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Departments of
*
Medicine,
Microbiology and Immunology, and
Ophthalmology, University of Kentucky Medical Center, Lexington, KY 40536; and
§
Microbiology and Immunology, University of Rochester Cancer Center, Rochester, NY 14642
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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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4ß7
integrin-expressing T cells (6, 7). The mechanism of Ag
transport across the epithelial cells into the conjunctival mucosa is
unknown. Although some investigators have discussed the presence of M
cells in the conjunctival epithelium (5), convincing data
demonstrating their existence in CALT have not yet been forthcoming
(8). Nevertheless, the conjunctiva contain lymphoid
elements common to other mucosal tissues and appear to be a part of a
common mucosal immune system.
Infectious agents entering through mucosal sites generally elicit
immune responses with a Th2 characteristic and dominated by IgA Ab
production (6, 9). On the other hand, the predominant
response to inert, T-dependent Ags is one of tolerance (10, 11). The phenomenon of oral tolerance has been extensively
studied, and multiple mechanisms appear to be involved, including
active suppression and anergy (12). Ag delivered to the
nasal-associated lymphoid tissue (NALT) (13) or the
bronchial-associated lymphoid tissue (14, 15) has also
been shown to induce a state of tolerance. This has led to the
suggestion that the common mucosal-associated lymphoid tissues have
evolved specialized mechanisms to generate T cell tolerance toward
mucosally delivered Ags. Although extensive analysis has been done on T
cells tolerized as a result of oral tolerance, studies are hampered by
the difficulty in controlling the site and dosage of Ag coming in
contact with the intestinal mucosa. Nevertheless, two classes of
tolerized T cells have been described depending on the dose of Ag used
to feed the mice. Low dose feeding induces regulatory cells capable of
suppression mediated by Th2 cytokines and TGF-, while high dose
feeding induces a state of anergy (12). One study using
TCR transgenic T cells in an adoptive transfer model provided more
direct evidence of clonal anergy among the Ag-specific T cells, but
failed to demonstrate active regulatory cells (16).
Despite these studies, major questions still remain concerning the
sequence of events resulting in Ag-specific T cell tolerance in mucosal
tissues. Furthermore, it is not clear whether the mechanisms of
tolerance induction are the same for all mucosal sites. Clearly, the
intestinal mucosa differs from other mucosal sites in terms of the
shear load of antigenic stimulus, and therefore, it is important to
evaluate other mucosal sites as well.
In this study, we demonstrate that application of Ag to the conjunctival mucosa induces potent immunological tolerance at doses considerably less than required for oral tolerance. Using a T cell hybridoma assay for APC and a TCR adoptive transfer system, we localized the major anatomical location of APC presentation and T cell recognition of conjunctival administered Ag to the submandibular lymph node (LN). Finally, we demonstrate that the continuous toleragenic application of Ag to the conjunctiva induces initial T cell priming and clonal expansion in the submandibular LN over a 10-day period, but ultimately results in a population of T cells that are anergic in vivo and in vitro. These findings suggest that tolerance in the form of T cell anergy can result from the continuous presentation of Ag in a specific LN draining the mucosal site of Ag contact.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DO11.10 transgenic mice were originally produced by Murphy et al. (17) and kindly provided by Dr. Marc Jenkins (University of Minnesota, Minneapolis, MN) with permission from Dr. Dennis Loh (Hoffman-LaRoche, Nutley, NJ). These mice have been backcrossed over 15 generations to BALB/c, and are currently being bred in our specific pathogen-free facility according to National Institutes of Health and American Association for the Accreditation of Laboratory Animal Care (AAALAC) guidelines. BALB/c mice were purchased from Charles River (Raleigh, NC) under a contract with the National Cancer Institute. The s.c. Ag injections described below were conducted under general anesthesia using Metophane (Pittman-Moore, Mundelein, IL).
Conjunctival administration of Ag and proliferative responses in BALB/c mice
BALB/c mice received various doses of OVA or PBS administered topically in a volume of 2.5 µl to the conjunctiva of both eyes twice per day for 10 days, or a single application, as indicated in the figure legends. Four days following the last Ag administration, or 14 days following a single injection, OVA (100 µg) in 0.1 ml of 1:1 CFA was injected s.c. in the back evenly distributed at the nape of the neck and both flanks. Seven days post-OVA/CFA immunization, cells from axillary, brachial, and inguinal LN were combined and incubated at 5 x 105 cells/well in 96-well plates containing OVA (25–100 µg/ml) in RPMI media supplemented with 10% FCS, 10 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5 x 10-5 M 2-ME in a humidified incubator with 7% CO2 at 37°C. Cultures were incubated for 5 days with the addition of 1 µCi/well [3H]thymidine for the final 24 h. Cells were harvested onto filters, and the incorporated cpm determined on a Packard Matrix 96 direct beta counter (Downers Grove, IL). Results are expressed as the mean cpm ± SEM of triplicate cultures.
Measurement of delayed-type hypersensitivity (DTH) responses by ear swelling
OVA or PBS was administered to the conjunctiva of groups of five mice, as described in the figure legends. On day 14, OVA (100 µg) in 0.1 ml of 1:1 CFA was injected s.c. in the back and flanks. A group of unmanipulated (naive) BALB/c mice served as a negative control. On day 28 (14 days after the CFA injection), OVA (100 µg in PBS) or PBS alone was injected in a volume of 10 µl into the pinnae of the right or left ear, respectively. Ear swelling was measured 24 and 48 h later and expressed as the mean (±SD) difference between right and left ears for groups of five mice.
Measurement of APC-presenting OVA in vivo
A T cell hybridoma specific for
OVA323–339/I-Ad and
harboring an IL-2-lacZ fusion gene was produced by fusion of
the BWZ.36 T cell hybridoma line with CD4+ T
cells derived from the DO11.10 TCR transgenic mouse. BWZ.36 is a
variant of BW5147 transfected with the IL-2 promoter-lacZ
fusion construct (18). A hybridoma subclone, BDZ 26.2, was
selected and was confirmed to express lacZ only in the
presence of OVA323–339 and
I-Ad-bearing APC (19). To assay for
APC-presenting OVA, 5 x 105 BDZ
cells/well were plated in triplicate in 96-well plates along with
5 x 105 LN or spleen cells/well or
dissociated tissue from mice injected with
OVA323–339. As a positive control,
OVA323–339 (10 µg/ml) was added to wells
containing BDZ cells only, or wells containing BDZ cells plus LN,
spleen, or tissue. The cells were cultured for 18 h at 37°C and
then fixed and stained with 5-bromo-4 chloro-3-indolyl
-D-galactoside (X-Gal), as
previously described (19, 20). Activated cells were
identified as blue staining cells, indicating expression of
ß-galactosidase, and quantitated by counting under an inverted
microscope. The number of blue staining cells/well varies linearly with
the number of added spleen cells over a three-log range in the presence
of OVA323–339, indicating that this method is
semiquantitative (data not shown).
Adoptive transfer of transgenic T cells
Adoptive transfer of CD4+, KJ1-26+ T lymphocytes from transgenic mice was performed as previously described (21, 22). Briefly, LN and spleen cells from D011.10 transgenic mice were treated with anti-CD8 mAb (hybridoma supernatant from clone 83.12.5) and rabbit complement, and the percentage of KJ1-26+, CD4+ T cells was determined by flow cytometry. A cell suspension containing 2.5 x 106 KJ1-26+, CD4+ T cells was injected i.v. by tail vein in a volume of 0.2 ml of HBSS into unirradiated BALB/c mice. Adoptive transfer recipients were used within 10 days of transfer. In some experiments, DO11.10 cells were stained immediately before transfer with the fluorescein-based dye 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), as previously described (23).
Inoculation of Ag into adoptive transfer recipients
For s.c. injections, anesthetized mice (three in each group) were injected in the nape of the neck and in both flanks with a total of 100 µg of OVA323–339 (synthesized by the University of Southern California Microchemical Facility, Los Angeles, CA) in 0.1 ml 1:1 CFA or IFA on day 0. For i.v. administration, 100 µg OVA323–339 in 0.2 ml dH2O was administered by tail vein on day 0. For conjunctival application, PBS or 25 µg OVA323–339 in 2.5 µl dH2O was applied to the conjunctiva once per day. This was accomplished by restraining an unanesthetized mouse and applying 2.5 µl of the OVA solution directly to the open eye using a microliter pipettor. In some cases, 100 µg of OVA323–339 in 0.1 ml CFA was administered s.c. in the back on day 14.
On the days indicated in the text, mice were euthanized, and the submandibular, cervical, axillary, inguinal, and mesenteric LN or spleen removed separately for analysis. The individual LN and spleen of each of the three mice were combined for flow cytometry and proliferation assays.
Flow cytometry
KJ1-26 mAb (clonotypic, anti-TCR) (24) was purified from KJ1-26 hybridoma supernatant using a protein A-Sepharose column. KJ1-26 mAb was either biotinylated using a protein biotinylation kit (Amersham, Arlington Heights, IL), or FITC conjugated (25). All other Abs were obtained from PharMingen (San Diego, CA). For two-color flow cytometry, LN or spleen cells pooled from three mice were incubated with anti-CD4 PE and KJ1-26 FITC, washed twice, and analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) collecting 50,000 events. For three-color flow cytometry, LN or spleen cells were incubated with KJ1-26 FITC, anti-CD4 PE, and anti-CD44 biotin, followed by incubation with quantum red-streptavidin (Sigma, St. Louis, MO). A gate was drawn around the CD4+, KJ1-26+ cells or the CD4+, KJ1-26- cells, and the fluorescence intensity of the CD44 staining was then determined on these populations. For CFSE experiments, cells were stained with CD4 PE and biotinylated KJ1-26, followed by avidin-quantum red.
Proliferation and IL-2 assays
For adoptive transfer recipients, proliferation assays were performed as described for BALB/c mice alone, except that 2 x 105 cells/well were used. For the measurement of IL-2, cells were incubated as above, except that Neutridoma (Boehringer Mannheim, Indianapolis, IN) was substituted for FCS. At 24 and 48 h, supernatant was analyzed for IL-2 production by ELISA (22).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether conjunctival application of Ag could induce
tolerance, we applied different doses of OVA directly to the
conjunctiva and measured OVA-specific T cell responsiveness. Low doses
of OVA or PBS were applied to the conjunctiva twice per day for 10
days, or a single large dose was applied once. All mice were then
immunized with OVA/CFA s.c. on day 14, and T cell proliferation to
either OVA or PPD in the draining LN was assessed on day 21. The data
in Fig. 1
show the proliferative
responses of individual mice to optimal concentrations of either OVA or
PPD. Because the degree of proliferation in response to CFA
immunization can vary significantly in individual mice, the PPD
response serves as an internal control for the response to OVA/CFA
immunization in each mouse. The average proliferative response to OVA
was significantly reduced compared with the average PPD response for
all groups of mice that received conjunctival OVA, but not in mice
receiving PBS. Surprisingly, we found that quite low doses (2.5 or 25
µg) of OVA induced potent tolerance if applied twice daily for 10
days (Fig. 1, A–C). To determine whether a single dose of
OVA could induce tolerance, larger doses of OVA were applied to both
eyes one time, and the mice were immunized 14 days later. A single
application of 2.5 mg induced partial tolerance, while 10 mg induced
almost complete tolerance (Fig. 1, D and E). The
DTH response to OVA was also investigated. Application of 25 µg of
OVA to the conjunctiva daily for 10 days resulted in a significantly
depressed DTH response following immunization and challenge (Fig. 1F). These data demonstrate that application of Ag to the
conjunctival mucosa induces a state of tolerance to a subsequent
immunizing dose of Ag.
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Given that Ag introduction through the conjunctival mucosa induces
tolerance, we wanted to determine the anatomic location of Ag
presentation to T cells following conjunctival OVA application. To do
this, we utilized a T cell hybridoma, BDZ 26.2, that expresses
ß-galactosidase only upon specific recognition of
OVA/I-Ad complexes on APC (19).
OVA323–339 peptide was applied to the
conjunctiva of both eyes, and the LN and spleen were then tested for
the presence of OVA323–339-bearing APC 24 h
later using the BDZ 26.2 cell line. In addition, because there is
drainage from the conjunctiva to the nasal sinuses via the
naso-lacrimal duct, we evaluated the presence of
OVA323–339-bearing APC in the NALT
(26), and the conjunctiva itself. The only tissue showing
significant numbers of APC-presenting OVA was the submandibular LN
(Fig. 2). We also investigated other time
points ranging from 2 to 96 h after administration of
OVA323–339 and, while 24 h was optimum for
OVA-bearing APC in the submandibular LN, we did not observe
APC-presenting OVA323–339 in the other tissues
at any of these time points (data not shown). Thus, these data
establish the submandibular LN as the major site of Ag presentation
following Ag application to the conjunctiva.
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To determine the effect of conjunctival Ag administration on the
Ag-specific T cell, we utilized an adoptive transfer system in which
OVA323–339-specific T cells derived from the
DO11.10 transgenic mouse can be physically tracked using the clonotypic
KJ1-26 mAb (21, 22). BALB/c mice received 2.5 x
106 KJ1-26+,
CD4+ T cells from DO11.10 TCR transgenic mice
i.v. and were used for experiments within 10 days. Adoptive transfer
recipients received either daily OVA323–339 (25
µg) applied conjunctivally, OVA323–339/CFA
s.c. in the back, or no Ag. LN and spleen cells were obtained on days 3
and 6 following each route of OVA323–339
administration, and the presence of CD4+,
KJ1-26+ T cells analyzed by flow cytometry (Figs. 3 and 4).
KJ1-26+, CD4+ T cells were
not detected in normal BALB/c mice and represented 
0.3% of LN or
spleen cells of naive adoptive transfer recipients up to 10 days
posttransfer (Fig. 3, A and B). As we
(22) and others (21) have previously shown,
s.c. administration of OVA323–339/CFA in the
back resulted in a large increase in KJ1-26+ T
cells in the draining axillary and inguinal LN at day 6 (Fig. 3C). Following OVA323–339 applied
conjunctivally, we observed Ag-specific T cell accumulation in the
submandibular LN at day 3, with a persistence of the accumulation at
day 6. However, no increase in KJ1-26+ cells was
observed in cervical LN (Fig. 3, D–F). A similar analysis
was performed on other distant LN or spleen and the results presented
as the percentage of KJ1-26+ cells in those
tissues at days 3 and 6 (Fig. 4). No increase in
KJ1-26+ cells was found in other lymphoid tissue
other than the submandibular LN.
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the CFSE staining in the naive CD4+,
KJ1-26+ cells found within the submandibular LN
was extremely bright and consisted of a single major peak. Following
conjunctival OVA application, the CD4+,
KJ1-26+ cells within the submandibular LN
increased in number 6.5-fold in 3 days, and the CFSE staining showed
a pattern indicative of multiple cell divisions. By counting the peaks,
it is possible to resolve discreet cell populations that range in the
number of cell divisions from 1 to 7, with an average of 5 to 6 cell
divisions. Thus, application of OVA to the conjunctival mucosa resulted
in OVA-specific T cell clonal expansion within the submandibular LN,
rather than redistribution of cells to that site. Furthermore, the
location of KJ1-26+, CD4+ T
cell clonal expansion coincides exactly with the location of Ag-bearing
APC identified by the T cell hybridoma assay, further defining the
submandibular LN as the primary draining LN from the conjunctival
mucosa.
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). As previously shown, a
single i.v. injection of OVA induced a rapid increase in the number of
KJ1-26+ cells, which peaked on day 3, followed by
a rapid decline and reaching the level of the uninjected adoptive
transfer mice by day 10. Also confirming previous results, injection of
OVA323–339 in CFA s.c. induced clonal expansion
that peaked at a later time than the i.v. group, but was maintained at
a significantly elevated level through day 14. Conjunctival application
of OVA induced a rapid increase in KJ1-26+ cells
like the i.v. group, but the expanded numbers of
KJ1-26+ cells were maintained through day 14,
like the CFA group. Thus, even though no adjuvant was present, the
continuous daily application of OVA323–339
resulted in a sustained clonal expansion of the
KJ1-26+ cells. In agreement with this, these
cells were found to show an activated phenotype, as assessed by an
increase in CD44 staining (Fig. 7).
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To assess the functional capacity of the expanded
KJ1-26+ cells following conjunctival
administration of OVA323–339, LN or spleen cells
were tested for their ability to proliferate and secrete IL-2 in
response to OVA323–339 stimulation in vitro.
Various LN and spleen cells from adoptive transfer mice were analyzed 6
days after initiation of daily conjunctival application of
OVA323–339. The results in Fig. 8 show strong proliferation and IL-2
secretion from submandibular and cervical LN cells in response to
OVA323–339. At the cell density used (2 x
105 per well), proliferation in this system
is due to the transferred KJ1-26+ cells and not
to other host-derived OVA-specific T cells present in the adoptive
transfer recipient (21, 22). The fact that the cervical
and, to a lesser extent, inguinal LN cells showed enhanced
proliferation in this assay but no clonal expansion by day 6 may
indicate that some of the conjunctivally applied
OVA323–339 is reaching more distant LN at levels
sufficient to prime the DO11.10 cells, but not induce perceptible
clonal expansion at the time points analyzed. Alternatively,
KJ1-26+ cells initially activated in the
submandibular LN may have recirculated to other LN following initial
clonal expansion. These results demonstrate that primary exposure of Ag
applied to the conjunctiva in a manner that induces tolerance
nevertheless results in the stimulation of Ag-specific T cells in the
draining LN that are functional in vitro, at least at this early time
point.
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Our results in Fig. 1
showed that proliferative responses to s.c.
OVA/CFA were diminished in BALB/c mice that had previously received
soluble OVA323–339 conjunctivally. To determine
the in vivo behavior of the Ag-specific T cells under similar
conditions, adoptive transfer mice were given
OVA323–339 conjunctivally for 10 days, or as a
single i.v. injection, and then immunized with
OVA/CFA323–339 14 days later. The accumulation
of KJ1-26+ cells in the LN draining the site of
OVA/CFA injection was then determined 7 days later (Fig. 9). In adoptive transfer mice that
received PBS conjunctivally, normal clonal expansion of the
KJ1-26+ cells was observed in the DLN following
OVA/CFA s.c. However, clonal expansion of KJ1-26+
cells in the LN draining the site of OVA/CFA injection was dramatically
reduced in adoptive transfer mice that received prior
OVA323–339 conjunctivally. In agreement with
previous studies, clonal expansion of KJ1-26+
cells was also reduced in adoptive transfer mice that received a prior
i.v. injection of OVA323–339, a
well-characterized tolerogenic route of injection (21). To
determine whether the remaining Ag-specific T cells in the LN draining
the site of OVA/CFA immunization were functionally competent, their
ability to proliferate to OVA323–339 in
vitro was determined and the results calculated per
KJ1-26+ cell added to the culture (Table I). In mice tolerized by administration
of OVA323–339 conjunctivally or by i.v.
injection, the KJ1-26+ T cells in the LN draining
the site of OVA/CFA immunization proliferated poorly to
OVA323–339 compared with mice that received PBS
conjunctivally. In contrast, LN cells from mice that received a primary
injection of OVA/IFA s.c. showed a heightened ability to proliferate,
indicating priming in this case. The fact that
KJ1-26+ cells were present, but unable to
proliferate, suggests that those cells were functionally impaired,
i.e., anergic.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The fact that APC-presenting OVA/I-Ad in vivo were only found in the submandibular LN and not NALT or mesenteric LN rules out that tolerance in our system was due to drainage of Ag through the naso-lacrimal duct and association with NALT or GALT. Rather, our results suggest that conjunctivally applied Ag drains directly to the submandibular LN and, to a lesser extent, the cervical LN. Recently, Wolvers et al. (27) have identified the same two LN as the principal LN draining the nasal cavity. (Although we have given them different names, our designation of submandibular LN corresponds to their designation of superficial cervical, and our designation of cervical corresponds to their designation of internal jugular.) Furthermore, they have shown that these two LN possess intrinsic properties promoting nasal tolerance not shared with other peripheral LN. Thus, it is possible that conjunctival tolerance and nasal tolerance operate via the same mechanism, by priming T cells within the same draining LN.
Although it ultimately results in tolerance, conjunctival
administration of OVA peptide induces initial priming and clonal
expansion of the KJ1-26+ cells within the
draining LN of adoptive transfer mice. The CFSE experiments clearly
showed that these cells underwent multiple rounds of cell division in
vivo. They were also functional in vitro, as assessed by proliferation
and IL-2 secretion. Their activated phenotype was also confirmed by
CD44 staining. These results are also in agreement with our previous
study showing priming of the KJ1-26+ cells
following intraocular injection of OVA323–339
(22). Why then does tolerance develop after repeated
conjunctival application of Ag? Our results demonstrate that the
KJ1-26+ T cells remain partially responsive to
repeated Ag stimulation via the conjunctiva for a short period of time
(Fig. 6). However, the fact that the number of
KJ1-26+ cells in the submandibular LN steadily
declined over the 10-day period, in spite of continuous peptide
application, indicates that these cells were progressively becoming
refractory to Ag stimulation. By day 14, these cells were largely
unresponsive to Ag challenge, whether in soluble form i.v. (data not
shown) or in CFA s.c. (Table I). This unresponsiveness to secondary
challenge in a distant s.c. site indicates that the conjunctival Ag
treatment was effective in tolerizing the majority of
KJ1-26+ cells in the adoptive transfer mouse,
presumably due to recirculation of the T cells from the submandibular
LN to other sites, and the likely systemic dispersion of some of the
peptide during the 10-day treatment.
In their initial description of this adoptive transfer model, Kearney et al. (21) demonstrated that i.v. administration of peptide induced early clonal expansion of the KJ1-26+ cells, but that these cells became refractory to secondary Ag stimulation in vivo and in vitro. Similar results were also demonstrated following peptide administration in IFA i.p. (28). Also using the same adoptive transfer model, Van Houten and Blake (16) showed that feeding of large quantities of OVA resulted in the systemic clonal expansion of KJ1-26+ cells, but that these cells subsequently became refractory to secondary stimulation. Similarly, several studies in other systems have also provided evidence that CD4+ T cell tolerance requires initial Ag-specific T cell priming (21, 22, 29, 30). Our results extend these studies to the conjunctival mucosa and collectively suggest that tolerance can result from systemic exposure and priming of Ag-specific T cells to soluble Ag, regardless of the route of Ag administration. In light of recent data showing that tolerance can be induced in B cell knockout mice (31), this initial priming most likely results from Ag presentation by dendritic cells in the LN and spleen.
In contrast to the initial priming and clonal expansion of the
KJ1-26+ T cells in response to conjunctival OVA
application, the expansion of KJ1-26+ T cells in
response to a secondary immunogenic OVA/CFA immunization was suppressed
and the remaining KJ1-26+ T cells were refractory
to stimulation in vitro. This phenomenon has been noted previously for
i.v. and i.p. tolerance (28, 32). The suppression of
secondary clonal expansion following initial priming could be due to
several, not mutually exclusive mechanisms. First, the initial primed
cell population could induce regulatory T cells capable of
down-regulating the secondary response to Ag in CFA. These cells could
either be KJ1-26+ cells polarized to a Th2
phenotype (30, 31, 33, 34, 35, 36, 37), a TGF-ß-secreting cell type
(11), or a separate suppressor cell derived from the host
that directly suppresses the KJ1-26+ cell.
However, using a variety of in vitro and in vivo approaches, we have
not detected any regulatory cells, suppressive cytokines, or bystander
suppression during conjunctival tolerance (data not shown). Similarly,
bystander suppression was not observed in the DO11.10 adoptive transfer
system after i.v. tolerance (32, 38). Second, the initial
priming of the Ag-specific cells could alter the traffic pattern of the
cells due to alterations in the levels of adhesion molecules such as
L-selectin. This could result in the increased migration of Ag-specific
T cells from the LN draining the site of secondary immunization to the
peripheral tissues in tolerized mice. However, this scenario would not
explain the observed inhibition of the DTH response, nor the defect in
the proliferative ability of cells from tolerized mice. Another
possibility is that the repeated exposure of the
KJ1-26+ T cells to Ag results in continuous
apoptosis of the KJ1-26+ cells, which has been
shown to be important in tolerance induction (39).
Although it is likely that apoptosis of the
KJ1-26+ T cells occurs as a normal consequence of
activation-induced cell death, we have found no evidence that apoptosis
contributes to tolerance in this system. Thus, we have been unable to
induce tolerance by the injection of large numbers of irradiated
(apoptotic) or unirradiated KJ1-26+ cells (R.
Egan and J. Woodward, unpublished observations). The trivial
explanation that continuous apoptosis depletes available
KJ1-26+ cells is ruled out by our demonstration
that KJ1-26+ cells are present at an elevated
level in the tolerized mice before immunization with OVA/CFA (Fig. 6).
Finally, repeated priming of the Ag-specific T cells with soluble Ag in
the absence of adjuvant could have the effect of anergizing the T cells
in vivo. Although T cell clonal anergy has been clearly demonstrated to
occur in vitro as a consequence of TCR signaling in the absence of
costimulation (40), it is much more difficult to
demonstrate in vivo. Recently, using the DO11.10 adoptive transfer
system, the Jenkins group has provided compelling evidence that the
cells that are unresponsive following i.v. tolerance have in fact
undergone prior clonal expansion and persist in vivo (32, 38). These results provide the strongest evidence to date that
the Ag-specific, CD4+ T cell can be anergized in
vivo as a consequence of soluble peptide exposure. A similar conclusion
was reached by Van Houten et al. in their model of oral tolerance
(16). Our results showing that the
KJ1-26+ cells were indeed present but
hyporesponsive in vivo and in vitro following secondary peptide
exposure extend this model to a system in which the in vivo clonal
expansion and subsequent anergy are largely restricted to a single pair
of LN draining the conjunctiva.
Collectively, our results and those of others suggest that repeated, systemic exposure of T cells to their cognate Ag in the absence of adjuvant or "danger" signals (41) induces initial T cell priming and clonal expansion, but eventually results in an anergic phenotype. Our results support a model whereby Ag applied to the conjunctiva is rapidly absorbed and drains to the submandibular LN. We propose that the subsequent mechanism of tolerance induction is similar if not identical to that which occurs following other routes of systemic Ag delivery, i.e., i.v. (21), i.p. (28), oral (42), and intraocular (43) routes. Thus, we support a model whereby these different anatomical sites of Ag delivery are viewed as facilitating systemic Ag exposure to T cells, primarily within LN and spleen. In this model, the mechanism of T cell tolerance would be the same, and only depends on the dose, timing, and affinity of Ag. It will therefore be critical to understand the signals in vivo that render T cells unresponsive after initial priming with Ag.
Dua et al. demonstrated that conjunctival instillation of retinal Ags in rats reduced the severity of experimental autoimmune ureitis and suggested that this was due to Ag-specific tolerance (44). The present study demonstrates that CD4+ T cell tolerance does indeed occur through a mechanism involving clonal anergy. Our results show that the Ag dosage required to induce conjunctival tolerance is considerably less than required for oral tolerance (12). Furthermore, the dosage can be controlled much more precisely and Ag application is much easier. Therefore, it is possible that conjunctival tolerance may have some clinical usefulness in inducing Ag-specific tolerance in autoimmune disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jerold G. Woodward, Department of Microbiology and Immunology, University of Kentucky Medical Center, Lexington, KY 40536-0084.
3 Abbreviations used in this paper: CALT, conjunctival-associated lymphoid tissue; CFSE, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester; DTH, delayed-type hypersensitivity; IFA, immunofluorescence assay; LN, lymph node; NALT, nasal-associated lymphoid tissue; PPD, purified protein derivative; X-Gal, 5-bromo-4-chloro-3-indolyl -D-galactoside.
Received for publication August 16, 1999. Accepted for publication February 22, 2000.
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P. Murapa, S. Gandhapudi, H. S. Skaggs, K. D. Sarge, and J. G. Woodward Physiological Fever Temperature Induces a Protective Stress Response in T Lymphocytes Mediated by Heat Shock Factor-1 (HSF1) J. Immunol., December 15, 2007; 179(12): 8305 - 8312. [Abstract] [Full Text] [PDF]
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Z. F. H. M. Boonman, G. J. D. van Mierlo, M. F. Fransen, R. J. W. de Keizer, M. J. Jager, C. J. M. Melief, and R. E. M. Toes Maintenance of Immune Tolerance Depends on Normal Tissue Homeostasis J. Immunol., October 1, 2005; 175(7): 4247 - 4254. [Abstract] [Full Text] [PDF]
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S. Camelo, A. Shanley, A. S. P. Voon, and P. G. McMenamin The Distribution of Antigen in Lymphoid Tissues following Its Injection into the Anterior Chamber of the Rat Eye J. Immunol., May 1, 2004; 172(9): 5388 - 5395. [Abstract] [Full Text] [PDF]
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Z. F. H. M. Boonman, G. J. D. van Mierlo, M. F. Fransen, K. L. M. C. Franken, R. Offringa, C. J. M. Melief, M. J. Jager, and R. E. M. Toes Intraocular Tumor Antigen Drains Specifically to Submandibular Lymph Nodes, Resulting in an Abortive Cytotoxic T Cell Reaction J. Immunol., February 1, 2004; 172(3): 1567 - 1574. [Abstract] [Full Text] [PDF]
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S. Camelo, A. S. P. Voon, S. Bunt, and P. G. McMenamin Local Retention of Soluble Antigen by Potential Antigen-Presenting Cells in the Anterior Segment of the Eye Invest. Ophthalmol. Vis. Sci., December 1, 2003; 44(12): 5212 - 5219. [Abstract] [Full Text] [PDF]
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W. W. J. Unger, F. Hauet-Broere, W. Jansen, L. A. van Berkel, G. Kraal, and J. N. Samsom |
) or PPD (
) for 5 days, and proliferation was
determined by [3H]thymidine incorporation during the last
18 h. Results represent mean cpm ± SEM of triplicate
cultures. Each pair of bars represents the response of an individual
mouse. All samples had proliferative responses of less than 2500 cpm in
the absence of Ag (not shown). LN cells from mice immunized with CFA
alone did not show proliferation to OVA (not shown). The average
response of all mice within a group is indicated by solid (PPD) and
dotted (OVA) lines. The average OVA response was significantly
(p < 0.001) reduced compared with the PPD response
in all groups except A (denoted by *).
F, Groups of five mice received either primary
(1o) conjunctival applications of OVA (+) or PBS (-) as in
group B above, followed by a secondary (2o)
immunization with OVA/CFA (+) or nothing (-) on day 14. Mice were then
challenged in the ear with OVA on day 28, and ear swelling was measured
24 h later. *, Significant (p < 0.001)
decrease in ear swelling compared with the control.
)
or a single dose of 100 µg OVA323–339 applied to the
conjunctiva of both eyes (
), conjunctivally 25 µg/eye
twice per day (
), or no Ag (
). LN cells were prepared from pooled
axillary and inguinal LN (•, 
, 
, 