Download Cells in Gut-Oriented Immune Responses The Role of Dendritic

The Role of Dendritic Cells, B Cells, and M
Cells in Gut-Oriented Immune Responses
Oral Alpan, Gregory Rudomen and Polly Matzinger
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1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2001 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
J Immunol 2001; 166:4843-4852; ;
doi: 10.4049/jimmunol.166.8.4843
http://www.jimmunol.org/content/166/8/4843
The Role of Dendritic Cells, B Cells, and M Cells in
Gut-Oriented Immune Responses
Oral Alpan,1* Gregory Rudomen,† and Polly Matzinger*
I
mmune responses generated to orally administered Ags have
certain local and systemic features that distinguish them from
other immune responses. For example, secretory IgA, a characteristic local feature of the mucosa, generates a barrier that prevents Ags from attaching to and penetrating the mucosal epithelium and forms immune complexes with Ags within epithelial cells
of the gut lamina propria (1). Immune responses to fed Ags are
also characterized by specific types of local and systemic T cell
responses. Locally, in the Peyer’s patches that line the gut, oral
administration of Ag leads to the induction of Th3 cells that produce TGF-␤ (2), a cytokine that promotes B cell switching to the
production of IgA (3). Systemically, the typical response to fed Ag
is characterized by a decrease in both delayed-type hypersensitivity (DTH)2 and Th cell proliferation upon a subsequent immunization with the same Ag (4). Other characteristics of the systemic
T cell response depend on the dose of fed Ag. High doses can lead
to deletion of the Ag-specific T cells within Peyer’s patches (5, 6),
whereas low doses can lead to a state in which the immune response has switched from the production of a DTH/Th1 response
to the production of Th2/Th3-type responses (7–11) and, in some
cases, the production of IgA (12–15). In addition, T cells from
animals fed low doses can be adoptively transferred to depress the
DTH and proliferative responses of normal naive recipients (16).
In the original host, fed T cells have suppressive bystander effects
on DTH responses to new Ags when given together in an immu-
*Ghost Lab, Section on T-Cell Tolerance and Memory, Laboratory of Cellular and
Molecular Immunology, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and †University Microscopy Imaging Center, University Hospital Medical Center, State University of New York at
Stony Brook, Stony Brook, NY 11794
Received for publication June 22, 2000. Accepted for publication January 23, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. Oral Alpan, Building 4, Room
111, Laboratory of Cellular and Molecular Immunology, National Institute of Allergy
and Infectious Diseases, National Institutes of Health, 9000 Rockville Pike, Bethesda,
MD 20892. E-mail address: [email protected]
2
Abbreviations used in this paper: DTH, delayed-type hypersensitivity; DC, dendritic
cell; EM, electron microscopy; FAE, follicle-associated epithelium; GALT, gut-associated lymphoid tissue; MLN, mesenteric lymph node; PCC, pigeon cytochrome c;
pLN, popliteal lymph node; Tg, transgenic; WT, wild type.
Copyright © 2001 by The American Association of Immunologists
nization with the original fed Ag (17). Both the transferable suppression and the bystander effect have been associated with Th3
cells producing TGF-␤, as well as with Th2 cells that produce IL-4
and IL-10 (18), although subsequent high-dose feeding may
change these characteristics (19). Thus, the response to fed Ag,
whether called oral vaccination (because it produces IgA and protects against pathogens) or oral tolerance (because it reduces systemic DTH responses and ameliorates the symptoms of experimental autoimmune diseases), appears to differ in well-defined
characteristics from responses induced at other sites.
There are several ways in which the intestinal environment
might influence an immune response. First, the cells of the gut
itself produce TGF-␤ (20), vasoactive intestinal peptide (which
controls the secretory piece of IgA) (21), and other immunologically relevant molecules (22). Second, the luminal side of the intestinal epithelium contains a set of specialized Ag-handling cells
called M (microfold) cells (23). These cells overlay intestinal lymphoid follicles and Peyer’s patches (Fig. 1A), sending down long
protrusions that form pockets in which T cells, B cells, and macrophages can be found (24). M cells can take up and transport Ags
such as ferritin (25) and pathogens such as Salmonella (26) and
HIV (27) by active transport. By their Ag handling, or perhaps by
the secretion of specialized cytokines, the M cells might influence
the effector class of local immune responses. Third, in contrast to
other lymph nodes, B cells far outnumber all other lymphoid cells
in the Peyer’s patches (28). They might therefore serve as a specialized set of APCs to induce tolerance or to drive immune responses toward particular effector classes.
To see whether B or M cells were required for the specialized
response to oral Ag, we studied the responses of ␮MT mice, which
contain no B cells due to a targeted mutation in the transmembrane
region of the IgM heavy chain (29) and no M cells or Peyer’s
patches because of the lack of B cells (30). Despite these deficiencies, the ␮MT mice made perfectly normal systemic T cell responses to oral Ag. Aside from a lack of IgA, their in vivo and in
vitro responses were identical to those of wild-type (WT) mice
over a large range of Ag doses. Thus, systemic Th cell responses
to orally administered soluble Ags require neither the specialized
Ag presentation properties of B cells, nor the microenvironment
provided by M cells or Peyer’s patches.
0022-1767/01/$02.00
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Although induction of T cell responses to fed Ag (oral tolerance) is thought to happen within the organized lymphoid tissue of the
gut, we found that mice lacking Peyer’s patches, B cells, and the specialized Ag-handling M cells had no defect in the induction
of T cell responses to fed Ag, whether assayed in vitro by T cell proliferation or cytokine production, or in vivo by delayed-type
hypersensitivity or bystander suppression against mycobacterial Ags in CFA. Feeding of Ag had a major influence on dendritic
cells from fed wild-type or ␮MT mice, such that these APCs were able to elicit a different class of response from naive T cells in
vitro. These results suggest that systemic immune responses to soluble oral Ags do not require an organized gut-associated
lymphoid tissue but are most likely induced by gut-conditioned dendritic cells that function both to initiate the gut-oriented
response and to impart the characteristic features that discriminate it from responses induced parenterally. The Journal of
Immunology, 2001, 166: 4843– 4852.
4844
When we tested the effects of Ag feeding on the dendritic cells
(DCs) of ␮MT and WT mice, we found that naive T cells responded to these fed APCs by proliferating less than to normal
APCs and producing high levels of IL-4 and IL-10. We suggest
that the special features of systemic immune responses to orally
fed Ag are therefore most likely due to characteristics of the professional APCs (DCs) that capture Ags entering through the gut.
Materials and Methods
Mice
We obtained C57BL/10 (B10 WT), C57BL/10 IgM⫺/⫺ ␮MT (B10 ␮MT),
B10.A IgM⫺/⫺ ␮MT (B10.A ␮MT), and B10.A (transgenic (Tg)) TCRCyt-5CC7 RAG2⫺/⫺ (5CC7) mice from Taconic Farms (Germantown,
NY). The mice were housed at the National Institute of Allergy and Infectious Diseases facility, which is fully accredited by the American Association of Laboratory Animal Care. In our colony, ⬍0.01% of the lymph
node T cells from the 5CC7 mice express high levels of CD44, suggesting
that the T cells are naive.
Feeding and immunization
Footpad swelling and DTH responses
Seven-day swelling. We measured footpad thickness 7 days after OVACFA immunization using a dial thickness gauge recorder (Popper and
Sons, New York, NY).
DTH. In mice immunized only at the base of the tail, we injected 50 ␮g
OVA intradermally into the footpad 7 days after the immunization and
measured the footpad thickness 48 h later.
Proliferation assays
Responders. To assay Th proliferation, we purified CD4 T cells from the
draining lymph nodes of mice. For this purpose, we labeled lymph node
cells with anti-B220, anti-CD8, and anti-MHC class II magnetic beads
(Miltenyi Biotec, Auburn, CA) for 15 min at 4°C, washed once, passed the
cells through Midi-MACS columns (Miltenyi Biotec), and collected the
effluent, which consisted of ⬎96% CD4 cells. To prepare 5CC7 responders, we labeled lymph node cells from 5CC7 mice with anti-MHC class II
magnetic beads and collected the effluent. More than 97% of the resultant
cells were T cells.
Stimulators. We depleted T cells from spleens of B10 (WT) mice using
anti-Thy-1.2 (CD90) beads (Miltenyi Biotec) and used the remaining cells
as stimulators in proliferation assays. To purify DCs for the experiments in
Fig. 7, we labeled the MLN or pLN cells with CD11c magnetic beads,
passed them through Midi-MACS columns, and collected the fraction
within the column. The final population consisted of ⬎94% CD11c⫹ DCs.
Proliferation and cytokine-producing cultures. We incubated 2 ⫻ 105
responder CD4 cells with 3 ⫻ 105 irradiated (1500 rad) T-depleted spleen
stimulators in triplicate in 96-well round-bottom microtiter plates, each
well containing a total of 200 ␮l of culture medium (IMDM supplemented
with10% FCS, 50 U/ml penicillin, 50 ␮g/ml streptomycin sulfate, 50
␮g/ml gentamicin sulfate, 4 mM glutamine, and 50 ␮M 2-ME) with or
without graded doses of OVA. The cells remained in culture for 4 days and
were pulsed with [3H]thymidine for the last 8 h. For the experiments shown
in Fig. 7, we incubated 2 ⫻ 104 responder 5CC7 T cells with 1 ⫻ 105
irradiated (1500 rad) enriched DC stimulators. The cells remained in culture for 3 days and were pulsed with [3H]thymidine for the last 8 h.
Cytokine assays
We assayed culture supernatants for IL-4 and IFN-␥ at 48 h using specific
sandwich ELISA (Endogen, Woburn, MA). In the experiments shown in
Fig. 7, we measured IL-4 and IFN-␥ (Endogen) at 72 h. To measure TGF-␤
(Promega, Madison, WI), we cultured cells in vitro for 4 days, washed and
restimulated them in serum-free medium (QBSF 56; Sigma, St. Louis, MO)
with irradiated APCs (T-depleted spleen cells) and Ag, and collected supernatants at 72 h, acid treated them to measure total TGF-␤, and used the
standard ELISA technique to quantify TGF-␤ levels. The results are shown
in pg/ml. We calculated these amounts using standard curves with known
amounts of recombinant cytokines. For intracellular IL-4 staining, we used
the anti-IL-4 Ab 11B11 either unlabeled (to block) or conjugated to allophycocyanin, and the protocol provided by PharMingen (San Diego, CA).
Adoptive transfer experiments
We prepared single-cell suspensions of splenocytes from PBS- or PBS/
OVA-fed (and otherwise unimmunized) WT and ␮MT mice and adoptively transferred them into the peritoneal cavities of unirradiated syngeneic mice (1 ⫻ 108 WT cells into WT mice and 3 ⫻ 107 ␮MT cells into
␮MT mice, which equals about one spleen equivalent each). One day after
the adoptive transfer, we immunized the recipient mice at the base of the
tail and one footpad with CFA-OVA, as described above.
Histology and electron microscopy (EM)
Histology. We fixed the entire intestinal tissue from WT and ␮MT mice
in 4% paraformaldehyde and then embedded it in paraffin. Thin sections (6
␮m) from these paraffin blocks were than stained with hematoxylin and
eosin.
M cell staining. We incubated unstained thin sections with biotinylated
Ulex europaeus 1 (UEA 1) for 60 min at room temperature. We then
washed the tissue sections in PBS and incubated them for 30 min with the
avidin-biotin complex (ABC) linked to HRP (Vectastain Elite ABC kit;
Vector Laboratories, Burlingame, CA), prepared according to the manufacturer’s instructions. After a 5-min wash, tissue sections were incubated
with 0.05% (w/v) 3,3-diaminobenzidine (Sigma), 0.05% NiCl, and 0.03%
H2O2, and examined under a light microscope.
EM. Intestinal tissue was fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in PBS (pH 7.3). After washing, the tissue was postfixed in 2%
OsO4, dehydrated in graded series of ethanol, and embedded in Spurr’s
resin blocks. From these blocks, 70-nm ultrathin sections were cut. The
sections were then mounted on 200-mesh copper grids and stained with
uranyl acetate and Reynolds lead citrate. The sections were examined with
a JEOL 1200EX at 80 kV.
Statistics
The two-tailed Student t test was used.
Results
Differences in intestinal architecture between ␮MT
and WT mice
Because orally administered Ag comes into contact with several
cell types that are not available to parenterally administered Ag,
we asked whether such cells might be involved in setting the
unique characteristics of the response to oral Ag. First, a large
majority of the cells found in the Peyer’s patches that line the gut
are B cells, known to be semiprofessional APCs that do not stimulate, but instead tolerize naive T cells (31–33). A second of these
structures peculiar to the gut are M cells, a specialized set of epithelial cells that are found throughout the intestine that are especially concentrated in the epithelial layer overlying the follicles of
Peyer’s patches (named follicle-associated epithelium or FAE),
where they can make up to 50% of the epithelial lining, whereas in
other parts of the gut, such as villus epithelium, their frequency can
be as low as 1% (23). They are easily recognized by a set of
features such as short microvilli, basally oriented nuclei, abundant
mitochondria, and long cytoplasmic extensions into the lamina
propria that can enfold several leukocytes (Fig. 1A) (34). Ultrastructural studies have shown that M cells contain IgA-immune
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We chose OVA for feeding because it is the Ag most commonly used in
oral tolerance experiments and because OVA feeding protocols are well
established. We fed mice, using a 20-gauge blunt-ended animal feeding
needle, either PBS or low (0.1 mg) or high (10 mg) doses of OVA every
other day for five feeds. In the experiment shown in Fig. 7, we added OVA
to the drinking water at concentrations of 0.02, 0.2, and 2 mg/ml, resulting
in ingestion of 0.1, 1, and 10 mg OVA/day/mouse, respectively (our mice
drank ⬃5.2 ml/day, calculated from a decrease of 26 ml of water over 24 h
in a cage of five mice). Two days after the last OVA feeding (or drinking),
we immunized the mice with 50 ␮g OVA emulsified in CFA in a total of
50 ␮l, divided equally between the base of the tail and one footpad. For
DTH experiments, we immunized only at the base of the tail (Fig. 3A).
When assaying the number of cells in draining nodes, we immunized the
mice only in the footpad (Fig. 3B). In the experiments shown in Fig. 7, we
added pigeon cytochrome c (PCC) to the drinking water of B10.A or
B10.A ␮MT mice at a concentration of 0.02 mg/ml and harvested the
draining mesenteric lymph nodes (MLNs) on day 4, or injected 0.1 mg
PCC into the right footpad and harvested the popliteal lymph nodes (pLNs)
on day 4.
GUT-ORIENTED IMMUNE RESPONSES
The Journal of Immunology
4845
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FIGURE 1. Comparison of M cells in WT and uMT mice. A, A schematic of M cells within the FAE. B, Comparison of M cells in WT and ␮MT mice.
Magnifications are shown to the right of each lane.
4846
FIGURE 2. The effect of feeding OVA is similar
in WT and ␮MT mice over a wide dose range. Mice
were fed either PBS (dashed lines) or graded doses
of OVA (solid lines), immunized with OVA-CFA
into the footpad and the base of the tail 2 days after
the last feeding, and tested for T cell proliferation
and cytokine production 7 days later. A, CD4 T cell
proliferation 7 days after immunization. Each line
represents one mouse. Doses of fed OVA are represented as circles with increasing sizes. Each circle
corresponds to the proliferation curve to its left. B,
Supernatants from wells in A containing 100 ␮g/ml
OVA were assayed for IFN-␥ and IL-4 at 48 h. Each
symbol represents one mouse.
The in vitro Th cell response in WT and ␮MT mice is similar
over a wide range of feeding doses
Because a decrease in systemic Th cell proliferation is a standard
characteristic of the response to immunization with a previously
fed Ag (40), we started our analysis by examining Th cell proliferation after feeding Ag. To take into account potential differences
in Ag handling that might occur in mice with and without B cells,
M cells, and Peyer’s patches, we tested a dose titration in vivo,
feeding amounts of OVA that differed over a 10,000-fold range
(from 0.001 to 10 mg), every other day for a total of five feeds,
immunized the mice 2 days after the last feeding with 50 ␮g of
OVA in CFA, and harvested the draining lymph nodes 7 days later
to test for OVA responsiveness in vitro. Surprisingly, the WT and
␮MT mice responded in the same way, as assayed both by proliferation of CD4 cells (Fig. 2A) and by cytokine production (Fig.
2B). As we increased the dose of fed OVA, the in vitro Th cell
proliferation to OVA decreased in a graded manner; the higher the
dose, the less the proliferation. We found similar results for IFN-␥
and opposite results for IL-4, in which feeding resulted in a
dose-dependent increase in IL-4 levels in both WT and ␮MT
mice (Fig. 2B).
DTH responses are similarly reduced in fed WT and ␮MT mice
From the data above, we concluded that the systemic effects of oral
immunization, as measured by in vitro tests, were induced with
equal efficacy in the presence or absence of B cells, M cells, and
Peyer’s patches. We next asked whether we could find a difference
using in vivo functions.
We first looked at DTH responses in mice given the same feeding and immunization protocol as described in Fig. 2, except that
they were immunized only at the base of the tail. Seven days after
immunization, we tested DTH responses by injecting OVA in PBS
intradermally into the footpad and measuring the footpad thickness
48 h later. Fig. 3A, representing a summary of two different experiments, shows that both WT and ␮MT mice responded to feeding by producing smaller DTH responses than PBS-fed controls. In
this assay, as in the in vitro tests, we found no significant difference
between the two types of mice.
Because a decreased DTH response may reflect decreased cellular infiltration and/or proliferation in vivo, we asked whether we
could see the same picture in the lymph nodes draining the immunization site. We fed WT and ␮MT mice either PBS or low (0.1
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complexes, suggesting that they may be involved in Ag transport
(35). Although the direction of transport cannot be known from
these static pictures, M cells are unlikely to secrete IgA into the
intestinal lumen because, unlike other intestinal epithelial cells,
they do not have receptors for polymeric Ig (36). A hint that the
transport of these Ag-Ab complexes might be from the lumen to
the lymphoid follicle comes from studies showing that M cells can
carry ferritin (24), liposomes (37), and latex beads (38) from the
gut lumen and that several kinds of bacteria and viruses (reovirus,
HIV) use M cells as entry points into the body from the gut (24).
Using transmission and scanning EM, Golovkina et al. (30) recently showed that M cells are absent in mice that lack B cells. Fig.
1 shows the typical patterns seen by EM in WT and ␮MT mice.
Fig. 1B shows that the guts of WT mice contain Peyer’s patches
covered with smooth FAE, rather than the villus epithelium found
in the rest of the gut. In contrast, the ␮MT mice do not have any
Peyer’s patches, but only a few, rare, lymphoid aggregates that do
not have significant FAE. In EM photographs of the FAE of WT
mice, one can easily detect M cells both in the FAE and within
villus epithelium. Fig. 1C, for example, depicts M cells within the
FAE of WT mice in contact with a lymphocyte. Among the aggregates in the ␮MT mice, however, we could detect no M cells by
EM, in agreement with the results reported by Golovkina et al. (30).
Searching for M cells using EM, however, has two problems.
First, because each field covers such a small area, it does not give
information on the overall frequency of these cells. Second, EM
only identifies M cells based on their structural properties and may
miss M cells that look structurally very similar to intestinal epithelial cells. To determine whether M cells are involved in the
response to oral Ag, we needed to be sure that the ␮MT mice had
no cryptic M cells that might have a different shape due to the lack
of interactions with B cells. We therefore used a method to search
for M cells, using the lectin U. europaeus 1 (UEA 1) to stain for
␣-L-fucose, which is expressed on the surface of M cells, but not
the epithelial cells of the gut (39). By this technique, we also found
no evidence for M cells in the ␮MT gut (Fig. 1D).
Thus, it appears that ␮MT mice lack both M cells and Peyer’s
patches, enabling us to ask whether their absence would have any
effect on systemic responses to Ags entering through the gut. For
this purpose, we undertook an extensive series of in vitro and in
vivo experiments to compare the effects of OVA feeding in WT
and ␮MT mice, focusing on the well-documented characteristics
of the systemic T cell response to oral Ag.
GUT-ORIENTED IMMUNE RESPONSES
The Journal of Immunology
4847
FIGURE 3. DTH response and draining lymph
node cell counts in fed WT and ␮MT mice. A, Mice
were fed either PBS or low- or high-dose OVA and
immunized only at the base of the tail. Seven days
after immunization, we injected 50 ␮g OVA in PBS
intradermally into the left footpad and measured
footpad thickness at 48 h. Each mouse is represented
by two symbols, filled for the injected left foot and
unfilled for the uninjected right foot. Different types
of symbols represent separate experiments. B, Mice
were fed as described in Fig. 4A, but immunized
only in the footpad. Seven days later, pLNs draining
the footpads were harvested, and total number of
living cells were counted. Symbols as in A, but representing left and right popliteal nodes. Short horizontal line to the left of each group ⫽ average. Statistical significance of difference between control
(PBS fed) and experimental (OVA fed): ⴱ, p ⬍
0.0001.
Bystander effects of T cells from fed mice
Having found that WT and ␮MT mice showed no differences in
their direct responses to fed Ag, we next asked about their ability
to mediate the bystander effects that have classically been associated with oral tolerance. There are two types of situations in which
fed cells have been shown to have an influence on other T cells.
First, in adoptive transfer systems, T cells from fed animals can
have an Ag-specific suppressive effect on the DTH, proliferation
(10), and cytokine (40) responses of naive host T cells. Second, in
the original host, orally immunized T cells can affect the naive
systemic T cell response against new Ags, as long as the immunizing mixture also contains the fed Ag (10). We compared both of
these bystander effects in WT and ␮MT mice.
Bystander effect on naive T cells against the same Ag. Earlier
studies show that the systemic effect of fed T cells on naive T cells
is dose dependent. It is mediated only by T cells in animals that
have been fed low doses of Ag. High doses induce deletion and
abrogate both the direct response and the bystander suppression to
the fed Ag (5, 6). To compare the Ag-specific bystander effect of
WT and ␮MT mice, we therefore harvested spleen cells from low
(0.1 mg) and high (10 mg) dose OVA-fed (five times every other
day) animals and transferred them to the peritoneal cavities of
unirradiated syngeneic hosts (WT spleen cells into WT hosts and
FIGURE 4. Bystander effect on naive cells to the fed Ag. Mice were fed either low or high doses of OVA or PBS. Two days after the last feeding,
splenocytes were harvested and adoptively transferred into the peritoneal cavity of naive hosts (fed WT to naive WT and fed ␮MT to naive ␮MT) and
immunized the next day. CD4 T cell proliferation 7 days after immunization. Supernatants from wells containing 100 ␮g/ml OVA were assayed for IFN-␥
and IL-4 at 48 h. Short horizontal line to left of each group ⫽ average. Statistical significance of difference between control (PBS fed) and experimental
(OVA fed): ⴱⴱ, p ⬍ 0.005; ⴱ, p ⬍ 0.02.
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mg) or high (10 mg) dose OVA, immunized at the footpad, and
counted the number of cells in the pLNs 7 days after immunization. Fig. 3B shows that both WT and ␮MT mice that had been fed
OVA responded to OVA-CFA immunization with a decreased cellular infiltration of the draining pLNs.
4848
GUT-ORIENTED IMMUNE RESPONSES
FIGURE 5. Bystander effect on the response
of naive cells to a new Ag, in this case mycobacteria in adjuvant. A, Mice were fed either OVA or
PBS and immunized with either PBS, PBS-CFA,
or OVA-CFA in the left footpad. Footpad swelling was measured 7 days later. B, Mice were fed
either low (0.1 mg) or high (10 mg) doses of OVA
or PBS. Two days after the last feeding, splenocytes were harvested and adoptively transferred
into the peritoneal cavity of naive hosts (fed WT
to naive WT and fed ␮MT to naive ␮MT) and
immunized the next day. Different types of symbols represent different experiments. Short horizontal line to the left of each group ⫽ average.
Statistical significance of difference between control mice (fed PBS and injected with OVA-CFA)
and experimental mice (fed OVA and immunized
with OVA-CFA): ⴱⴱ, p ⬍ 0.0001; ⴱ, p ⬍ 0.01.
Spleen cells from low dose fed animals had a strong effect on their
adoptive hosts, whereas spleen cells from high-dose fed mice were
only minimally suppressive. Fig. 4 shows that the effects were
evident in both the proliferation and cytokine assays
FIGURE 6. The bystander effect occurs in mice that drink OVA. A, Mice were fed either by a feeding needle once every other day for five feeds or by
adding OVA to their drinking water for 8 days (refreshing the bottles at day 4 and changing to Ag-free water at day 9). OVA was added to the water at
concentrations of 0.02, 0.2, and 2 mg/ml. Because the mice drank an average of 5.2 ml/mouse/day, this results in doses of 0.1, 1, and 10 mg/mouse/day
of OVA. Two days after the last feeding, mice were immunized with OVA in CFA in the left footpad. Footpad swelling was measured 7 days later. Different
types of symbols represent different experiments. B, Mice were fed 0.1 mg OVA, through the drinking water, and immunized. Draining lymph node cells
were harvested and cultured in vitro with 100 ␮g/ml OVA. For IL-4, supernatants were assayed at day 4. For TGF-␤, after 4 days in culture, cells were
washed and restimulated with APC and OVA in serum-free medium, and supernatants were assayed at day 3. Statistical significance of difference betwen
control (water fed) and experimental (OVA fed): ⴱ, p ⬍ 0.0001.
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␮MT spleen cells into ␮MT hosts). One day after the transfer, we
immunized the recipients with OVA in CFA and assayed for in
vitro Th cell proliferation 7 days later. We found that the fed WT
and ␮MT spleen cells behaved identically and characteristically.
The Journal of Immunology
4849
after injection of OVA in CFA. Spleen cells from low-dose fed
animals induced approximately a 100-fold decrease in the titrated
proliferative response to Ag, a 10- to 20-fold decrease in IFN-␥
production and an 8- to 10-fold increase in IL-4. In contrast, spleen
cells from animals that were fed high doses of OVA (10 mg) had
only a slight effect when compared with spleen cells from PBS or
low-dose fed donors. Here again, transfer of low-dose fed cells
was the most effective, and ␮MT mice behaved similarly to WT.
Bystander effect on naive T cells against new Ags that are given
concomitantly with the fed Ag. To compare the bystander effect
against new Ags, we looked at footpad swelling 7 days after immunizing with OVA in CFA, asking whether a shift in response to
a fed Ag can affect the strong primary response normally seen to
whole mycobacteria emulsified in oil. Fig. 5A shows that the injected feet of naive mice fed only PBS swelled to about twice their
size after injection of PBS-CFA and that prior OVA feeding had
no effect. In contrast, prior OVA feeding markedly reduced the
response if the OVA was also given along with the CFA injection.
This bystander effect could also be adoptively transferred. Splenocytes from mice fed OVA, when transferred into naive mice, were
able to suppress footpad swelling induced by immunization with
OVA-CFA (Fig. 5B). The effect waned, as would be expected
because of the deletional effects, at higher doses of OVA. In both
of these types of bystander assays in vivo, as we had previously
seen in all other tests, the ␮MT mice behaved identically to the
WT mice.
The bystander effect occurs in mice that drink OVA
A possible complication resulting from Ag administration using a
feeding needle is accidental abrasion to the esophagus, which may
lead to spillage of Ag into the blood. There have been reports that
i.v. administration of Ag can lead to responses with characteristics
similar to those seen with low-dose oral administration, such as a
decrease in DTH (41, 42), proliferation, and cytokine production
(43) concomitant with an increase in bystander suppression (39)
and, occasionally, production of secretory IgA (42). It has also
been reported that a large bolus of fed OVA can lead to low levels
of blood-borne OVA (44). To determine whether the T cell response and the bystander suppression we had seen after feeding an
Ag were due to abrasion-induced systemic spill, we fed titrated
amounts of OVA to both WT and ␮MT mice by adding it into their
drinking water and then compared their footpad-swelling responses with those of animals fed using a feeding needle. Fig. 6A
shows that both groups responded the same way, and that there
was little difference between WT and ␮MT mice. Furthermore,
both WT and ␮MT Th cells from mice that drank OVA made high
levels of TGF-␤ and IL-4 in vitro compared with unfed controls
(Fig. 6B).
Th cell response induced by APCs from fed ␮MT mice
The finding that there were no discernible differences between WT
and ␮MT mice suggested that the characteristic properties of the
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FIGURE 7. Th cell response induced by DCs
from fed B10.A WT and ␮MT mice. Mice were
either injected with PCC in one rear footpad (inj.)
or fed PCC for 4 days by adding it into the feeding
water (fed). On day 4, MLNs of fed mice and unfed
controls, and draining pLNs of injected mice and
uninjected (un-inj) controls were harvested, and
CD11c⫹ DCs were purified, irradiated, and cultured together with 5CC7 T cells in vitro. A, Cytokine production. This figure is a summary of two
different experiments in which the cells were cultured either with (F) or without (E) 10 ␮g/ml PCC
in vitro. Each circle represents data from one experiment. B, Tg TCR V␣11.1 naive 5CC7 cells are
the source of IL-4 in experiments in which these
cells are cultured with fed MLN DCs. The staining
is specific for IL-4 since it can be blocked by the
unlabeled ␣IL-4 Ab, 11B11, during staining.
4850
Discussion
This study demonstrates two aspects of immunity to Ags that enter
through the gut. The first relates to the systemic immune response,
showing that the absence of GALT and the consequent inability to
coordinate the local gut-oriented immune response have no effect
on the characteristic systemic effects of orally administered Ag.
The second relates to the APCs involved in gut-oriented immunity
and shows that APCs from the MLNs of Ag-fed mice are able to
elicit copious amounts of IL-4 from naive T cells, a surprising
finding in light of the fact that naive T cells are thought to be able
to produce only IL-2 and not IL-4. We will discuss both of these
aspects separately.
B cells, M cells, and Peyer’s patches are not necessary for the
characteristic systemic response to orally administered Ags
The immune response to fed Ags has both local and systemic components. To determine whether the GALT is involved in the systemic response, we analyzed the response of ␮MT mice to orally
administered OVA. We had previously reported that parenterally
immunized systemic T cell responses seem to be normal in ␮MT
mice, showing that neither B cells nor M cells are necessary for in
vitro cytokine production or proliferation, the in vivo production of
granulomas against parasite eggs, the rejection of skin grafts, priming for CTL function, or the maintenance of CTL memory (46 –
48). In effect, we found that ␮MT T cells respond normally to
several different types of Ags, given by several different routes. We
had not, however, tested responses to oral Ag, and it could be
argued that the lack of B cells, M cells, and Peyer’s patches might
have a serious effect on responses to Ags that enter through the gut.
We therefore undertook an extensive series of tests to determine
whether the lack of B cells, M cells, and Peyer’s patches would
have any effect on the systemic T cell response to fed Ags. We
found that all of the well-described features of oral tolerance were
intact. Whether tested in DTH or 7-day footpad-swelling tests in
vivo or by proliferation and cytokine production in vitro, over a
10,000-fold dose range, the T cells from fed WT and ␮MT mice
behaved identically, both in their own responses to the fed Ag and
in their ability to influence the responses of naive T cells to the fed
Ag and to bystander Ags. From these data, we conclude that neither the lack of B cells nor the absence of M cells or Peyer’s
patches had any measurable effect on the systemic T cell response
to fed Ag.
Because feeding by gavage may cause damage to the esophagus,
and because the systemic effects of oral tolerance are often similar
to those generated by i.v. administration of Ag, we considered the
possibility that systemic blood-borne Ag, rather than the GALT
response, was the basis of the feeding effect. The lack of an organized GALT would then be expected to have little influence. We
found, however, that the immune response to fed Ag is the same
whether the Ag is fed by gavage or delivered in the water, suggesting that Ag delivery without trauma, and thus without major
overt spillage into the blood, also results in the same effect. In fact,
we found (Fig. 6) that Ag delivered daily through the drinking
water seemed to be slightly more effective than Ag given every 2
days by gavage. As the final doses were the same, this difference
may be due to the increased frequency with which Ag was delivered. One might even envisage the contrasting scenario that the
effect of i.v. administration of Ag is actually due to seepage of Ag
into the gut environment and activation of a gut-oriented immune
response. In either case, however, whether the skewing of the response is due to leakage out of the gut or into it, it appears that
neither B cells, M cells, or Peyer’s patches are necessary.
These data are in line with the earlier studies showing that B
cells are semiprofessional (49), rather than professional APCs
(31), that are able to stimulate memory, but not naive T cells (32,
33, 50, 51). They also eliminate one role for M cells, leaving open
the possibility that, contrary to all of the structural evidence (52),
M cells may have no role in initiating T cell immunity to soluble
Ags. M cells and Peyers patches may only be involved in local gut
responses to particulate Ags and microorganisms, leaving other
cells to deal with the systemic response to fed Ag.
DCs from Ag-fed animals are educated to induce a primary IL-4
response and high levels of TGF-␤
If B cells, M cells, and Peyer’s patches are not necessary for the
systemic response to Ags entering through the gut, then what initiates the response?
Gut epithelial cells are unlikely candidates. Although these express MHC class II constitutively (53), they are not professional
APCs (54) and should therefore induce deletion rather than activation of T cells. They are thus apt to be involved in the Agspecific deletion of T cells that occurs after high-dose administration of oral Ag. This leaves us with macrophages and DCs. The
observation that oral tolerance is enhanced by in vivo treatment
with Flt3 ligand (which expands the number of DCs) (55) supports
the view that, as with other responses, the immune response to oral
Ags is most likely initiated by DCs.
But the initiation of the response is not the whole story. Gutoriented immune responses are characterized by a particular class
of effector function such as IgA (56, 57), TGF-␤ (4), IL-4, and
IL-10 (58), which are known alternatively as oral tolerance (4) or
immune deviation (8). What gives rise to these qualities?
We would like first to suggest that the gut-oriented immune
response is neither tolerance nor deviation. It is simply an immune
response tailored to the environment of the gut, which has specific
immunological needs (the production of IgA) as well as certain
sensitivities (it can be harmed by a DTH response (59 – 62)). In
these respects, it is similar to the eye (63). To protect itself and to
ensure that a local immune response is nevertheless effective, the
gut suppresses DTH responses (61) and enhances IgA production
(20, 21). It appears that it may not do this through specialized Ag
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systemic response to fed Ag might be established by an APC common to both strains, e.g., the DCs that drain from the gut to the
mesenteric nodes. We therefore fed PCC to B10.A WT and ␮MT
mice by adding it into their drinking water, harvested the MLNs on
day 4, and purified the CD11c⫹ DCs by positive selection using
magnetic columns. We then irradiated these DCs and used them as
APCs in vitro for a monoclonal population of naive TCR Tg T
cells specific for PCC (5CC7 TCR Tg bred onto a B10.A
RAG2⫺/⫺ background) and compared them to APCs from the
MLNs of unfed mice or from pLNs from mice immunized in the
footpads to PCC or from control mice. Fig. 7A shows that MLN
DCs from the fed mice induced the naive T cells to make higher
levels of IL-4 and IL-10 and less IFN-␥ than did APCs from control mice or APCs draining a peripheral site injected with PCC. In
contrast, APCs from the pLNs of mice injected at the footpad with
PCC induced strong IFN-␥ production, low level IL-10 production, and no IL-4. To confirm that the IL-4 measured by ELISA
was being made by the naive Tg T cells rather than the DCs themselves, we stained the in vitro cultured T cells for intracellular
IL-4. Fig. 7B shows that, after 3 days in culture, when stimulated
with fed DC and Ag, the TCR V␣11.1-positive Th against PCC are
themselves staining for intracellular IL-4. This is a striking result,
as it demonstrates for the first time that naive T cells can make
large quantities of IL-4 in vitro, without a requirement for restimulation (45), if they receive appropriate stimuli (e.g., DCs from the
MLNs of Ag-fed mice).
GUT-ORIENTED IMMUNE RESPONSES
The Journal of Immunology
Acknowledgments
We are indebted to T. Kamala for her ideas, suggestions, critiques, and help
throughout the study and the preparation of the manuscript. We thank Ron
Schwartz, Albert Bendelac, Bana Jabri, Warren Strober, and David Volkman for reading and improving the manuscript, and Ron Germain for suggesting the “drinking” experiment. We also thank Zeynep Melis Alpan for
all of her inspirations.
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GUT-ORIENTED IMMUNE RESPONSES