The ICOS molecule stimulates production of the immunoregulatory cytokine IL-10, suggesting an important role for ICOS in controlling IL-10-producing regulatory T cells and peripheral T cell tolerance. In this study we investigate whether ICOS is required for development of oral, nasal, and high dose i.v. tolerance. Oral administration of encephalitogenic myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide to ICOS-deficient (ICOS−/−) mice did not inhibit experimental autoimmune encephalomyelitis (EAE), T cell proliferation, or IFN-γ production, in striking contrast to wild-type mice. Similarly, intranasal administration of MOG35–55 before EAE induction suppressed EAE and T cell responses in wild-type, but not in ICOS−/−, mice. In contrast, ICOS−/− mice were as susceptible as wild-type mice to high dose tolerance. These results indicate that ICOS plays an essential and specific role in mucosal tolerance and that distinct costimulatory pathways differentially regulate different forms of peripheral tolerance. Surprisingly, CD4+ cells from MOG-fed wild-type and ICOS−/− mice could transfer suppression to wild-type recipients, indicating that functional regulatory CD4+ cells can develop in the absence of ICOS. However, CD4+ T cells from MOG-fed wild-type mice could not transfer suppression to ICOS−/− recipients, suggesting that ICOS may have a key role in controlling the effector functions of regulatory T cells. These results suggest that stimulating ICOS may provide an effective therapeutic approach for promoting mucosal tolerance.

The B7-1/7–2:CD28/CTLA-4 pathway is the best-characterized T cell costimulatory pathway and has a critical role in regulating T cell activation and tolerance (1, 2, 3). The B7-1 (CD80) and B7-2 (CD86) costimulatory molecules provide a major signal for augmenting and sustaining T cell responses via interaction with CD28, but deliver inhibitory signals when they engage a second, higher affinity receptor on T cells, CTLA-4 (CD152) (4). In addition, CTLA-4 has been shown to be important in the induction of high dose tolerance (5, 6). Over the past several years, additional B7 and CD28 family members have been identified, and new costimulatory pathways have been delineated (2, 7, 8, 9). The discovery of the CD28 homologue ICOS together with its ligand (originally identified as B7h, but also called B7RP-1, GL-50, B7-H2, and LICOS) has raised questions about whether this pathway has unique or overlapping functions with the B7:CD28/CTLA-4 pathway for regulating T cell activation and tolerance (10, 11, 12, 13, 14). In contrast to the constitutive expression of CD28, ICOS is inducible, requiring previous activation of T cells. ICOS is up-regulated on most CD4+ and CD8+ T cells after TCR stimulation (10, 11). In addition, cross-linking of CD28 can stimulate ICOS expression (15). The ligand for ICOS (ICOSL)4 is expressed more broadly than B7-1 and B7-2 in lymphoid and nonlymphoid organs (13, 16). Although B7-1 and B7-2 are expressed predominately on APCs, ICOSL is expressed not only on cells of hemopoietic origin, but also on fibroblasts, endothelial cells, and some epithelial cells. ICOSL is expressed constitutively at low levels on resting B cells, on some macrophages and dendritic cells, and on a small subset of CD3+ T cells (11, 14, 16). Proinflammatory cytokines and CD40 cross-linking can up-regulate ICOSL expression. IFN-γ induces ICOSL expression on B cells, monocytes, and some dendritic cells, and TNF-α/IL-1β up-regulate ICOSL on endothelial cells (17).

Like CD28, ICOS has positive costimulatory activities, including enhancing cytokine production, up-regulating CD40L expression, and providing help for Ig isotype class switching by B cells (18, 19, 20). However, although CD28 has a key role in stimulating IL-2 production, ICOS does not significantly enhance IL-2 production (10, 21, 22, 23, 24). Initial studies showed that ICOS has an important role in costimulating production of IL-10 (10), a key immunoregulatory cytokine involved in the induction of regulatory T type 1 (Tr1) cells and suppression of autoimmunity (25). Although some early studies suggested a preferential role for ICOS in regulating Th2 responses, subsequent work has indicated that ICOS can stimulate both Th1 and Th2 cytokines (17, 26, 27, 28), and that it may be particularly important for the induction of IL-10-producing Tr cells that can inhibit Th2 responses and acute airway hyperreactivity (29).

The role of ICOS in the induction and regulation of autoimmunity, particularly in experimental autoimmune encephalomyelitis (EAE), has been investigated. There have been opposing outcomes of ICOS blockade during the induction vs effector phase of EAE (20, 30, 31). ICOS blockade during the effector phase of EAE ameliorated clinical disease, whereas ICOS blockade during the induction of EAE resulted in severely exacerbated and accelerated disease (30, 31). Similarly, ICOS-deficient (ICOS−/−) mice on the 129 × B6 mixed background developed more severe clinical disease in myelin oligodendrocyte glycoprotein 33–55 (MOG35–55)-induced EAE (20). There were more inflammatory foci in the brain and more CD4+ T cells in the CNS producing IFN-γ in ICOS−/− mice than in wild-type littermate controls. The increased severity of EAE in the ICOS−/− mice was attributed to a decrease in IL-13 and an increase in IFN-γ production by the ICOS−/− T cells (20). The mechanism by which blockade or elimination of ICOS results in the exacerbation of EAE has not been studied in any detail.

The importance of IL-10 as an immunoregulatory cytokine in EAE (32, 33) together with the role of ICOS in stimulating IL-10 production (10, 23, 27) suggest that ICOS may be a critical costimulatory molecule responsible for the induction and/or function of IL-10-producing Tr cells and peripheral T cell tolerance. Previous studies have shown that IL-10 is crucial for the generation of not only Tr1 cells, but also a critical cytokine for the induction of both oral and nasal (mucosal) tolerance (34, 35). In this study we investigate the obligatory role of ICOS in the development of various forms of peripheral T cell tolerance using ICOS−/− mice. We first examined whether ICOS is required for the induction of mucosal T cell tolerance. ICOS−/− mice were resistant to the induction of oral and nasal tolerance. To determine whether there were more global defects in induction of peripheral tolerance in ICOS−/− mice, we also examined tolerance induction by high dose i.v. administration of aqueous Ag. High dose tolerance was intact in ICOS−/− mice. These findings suggest that ICOS plays a crucial role in the development of mucosal tolerance, and that distinct costimulatory pathways may differentially regulate different forms of peripheral tolerance. Our data also demonstrate that ICOS−/− mice are not defective in the generation of Tr cells, but that Tr cells do not induce tolerance in the ICOS−/− host. These findings suggest that ICOS may have a key role in maintaining or controlling effector functions of Tr cells.

Wild-type and ICOS-deficient mice on the inbred 129 SvS4/Jae background were bred in our facility and have been described previously (18). ICOS-deficient mice were backcrossed onto the C57BL/6 background for 10 generations. Genotyping of ICOS−/− mice was performed by PCR as previously described (18). All mice for experiments were 8–12 wk old. Harvard Medical School and Brigham and Women’s Hospital are accredited by the American Association of Accreditation of Laboratory Animal Care, and mice were cared for in accordance with institutional guidelines in a pathogen-free facility.

MOG35–55 (single letter code of amino acids: MEVGWYRSPFSRVVHLYRNGK) and OVA323–339 (ISQAVHAAHAEINEAGR) were synthesized by Dr. D. Teplow (Biopolymer Facility, Center for Neurologic Diseases, Brigham and Women’s Hospital, Boston, MA) on an Applied Biosystems 430A peptide synthesizer using F-moc chemistry. The peptides were >90% pure, as determined by HPLC.

Mice maintained under specific pathogen-free conditions were injected in the flank bilaterally with 200 μl of inoculum containing 100 μg of MOG35–55 and 0.4 mg of Mycobacterium tuberculosis H37Ra (Difco) in IFA. Pertussis toxin (List Biological Laboratories; 200 ng) was injected i.v. on days 0 and 2 after immunization. The clinical signs were scored as follows: 1, limp tail; 2, partial hind leg paralysis; 3, total hind leg or partial hind and front leg paralysis; 4, total hind leg and partial front leg paralysis; and 5, moribund or dead. Mice were examined daily for signs of EAE in a blind fashion. On day 35 after immunization, mice were killed. Brains and spinal cords were harvested and fixed in 10% neutral buffered formalin. Paraffin sections were stained with H&E-Luxol Fast Blue stain to assess inflammation and demyelination. The numbers of inflammatory foci in meninges and parenchyma were counted for each sample by a blinded observer as described previously (36, 37).

To assess the role of ICOS in the induction of tolerance, we tolerized wild-type and ICOS−/− 129SvS4/Jae mice via different routes as described below.

Oral tolerance.

For induction of oral tolerance, mice were fed 250 μg of MOG35–55 in 200 μl of PBS by a cannula on days 7, 6, 5, 4, and 3 before EAE induction. EAE was induced in the tolerized mice by immunization with MOG35–55/CFA as described previously (38).

Nasal tolerance.

Nasal tolerance was induced by slowly instilling ∼100 μg of MOG35–55 in 10 μl of PBS into both nostrils on days 7, 5, and 3 before EAE induction as described above.

High dose tolerance.

Induction of high dose tolerance was performed by the injection i.v. of 500 μg of MOG35–55 dissolved in PBS on the same day that the mice were immunized for EAE induction. As a control, a separate set of mice was injected with 500 μg of OVA323–339 i.v. and immunized for EAE induction on the same day (39).

For adoptive transfer studies using CD4+ T cells from MOG peptide-fed mice, ICOS−/− and wild-type 129 or C57B6 mice were fed 250 μg of MOG35–55 in 0.1 ml of PBS by a cannula on days −7, −6, −5, −4, and −3. On day 0, mesenteric lymph nodes and spleens were harvested from mice, and CD4+ T cells were purified by flow cytometry using a FACSAria or FACSVantage high-speed cell sorter (BD Biosciences). After cell sorting, >99% cells were CD4+. CD4+ T cells (1 × 106) from either ICOS−/− or wild-type mice were adoptively transferred i.v. into wild-type or ICOS−/− recipients immediately after immunization with MOG35–55/CFA.

For proliferation assays, mice were immunized with peptide/CFA as described above, but were not given any pertussis toxin. A single-cell suspension was prepared from the draining lymph nodes on day 11 after immunization. Cells were cultured in HL-1 medium (Invitrogen Life Technologies) supplemented with 2 mM l-glutamine and seeded onto 96-well, flat-bottom plates (1 × 106 cells/well). The cells were restimulated with peptide for 72 h at 37°C in a humidified air condition with 5% CO2. To measure cellular proliferation, [3H]thymidine was added (1 μCi/well), and uptake of the radioisotope during the final 18 h of culture was counted with a beta-1205 counter (Pharmacia Biotech).

In parallel, the lymph nodes cells from immunized mice were cultured with peptide concentrations of 0, 1, 10, and 100 μg/ml. Supernatant from the cultures were harvested 48 h after activation and tested for the presence of various cytokines. The concentrations of IFN-γ, IL-2, IL-4, and IL-10 in the supernatants were measured by a sandwich ELISA according to the manufacturer’s guideline (BD Biosciences). Limits of detection were 195 pg/ml for IFN-γ, 25 pg/ml for IL-2, 12.5 pg/ml for IL-4, and 50 pg/ml for IL-10.

Three-color flow cytometry was performed as previously described (40). Briefly, 0.5–1 × 106 cells in 50 μl from spleens or lymph nodes were incubated in staining buffer (PBS with 4% BSA and 0.1% sodium azide) for 5 min. The cells were then stained with a mixture of PE- and FITC-conjugated mAb (0.5 μg/sample) for 30 min, washed twice, then fixed in PBS with 1% formaldehyde. The analysis was performed on a FACScan flow cytometer (BD Biosciences) with CellQuest software (BD Biosciences). All procedures were performed on ice until analysis. The mAbs for flow cytometry were purchased from BD Biosciences: biotinylated anti-CD25 (7D4), PE- or FITC-conjugated anti-CD4 (L3T4), and FITC-conjugated anti-ICOS (7E.17G9).

Because ICOS has an important role in regulating IL-4 and IL-10 production, and these two cytokines have been shown to be crucial in the induction of oral tolerance, we first investigated whether oral tolerance could be induced in ICOS−/− mice. ICOS−/− and wild-type 129SvS4/Jae mice were orally tolerized with MOG35–55 peptide and then immunized with MOG35–55/CFA to induce EAE. When wild-type mice were tolerized with vehicle only (PBS) and subsequently immunized with MOG35–55/CFA, they developed typical EAE. EAE could be suppressed in the wild-type mice by feeding MOG35–55 peptide before EAE induction. ICOS−/− mice orally tolerized with vehicle only (PBS) and subsequently immunized with MOG35–55/CFA developed EAE. In contrast to wild-type mice, the feeding of MOG35–55 to ICOS−/− mice before EAE induction did not inhibit EAE (Fig. 1,a and Table I). These results indicate that ICOS is necessary for the development of oral tolerance.

FIGURE 1.

Lack of ICOS prevents induction of oral tolerance. Control PBS solution or 100 μg of MOG35–55 peptide diluted in PBS was orally administered via a feeding cannula on days 7, 5, and 3 before immunization. ICOS−/− and wild-type mice were immunized with MOG35–55 for induction of EAE as described in Materials and Methods. EAE clinical signs in wild-type MOG35–55-fed mice (wild-type and ICOS−/−) were assessed daily and are presented over time (a). MOG35–55-reactive T cells from tolerized mice were tested for MOG35–55-specific proliferation (b) by [3H]thymidine incorporation in triplicate wells represented as the mean cpm. Cytokines (c–e) were analyzed in the supernatants by cytokine ELISA from activated cultures. Each data point represents the mean value representative of at least three individual experiments. ∗, p < 0.05; ∗∗, p < 0.01 (vs wild-type PBS-treated mice, by Mann-Whitney U test). Each experimental group consisted of four to six mice.

FIGURE 1.

Lack of ICOS prevents induction of oral tolerance. Control PBS solution or 100 μg of MOG35–55 peptide diluted in PBS was orally administered via a feeding cannula on days 7, 5, and 3 before immunization. ICOS−/− and wild-type mice were immunized with MOG35–55 for induction of EAE as described in Materials and Methods. EAE clinical signs in wild-type MOG35–55-fed mice (wild-type and ICOS−/−) were assessed daily and are presented over time (a). MOG35–55-reactive T cells from tolerized mice were tested for MOG35–55-specific proliferation (b) by [3H]thymidine incorporation in triplicate wells represented as the mean cpm. Cytokines (c–e) were analyzed in the supernatants by cytokine ELISA from activated cultures. Each data point represents the mean value representative of at least three individual experiments. ∗, p < 0.05; ∗∗, p < 0.01 (vs wild-type PBS-treated mice, by Mann-Whitney U test). Each experimental group consisted of four to six mice.

Close modal
Table I.

ICOS−/− mice are resistant to induction of mucosal tolerancea

Mice ToleranceClinical DiseasebHistologic Lesionsc
IncidenceDay of onsetMean maximal scoreCumulative scoreMeningeal fociParenchymal fociTotal foci
Oral administration        
 ICOS−/− MOG 11/11 10.3 ± 0.8 3.18 ± 0.32 45.0 ± 6.7 9.3 ± 3.8 9.8 ± 4.5 19.1 ± 8.2 
  PBS 11/11 10.1 ± 0.9 3.09 ± 0.39 42.2 ± 8.7 20.4 ± 9.1 26.3 ± 11.3 46.6 ± 20.2 
 Wild type MOG 11/11 13.0 ± 1.9 1.23 ± 0.18d 12.8 ± 3.5e 2.6 ± 1.4 4.0 ± 3.1 6.6 ± 4.4 
  PBS 11/11 10.4 ± 0.8 2.91 ± 0.32 31.1 ± 5.5 6.7 ± 2.1 4.5 ± 2.1 11.3 ± 4.1 
        
Nasal administration        
 ICOS−/− MOG 11/11 13.7 ± 0.8 2.86 ± 0.21 33.6 ± 4.4 10.7 ± 3.7 15.9 ± 5.2 23.1 ± 7.5 
  PBS 11/11 13.5 ± 0.7 3.27 ± 0.26 43.3 ± 5.4 24.1 ± 7.1 32.1 ± 9.0 56.3 ± 15.7 
 Wild type MOG 9/10 15.6 ± 0.9e 1.20 ± 0.28f 15.3 ± 4.5g 4.7 ± 2.8 3.9 ± 2.8 8.6 ± 5.5 
  PBS 10/10 12.8 ± 1.0 3.05 ± 0.37 39.0 ± 5.8 13.2 ± 6.0 19.3 ± 10.7 32.5 ± 16.1 
        
High-dose i.v. injection        
 ICOS−/− MOG 7/12 13.7 ± 1.4 0.58 ± 0.17h 2.3 ± 0.9i 0.5 ± 0.3 2.2 ± 1.8 2.7 ± 1.9 
  OVA 10/12 14.1 ± 1.3 2.71 ± 0.48 25.3 ± 7.0 19.3 ± 15.8 25.0 ± 22.8 44.3 ± 38.6 
 Wild type MOG 6/10 19.8 ± 3.9 0.50 ± 0.24h 2.1 ± 1.3j 1.8 ± 1.4k 2.8 ± 2.1 4.6 ± 3.5 
  OVA 9/10 16.1 ± 2.2 2.60 ± 0.43 20.0 ± 4.0 34.2 ± 12.3 23.8 ± 13.5 58.0 ± 25.1 
Mice ToleranceClinical DiseasebHistologic Lesionsc
IncidenceDay of onsetMean maximal scoreCumulative scoreMeningeal fociParenchymal fociTotal foci
Oral administration        
 ICOS−/− MOG 11/11 10.3 ± 0.8 3.18 ± 0.32 45.0 ± 6.7 9.3 ± 3.8 9.8 ± 4.5 19.1 ± 8.2 
  PBS 11/11 10.1 ± 0.9 3.09 ± 0.39 42.2 ± 8.7 20.4 ± 9.1 26.3 ± 11.3 46.6 ± 20.2 
 Wild type MOG 11/11 13.0 ± 1.9 1.23 ± 0.18d 12.8 ± 3.5e 2.6 ± 1.4 4.0 ± 3.1 6.6 ± 4.4 
  PBS 11/11 10.4 ± 0.8 2.91 ± 0.32 31.1 ± 5.5 6.7 ± 2.1 4.5 ± 2.1 11.3 ± 4.1 
        
Nasal administration        
 ICOS−/− MOG 11/11 13.7 ± 0.8 2.86 ± 0.21 33.6 ± 4.4 10.7 ± 3.7 15.9 ± 5.2 23.1 ± 7.5 
  PBS 11/11 13.5 ± 0.7 3.27 ± 0.26 43.3 ± 5.4 24.1 ± 7.1 32.1 ± 9.0 56.3 ± 15.7 
 Wild type MOG 9/10 15.6 ± 0.9e 1.20 ± 0.28f 15.3 ± 4.5g 4.7 ± 2.8 3.9 ± 2.8 8.6 ± 5.5 
  PBS 10/10 12.8 ± 1.0 3.05 ± 0.37 39.0 ± 5.8 13.2 ± 6.0 19.3 ± 10.7 32.5 ± 16.1 
        
High-dose i.v. injection        
 ICOS−/− MOG 7/12 13.7 ± 1.4 0.58 ± 0.17h 2.3 ± 0.9i 0.5 ± 0.3 2.2 ± 1.8 2.7 ± 1.9 
  OVA 10/12 14.1 ± 1.3 2.71 ± 0.48 25.3 ± 7.0 19.3 ± 15.8 25.0 ± 22.8 44.3 ± 38.6 
 Wild type MOG 6/10 19.8 ± 3.9 0.50 ± 0.24h 2.1 ± 1.3j 1.8 ± 1.4k 2.8 ± 2.1 4.6 ± 3.5 
  OVA 9/10 16.1 ± 2.2 2.60 ± 0.43 20.0 ± 4.0 34.2 ± 12.3 23.8 ± 13.5 58.0 ± 25.1 
a

Data are presented as mean ± SE (by Mann-Whitney U-test).

b

Day of onset, mean day of the onset of clinical disease. Mean maximal score, mean maximal clinical scores for the mice showing clinical disease. Cumulative score, summation of the clinical scores from day 0 to day 35.

c

Histological findings, data are presented as mean number of meningeal and parenchymal inflammatory lesions in brain and spinal cord.

d

1, p < 0.001;

e

2, p < 0.05;

f

3, p < 0.0001;

g

4, p < 0.01; (versus wild type, PBS);

h

5, p < 0.0001;

i

6, p < 0.001;

j

7, p < 0.01;

k

8, p < 0.05 (versus wild type, OVA).

To define the mechanism for the resistance of ICOS−/− mice to oral tolerance induction, we compared T cell responses in wild-type and ICOS−/− mice that had been fed MOG35–55 or PBS before EAE induction. Draining lymph cells were harvested and assessed for T cell proliferative responses and cytokine production after in vitro restimulation with MOG35–55 peptide. The PBS-fed and MOG35–55-immunized wild-type mice exhibited an in vitro proliferative response to MOG35–55 with concomitant production of IFN-γ. Oral administration of MOG35–55 before EAE induction in wild-type mice suppressed T cell proliferation (Fig. 1,b) and IFN-γ production (Fig. 1,c). In contrast, oral administration of MOG35–55 before EAE induction did not suppress T cell responses in ICOS−/− mice (Fig. 1, b–e). Both PBS- and MOG35–55-fed ICOS−/− mice that were immunized with MOG35–55 exhibited strong in vitro recall responses to MOG35–55 peptide associated with T cell proliferation and substantial IFN-γ production. There was little or no IL-2 or IL-4 detected in the culture supernatants (Fig. 1,d and data not shown). MOG feeding followed by MOG immunization induced low levels of IL-10 after in vitro activation in both wild-type and ICOS−/− mice. However, the production of IL-10 was relatively higher in wild-type MOG-fed and -immunized mice than in ICOS−/− mice that were fed and immunized with MOG (Fig. 1 e). These experiments demonstrate that low dose oral tolerance cannot be induced in ICOS−/− mice.

Because ICOS−/− mice were resistant to oral tolerance, we next investigated whether ICOS also had a significant role in another form of mucosal tolerance, nasal tolerance. Wild-type and ICOS−/− mice were nasally tolerized with MOG35–55 or a vehicle control, subsequently immunized with MOG35–55/CFA, and observed for the development of clinical EAE. Similar to the oral administration of MOG35–55, nasal administration of MOG35–55 to wild-type mice before EAE induction significantly suppressed EAE. However, when ICOS−/− mice were given MOG35–55 intranasally before EAE induction, EAE was not suppressed (Fig. 2,a and Table I). Wild-type mice that were nasally tolerized and then immunized with MOG35–55 exhibited reduced in vitro T cell proliferation and IFN-γ production in response to MOG35–55 compared with wild-type mice that had been given vehicle control before EAE induction. In contrast, in vitro T cell proliferation and IFN-γ production in response to MOG35–55 were not suppressed in ICOS−/− mice that had been given MOG35–55 intranasally and immunized with MOG35–55, similar to the resistance of ICOS−/− mice to oral tolerance. The production of IL-4 and IL-10, the two anti-inflammatory cytokines generally induced by nasal tolerance, was less in ICOS−/− mice than in wild-type mice that had been given MOG35–55 intranasally before EAE induction; however, nasal tolerance only modestly increased the production of these cytokines in wild-type and ICOS−/− mice. Although there was only a low level production of IL-2 in cells from both wild-type and ICOS−/− mice, nasal administration of Ag suppressed the in vitro production of IL-2 by cells from wild-type mice, but not that in ICOS−/− mice (Fig. 2, b–f).

FIGURE 2.

Lack of ICOS prevents induction of nasal tolerance. Mice were nasally administered MOG35–55 or PBS on days 7, 5, and 3 before the induction of EAE with MOG35–55. EAE was induced in wild-type and ICOS−/− mice with MOG35–55 as described in the text. The clinical EAE score was checked daily. Wild-type mice that received MOG35–55 nasally were resistant to EAE, whereas control mice developed EAE (a). Proliferation was detected by [3H]thymidine incorporation in triplicate wells after in vitro activation for 72 h. The data are presented as the mean cpm of triplicate wells (b). Cytokines from activated cultures were detected by quantitative ELISA (c–e). ICOS−/− mice that received MOG35–55 or PBS before EAE induction remained susceptible to EAE. T cells from tolerized MOG35–55-indued wild-type mice showed less proliferation (b), less IFN-γ (c) and IL-2 (d) production, and more IL-10 production compared with control and ICOS−/− mice. IL-4 production was greater in wild-type than ICOS−/− mice (e). Each data point represents mean value. A representative of at least three individual experiments is shown. ∗, p < 0.05; ∗∗, p < 0.01 (vs wild-type PBS-treated mice, by Mann-Whitney U test). Each experimental group consisted of four to six mice.

FIGURE 2.

Lack of ICOS prevents induction of nasal tolerance. Mice were nasally administered MOG35–55 or PBS on days 7, 5, and 3 before the induction of EAE with MOG35–55. EAE was induced in wild-type and ICOS−/− mice with MOG35–55 as described in the text. The clinical EAE score was checked daily. Wild-type mice that received MOG35–55 nasally were resistant to EAE, whereas control mice developed EAE (a). Proliferation was detected by [3H]thymidine incorporation in triplicate wells after in vitro activation for 72 h. The data are presented as the mean cpm of triplicate wells (b). Cytokines from activated cultures were detected by quantitative ELISA (c–e). ICOS−/− mice that received MOG35–55 or PBS before EAE induction remained susceptible to EAE. T cells from tolerized MOG35–55-indued wild-type mice showed less proliferation (b), less IFN-γ (c) and IL-2 (d) production, and more IL-10 production compared with control and ICOS−/− mice. IL-4 production was greater in wild-type than ICOS−/− mice (e). Each data point represents mean value. A representative of at least three individual experiments is shown. ∗, p < 0.05; ∗∗, p < 0.01 (vs wild-type PBS-treated mice, by Mann-Whitney U test). Each experimental group consisted of four to six mice.

Close modal

The resistance of ICOS−/− mice to the generation of both oral and nasal tolerance (mucosal tolerance) prompted us to investigate whether ICOS−/− mice have global defects in the development of tolerance. Therefore, we investigated whether ICOS−/− mice could be tolerized by i.v. administration of a high dose of MOG35–55 before immunization with MOG35–55. Wild-type and ICOS−/− mice developed EAE, which was suppressed equally well by previous administration of MOG35–55 i.v. (Fig. 3 and Table I). These findings indicate that although ICOS−/− mice have defects in the development of mucosal tolerance, they can develop high dose tolerance. Thus, ICOS−/− mice have selective defects in the development of two forms of mucosal tolerance tested.

FIGURE 3.

Lack of ICOS dose not prevent high dose tolerance. Both ICOS−/− and wild-type (Wt) mice were immunized with MOG35–55, to develop EAE and were injected i.v. simultaneously with 500 μg of MOG35–55 or OVA323–339 as a control. The mice were observed daily for the signs of EAE, and EAE scores were determined. Each data point represents the mean ± SEM. Shown is a representative of two separate experiments. Each experimental group consisted of four to six mice.

FIGURE 3.

Lack of ICOS dose not prevent high dose tolerance. Both ICOS−/− and wild-type (Wt) mice were immunized with MOG35–55, to develop EAE and were injected i.v. simultaneously with 500 μg of MOG35–55 or OVA323–339 as a control. The mice were observed daily for the signs of EAE, and EAE scores were determined. Each data point represents the mean ± SEM. Shown is a representative of two separate experiments. Each experimental group consisted of four to six mice.

Close modal

The selective role of ICOS in regulating mucosal tolerance led us to further investigate the mechanism by which ICOS influences mucosal tolerance, particularly whether ICOS−/− recipients had defects in the generation or the effector functions of regulatory T cells. CD4+ T cells can transfer suppression from orally tolerized animals to syngeneic recipients. To investigate whether ICOS−/− CD4+ T cells have defects in transferring suppression, we compared transfer of protection by CD4+ T cells from MOG-fed wild-type and ICOS−/− mice to syngeneic wild-type mice. Wild-type and ICOS−/− mice were fed five times with 0.25 mg of MOG35–55 over a 7-day period. Three days after the last feeding, spleen cells and mesenteric lymph nodes were removed, and their CD4+ T cells were purified, then adoptively transferred into syngeneic wild-type mice immediately after immunization with MOG35–55/CFA. As shown in Fig. 4, wild-type and ICOS−/− CD4+ T cells were comparably effective in suppressing EAE in wild-type MOG35–55-immunized recipients. These findings indicate that in the absence of ICOS, CD4+ Tr cells can be generated and are functional in vivo. However, wild-type CD4+ T cells from MOG35–55-fed wild-type mice failed to transfer protection to ICOS−/− MOG35–55-immunized recipients, and the transfer of CD4+ T cells from MOG-fed ICOS−/− mice to ICOS−/− MOG35–55-immunized recipients resulted in greatly exacerbated clinical disease. These studies suggest that ICOS has a key role in controlling the survival or effector function of Tr cells.

FIGURE 4.

Adoptive transfer of CD4+ T cells from MOG peptide-fed mice. Both ICOS−/− and wild-type (WT) mice were reconstituted with 1 × 106 CD4+ T cells isolated from MOG35–55 peptide-fed mice (ICOS−/− or wild type) and immunized with MOG35–55/CFA to develop EAE. Control mice were immunized, but not reconstituted with CD4+ T cells. Mice were scored daily. These data are representative of four independent experiments in which there were four to six mice per group. WT CD4+ T cells into WT mice vs WT CD4+ T cells into ICOS−/− mice, p < 0.05. ICOS−/− CD4+ T cells into WT mice vs ICOS−/− CD4+ T cells into ICOS−/− mice, p < 0.05 (by Mann-Whitney U test).

FIGURE 4.

Adoptive transfer of CD4+ T cells from MOG peptide-fed mice. Both ICOS−/− and wild-type (WT) mice were reconstituted with 1 × 106 CD4+ T cells isolated from MOG35–55 peptide-fed mice (ICOS−/− or wild type) and immunized with MOG35–55/CFA to develop EAE. Control mice were immunized, but not reconstituted with CD4+ T cells. Mice were scored daily. These data are representative of four independent experiments in which there were four to six mice per group. WT CD4+ T cells into WT mice vs WT CD4+ T cells into ICOS−/− mice, p < 0.05. ICOS−/− CD4+ T cells into WT mice vs ICOS−/− CD4+ T cells into ICOS−/− mice, p < 0.05 (by Mann-Whitney U test).

Close modal

We have examined the role of ICOS in the development of three forms of peripheral tolerance, i.e., oral, nasal, and high dose i.v. tolerance. ICOS−/− mice were resistant to the development of both oral and nasal tolerance, but not to high dose tolerance, suggesting that ICOS plays a specific role in mucosal tolerance. Adoptive transfer studies indicate that CD4+ Tr cells can be induced by oral administration of Ag in the absence of ICOS and that these CD4+ T cells can transfer protection to wild-type mice as effectively as CD4+ T cells induced in Ag-fed wild-type mice. However, CD4+ T cells induced in Ag-fed wild-type mice cannot transfer protection to ICOS−/− mice, suggesting that ICOS has a critical role in controlling the effector functions of these CD4+ Tr cells.

Oral tolerance has been used successfully to prevent a number of experimental autoimmune diseases, including EAE, experimental autoimmune neuritis, experimental autoimmune uveitis, arthritis, and diabetes in the NOD mouse (41, 42, 43, 44, 45, 46, 47). The effector mechanisms involved in oral tolerance have been extensively studied, and there appear to be multiple mechanisms involved in the generation of oral tolerance. Deletion and anergy of Ag-specific T cells occurs after the administration of high Ag doses, whereas the induction of regulatory CD4+ T cells, which produce anti-inflammatory cytokines such as IL-4, IL-10, and TGF-β, with a unique role for TGF-β, follows low dose Ag administration (35, 42, 43). Recent data suggest that CD4+CD25+ Tr cells may be partly responsible for the induction of oral tolerance in addition to regulatory Th2 and Th3 cells (45). In the present series of experiments ICOS was crucial for tolerance induction using a low dose multiple feeding regimen. High dose oral tolerance is intact in ICOS−/− mice (data not shown). Similar to high dose i.v. tolerance, high dose oral tolerance is primarily mediated by the induction of anergy and/or the deletion of responding T cells.

The nasal route appears equally efficient and, in some instances, even more effective than oral tolerance induction in suppressing autoimmune diseases in animal models (48, 49, 50). A recent report has shown that Tr cells and the ICOS-ICOSL signaling pathway are critically involved in down-regulating pulmonary inflammation in asthma (29). T cell tolerance induced by respiratory exposure to allergen can inhibit the development of airway hyperreactivity, a cardinal feature of asthma (51). In this study the Tr cells produced IL-10 and had potent inhibitory activity. Both the development and the inhibitory function of the regulatory cells were dependent on the presence of IL-10 and ICOS-ICOSL interactions (29).

Our present findings demonstrate that ICOS may play a broader role in the induction of mucosal tolerance than previously appreciated. The critical role for the ICOS-ICOSL pathway in mucosal tolerance might be related in part to the expression of ICOSL at mucosal sites. ICOSL is expressed by epithelial cells in mucosal tissue and by the APCs derived from mucosal tissue (51, 52). ICOSL expression has been detected on a human airway epithelial cell line, primary bronchial epithelial cells, as well as on colonic epithelial cell lines and primary colonic epithelial cells (53). Dendritic cells from pulmonary mucosa also have been shown to express high levels of ICOSL on their surface. This expression of ICOSL on dendritic cells and epithelial cells suggests a means by which the ICOS-ICOSL pathway could costimulate IL-10 production and favor generation of ICOS+ Tr cells at the mucosal sites.

Our data, however, suggest that ICOS does not control the induction of Tr cells, but, instead, appears to be necessary for sustaining or eliciting effector functions from Tr cells during mucosal tolerance. The distinct outcomes of transfer of CD4+ T cells from MOG-fed mice into wild-type vs ICOS−/− recipients suggests that the presence of ICOS in the recipient mice controls the regulatory functions of the transferred Tr cells. How does ICOS exert these effects? ICOS has the unique ability to costimulate IL-10 (10, 23, 27). During T cell expansion and differentiation, ICOS is up-regulated and maintained on Th2 cells, but is lost or down-regulated on Th1 cells (15). IL-10 and Th2 cells are crucial for the induction of both oral and nasal tolerance and the generation of IL-10-producing Tr1 cells and TGF-β-producing Th3 cells (37, 44, 49, 54). However, once induced, it not clear whether IL-10 also plays a role in the survival and maintenance of Tr1 and Th3 cells. ICOS may regulate these forms of peripheral tolerance through its effects on regulating IL-10 production and thus affect effector functions of the Tr cells. Transcriptional profiling studies indicate that ICOS blockade leads to marked changes in gene expression profiles of the Tr cells obtained from pancreatic infiltrates of 3- to 4-wk-old BCDC2.5 mice, suggesting that ICOS may be necessary for maintaining the Tr cell gene program or regulating the balance between effector T cells and Tr cells (55). Another potential explanation for the adoptive transfer data is that the Tr cells generated by oral or nasal tolerance are not able to control effector T cells that are deficient in ICOS expression. In the ICOS−/− recipient, the balance between Tr cells and T effector cells may be altered because of a change in the cytokine milieu, in particular, a reduction in IL-10. Without ICOS, the balance is shifted in the favor of autopathogenic Th1 cells, which may expand faster or produce more proinflammatory cytokines, such that these effector cells cannot be controlled by Tr cells, thereby resulting in more severe clinical disease (Fig. 4). To address these issues, additional studies are needed to investigate the relative expansion and cytokine profiles of regulatory vs effector cells in ICOS−/− recipients.

CTLA-4 has been shown to play an important role in the induction of high dose tolerance, whereas ICOS appears to be critical for mucosal, but not high dose, tolerance (5, 6). The different roles played by ICOS and CTLA-4 in mucosal tolerance vs high dose tolerance indicate that different costimulatory pathways may differentially regulate these different forms of tolerance. CTLA-4 inhibits IL-2 production and cell cycle progression, whereas ICOS does not affect IL-2 production, but instead has a unique role in promoting IL-10 production. The different effects of CTLA-4 and ICOS in regulating IL-2 and IL-10 production may explain why CTLA-4, but not ICOS, is required for tolerance induced by i.v. administration of high dose aqueous Ag.

In conclusion, we have found that ICOS has an essential and specific role in regulating mucosal tolerance. Stimulation of ICOS signals may provide an effective means to promote the function of IL-10-producing Tr cells and the induction of oral and nasal tolerance. Therefore, these results suggest that the ICOS-ICOSL pathway may be a novel target for immunotherapy.

We thank John Burgess and Yin Wu for outstanding technical support.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health Grants P01AI39671, R01NS35685 (to V.K.K. and A.H.S.), R01AI38310 (to A.H.S), R01NS38037 (to V.K.K. and H.L.W.), and NS046414 (to R.A.S.) and fellowships from the Japan Society for the Promotion of Science Postdoctoral Fellowships for Research Abroad (to K.M.) and the National Multiple Sclerosis Society (to C.K.). V.K.K. is the recipient of the Javitz Neuroscience Investigator Award from the National Institutes of Health.

4

Abbreviations used in this paper: ICOSL, ICOS ligand; EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; Tr1, regulatory T type 1.

1
Alegre, M. L., K. A. Frauwirth, C. B. Thompson.
2001
. T-cell regulation by CD28 and CTLA-4.
Nat. Rev. Immunol.
1
:
220
-228.
2
Sharpe, A. H., G. J. Freeman.
2002
. The B7-CD28 superfamily.
Nat. Rev. Immunol.
2
:
116
-126.
3
Chikuma, S., J. A. Bluestone.
2003
. CTLA-4 and tolerance: the biochemical point of view.
Immunol. Res.
28
:
241
-253.
4
Noel, P. J., L. H. Boise, C. B. Thompson.
1996
. Regulation of T cell activation by CD28 and CTLA4.
Adv. Exp. Med. Biol.
406
:
209
-217.
5
Perez, V. L., L. Van Parijs, A. Biuckians, X. X. Zheng, T. B. Strom, A. K. Abbas.
1997
. Induction of peripheral T cell tolerance in vivo requires CTLA-4 engagement.
Immunity
6
:
411
-417.
6
Greenwald, R. J., V. A. Boussiotis, R. B. Lorsbach, A. K. Abbas, A. H. Sharpe.
2001
. CTLA-4 regulates induction of anergy in vivo.
Immunity
14
:
145
-155.
7
Coyle, A. J., J. C. Gutierrez-Ramos.
2003
. More negative feedback?.
Nat. Immunol.
4
:
647
-648.
8
Khoury, S. J., M. H. Sayegh.
2004
. The roles of the new negative T cell costimulatory pathways in regulating autoimmunity.
Immunity
20
:
529
-538.
9
Chen, L..
2004
. Co-inhibitory molecules of the B7-CD28 family in the control of T-cell immunity.
Nat. Rev. Immunol.
4
:
336
-347.
10
Hutloff, A., A. M. Dittrich, K. C. Beier, B. Eljaschewitsch, R. Kraft, I. Anagnostopoulos, R. A. Kroczek.
1999
. ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28.
Nature
397
:
263
-266.
11
Yoshinaga, S. K., J. S. Whoriskey, S. D. Khare, U. Sarmiento, J. Guo, T. Horan, G. Shih, M. Zhang, M. A. Coccia, T. Kohno, et al
1999
. T-cell co-stimulation through B7RP-1 and ICOS.
Nature
402
:
827
-832.
12
Mages, H. W., A. Hutloff, C. Heuck, K. Buchner, H. Himmelbauer, F. Oliveri, R. A. Kroczek.
2000
. Molecular cloning and characterization of murine ICOS and identification of B7h as ICOS ligand.
Eur. J. Immunol.
30
:
1040
-1047.
13
Swallow, M. M., J. J. Wallin, W. C. Sha.
1999
. B7h, a novel costimulatory homolog of B7.1 and B7.2, is induced by TNFα.
Immunity
11
:
423
-432.
14
Ling, V., P. W. Wu, H. F. Finnerty, K. M. Bean, V. Spaulding, L. A. Fouser, J. P. Leonard, S. E. Hunter, R. Zollner, J. L. Thomas, et al
2000
. Cutting edge: identification of GL50, a novel B7-like protein that functionally binds to ICOS receptor.
J. Immunol.
164
:
1653
-1657.
15
McAdam, A. J., T. T. Chang, A. E. Lumelsky, E. A. Greenfield, V. A. Boussiotis, J. S. Duke-Cohan, T. Chernova, N. Malenkovich, C. Jabs, V. K. Kuchroo, et al
2000
. Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4+ T cells.
J. Immunol.
165
:
5035
-5040.
16
Aicher, A., M. Hayden-Ledbetter, W. A. Brady, A. Pezzutto, G. Richter, D. Magaletti, S. Buckwalter, J. A. Ledbetter, E. A. Clark.
2000
. Characterization of human inducible costimulator ligand expression and function.
J. Immunol.
164
:
4689
-4696.
17
Khayyamian, S., A. Hutloff, K. Buchner, M. Grafe, V. Henn, R. A. Kroczek, H. W. Mages.
2002
. ICOS-ligand, expressed on human endothelial cells, costimulates Th1 and Th2 cytokine secretion by memory CD4+ T cells.
Proc. Natl. Acad. Sci. USA
99
:
6198
-6203.
18
McAdam, A. J., R. J. Greenwald, M. A. Levin, T. Chernova, N. Malenkovich, V. Ling, G. J. Freeman, A. H. Sharpe.
2001
. ICOS is critical for CD40-mediated antibody class switching.
Nature
409
:
102
-105.
19
Tafuri, A., A. Shahinian, F. Bladt, S. K. Yoshinaga, M. Jordana, A. Wakeham, L. M. Boucher, D. Bouchard, V. S. Chan, G. Duncan, et al
2001
. ICOS is essential for effective T-helper-cell responses.
Nature
409
:
105
-109.
20
Dong, C., A. E. Juedes, U. A. Temann, S. Shresta, J. P. Allison, N. H. Ruddle, R. A. Flavell.
2001
. ICOS co-stimulatory receptor is essential for T-cell activation and function.
Nature
409
:
97
-101.
21
Parry, R. V., C. A. Rumbley, L. H. Vandenberghe, C. H. June, J.L. Riley.
2003
. CD28 and inducible costimulatory protein Src homology 2 binding domains show distinct regulation of phosphatidylinositol 3-kinase, Bcl-xL, and IL-2 expression in primary human CD4 T lymphocytes.
J. Immunol.
171
:
166
-174.
22
Riley, J. L., P. J. Blair, J. T. Musser, R. Abe, K. Tezuka, T. Tsuji, C. H. June.
2001
. ICOS costimulation requires IL-2 and can be prevented by CTLA-4 engagement.
J. Immunol.
166
:
4943
-4948.
23
Okamoto, N., K. Tezuka, M. Kato, R. Abe, T. Tsuji.
2003
. PI3-kinase and MAP-kinase signaling cascades in AILIM/ICOS- and CD28-costimulated T-cells have distinct functions between cell proliferation and IL-10 production.
Biochem. Biophys. Res. Commun.
310
:
691
-702.
24
Rudd, C. E., H. Schneider.
2003
. Unifying concepts in CD28, ICOS and CTLA4 co-receptor signalling.
Nat. Rev. Immunol.
3
:
544
-556.
25
Groux, H., A. O’Garra, M. Bigler, M. Rouleau, S. Antonenko, J. E. de Vries, M. G. Roncarolo.
1997
. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis.
Nature
389
:
737
-742.
26
Greenwald, R. J., A. J. McAdam, D. Van der Woude, A. R. Satoskar, A. H. Sharpe.
2002
. Cutting edge: inducible costimulator protein regulates both Th1 and Th2 responses to cutaneous leishmaniasis.
J. Immunol.
168
:
991
-995.
27
Lohning, M., A. Hutloff, T. Kallinich, H. W. Mages, K. Bonhagen, A. Radbruch, E. Hamelmann, R. A. Kroczek.
2003
. Expression of ICOS in vivo defines CD4+ effector T cells with high inflammatory potential and a strong bias for secretion of interleukin 10.
J. Exp. Med.
197
:
181
-193.
28
Wassink, L., P. L. Vieira, H. H. Smits, G. A. Kingsbury, A. J. Coyle, M. L. Kapsenberg, E. A. Wierenga.
2004
. ICOS expression by activated human Th cells is enhanced by IL-12 and IL-23: increased ICOS expression enhances the effector function of both Th1 and Th2 cells.
J. Immunol.
173
:
1779
-1786.
29
Akbari, O., G. J. Freeman, E. H. Meyer, E. A. Greenfield, T. T. Chang, A. H. Sharpe, G. Berry, R. H. DeKruyff, D. T. Umetsu.
2002
. Antigen-specific regulatory T cells develop via the ICOS-ICOS-ligand pathway and inhibit allergen-induced airway hyperreactivity.
Nat. Med.
8
:
1024
-1032.
30
Rottman, J. B., T. Smith, J. R. Tonra, K. Ganley, T. Bloom, R. Silva, B. Pierce, J. C. Gutierrez-Ramos, E. Ozkaynak, A. J. Coyle.
2001
. The costimulatory molecule ICOS plays an important role in the immunopathogenesis of EAE.
Nat. Immunol.
2
:
605
-611.
31
Sporici, R. A., R. L. Beswick, C. von Allmen, C. A. Rumbley, M. Hayden-Ledbetter, J. A. Ledbetter, P. J. Perrin.
2001
. ICOS ligand costimulation is required for T-cell encephalitogenicity.
Clin. Immunol.
100
:
277
-288.
32
Bettelli, E., M. Prabhu Das, E. D. Howard, H. L. Weiner, R. A. Sobel, V. K. Kuchroo.
1998
. IL-10 is critical in the regulation of autoimmune encephalomyelitis as demonstrated by studies of IL-10- and IL-4-deficient and transgenic mice.
J. Immunol.
161
:
3299
-3306.
33
Cua, D. J., B. Hutchins, D. M. LaFace, S. A. Stohlman, R. L. Coffman.
2001
. Central nervous system expression of IL-10 inhibits autoimmune encephalomyelitis.
J. Immunol.
166
:
602
-608.
34
Massey, E. J., A. Sundstedt, M. J. Day, G. Corfield, S. Anderton, D. C. Wraith.
2002
. Intranasal peptide-induced peripheral tolerance: the role of IL-10 in regulatory T cell function within the context of experimental autoimmune encephalomyelitis.
Vet. Immunol. Immunopathol.
87
:
357
-372.
35
Faria, A. M., R. Maron, S. M. Ficker, A. J. Slavin, T. Spahn, H. L. Weiner.
2003
. Oral tolerance induced by continuous feeding: enhanced up-regulation of transforming growth factor-β/interleukin-10 and suppression of experimental autoimmune encephalomyelitis.
J. Autoimmun.
20
:
135
-145.
36
Sobel, R. A., V. K. Tuohy, Z. J. Lu, R. A. Laursen, M. B. Lees.
1990
. Acute experimental allergic encephalomyelitis in SJL/J mice induced by a synthetic peptide of myelin proteolipid protein.
J. Neuropathol. Exp. Neurol.
49
:
468
-479.
37
Weiner, H. L..
1997
. Oral tolerance: immune mechanisms and treatment of autoimmune diseases.
Immunol. Today
18
:
335
-343.
38
Gaupp, S., H. P. Hartung, K. Toyka, S. Jung.
1997
. Modulation of experimental autoimmune neuritis in Lewis rats by oral application of myelin antigens.
J. Neuroimmunol.
79
:
129
-137.
39
Friedman, A., H. L. Weiner.
1994
. Induction of anergy or active suppression following oral tolerance is determined by antigen dosage.
Proc. Natl. Acad. Sci. USA
91
:
6688
-6692.
40
Critchfield, J. M., M. K. Racke, J. C. Zuniga-Pflucker, B. Cannella, C. S. Raine, J. Goverman, M. J. Lenardo.
1994
. T cell deletion in high antigen dose therapy of autoimmune encephalomyelitis.
Science
263
:
1139
-1143.
41
Chen, Y., V. K. Kuchroo, J. Inobe, D. A. Hafler, H. L. Weiner.
1994
. Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis.
Science
265
:
1237
-1240.
42
Chen, Y., J. Inobe, H. L. Weiner.
1995
. Induction of oral tolerance to myelin basic protein in CD8-depleted mice: both CD4+ and CD8+ cells mediate active suppression.
J. Immunol.
155
:
910
-916.
43
Chen, Y., J. Inobe, R. Marks, P. Gonnella, V. K. Kuchroo, H. L. Weiner.
1995
. Peripheral deletion of antigen-reactive T cells in oral tolerance.
Nature
376
:
177
-180.
44
Weiner, H. L..
2001
. Induction and mechanism of action of transforming growth factor-β-secreting Th3 regulatory cells.
Immunol. Rev.
182
:
207
-214.
45
Zhang, X., L. Izikson, L. Liu, H. L. Weiner.
2001
. Activation of CD25+CD4+ regulatory T cells by oral antigen administration.
J. Immunol.
167
:
4245
-4253.
46
Liu, J. Q., X. F. Bai, F. D. Shi, B. G. Xiao, H. L. Li, M. Levi, M. Mustafa, B. Wahren, H. Link.
1998
. Inhibition of experimental autoimmune encephalomyelitis in Lewis rats by nasal administration of encephalitogenic MBP peptides: synergistic effects of MBP 68–86 and 87–99.
Int. Immunol.
10
:
1139
-1148.
47
Xu, L. Y., J. S. Yang, Y. M. Huang, M. Levi, H. Link, B. G. Xiao.
2000
. Combined nasal administration of encephalitogenic myelin basic protein peptide 68–86 and IL-10 suppressed incipient experimental allergic encephalomyelitis in Lewis rats.
Clin. Immunol.
96
:
205
-211.
48
Anderton, S. M., D. C. Wraith.
1998
. Hierarchy in the ability of T cell epitopes to induce peripheral tolerance to antigens from myelin.
Eur. J. Immunol.
28
:
1251
-1261.
49
Tian, J., M. A. Atkinson, M. Clare-Salzler, A. Herschenfeld, T. Forsthuber, P. V. Lehmann, D. L. Kaufman.
1996
. Nasal administration of glutamate decarboxylase (GAD65) peptides induces Th2 responses and prevents murine insulin-dependent diabetes.
J. Exp. Med.
183
:
1561
-1567.
50
Staines, N. A., N. Harper, F. J. Ward, V. Malmstrom, R. Holmdahl, S. Bansal.
1996
. Mucosal tolerance and suppression of collagen-induced arthritis (CIA) induced by nasal inhalation of synthetic peptide 184–198 of bovine type II collagen (CII) expressing a dominant T cell epitope.
Clin. Exp. Immunol.
103
:
368
-375.
51
Gonzalo, J. A., J. Tian, T. Delaney, J. Corcoran, J. B. Rottman, J. Lora, A. Al-garawi, R. Kroczek, J. C. Gutierrez-Ramos, A. J. Coyle.
2001
. ICOS is critical for T helper cell-mediated lung mucosal inflammatory responses.
Nat. Immunol.
2
:
597
-604.
52
Bonhagen, K., O. Liesenfeld, M. J. Stadecker, A. Hutloff, K. Erb, A. J. Coyle, M. Lipp, R. A. Kroczek, T. Kamradt.
2003
. ICOS+ Th cells produce distinct cytokines in different mucosal immune responses.
Eur. J. Immunol.
33
:
392
-401.
53
Nakazawa, A., I. Dotan, J. Brimnes, M. Allez, L. Shao, F. Tsushima, M. Azuma, L Mayer.
2004
. The expression and function of costimulatory molecules B7H and B7–H1 on colonic epithelial cells.
Gastroenterology
126
:
1347
-1357.
54
Slavin, A. J., R. Maron, H. L. Weiner.
2001
. Mucosal administration of IL-10 enhances oral tolerance in autoimmune encephalomyelitis and diabetes.
Int. Immunol.
13
:
825
-833.
55
Herman, A. E., G. J. Freeman, D. Mathis, C. Benoist.
2004
. CD4+CD25+ T regulatory cells dependent on ICOS promote regulation of effector cells in the prediabetic lesion.
J. Exp. Med.
199
:
1479
-1489.