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*
Division of Immunology and Transplantation Biology, Department of Pediatrics, Stanford University, Stanford, CA 94305; and
Respiratory Medicine Unit, University of Edinburgh Medical School, Edinburgh, United Kingdom
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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 immune system has evolved several mechanisms to maintain in the
periphery a state of tolerance against innocuous Ags. In particular,
clonal deletion, anergy, and active suppression mediated by regulatory
cells secreting TGF-ß and Th2-like cytokines have been implicated, in
an Ag dose-dependent fashion, in the down-regulation of immune
responses in the gut mucosa (4, 5, 6). However, the
immunological parameters underlying the natural immunity to inhaled
proteins are poorly defined. It has been suggested that the default
immune response to aeroallergens includes a strong component of
Th2-like reactivity, and avoidance of allergic sensitization is
achieved through early immune deviation toward the Th1 pathway
(1, 2, 7). This shift in the phenotypic profile of the
immune response has been mainly attributed to Ag-specific
CD8+ T cells that become activated following Ag
inhalation and secrete enhanced amounts of IFN-
(1, 2).
Nevertheless, the experimental evidence for this process remains
controversial. Normal individuals do not exhibit vigorous Th1-type
reactivity against aeroallergens, but, rather, they are characterized
by a lack of productive immunity. Furthermore, recent studies have
concluded that CD8+ T cells also play a
pathogenic role in the development of airway inflammation and
hyper-responsiveness (8, 9).
In contrast to CD8+ T cells, the direct effect of inhaled proteins on CD4+ T cell functions has not been analyzed. It is well documented that the induction of long-lasting immunity to protein Ags requires the expansion and differentiation of Ag-specific, effector CD4+ T cells, while unresponsiveness has been correlated with their elimination or functional inactivation. The quality of costimulatory signaling at the time of priming has been shown to differentially regulate this process (10, 11, 12). Therefore, the principal aim of this study was to investigate in detail the effect of intranasal (i.n.)3 (3) exposure to OVA on T cell responses in vivo and define the requirements for the development of protective immunity. To address this question, we analyzed immune responses in wild-type mice as well as in mice bearing a small population of adoptively transferred OVA-TCR transgenic T cells. The latter allowed us to follow the fate of Ag-specific CD4+ T cells in vivo (13). The results of our study indicate that i.n. exposure to OVA induces unresponsiveness to subsequent immunogenic challenges that is not dependent on the presence of regulatory CD8+ T cells or inhibitory cytokines, but is mediated by functionally impaired Ag-specific CD4+ T cells. An initial, transient phase of activation, during which the T cells produce Th1 and Th2 cytokines, precedes the development of unresponsiveness. The functional differentiation of the CD4+ T cells in response to i.n. OVA appears to be selectively dependent on CD86 (B7.2), but not CD80 (B7.1), costimulatory signals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). OVA-TCR transgenic D011.10 mice (14) were obtained from Dr. D. Y. Loh (Washington University, St. Louis, MO). D011.10 mice were backcrossed with normal BALB/c mice, and the progeny were screened by flow cytometric analysis of peripheral blood leukocytes stained with anti-CD4 and the clonotype-specific KJ1-26 mAbs (14). All mice were housed in pathogen-free conditions at the laboratory animal facilities of Stanford University, in accordance with the guidelines of National Institutes of Health. Mice used for experiments were sex and age matched.
Media, reagents, and mAbs
All cells were cultured in DMEM (Sigma, St. Louis, MO) supplemented with 10% FCS (Gemini Bioproducts, Calabasas, CA), 2 mM glutamine, 20 µg/ml gentamicin, and 5 x 10-5 M 2-ME. The hybridoma cell lines secreting the anti-CD8 (53.6.7), anti-CD80 (1G10), and anti-CD86 (GL1) mAbs were obtained from American Type Culture Collection (Manassas, VA). The cell lines secreting anti-CTLA-4 (UC10-4F10) and anti-IL-10 (JES5-2A5) mAbs were gifts from Dr. J. Bluestone (University of Chicago, Chicago, IL) and Dr. J. Abrams (DNAX, Palo Alto, CA), respectively. Hybridoma cells producing the anti-clonotypic Ab KJ1-26 were provided by Dr. P. Marrack (National Jewish Medical Center, Denver, CO). Each of the above-mentioned Abs were grown as ascites in pristane-primed BALB/c-nu/nu mice (0.5 ml/mouse i.p. of 98% 2,6,10,14-tetramethylpentadecane; Sigma) and were subsequently purified by ammonium sulfate precipitation and ion-exchange chromatography. The neutralizing anti-TGF-ß mAb was obtained from Genzyme (Cambridge, MA). The Ab doses we used in our experiments have been previously shown to be effective in vivo (15, 16, 17, 18, 19). The depletion of CD8+ T cells following in vivo treatment with anti-CD8 mAb was confirmed by flow cytometry of spleen or lymph nodes cells from the treated mice.
Induction of tolerance to i.n. Ag
Mice lightly anesthetized with methoxuflurane received i.n. 30 µl of PBS containing OVA (grade V; Sigma) on 3 consecutive days. Control mice received i.n. PBS. To confirm that the i.n. administered Ag was delivered to the airways, mice were given i.n. OVA in Evans Blue dye. We found that the dye was deposited only in the respiratory tract, while there were no detectable traces in the esophagus or stomach (unpublished data) (16). Ten days later the mice were immunized i.p. with 10 µg of OVA in 2 mg of aluminum hydroxide (alum) in a volume of 0.5 ml. For the experiments with chimeric OVA-transgenic mice 100 µg of OVA in alum were used for the i.p. injection. To examine bystander suppression to an unrelated Ag, mice pretreated with i.n. OVA or PBS were coimmunized i.p. with 10 µg of OVA and 20 µg of hen egg lysozyme (HEL; Sigma) in alum.
Adoptive transfer of TRC-transgenic T cells
Lymph node and spleen cells from D011.10 mice were pooled and enriched for CD4+ T cells. In brief, erythrocytes were removed by hypotonic lysis, resting B cells were depleted by adherence to goat anti-mouse Ig-coated plates; CD8+ cells and APC were depleted by treatment with anti-CD8 (HB130), anti-HSA(J11d.2), and anti-MHC class II (MKD6) mAbs in the presence of rabbit complement (Pel-Freez, Brown Deer, WI). The percentage of CD4+, KJ1-26+ cells in the final population was determined by flow cytometric analysis, and 5–6 x 106 CD4+, KJ1-26+ cells were injected i.v. into unirradiated syngeneic BALB/c recipients, in a volume of 0.3 ml of normal saline.
In vitro proliferation and cytokine assays
Lymph node or spleen cells were harvested, passed through a
nylon mesh, and cultured (5 x 105
cells/well) with or without OVA in 0.2 ml of complete DMEM. After
72 h, the cultures were pulsed with 0.25 µCi of
[3H]TdR thymidine for 12–16 h, and the
incorporated radioactivity was measured in a Betaplate scintillation
counter (MicroBeta Trilux, Wallac, Turku, Finland). The
clonotype-specific proliferation was calculated by dividing the total
OVA-specific proliferation in counts per minute by the number of
KJ1-26+ CD4+ T cells in the
culture (assessed by flow cytometry). To determine the secretion of
cytokines in the cultures, supernatants were collected on day 4. The
levels of IL-4, IL-5, IL-10, and IFN- in the supernatants were
assayed by ELISA. The presence of TGF-ß was assessed in a bioassay
using the mink lung epithelial cell line Mv1Lu (American Type Culture
Collection).
Flow cytometry
Lymph node or spleen cells were harvested at various times, and 1 x 106 cells were incubated on ice with anti-CD4-FITC (PharMingen, San Diego, CA) and the biotinylated KJ1-26 mAbs followed by streptavidin Cy-chrome (PharMingen). For some experiments the cells were also stained with anti-CD45RB-PE (PharMingen). After the final washing the cells were fixed with 1% paraformaldehyde and analyzed on a Becton Dickinson FACScan (Rutherford, NJ). Analysis was performed on 15,000 collected events. CD45RB expression and cell size were assessed on the gated CD4+, KJ1-26+ population. The total number of DO11.10 cells present in the lymphoid organs was calculated by multiplying the total number of viable lymph node or spleen cells by the percentage of CD4+, KJ1-26+ cells obtained by flow cytometry.
Measurement of OVA-specific Igs
Mice were bled, and OVA-specific Abs were measured using modified OVA-specific ELISAs. For the measurement of OVA-specific IgG1 and IgG2a, plates were coated overnight with 5 µg/ml OVA. After washing and blocking, serial dilutions of sera were added for 24 h. Subsequently, the plates were incubated with HRP-conjugated goat anti-IgG subclass-specific Abs (Southern Biotechnology Associates, Birmingham, AL), washed, and developed by adding o-phenylenediamine. The OD was determined at 492 nm. Anti-OVA IgG1 and IgG2a mAbs 6C1 and 3A11, respectively, were used as standards for the quantification of each IgG subclass. For the determination of OVA-specific IgE, the rat anti-mouse IgE mAb EM95 (5 µg/ml) was used to coat the plates overnight. After the samples were applied for 24 h, biotinylated OVA (10 µg/ml) was added for 2 h, followed by 1-h incubation with HRP-conjugated streptavidin (Southern Biotechnology Associates). Plates were developed with o-phenylenediamine substrate, and the OD was determined at 492 nm. The standard for the OVA-specific IgE ELISA was sera from mice hyperimmunized with OVA that had been quantitated for IgE.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the effect of exposure to i.n. protein Ag on
subsequent antigenic challenges, BALB/c mice were treated with i.n. OVA
or PBS and then immunized i.p. with OVA in alum. Splenic T cells from
the OVA-pretreated mice failed to expand in response to OVA in vitro in
contrast to cells from mice that had received i.n. PBS, indicating that
prior exposure to i.n. Ag impaired subsequent Ag priming (Fig. 1
A). The inhibition of T cell
responsiveness was Ag specific, because spleen cells from mice exposed
to i.n. OVA and immunized i.p. with both OVA and HEL in alum
proliferated normally when restimulated in vitro with HEL (Fig. 1B), but not with OVA (Fig. 1A). The T cell
unresponsiveness waned with time, as mice exposed to i.n. OVA and
rechallenged 2, 3, and 4 mo after the last administration of i.n. Ag
slowly regained their capacity to proliferate following in vivo
restimulation with OVA (Fig. 2).
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). Analogous
reduction was observed in the levels of secreted cytokines, including
IFN-, IL-4, and IL-5 (Table I). Production of IL-10 and TGF-ß was
not detected in any of the cultures. Maximal inhibition of T cell
function was noticed in mice pretreated with 100 µg of i.n. OVA.
Exposure to low quantities of i.n. OVA, such as 0.1 µg/dose, failed
to down-regulate the T cell responsiveness.
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To determine the contribution of CD8+ T
cells in the development of the unresponsive state observed in our
system, mice were depleted of CD8+ cells, by
injection with anti-CD8 mAb, before i.n. administration of OVA or
PBS. The depletion of CD8+ T cells failed to
abrogate the induction of unresponsiveness, because mice given i.n. OVA
displayed decreased T cell reactivity when rechallenged i.p. with OVA
in alum regardless of whether they were treated with anti-CD8 Ab
(Fig. 4a). Similar results
were obtained when the mice were treated with anti-CD8 Ab before
the administration of i.n. Ag as well as at the time of i.p.
immunization (data not shown). This observation suggests that other
mechanisms potentially mediated by the CD4+ T
cell compartment may be responsible for the down-regulation of Th2
immunity that is associated with i.n. exposure to a protein
Ag.
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, b and c),
indicating that the absence of IL-10 or TGF-ß did not influence the
development of T cell unresponsiveness to secondary antigenic
challenge. Exposure to i.n. Ag induces a persistent activated population of CD4+ T cells
To examine the involvement of CD4+ T cells
in the down-regulation of T cell responses after i.n. exposure to OVA,
we used a chimeric model in which a small population of OVA-TCR
transgenic CD4+ T cells from D011.10 mice was
adoptively transferred into syngeneic BALB/c mice. The recipient mice
were subsequently treated with i.n. OVA or PBS, and the fate of D011.10
cells was monitored by flow cytometry using the clonotype Ab KJ1-26.
CD4+, KJ1-26+ cells are
undetectable in normal BALB/c mice even after immunization with OVA in
adjuvant (data not shown). Exposure of the chimeric mice to i.n. OVA
three times resulted in considerable expansion of the D011.10 T cells
in vivo. In the peribronchial lymph nodes, the main site of
accumulation of inhaled Ags (16), a considerable increase
in cellularity occurred 2 days after the last administration of OVA,
with a 15-fold increase in the percentage of
CD4+, KJ1-26+ T cells (Fig. 5a). This change represents a
49-fold expansion of the absolute number of OVA-transgenic T cells per
mouse. On day 5 the total number of CD4+,
KJ1-26+ T cells bronchial lymph nodes was
3.5 x 104 in the PBS-pretreated mice
compared with 171 x 104 in the OVA-treated
group (Fig. 6a). The
CD4+, KJ1-26+ T cells in
the OVA-exposed mice presented an activated phenotype, as demonstrated
by an increase in cell size (Fig. 5b) and down-regulation of
CD45RB expression, which remained reduced 1 wk later (Fig. 5c). Similar expansion of D011.10 cells was also observed in
the spleen (Fig. 6b) and other nondraining lymph nodes (data
not shown), providing evidence for systemic activation of the
OVA-specific CD4+ T cells. To examine whether the
in vivo activated Ag-specific T cells from mice exposed to i.n. OVA
were initially functionally responsive, bronchial lymph node cells were
restimulated in vitro with OVA. The cells showed strong proliferation
accompanied by the production of both Th1-type (IFN-) and Th2-type
(IL-4 and IL-5) cytokines (Table II). The
initial phase of T cell reactivity following exposure to i.n. OVA was
also confirmed in normal BALB/c mice (data not shown).
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Nevertheless, not all CD4+,
KJ1-26+ cells were eliminated, and on day 13, the
time of the i.p. immunogenic challenge, a substantially larger
population of D011.10 cells remained in the OVA-pretreated mice
compared with their control counterparts (Fig. 6). Particularly in the
peribronchial area, these mice had 16-fold higher numbers of D011.10
cells with an activated phenotype (Fig. 5c). Thus, exposure
to i.n. OVA results in an initial phase of T cell activation followed
by some degree of deletion, possibly due to apoptosis. However, a small
activated population of OVA-specific CD4+ T cells
persists, implying that deletion is not the primary cause underlying
the unresponsiveness to further in vivo immunogenic challenges
with OVA. The development of i.n. tolerance is due to the presence of functionally impaired Ag-specific CD4+ T cells
The immunological basis underlying the failure of T cells to
respond to antigenic restimulation after i.n. exposure to OVA was
investigated. Mice injected with D011.10 cells and then exposed to i.n.
OVA or PBS were immunized i.p. with OVA in alum 10 days later. Spleen
or mesenteric lymph node cells from these mice were retrieved after 4
days and stained with KJ1-26 and anti-CD4+
Abs. The accumulation of D011.10 cells in lymphoid tissues in response
to immunization varied for the i.n. OVA- and PBS-treated groups. Thus,
an 8- to 9-fold expansion of CD4+,
KJ1-26+ T cells in the spleens of previously
naive mice was observed, while in the i.n. OVA-pretreated mice the
increase was <2-fold (Fig. 7A). Similar differences were
found in the numbers of OVA-transgenic cells in mesenteric lymph nodes
(data not shown). The lack of efficient accumulation of D011.10 cells
in the tolerized mice may reflect a defect in their capacity to
proliferate. To examine this possibility, spleen cells from these mice
were cultured in vitro with OVA, and the proliferative response per
transgenic T cell was estimated. Our results revealed a substantial
decrease in the proliferative potential of D011.10 cells recovered from
mice preexposed to i.n. OVA (Fig. 7B). The ability of the
tolerized T cells to secrete cytokines was also reduced (data not
shown). Similar results were obtained when these cultures were
performed using purified CD4+ T cells from the
spleens of tolerized mice (data not shown).
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), suggesting that a
suppressive environment was not created. Moreover, adoptive transfer of
bronchial lymph node and spleen cells or purified
CD4+ T cells (up to 20 x
106 cells) from i.n. tolerized mice (wild type,
as well as chimeric) into naive BALB/c recipients failed to confer a
state of OVA-specific hyporesponsiveness to subsequent immunization
analogous to that of the original donors (data not shown).
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Our observation that activation of Ag-specific
CD4+ T cells occurs initially following i.n.
exposure to OVA prompted us to examine the contribution of
costimulation in the induction of tolerance. BALB/c mice exposed to
i.n. OVA or PBS also received injections of anti-CD80 (B7.1) or
anti-CD86 (B7.2) mAbs and were rechallenged i.p. with OVA in alum.
The administration of anti-B7 Abs did not prevent T cell responses
to i.p. immunization with OVA 10 days after the completion of the Ab
treatment (Fig. 9). Mice receiving i.n.
OVA and anti-CD80 Ab were still unresponsive to secondary challenge
(Fig. 9A). In contrast, treatment with anti-CD86 Ab
resulted in abrogation of tolerance induction (Fig. 9B). The
possibility that selective interaction of CD86 with CTLA-4 was required
for the development of i.n. tolerance was also examined by treating the
mice with anti-CTLA-4 Ab at the time of administration of i.n. OVA.
Blocking the CTLA-4-mediated costimulation, however, failed to abolish
the induction of i.n. unresponsiveness, and no marked difference in the
proliferation was observed for splenic T cells from mice treated before
rechallenge with anti-CTLA-4 and i.n. OVA or i.n. OVA alone (Fig. 9C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several independent groups have reported that IgE Ab induction to an
immunogenic challenge is inhibited by prior exposure to aerosolized
OVA, whereas specific IgG1 and IgG2a responses remain intact (3, 20). Much attention has focused on the role of
CD8+ T cells in both the regulation of Th2
responses and peripheral tolerance to inhaled Ags. However, the
experimental evidence has been controversial. Initial studies in
rodents led to the hypothesis that immunologic homeostasis to inhaled
proteins is mediated by a population of Ag-specific
CD8+ T cells secreting IFN- (1, 2, 21). In contrast, in other models of aeroallergen sensitization
the activated CD8+ T cells participated in the
development of airway inflammation and hyper-responsiveness (8, 9). In our study the failure to prevent the induction of i.n.
tolerance when CD8+ T cells were deleted in vivo,
by Ab treatment, suggests that they do not contribute to the
development of the unresponsive state. Similarly, it was recently shown
that administration of nebulized OVA to CD8- and
-deficient mice reduced IgE responses and blood eosinophilia to
subsequent challenges to the same degree as in normal wild-type mice
(3). Holt and co-workers have suggested that the
down-regulation of Th2 immunity following exposure to inhaled Ags is
due to immune deviation toward the Th1 pathway as a result of enhanced
production of IFN- (1, 7, 20). However, this was not
confirmed in our model, because the dose-dependent inhibition of T cell
expansion in the i.n. tolerized mice was associated with the reduction
of both Th1- and Th2-type cytokine secretion. Moreover, we did not
detect TGF-ß or IL-10 secretion by T cells from the spleens or lymph
nodes of OVA-unresponsive mice. The lack of a substantial role for
these immunosuppressive cytokines was further confirmed by the finding
that i.n. tolerance induction was not abrogated by the in vivo
administration of neutralizing Abs specific for either TGF-ß or
IL-10. Inhibitory cytokines are involved in the regulation of immune
responses in gut mucosa. It has been reported that oral administration
of low doses of protein Ag leads to tolerance mediated by T
cell-derived TGF-ß and Th2 cytokines, while feeding high doses of Ag
promotes deletion or anergy (4, 5, 6). Nevertheless, oral
tolerance could be generated in TGF-ß knockout mice regardless of the
amount of the ingested Ag, indicating redundancy and that multiple
mechanisms of tolerance may coexist (22). In
general, much lower levels of Ag exposure are sufficient to initiate a
tolerogenic response in the lungs compared with the gut. This may be
due to differences in the local microenvironment in which the immune
system first encounters the Ag and may differentially influence the
mechanisms employed for the maintenance of nonresponsiveness to
innocuous Ags.
To establish whether the CD4+ T cell functions
were affected by prior exposure to inhaled protein Ags, we followed the
fate of a small population of OVA-specific TCR transgenic
CD4+ T cells in mice treated with i.n. OVA. We
observed that exposure to i.n. Ag resulted in activation and initial
expansion of the specific CD4+ T cells both in
the draining lymph nodes and systemically. Systemic activation takes
place as early as 6 h after ingestion, indicating that oral Ag,
intact or degraded, enters the bloodstream and is rapidly distributed
to secondary lymphoid organs (23). It is possible that
inhaled proteins also enter the blood. OVA-specific
CD4+ T cells isolated from the peribronchial
lymph nodes 2 days after the i.n. treatment were capable of secreting
IFN- and IL-4 when restimulated. Many recent reports support the
idea that activation before the loss of responsiveness is a necessary
step for T cell tolerance induction (13, 24, 25). In our
model the number of OVA-specific CD4+ T cells
initially expanded in the tolerant mice, declined rapidly with time,
presumably due to apoptosis. Nevertheless, a population of cells with
an activated phenotype persisted, but was refractory to further
antigenic stimulation both in vitro and in vivo. It has been proposed
that the presence of inflammatory stimuli at the time of priming is the
deterministic factor for the functional outcome of Ag-T cell
interaction. Inflammatory cytokines, such as TNF-
and IL-1, can
rescue CD4+ T cells from deletion after exposure
to superantigens or soluble peptide and promote the acquisition of an
effector phenotype (17, 26). Costimulation alone is
insufficient to abrogate tolerance in the absence of inflammation, and
this is further illustrated by the observation that tolerance can be
induced in transgenic mice overexpressing CD80 or CD86 on their APCs
(17, 27). We also have found that coadministration of OVA
with cytokines i.n. prevents the induction of nonresponsiveness
to further antigenic challenges (D. C. Tsitoura, unpublished
observations). It is possible that in atopic individuals a
genetic predisposition for enhanced secretion of IL-4 contributes to
the breaking of mucosal tolerance and the development of Th2 immunity.
Proinflammatory molecules, such as IL-6, IL-8, and GM-CSF, present in
the airways of atopic subjects (28, 29) may facilitate
this process.
It is not clear why a population of unresponsive CD4+ T cells survives deletion. We noted, however, that these cells could not maintain the state of peripheral tolerance for periods longer than 3 mo. This may simply reflect that in the absence of Ag the frequency of tolerant T cells decreases with time, while the number of naïve, potentially reactive T cells increases. Alternatively, it is possible that tolerance is reversible. Similar to our findings Pape and colleagues recently reported that an unresponsive population of Ag-specific CD4+ T cells with an intrinsic defect in cytokine production is induced by i.v. injection of a high dose OVA peptide (25). The cells could retain their unresponsive status only as long as some peptide persisted in vivo, implying that some TCR-transduced signals are required to maintain tolerance. It has been suggested that in the absence of inflammation TCR signaling is perturbed, leading eventually to abortive cell differentiation with defective transcription of growth factor and other lymphokine genes (30, 31). Production of repressor proteins, within the tolerized cells, that are responsible for the unresponsive state has also been postulated (32).
The fact we did not observe bystander suppression, immunosuppressive cytokines, or transfer tolerance into naive mice suggests that regulatory cells are not induced in our model. Indeed, not all forms of peripheral tolerance occur due to suppression (33). Regulatory T cells with a suppressor phenotype have been shown to be involved in protection from autoimmunity (34). How these cells develop in the periphery and what their exact phenotype is remain to be determined. They may appear early during thymic development and constitute specialized subsets of the T cell repertoire that mainly function to maintain homeostasis against self Ags (35, 36). Alternatively, the localization, amount, and duration of availability of the Ag may regulate their selection and expansion in the periphery (37). Brief exposure of T cells to exogenous, innocuous Ags may lead to transient immune responses, while Ag persistence in the lymphoid tissues may be necessary for the generation of protective regulatory cells. Furthermore, there is evidence that repeated exposure of CD4+ T cells to inhibitory cytokines, such as IL-10, can generate a suppressor phenotype associated with reduced capacity of the cells to expand (38).
In vitro studies with cell lines led to the conclusion that T cell tolerance arises as a result of TCR activation in the absence of costimulation (10, 11). However, recent experimental evidence suggests that this concept may not be applicable in vivo. B7 antagonists, such as CTLA-4-Ig, do not promote peptide-induced anergy, but keep the T cells in a naive, functionally competent state (12). It was proposed that tolerance may arise from engagement of B7 molecules by the CTLA-4 receptor, instead of CD28, and not from lack of costimulation (12). The initial activation of CD4+ T cells following i.n. delivery of OVA implies that costimulatory signals derived from APCs are at least partially intact during the primary encounter with Ag. Potentially altered kinetics or dysregulation of the expression of CTLA-4 and the ligands, CD80 and CD86, may contribute to the development of i.n. tolerance. However, the in vivo administration of anti-CTLA-4 or anti-CD80 Abs neither enhanced nor diminished the inhibitory effects of the OVA protein in our system, whereas an anti-CD86 Ab abrogated tolerance completely. Distinct inhibitory effects of anti-CD80 and -CD86 Abs have been described for T cell responses to both nominal and allo-Ags (39, 40, 41). Signaling through CD86 is associated with Th2 differentiation (39, 40), and inhibition of this costimulatory pathway has been shown to result in decreased expression of IL-4, IL-5, and IL-13 genes (42, 43). In the respiratory tract, allergen-induced Th2-type inflammation and airway hyper-responsiveness have been found to be susceptible to selective blockade of CD86-mediated, but not CD80-mediated, costimulation in animal models (16) as well as in patients with atopic asthma (44). CD86, in contrast to CD80, is constitutively expressed on mature dendritic cells, macrophages, and resting B cells and can, therefore, mediate some costimulatory signals to T cells even in the absence of an inflammatory environment (45, 46). Thus, it is possible that the selective involvement of CD86 costimulation in the induction of i.n. tolerance is due to its prominent role in the initiation of immune responses. Furthermore, CD86 might be implicated in the early, albeit transient in the case of i.n. tolerance, synthesis of Th2-type cytokines that characterizes mucosal immunity. Blocking the interaction of B7 with CTLA-4 did not prevent the induction of unresponsiveness in our system. However, CTLA-4-mediated signaling appeared to negatively control the initial expansion of CD4+ T cells following i.n. exposure to OVA, because we found that the number of OVA-specific CD4+ T cells in the bronchial lymph nodes was markedly enhanced when anti-CTLA-4 Ab was also administered (D. C. Tsitoura, unpublished observations). In contrast, it has been reported that CTLA-4 signaling may be involved in the induction of high dose oral tolerance (47).
In conclusion, our results suggest that i.n. administration of protein Ags prevents the induction of Th2 immunity to subsequent immunogenic challenges. The lack of responsiveness is associated with an early CD86-dependent activation of specific CD4+ T cells, a proportion of which survive deletion. There is no evidence for CD8+ T cells, immunosuppressive cytokines, or immune deviation being the mechanisms leading to tolerance in this system. It is likely that unresponsiveness arises from the failure of CD4+ T cells to develop normal effector function in the tolerant mice. The initial phase of activation has many similarities with allergic respiratory responses. However, in contrast to the situation in health, in allergic individuals the inflammatory response persists, and tolerance is not generated. The functional outcome of immunity or tolerance in response to inhaled Ags can be influenced by both genetic and environmental factors that remain to be defined. The information reported in this study is relevant to understanding the regulation of Th2 immunity to aeroallergens and may be of practical value in the advancement of immunotherapy.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Daphne C. Tsitoura, Division of Immunology and Transplantation Biology, Department of Pediatrics (Room S303), Stanford University Medical Center, Stanford, CA 94305-5208. E-mail address:
3 Abbreviations used in this paper: i.n., intranasal; HEL, hen egg lysozyme.
Received for publication May 5, 1999. Accepted for publication June 25, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells. Science 265:1869./+ T cells or interferon (IFN)- in a murine model of allergen sensitization. J. Exp. Med. 187:721./ß-T cell receptor (TCR)+CD4-CD8- (NKT) thymocytes prevent insulin-dependent diabetes mellitus in nonobese diabetic (NOD)/Lt mice by the influence of interleukin (IL)-4 and/or IL-10. J. Exp. Med. 187:1047. R, and HLA-DR in nasal epithelial cells in allergic rhinitis. Am. J. Respir. Crit. Care Med. 153:1675.[Abstract]
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 Y. I. Koh, H. Y. Kim, E. H. Meyer, M. Pichavant, O. Akbari, T. Yasumi, P. B. Savage, R. H. DeKruyff, and D. T. Umetsu Activation of Nonclassical CD1d-Restricted NK T Cells Induces Airway Hyperreactivity in {beta}2-Microglobulin-Deficient Mice J. Immunol., October 1, 2008; 181(7): 4560 - 4569. [Abstract] [Full Text] [PDF]
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C. von Garnier, M. E. Wikstrom, G. Zosky, D. J. Turner, P. D. Sly, M. Smith, J. A. Thomas, S. R. Judd, D. H. Strickland, P. G. Holt, et al. Allergic Airways Disease Develops after an Increase in Allergen Capture and Processing in the Airway Mucosa J. Immunol., November 1, 2007; 179(9): 5748 - 5759. [Abstract] [Full Text] [PDF]
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C. Ebeling, T. Lam, J. R. Gordon, M. D. Hollenberg, and H. Vliagoftis Proteinase-Activated Receptor-2 Promotes Allergic Sensitization to an Inhaled Antigen through a TNF-Mediated Pathway J. Immunol., September 1, 2007; 179(5): 2910 - 2917. [Abstract] [Full Text] [PDF]
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 M. M. Anis, S. A. Fulton, S. M. Reba, C. V. Harding, and W. H. Boom Modulation of Naive CD4+ T-Cell Responses to an Airway Antigen during Pulmonary Mycobacterial Infection Infect. Immun., May 1, 2007; 75(5): 2260 - 2268. [Abstract] [Full Text] [PDF]
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M. E. Wikstrom, E. Batanero, M. Smith, J. A. Thomas, C. von Garnier, P. G. Holt, and P. A. Stumbles Influence of Mucosal Adjuvants on Antigen Passage and CD4+ T Cell Activation during the Primary Response to Airborne Allergen J. Immunol., July 15, 2006; 177(2): 913 - 924. [Abstract] [Full Text] [PDF]
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P. J. Dunford, N. O'Donnell, J. P. Riley, K. N. Williams, L. Karlsson, and R. L. Thurmond The Histamine H4 Receptor Mediates Allergic Airway Inflammation by Regulating the Activation of CD4+ T Cells. J. Immunol., June 1, 2006; 176(11): 7062 - 7070. [Abstract] [Full Text] [PDF]
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D. Alvarez, F. K. Swirski, T.-C. Yang, R. Fattouh, K. Croitoru, J. L. Bramson, M. R. Stampfli, and M. Jordana Inhalation Tolerance Is Induced Selectively in Thoracic Lymph Nodes but Executed Pervasively at Distant Mucosal and Nonmucosal Tissues J. Immunol., February 15, 2006; 176(4): 2568 - 2580. [Abstract] [Full Text] [PDF]
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S.-Y. Ko, H.-J. Ko, W.-S. Chang, S.-H. Park, M.-N. Kweon, and C.-Y. Kang {alpha}-Galactosylceramide Can Act As a Nasal Vaccine Adjuvant Inducing Protective Immune Responses against Viral Infection and Tumor J. Immunol., September 1, 2005; 175(5): 3309 - 3317. [Abstract] [Full Text] [PDF]
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C. von Garnier, L. Filgueira, M. Wikstrom, M. Smith, J. A. Thomas, D. H. Strickland, P. G. Holt, and P. A. Stumbles Anatomical Location Determines the Distribution and Function of Dendritic Cells and Other APCs in the Respiratory Tract J. Immunol., August 1, 2005; 175(3): 1609 - 1618. [Abstract] [Full Text] [PDF]
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J. Padilla, E. Daley, A. Chow, K. Robinson, K. Parthasarathi, A. N. J. McKenzie, T. Tschernig, V. P. Kurup, D. D. Donaldson, and G. Grunig IL-13 Regulates the Immune Response to Inhaled Antigens J. Immunol., June 15, 2005; 174(12): 8097 - 8105. [Abstract] [Full Text] [PDF]
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 Y. Chung, S.-H. Lee, D.-H. Kim, and C.-Y. Kang Complementary role of CD4+CD25+ regulatory T cells and TGF-{beta} in oral tolerance J. Leukoc. Biol., June 1, 2005; 77(6): 906 - 913. [Abstract] [Full Text] [PDF]
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M. Inobe and R. H. Schwartz CTLA-4 Engagement Acts as a Brake on CD4+ T Cell Proliferation and Cytokine Production but Is Not Required for Tuning T Cell Reactivity in Adaptive Tolerance J. Immunol., December 15, 2004; 173(12): 7239 - 7248. [Abstract] [Full Text] [PDF]
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 H. J. de Heer, H. Hammad, T. Soullie, D. Hijdra, N. Vos, M. A.M. Willart, H. C. Hoogsteden, and B. N. Lambrecht Essential Role of Lung Plasmacytoid Dendritic Cells in Preventing Asthmatic Reactions to Harmless Inhaled Antigen J. Exp. Med., July 6, 2004; 200(1): 89 - 98. [Abstract] [Full Text] [PDF]
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M. Apostolaki and N. A. Williams Nasal Delivery of Antigen with the B Subunit of Escherichia coli Heat-Labile Enterotoxin Augments Antigen-Specific T-Cell Clonal Expansion and Differentiation Infect. Immun., July 1, 2004; 72(7): 4072 - 4080. [Abstract] [Full Text] [PDF]
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 J.-G. Chai, E. James, H. Dewchand, E. Simpson, and D. Scott Transplantation tolerance induced by intranasal administration of HY peptides Blood, May 15, 2004; 103(10): 3951 - 3959. [Abstract] [Full Text] [PDF]
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Y.-S. Hahn, C. Taube, N. Jin, L. Sharp, J. M. Wands, M. K. Aydintug, M. Lahn, S. A. Huber, R. L. O'Brien, E. W. Gelfand, et al. Different Potentials of {gamma}{delta} T Cell Subsets in Regulating Airway Responsiveness: V{gamma}1+ Cells, but Not V{gamma}4+ Cells, Promote Airway Hyperreactivity, Th2 Cytokines, and Airway Inflammation J. Immunol., March 1, 2004; 172(5): 2894 - 2902. [Abstract] [Full Text] [PDF]
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 M. A. Aronica, S. McCarthy, S. Swaidani, D. Mitchell, M. Goral, J. R. Sheller, and M. Boothby Recall Helper T Cell Response: T Helper 1 Cell-resistant Allergic Susceptibility without Biasing Uncommitted CD4 T Cells Am. J. Respir. Crit. Care Med., March 1, 2004; 169(5): 587 - 595. [Abstract] [Full Text] [PDF]
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C. M. Schramm, L. Puddington, C. Wu, L. Guernsey, M. Gharaee-Kermani, S. H. Phan, and R. S. Thrall Chronic Inhaled Ovalbumin Exposure Induces Antigen-Dependent but Not Antigen-Specific Inhalational Tolerance in a Murine Model of Allergic Airway Disease Am. J. Pathol., January 1, 2004; 164(1): 295 - 304. [Abstract] [Full Text] [PDF]
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Z.-H. Cui, A. Joetham, M. K. Aydintug, Y.-S. Hahn, W. K. Born, and E. W. Gelfand Reversal of Allergic Airway Hyperreactivity after Long-term Allergen Challenge Depends on {gamma}{delta} T Cells Am. J. Respir. Crit. Care Med., December 1, 2003; 168(11): 1324 - 1332. [Abstract] [Full Text] [PDF]
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Y.-S. Hahn, C. Taube, N. Jin, K. Takeda, J.-W. Park, J. M. Wands, M. K. Aydintug, C. L. Roark, M. Lahn, R. L. O'Brien, et al. V{gamma}4+ {gamma}{delta} T Cells Regulate Airway Hyperreactivity to Methacholine in Ovalbumin-Sensitized and Challenged Mice J. Immunol., September 15, 2003; 171(6): 3170 - 3178. [Abstract] [Full Text] [PDF]
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M. K. Brimnes, L. Bonifaz, R. M. Steinman, and T. M. Moran Influenza Virus-induced Dendritic Cell Maturation Is Associated with the Induction of Strong T Cell Immunity to a Coadministered, Normally Nonimmunogenic Protein J. Exp. Med., July 7, 2003; 198(1): 133 - 144. [Abstract] [Full Text] [PDF]
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U. Raap, T. Brzoska, S. Sohl, G. Path, J. Emmel, U. Herz, A. Braun, T. Luger, and H. Renz {alpha}-Melanocyte-Stimulating Hormone Inhibits Allergic Airway Inflammation J. Immunol., July 1, 2003; 171(1): 353 - 359. [Abstract] [Full Text] [PDF]
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K. Takabayashi, L. Libet, D. Chisholm, J. Zubeldia, and A. A. Horner Intranasal Immunotherapy Is More Effective Than Intradermal Immunotherapy for the Induction of Airway Allergen Tolerance in Th2-Sensitized Mice J. Immunol., April 1, 2003; 170(7): 3898 - 3905. [Abstract] [Full Text] [PDF]
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S. C. Eisenbarth, D. A. Piggott, J. W. Huleatt, I. Visintin, C. A. Herrick, and K. Bottomly Lipopolysaccharide-enhanced, Toll-like Receptor 4-dependent T Helper Cell Type 2 Responses to Inhaled Antigen J. Exp. Med., December 16, 2002; 196(12): 1645 - 1651. [Abstract] [Full Text] [PDF]
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F. Kheradmand, A. Kiss, J. Xu, S.-H. Lee, P. E. Kolattukudy, and D. B. Corry A Protease-Activated Pathway Underlying Th Cell Type 2 Activation and Allergic Lung Disease J. Immunol., November 15, 2002; 169(10): 5904 - 5911. [Abstract] [Full Text] [PDF]
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J.-i. Masuyama, S. Kaga, S. Kano, and S. Minota A Novel Costimulation Pathway Via the 4C8 Antigen for the Induction of CD4+ Regulatory T Cells J. Immunol., October 1, 2002; 169(7): 3710 - 3716. [Abstract] [Full Text] [PDF]
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 D. C. Tsitoura, V. P. Yeung, R. H. DeKruyff, and D. T. Umetsu Critical role of B cells in the development of T cell tolerance to aeroallergens Int. Immunol., June 1, 2002; 14(6): 659 - 667. [Abstract] [Full Text] [PDF]
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K. A. Frauwirth, M.-L. Alegre, and C. B. Thompson CTLA-4 Is Not Required for Induction of CD8+ T Cell Anergy In Vivo J. Immunol., November 1, 2001; 167(9): 4936 - 4941. [Abstract] [Full Text] [PDF]
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A. G. Jarnicki, T. Tsuji, and W. R. Thomas Inhibition of mucosal and systemic Th2-type immune responses by intranasal peptides containing a dominant T cell epitope of the allergen Der p 1 Int. Immunol., October 1, 2001; 13(10): 1223 - 1231. [Abstract] [Full Text] [PDF]
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 A. D Dick, V. Sharma, and J. Liversidge Single dose intranasal administration of retinal autoantigen generates a rapid accumulation and cell activation in draining lymph node and spleen: implications for tolerance therapy Br J Ophthalmol, August 1, 2001; 85(8): 1001 - 1006. [Abstract] [Full Text] [PDF]
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H.-R. Jiang, N. Taylor, L. Duncan, A. D Dick, and J. V Forrester Total dose and frequency of administration critically affect success of nasal mucosal tolerance induction Br J Ophthalmol, June 1, 2001; 85(6): 739 - 744. [Abstract] [Full Text] [PDF]
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D. M. Walter, C. P. Wong, R. H. DeKruyff, G. J. Berry, S. Levy, and D. T. Umetsu IL-18 Gene Transfer by Adenovirus Prevents the Development of and Reverses Established Allergen-Induced Airway Hyperreactivity J. Immunol., May 15, 2001; 166(10): 6392 - 6398. [Abstract] [Full Text] [PDF]
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 L. Cohn Food for Thought . Can Immunological Tolerance Be Induced to Treat Asthma? Am. J. Respir. Cell Mol. Biol., May 1, 2001; 24(5): 509 - 512. [Full Text]
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 A. Hänninen, A. Braakhuis, W. R. Heath, and L. C. Harrison Mucosal Antigen Primes Diabetogenic Cytotoxic T-Lymphocytes Regardless of Dose or Delivery Route Diabetes, April 1, 2001; 50(4): 771 - 775. [Abstract] [Full Text]
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E. Prager, G. Staffler, O. Majdic, M. D. Saemann, S. Godar, G. J. Zlabinger, and H. Stockinger Induction of Hyporesponsiveness and Impaired T Lymphocyte Activation by the CD31 Receptor:Ligand Pathway in T Cells J. Immunol., February 15, 2001; 166(4): 2364 - 2371. [Abstract] [Full Text] [PDF]
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K. Dabbagh, Y. Xiao, C. Smith, P. Stepick-Biek, S. G. Kim, W. J. E. Lamm, D. H. Liggitt, and D. B. Lewis Local Blockade of Allergic Airway Hyperreactivity and Inflammation by the Poxvirus-Derived Pan-CC-Chemokine Inhibitor vCCI J. Immunol., September 15, 2000; 165(6): 3418 - 3422. [Abstract] [Full Text] [PDF]
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D. C. Tsitoura, S. Kim, K. Dabbagh, G. Berry, D. B. Lewis, and D. T. Umetsu Respiratory Infection with Influenza A Virus Interferes with the Induction of Tolerance to Aeroallergens J. Immunol., September 15, 2000; 165(6): 3484 - 3491. [Abstract] [Full Text] [PDF]
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M. Astori, C. von Garnier, A. Kettner, N. Dufour, G. Corradin, and F. Spertini Inducing Tolerance by Intranasal Administration of Long Peptides in Naive and Primed CBA/J Mice J. Immunol., September 15, 2000; 165(6): 3497 - 3505. [Abstract] [Full Text] [PDF]
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K. A. Frauwirth, M.-L. Alegre, and C. B. Thompson Induction of T Cell Anergy in the Absence of CTLA-4/B7 Interaction J. Immunol., March 15, 2000; 164(6): 2987 - 2993. [Abstract] [Full Text] [PDF]
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) or 100 µg of OVA
on 3 consecutive days were immunized i.p. with OVA in alum 10 days
(
), 2 mo (
), 3 mo (
), and 4 mo
(
) later. Spleen cells were taken form the mice 6 days after the
secondary challenge and assayed for proliferation in response to
increasing concentrations of OVA in vitro. Results are expressed as the
mean counts per minute (±SD) of triplicate cultures.
