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*
Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia; and
Department of Pathology and Immunology, Monash Medical School, Prahran, Victoria, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1
). By day 7, CD8+
T cells that had undergone proliferation were present in all lymphoid
organs (Fig. 2). 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. 1 and Fig. 2).
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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. 3). 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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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. 4). 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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). This showed that 6 mg was also the
minimum dose able to cause proliferation of CD4+
T cells.
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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. 6). 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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). OVA-specific CTLs were only induced
in mice when the bone marrow compartment was of B6 origin (Fig. 7, 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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). 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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 6
and 8).
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.
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Acknowledgments |
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Footnotes |
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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:
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.
Received for publication August 12, 1999. Accepted for publication January 3, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and ß-genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 76:34.[Medline]
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