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The Journal of Immunology, 2000, 165: 3145-3153.
Copyright © 00 by The American Association of Immunologists

An Intra-Peyer’s Patch Gene Transfer Model for Studying Mucosal Tolerance: Distinct Roles of B7 and IL-12 in Mucosal T Cell Tolerance1

Yiguang Chen*, Kaimei Song*, Stephen L. Eck{dagger} and Youhai Chen2,*

* Department of Molecular and Cellular Engineering, Institute for Human Gene Therapy, and {dagger} Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussions
 References
 
Development of mucosal immunity and tolerance requires coordinated expression of a number of genes within the mucosa-associated lymphoid tissue (MALT). To study the roles of these genes in the MALT, we have established a MALT-specific gene transfer model using replication-defective adenovirus as vector. In this model, the target gene of interest is directly delivered into the Peyer’s patch by intra-Peyer’s patch injection of the recombinant virus. Using this gene transfer model, we investigated the roles of B7-1 and IL-12 in the development of mucosal tolerance. We found that intra-Peyer’s patch injection of OVA induced Ag-specific T cell hyporesponsiveness, as manifested by decreased T cell proliferation and IL-2/IFN-{gamma} production upon subsequent immune challenge. Intra-Peyer’s patch B7-1 gene transfer at the time of OVA administration partially reversed the inhibition of T cell proliferation and IL-2 secretion, but had no effect on IFN-{gamma} production. By contrast, intra-Peyer’s patch IL-12 gene transfer completely restored T cell proliferation and IFN-{gamma} secretion and partially reversed IL-2 inhibition. Using an adoptive TCR transgenic model, we further demonstrated that B7 and IL-12 played distinct roles during the inductive phase of mucosal tolerance. B7 selectively increased T cell proliferation and IL-2 secretion without affecting IFN-{gamma} production, whereas IL-12 increased both IL-2 and IFN-{gamma} production. These results indicate that B7 alone may not be sufficient to abrogate mucosal tolerance, and that cytokines such as IL-12 may also be required. Based on these findings, we propose a new model to explain the paradoxical roles of B7 in mucosal immunity and tolerance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussions
 References
 
Both mucosal immunity and tolerance are crucial for maintaining the integrity of the self. On the one hand, the mucosae are the major ports of entry for pathogens that ought to be expelled; in contrast, the gut contains an enormous amount of dietary Ags that must be tolerated. Mucosal immunity to pathogens prevents infections, whereas mucosal tolerance to dietary Ags averts hypersensitivity. This unique feature of the mucosal immune system has made it possible to either up- or down-regulate immune function through mucosal administration of Ags. Thus, mucosal vaccination has long been used to prevent infections (such as poliovirus-induced myelitis and meningitis), and mucosal tolerization has now been vigorously exploited for the treatment of allergy and autoimmune diseases (1, 2, 3). However, the dual functions of the mucosal immune system have also made it difficult to fully realize the potential of mucosal therapies, because both immunity and tolerance may develop following mucosal exposure of Ags. Indeed, overcoming mucosal tolerance has been the long term objective for oral vaccine development, and overcoming mucosal immunization is crucial for effective therapy of autoimmune diseases using oral Ags. Therefore, elucidation of the mechanisms by which mucosal immunity and tolerance are regulated is not only crucial for our understanding of the biology of the mucosal immune system but also for developing novel strategies for the treatment of immune-related disorders.

A central step in the development of adaptive immunity is the activation and differentiation of naive precursor T cells by specific Ags. This requires a minimum of two signals, an Ag-specific signal provided by peptide-MHC complex and a nonspecific costimulatory signal provided by the APCs. The best-studied costimulatory molecules to date are the B7 family of proteins, which include at least two members: B7-1 (CD80) and B7-2 (CD86) (4, 5). In naive animals, low levels of B7-2 are constitutively expressed on macrophages and dendritic cells. However, resting B cells express little or no B7-1 or B7-2 (6, 7, 8, 9, 10, 11, 12). Upon cell activation, both B7-1 and B7-2 are up-regulated, although expression of B7-1 often follows that of B7-2 (13). Both B7-1 and B7-2 bind to the same set of receptors: CD28 and CTLA4, which may deliver different signals (4, 5, 14, 15). B7:CD28 interaction is crucial for the activation of T cells; Ag presentation in the absence of CD28 costimulatory signal can lead to anergy in vitro (16, 17, 18). The precise roles of CTLA4 are not clear, although most studies suggest that it serves as a feedback negative regulator of CD28-mediated costimulation (19, 20, 21). Germline disruption of CTLA4 gene leads to systemic lymphoproliferative disorders and death of animals (22, 23). CTLA4 binds to B7 with >20-fold higher affinity than CD28, and a fusion protein carrying the extracellular domain of CTLA4 and human Ig C{gamma} chain (CTLA4-Ig) effectively blocks the interaction of B7 with CD28 and CTLA4 (15). CTLA4 may down-regulate immune responses through 1) directly competing with CD28 for the limited number of B7 molecules, or 2) interfering with the proximal CD3 and/or CD28 signal transduction through interaction with TCR/CD28 activation cap (24, 25), or 3) directly transmitting signals through interaction with phosphotyrosine phosphatase, PTP-1D (26).

It has long been speculated that peripheral T cell tolerance may be induced as a result of TCR signaling (first signal) in the absence of a second signal (27, 28, 29, 30). Although the nature of the second signal is still under intense investigation, B7-mediated costimulation has been considered to be the most likely candidate. However, recent reports from several laboratories including ours indicate that B7 may also be required for the induction of T cell tolerance, and that B7:CTLA4 interaction is essential for maintaining peripheral T cell tolerance (31, 32, 33, 34, 35). Thus, it appears that the costimulatory molecule B7 may be required for the development of both immunity and tolerance. To address this B7 paradox, we have developed a unique mucosal gene transfer model to selectively up-regulate B7 gene expression in the Peyer’s patches. We report here that up-regulating B7 in the mucosal tissue has no effect on the IFN-{gamma} pathway of T cell tolerance. By contrast, up-regulating inflammatory cytokine IL-12 completely reversed the inhibition of IFN-{gamma} and T cell proliferation in tolerized animals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussions
 References
 
Mice

Female BALB/c mice, 6–8 wk of age, were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice transgenic for a TCR specific for OVA323–339 peptide were provided by Dr. Dennis Y. Loh (36). The transgene-positive mice were extensively backcrossed to BALB/c background. Mice were screened for the expression of OVA-specific TCR by flow cytometry using anti-clonotypic mAb KJ1-26 (37). All mice were housed at the University of Pennsylvania animal care facilities.

Recombinant adenoviruses

The recombinant adenoviruses that carry mouse B7-1 (Ad-B7-1), IL-12 (Ad-IL-12), or Escherichia coli lacZ (Ad-lacZ) gene have been described previously (38, 39, 40, 41, 42, 43). The genomic sequences of these viruses are essentially the same, except for the transgenes that they carry (Fig. 1Go). They all possess the CMV enhancer and promotor and the SV40 late gene polyadenylation signal. The recombinant adenoviruses are devoid of the immediate early (E)3 1a, E1b, and a portion of E3 sequences and, therefore, are incapable of replicating themselves (38, 39, 40). In vitro and in vivo, the recombinant adenoviruses effectively transfect a variety of cell types and confer high levels of transgene expression (38, 39, 40).



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  		FIGURE 1. The genomic structure of the recombinant adenoviruses. PstI, XbaI, and XhoI are representative restriction sites in the viral genome. CMV, CMV promoter; poly(A), the poly adenylation tail.

 
Intra-Peyer’s patch injection of Ags and recombinant adenoviruses

Mice were first anesthetized by i.p. injection of ketamine/xylazine (50/1, 60 mg/kg) 10 min before surgery. Their flanks were shaved, and treated with 70% ethanol and Clinidine solution (Clinipad, Rocky Hill, CT). A middle skin incision (~1 cm in length) was then created, and Peyer’s patches located at the proximal end of the small intestine were gently exposed. Ags and/or adenoviruses, which were suspended in PBS, were injected directly into Peyer’s patches using a 33-gauge needle (Hamilton, Reno, Nevada). A total of four Peyer’s patches per mouse were injected, with each Peyer’s patch receiving 1 µl of the solution. The peritoneal wall was then closed, and mice were allowed to recover from anesthesia while resting on warm water pads.

Histochemistry

Peyer’s patches were removed surgically from small intestine, snap-frozen in OCT (Miles, Elkhart, IN), and cryosectioned at 6 µm. For B7-1 staining, Peyer’s patch sections were first treated with periodic acid and avidin/biotin blocking solution (Vector, Burlingame, CA) to block endogenous enzyme and biotin activities, and then were stained with biotinylated anti-mouse B7-1 mAb and peroxidase-streptavidin (PharMingen, San Diego, CA) according to the manufacturer’s instructions. Diaminobenzidine was used as the substrate for peroxidase, and methyl green was used for counterstaining. Isotype-matched Abs were routinely used as controls. For detecting ß-galactosidase activity, X-gal histochemistry was performed as previously described (43, 44).

Cell culture (45)

Splenocytes or mesenteric lymph node cells (1.5 x 106 cells/well) were cultured in 0.2 ml of serum-free medium (X-vivo 20; BioWhittaker, Walkersville, MD) containing various concentrations of OVA (grade V; Sigma, St. Louis, MO). Culture supernatants were collected 40 h later, and cytokine concentrations were determined by ELISA. For proliferation assays, splenocytes were cultured at 5 x 105 cells/well for 72 h and pulsed with 1 µCi/well of [3H]thymidine for an additional 16 h. Cells were harvested, and radioactivity was determined using a flatbed beta counter (Wallac, Gaithersburg, MD).

ELISA for cytokines

Quantitative ELISAs for IL-2 and IFN-{gamma} were performed using paired mAbs specific for corresponding cytokines according to the manufacturer’s recommendations (PharMingen). The following reagents were purchased from PharMingen: purified rat anti-mouse IL-2 (clone JES-1A12) and IFN-{gamma} (clone R4-6A2) mAb, biotinylated rat anti-mouse IL-2 (clone JES6-5H4) and IFN-{gamma} (clone XMG1.2) mAb, and recombinant mouse IL-2 and IFN-{gamma}.

Statistical analysis

Statistical significance of differences among various groups was determined by ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussions
 References
 
An intra-Peyer’s patch gene transfer model for studying mucosal tolerance

To manipulate the levels of gene expression in the mucosa, we developed an intra-Peyer’s patch gene transfer model using replication-defective human adenovirus type 5 (Ad5) vector. Thus, 108–1010 particles of recombinant viruses were injected directly into Peyer’s patches, and transgene expression in the Peyer’s patch, small intestine, liver, mesenteric lymph node, and spleen was examined by histochemistry at different time points after the viral injection. We found that intra-Peyer’s patch injection of recombinant adenoviruses conferred specific transgene expression in the Peyer’s patches. The levels of transgene expression correlated to the doses of the virus used. Transgene expression was primarily localized in the injected Peyer’s patches and was not found in the liver, spleen, peritoneum, or other parts of the intestine regardless of the doses of viruses used. When 1010 viral particles were injected, weak transgene expression could also be detected in mesenteric lymph nodes. Transgene expression in the Peyer’s patches peaked on day 1 after the viral injection and gradually disappeared 5–7 days later. Fig. 2Go, A–D, illustrates lacZ gene expression 1, 3, 5, and 7 days after intra-Peyer’s patch injection of 109 particles of Ad-lacZ, and Fig. 2Go, E and F, illustrates B7-1 gene expression before and after intra-Peyer’s patch injection of 109 particles of Ad-B7-1. B7-1 was barely detectable in nontreated Peyer’s patches (Fig. 2GoE), or Peyer’s patches injected with control vector viruses or Ad-lacZ (data not shown). By contrast, high levels of B7-1 expression were detected in Peyer’s patches injected with B7-1 virus (Fig. 2GoF). Similar patterns of transgene expression were observed following intra-Peyer’s patch injection of 109 particles of recombinant adenoviruses carrying IL-12 (data not shown).



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  		FIGURE 2. Adenovirus-mediated gene transfer in the Peyer’s patches. Peyer’s patches located at the proximal end of the small intestine were injected with 109 particles of recombinant adenovirus carrying either the lacZ or the B7-1 gene as described in Materials and Methods. One to 7 days later, the injected Peyer’s patches were removed, sectioned, and tested for 1) ß-galactosidase activity by X-gal histochemistry (A–D), and 2) B7-1 gene expression by immunohistochemistry (E and F) or flow cytometry (G and H). Results shown are representative of two similar experiments. A–D, Peyer’s patches injected with Ad-lacZ were stained by X-gal histochemistry. E, A control Peyer’s patch that was not injected with adenovirus but stained for B7-1. GC, germinal center; IFR, interfollicular region; LU, lumen; SED, subepithelial dome. F, A Peyer’s patch 1 day after Ad-B7-1 injection, which was stained for B7-1. G and H, Peyer’s patch cells from an Ad-lacZ-treated mouse (G) and from an Ad-B7-1-treated mouse were stained with 1) FITC-labeled anti-mouse B7-1 mAb, and 2) PE-labeled anti-mouse Mac-1, B220, or CD4 mAb (Caltag, Burlingame, CA). Histograms illustrate B7-1 expression by Peyer’s patch macrophages.

 
To determine which cell in the Peyer’s patch expresses the transgene, we performed flow cytometric analysis of Peyer’s patch cells following intra-Peyer’s patch B7-1 gene transfer. Isolated Peyer’s patch cells were stained with fluorescein-labeled Abs to Mac-1, B220, and CD4 as well as B7-1. B7-1 expression on different cell types was then analyzed as shown in Fig. 2Go, G and H. We found that >95% of B7-1+ cells in Ad-B7-1-treated Peyer’s patches were Mac-1+ (Fig. 2GoH). B cells and T cells expressed little or no B7-1 (<0.5% of B and T cells were positive for B7-1; data not shown). In Peyer’s patches injected with Ad-lacZ, <0.1% of Mac-1+ cells were positive for B7-1 (Fig. 2GoG). These results are consistent with reports that adenoviruses preferentially infect macrophages and epithelial cells, but not lymphocytes (43, 46, 47).

Thus, gene delivery to Peyer’s patches can be achieved by intra-Peyer’s patch injection of recombinant adenoviruses. In theory, any gene of interest can be delivered into mucosal tissues using the strategies described here. When animals treated with the recombinant Ad5 are compared with those treated with control Ad5, an obligatory role, if any, of the transgene can be deduced. Although highly artificial, this model allows direct evaluation of the functions of a cloned gene in vivo. Although the transient nature of gene expression conferred by viral vectors poses a formidable challenge for gene therapy of genetic diseases (for which permanent gene expression is desired), the viral gene transfer model described here provides a unique opportunity to study the roles of genes that are transiently expressed during mucosal immune responses.

Intra-Peyer’s patch injection of OVA induces Ag-specific Th1 cell tolerance

Although oral tolerance is a well-established phenomenon, the precise sites of tolerance induction following oral or intragastric administration of Ag are unknown. Because our intra-Peyer’s patch gene transfer model targets genes directly into Peyer’s patches, we investigated whether Ags injected into Peyer’s patches could induce specific T cell tolerance and, if so, whether it would be affected by the adenoviral vector used for gene transfer. Therefore, intra-Peyer’s patch injection of OVA was performed in BALB/c mice with or without Ad-lacZ. Seven days after intra-Peyer’s patch injection, mice were immunized with OVA and were tested for specific anti-OVA T cell responses 2 wk later. As shown in Fig. 3Go, intra-Peyer’s patch injection of 50–250 µg of OVA induced specific T cell tolerance, as manifested by decreases in T cell proliferation and IL-2/IFN-{gamma} production in mice treated with OVA. This effect was OVA specific, because T cell responses to hen egg lysozyme were not affected in these animals (data not shown). Injection of 108–1010 particles of Ad-lacZ did not significantly affect T cell proliferation or IL-2/IFN-{gamma} production. In addition to IL-2 and IFN-{gamma}, we tested IL-4 production in the same splenocyte cultures as those described in Fig. 3Go. In the PBS-treated group, a low, but detectable, amount of IL-4 was present in cultures stimulated with 10–1000 µg/ml of OVA, which ranged from 25–40 pg/ml (no IL-4 was detected in cultures containing no OVA). However, intra-Peyer’s patch injection of 50–250 µg of OVA neither significantly increased nor decreased IL-4 production in these cultures (data not shown). Thus, intra-Peyer’s patch injection of OVA induces Ag-specific Th1 cell tolerance, with little or no effect on Th2 cells. This is reminiscent of reports that oral or i.v. administration of soluble Ags, especially at low doses, selectively tolerizes Th1 cells, but not Th2 or B cells (48, 49, 50, 51). Additionally, the results presented in Fig. 3Go suggest that Peyer’s patches can serve as inductive sites for Th1 cell tolerance following mucosal exposure of Ags and that replication-defective adenoviruses themselves (at least at the doses tested here) do not affect the induction of T cell tolerance.



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  		FIGURE 3. T cell tolerance induced by intra-Peyer’s patch injection of OVA in the absence or the presence of replication-defective adenoviruses. Groups of BALB/c mice, four mice per group, had 0–250 µg of OVA (A–C) or 50 µg of OVA plus 108–1010 particles of recombinant Ad-lacZ (D–F) injected into their Peyer’s patches as described in Materials and Methods. A total of four Peyer’s patches per mouse were injected. Seven days after intra-Peyer’s patch injection, all mice were immunized s.c. with 100 µg of OVA emulsified in CFA containing 100 µg of Mycobacterium tuberculosis H37 RA (Difco, St. Louis, MO) (59 ). Mice were sacrificed 2 wk after the immunization, and their splenocytes were prepared and cultured with 0–1000 µg/ml of OVA. Results shown are from one representative experiment of five performed. The error bars represent the SDs calculated from triplicate cultures.

 
Intra-Peyer’s patch B7-1 gene transfer has no effect on the IFN-{gamma} pathway of T cell tolerance, but partially reverses inhibition of proliferation and IL-2 secretion

To test the roles of B7-1 in mucosal T cell tolerance, we injected OVA into Peyer’s patches with or without B7-1 virus. Mice were then immunized with OVA and tested for anti-OVA immune responses 2 wk later. As shown in Fig. 4Go, A–C, intra-Peyer’s patch injection of OVA dramatically inhibited IFN-{gamma}/IL-2 production and T cell proliferation. This was not affected by coadministration of lacZ virus. By contrast, injection of B7-1 virus partially reversed the inhibition of IL-2 secretion and T cell proliferation in tolerized animals. Surprisingly, B7-1 virus had no effect on IFN-{gamma} pathway of T cell tolerance, because inhibition of IFN-{gamma} production in tolerized animals was not affected by B7-1 virus. In parallel experiments, a higher dose of virus (1010 particles/Peyer’s patch) was also tested. No effect on IFN-{gamma} production was ever observed, and only partial reversal of IL-2 production and T cell proliferation was detected following intra-Peyer’s patch injection of the B7 virus. The B7-1 effect depended on coadministration of OVA and Ad-B7-1 to Peyer’s patches, because injection of Ad-B7-1 into non-Peyer’s patch regions of the small intestine had no effect on T cell tolerance induced by intra-Peyer’s patch injection of OVA (data not shown).



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  		FIGURE 4. Effects of intra-Peyer’s patch B7-1 gene transfer. Groups of BALB/c mice, four mice per group, had 50 µg of BSA or OVA with or without 109 particles of recombinant Ad-lacZ or Ad-B7-1 injected into their Peyer’s patches. Mice were immunized and tested for cytokine production (A and B) and T cell proliferation (C) as described in Fig. 3Go. For humoral immune responses, sera were collected 10 days after immunization and tested for anti-OVA IgG1 (D) and IgG2a (E) by isotype-specific ELISA using OVA-coated plates (PharMingen). ODs of sera at various dilutions are shown. Results are from one representative experiment of four performed. For IL-2 and T cell proliferation, the differences between OVA- and OVA/Ad-B7-1-treated groups are statistically significant (p < 0.01 for cultures with 100-1000 µg/ml of OVA). For humoral immune responses, only anti-OVA IgG1 in the BSA-treated group is significantly different from those in other groups (p < 0.01).

 
To determine whether intra-Peyer’s patch injection of Ag regulates humoral immune responses, we also tested anti-OVA Abs in mouse sera. As shown in Fig. 4Go, D and E, both anti-OVA IgG2a and anti-OVA IgG1 Abs were detected in the sera of mice 10 days after immunization, as measured by OVA-specific ELISA (52). Interestingly, intra-Peyer’s patch injection of OVA had no significant effect on anti-OVA IgG2a production, but increased anti-OVA IgG1 responses. Intra-Peyer’s patch B7-1 gene transfer had no effect on anti-OVA Ab responses. These results suggest that B7-1 alone may not be sufficient to abrogate mucosal Th1 tolerance and that other factors, such as cytokines, may be required.

Intra-Peyer’s patch IL-12 gene transfer completely reverses the IFN-{gamma} pathway of T cell tolerance

IL-12 may be important not only for the development of cellular immunity, but also for preventing or reversing T cell tolerance (53, 54, 55, 56, 57, 58). To determine the roles of IL-12 in mucosal tolerance, we performed a gene transfer experiment similar to that described above using rIL-12 adenovirus. As shown in Fig. 5Go, A–C, intra-Peyer’s patch injection of OVA induced OVA-specific T cell tolerance in mice, which was not affected by coadministration of 109 particles of lacZ virus. Remarkably, coadministration of rIL-12 virus completely reversed the inhibition of IFN-{gamma} production and T cell proliferation in tolerized animals. Interestingly, inhibition of IL-2 was only partially reversed by the IL-12 virus. Thus, unlike B7, IL-12 primarily affects the IFN-{gamma} pathway of T cell tolerance. Consistent with the enhanced IFN-{gamma} production, anti-OVA IgG2a responses were also significantly increased in mice treated with Ad-IL-12 (Fig. 5GoE). By contrast, anti-OVA IgG1 responses were not affected by Ad-IL-12 (Fig. 5GoD).



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  		FIGURE 5. Effects of intra-Peyer’s patch IL-12 gene transfer. Groups of BALB/c mice, four mice per group, had 50 µg of BSA or OVA with or without 109 particles of recombinant Ad-lacZ or Ad-IL-12 injected into their Peyer’s patches. Mice were immunized and tested as described in Fig. 4Go. Results are from one representative experiment of three performed. For cellular immune responses, the differences between OVA- and OVA/Ad-IL-12-treated groups are statistically significant (p < 0.01 for cultures with 100-1000 µg/ml of OVA). For anti-OVA IgG1 responses, the differences between the BSA-treated group and all other groups are statistically significantly (p < 0.01). For anti-OVA IgG2a responses, the differences between the OVA/Ad-IL-12 group and all other groups are statistically significantly (p < 0.001).

 
It should be noted that because IL-12 is a soluble cytokine, the experiments described above do not exclude the possibility that IL-12 produced in Peyer’s patches might be transported into other organs and affect T cell differentiation at nonmucosal sites. However, the following observations do not support this view. First, when injected at different sites of the small intestine, Ad-IL-12 did not abrogate T cell tolerance induced by intra-Peyer’s injection of OVA. Secondly, IL-12 was not detectable (<10 pg/ml) in the blood of mice treated with Ad-IL-12 (as described in Fig. 5Go). Lastly, intra-Peyer’s patch injection of Ad-IL-12 in the absence of OVA did not affect systemic T cell responses in mice immunized with OVA (data not shown).

Differential roles of B7-1 and IL-12 in the inductive phase of mucosal tolerance

To further elucidate the roles of B7-1 and IL-12 in mucosal tolerance, we studied the effects of B7-1 and IL-12 gene transfer during the inductive phase of mucosal tolerance. This was achieved by combining the intra-Peyer’s patch gene transfer model with a TCR transgenic adoptive transfer model as we described previously (59). In the TCR transgenic adoptive transfer model, T cell responses to specific Ags during the inductive phase of immune tolerance can be studied directly (59). Thus, mice adoptively transferred with OVA-specific TCR transgenic cells were injected, into their Peyer’s patches, with OVA in the presence or the absence of recombinant adenoviruses. Anti-OVA T cell responses in mesenteric lymph node and spleen were determined ex vivo 3–7 days later. As shown in Fig. 6Go, intra-Peyer’s patch injection of OVA induced detectable T cell proliferation and IL-2 secretion in both mesenteric lymph node and spleen, which were most evident on day 3. Coadministration of B7-1 or IL-12 virus moderately increased these responses, especially in the mesenteric lymph nodes. Interestingly, the IFN-{gamma} pathway was not significantly activated by OVA or OVA plus B7 virus, but was dramatically up-regulated when IL-12 signal was provided. These results strongly suggest that IL-12 is crucial for abrogating T cell tolerance and that the differential roles of B7 and IL-12 in mucosal T cell tolerance may result from their distinct effects on T cell activation during the inductive phase of mucosal tolerance.



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  		FIGURE 6. Roles of B7-1 and IL-12 in the inductive phase of immune tolerance. Groups of BALB/c mice, four mice per group, were transfused vial the tail vein with 2.5 x 106 DO11 TCR transgenic T cells as we previously described (59 ). One week later, mice were given intra-Peyer’s patch injections of 50 µg of BSA or OVA with or without 109 particles of Ad-lacZ , Ad-B7-1, or Ad-IL-12. Three and 7 days after the intra-Peyer’s patch injection, mice were sacrificed, and their splenocytes (A–F) and mesenteric lymph node cells (G–L) were cultured with or without 100 µg/ml of OVA. Cytokine secretion and proliferative responses were assessed as described in Materials and Methods. Results shown are from one representative experiment of three performed. *, Groups that are significantly different from the control groups ({square}, BSA-treated groups) as determined by ANOVA (p < 0.01).

 
Consistent with these results, direct tracking of transgenic T cells in vivo revealed that both B7-1 and IL-12 were capable of promoting T cell expansion. Fig. 7Go is a representative of such experiments performed 7 days after intra-Peyer’s patch injection. Transgenic T cells were not detectable in control mice (<0.1%; Fig. 7GoA), but made up 0.31% of the mesenteric cell pool in mice that received injections of transgenic cells. Injection of OVA alone did not significantly affect the frequency of the transgenic cells at this time point (Fig. 7GoC), but injection of either Ad-B7-1 or Ad-IL-12 increased the frequency of the transgenic cells by 3-fold (Fig. 7Go, E and F). Thus, the increased T cell responses in Ad-B7-1- and Ad-IL-12-treated mice, as demonstrated in Fig. 6Go, were at least partially due to the increase in the frequency of transgenic cells in these animals.



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  		FIGURE 7. Increased expansion of OVA-specific T cells following intra-Peyer’s patch B7-1 or IL-12 gene transfer. Groups of BALB/c mice, four mice per group, were treated as described in Fig. 6Go, and sacrificed 7 days after intra-Peyer’s patch injection. Single-cell suspensions of mesenteric lymph nodes were prepared and stained for CD4 and the transgenic TCR (KJ-126) and were analyzed using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, Mountain View, CA) (59 ). Data represent 10,000 live cell events, with the percentages of cells in each quadrant presented. A, Control BALB/c mice that did not receive injections of transgenic cells. B–F, BALB/c mice that received injections of transgenic cells plus BSA (B) or OVA (C–F) with or without recombinant adenoviruses.

 

   Discussions
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussions
References
 
The mucosae are the major ports of entry for foreign Ags. More than 100 kilograms of food Ags are processed each year by our gastrointestinal mucosa, and an estimated 0.1–1% of these (100–1000 g) are absorbed in nondegraded forms (60, 61, 62). To meet up with this challenge, >60% of our peripheral lymphoid tissues are deployed at the gut mucosa, where they protect us against various microbial or parasitic infections (61, 63). Yet, not all foreign Ags are infectious pathogens. In fact, most food Ags are beneficial nutrients that must be tolerated. Thus, the gut immune system has to differentiate two types of Ags: beneficial dietary Ags and infectious pathogens. Failure to tolerate dietary Ags may lead to intestinal hypersensitivity, as exemplified by food-sensitive enteropathies, and failure to expel infectious pathogens may contribute to infections (2, 63). Indeed, mucosal infection is still the major killer of infants today and claims >14 million deaths in children annually (among which diarrhea alone accounts for ~5 million deaths) (1). Mucosal vaccination may be the only logical solution to these problems.

However, despite recent developments in mucosal immunology, effective manipulation of the mucosal immune system, either for vaccination or for tolerance induction, remains extremely challenging. This is primarily due to the lack of understanding of the fundamental principles governing mucosal immunity vs tolerance. For instance, it is not known how the gut-associated lymphoid tissue differentiates pathogenic from beneficial dietary Ags. Is it based on the signals induced by the Ag but produced by the immune system as many of us believe (18, 27, 30)? If so, what are those signals? Are they costimulatory signals or cytokine signals? Experiments reported here were designed to address these issues. Although B7 and IL-12 may be both involved in tilting the balance between immunity and tolerance, our data suggest that they do so through different mechanisms. B7 enhances IL-2 secretion, but does not affect IFN-{gamma} production, whereas IL-12 promotes both IFN-{gamma} and IL-2 production as well as T cell proliferation. This is consistent with the report that IL-12, but not CTLA-4, affects the IFN-{gamma} pathway of T cell tolerance following i.v. administration of Ag (53).

The data reported here also suggest that the nonmanipulated level, the default level, of B7 in the gut may be the optimal level for tolerance induction. It has been reported that when the default level of B7 is blocked (e.g., by CTLA4-Ig or anti-B7 Ab), T cell tolerance is partially abrogated (31, 53). This is presumably due to the blockade of B7:CTLA-4, but not B7:CD28, interaction (31, 53). Similarly, when the default level of B7 is increased, as shown in Fig. 2Go, T cell tolerance is also diminished. Based on these new findings, we propose the following model to explain the paradoxical roles of B7 in mucosal immunity and tolerance (Table IGo).


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  		Table I. Levels of B7 costimulation in mucosal immunity and tolerance1

 
1) In the total absence of B7, naive T cells interact with APC through the TCR-peptide-MHC complex without engaging the CD28 molecule. This leads to no activation of T cells and no up-regulation of the CTLA4 molecule (53). T cells will enter and leave the Peyer’s patch through lymph as naive T cells. The presence of the specific Ag in the Peyer’s patch will be simply ignored; neither immunity nor tolerance develops.

2) In the presence of the default level of B7, naive T cells recognizing Ags present in the Peyer’s patch will receive both the TCR and the CD28 signals. This leads to partial T cell activation, cytokine secretion, and up-regulation of CTLA4 molecule. As the affinity of CTLA4 to B7 is >20 times higher than CD28, the up-regulated CTLA4 will compete with CD28 for the limited number of B7 molecules. This effectively blocks the CD28 signal, not unlike that seen when B7 is blocked by CTLA4-Ig. Additionally, engagement of CTLA4 may directly transmit negative signals to T cells, leading to their inactivation. Thus, the default level of B7 costimulation may initiate a T cell response in the Peyer’s patch, but is unable to sustain it. The inactivated T cells will not be able to enter the B cell-rich follicle. They may either die in the Peyer’s patch or leave as anergic or deviated Th3 cells (18, 64). Therefore, specific peripheral T cell tolerance is established.

3) In the presence of high levels of B7 (e.g., during bacterial infection), T cell activation in the Peyer’s patches also leads to CTLA4 up-regulation. However, due to the abundance of B7 on APC, B7:CD28 interaction will not be totally blocked by CTLA4. The persistence of CD28 signal neutralizes the CTLA4 effect and enhances T cell activation. However, an increased B7 level alone may not be sufficient to completely abrogate T cell tolerance, because B7 does not affect the IFN-{gamma} pathway of T cell tolerance. Other factors, such as cytokines, are required.

Among cytokines that may help to prevent mucosal tolerance, IL-12 may be one of the most important (53, 54, 55, 56, 57, 58). Up-regulating IL-12 alone is sufficient to completely reverse inhibition of IFN-{gamma} and T cell proliferation in tolerized animals (Fig. 5Go). This is consistent with other reports that injection of rIL-12 at nonmucosal sites reverses T cell tolerance and that neutralization of IL-12 enhances oral tolerance, presumably by enhancing apoptosis or TGF-ß secretion (58). Our model is also consistent with those proposed by several other laboratories with respect to the roles of B7 in the inductive phase of T cell tolerance (32, 33).

In summary, we have developed a unique intra-Peyer’s patch gene transfer model for studying mucosal tolerance. Using this model, we found that intra-Peyer’s patch injection of Ag induces specific T cell tolerance, and that intra-Peyer’s patch B7 and IL-12 gene transfer selectively reverses it.


   Acknowledgments
 
We thank Dr. James M. Wilson for providing Ad-lacZ, Dr. Frank L. Graham for supplying Ad-IL-12, and Dr. Dennis Y. Loh for providing OVA-specific TCR transgenic mice.


   Footnotes
 
1 This work was supported by Grants AI41060 and CA74294 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Youhai Chen, BRB-II/III, Room 511, Institute for Human Gene Therapy and Department of Molecular and Cellular Engineering, University of Pennsylvania School of Medicine, 421 Curie Boulevard, Philadelphia, PA 19104. Back

3 Abbreviations used in this paper: E, early; Ad5, adenovirus type 5. Back

Received for publication January 4, 2000. Accepted for publication June 27, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussions
 References
 

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