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The Journal of Immunology, 2000, 164: 2890-2896.
Copyright © 2000 by The American Association of Immunologists

A Bone Marrow-Derived APC in the Gut-Associated Lymphoid Tissue Captures Oral Antigens and Presents Them to Both CD4+ and CD8+ T Cells1

Effrossini Blanas*, Gayle M. Davey*, Francis R. Carbone{dagger} and William R. Heath2,*

* Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia; and {dagger} Department of Pathology and Immunology, Monash Medical School, Prahran, Victoria, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously reported that feeding OVA to C57BL/6 mice can lead to a weak CTL response that is dependent on CD4+ T cell help and is capable of causing autoimmunity. In this study, we investigated the basis of the class I and class II-restricted Ag presentation required for such CTL induction. Two days after feeding OVA, Ag-specific CD4+ and CD8+ T cells were seen to proliferate in the Peyer’s patches and mesenteric lymph nodes. Little proliferation was evident in other lymphoid tissues, except at high Ags doses, in which case some dividing CD4+ T cells were observed in the spleen and peripheral lymph nodes. Using chimeric mice, the APC responsible for presenting orally derived Ags was shown to be derived from the bone marrow. Examination of the Ag dose required to activate either CD4+ or CD8+ T cells indicated that a single dose of 6 mg OVA was the minimum dose that consistently stimulated either T cell subset. These data indicate that oral Ags can be transported from the gut into the gut-associated lymphoid tissue, where they are captured by a bone marrow-derived APC and presented to both CD4+ and CD8+ T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Orally administered Ags gain access to the body via the mucosal system known as the gut-associated lymphoid tissue (GALT)3 (1). The GALT consists of organized lymphoid tissues (Peyer’s patches and mesenteric lymph nodes) and a large number of lymphoid cells distributed diffusely throughout the lamina propria and epithelium. The principal inductive site in the GALT is the Peyer’s patches, which acquire Ag from the intestinal lumen via specialized epithelial cells called M cells overlying the patches’ dome (2). M cells take up and transport Ags, without degradation, into the Peyer’s patches. Once Ag enters the Peyer’s patches, it is processed and presented to T cells by APCs that stimulate either tolerance or immunity. It is generally recognized that oral delivery of soluble Ag induces a state of Ag-specific tolerance (3). However, we have shown that feeding OVA to C57BL/6 (B6) mice can generate a CTL response (4).

The ability to generate CTL immunity in this way indicates that soluble Ags are able to be processed and presented in association with MHC class I molecules for recognition and subsequent activation of CD8+ T cells. This is unlikely to occur by simple degradation of whole protein into peptides within the gut because oral administration of peptide does not induce CTL immunity (4, 5). The ability to generate CTL immunity with whole but not peptide forms of OVA implies that processing of whole protein is necessary for the induction of OVA-specific CTLs. However, where this processing takes place, and in what type of APC, is unknown. In this report we examine the site of Ag presentation of orally administered OVA and determine the nature of the APC involved in this presentation.


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

All mice were bred and maintained at the Walter and Eliza Hall Institute of Medical Research. RAG-1-/- OVA-specific class I-restricted T cell receptor (OT-I) transgenic mice (6) and OVA-specific class II-restricted T cell receptor (OT-II) transgenic mice (7) and class II-deficient mice (8) have been described. For all experiments, mice were aged between 8 and 12 wk, except for bone marrow chimeras for which mice were reconstituted at 8–12 wk and used 4–9 wk later.

Antigens

Chicken egg OVA, grade V, was purchased from Sigma (St. Louis, MO). BSA, fraction V, was purchased from Flow Laboratories (North Ryde, Australia).

Induction of oral priming

Mice were given various doses of OVA dissolved in mouse-tonicity PBS (pH 7.2) by gastric intubation (4). Briefly, under light penthrane anesthesia, a feeding tube (made from a capillary tube) was inserted into the stomach and 0.5 ml OVA in PBS was delivered by means of an attached syringe.

Adoptive transfer of 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE)-labeled cells and flow cytometry

Preparation and CFSE labeling of OT-I and OT-II cells was performed as previously described (9). Briefly, for OT-I cells, the spleen and lymph nodes were removed from RAG-1-/- OT-I mice and single cells were prepared. These cells were then treated with a CD4-specific mAb (RL172) and a heat stable Ag-specific mAb (J11d) and then with complement. For OT-II cells, lymph nodes (including inguinal, axillary, brachial, cervical, and mesenteric) were removed from OT-II transgenic mice and single cells were prepared. These were treated with a CD8-specific mAb (3.168) and J11d, followed by complement. These single-cell populations were washed three times before use.

For flow cytometric analysis of lymphoid organs after adoptive transfer, Peyer’s patches, mesenteric lymph nodes, pooled peripheral lymph nodes (which included the inguinal, axillary, and brachial lymph nodes), and spleen were removed, and single cells were stained using the following mAbs: PE-conjugated anti-CD8 (YTS 169.4) or anti-CD4 (YTS 191.1; Caltag, San Francisco, CA) (9). Dead cells were excluded based on propidium iodide staining (Calbiochem, La Jolla, CA). Three-color flow cytometry was performed on a FACScan (Becton Dickinson, Mountain View, CA) using Lysis II software. Gates were set on lymphocytes based on forward and side scatter profiles. A total of 10,000–40,000 live CD8+ or CD4+ cells were collected. Analysis was conducted using WEASEL software (F. Battye, Walter and Eliza Hall Institute, Melbourne, Australia).

OVA-loaded spleen cells

Spleen cells from B6 mice were cytoplasmically loaded with OVA protein (10). Briefly, 2 x 108 spleen cells were suspended in 1 ml hypertonic solution (0.5 M sucrose, 10% w/v polyethylene glycol 1000, and 10 mM HEPES (pH 7.2) in mouse tonicity RPMI 1640 medium MT-RPMI) containing 10 mg/ml of OVA protein for 10 min at 37°C. The cells were then rapidly diluted with 14 ml of prewarmed hypotonic solution (60:40 MT-RPMI:H20) for 2 min at 37°C and then centrifuged for 5 min at 4°C. Cells were irradiated 1500 centiGrey (cGy) before their use. Mice were immunised i.v. with a 0.5 ml suspension of 25 x 106 irradiated spleen cells.

In vitro stimulation of effector populations

All nucleated spleen cells (~ 1 x 108 cells) from individual mice primed 14 days earlier were incubated for 6 days with 1 x 108 OVA-loaded spleen cells as previoulsy described (4). Cultures were maintained at 37°C and 5% CO2 in 30 ml MT-RPMI supplemented with 10% FCS, 2 mM L-glutamine, 5 x 10-5 2-ME, and antibiotics. Cytotoxicity was assessed in a conventional 51Cr-release assay using the H-2b cell line EL4 cells alone and EL4 cells coated with 1 µg/ml with OVA257–264 as target cells. Results are shown either as percentage of OVA-specific lysis or as LU/spleen. The percentage of OVA-specific lysis was determined by subtracting the percentage of specific lysis of the OVA257–264-coated EL4 targets from the percentage of specific lysis of EL4 targets. Lytic units were calculated by determining the minimum number of effectors required to generate 10% OVA-specific lysis (in this case, background nonspecific lysis of EL4 targets was subtracted from that of the OVA257–264-coated EL4 targets) and then dividing this into the total number of effectors generated from each responder spleen. Each point represents an individual mouse. Twelve lytic units was the minimum detectable response. Nonresponders are represented by points below the line drawn at 12 LU.

Bone marrow reconstitution

Two types of bone marrow chimeras were used. For the first type of chimeric mice, adult B6 mice were lethally irradiated at 900 cGy and reconstituted with 5 x 106 T-depleted B6 or class II-deficient (8) bone marrow cells. For the second type of chimeric mice, adult (B6 x bm1)F1 mice were lethally irradiated at 900 cGy and reconstituted with 5 x 106 T-depleted B6 or bm1 bone marrow cells (11). All mice were injected i.p. with 0.1 ml of T24 ascites (Thy-1 specific) 1 day after bone marrow reconstitution to eliminate radioresistant host T cells. The mice were left for 4–9 wk before use.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Oral OVA is presented to CD8+ T cells in the GALT

In our previous studies, we showed that feeding OVA to B6 mice led to the induction of a CTL response (4). To determine the site of OVA presentation to CD8+ T cells in vivo, OVA-specific CD8+ T cells from the RAG-1-/- OT-I transgenic line (OT-I cells) were labeled with the fluorescent dye CFSE and injected into unirradiated B6 mice. The next day, these mice were fed 60 mg OVA. At later time points, their secondary lymphoid organs (Peyer’s patches, mesenteric lymph nodes, pooled peripheral lymph nodes (inguinal, axillary, and brachial) and spleen) were removed and analyzed by flow cytometry for the presence of proliferating OT-I cells. Detection of proliferating cells in any particular site, which is evidenced by separate peaks on a FACS histogram, is indicative of Ag presentation. Analysis of the various populations by flow cytometry 2 days after feeding revealed that CD8+ T cells proliferated in the Peyer’s patches and mesenteric lymph nodes but not in the peripheral lymph nodes or spleen (Fig. 1Go). By day 7, CD8+ T cells that had undergone proliferation were present in all lymphoid organs (Fig. 2Go). Whether this latter observation was due to recirculation of proliferated cells from the GALT to peripheral sites or to delayed trafficking of Ag from the gut to more distant regions could not be distinguished. Mice that were left unfed did not show CD8+ T cell proliferation in any sites (Fig. 1Go and Fig. 2Go).



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  		FIGURE 1. CD8+ cells are activated in the Peyer’s patches and mesenteric lymph nodes 2 days after oral Ag delivery. A total of 10.5 x 106 CFSE-labeled CD8+ V{alpha}2+ RAG-1-/- OT-I cells was injected i.v. into B6 mice. The next day, the mice were fed 60 mg OVA or were left unfed. Two days later, the Peyer’s patches, mesenteric lymph nodes, peripheral lymph nodes, and spleen from each mouse were analyzed by flow cytometry. Histograms were gated on CFSE+CD8+ live cells. The percentages indicate the proportion of cells that had proliferated.

 


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  		FIGURE 2. Activated CD8+ cells are evident in the Peyer’s patches, mesenteric lymph nodes, and other peripheral lymphoid tissues 7 days after oral Ag delivery. A total of 11.4 x 106 CFSE-labeled CD8+ V{alpha}2+ RAG-1-/- OT-I cells was injected i.v. into B6 mice. The next day, the mice were fed 60 mg OVA or were left unfed. Seven days later, the Peyer’s patches, mesenteric lymph nodes, peripheral lymph nodes, and spleen from each mouse were analyzed by flow cytometry. Histograms were gated on CFSE+CD8+ live cells. The percentages indicate the proportion of cells that had proliferated.

 
Oral OVA is presented to CD4+ T cells in the GALT

Next, we examined the site of Ag presentation to CD4+ T cells by using the same approach, but we substituted OT-I cells with OVA-specific CD4+ T cells from the OT-II transgenic line (OT-II cells) (7). Five independent experiments showed that 2 days after feeding 60 mg OVA, proliferating CD4+ T cells were always detected in the GALT (Peyer’s patches and mesenteric lymph nodes). In three of five of these experiments (five of nine mice tested), some proliferating cells were also seen in the peripheral lymph nodes and the spleen (Fig. 3Go). The failure to consistently see proliferating CD4 cells in the peripheral sites on day 2 might be explained if the Ag dose was at a threshold for this to be achieved. Consistent with this view, feeding 20 mg OVA led to proliferation in the GALT only (data not shown). Analysis 6 days after feeding 60 mg OVA revealed divided CD4+ T cells in the GALT and in other peripheral lymphoid organs (data not shown).



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  		FIGURE 3. CD4+ cells are activated in the Peyer’s patches and mesenteric lymph nodes (and sometimes at other sites) 2 days after oral Ag delivery. A total of 3 x 106 CFSE-labeled CD4+ V{alpha}2+ OT-II cells was injected i.v. into B6 mice. The next day, the mice were fed 60 mg OVA or were left unfed. Two days later, the Peyer’s patches, mesenteric lymph nodes, peripheral lymph nodes, and spleen from each mouse were analyzed by flow cytometry. Histograms were gated on CFSE+CD4+ live cells. The percentages indicate the proportion of cells that had proliferated.

 
Various doses of oral OVA can activate T cells in the GALT

To examine the dose of OVA required to activate CD8+ T cells in the GALT, B6 mice were injected with CFSE-labeled OT-I cells and then fed various doses of OVA protein. Flow cytometric analysis of their secondary lymphoid organs on day 3 revealed divided CD8+ T cells in the Peyer’s patches and mesenteric lymph nodes after a range of OVA doses down to as little as 1 mg (Fig. 4Go). It appeared that 6 mg of OVA was the minimum dose that could routinely activate OT-I cells because 1 mg OVA only stimulated proliferation in two of five mice tested.



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  		FIGURE 4. Various doses of oral OVA can activate CD8+ cells in the Peyer’s patches and mesenteric lymph nodes. Approximately 15 x 106 CFSE-labeled CD8+ V{alpha}2+ RAG-1-/- OT-I cells were injected i.v. into B6 mice. The next day, the mice were fed different doses of OVA or were left unfed. Three days later, the Peyer’s patches and mesenteric lymph nodes from each mouse were analyzed by flow cytometry. Histograms were gated on CFSE+CD8+ live cells. The percentages indicate the proportion of cells that had proliferated. For 1 mg OVA, activation of CD8+ cells was only evident in two of the five experiments.

 
The dose response was also examined for OT-II cells. Two days after feeding various doses of OVA protein to B6 mice injected with CFSE-labeled OT-II cells, proliferation was examined in the Peyer’s patches and mesenteric lymph nodes (Fig. 5Go). This showed that 6 mg was also the minimum dose able to cause proliferation of CD4+ T cells.



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  		FIGURE 5. Various doses of oral OVA can activate CD4+ cells in the Peyer’s patches and mesenteric lymph nodes. A total of 7.5 x 106 CFSE-labeled CD4+ V{alpha}2+ OT-II cells was injected i.v. into B6 mice. The next day, the mice were fed different doses of OVA or were left unfed. Two days later, the Peyer’s patches and mesenteric lymph nodes from each mouse were analyzed by flow cytometry. Histograms were gated on CFSE+CD4+ live cells. The percentages indicate the proportion of cells that had proliferated.

 
The APC responsible for inducing oral CTLs is bone marrow-derived

The above results demonstrated that oral OVA can be presented to CD8+ T cells in GALT. To examine whether the APC responsible for CTL priming via the oral route was of bone marrow origin, we took advantage of the fact that bm1 mice, which differ from B6 mice only at the H-2K locus, are unable to present OVA to CD8+ T cells (12). Adult (B6 x bm1)F1 mice were lethally irradiated (900 cGy) and then reconstituted with either B6 or bm1 bone marrow cells. In the B6 -> (B6 x bm1)F1 chimeric mice, Kb is expressed by all cells, whereas bm1 -> (B6 x bm1)F1 chimeric mice express Kb only on non-bone marrow-derived cells. Therefore, proliferation of CD8+ T cells in the B6 -> (B6 x bm1)F1 mice but not in the bm1 -> (B6 x bm1)F1 mice would indicate that a bone marrow-derived APC was involved in the process. After bone marrow reconstitution, the mice were left for 9 wk to allow APC reconstitution. The origin of the APC was then determined by adoptively transferring CFSE-labeled OT-I cells and then feeding 60 mg OVA the next day. Two days after feeding, lymphoid tissues were analyzed by flow cytometry. Analysis of the B6 -> (B6 x bm1)F1 chimeras fed OVA revealed that there was proliferation of the CD8+ T cells in the mesenteric lymph nodes but not in the peripheral lymph nodes and spleen (Fig. 6Go). By contrast, in the bm1 -> (B6 x bm1)F1 chimeras, there was no proliferation of CD8+ T cells in any of the lymphoid tissues examined. Proliferation of CD8+ T cells was not evident in unfed bone marrow chimeras (data not shown). Detection of proliferation in B6 -> (B6 x bm1)F1 chimeras but not in bm1 -> (B6 x bm1)F1 chimeras indicated that a bone marrow-derived APC was required for Ag presentation to CD8+ T cells.



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  		FIGURE 6. The APC responsible for CD8 T cell activation is bone marrow-derived. Adult (B6 x bm1)F1 mice were lethally irradiated and then reconstituted with either B6 or bm1 bone marrow. Nine weeks later, the chimeric mice were injected i.v. with 9.6 x 106 CFSE-labeled CD8+ V{alpha}2+ RAG-1-/- OT-I cells. The next day, the mice were fed 60 mg OVA or were left unfed. Two days later, the mesenteric lymph nodes, peripheral lymph nodes, and spleen from each mouse were analyzed by flow cytometry. Histograms were gated on CFSE+CD8+ live cells. The percentages indicate the proportion of cells that had proliferated.

 
We also investigated whether a bone marrow-derived APC was necessary for the generation of OVA-specific CTL immunity after the oral delivery of OVA. These studies did not use transgenic T cells but examined the CTL response of a normal T cell repertoire. Chimeric (B6 x bm1)F1 mice were fed 60 mg OVA or were left unfed and then analyzed for CTL induction 14 days later (Fig. 7Go). OVA-specific CTLs were only induced in mice when the bone marrow compartment was of B6 origin (Fig. 7Go, A and C). However, as observed for normal mice (4), not all chimeras reconstituted with B6 bone marrow generated CTL. Overall, these data indicate that the APC responsible for the oral priming of OVA-specific CTL immunity is of bone marrow origin.



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  		FIGURE 7. A bone marrow-derived APC is required for the induction of a CTL response by oral Ag. Chimeric (B6 x bm1)F1 mice generated as described in Fig. 6Go were either fed 60 mg OVA, left unfed, or injected i.v. with 25 x 106 irradiated OVA-loaded B6 spleen cells. Fourteen days later, their spleens were removed and stimulated in vitro with OVA-loaded B6 spleen cells for 6 days before they were analyzed for OVA-specific lytic activity. Chimeras reconstituted with B6 bone marrow (A and C). Chimeras reconstituted with bm1 bone marrow (B and D). A and B, Results are shown as a percentage of OVA-specific lysis. C and D, These results are representative of three experiments and are shown as LU/spleen. Each point represents an individual mouse and the bar represents the mean within that group.

 
Although the above data showed that proliferation of CD8+ T cells and CTL induction were dependent on a bone marrow-derived APC, they did not formally prove that such an APC was responsible for CD4+ T cell activation. To address this issue, we examined CD4+ T cell activation in chimeric mice in which the bone marrow compartment did not express class II molecules. This was achieved by injecting class II-deficient bone marrow into lethally irradiated B6 mice. After 4 wk, these mice were injected with CFSE-labeled OT-II cells and then fed 60 mg OVA. Two days later, their lymphoid compartment was examined for proliferating OT-II cells (Fig. 8Go). As indicated, OT-II cells failed to respond to OVA in class II-/- -> B6 chimeras but were activated in the mesenteric lymph nodes of control B6 -> B6 chimeras. This showed that the APC responsible for stimulating OVA-specific CD4+ T cells was bone marrow-derived.



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  		FIGURE 8. The APC responsible for CD4+ T cell activation is bone marrow-derived. Adult B6 mice were lethally irradiated and then reconstituted with either B6 or class II-/- bone marrow. Four weeks later, the chimeric mice were injected i.v. with 3.0 x 106 CFSE-labeled CD4+ V{alpha}2+ OT-II cells. The next day, the mice were fed 60 mg OVA or were left unfed. Two days later, the mesenteric lymph nodes, peripheral lymph nodes, and spleen from each mouse were analyzed by flow cytometry. Histograms were gated on CFSE+CD4+ live cells. The percentages indicate the proportion of cells that had proliferated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously shown that oral administration of OVA is able to generate an OVA-specific, CD4-dependent, CTL response in B6 mice (4). In the current study, we investigated the site of Ag presentation after oral delivery of OVA by examining the proliferation of adoptively transferred, CFSE-labeled, OVA-specific CD8+ or CD4+ T cells. Using this model, we demonstrated that Ag was primarily presented in the Peyer’s patches and mesenteric lymph nodes after feeding and that the cell responsible for presentation to both CD4+ and CD8+ T cells was of bone marrow origin, most likely a dendritic cell or macrophage.

One limitation when using CFSE-labeled cells to detect proliferation is that it is impossible to distinguish between cells that proliferated directly in the tissue examined and those that proliferated somewhere else but then migrated to that tissue. To overcome this problem, we examined proliferation very early after feeding (day 2). This revealed that proliferation occurred primarily in the GALT. Analysis of later time points indicated that divided cells could be found in other peripheral sites. However, whether this proliferation was due to delayed responses occurring in these distant sites or to migration of divided cells after proliferation in the GALT could not be determined. With the highest dose of OVA examined (60 mg), we sometimes observed proliferation of CD4 cells in the spleen and peripheral lymph nodes on day 2. This suggested that orally delivered Ags can be presented in sites other than the GALT but that the efficiency is much reduced. It is of note that i.v. administration of soluble OVA results in proliferation of both OT-I and OT-II cells in all secondary lymphoid tissues (M. Li, F. R. Carbone, and W. R. Heath, unpublished observations), indicating that soluble OVA can be presented in both the class I- and class II-restricted pathways in the spleen and peripheral lymph nodes, provided enough Ag is delivered.

The preferential activation of CD4+ T cells in the Peyer’s patches and mesenteric lymph nodes seen in our study is consistent with a recent report showing activation of DO11.10 transgenic CD4+ T cells in the mesenteric lymph nodes but rarely in the spleen after feeding OVA to BALB/c mice (13). In contrast to these findings, activation of cytochrome c-specific CD4+ T cells was evident in the spleen as well as in the Peyer’s patches and mesenteric lymph nodes within 6 h of feeding 0.5 mg cytochrome c to mice transgenic for the ß-chain of a cytochrome c-specific T cell (14). This latter report used up-regulation of CD69 rather than CFSE-measured proliferation to indicate T cell activation. Thus, perhaps this is a more sensitive measure for activation or, alternatively, cytochrome c may be more easily transported throughout the body. Because all three studies, including our own, have some evidence for Ag presentation in the spleen, it seems clear that orally administered Ag can reach this distant site. However, the preferential activation of cells in the GALT probably reflects more efficient local presentation. How orally delivered Ags reach distant sites such as the spleen is unclear, but there is some evidence that they can be found in the serum 24 h after feeding (14, 15, 16).

Activation of CD8+ T cells within the GALT but not in other sites at day 2 suggested that although Ag could be captured by APCs of the spleen and peripheral lymph nodes for class II-restricted presentation, it could not easily gain access to the class I presentation pathway in these sites. This implied that there is a specialized APC in the GALT that is able to capture exogenous, fed Ag and to process it into the class I presentation pathway for subsequent recognition by CD8+ T cells. It is notable that we found proliferation of CD8+ T cells in all lymphoid sites tested 7 days after feeding OVA. This suggests that CD8+ T cells migrate from the GALT into other peripheral lymphoid sites after activation. However, it is formally possible that specialized APCs capture Ag in the GALT and then migrate to other lymphoid sites and present the Ag to CD8+ T cells. Studies by Liu and MacPherson (17, 18) used mesenteric lymphadenectomy in the rat to show that dendritic cells in the intestinal wall can acquire soluble protein Ag injected directly into the intestinal lumen. Within a few hours, these intestinal dendritic cells migrate into peripheral lymph, carrying the Ag in a form that can prime T cells in vitro as well as in vivo. However, it is unlikely that such migratory cells normally travel further than the mesenteric lymph nodes.

Several distinct potential APCs are present within the GALT. These include different types of conventional APCs like dendritic cells (17, 18, 19, 20, 21, 22, 23, 24, 25), macrophages (26, 27), and B cells (28) along with other putative APCs such as intestinal epithelial cells (29). We have found that the APC responsible for the presentation of oral OVA to both CD4+ and CD8+ T cells in the GALT is of bone marrow origin because T cell proliferation was evident only in chimeric mice in which the bone marrow-derived compartment expressed the correct MHC haplotype (Figs. 6Go and 8Go). Furthermore, a bone marrow-derived APC was required for the generation of an OVA-specific CTL response in the spleen 14 days after oral delivery of OVA protein. Likewise, in other model systems it has been reported that cross-presentation of exogenous Ag involves a bone marrow-derived APC (11, 30, 31, 32). Therefore, our data clearly exclude the possibility that gut epithelial cells or more specialized M cells are responsible for presentation. Although the actual bone marrow-derived APC involved in the presentation of oral OVA remains to be identified, now that the site of Ag presentation is defined, experiments can be designed to classify this APC. Both dendritic cells and macrophages have been implicated in the generation of CTL responses to exogenous Ag in vivo (33, 34, 35, 36, 37, 38, 39).

It has been proposed that, when local Ag concentrations are high, exogenous Ag can enter the class I pathway of APCs by disruption of the phagosomal membrane and entry into the cytoplasm (40). Although we originally used a relatively high dose of OVA to induce CTLs, our finding that feeding 6 mg of OVA is capable of activating CD8+ T cells suggests a more natural route of entry into the class I pathway. Furthermore, evidence that the APC is bone marrow-derived indicates that cells such as epithelial cells responsible for Ag transport from the gut are not forced to present Ag via endosomal damage and release of OVA.

In conclusion, we have shown that oral Ags can be captured by a bone marrow-derived APC capable of efficient processing and presentation into both the class I-restricted and class II-restricted pathways for the activation of CD8+ and CD4+ T cells, respectively, in the GALT. Such activation can lead to CTL activation, even at Ag doses as low as 6 mg.


   Acknowledgments
 
We thank Tatiana Banjanin, Jenny Falso, Freda Karamalis, and Paula Nathan for their technical assistance and Christian Kurts for his helpful discussions.


   Footnotes
 
1 This work was supported by the National Heath and Medical Research Council of Australia. Back

2 Address correspondence and reprint requests to Dr. William R. Heath, Immunology Division, Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Parkville 3050, Victoria, Australia. E-mail address: Back

3 Abbreviations used in this paper: GALT, gut-associated lymphoid tissue; B6, C57BL/6; OT-I, OVA-specific class I-restricted T cell receptor; OT-II, OVA-specific class II-restricted T cell receptor; CFSE, 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester; cGy, centiGrey; MT-RPMI, mouse tonicity-RPM1 1640 medium. Back

Received for publication August 12, 1999. Accepted for publication January 3, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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