Gastroenterology
Volume 133, Issue 2 , Pages 517-528, August 2007

Induction of Ovalbumin-Specific Tolerance by Oral Administration of Lactococcus lactis Secreting Ovalbumin

  • Inge L. Huibregtse

      Affiliations

    • Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
    • I.L. Huibregtse, and V. Snoeck contributed equally to this manuscript.
    • ,
  • Veerle Snoeck

      Affiliations

    • Department for Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology, Ghent, Belgium
    • Department of Molecular Biology, Ghent University, Ghent, Belgium
    • I.L. Huibregtse, and V. Snoeck contributed equally to this manuscript.
    • ,
  • An de Creus

      Affiliations

    • Department for Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology, Ghent, Belgium
    • Department of Molecular Biology, Ghent University, Ghent, Belgium
    • ,
  • Henri Braat

      Affiliations

    • Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
    • ,
  • Ester C. de Jong

      Affiliations

    • Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
    • ,
  • Sander J.H. van Deventer

      Affiliations

    • Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
    • ,
  • Pieter Rottiers

      Affiliations

    • Department for Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology, Ghent, Belgium
    • Department of Molecular Biology, Ghent University, Ghent, Belgium
    • Corresponding Author InformationAddress requests for reprints to: Pieter Rottiers, PhD, Technologiepark 4 B-9052 Zwijnaarde, Ghent, Belgium. fax: (32) 9 2610619.

Received 22 January 2007; accepted 19 April 2007. published online 04 May 2007.

Article Outline

Background & Aims: Obtaining antigen-specific immune suppression is an important goal in developing treatments of autoimmune, inflammatory, and allergic gastrointestinal diseases. Oral tolerance is a powerful means for inducing tolerance to a particular antigen, but implementing this strategy in humans has been difficult. Active delivery of recombinant autoantigens or allergens at the intestinal mucosa by genetically modified Lactococcus lactis (L lactis) provides a novel therapeutic approach for inducing tolerance. Methods: We engineered the food grade bacterium L lactis to secrete ovalbumin (OVA) and evaluated its ability to induce OVA-specific tolerance in OVA T-cell receptor (TCR) transgenic mice (DO11.10). Tolerance induction was assessed by analysis of delayed-type hypersensitivity responses, measurement of cytokines and OVA-specific proliferation, phenotypic analysis, and adoptive transfer experiments. Results: Intragastric administration of OVA-secreting L lactis led to active delivery of OVA at the mucosa and suppression of local and systemic OVA-specific T-cell responses in DO11.10 mice. This suppression was mediated by induction of CD4+CD25− regulatory T cells that function through a transforming growth factor β-dependent mechanism. Restimulation of splenocytes and gut-associated lymph node tissue from these mice resulted in a significant OVA-specific decrease in interferon γ and a significant increase in interleukin-10 production. Furthermore, Foxp3 and CTLA-4 were significantly up-regulated in the CD4+CD25− population. Conclusions: Mucosal antigen delivery by oral administration of genetically engineered L lactis leads to antigen-specific tolerance. This approach can be used to develop effective therapeutics for systemic and intestinal immune-mediated inflammatory diseases.

Abbreviations used in this paper: APC, antigen presenting cell, BM, bone marrow, DC, dendritic cells, DTH, delayed type hypersensitivity, GALT, gut-associated lymphoid tissue, IFN, interferon, IL, interleukin, LL, Lactococcus lactis, LL-OVA, OVA-secreting L lactis, OT, oral tolerance, OVA, ovalbumin, TCR, T-cell receptor, Treg, regulatory T cell

 

See editorial on page 706.

The mucosal immune system maintains an equilibrium between tolerance toward commensals and harmless agents (eg, food antigens) on the one hand and active immunity toward pathogenic agents on the other.1 Disturbance of this balance is an important pathogenic mechanism in many different autoimmune, allergic, and inflammatory gastrointestinal diseases. Induction of antigen-specific oral tolerance (OT) is an attractive therapeutic approach because it generally lacks toxicity, can be easily administered over time, and avoids adverse effects associated with generalized immune suppressive intervention. Oral administration of (auto)antigens or allergens can effectively prevent the induction of autoimmune and allergic diseases in animal models, but several clinical attempts to induce OT for therapeutic purposes have failed.2, 3, 4 These failures may be related to the source, the purity, and the amount of (auto)antigen needed or to the way the antigen is presented to the mucosal immune system. Experimental data indicate that heterogeneous antigen mixtures are less effective inducers of OT than single purified antigens and that the antigen dose is critical. High doses of antigen can lead to clonal deletion or anergy of the T cells recognizing the antigen, whereas low doses can induce active suppression, eg, by inducing antigen-specific regulatory T cells secreting suppressive cytokines.5, 6, 7 When anergy or clonal deletion is desired,8 the antigen has to be known. However, if multiple pathogenic antigens are implicated, or when the causal antigen is unknown, therapeutic effects can be induced by generating “bystander” regulatory T cells.2, 9, 10 Different types of regulatory T cells can be induced or expanded by mucosal antigens, including CD4+CD25+, CD4+CD25−, and CD8+ regulatory T cells through a transforming growth factor (TGF)-β and/or interleukin (IL)-10-dependent mechanism.2

The Lactococcus lactis (L lactis)-mediated delivery system obviates the need for large scale purification of human autoantigens or allergens and enables delivery of antigens to the intestinal mucosa. L lactis is a nonpathogenic, noninvasive, noncolonizing gram-positive bacterium, and, according to the US Food and Drug Administration, it is generally regarded as safe. Previously genetically modified L lactis strains have been produced for synthesis and delivery of immunomodulatory proteins at the intestinal mucosa, and an adequate biologic containment system has been established.11, 12, 13 A biologically contained L lactis strain secreting human IL-10 was used in a phase I, open label clinical trial on Crohn’s disease patients. This trial demonstrated that treatment of humans with viable L lactis secreting IL-10 is clinically and biologically safe and gave indications of its clinical efficacy.14 The use of genetically modified L lactis for intestinal delivery of proteins in humans warrants further investigation.

In the present study, we evaluated the efficacy of L lactis as a vehicle for intestinal delivery of antigens for the induction of antigen-specific peripheral tolerance. We fed ovalbumin (OVA)-immunized DO11.10 mice, which bear transgenic OVA-specific CD4+ T-cell receptors, with OVA-secreting L lactis (LL-OVA). Our data indicate that LL-OVA leads to OVA-specific tolerance by inducing CD4+CD25− regulatory T cells that function through a TGF-β-dependent mechanism. These cells can transfer tolerance to OVA-immunized wild-type Balb/c recipients. This intestinal delivery system is more effective than oral administration of purified antigen. Thus, live, genetically modified L lactis holds promise as a tool for efficient induction of antigen-specific peripheral tolerance in humans suffering from antigen-driven immune diseases.

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Materials and Methods 

Bacteria and Media 

The Lactococcus lactis MG1363 (LL) strain15 was genetically modified and used throughout this study. Bacteria were cultured in GM17E medium consisting of M17 broth (Difco Laboratories, Detroit, MI) supplemented with 0.5% glucose and 5 μg/mL erythromycin (Abbott B.V., Hoofddorp, The Netherlands). Stock suspensions of the Lactococcus lactis MG1363 strains were stored at −20°C in 50% glycerol in GM17E medium. Stock suspensions were diluted 200-fold in GM17E medium and incubated at 30°C overnight. Within 16 hours, they reached a saturation density of 2 × 109 colony-forming units (CFU) per mL. Bacteria were harvested by centrifugation and resuspended in BM9 medium at 2 × 1010 bacteria/mL. Each mouse received 100 μL of this suspension daily through an intragastric catheter.

Plasmids 

The messenger RNA (mRNA) sequence encoding Gallus gallus ovalbumin was retrieved from Genbank (accession number AY223553) and from published data.16 Total RNA was isolated from chicken uterus and complementary DNA (cDNA) was synthesized using 2 μg total RNA, 2 μmol/L oligo dT primers (Promega Corporation Benelux, Leiden, The Netherlands), 0.01 mmol/L DTT (Sigma-Aldrich, Zwijndrecht, The Netherlands), 0.5 mmol/L dNTP (Invitrogen, Merelbeke, Belgium), 20 U Rnasin (Promega Incorporation Benelux), and 100 U superscript II reverse transcriptase (Invitrogen) in a volume of 25 μL. An OVA cDNA fragment was amplified by polymerase chain reaction (PCR) using the following primers: forward 5′-GGCTCCATCGGTGCAGCAAGCATGGAATT-3′ and reverse 5′-ACTAGTTAAGGGGAAACACATCTGCCAAAGAAGAGAA-3′. Reaction conditions were 94°C for 2 minutes followed by 30 cycles at 94°C for 45 seconds, 62°C for 30 seconds, and 72°C for 90 seconds. The amplified fragment was fused to the Usp45 secretion signal17 of the erythromycin resistant pT1NX vector, downstream of the lactococcal P1 promotor.18 MG1363 strains transformed with plasmids carrying OVA cDNA were designated L lactis secreting OVA (LL-OVA). The L lactis-pT1NX, which is MG1363 containing the empty vector pT1NX, served as control (LL-pT1NX).15

Mice 

Seven-week-old female Balb/c mice were obtained from Charles River Laboratories (Calco, Italy). OVA-specific T-cell receptor transgenic mice (DO11.10) on a BALB/c background were kindly provided by Dr J. Samson (Vrije Universiteit, Amsterdam, The Netherlands) and bred at the Academic Medical Center, Amsterdam. DO11.10 mice were used at the age of 8–10 weeks. All mice were housed in a conventional animal facility under routine laboratory conditions. All experiments were approved by the Animal Experiments Committee of the Academic Medical Center.

Antigen and Antibodies 

Intact, lipopolysaccharide (LPS)-free OVA grade V protein was used as antigen in all experiments (Sigma Aldrich). Anti-CD4, anti-CD25, and anti-CTLA-4 antibodies were purchased from BD-Pharmingen (Woerden, The Netherlands), and anti-Foxp3 antibodies were obtained from eBiosciences (San Diego, CA).

In Vitro and In Vivo Quantification of OVA Secreted by L lactis 

Female BALB/c mice were fed 10 serial inoculates of 2 × 109 CFU of LL-OVA in 100 μL BM9 medium, or 100 μL BM9 medium alone (control), once every 30 minutes. One hour after the final inoculation, intestinal segments were homogenized in phosphate-buffered saline (PBS) containing 1% fetal calf serum (FCS) for analyses. OVA production was quantified by enzyme-linked immunosorbent assay (ELISA) and live L lactis bacterial count by plating 10-fold dilutions of the homogenates on GM17E agar plates containing 5 μg/mL erythromycin. OVA secreted in vivo was quantified by sampling the proximal and distal small intestines, the cecums, and the colons in 2 different ways: (1) samples of the entire intestine, including luminal content, and (2) extensively washed intestinal tissue samples for measurement of mucosal OVA concentrations. Samples were homogenized, and OVA was quantified by ELISA. OVA was captured from the saturated culture supernatant by immobilized polyclonal rabbit anti-OVA antibody (2 μg/mL; Research Diagnostics Inc), quantified by anti-OVA biotin-conjugated polyclonal rabbit antibody (2.5 μg/mL; Research Diagnostics Inc), and stained with horseradish peroxidase-conjugated streptavidin (1/1000; BD Pharmingen, San Diego, CA) followed by 3,3’, 5,5’ tetramethylbenzidine substrate (BD Pharmingen).

Oral Feeding and Delayed-Type Hypersensitivity 

DO11.10 mice were immunized by subcutaneous (SC) injection of 100 μg OVA in 50 μL of a 1:1 mixture of Complete Freund’s adjuvant (Difco, BD, Alphen aan de Rijn, The Netherlands) and saline solution at the base of the tail on the first day.19 Mice were fed purified OVA dissolved in 100 μL BM9 as follows: 200 mg on day 8, 1 mg on days 1–5, and 1 μg on days 1–5 and 8–12. LL-OVA or LL-pT1NX was administered on days 1–5 and 8–12. Control mice received only BM9. Antigen or bacterial suspensions were introduced into the stomach using an 18-gauge stainless animal feeding needle. Eleven days after sensitization, antigen-specific, delayed-type hypersensitivity (DTH) responses were assessed by injection of OVA. Twenty-four hours later, DTH measurements were performed, or spleen and lymph nodes were harvested and the cells assessed for OVA-specific proliferation and cytokine production. For measurement of antigen-specific DTH responses, mice were challenged with 10 μg OVA in 10 μL saline in the auricle of one ear and 10 μL saline in the other. Ear swelling, defined as the increase in ear thickness because of challenge, was measured in a blinded fashion 24 hours after challenge using a micrometer (Mitutoyo, Tokyo, Japan). DTH responses were expressed as the difference in swelling between the OVA-injected and the saline-injected ears (swelling because of OVA minus swelling because of saline).

Cell Cultures and Proliferation and Cytokine Assays 

Single cell suspensions of spleen and lymph nodes were prepared by passing the cells through 70-μm cell strainers (Becton/Dickinson Labware). Erythrocytes in the spleen cell suspensions were lysed by incubation with red cell lysis buffer. For IL-10 measurements, 1 × 105 lymphocytes isolated 1 day after DTH challenge were incubated in triplicate 96-well plates (Costar, Cambridge, MA) in 200 μL complete medium. They were either left unstimulated or were stimulated in the presence of plate-bound anti-CD3 (1:30 concentration; clone 145.2C11, a gift from Dr R. Mebius) and soluble anti-CD28 (1:1000 concentration; BD Biosciences) for 48 hours or with 500 μg/mL OVA for 40 hours. CD4+ T cells and CD4+CD25− T cells were enriched using CD4+ T-cell isolation kit or CD4+CD25+ regulatory T-cell isolation kit and midiMACS columns (all materials from Miltenyi Biotec, Germany).

A T-cell proliferation assay was performed using DO11.10 CD4+ T cells and bone marrow (BM)-derived CD11c+ dendritic cells (DC) cultured with LL-OVA or LL-pT1NX. BM-derived DCs were generated as described20 and positively selected using anti-CD11c magnetic microbeads and MACS columns (Miltenyi Biotec, Utrecht, The Netherlands) according to manufacturer’s instructions. The CD11c+ cells were plated at 2 × 106 per well (24-well; Costar) with 4 × 105 CFU L lactis or with 1 μg or 1 mg OVA per milliliter in RPMI 1640 supplemented with 10% FCS, 2 mmol/L L-glutamax, 0.1 mmol/L nonessential amino acids, 0.4 mol/L sodium pyruvate, 25 mmol/L HEPES (all 4 from Invitrogen), 50 μmol/L β-mercaptoethanol (2-ME), and 5 μg/mL erythromycin (Abbott). After 4-hour incubation, the bacteria were killed by adding 75 μg/mL gentamycin. Twenty hours later, cells were harvested. Two × 105 CD4+ T cells were then cultured with the harvested DCs at ratios of 1/0.3, 1/0.11, 1/0.03, and 1/0.01, respectively. The cultures were grown in 96-well U-bottom plates (Becton Dickinson, Alphen aan de Rijn, The Netherlands) in a total volume of 200 μL complete medium consisting of RPMI-1640 with 10% FCS, 2 mmol/L L-glutamax (Invitrogen), 0.1 mmol/L nonessential amino acids, 0.4 mol/L sodium pyruvate, 25 mmol/L HEPES, 50 μmol/L 2-ME, 10 U/mL penicillin (Invitrogen), and 10 μg/mL streptomycin (Invitrogen).

To assay proliferation of total splenocyte populations, 2 × 105 cells were cultured in 96-well U-bottom plates in a total volume of 200 μL complete medium either alone or with OVA and either with or without anti-IL-10 neutralizing monoclonal antibody (1 μg/mL, clone JES052A5; R&D Systems, Abingdon, United Kingdom). OVA was added at concentrations ranging from 1.2 to 100 μg/mL. The anti-IL-10 antibody was added at 1.0, 0.1, and 0.01 μg/mL. To assay proliferation of CD4+ T cells and CD4+CD25− T-cell populations, 0.2 × 105 CD4+ T cells or CD4+CD25− T cells were cultured in 96-well U-bottom plates with 1 × 105 irradiated CD4− cells, acting as antigen presenting cells (APC), and OVA (0 or 100 μg/mL) in a total volume of 200 μL complete medium either with or without TGF-β neutralizing monoclonal antibody (1 μg/mL, clone 1D11; R&D Systems). Cells were cultured for 90 hours at 37°C in 5% CO2 in a humidified incubator. For proliferation assays, 1 μCi/well [3H]-thymidine was added for the last 18 hours of culture, DNA was harvested on glass fiber filter mats (Perkin Elmer, Boston, MA), and DNA-bound radioactivity was measured on a scintillation counter (Perkin Elmer). For cytokine measurements, supernatants of the cell cultures used in the different proliferation assays were collected after 72 hours of culture and frozen at −20°C. Cytokine production was quantified using the Mouse Inflammation Cytometric Bead Assay (BD Biosciences, Mountain View, CA).

Flow Cytometry Analysis 

On day 12, spleens of mice treated with BM9, 1 μg OVA, LL-pT1NX, or LL-OVA were isolated and enriched for CD4+CD25− T cells using CD4+ T-cell isolation kit or CD4+CD25+ regulatory T-cell isolation kit, as described above. To determine the regulatory phenotype of the CD4+CD25− T-cell population, cells were stained for Foxp3 and CTLA-4 and analyzed by flow cytometry (FACScan; Becton Dickinson, Woerden, The Netherlands).

Adoptive Transfer Experiments 

Wild-type Balb/c donor mice were immunized by SC injection with 100 μg OVA in 50 μL of a 1:1 mixture of CFA (Difco; Becton Dickinson, Alphen aan de Rijn, The Netherlands) and saline solution at the base of the tail on days 0 and 1. On day 7, CD25 subsets isolated from spleens of DO11.10 mice fed with LL-OVA (as previously described) were adoptively transferred in the sensitized Balb/c mice. To obtain the CD25 subsets, spleen cells were stained using the CD4+CD25+ regulatory T-cell isolation kit (Miltenyi Biotec) and sorted by MACS cell sorting. The CD4+CD25+ and CD4+CD25− populations were sorted again using a FACS diva (BD, Becton Dickinson, Erembodegem, Belgium), which separated the positive population into CD25high and CD25intermediate subpopulations. Immediately afterwards, 5 × 104 CD25high, CD25intermediate, or CD25− cells in 200 μL PBS were adoptively transferred by intravenous (IV) injection into the OVA-immunized Balb/c mice. These mice were then challenged on day 12 with 10 μg OVA in 10 μL saline in the auricle of one ear and 10 μL saline in the other ear, and DTH responses were measured 24 hours later, as described previously.

Statistical Analysis 

Results from cytokine measurements are expressed as means ± SEM. Significance of differences between groups in ear thickness and cytokine measurements were tested using 1-way ANOVA followed by Student t test. Significance of OVA-specific proliferation was evaluated using a general linear model with repeated measurements. For both tests, statistical significance is indicated as *P < .05 or **P < .01.

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Results 

LL-OVA Induces APC-Mediated T-Cell Proliferation In Vitro 

A L lactis strain that secretes chicken OVA, designated LL-OVA, was constructed. In vitro synthesis of OVA was evaluated by ELISA. OVA secretion did not alter the growth rate of L lactis, and, after 16 hours of growth, OVA was detected in the culture supernatant at a concentration of 7 ± 2 ng/mL. No intracellular OVA could be detected, demonstrating efficient secretion of OVA.

LL-OVA were able to induce BM-DC maturation (data not shown) and proliferation of DO11.10 CD4+ T cells that were cocultured with BM-DC. Less than 0.5 ng/mL of OVA was produced by LL-OVA during a 4-hour coculture of BM-DC with LL-OVA. Purified OVA at 1 mg/mL induced significantly more proliferation than LL-OVA, but, at 1 μg/mL, it failed to induce any proliferation (Figure 1). This clearly demonstrates that L lactis-derived OVA is bioactive and suggests that the LL-OVA-induced T-cell proliferation is mediated by APC.

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  • Figure 1. 

    LL-OVA induces APC-mediated T-cell proliferation. L lactis-derived OVA is bioactive because it induces proliferation of DO11.10 CD4+ T cells after preincubation of BM-DC with LL-OVA. BM-derived DC were generated as described in the Materials and Methods section. Two × 106 BM-DC were cultivated with 4 × 105 CFU LL-OVA or LL-pT1NX, with 1 μg or 1 mg OVA per mL or with no additive (/). After 4 hours, bacteria were killed with gentamycin. Twenty hours later, BM-DC were harvested, and their proliferation was assayed using DO11.10 CD4+ T cells. Two × 105 DO11.10 CD4+ T cells were incubated with 6.67 × 104, 2.22 × 104, 7.41 × 103, and 2.47 × 103 BM-DC, corresponding to 1/0.33, 1/0.11, and 1/0.01, respectively, CD4+ T cells/BM-DC. Given the considerable higher proliferation after stimulation with 1 mg/mL OVA, this proliferation is shown in a separate graph.

Active Intestinal Delivery of OVA by L lactis 

To quantify the in vivo secretion of OVA, small intestines, cecums, and colons were obtained 1 hour after the final inoculation. CFU numbers and OVA concentrations were determined within the entire intestine, including its luminal content as well as within the intestinal tissue, ie, the mucus and mucosa. No live L lactis bacteria were found in the proximal small intestine, whereas, in samples of luminal content from the distal small intestine, the cecum, and the colon, CFU averaged 7.25 × 109, 2.49 × 109, and 3.49 × 109, respectively. Relatively few viable L lactis were found in contact with mucus or within the mucosa of the distal small intestine (4.16 × 106 CFU), cecum (7.03 × 107 CFU), and colon (1.41 × 106 CFU) (Table 1). The largest amount of OVA derived from LL-OVA was detected in the colon (48 ng per colon), but only a small fraction of the colon-delivered OVA (3 ng per colon) was found in the intestinal tissue. A smaller amount of OVA was found in the distal part of the small intestine, but this was localized entirely in the mucus and/or mucosa. No OVA was detected in the proximal small intestine (Figure 2). This indicates that L lactis actively produced OVA in vivo, which was mostly recovered from the mucus and/or mucosa of the small bowel and in the lumen of the large intestine.

Table 1. In Vivo Detection of Live L Lactis Bacteria
CFU LL-OVADistal small intestineCecumColon
Entire intestine, including luminal content7.25±0.14×1092.49±0.15×1093.49±0.015×109
Entire intestine, excluding luminal content4.16±0.25×1067.03±0.17×1071.41±0.10×106
  • View full-size image.
  • Figure 2. 

    OVA is actively produced by LL-OVA in the intestine and is delivered to the small and large intestines. Balb/c mice (n = 4) received 10 serial inocula of 2 × 109 CFU of LL-OVA in 100 μL suspension at intervals of 30 minutes. One hour after the final inoculation, live bacteria (Table 1) and OVA (Figure 2) were quantified in the entire small intestines, cecums, and colons, including their luminal contents (solid bar), and in the intestinal tissue (open bar), as described in the Materials and Methods section. Detection limit of the ELISA = 1.5 ng. Results are representative of 2 individual experiments.

Administration of LL-OVA Significantly Suppresses OVA-Induced DTH 

OVA-specific DTH reactions in the ear are accurately reflected in ear swelling.21, 22 Using an intragastric catheter, OVA-immunized mice were fed BM9 (as a negative control), 200 mg OVA on day 8, 1 mg OVA on days 1–5, 1 μg OVA on days 1–5 and 8–12, or LL-pT1NX (as vector control) or LL-OVA on days 1–5 and 8–12. On day 11, mouse ears, after measurement of thickness, were injected with 10 μg OVA. Ear thickness was measured again after 24 hours, and swelling was measured as the increase in ear thickness. Control mice were significantly sensitized to OVA, as evidenced by ear swelling, but daily intragastric administration of LL-OVA significantly reduced this swelling (15 × 10−2 mm vs 1 × 10−2 mm, respectively, P = .0152) (Figure 3). Ear swelling was also somewhat reduced in LL-pT1NX-treated mice compared with controls (BM9) (6.5 × 10−2 mm vs 15 × 10−2 mm, respectively). Remarkably, mice that were fed either a low dose of OVA (10 × 1 μg), comparable with the amount of OVA secreted by the LL-OVA, or a high dose (1 × 200 mg or 5 × 1 mg) did not show a significant reduction in swelling compared with the control group. These data strongly indicate that LL-OVA can suppress systemic T-cell responses in DO11.10 mice and that this effect is in part because of the apparent intrinsic tolerogenic effect of L lactis.

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  • Figure 3. 

    Oral feeding of LL-OVA significantly reduces DTH responses. DO11.10 mice were sensitized by subcutaneous injection of 100 μg OVA in CFA on day 1. Mice were orally treated with 200 mg OVA on day 8, 1 mg OVA on days 1–5, 1 μg OVA at days 1–5 and 8–12, or with LL-OVA or LL-pT1NX on days 1–5 and 8–12. Control mice received BM9. On day 11, mice were challenged with 10 μg OVA in 10 μL saline in the auricle of 1 ear and with 10 μL saline in the other. DTH responses are expressed as the mean difference in ear swelling between the OVA-injected and the saline-injected ears. Results summarize data of 4 independent experiments including 6 mice per group per experiment.

Reduction of DTH Response by Treatment With LL-OVA Is Accompanied by an OVA-Specific Increase in IL-10 Production 

Immediately after measuring DTH, cervical lymph node cells draining the ears of OVA-challenged mice were pooled and restimulated with OVA. This led to higher IL-10 production in the LL-OVA-treated mice than in the BM9 (control) and LL-pT1NX mice (Figure 4A).

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  • Figure 4. 

    OVA-specific increase in IL-10 production after restimulation of cervical lymph node, spleen, and GALT cells. IL-10 secretion of pooled cervical lymph node cells was determined in the supernatants 24, 48, and 72 hours after restimulation. Data are represented as means of IL-10 secretion in pg/mL of at least 2 separate experiments (A). IL-10 production by bulk spleen and GALT cells was measured after ex vivo restimulation. Mice were fed BM9, 1 μg OVA, LL-pT1NX, or LL-OVA as described in Figure 3, and DTH was determined. Thereafter, bulk cell populations were isolated, and 1 × 105 cells were restimulated ex vivo with 0.5 mg/mL OVA (B). To determine antigen-specific IL-10 secretion, cells were restimulated with anti-CD3/anti-CD28 (C). Data summarize 3 independent experiments including 6 mice per experiment.

IL-10 production by isolated bulk spleen and gut-associated lymph node cells (GALT) following 40-hour ex vivo OVA restimulation was also assessed immediately after DTH measurement. Production of IL-10 was significantly higher in restimulated bulk spleen cells from LL-OVA-treated mice than in the negative control and in the groups receiving 1 μg OVA or LL-pT1NX. Similar results were obtained using restimulated GALT cells (Figure 4B). The observed production of IL-10 was antigen specific because no differences in IL-10 production were observed after restimulation with anti-CD3/anti-CD28 (Figure 4C). In summary, we found that the production of IL-10 in OVA-stimulated cervical lymph nodes, splenocytes, and GALT cells was significantly higher in mice that were treated with LL-OVA.

Oral Feeding With L lactis Suppresses the OVA-Specific Proliferative Capacity of Splenocytes and Decreases IFN-γ Production 

Analysis of OVA-specific proliferation of splenocytes was used to examine peripheral immune responses. Bulk splenocytes of mice treated with BM9, LL-pT1NX, or LL-OVA were isolated on day 12, and the OVA-specific proliferative response was assessed. Control mice were clearly sensitized and showed a high proliferative response. Daily intragastric administration of LL-OVA significantly reduced the OVA-specific proliferative response (P = .002) (Figure 5). Noteworthy, the OVA-induced proliferative response was also significantly reduced (P = .022) after intragastric administration of LL-pT1NX in comparison with the BM9 control group.

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  • Figure 5. 

    Feeding of LL-OVA or LL-pT1NX significantly reduces the OVA-specific proliferation and IFN-γ production of bulk splenocytes. DO11.10 mice were immunized by subcutaneous injection of 100 μg OVA in CFA on day 1. Mice were treated orally with BM9 (control), LL-OVA, or LL-pT1NX on days 1–5 and 8–12. On day 12, bulk splenocytes were isolated and tested for OVA-specific proliferation, which is expressed as the mean cpm at different OVA concentrations, and for IFN-γ production after 72-hour ex vivo stimulation with 33 μg/mL OVA (upper panel). Data represent at least 3 separate experiments, including 4 mice per group per experiment.

The lower proliferative response of spleen cells was accompanied by a significant down-regulation of IFN-γ production after ex vivo OVA restimulation, both in the LL-pT1NX- and the LL-OVA-treated mice (Figure 5, upper panel). These data indicate that LL-OVA and LL-pT1NX treatments are both able to suppress systemic T-cell responses in DO11.10 mice.

Feeding With LL-OVA Suppresses the OVA-Specific Proliferative Responses of CD4+ Splenic T Cells Mediated by TGF-β and CD4+CD25− T Cells 

To determine whether the reduced proliferative response of bulk splenocytes was related to reduced responsiveness of the CD4+ T cells, splenic CD4+ T cells from the mice treated with BM9, LL-pT1NX, or LL-OVA were isolated on day 12, and OVA-specific proliferative responses were assessed. The OVA-specific proliferative response of splenic CD4+ T cells was lower after LL-OVA treatment than in the BM9 and LL-pT1NX control groups (Figure 6A). Because blocking with neutralizing anti-IL-10 did not reverse the reduction in the proliferative response of bulk splenocytes (data not shown), we investigated whether TGF-β was involved. Blocking with TGF-β neutralizing monoclonal antibody did not significantly alter the proliferative response of the CD4+ splenic T cells in the BM9 and LL-pT1NX groups but reversed the reduction in proliferation in the LL-OVA group.

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  • Figure 6. 

    Feeding with LL-OVA reduces the OVA-specific proliferation of CD4+ splenic T cells. The reduction is mediated by CD4+CD25− cells and TGF-β. DO11.10 mice were sensitized by subcutaneous injection of 100 μg OVA in CFA at day 1. They were treated orally with BM9, LL-OVA, or LL-pT1NX on days 1–5 and 8–12. On day 12, spleens were isolated and splenic CD4+ T, CD4+CD25high, CD4+CD25intermediate, and CD4+CD25− T cells were isolated. OVA-specific proliferation of CD4+ and CD4+CD25− T cells from all groups were assayed as described in the Materials and Methods section. Proliferative response of CD4+ and CD4+CD25− T cells was studied while blocking TGF-β with anti-TGF-β neutralizing antibody (1 μg/mL). Proliferative responses are expressed as the proliferation relative to that of CD4+ T cells of the BM9 group (A). After 72 hours of culture, supernatants were collected from the proliferation assays and tested for IFN-γ production (B). Data represent at least 2 separate experiments. On day 12, isolated CD4+CD25− T-cell subsets of the mice treated with BM9, 1 μg OVA, LL-pT1NX, or LL-OVA were intracellularly stained for Foxp3 and CTLA-4, and flow cytometry was performed (C).

Importantly, the reduction in proliferation because of LL-OVA treatment was maintained after depleting CD4+CD25+ T cells (Figure 6A). Moreover, the proliferative response of the CD4+CD25− T cells in the LL-OVA group significantly increased after blocking TGF-β with a specific neutralizing monoclonal antibody (P = .028). These proliferation data are supported by the IFN-γ production data (Figure 6B). To analyze further the induced CD4+CD25− regulatory T cell (Treg) population, we defined the percentage of CD4+CD25− lymphocytes expressing 2 major Treg markers: Foxp3 and CTLA4. For this purpose, we stained the isolated CD4+CD25− T cells from mice treated with BM9 or 1 μg OVA, LL-pT1NX, or LL-OVA on day 12 and gated them on the Foxp3 and CTLA4+ subpopulation. A de novo expansion of CD4+CD25− Foxp3+ and CD4+CD25− CTLA4+ is observed only in the LL-OVA-treated group (Figure 6C). This indicates that CD4+CD25− regulatory T cells expressing Foxp3 and CTLA4 are induced following LL-OVA treatment and that, in vitro, the CD25− cells mediate OVA-specific tolerance through a TGF-β-dependent mechanism.

CD4+CD25− Regulatory T Cells Induced After LL-OVA Treatment Can Transfer OVA Tolerance in vivo 

To assess the functional activity of the different CD4+CD25 subsets in vivo, we fed DO11.10 mice with LL-OVA as described above, and we adoptively transferred CD4+CD25high, CD4+CD25intermediate, and CD4+CD25− subsets from their spleens into Balb/c mice sensitized by SC injection with 100 μg OVA in CFA on days 0 and 1. Five days thereafter, mouse ears were injected with 10 μg OVA, and ear thickness was measured 24 hours later. Remarkably, only adoptive transfer of the CD4+CD25− T cells caused a reduction of DTH (P = .0317) (Figure 7). Together, these data strongly suggest that intragastric administration of OVA-secreting L lactis induces OVA-specific immune tolerance that is mediated by CD4+CD25− regulatory T cells.

  • View full-size image.
  • Figure 7. 

    Adoptive transfer of CD4+CD25− T cells of LL-OVA-treated mice into sensitized Balb/c mice induces OVA-specific tolerance in vivo. Three CD4+CD25 subpopulations were sorted: CD4+CD25high, CD4+CD25intermediate, and CD4+CD25−. On day 7, 5 × 104 cells of each subpopulation were adoptively transferred into Balb/c mice that had been sensitized by subcutaneous injection of 100 μg OVA in CFA on days 0 and 1. Five days later, DTH measurements of the ear were performed.

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Discussion 

Our data indicate that genetically modified L lactis can be used for mucosal delivery of antigens and that this suppresses local and systemic antigen-specific T-cell responses in both antigen-specific and nonspecific manners. Antigen-specific suppression induced by OVA-secreting L lactis is mediated by CD4+CD25− “adaptive” Treg and seems to be dependent on TGF-β. Importantly, OVA dose feeding alone (either high dose or low dose) was less efficient than LL-OVA in reducing the DTH response in our model, indicating that the mode of mucosal delivery of an antigen to the immune system critically determines subsequent immune responses.

Induction of Treg is a major goal for immunotherapy for autoimmune diseases and several inflammatory diseases, and it can be achieved by exposing the mucosal immune system to low doses of antigen. It has been difficult to apply antigen-specific mucosal tolerance for the treatment of human diseases. Mucosal tolerance depends critically on several factors, including the purity, source, and dose of antigen and the mode of antigen presentation to the mucosal immune system.4 Here, we propose a novel therapeutic strategy: active intestinal synthesis and delivery of an antigen by genetically engineered L lactis, which obviates the need for large scale purification of human (auto)antigens and circumvents current issues related to induction of oral tolerance in humans.

The in situ fate of the bacteria as well as the distribution of OVA produced by the LL-OVA was determined by multiple intragastric administrations to Balb/c mice. Except for the proximal part of the small intestine, viable L lactis were found throughout the intestine, with most bacteria located in the distal part of the small intestine and in the cecum. In a study performed by Droualt et al, only 10%–30% of the L lactis administered survived the duodenal transit.23 Here, we show that more than 50% of the administered L lactis was recovered from the intestine. We believe that the BM9 inoculation buffer used in our experiments partially protects the bacteria against the extreme gastrointestinal conditions. The largest amounts of OVA were detected in the colonic lumen, with only small amounts in the intestinal tissue. Less OVA was found in the distal part of the small intestine, but much of it was in the intestinal tissue, indicating that the OVA is efficiently taken up by the small intestine. We have not studied uptake of OVA and/or L lactis by the distal small intestine in detail, but it is possible that OVA peptide and/or lactococcal antigens were either taken up by M cells located in Peyer’s patches or that whole L lactis were directly sampled by intraluminal extensions of mucosal DC.24 Interestingly, most viable bacteria and the largest amounts of mucosal OVA were present in the distal small intestine, which is the predominant location of the intestinal sampling DC network.25, 26 In addition, L lactis might serve as a bioadhesive delivery vehicle that delivers the antigen at the intestinal sampling network or intensifies contact with the mucosa. This would increase the antigen concentration gradient and ensure immediate absorption without dilution or degradation in the luminal fluid. The large difference between the immune responses to oral OVA and L lactis-delivered OVA indicates that these routes may be pivotal for induction of L lactis-mediated oral tolerance.

Both DC and Treg are critically involved in tolerance induction.27, 28, 29, 30, 31 Our experiments demonstrate that L lactis influences APC-mediated, OVA-specific, T-cell proliferation because LL-OVA could induce OVA-specific proliferation, but OVA protein could not even at a concentration that was 2000-fold higher than that secreted by LL-OVA. Recently, we demonstrated that L lactis can imprint a regulatory phenotype on human monocyte-derived DC and that this effect is greatly boosted by engineered L lactis secreting IL-10 (Braat H, Steidler L, Neirynck S, Huibregtse I, Smits H, Zaat B, Kapsenberg ML, van Deventer S, de Jong E, manuscript submitted). Together, these data suggest that exposure to L lactis alters DC function, which in the presence of simultaneous exposure to a DC-presented antigen might result in the generation of an antigen-specific Treg subset.

We further demonstrated that both LL-pT1NX and LL-OVA reduce the DTH response and OVA-specific proliferation of bulk splenocytes and splenic CD4+ T cells. This reduction was not observed after feeding OVA protein, and it was much more pronounced in the LL-OVA- than in the LL-pT1NX-treated mice. Moreover, it was only in the LL-OVA-treated mice that we observed antigen-specific production of IL-10 by OVA-stimulated cervical lymph node cells, splenocytes, and GALT cells, as well as TGF-β-dependent, OVA-specific, CD4+ T-cell suppression. Moreover, LL-OVA treatment induced within the CD4+CD25− population a T-cell subset that expresses the typical Treg markers Foxp3 and CTLA-4. We also proved that the suppressive activity resident in the CD4+CD25− Treg population could be transferred to sensitized Balb/c mice. These data indicate that L lactis can condition the mucosal immune system toward tolerance induction and boost antigen-specific induction of Treg by the codelivered antigen.

Different studies have demonstrated the efficacy of genetically modified L lactis to abrogate Th2-type responses induced in allergic mice models using nontransgenic Balb/c mice. However, in these studies the allergic responses were diminished by the induction of counter-regulatory Th1 immune responses.32, 33, 34, 35 In fact oral pretreatment of mice with natural L lactis plus soluble antigen or antigen-secreting L lactis abrogated the oral tolerance induced by antigen alone, demonstrating a Th1 adjuvant effect of these noncolonizing bacteria.34 Apparently, L lactis has not such a Th1 adjuvant effect in DO11.10 OVA TCR transgenic mice. Recently, we demonstrated that the Th1 adjuvant effect of L lactis observed in nontransgenic Balb/c mice is absent in Balb/c mice fed over a long period with natural L lactis (Snoeck et al, manuscript in preparation). L lactis is not a normal constituent of the microflora or diet of laboratory mice. As such, it can be considered as a foreign microorganism in mice. In contrast, L lactis has been extensively consumed by humans and has never been associated with any form of pathology. Its main use lies in the manufacture of fermented milk, vegetable, and meat production. It is therefore granted a “generally regarded as safe” status, and, thus, it is acceptable to believe that L lactis will not have any Th1 adjuvant effect in humans. Currently, we are evaluating the L lactis delivery vehicle for the induction of antigen-specific immune tolerance in L lactis conditioned nontransgenic Balb/c mice.

Several phenotypically and functionally distinct Treg subsets have been implicated in suppression of intestinal inflammation and induction of oral tolerance.2, 36, 37 TGF-β has long been known to have a pivotal role in the induction of oral tolerance, both as a secreted cytokine and in the form of a latency-associated peptide corresponding to the aminoterminal domain of the TGF-β precursor protein.38, 39, 40, 41, 42 Moreover, TGF-β-dependent Treg are efficiently induced by low doses of antigen.5 Previously, it has been demonstrated that oral tolerance induced by feeding OVA was only partly blocked by CD25 depletion but completely abrogated by blocking of TGF-β.43 Recently, it was shown that antigen-specific, TGF-β-producing Th3 cells drive the differentiation of antigen-specific Foxp3+ regulatory cells in the periphery and play a crucial role in inducing and maintaining peripheral tolerance.44 Although TGF-β partially mediates the suppressive effects of several types of Treg, both latency-associated peptide and (membrane-associated) TGF-β are intimately involved in the suppression mediated by a specific subset of GALT-derived CD4+CD25− Tregs that can induce oral tolerance.45 These cells suppress the colitis induced by transfer of CD4+CD45RBhigh T cells into SCID mice,46 and they are expanded after oral administration of low-dose anti-CD3 antibody to mice. Here, we demonstrate that oral administration of OVA-secreting L lactis induces a similar Treg population residing within the CD4+CD25− compartment; but, in contrast to oral administration of anti-CD3 antibodies, the induced regulatory cells are antigen specific and Foxp3 and CTLA-4 positive.

The exact mechanism by which intestinal CD4+CD25− Treg interfere with antigen-induced inflammatory reactions needs to be determined, and different possible mechanisms have been proposed. Effector T cells may be directly antagonized through the induction of immunosuppressive cytokines or by cell-cell contact, ie, by the activity of membrane-bound TGF-β.45, 47 Suppression can also result via induction of “secondary” Treg that may depend on IL-10 production.48, 49 Foxp3+CD25− Th3 regulatory T cells represent a different cell lineage from thymus-derived CD25+ Tregs in the periphery but may play an important role in their maintenance.50 Moreover, TGF-β-dependent conversion of peripheral CD4+CD25− T cells into CD25+CD45RB−/low suppressor cells has also been reported.51 The mechanism by which CD4+CD25− cells function needs to be determined, but both orally administered anti-CD3 and LL-OVA-induced CD4+CD25− regulatory cells clearly can suppress systemically induced inflammatory responses.

Therapeutic induction of Treg is a promising strategy for treating or restoring tolerance in patients suffering from autoimmune diseases. Current strategies for therapeutic induction of antigen-specific suppressor cells face considerable hurdles and usually require techniques to isolate, handle, and transfer adequate numbers of regulatory cells.9, 10, 52 The L lactis antigen delivery system circumvents these problems and effectively induces antigen-specific Treg.

In conclusion, our data demonstrate that mucosal delivery of OVA by genetically modified L lactis induces suppression of local and systemic OVA-specific T-cell responses in DO11.10 mice and that this effect does not depend solely on the secreted OVA but also on the presence of L lactis. Oral administration of an antigen by L lactis resulted in the induction of systemic tolerance mediated by CD4+CD25− regulatory T cells that seem to function through a TGF-β-dependent mechanism. Importantly, the intestinal delivery system by L lactis is superior to oral administration of soluble antigen because both low- and high-dose antigen feeding were not able to diminish the DTH response as significant as LL-OVA treatment in this model. These data indicate that engineered L lactis could be an effective tool for inducing antigen-specific tolerance, with possible application in the treatment of antigen-induced autoimmune diseases.

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The authors thank Dr J. Samson for kindly providing the DO11.10 mice and T. van Capel, I. Pronk, K. van Laer, and A.P. Verhaar for technical support and B. Amin for critical reading of the manuscript.

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References 

  1. Abreu-Martin MT, Targan SR. Regulation of immune responses of the intestinal mucosa. Crit Rev Immunol. 1996;16:277–309
  2. Faria AM, Weiner HL. Oral tolerance. Immunol Rev. 2005;206:232–259
  3. Kraus TA, Mayer L. Oral tolerance and inflammatory bowel disease. Curr Opin Gastroenterol. 2005;21:692–696
  4. Weiner HL. Current issues in the treatment of human diseases by mucosal tolerance. Ann N Y Acad Sci. 2004;1029:211–224
  5. Friedman A, Weiner HL. Induction of anergy or active suppression following oral tolerance is determined by antigen dosage. Proc Natl Acad Sci U S A. 1994;91:6688–6692
  6. Mowat AM, Strobel S, Drummond HE, Ferguson A. Immunological responses to fed protein antigens in mice (I. Reversal of oral tolerance to ovalbumin by cyclophosphamide). Immunology. 1982;45:105–113
  7. Yoshida T, Hachimura S, Kaminogawa S. The oral administration of low-dose antigen induces activation followed by tolerization, while high-dose antigen induces tolerance without activation. Clin Immunol Immunopathol. 1997;82:207–215
  8. Sun J, Alison SM, Thompson KL, Fisher VH. Cell cycle block in anergic T cells during tolerance induction. Cell Immunol. 2003;225:33–41
  9. Huibregtse IL, van Lent AU, van Deventer SJ. Immunopathogenesis of IBD: insufficient suppressor function in the gut?. Gut. 2007;56:584–592
  10. Steidler L, Hans W, Schotte L, Neirynck S, Obermeier F, Falk W, et al. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science. 2000;289:1352–1355
  11. Vandenbroucke K, Hans W, Van Huysse J, Neirynck S, Demetter P, Remaut E, et al. Active delivery of trefoil factors by genetically modified Lactococcus lactis prevents and heals acute colitis in mice. Gastroenterology. 2004;127:502–513
  12. Steidler L, Neirynck S, Huyghebaert N, Snoeck V, Vermeire A, Goddeeris B, et al. Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10. Nat Biotechnol. 2003;21:785–789
  13. Braat H, Rottiers P, Hommes DW, Huyghebaert N, Remaut E, Remon JP, et al. A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn’s disease. Clin Gastroenterol Hepatol. 2006;4:754–759
  14. Gasson MJ. Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol. 1983;154:1–9
  15. McReynolds L, O’Malley BW, Nisbet AD, Fothergill JE, Givol D, Fields S, et al. Sequence of chicken ovalbumin mRNA. Nature. 1978;273:723–728
  16. van Asseldonk M, Rutten G, Oteman M, Siezen RJ, de Vos WM, Simons G. Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp. lactis MG1363. Gene. 1990;95:155–160
  17. Waterfield NR, Le Page RW, Wilson PW, Wells JM. The isolation of lactococcal promoters and their use in investigating bacterial luciferase synthesis in Lactococcus lactis. Gene. 1995;165:9–15
  18. Tobagus IT, Thomas WR, Holt PG. Adjuvant costimulation during secondary antigen challenge directs qualitative aspects of oral tolerance induction, particularly during the neonatal period. J Immunol. 2004;172:2274–2285
  19. Morelli AE, Zahorchak AF, Larregina AT, Colvin BL, Logar AJ, Takayama T, et al. Cytokine production by mouse myeloid dendritic cells in relation to differentiation and terminal maturation induced by lipopolysaccharide or CD40 ligation. Blood. 2001;98:1512–1523
  20. Hauet-Broere F, Unger WW, Garssen J, Hoijer MA, Kraal G, Samsom JN. Functional CD25− and CD25+ mucosal regulatory T cells are induced in gut-draining lymphoid tissue within 48h after oral antigen application. Eur J Immunol. 2003;33:2801–2810
  21. Unger WW, Hauet-Broere F, Jansen W, van Berkel LA, Kraal G, Samsom JN. Early events in peripheral regulatory T-cell induction via the nasal mucosa. J Immunol. 2003;171:4592–4603
  22. Drouault S, Corthier G, Ehrlich SD, Renault P. Survival, physiology, and lysis of Lactococcus lactis in the digestive tract. Appl Environ Microbiol. 1999;65:4881–4886
  23. Rescigno M, Urbano M, Valzasina B, Francolini M, Rotta G, Bonasio R, et al. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat Immunol. 2001;2:361–367
  24. Niess JH, Brand S, Gu X, Landsman L, Jung S, McCormick BA, et al. CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance. Science. 2005;307:254–258
  25. Niess JH, Reinecker HC. Dendritic cells: the commanders-in-chief of mucosal immune defenses. Curr Opin Gastroenterol. 2006;22:354–360
  26. Kapsenberg ML. Dendritic-cell control of pathogen-driven T-cell polarization. Nat Rev Immunol. 2003;3:984–993
  27. Kelsall BL, Leon F. Involvement of intestinal dendritic cells in oral tolerance, immunity to pathogens, and inflammatory bowel disease. Immunol Rev. 2005;206:132–148
  28. Read S, Powrie F. CD4(+) regulatory T cells. Curr Opin Immunol. 2001;13:644–649
  29. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor α-chains (CD25) (Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases). J Immunol. 1995;155:1151–1164
  30. Steinman RM, Hawiger D, Nussenzweig MC. Tolerogenic dendritic cells. Annu Rev Immunol. 2003;21:685–711119:952–959
  31. Frossard CP, Steidler L, Eigenmann PA. Oral administration of an IL-10-secreting Lactococcus lactis strain prevents food-induced IgE sensitization. J Allergy Clin Immunol. 2007;
  32. Daniel C, Repa A, Wild C, Pollak A, Pot B, Breiteneder H, et al. Modulation of allergic immune responses by mucosal application of recombinant lactic acid bacteria producing the major birch pollen allergen Bet v 1. Allergy. 2006;61:812–819
  33. del-Patient K, Ah-Leung S, Creminon C, Nouaille S, Chatel JM, Langella P, et al. Oral administration of recombinant Lactococcus lactis expressing bovine β-lactoglobulin partially prevents mice from sensitization. Clin Exp Allergy. 2005;35:539–546
  34. Repa A, Grangette C, Daniel C, Hochreiter R, Hoffmann-Sommergruber K, Thalhamer J, et al. Mucosal co-application of lactic acid bacteria and allergen induces counter-regulatory immune responses in a murine model of birch pollen allergy. Vaccine. 2003;22:87–95
  35. Lider O, Santos LM, Lee CS, Higgins PJ, Weiner HL. Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein (II. Suppression of disease and in vitro immune responses is mediated by antigen-specific CD8+ T lymphocytes). J Immunol. 1989;142:748–752
  36. Zhang X, Izikson L, Liu L, Weiner HL. Activation of CD25(+)CD4(+) regulatory T cells by oral antigen administration. J Immunol. 2001;167:4245–4253
  37. Chen Y, Kuchroo VK, Inobe J, Hafler DA, Weiner HL. Regulatory T-cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science. 1994;265:1237–1240
  38. Faria AM, Weiner HL. Oral tolerance and TGF-β-producing cells. Inflamm Allergy Drug Targets. 2006;5:179–190
  39. Miller A, Lider O, Roberts AB, Sporn MB, Weiner HL. Suppressor T cells generated by oral tolerization to myelin basic protein suppress both in vitro and in vivo immune responses by the release of transforming growth factor β after antigen-specific triggering. Proc Natl Acad Sci U S A. 1992;89:421–425
  40. Nakamura K, Kitani A, Strober W. Cell contact-dependent immunosuppression by CD4(+)CD25(+) regulatory T cells is mediated by cell surface-bound transforming growth factor β. J Exp Med. 2001;194:629–644
  41. Nakamura K, Kitani A, Fuss I, Pedersen A, Harada N, Nawata H, et al. TGF-β 1 plays an important role in the mechanism of CD4+CD25+ regulatory T cell activity in both humans and mice. J Immunol. 2004;172:834–842
  42. Chung Y, Lee SH, Kim DH, Kang CY. Complementary role of CD4+CD25+ regulatory T cells and TGF-β in oral tolerance. J Leukoc Biol. 2005;77:906–913
  43. Carrier Y, Yuan J, Kuchroo VK, Weiner HL. Th3 cells in peripheral tolerance (I. Induction of Foxp3-positive regulatory T cells by Th3 cells derived from TGF-β T-cell-transgenic mice). J Immunol. 2007;178:179–185
  44. Ochi H, Abraham M, Ishikawa H, Frenkel D, Yang K, Basso AS, et al. Oral CD3-specific antibody suppresses autoimmune encephalomyelitis by inducing CD4+. Nat Med. 2006;12:627–635
  45. Oida T, Zhang X, Goto M, Hachimura S, Totsuka M, Kaminogawa S, et al. CD4+CD25- T cells that express latency-associated peptide on the surface suppress CD4+CD45RBhigh-induced colitis by a TGF-beta-dependent mechanism. J Immunol. 2003;170:2516–2522
  46. Coombes JL, Robinson NJ, Maloy KJ, Uhlig HH, Powrie F. Regulatory T cells and intestinal homeostasis. Immunol Rev. 2005;204:184–194
  47. Fuss IJ, Boirivant M, Lacy B, Strober W. The interrelated roles of TGF-β and IL-10 in the regulation of experimental colitis. J Immunol. 2002;168:900–908
  48. Kitani A, Fuss I, Nakamura K, Kumaki F, Usui T, Strober W. Transforming growth factor (TGF)-β1-producing regulatory T cells induce Smad-mediated interleukin 10 secretion that facilitates coordinated immunoregulatory activity and amelioration of TGF-β1-mediated fibrosis. J Exp Med. 2003;198:1179–1188
  49. Carrier Y, Yuan J, Kuchroo VK, Weiner HL. Th3 cells in peripheral tolerance (II. TGF-β-transgenic Th3 cells rescue IL-2-deficient mice from autoimmunity). J Immunol. 2007;178:172–178
  50. Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, et al. Conversion of peripheral CD4+. J Exp Med. 2003;198:1875–1886
  51. Tang Q, Henriksen KJ, Bi M, Finger EB, Szot G, Ye J, et al. In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes. J Exp Med. 2004;199:1455–1465
  52. Tang Q, Bluestone JA. Regulatory T-cell physiology and application to treat autoimmunity. Immunol Rev. 2006;212:217–237

 Supported by the Research Fund of Ghent University (GOA, 01G01205).

 No conflict of interest to disclose.

PII: S0016-5085(07)00931-6

doi:10.1053/j.gastro.2007.04.073

Refers to article:

  • Teaching Tolerance With a Probiotic Antigen Delivery System

    Michel H. Maillard, Scott B. Snapper
    Gastroenterology August 2007 (Vol. 133, Issue 2, Pages 706-709)

Gastroenterology
Volume 133, Issue 2 , Pages 517-528, August 2007

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