells (2x106) isolated from TCR
/ mice fed low doses of OVA in the presence of 1 mg of OVA and feeder cells for 7 or 14 days. In some experiments, a double-chamber culture system (Becton Dickinson, Franklin Lakes, NJ) was employed. The purified 
T cells (2.5x1051x106) were added to the upper well of the double chamber with CD4+ T cells, feeder cells and OVA as described above.
Cytokine-specific ELISA and ELISPOT assays
Levels of cytokines in culture supernatants were measured by an ELISA, while the number of cytokine-secreting cells in CD4+ T cell fractions was determined by cytokine-specific ELISPOT assays. The details of the ELISA and ELISPOT assays for IFN-
, IL-2, IL-4, IL-5, IL-6 and IL-10 have been described previously (2527). Combinations of anti-IFN-
: R4-6A2 and XMG 1.2 mAb; anti-IL-2: JES6-1A12 and JES6-5H4 mAb; anti-IL-4: BVD4-1D11 and BVD6-24G2 mAb; anti-IL-5: TRFK-5 and TRFK-4 mAb; anti-IL-6: MP5-20F3 and MP5-32C11 mAb; and anti-IL-10: JES5-2A5 and JES5-16E3 mAb were used as coating and detection antibodies respectively. A mouse IL-10 immunoassay kit (Quantikine M; R & D systems, Minneapolis, MN) was used to detect IL-10 in the culture supernatants. The levels of antigen-specific cytokine production were calculated by subtracting the results of control cultures (e.g. without antigen stimulation) from those of antigen-stimulated cultures. This ELISA was capable of detecting 0.8 ng/ml of IFN-
, 0.4 U/ml of IL-2, 25 pg/ml of IL-4, 0.8 U/ml of IL-5, 200 pg/ml of IL-6 and 4 pg/ml of IL-10.
Antibody assays
Antibody titers in serum and fecal extracts were determined by an ELISA (21,28). Falcon Microtest assay plates (Becton Dickinson) were coated with an optimal concentration of OVA or HEL (100 µl of 1 mg/ml) in PBS. Endpoint titers were expressed as the last dilution yielding an optical density at 414 nm (OD414) > 0.1 U above negative control values after a 15 min incubation. Total IgE levels were determined by ELISA using mouse IgE mAb (clone 27-74; PharMingen) as the standard. Rat anti-mouse IgE mAb (clone R35-72; PharMingen) and biotinylated rat anti-mouse IgE mAb (clone R35-92; PharMingen) were employed respectively as coating and detection antibodies. OVA-specific serum IgE was detected by a modified IgE-capture luminometric assay (28). Light development was carried out in a Dynetech ML-3000 luminometer by injection of Ca2+ buffer (50 mM Tris, 20 mM calcium acetate, pH 7.5). Endpoint titers were determined as the dilution of each sample showing a 2-fold higher level of luminometric units above background.
Enumeration of antibody-forming cells (AFC)
Spleens were aseptically removed and single-cell suspensions prepared (21). Mononuclear cells in the lamina propria were isolated after removal of Peyer's patches from the small intestine by the use of a combination of enzymatic dissociation and discontinuous Percoll gradients (Pharmacia, Uppsala, Sweden) (21,28). Mononuclear cells in the interface between the 40 and 75% layers were removed, washed, and resuspended in RPMI 1640 containing 10% FCS. An ELISPOT assay was employed to detect cells producing IgM, IgG and IgA antibodies (21). Ninety-six well nitrocellulose plates (Millititer HA; Millipore, Bedford, MA) were coated with 1 mg/ml of OVA (100 µl/well) in order to detect anti-OVA specific AFC (28).
Statistics
The significance of the difference (e.g. P values) between groups was evaluated by the MannWhitney U-test using a Statview II program designed for Macintosh computers.
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Results
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Systemic B cell unresponsiveness in TCR
/ mice immunized orally with a large dose of OVA
In order to investigate the potential role of 
T cells in oral tolerance induction, we initially examined the effects of TCR
gene disruption on the efficiency with which oral administration of OVA induces unresponsiveness. When OVA-specific antibody levels in serum of TCR
/ mice and control mice of the same background (TCR
+/+; C57BL/6) were examined, both groups of mice given 25 mg of OVA orally had significantly reduced IgG responses following i.p. challenge when compared with mice given PBS only (Fig. 1A
, P < 0.004). Similar results were obtained when mice were challenged by the s.c. route (data not shown). On the other hand, reduced levels of fecal IgA antibodies were seen in TCR
/ mice given OVA orally when compared with fecal IgA levels in TCR
+/+ mice fed the same antigen (Fig. 1B
, P < 0.003). These findings were confirmed at the single-cell level by using the ELISPOT assay. Prior oral administration of OVA also significantly inhibited splenic IgG responses in TCR
/ and TCR
+/+ mice to subsequent parenteral immunization (Fig. 2A
, P < 0.001). The small intestinal lamina propria in both TCR
/ and TCR
+/+mice contained OVA-specific IgA-producing cells, although the number of these was significantly lower in TCR
/ mice than in normal controls (Fig. 2B
, P < 0.001). Oral administration of a large dose of protein antigen thus elicits reduced antigen-specific B cell responses in both systemic and mucosal compartments of TCR
/ mice.
When the serum levels of IgE were examined in TCR
/ and TCR
+/+ mice fed either OVA or PBS, the TCR
+/+ mice that ingested OVA (25 mg) showed lower total IgE levels than did those given PBS (Fig. 3A
, P < 0.004). IgE levels were similarly diminished in TCR
/ mice given OVA orally (Fig. 3A
, P < 0.002). However, OVA-specific IgE antibody responses were comparably reduced in the TCR
/ and control mice fed OVA prior to systemic immunization (Fig. 3B
, P < 0.002). Systemic IgE responses to protein antigens can therefore be inhibited in TCR
/ as well as in TCR
+/+ mice by prior oral administration of a protein antigen, at least in relatively high doses.
Oral OVA (high-dose) administration diminishes DTH and T cell proliferative responses in TCR
/ mice
We next determined whether or not oral administration of a protein antigen in relatively high doses similarly reduces subsequent DTH responsiveness in TCR
/ mice. Twenty-four hours after intradermal OVA challenge, DTH responses were determined by measuring the ear swelling reaction in systemically immunized mice. Both TCR
/ and TCR
+/+ mice given PBS orally and then immunized parenterally showed significant ear swelling (5060 µm) when the challenge dose of OVA (10 µg) was injected into the pinna. DTH responses were reduced comparably in both groups of mice given 25 mg of OVA via the oral route prior to systemic immunization (Fig. 4A
, P < 0.02). Having established that DTH responses were compromised in TCR
/ mice fed a relatively high dose of OVA, we examined the proliferative response of their T cells to OVA. Splenic CD4+ T cells were cultured for 4 days with antigen-presenting cells (APC) in the presence of OVA. Relatively low levels of T cell proliferation were observed for splenic CD4+ T cells from OVA-tolerized TCR
/ mice and TCR
+/+ mice, whereas relatively vigorous proliferative responses were observed for splenic CD4+ T cells from control TCR
/ and TCR
+/+ mice given saline orally (Fig. 4B
). The systemic hyporesponsiveness induced by the oral administration of a large dose of OVA is thus evident at the T cell level in TCR
/ mice.
OVA-specific Th1 and Th2 cytokine responses of splenic CD4+ T cells
Since T cell unresponsiveness was induced by oral administration of OVA to TCR
/ as well as in control mice, it was of interest to evaluate the Th1 and Th2 cytokine responses in orally tolerized mice. Purified CD4+ T cells from the spleens of TCR
/ mice previously fed OVA or PBS were incubated with or without 1 mg of OVA in the presence of autologous APC. Four days later, when the culture supernatants were harvested and examined by cytokine-specific ELISA, antigen-specific Th1 (IFN-
and IL-2) and Th2 ( IL-4, IL-5, IL-6 and IL-10) cell responses were significantly reduced in TCR
+/+ mice given OVA (Table 1
). A virtually identical profile of down-regulation in cytokine synthesis was seen in the TCR
/ mice following oral administration of OVA. To confirm these findings at the cellular level, the number of Th1 and Th2 cytokine-producing cells was determined by cytokine-specific ELISPOT assays (Table 2
). Splenic CD4+ T cells from TCR
/ mice systemically immunized with OVA in CFA included significant numbers of IFN-
-producing Th1 type cells as well as IL-4-, IL-5-, IL-6- and IL-10-secreting Th2-type cells 4 days after in vitro antigen stimulation (Table 2
). Conversely, when TCR
/ mice received a tolerizing dose of OVA before parenteral challenge, the numbers of antigen-specific cytokine-producing cells were greatly reduced for both Th1 and Th2 types of cytokines, and similar results were obtained for the TCR
+/+ control mice (Table 2
). These data indicate that Th1 and Th2 types of systemic responses are comparably down-regulated in TCR
/ and control mice by prior oral administration of a large dose of protein antigen.
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Table 2. The frequencies of splenic CD4+ Th1 and Th2 cytokine producing cells from TCR / mice orally tolerized with OVAa
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Low doses of oral OVA fail to induce systemic unresponsiveness in TCR
/ mice
Since repeated oral administration of relatively low doses of antigen can elicit systemic tolerance, the role of 
T cells was assessed in this form of oral tolerance. Both TCR
/ and TCR
+/+ mice were fed three doses of 2.5 mg of OVA prior to challenge by the parenteral (s.c.) route. TCR
+/+ mice showed lower anti-OVA IgG antibody responses in serum than did mice given oral PBS (Fig. 5A
, P < 0.02). Splenic IgG AFC were also reduced in the TCR
+/+ mice (Fig. 5B
, P < 0.004). In contrast, no differences in serum IgG anti-OVA antibody or splenic IgG AFC responses were observed between control and oral feeding groups of TCR
/ mice.
OVA-specific T cell responses were also assessed in TCR
/ and TCR
+/+ mice given small oral doses of antigen. Oral administration of small doses of OVA prior to systemic challenge resulted in diminished ear swelling or DTH (P < 0.004) in TCR
+/+ mice but not in TCR
/ mice (Fig. 6A
). A similar pattern was revealed by analysis of the CD4+ T cell proliferative responses. Thus, splenic CD4+ T cells from TCR
/ mice fed either PBS or small doses of OVA responded with significant proliferation after OVA stimulation, while the CD4+ T cell proliferative responses were significantly reduced in OVA pre-fed TCR
+/+ mice (Fig. 6B
, P < 0.02). Collectively, these results suggest that the 
T cells are essential for the induction of mucosally induced tolerance established by low doses of oral antigen.
Similar results were also obtained when HEL was used as antigen. TCR
+/+ mice fed relatively low doses of HEL showed significant reductions in anti-HEL IgG antibody responses in serum when compared with mice fed PBS (reciprocal log2 titer, HEL fed: 8.2 ± 0.2 versus PBS fed: 14.4 ± 0.5, P < 0.001). On the other hand, TCR
/ mice fed low doses of HEL had no reductions in HEL-specific IgG antibody responses (reciprocal log2 titer, HEL fed: 14.2 ± 0.6 versus. PBS fed: 14.5 ± 0.5 ). These results, indicating a lack of mucosally induced low-dose tolerance in TCR
/ mice, were confirmed by the observation of normal DTH and T cell proliferative responses in these mice (data not shown). These findings indicate the absence of low-dose oral tolerance in TCR
/ mice is antigen-specific.
Dose-dependent mucosally induced tolerance in anti-
mAb-treated TCR
+/+ mice
In order to examine whether or not dose-dependent mucosally induced tolerance is related to ontogenetic abnormalities in TCR
/ mice, a series of experiments were performed with TCR
+/+ mice treated with anti-
mAb. The anti-
treated TCR
+/+ mice fed a high dose of OVA prior to systemic immunization showed reduced IgG antibody responses when compared with anti-
treated TCR
+/+ mice given oral PBS only (Fig. 7A
, P < 0.02). In contrast, when anti-
treated TCR
+/+ mice were fed low doses of OVA, no reduction of the anti-OVA IgG antibody response was seen. A similar pattern of responses was noted at the T cell level. Thus, anti-
treated TCR
+/+ mice fed low doses of OVA resulted in the maintenance of DTH and T cell proliferative responses after systemic challenge with OVA/CFA. On the other hand, these T cell responses were markedly reduced in anti-
treated TCR
+/+ mice given a high oral dose of OVA (Fig. 7B and C
, P < 0.004 and P < 0.02 respectively). These results confirmed the dose-dependent 
T cell requirement for mucosally induced tolerance observed in TCR
/ mice.
Elevated levels of IL-10 in TCR
+/+ but not TCR
/ mice given low oral doses of OVA
The Th1 and Th2 cytokine responses in TCR
/ and TCR
+/+mice given small doses of OVA orally were assessed by culturing CD4+ T cells for 4 days followed by measurement of cytokines in the supernatants. The major difference observed in cytokine responses of TCR
/ and TCR
+/+ mice involved IL-10 production. Although none of the experimental groups produced IL-10 without in vitro antigen stimulation, splenic CD4+ T cells from TCR
+/+ mice fed multiple small doses of OVA prior to systemic immunization produced elevated levels of IL-10 when compared with TCR
+/+ mice given PBS only (Table 3
, P < 0.03). In contrast, CD4+ T cell IL-10 synthesis was significantly low in TCR
/ mice given small oral doses of OVA, although IL-10 production by CD4+ T cells from control TCR
/ and TCR
+/+ mice was comparable. These results were confirmed at the RNA level by quantitative RT-PCR analysis of IL-10 transcripts (data not shown). When production of other cytokines was examined, significant differences were not seen between the levels produced by CD4+ T cells from TCR
/ and TCR
+/+ mice. These findings suggest that IL-10 production may play an important role in low-dose oral tolerance induction and that 
T cells may regulate this response by CD4+ T cells.
In order to examine a possible role for TGF-ß in the induction and maintenance of low-dose oral tolerance, we measured levels of TGF-ß in CD4+ T cell cultures from orally treated TCR
/ and TCR
+/+ mice. The levels of TGF-ß did not increased in culture supernatants of splenic CD4+ T cells after OVA stimulation, suggesting that TGF-ß may not have an effect on the induction of systemic unresponsiveness by low doses of oral OVA.

T cells can up-regulate IL-10 synthesis in CD4+ T cells from TCR
/ mice fed low doses of OVA
In order to address the potential role of 
T cells in the induction of IL-10-mediated low-dose tolerance, 
T cells and CD4+ T cells respectively were isolated from TCR
+/+ and TCR
/ mice previously fed low doses of OVA. These T cell subsets were cultured separately, mixed together or separated by filters in the presence of antigen and feeder cells for 7 or 14 days. The culture supernatants were harvested and IL-10 levels were examined by ELISA. The control wells without antigen stimulation and those with 
T cells alone contained 13.1 ± 4.1 and 4.4 ± 0.2 pg/ml of IL-10 respectively. The levels of IL-10 production by CD4+ T cells were increased in the presence of 
T cells from spleen or intestinal epithelium of OVA fed and immunized (Table 4
, P < 0.03), but not by 
T cells taken from naive mice or from high-dose tolerant TCR
/ mice. Increased levels of IL-10-specific mRNA expression in CD4+ T cells but not in 
T cells after 7 days of culture further confirmed our findings (data not shown). Interestingly, when direct cellcell interactions between 
T cells and CD4+ T cells were prevented by using a double-chamber well system, reduced IL-10 production was noted (Table 4
). Splenic 
T cells in the trans-well system showed marked decreases in IL-10 production when compared with direct cellcell contact cultures (Table 4
, P < 0.02). These results provide direct evidence that 
T cells serve an immunoregulatory role for CD4+ T cells of IL-10 production under conditions of low-dose oral tolerance. The present findings further suggest that both soluble factors produced by 
T cells and cellcell interactions between 
T cells and CD4+ T cells are essential for maximum IL-10 synthesis which leads to an inhibition of systemic immune responses.
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Discussion
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The results of the present study indicate that the role of 
T cells in oral tolerance depends on the antigen dose. The 
T cells are not required for the systemic B or T cell unresponsiveness by relatively high doses of orally administered antigen. To the contrary, this T cell subset regulates low-dose oral tolerance via modulation of IL-10 production by CD4+ T cells. This antigen dose-dependent regulatory dichotomy provides an interesting paradox with regard to the local function of intestinal 
T cells in oral antigen responses. The sentinel location and increased numbers of 
T cells in the intestinal epithelium intuitively suggest a significant role in the responses to orally administered antigens. Their absence has been associated with the loss of certain epithelial cell functions (29) and to the diminishment of mucosal IgA responses to oral antigens (21), a finding confirmed in the present studies. The absence of the 
T cell subset would therefore be expected to influence the immunologic homeostasis pathway involved in mucosally induced hyporesponsiveness and our present findings indeed indicate compromised oral tolerance in TCR
/ mice given relatively low doses of OVA. We further demonstrated that 
T cells are able to up-regulate IL-10 synthesis in TCR
+/+ mice tolerized with low oral antigen doses. This finding provides direct evidence that 
T cells regulate IL-10 synthesis by CD4+ T cells in the induction of low-dose oral tolerance. Our results also confirm studies in which 
T cells were shown to regulate the induction and maintenance of systemic tolerance to antigens administered via oral (22,23), respiratory mucosa (4) or portal vein routes (30,31).
In striking contrast, the present results indicate that systemic tolerance following oral antigen administration can be induced normally by relatively high antigen doses in the complete absence of 
T cells. Equivalent reductions in systemic IgG and IgE antibody responses, DTH, T cell proliferation, and both Th1 and Th2 cytokine responses to OVA were all seen in both TCR
/ and TCR
+/+ mice fed with this antigen prior to parenteral immunization. The same results were observed in experiments in which TCR
+/+ mice were given anti-
mAb treatment. Moreover, TNBS and oxazolone feedings induced tolerance to contact sensitization in TCR
/ mice (data not shown). Although these results differ from those of similar studies in a previous report (22), others have shown that 
T cells are not essential for the induction of aerosol-induced IgE unresponsiveness to OVA (32). We conclude that, whereas the 
T cells are required for normal mucosal epithelial function and IgA responsiveness (21,31), they do not play an obligatory role in the establishment of high-dose oral or mucosally induced tolerance.
A major difference between earlier studies of 
T cell involvement in oral tolerance and the present ones involved the manner in which the 
T cells were manipulated. Some studies have relied primarily on anti-
mAb treatment of mice (22,23), while others involved the transfer of antigen-stimulated 
T cells to naive recipients (4,23,30,31). Engagement of the 
T cells via their TCR was a common feature in most of these experiments and one proposed mechanism for the tolerization was the production of inhibitory cytokines by the 
T cells (31). This possibility gains support from the demonstration that treatment of mice with anti-
TCR antibodies does not eliminate 
T cells, but instead leads indirectly to the activation of recipient
ß T cells (33,34). It is therefore possible that while 
T cells are non-essential for the induction of tolerance to antigens administered by mucosal routes, they may still play an active role in this process under certain natural and experimental conditions. Our results in TCR
/ mice and in an anti-
mAb treatment system have provided evidence that 
T cells may indeed play distinct roles in the induction of orally induced tolerance depending on the dose of antigen. Specifically, our data indicates that low-dose oral tolerance requires 
T cells for development of the systemic unresponsiveness and high-dose oral tolerance does not.
The difference in outcomes between the present studies and those of Ke et al. (23), are not easily reconciled. These investigators found that TCR
/ mice were refractory to tolerance induction by various doses of oral antigen as assessed by reduction in IgG antibody responses, and T cell proliferative and cytotoxic T lymphocyte responses to subsequent systemic immunization. The resolution of this discrepancy must await additional studies. However, a second potentially important difference in the present study and those of other investigations (23) is the manner in which oral tolerance was induced by oral feeding. This is a significant consideration since the mechanisms involved in mucosally induced tolerance may differ according to the antigen dose (1). Indeed, we found systemic tolerance was not induced in TCR
/ mice pre-administered orally with multiple small doses of OVA, but was induced in identically treated TCR
+/+ mice. Prolonged oral administration or feeding with doses of antigen can selectively activate CD4+ or CD8+ T cells which secrete suppressive cytokines, e.g. TGF-ß, IL-4 and IL-10, that may down-regulate antigen-specific immune responses (1). In tolerance induced by small doses of oral antigen, 
T cells may contribute to unresponsiveness by the production of TGF-ß, considered to be a key cytokine for the induction of active suppression (35). However, we could not demonstrate an inhibitory effect for TGF-ß for the induction of low-dose oral tolerance. Instead, in the present study, splenic CD4+ T cells in TCR
+/+ mice given low doses of OVA produced higher levels of IL-10 than did those of non-tolerized TCR
+/+mice, while those from TCR
/ mice fed low doses of OVA failed to produce IL-10. A recent study showed that regulatory CD4+ T cell clones which selectively produce IL-10, termed T regulatory cell 1 (Tr1), can dramatically reduce T cell proliferative responses (36). This T cell subset was found to be responsible for preventing the development of inflammatory bowel disease in a mouse model (36). These results support the importance of IL-10 production for induction of low-dose oral tolerance. The present results indicate that 
T cells are able to up-regulate the synthesis of this important cytokine by CD4+ T cells. The 
T cells may thus induce IL-10 producing regulatory CD4+ T cells that can limit systemic immune responses in mice primed with low oral antigen doses.
The findings obtained in a trans-well response system provide evidence indicating that both soluble factors produced by 
T cells and direct cellcell interactions are essential for maximum IL-10 production. Monocyte chemotactic protein (MCP)-1, a CC chemokine, is a candidate soluble factor for the induction of IL-10 synthesis. In support of this idea, MCP-1 enhanced Th2-type cytokine responses when transgenic T cells were stimulated via the TCRCD3 complex (37). MCP-1 may also play an immunoregulatory role in the induction of oral tolerance by inhibition of Th1-type responses (38). The 
T cells in spleen and in intestinal epithelium of low-dose tolerized mice could produce MCP-1 since 
T cells from spleen, intestinal epithelium or epidermis have been reported to exhibit distinct mRNA levels for chemokines such as macrophage inflammatory protein (MIP)-1
, MIP-1ß, RANTES and lymphotactin (39). Indeed, our separate study has shown that intestinal intraepithelial 
T cells stimulated with IL-7 and stem cell factor expressed mRNA specific for MCP-1 (manuscript in preparation). Our current findings provide direct evidence that 
T cells can regulate CD4+ T cell synthesis of IL-10, a cytokine that is important in the induction of low-dose oral tolerance, and the mechanism by which 
T cells regulate IL-10 synthesis by CD4+ T cells is currently under investigation. Interestingly, levels of IL-6, an important cytokine for terminal B cell differentiation, was higher in TCR
/ mice given low oral doses of OVA than in TCR
/ mice fed PBS. The findings that 
T cells can play a regulatory role in the induction of Th2-type cytokine production by CD4+ T cells complements those in previous studies, indicating that 
T cells may modulate Th2-type cytokine-mediated B cell responses (e.g. for IgE and IgA) (4,21).
A large single oral dose of protein can lead to T cell anergy or clonal deletion (1,40). In accordance with this outcome, our studies indicate that both Th1- and Th2-type OVA responses of splenic T cells were reduced when a large oral dose of OVA was given prior to parenteral challenge. A recent study involving adoptive transfer of OVA-specific transgenic T cells also indicated that a large oral dose of OVA elicited T cell anergy (41). In addition, mice rendered deficient in IL-4 production and thus Th2 defective could be tolerized with a large oral dose of OVA, and IL-4-regulated IgG1 and IFN-
-induced IgG2a antibody responses were both down-regulated (9). On the other hand, our previous study had shown that elevated levels of IFN-
production occur in orally tolerized BALB/c mice (25). The difference between the previous study (25) and our present investigation can be explained by the use of different experimental systems and strains of mice. The previous study was performed by using BALB/c mice which are known to be a Th2 prone strain. Further, these mice were immunized twice with OVA and CFA after OVA feeding. Thus, it is possible and even likely that high IFN-
synthesis was required in orally tolerized BALB/c mice in order to down-regulate preferential Th2-type responses. In addition, although purified CD4+ T cells were used in the in vitro system for Th1 and Th2 cytokine analysis in the present study, our previous work was performed with whole T cell populations. This could be an additional explanation for the high level of IFN-
synthesis in the previous report since both CD4+ and CD8+ T cells are sources of this cytokine.
In summary, the present studies also indicate that 
T cells regulate IL-10-producing CD4+ T cells for subsequent inhibition of systemic IgG responses in mice orally tolerized with low doses of antigen. Thus, 
T cells regulate the induction of low-dose oral tolerance, but are not essential participants in the induction of systemic unresponsiveness to high doses of orally ingested antigen.
 |
Acknowledgments
|
---|
We thank Dr Howard L. Weiner for his critical review and Dr Leo Lafranciois for providing the GL3 cell line. We thank Dr Kimberly McGhee for editorial assistance and Ms Sheila D. Turner for the final preparation of this manuscript. This work is supported by US Public Health Service Grants AI 35932, DE 09837, AI 18958, DK 44240, AI 43197, DE 12242, AI 65298 and AI 65299, as well as grants from the Ministry of Education, Science, Sports and Culture, the Ministry of Health and Welfare, an OPSR in Japan.
 |
Abbreviations
|
---|
AFC antibody-forming cells |
APC antigen-presenting cell |
CFA complete Freund's adjuvant |
DTH delayed-type hypersensitivity |
HEL hen egg lysozyme |
MCP monocyte chemotactic protein |
MIP macrophage inflammatory protein |
OVA ovalbumin |
TGF transforming growth factor |
 |
Notes
|
---|
Transmitting editor: S.-i. Nishikawa
Received 6 May 1999,
accepted 13 August 1999.
 |
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