| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Institut National de la Santé et de la Recherche Médicale Unité 404, Lyon, France; and
Institut National de la Santé et de la Recherche Médicale Unité 503, Immunodermatology, Faculté Laennec, Lyon, France
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-producing CD8+ T cells, and inability of
CD8+ T cells to transfer CS in RAG20/0 mice.
RT-PCR and immunohistochemical analysis confirmed that recruitment of
CD8+ effectors of CS in the skin at the site of hapten
challenge was impaired in orally tolerized mice. Sequential
anti-CD4 Ab treatment showed that only depletion of
CD4+ T cells during the afferent phase of CS abrogated oral
tolerance induction by restoring high numbers of specific
CD8+ effectors in lymphoid organs, whereas CD4 depletion
during the efferent phase of CS did not affect oral tolerance. These
data demonstrate that a single intragastric administration of hapten
can block in vivo induction of DNFB-specific CD8+ CTL
responsible for tissue inflammation and that a subset of regulatory
CD4+ T cells mediate oral tolerance by inhibiting expansion
of specific CD8+ effectors in lymph
nodes. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Numerous studies in rodents have documented that oral tolerance is an efficient mean to inhibit autoimmune diseases such as experimental autoimmune encephalomyelitis, arthritis, experimental autoimmune uveitis, or diabetes (2, 3, 4, 5) and DTH reactions to exogenous protein Ags (6), all mediated by Ag-specific Th1-type CD4+ T cells. More recently, intragastric administration of protein allergens was reported to prevent differentiation of Th2-type CD4+ T cells and to inhibit specific IgE production (7, 8). In these experimental models, the immune mechanisms underlying oral tolerance have been reported to include either anergy/deletion of Ag-specific effector T cells or bystander suppression through TGF-ß/IL-10 production by regulatory T cells, at high or low dose of Ag, respectively. However, little is known about the ability of oral tolerance to prevent inflammatory diseases mediated by CD8+ effector T cells.
We have recently documented that contact sensitivity (CS), a DTH reaction induced by epicutaneous application of haptens, is mediated by hapten-specific class I-restricted CD8+ cytotoxic T cells, which initiate cellular infiltration and the development of skin lesions (9, 10). In humans, the CS response secondary to skin exposure to metals or chemicals (i.e., haptens) in previously sensitized hosts, is the most frequent inflammatory dermatosis, also known as contact dermatitis (reviewed in Ref. 11).
Haptens are low m.w. chemicals, which become immunogenic after binding to discrete amino acids of self proteins (12) and are presented as modified peptides by MHC class I and class II molecules to CD8+ and CD4+ T cells, respectively (13, 14). The afferent phase of the CS response is initiated by hapten capture by epidermal Langerhans cells that migrate through afferent lymph to the paracortical zone of draining lymph nodes and present hapten/MHC complexes to naive T cells (15, 16). Expansion of hapten-specific T cells, which peaks at days 4–5 after sensitization is followed by T cell emigration through efferent lymph and seeding to peripheral tissues through the blood flow. The efferent phase is characterized by the recruitment of hapten-specific T cells into the skin at the site of hapten challenge and results within 24–48 h in skin infiltration with inflammatory cells responsible for the edematous lesion (reviewed in Ref. 17).
We have previously reported that a single intragastric administration of DNFB, before epicutaneous sensitization of mice with the same hapten, prevents the CS response to DNFB (18). In this study, we show that oral tolerance prevents the induction of the skin inflammatory response by inhibiting in vivo expansion of DNFB-specific CD8+ effector T cells in secondary lymphoid organs and requires the presence of regulatory CD4+ T cells during the afferent phase of CS.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
All mice were used at 7 to 10 wk of age and were on a C57BL/6 background (H-2b). Female C57BL/6 mice were purchased from Iffa Credo (l’Arbresle, France). Female RAG2 knockout mice (RAG20/0) were purchased from the CDTA (Orleans, France).
Haptens
2,4-Dinitrofluorobenzene (DNFB) and oxazolone (OXA) were used for in vivo studies and 2,4-dinitrobenzenesulfonate (DNBS) was used for in vitro studies (all reagents were from Sigma, St. Quentin Fallavier, France).
Contact sensitivity assay
CS to DNFB or OXA were determined by the mouse ear swelling test (19). Briefly, mice were sensitized epicutaneously on day 0 by application of 25 µl of either 0.5% DNFB or 2% OXA diluted in acetone-olive oil (4:1, v/v) onto 2 cm2 of shaved abdominal skin. Mice were challenged on day 5, with 4 µl of a nonirritant concentration of either 0.2% DNFB or 0.4% OXA, applied onto each side of the right ear. The left ear received the vehicle alone. Ear thickness was measured using a caliper (J15 Blet, Lyon, France) before and at various time after challenge. The ear swelling (micrometers) was calculated as (T - T0 of the right ear) - (T - T0 of the left ear), where T and T0 represent the values of ear thickness after and before challenge, respectively.
Induction of oral tolerance
Seven days before skin sensitization with DNFB, mice received a single intragastric administration of 300 µl of either 0.1% DNFB or 1% OXA in acetone-olive oil (1:10, v/v), or 300 µl of the vehicle alone as control, as previously reported (18).
Anti-CD4 mAb treatment
Mice received i.p. injections of 200 µl anti-CD4 mAb (GK1.5) (20) as 1/10 dilution of ascites fluid, either on days -1 and +4 of DNFB sensitization or on day +4 of sensitization. Depletion of CD4+ T cells was checked by direct immunofluorescence and FACS analysis using an anti-CD4-PE mAb (PharMingen, San Diego, CA).
Hapten-specific T cell proliferation in vitro
Spleen cells were harvested five days after epicutaneous DNFB
sensitization. T cells were purified by negative selection using
anti-Ig columns (Cedarlane, Tebu, France) as described
(21). The resulting cell suspensions contained >90%
viable CD3+ T cells, including 
35%
CD8+ T cells. CD8+ T cells
were isolated from spleen by depletion of CD4+ T
using columns coated with a goat anti-mouse Ig and a goat
anti-rat IgG and a rat anti-mouse CD4 mAb (YTS191.1) (Biotex,
Edmonton, Alberta, Canada). FACS analysis showed <0.5%
CD4+ T cells. Unfractionated T cells or
CD8+ T cells (2.5 x
105/well) purified from the spleen on day 5 after
epicutaneous sensitization, were cocultured for 3 days at 37°C in
96-well round-bottom plates with mitomycin C-treated syngeneic spleen
cells (106/well) from naive C57BL/6 mice,
previously derivatized with DNBS as described (18).
Briefly, cells were incubated for 20 min at 37°C with 4 mM DNBS, pH
8, in serum-free RPMI and washed in complete medium before use. The
proliferative response was assessed on day 3 of culture by
[3H]thymidine incorporation (1 µCi/well)
during the last 6 h of culture. The cultures were harvested and
the amount of [3H]thymidine incorporation was
counted in a ß-plate liquid scintillation counter. The results are
expressed as 
cpm ± SD, where cpm = (cpm in cultures of
T cells with DNBS-treated spleen cells) - (cpm in cultures of T
cells with untreated spleen cells).
Adoptive transfer of CD8+ T cells in RAG20/0 mice
Unfractionated T cells or purified CD8+ T cells were isolated using anti-Ig columns, as described above, from the spleen of DNFB-fed or vehicle-fed mice, on day 5 after sensitization. Cells were then resuspended at a concentration of 5 x 106 cells/100 µl PBS and transferred i.v. through the tail vein into female syngeneic RAG20/0 mice. Mice were challenged with DNFB 2 h after cell transfer, and CS was measured by ear swelling 24 h after challenge, as described above.
Hapten-specific CTL assay
Spleens recovered from DNFB-fed or vehicle-fed mice on day 5 after epicutaneous DNFB sensitization were tested for hapten-specific CTL activity as previously described (22). Briefly, spleen cells were restimulated in vitro with mitomycin C-treated DNBS-derivatized syngeneic spleen cells, at a 1:1 ratio. Viable cells recovered on day 5 were then assayed for cytolytic activity against either untreated or DNBS-derivatized EL-4 targets using a 4-h 51Cr release assay. The targets were simultaneously haptenated and chromium labeled by incubating for 1 h at 37°C with periodic mixing, 2 x 106 cells in 100 µl RPMI supplemented with 4 mM DNBS and 100 µCi Na251CrO4 (sodium chromate, 1 Ci/mmol). In some experiments, CD8+ T cells were depleted just before the CTL assay using the rat anti-CD8 YTS 169.4 mAb supernatant and anti-rat Ab-coated magnetic beads (Dynal, Oslo, Norway). Log dilutions of effector cells were plated in round-bottom microculture plates with 104 51Cr-labeled EL-4 targets. The plates were then incubated at 37°C for 4 h, and the radioactivity released in the supernatant was counted using a gamma counter. The results are expressed as percent specific lysis ± SD calculated as follows: (cpm test - spontaneous cpm)/(maximal cpm - spontaneous cpm) x 100, where maximal and spontaneous cpm represent the radioactivity released by targets exposed to 0.5 M HCl or medium, respectively.
IFN- enzyme-linked immunospot (ELISPOT) assay
Spleen cells harvested from DNFB-fed or vehicle-fed mice on day
5 after epicutaneous sensitization were restimulated overnight with 0.4
mM DNBS or medium alone. The number of hapten-specific
IFN--producing cells was determined by ELISPOT assay, as described
(10). Briefly, cells were incubated for 4 h at 37°C
in duplicate wells of nitrocellulose 96-well plates (MAHA 45,
Millipore, Bedford, MA) coated with the anti-IFN- mAb (R46A2).
The plates were washed three times with PBS/0.1% Tween before addition
of a biotinylated anti-IFN- Ab (AN18). The hybridomas producing
the mAbs R46A2 and AN18 were kindly provided by DNAX (Palo Alto, CA).
IFN- spot-forming cells (SFC) were developed using
streptavidin-alkaline phosphatase (Boehringer Mannheim, Mannheim,
Germany), incubated for 2 h, and washed extensively before
addition of substrate (5-bromo-4-chloro-3-indolyl phosphate, Sigma).
The number of IFN- SFC was counted in each well using a binocular,
and the results are expressed as the number of
IFN--SFC/106 cells.
RT-PCR analysis of CD8 and IFN- mRNA
Ear samples collected at different time after challenge were
frozen in liquid nitrogen. Total RNA was extracted using a RNAXEL kit
(Eurobio, Les Ulis, France) and treated with DNase I, and 1 µg RNA
was reverse transcribed using poly(dT)15 primers
and Superscript II RT (Life Technologies, France) for 90 min at 37°C.
RNA detection was normalized using the housekeeping gene HPRT
(hypoxanthine phosphoribosyltransferase) as standard. The cDNA was then
amplified using different sets of primers, including for HPRT
(5'-primer: 5'-GTA ATG ATC AGT CAA CGG GGG AC 3'; 3'-primer: 5'-CCA GCA
AGC TTG CAA CCT TAA CCA-3'), for CD8 (5'-primer: 5'-AGG ATG CTC TTG GCT
CTT CC-3'; 3'-primer: 5'-TCA CAG GCG AAG TCC AAT CC-3') and for IFN-
(5'-primer: 5'-GCT CTG AGA CAA TGA ACG CT-3'; 3'-primer: 5'-AAA GAG ATA
ATC TGG CTC TGC-3'). The amplifications were conducted with 29 cycles
for HPRT and 35 cycles for IFN- and 32 cycles for CD8 (1 min at
94°C, 1 min at 61°C, 1 min 30 s at 72°C), and the PCR
products were analyzed on 1.5% agarose gel, as described
(10).
Immunohistochemical staining of CD4+ and CD8+ T cells
Cryostat sections (5 µm thick) of the ears were incubated for 1 h with anti-CD4 (GK1.5) or anti-CD8 (H35) rat mAbs or an irrelevant rat mAb as control, followed by a biotinylated mouse adsorbed goat anti-rat IgG Ab (Biosys, Compiegne, France). Specific binding was revealed with a streptavidin-peroxidase kit (DAKO, Glostrup, Denmark) and AEC in the presence of H2O2 as substrate as described (22). Sections were counterstained with hematoxylin.
Statistical analysis
Statistical analysis were performed using Student’s t test. The difference was considered statistically significant when the p value was <0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Using two non-cross-reacting haptens, i.e., DNFB and OXA, we first
showed that CS to DNFB (Fig. 1
a) and CS to OXA (Fig. 1b) are inhibited by prior intragastric feeding with the
relevant, but not the irrelevant hapten. This was further confirmed in
mice double-sensitized with DNFB and OXA and challenged with either
DNFB or OXA alone (Fig. 1, c and d). Double
sensitization did not affect the ability of mice to develop a CS in
response to challenge with either DNFB (Fig. 1c) or OXA
(Fig. 1d). Inhibition of CS in double-sensitized mice
challenged with either DNFB or OXA was achieved by oral feeding with
the same hapten as that used for challenge (Fig. 1, c and
d).
|
Because CS to DNFB is exclusively mediated by
CD8+ T cells (9, 10), we examined
the effect of oral tolerance on the ability of
CD8+ T cells to transfer CS to DNFB to naive
RAG20/0 mice. Unfractionated T cells or purified
CD8+ T cells isolated from control mice fed with
vehicle alone 7 days before epicutaneous sensitization were able to
transfer CS to RAG20/0 mice as shown by
hapten-specific ear swelling at 24 h after ear challenge with
DNFB. In contrast, CS could not be induced by transfer of either total
T cells or purified CD8+ T cells from mice fed
DNFB 7 days before epicutaneous sensitization (Fig. 2). These data confirm that
CD8+ T cells are the effector cells in CS to DNFB
and indicate that their induction is impaired by intragastric
administration of DNFB before skin sensitization.
|
-producing cytotoxic CD8+ T cells
To determine whether the lack of CS in orally tolerized mice
resulted from impaired generation of specific
CD8+ T cells in secondary lymphoid organs, we
compared hapten-specific proliferative response, IFN- production,
and cytolytic function of spleen cells isolated from orally tolerized
and nontolerized mice on day 5 after epicutaneous DNFB
sensitization.
T cells from mice fed with vehicle alone before skin sensitization
proliferated to in vitro restimulation with syngeneic haptenated cells.
Feeding DNFB before skin sensitization induced 90% inhibition of the
hapten-specific proliferative response of either unfractionated T cells
or purified CD8+ T cells (Fig. 3a).
|
b).
To determine whether intragastric administration of DNFB affected the
frequency of DNFB-specific CD8+ T cells, we used
a IFN- ELISPOT assay that quantitates the number of hapten-specific
CD8+ T cells. We have previously reported that
hapten-specific IFN--producing cells were entirely comprised in the
CD8+ T cell subset (10). Spleen
cells from control vehicle-fed and skin-sensitized mice had a mean
frequency of 25 hapten-specific IFN--SFC/106
cells, whereas no spots were detected in mice orally tolerized by DNFB
feeding before skin sensitization (Fig. 3c). Kinetic studies
performed on days 1, 3, and 5 after DNFB sensitization showed a
frequency of 40 IFN--SFC/106 cells present as
early as day 3 in draining lymph nodes and appeared by day 5 in the
spleen of DNFB-sensitized mice. Alternatively, IFN--SFC were never
detected in either lymph nodes or spleen of orally tolerized mice.
Titration by ELISA of IFN- production in culture supernatant of
purified CD8+ T cells restimulated in vitro with
syngeneic haptenated spleen cells confirmed that IFN- production by
CD8+ T cells during CS was completely prevented
by oral DNFB feeding (not shown). These data demonstrate that oral
tolerance induced by DNFB feeding inhibited the development of
hapten-specific IFN--producing CD8+ T cells in
secondary lymphoid organs.
CD4+ T cells inhibit expansion of CD8+ T cells during the afferent phase of CS
We have previously documented that class II-restricted
CD4+ T are mandatory for oral tolerance induction
(18). To examine whether CD4+ T
cells are responsible for the inhibition of hapten-specific
CD8+ T cell development induced by oral DNFB
feeding, we compared the effect of anti-CD4 mAb injections during
the afferent phase (day -1 and day +4 after sensitization) (Fig. 4, c and d) or the
efferent phase (day +4 after sensitization) of CS (Fig. 4, e
and f), both on development of oral tolerance to CS (Fig. 4, a, c, and e) and on the frequency of
hapten-specific IFN--producing cells (Fig. 4, b, d, and
f). Treatment with control rat IgG did not affect the CS
response or oral tolerance to DNFB (Fig. 4a) as compared
with untreated mice (Fig. 1a) or the frequency of
hapten-specific IFN--SFC in spleen (Fig. 4b) as compared
with untreated mice (Fig. 3c). Depletion of
CD4+ T cells during the efferent of CS (i.e., 1
day before challenge) did not affect oral tolerance, as shown by the
complete inhibition of the CS response to DNFB (Fig. 4e)
associated with the absence of hapten-specific IFN--producing cells
in the spleen (Fig. 4f). Alternatively, injection of
anti-CD4 mAb during the afferent phase abrogated oral tolerance in
DNFB-fed mice, which developed an increased CS response (Fig. 4c) concomitant with enhanced frequency of hapten-specific
CD8+ T cells (Fig. 4d), comparable
with that of anti-CD4-treated vehicle-fed mice.
|
Oral tolerance is associated with lack of IFN--producing
CD8+ effector T cells in the challenged skin
To examine the outcome of oral tolerance at the level of the
target tissue (i.e., the skin), we analyzed the distribution of
CD4+ and CD8+ T cells at
the site of challenge in orally tolerized and sensitized mice. We
previously reported that the skin inflammatory response to DNFB in
sensitized mice is initiated by IFN--producing
CD8+ T cells which migrate to the skin by 6
h after challenge and is followed by recruitment of a inflammatory
cells including both CD8+ (10) and
CD4+ (H. Akiba et al., unpublished data) T cells,
24–48 h after challenge. RT-PCR analysis showed that CD8 and IFN-
mRNA were present in the challenged ear of vehicle-fed and
skin-sensitized mice but were both substantially decreased in orally
fed mice (Fig. 5). Immunohistochemical
analysis revealed that recruitment of both CD8+
and CD4+ T cells in the ear observed 24 h
after DNFB challenge in DNFB-sensitized mice (Fig. 6, a and b) does
not occur in orally tolerized mice (Fig. 6, c and
d). These data demonstrated that the lack of skin
inflammation in orally tolerized mice resulted from of lack of
infiltration IFN--producing CD8+ T cells at
the site of challenge and further confirmed that inhibition of
CD8+ T cells by CD4+ T
cells mediating oral tolerance does not occur during the elicitation
of CS.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We have demonstrated that a single intragastric administration of a
nontoxic dose of DNFB, before skin sensitization, blocks the outcome of
DNFB-specific CS by inhibiting in vivo development of DNFB-specific
cytolytic CD8+ T cells in secondary lymphoid
organs. This was revealed in vivo by the inability of splenic
CD8+ T cells from orally tolerized mice to
transfer DNFB-specific CS to RAG20/0 mice. In
vitro studies showed that CD8+ T cells from
tolerant mice failed to proliferate in response to hapten stimulation
and to differentiate into hapten-specific class I-restricted
CD8+ cytotoxic T cells. Analysis at the single
cell level of the frequency of hapten-specific
CD8+ T cells by a IFN- ELISPOT assay
(10) confirmed that the inhibition of hapten-specific
immune responses cells in orally tolerized mice was due to a lack of
expansion of hapten-specific CD8+ T cells during
the afferent phase of CS. Indeed, hapten-specific IFN--producing
CD8+ T cells were detected as early as day 3 of
sensitization in axillary and inguinal lymph nodes of control
vehicle-fed DNFB-sensitized mice, and by day 5 only in the spleen. In
contrast, these cells were not detected in either the lymph nodes or
spleen from orally tolerized mice. In addition, RT-PCR analysis of CD8
and IFN- mRNA in the ear skin revealed that the lack of CS in
DNFB-tolerized mice was correlated with a dramatic reduction in the
numbers of CD8+ T cells recruited in the skin on
DNFB challenge. Thus, inability of orally tolerized mice to mount a
skin inflammatory response in CS results from impaired development of
specific CD8+ effectors in lymph nodes during the
primary immune response rather than inability of the effector to
migrate at the site of inflammation during the secondary immune
response, i.e., the efferent phase of CS.
Previous studies documented that the CD8+ T cells
mediating CS are of the Tc1 type inasmuch as they produce IFN- but
not IL-4 in response to hapten stimulation (25). In our
studies, intragastric DNFB feeding inhibits IFN- production by
CD8+ T cells, indicating that oral tolerance
prevents the development of Tc1 CD8+ T cells.
Whether oral tolerance resulted from impaired activation of naive
CD8+ T cells, anergy, or deletion of
hapten-reactive CD8+ T cells is not clear.
Previous studies have reported that during oral tolerance Ag-specific T
cell activation and proliferation precedes T cell anergy (26, 27) or deletion (28). In our studies, although
DNFB-specific IFN--producing CD8+ T cells were
undetectable in the draining lymph nodes even at early time points
after sensitization, we observed that IL-2 partially restored the
ability of CD8+ T cells from DNFB-tolerized mice
to proliferate and secrete IFN- in response to in vitro
restimulation with the hapten (data not shown). This suggested that the
mechanisms involved could be attributed to anergy, deletion of a
fraction of hapten-specific CD8+ T cells, or
both.
Interestingly, we found that CD4+ T cells mediate
oral tolerance by inhibiting the development of
CD8+ effector T cell in lymph nodes. This was
demonstrated by the differential outcome of sequential
CD4+ T cell depletion during the afferent or the
efferent phase of CS. Indeed, depletion of CD4+ T
cells before and after sensitization was able to prevent tolerance
induction in DNFB-fed mice, which developed an exaggerated CS response
associated with a large increase in the frequency of hapten-specific
IFN--producing T cells in lymphoid organs. Alternatively, removal of
CD4+ T cells by injecting anti-CD4 mAb on day
4 after sensitization (a time by which effector
CD8+ T cells had already developed in control
skin-sensitized mice), was unable to induce hapten-specific
CD8+ effectors in lymphoid organs (Fig. 5f) and did not affect oral tolerance induction (Fig. 5e). That CD4+ T cells inhibited
priming and/or expansion of CD8+ T cells in the
lymph nodes is further strengthened by the fact that hapten-specific
CD8+ effectors were never detected from day 1 to
day 5 of sensitization in lymph nodes of DNFB-fed mice, whereas they
present in maximal numbers as early as day 3 in vehicle-fed controls.
These findings are in keeping with the lack of oral tolerance to DNFB
in mice deficient in either MHC class II or invariant chain which
exhibited increased in vitro proliferation of hapten-specific splenic T
cells and in which DNFB feeding could induce a CS response, even
without skin sensitization (18). More recently, we
observed that CD4+ T cells could restore
susceptibility to oral tolerance upon adoptive transfer to Ii-deficient
mice (our unpublished data). These observations, together with the lack
of both CD4+ and IFN--producing
CD8+ CS effectors at the challenge site in orally
tolerant mice, clearly demonstrate that CD4+ T
cells mediate oral tolerance by regulating the development of specific
CS effectors in lymphoid organs during the primary immune response and
not in the target tissue.
It should be emphasized that CD4+ T cells can
also modulate the intensity and duration of CS in DNFB-sensitized mice
(9). CD4+ T cells limit induction of
hapten-specific CD8+ effectors in lymph nodes
during the afferent phase of CS (Fig. 4b compared with Fig. 4d) but can also regulate the duration of the skin
inflammatory response (Fig. 4a compared with Fig. 4e) during the efferent phase. In this respect, RT-PCR and
immunohistochemical analysis of the ears 24 h after challenge
showed the presence of IFN--producing hapten-specific
CD8+ effector T cells in DNFB-sensitized mice
associated with an inflammatory infiltrate composed of both
CD4+ and CD8+ T cells.
Recruitment of CD4+ and
CD8+ T cells in the skin is conditioned by the
ability of cytolytic effectors to migrate at the challenge site (Ref.
10 , and I. Akiba et al., unpublished results). Therefore,
the lack of CD4+ and CD8+
in the challenged ear of orally tolerant mice is compatible with the
lack of hapten-specific effectors able to migrate into the
skin.
The fact that regulatory CD4+ T cells activated by oral DNFB are able to completely inhibit the development of CD8+ T cells, whereas CD4+ T cells activated after epicutaneous DNFB sensitization regulate but do not block the CS response, raises the issue of identity and origin of regulatory CD4+ T cells. It may be hypothesized either that regulatory CD4+ T cells activated by intragastric DNFB administration belong to a distinct CD4+ cell subset enriched in mucosal tissues or that feeding provides a pool of readily activated CD4+ T cells present in the lymph nodes at the time of sensitization. Alternatively, it is possible that the hapten is captured by intestinal dendritic cells which migrate to lymph nodes where they can prime regulatory CD4+ T cells. This is supported by the observation that 24 h after intragastric administration of the fluorescent hapten FITC, CD11c+ dendritic cells are recovered in mesenteric lymph nodes (D. Kaiserlian, unpublished data). In addition, studies showing that dendritic cells recovered from mesenteric lymph of OVA-fed rat can activate class II-restricted CD4+ T cell in vivo and in vitro (28) and that Flt3-L-treated mice are more susceptible to oral tolerance of DTH (29) have pointed to a role of dendritic cells in oral tolerance. Finally, we cannot exclude that regulatory CD4+ T cells are activated in extraintestinal sites by circulating DNFB reaching peripheral lymphoid organs, as recently documented in oral tolerance induced after intragastric administration cytochrome c in TCR Tg+ mice (30).
That CD4+ T cells mediate oral tolerance by inhibiting the function of CD4+ Th1 cells has been described in DTH to OVA (31, 32) and experimental autoimmune encephalomyelitis induced by myelin basic protein (33). It is currently considered that feeding high doses of protein induces tolerance by deletion of effector cells, while feeding low dose of protein induces bystander suppression, mediated by a subset of regulatory CD4+ T cells producing high levels of TGF-ß, named Th3 (33). More recent studies reported that regulatory CD4+ T cell clones (Tr1), established in vitro in the presence of IL-10, mediated standard suppression through production of IL-10 and prevented experimental colitis induced in SCID mice injected with pathogenic CD4+CD45RBhigh T cells (34). In our studies, oral tolerance was hapten specific inasmuch as CS to DNFB was inhibited only by feeding with DNFB but not with the irrelevant hapten OXA. However, we did not formally demonstrate that the regulatory CD4+ T cells mediating oral tolerance are hapten specific, and it is possible that they exert bystander suppression. Studies performed thus far with spleen cells from DNFB-tolerized mice could not ascribe a Th3 or a Tr1 phenotype to these CD4+ T cells. It should be emphasized that although the doses of hapten used for inducing tolerance (i.e., 300 µg DNFB and 3 mg OXA) would be considered low doses of oral tolerogen compared with proteins, they correspond to the highest dose of haptens nontoxic by the oral route, and reducing the dose also diminished oral tolerance induction in our system. This could be attributed to a highest immunogenicity of haptens compared with proteins, inasmuch as hapten binding to soluble and membrane proteins, (including MHC molecules) may generate a higher number of T cell epitopes than those generated by protein processing.
Inhibition of cytotoxic CD8+ T cells induced by OVA feeding has been illustrated in mice subsequently immunized parenterally with either OVA in ISCOMS (31) or mixed with CFA (32), or OVA-loaded spleen cells (35) although in this latter study OVA feeding by itself could induce a low CTL response in some mice, of similar intensity to that of tolerized mice. To our knowledge, our study is the first to demonstrate that oral tolerance can block development of specific CD8+ CTL, which do not require CD4+ T cell help and which are responsible for a pathophysiological inflammatory disease. Considerable evidence support the hypothesis that CD8+ CTL, which mediate DTH responses to contact sensitizers, including drug metabolites and chemical and protein allergens, are the inflammatory effector cells responsible for diseases such as allergy, asthma, and autoimmunity (36). We propose that oral tolerance may be a valuable approach for the treatment of inflammatory diseases mediated by Ag-specific cytotoxic CD8+ T cells independent of CD4 help. Further studies are necessary to determine whether CD4+ T cells could regulate the function of memory CD8+ T cells.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Dominique Kaiserlian, Institut National de la Santé et de la Recherche Médicale Unité 404, Avenue Tony Garnier, 69365 Lyon Cedex 07, France.
3 Abbreviations used in this paper: DTH, delayed-type hypersensitivity; CS, contact sensitivity; DNFB, 2,4-dinitrofluorobenzene; DNBS, 2,4-dinitrobenzenesulfonate; OXA, oxazolone; ELISPOT, enzyme-linked immunospot; SFC, spot-forming cells; HPRT, hypoxanthine phosphoribosyltransferase.
Received for publication July 8, 1999. Accepted for publication December 27, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-producing (Tc1) effector CD8+ T cells and interleukin (IL) 4/IL-10-producing (Th2) negative regulatory CD4+ T cells. J. Exp. Med. 183:1001.This article has been cited by other articles:
|
|
 |
|
  L. Lamy, A. Foussat, E. J. Brown, P. Bornstein, M. Ticchioni, and A. Bernard Interactions between CD47 and Thrombospondin Reduce Inflammation J. Immunol., May 1, 2007; 178(9): 5930 - 5939. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Cavani, F. Nasorri, C. Ottaviani, S. Sebastiani, O. De Pita, and G. Girolomoni Human CD25+ Regulatory T Cells Maintain Immune Tolerance to Nickel in Healthy, Nonallergic Individuals J. Immunol., December 1, 2003; 171(11): 5760 - 5768. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Dubois, L. Chapat, A. Goubier, M. Papiernik, J.-F. Nicolas, and D. Kaiserlian Innate CD4+CD25+ regulatory T cells are required for oral tolerance and inhibition of CD8+ T cells mediating skin inflammation Blood, November 1, 2003; 102(9): 3295 - 3301. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Saint-Mezard, C. Chavagnac, S. Bosset, M. Ionescu, E. Peyron, D. Kaiserlian, J.-F. Nicolas, and F. Berard Psychological Stress Exerts an Adjuvant Effect on Skin Dendritic Cell Functions In Vivo J. Immunol., October 15, 2003; 171(8): 4073 - 4080. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Akiba, J. Kehren, M.-T. Ducluzeau, M. Krasteva, F. Horand, D. Kaiserlian, F. Kaneko, and J.-F. Nicolas Skin Inflammation During Contact Hypersensitivity Is Mediated by Early Recruitment of CD8+ T Cytotoxic 1 Cells Inducing Keratinocyte Apoptosis J. Immunol., March 15, 2002; 168(6): 3079 - 3087. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Nakajima, H. Iijima, M. F. Neurath, T. Nagaishi, E. E. S. Nieuwenhuis, R. Raychowdhury, J. Glickman, D. M. Blau, S. Russell, K. V. Holmes, et al. Activation-Induced Expression of Carcinoembryonic Antigen-Cell Adhesion Molecule 1 Regulates Mouse T Lymphocyte Function J. Immunol., February 1, 2002; 168(3): 1028 - 1035. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Etchart, P.-O. Desmoulins, K. Chemin, C. Maliszewski, B. Dubois, F. Wild, and D. Kaiserlian Dendritic Cells Recruitment and In Vivo Priming of CD8+ CTL Induced by a Single Topical or Transepithelial Immunization Via the Buccal Mucosa with Measles Virus Nucleoprotein J. Immunol., July 1, 2001; 167(1): 384 - 391. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. K. Bishop, S. C. Wood, E. J. Eichwald, and C. G. Orosz Immunobiology of Allograft Rejection in the Absence of IFN-{{gamma}}: CD8+ Effector Cells Develop Independently of CD4+ Cells and CD40-CD40 Ligand Interactions J. Immunol., March 1, 2001; 166(5): 3248 - 3255. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 0.1% DNFB (•) or 1% OXA (
), were sensitized
epicutaneously 7 days later with 0,5% DNFB (a), 2% OXA
(b), or both DNFB and OXA (c and
d). Hapten-specific CS was determined at various time
points after ear challenge with either 0.2% DNFB (a and
c) or 0.4% OXA (b and
d).
,
) or CD8+ T
cells (
), purified
from the spleen of vehicle-fed (
) mice, on day 5 after epicutaneous
sensitization, were restimulated in vitro for 3 days with
106 syngeneic mitomycin C-treated spleen cells either
untreated or DNFB derivatized. T cell proliferation was determined by
[3H]thymidine incorporation during the last 6 h of
culture. Results are expressed as 
) mice or from either
vehicle-fed () or DNFB-fed
(
) mice, harvested on day
5 after epicutaneous sensitization, was assessed after in vitro
restimulation for 5 days with DNBS-derivatized mitomycin C-treated
spleen cells (25 x 106) from C57BL/6. Anti-CD8 depletion
of splenic effector cells from vehicle-fed DNFB-sensitized mice was
performed before use in the cytotoxicity assay
(
). Specific cytotoxicity
was determined by lysis of DNBS-derivatized 51Cr-labeled
EL-4 targets in a 4-h 51Cr release assay. Results are
representative of three experiments. c, Hapten-specific
IFN-
