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Departments of
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Pediatrics and
Internal Medicine and
Sealy Center for Molecular Sciences, University of Texas Medical Branch, Galveston, TX 77555
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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after OVA restimulation in vitro. Results from this study indicate that
the inductive phase of oral tolerance is preceded by Ag-specific T cell
activation in vivo, proliferation in the regional draining LNs, and
differentiation into a memory-like state. These results indicate that
Ag-directed differentiation occurs as a part of T cell tolerance
through anergy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study we focused on the induction phase of peripheral
tolerance resulting from orally encountered Ag. Due to a low T cell
precursor frequency in vivo, T cell proliferation in response to OVA
usually cannot be detected in unimmunized hosts. Therefore, we adopted
a system described by Kearney et al., in which limited numbers of
OVA-specific cells were transferred into syngeneic recipients (1). We
transferred lymph node (LN) cells from OVA TCR-transgenic (Tg) mice
into syngeneic BALB/c mice. As a result, 1–2% of the cells in the LNs
of recipient mice bear the clonotypic TCR encoded by the TCR-
and
-ß transgenes that together recognize OVA peptide 323–339, and their
presence was easily detected by a clonotypic mAb KJ126 (1, 3). The
responses of recipient mice immunized s.c. with OVA peptide in CFA were
compared with responses from mice fed with OVA in PBS. At several time
points after Ag encounter, lymphocytes from mesenteric and peripheral
(axillary and inguinal) LNs were harvested. Changes in the expression
of cell surface activation markers, proliferative responses, and
lymphokine synthesis of the Tg+ T cells were measured in
response to OVA stimulation. Results from this study demonstrate that
during the inductive phase of oral tolerance, anergy is preceded by
Ag-specific T cell activation, and that regional draining LNs
participate in Ag uptake and tolerance induction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DO11.10 (OVA-TCR) breeders were obtained from D. Y. Loh (4). They were maintained under specific pathogen-free conditions in the Animal Resource Facility at the University of Texas Medical Branch (Galveston, TX) and were fed an OVA-free diet. BALB/c mice (6–10 wk old from Harlan Sprague-Dawley, Indianapolis, IN) were used as recipients of Tg+ LN cells. The university animal use and care committee approved all procedures.
Adoptive transfer and 5,6-carboxy-succinimidyl-fluoresceine-ester (CFSE) labeling of donor cells
Peripheral (axillary, inguinal, and popliteal) and mesenteric LN lymphocytes from DO11.10 (OVA-TCR) Tg mice were isolated and washed in sterile PBS containing 1% normal mouse serum. An aliquot of cells was analyzed by flow cytometry to determine the percentage of CD4+ and Tg+ cells. Tg+ cells (3 x 106 in 100 µl of PBS) were transplanted into each recipient BALB/c mouse by tail vein injection. To directly follow the donor cells in recipient mice, CFSE (Molecular Probes, Eugene, OR) labeling was performed as previously described (5, 6). Briefly, lymphocytes from OVA TCR-Tg mice were resuspended in PBS containing 0.1% BSA (Sigma, St. Louis, MO) at 107 cells/ml and were incubated in a final concentration of 10 µM CFSE for 10 min at 37°C. Labeled cells were washed twice with PBS containing 0.1% BSA before they were resuspended in PBS for tail vein injection.
Feeding and immunization
Mice transplanted with Tg+ cells were divided into three groups. One day following the transfer, one group of mice was fed 100 mg of OVA (Sigma) in 250 µl of PBS. The second group was immunized s.c. with 125 µg of OVA peptide 323–339 in CFA. The peptide was synthesized by the Protein Chemistry Laboratory of the Sealy Center for Molecular Science (Galveston, TX). The control group received neither OVA preparation. Mice from each group were sacrificed at the indicated time intervals following Ag administration.
Proliferation assay
To quantify the in vitro proliferative response to OVA peptide 323–339, 105 lymphocytes from peripheral and mesenteric LNs were cultured in 200 µl of complete medium with 10% FCS (HyClone, Logan, UT), 50 µM 2-ME, 10 mg/ml folate, and 2 mM glutamine with the indicated concentrations of OVA peptide. Cells were cultured for 3 days, and 1 µCi of [3H]thymidine was added to the wells for the last 18 h of culture. Statistical analysis was performed by calculation of SDs, and p values were calculated by Student’s t test.
Determination of anergy was performed as described previously (2).
Since [3H]thymidine uptake per input cell reflects the
intensity of proliferation rather than the total number of
Tg+ cells, the counts per minute per input Tg+
cell was calculated to assess the level of anergy in T cells. For each
individual mouse, the percentage of Tg+ T cells, based on
FACS analysis (KJ126+ CD4+) was multiplied by
the number of cells per well (105) to give the total number
of Tg+ cells per well. The specific
[3H]thymidine incorporation (average of triplicate wells)
was divided by the number of Tg+ T cells per well to give
the counts per minute per Tg+ cell for each animal:
cpm/(% Tg+/100) x 105] =
cpm/Tg+ T cell.
mAbs and FACS analysis
Anti-transgene (Tg+) clonotypic mAb (KJ126) (3) was prepared from hybridoma cells. IgG purified by protein G chromatography (Pharmacia, Uppsala, Sweden) was FITC conjugated. Affinity-purified mouse IgG-FITC was used as a control for KJ126-FITC. Anti-CD4-Tricolor (clone CT-CD4) and control Rat-IgG2a Tricolor were purchased from Caltag (South San Francisco, CA). Anti-CD69 (clone H1.2F3), CD45RB (clone 16A), and CTLA-4 (clone UC10-4F10-11) were purchased from PharMingen (San Diego, CA). Hamster IgG-PE (clone A19-3) was used as an isotype control for intracellular staining of CTLA-4.
Most cell surface molecules were detected by staining 106 viable cells with the indicated conjugates for 20 min at 4°C in PBS/0.1% azide/1% BSA. Intracellular CTLA-4 (along with an isotype control mAb) was detected following 0.3% saponin permeabilization (7). Data from 50,000 size-gated lymphocytes were acquired on a FACScan (Becton Dickinson, Mountain View, CA) and analyzed with CellQuest software from Becton Dickinson.
ELISA for cytokines
IL-2 and IL-10 were measured by ELISA using paired mAbs specific
for corresponding cytokines according to the manufacturer’s
recommendations (PharMingen). Paired mAb clones specific for IL-2 and
IL-10 were JES6-1A12/JES6-5H4 and JES5-2A5/SXC-1, respectively. IFN-
was determined as follows: 96-well microtiter plates (Dynatech,
Chantilly, VA) were coated overnight at 4°C with 5 µg of purified
anti-IFN- (HB170) from PharMingen in 100 µl of carbonate
buffer, pH 8.0. The plates were washed with PBS containing 0.5%
Tween-20, blocked with 5% FBS in PBS, washed, and then incubated with
culture supernatants or rIFN- standard (PharMingen) overnight at
4°C. The plates were washed again and incubated with rabbit
anti-IFN- Ab (1/1000) for 1 h followed by peroxidase-conjugated
goat-anti-rabbit IgG (1/5000) for 45 min at 37°C. The enzyme
activity was determined colorometrically using
O-phenylenediamine dihydrochloride reagent (Sigma) and was
read at 492 nm.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When T cells from OVA TCR-Tg mice were transferred into normal histocompatible BALB/c mice, a small population of transgenic donor cells (CD4+ KJ126+) persisted in the LNs of recipient animals for an extended period (2, 8). We previously observed the induction of anergy following oral OVA feeding of the recipient mice. In the present study, we explored the early differentiative events and the role of regional draining LNs in the development of anergy.
BALB/c mice received 3 x 106 Tg+ cells
from OVA TCR-Tg mice (DO11.10) and were divided into groups that were
OVA/CFA immunized as positive controls, OVA-fed, or untreated. Mice
from each group were sacrificed at the indicated time intervals
following antigenic exposure, and their responses to antigenic
restimulation in vitro were determined using proliferation assays. Mice
from all three groups responded initially to OVA peptide restimulation
18 h after antigenic exposure (Fig. 1
). Lymphocytes from OVA-immunized mice
proliferated vigorously in response to antigenic exposure in vitro. By
72 h after antigenic stimulation, lymphocytes from OVA-immunized
mice were significantly more responsive than LN cells from animals
receiving OVA orally. No significant differences between OVA-fed and
control lymphocytes were observed until 96 h, when lymphocytes
from the OVA-fed mice were significantly less responsive to
restimulation in vitro than lymphocytes from untreated mice
(p < 0.05). The lack of response in the
OVA-fed group persisted despite increases in the proportion of
Tg+ T cells following feeding (Fig. 2A). The percentages of
Tg+ cells from pooled peripheral and mesenteric LNs in all
three groups were relatively uniform before cellular expansion (18 h).
The proportion increased steadily in OVA-immunized mice thereafter. In
OVA-fed animals, however, the proportion of Tg+ cells
peaked at 72 h and then began to decline (Fig. 2A).
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).
During the early priming phase T cells were not significantly deleted
in OVA-fed animals (Fig. 2A). To help elucidate the
mechanism(s) involved in the OVA-tolerance model, we devised a method
for measuring anergy by normalizing the T cell proliferation for the
number of Tg+ cells in each well (total counts per
minute/total number of Tg+ cells) (2). We reasoned that the
reduced proliferation per input Tg+ T cells should indicate
a state of anergy.
When proliferation was expressed as a function of input Tg+
cells in the in vitro cultures restimulated with 1 µM OVA peptide
323–339, Tg+ cells from immunized mice responded to a
greater extent than those of control and OVA-fed animals throughout the
study (Fig. 2B). While the proliferative responses of cells
from all three groups of animals gradually declined after the initial
antigenic priming, proliferation in OVA-fed mice decreased more quickly
than that in the immunized animals. These data suggest that oral
priming alone is sufficient to induce anergy as early as 48 h
after priming.
OVA-specific T cells undergo Ag-specific activation and proliferation in vivo in response to OVA feeding
Since activated T lymphocyte blasts are larger than resting T
lymphocytes, we examined the distribution of forward light scatter
(FSC) by T cells from recipient chimeric mice. Donor T cells that were
CD4+ Tg+ demonstrated a considerable increase
in frequency of large cells in response to OVA (Fig. 3A). Most lymphocytes from the
LNs that were derived from the recipient animal (Tg-) did
not respond with an increase in size whether they were CD4+
or CD4- (Fig. 3A). Kinetic analysis revealed
that the FSC increased in donor T cells (CD4+
Tg+) at 18, 48, and 96 h following antigenic exposure
(Fig. 3B). Recipient cells did not exhibit these changes at
those time points.
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and
Table I). Transgene-negative donor cells
in both immunized and fed groups served as internal controls and failed
to show cell division. Unprimed control mice did not respond to the Ag
with cell division. Thus, using both ex vivo and in vivo approaches in
this study, we were able to show that T cell activation and
proliferation accompanied functional unresponsiveness and was highly
specific to OVA (Figs. 3 and 4 and Table I).
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and Table I). Subcutaneous OVA
peptide stimulation caused T cells from peripheral LNs to divide
vigorously in all (six of six) immunized mice (Table I); this robust
proliferation was not found in the mesenteric LNs of any of these
animals. In contrast, T cells from OVA-fed mice proliferated
preferentially in mesenteric LNs (five of six) over their peripheral
LNs (two of six). The local Ag concentration may have an influence on
the degree of expansion in the different sites. However, tolerance is
achieved in the peripheral LNs as well as in mesenteric LNs in OVA-fed
animals. Rapid induction of T cell activation and memory markers after feeding OVA
Since the state of anergy is induced during the early priming
phase, we examined the initial events associated with tolerance.
Phenotypic changes in cell activation markers were determined on
OVA-specific T cells by flow cytometric analysis of the cell surface
expression of CD69 and CD45RB. Intracellular CTLA-4 levels in T cells
were also determined by flow cytometry (Fig. 5A).
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B). The expression
of this early T cell marker subsided gradually after 18 h in
OVA-immunized and OVA-fed mice (Table II). A subsequent decrease in the CD45RB
level also suggested a prior activation of T cells in both groups of
OVA-primed mice. Changes in CD69 and CD45RB in control animals were not
significant. These data suggest that T cells from both OVA-fed and
OVA-immunized animals were progressing to a phenotype of memory-like T
cells.
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A). Intracellular CTLA-4 levels were near
background in all three groups 18 h after antigenic exposure
(Table II). While CTLA-4 remained undetectable in control animals, its
levels were markedly increased in OVA-immunized and OVA-fed animals at
48 h after priming. Kinetic analysis of CTLA-4 expression
indicated that the up-regulation of CTLA-4 was transient and returned
to its initial level 96 h following antigenic exposure in all
groups. In summary, data from these experiments revealed rapid T cell
activation shortly after feeding. Sustained, but regulated, T cell
activation led these cells to progress to a memory-like phenotype. No
phenotypic differences between the OVA-immunized and OVA-fed mice were
detected that would aid in differentiation between the early events in
oral tolerance and systemic immunization. Production of cytokines during anergy induction
Since the proliferation and differentiation of resting T cells are
driven by IL-2 and are also influenced by many other cytokines (e.g.,
IFN-), the most important change in anergic T cells is their
inability to produce IL-2 when they encounter an Ag. To further examine
the early events after antigenic stimulation, we compared the cytokine
production by lymphocytes from OVA-immunized and -fed mice. T cells
from peripheral and mesenteric LNs (or pooled) were restimulated in
vitro with the OVA peptide. The production of IL-2, IFN-, and IL-10
was detected by sandwich ELISA and was sensitive down to approximately
32 pg/ml for IL-2, 45 pg/ml for IFN-, and 120 pg/ml for IL-10.
T cells from peripheral LNs in OVA-primed mice secreted large amounts
of IL-2 18 h following in vitro restimulation (Fig. 6A) whether fed OVA or
immunized s.c. with the peptide in CFA. In OVA/CFA-immunized animals,
IL-2 reached its peak concentration 72 h after priming and then
declined. By comparison, cells from OVA-fed mice produced much less
IL-2 at 72 h, resulting in levels no more than those in control
animals. While the kinetics of IL-2 secretion in mesenteric LNs (Fig. 6B) were reminiscent of those in peripheral LN responses,
mesenteric T cells from OVA-fed animals were more responsive to
restimulation at 72 and 96 h following feeding compared with their
peripheral counterpart. When T cells were pooled from peripheral and
mesenteric LNs, reduced IL-2 production upon restimulation was also
observed in OVA-fed mice compared with that in OVA/CFA-immunized mice
(data not shown).
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after in vitro restimulation,
reaching a peak concentration at 72 h, after which IFN-
production declined (Fig. 7). LN cells
from OVA-fed mice produced much less IFN- than the immunized mice,
although the kinetics of IFN- production in the OVA-fed mice was
similar to that of their immunized counterparts.
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). T cells from control and
OVA-fed mice did not secrete or accumulate great amounts of IL-10 in
response to restimulation. While mesenteric T cells in OVA-immunized
and fed animals appeared to secrete slightly more IL-10, the difference
between these groups and the control group was not significant by
Student’s t test. In another experiment lymphocytes were
pooled from peripheral and mesenteric LNs. In that case, again, only
OVA-immunized animals produced increased amounts of IL-10 (data not
shown).
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, and
IL-10 upon in vitro restimulation. While both OVA-immunized and -fed
animals underwent T cell activation and proliferation shortly upon
priming, LN T cells from OVA-fed mice secreted much less IL-2, IFN-,
and IL-10 than immunized mice (
Figs. 6–8). Normalization for
variations in the proportions of Tg+ T cells prior to
restimulation did not alter the results (data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The work presented here addresses the induction phase of peripheral
tolerance resulting from orally encountered OVA. By 48 h,
OVA-specific T cells from Ag-fed animals no longer responded in vitro,
and T cell proliferation became virtually undetectable by 96 h.
The reduced proliferative capacity of T cells from OVA-fed animals
cannot be attributed to a lack of priming, since sufficient priming was
indicated by activation and proliferation of Tg+ T cells in
tolerized animals (
Figs. 2–5 and Tables I and II). This was not due to
a lack of OVA-specific T cells, but rather resulted from the failure of
Tg+ T cells to actively proliferate in response to Ag in
vitro (Fig. 2). This observation is supported by a study in which T
cell tolerance was induced by i.v. injections of OVA peptide (8) as
well as by our own findings with oral tolerance challenged with OVA/CFA
immunization (2). OVA-specific T cells persisted for extended periods
of time in tolerized mice, and the poor accumulation of T cells in the
LNs correlated with reduced DNA synthesis, pointing to the functional
impairment rather than the death of proliferating T cells. In our
previous study we found that both clonal deletion and anergy
contributed to Ag-specific nonresponsiveness 12 days after initial OVA
feeding (2).
The initial stages of tolerance induction of CD4+ T cells involve a primary activation state, characterized by CD69 expression in 3–6 h as well as increases in cell size and cell division. A similar finding of CD69 induction has been observed in an elegant system using class II MHC/cytochrome c (Cyt c) tetramers to detect Cyt c-specific T cells in TCR transgenic mice (5C.C7) (15). Most transgenic T cells expressed CD69 within 6 h of a single fed dose of Cyt c. Both studies showed rapid up-regulation of CD69 in all lymphoid sites tested. We further demonstrate by CFSE labeling of transferred T cells that activation accompanied by cell division occurred primarily in the mesenteric LNs after feeding OVA, while functionally, lymphocytes in both peripheral and mesenteric LNs became tolerized. Results from this study suggest that anergy is preceded by Ag-specific T cell activation during the inductive phase of oral tolerance, supporting the view that the unresponsive state is more likely to be associated with an inherent response defect than with immune deviation or suppression.
Evidence of activation led to the question of the sustained phenotype of the remaining anergic T cells. CD45RB is a cell surface phosphatase expressed at high levels on naive T cells. Expression is decreased on memory and effector CD4+ T cells compared with that on their naive progenitors (16, 17). In our study OVA-activated T cells progress to a memory phenotype by virtue of the reduced CD45RB expression (17). Costaining of CFSE with CD45RB on Tg+ gated T cells from OVA-fed mice suggested that the loss of CD45RB expression is due to dilution during cell division, as cells with the least CFSE intensity exhibited concomitantly lower CD45RB expression (not shown).
The results from this study also reflect on the site where T cell tolerance takes place after OVA administration to the gastrointestinal tract. While the specific site of tolerance induction has not been clearly defined, the GALT, specifically Peyer’s patches (PP) and lamina propria, has been proposed (18). PP of OVA TCR-Tg mice have increased synthesis of Th2 cytokines IL-4, IL-10, and TGF-ß, indicating a predominately Th2-type response rather than unresponsiveness in GALT (19). However, these studies were performed in animals that had a high precursor frequency of OVA-specific T cells and thus differ from the environment in the DO11.10-BALB/c chimeras, which have only about 1% Tg+ T cells in lymphoid organs and even fewer in PP. In a normal precursor environment, tolerance induction may instead occur within local draining (mesenteric) LNs. We previously found no evidence of trafficking of OVA-specific CD4+ T cells to the small intestine intraepithelial or lamina propria compartments (2) or to PP (N. Van Houten unpublished observations). In the present study antigenic priming clearly took place in both mesenteric and peripheral LNs shortly after OVA feeding. Yet, T cell activation and proliferation in these sites were not accompanied by the production of IL-2 and IL-10 upon restimulation. This observation is consistent with the report that orally administered Ag can travel systemically (20) and induce tolerance at sites other than the GALT. Recent work using Cyt c TCR transgenic mice demonstrated that the orally administered Cyt c enters the bloodstream and is rapidly distributed to secondary lymphoid organs (mesenteric LNs and the spleen), where it can trigger T cell responses (15). Peripheral tolerance that is induced by orally administered OVA could also be explained by split tolerance, where Ags introduced solely to the gastrointestinal tract have distinctive local effects. Oral Ag frequently elicits a local Ab response in the intestinal lamina propria while at the same time producing a state of systemic tolerance manifest as a diminished response to the same Ag if it is administrated in immunogenic form elsewhere in the body. This unique antigenic tolerance may be important in avoiding allergy to Ags in food, while the inhibition of systemic response prevents self destruction of the host tissues processing Ag.
The mechanisms of oral tolerance induction are probably due to the nature of Ag presentation. Although dendritic cells are generally accepted as immunostimulatory APCs in the initiation of immune responses, the role of these cells in T cell sensitization and tolerization remains controversial. OVA-specific T cells can be sensitized by exposure to adoptively transferred OVA-loaded dendritic cells (21, 22). However, in situ expansion of dendritic cells in mice after administration of Flt3 ligand enhances tolerance induction (23). There is accumulating evidence that it is the qualitative nature of the interaction between APC and T cells that determines whether tolerance is induced. The role of B cells in T cell sensitization has also been examined (24). OVA-specific T cells can be sensitized by HEL-specific B cells supplied with Ag containing both T and B cell epitopes (OVA/HEL); however, this T/B interaction does not occur when Ags are administered in separate soluble forms (24). The form, site, and dose of the Ag dictate the fine microenvironmental influence of T cell migration and sensitization.
The two-signal theory of T cell stimulation (25) has led to the investigation of CD28 and its counterpart CTLA-4 in the induction of T cell signaling and tolerance (9). Given the immunosuppressive role of CTLA-4 engagement in T cell stimulation (26, 27), we hypothesized that the kinetics or quantity of expression of CTLA-4 would differ between anergic and sensitized T cells. Although blockage of CTLA-4 signaling prevents tolerance induction in i.v. and oral tolerance (28, 29), we found that the expression of CTLA-4 on OVA-specific T cells after in vivo exposure was similar in groups of animals that were fed OVA or sensitized with the Ag in adjuvant. Thus, it appears that the decision between anergy and sensitization in vivo depends on the level of the signal delivered and not on variation in the expression of the molecule receiving the signal.
It has not been established exactly how cytokines participate in the decision of T cells to become anergic or sensitized in vivo. The differentiated anergic or sensitized T cells may express differential cytokine gene products or cytokine receptors once the functional commitment has been made. Although IL-10 has been associated with immunosuppressive activity in some systems (30, 31, 32, 33), it can also restore proliferative responses in tolerized T cells (34). IL-10 is synthesized in the spleens of OVA-TCR recipient mice that were fed low, but not high, doses of OVA (35). It can also be expressed in PP in OVA TCR-Tg mice fed OVA (19). Conversely, oral tolerance has been achieved in mice treated with anti-IL-10 (36). Thus, our findings that IL-10 is not induced in peripheral LNs upon restimulation in lymphocytes from orally tolerized mice are consistent with its potential immunostimulatory role in immune responses (37, 38).
The most remarkable difference in lymphocytes between OVA-fed and
-immunized animals was the production of IFN- after restimulation in
vitro. Lymphocytes from OVA-immunized animals produced considerable
levels of IFN- upon restimulation in vitro, while those from OVA-fed
animals produced significantly less. In a study of splenocytes from
OVA-TCR recipient mice (35), IFN- was detected at 2 days after
feeding but was at much lower levels at 10 and 20 days. Their most
striking finding was the production of TGF-ß in the spleens of
animals fed both high and low doses of Ag.
We have found that the mechanism of oral tolerance induction is parallel in many way to that of i.v. induced tolerance. This finding provides a potential opportunity to draw from the results of other studies of i.v. tolerance in further defining oral tolerance. However, it should be appreciated that oral exposure is routine for dietary Ags and is being explored extensively for protective immunizations and potential tolerance against autoantigens and transplantation Ags (39, 40, 41, 42). Thus, further studies of the mechanisms of induction of oral tolerance have extensive practical implications in the development and application of mucosal vaccines and in the treatment of inflammation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nancy Van Houten, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0366. E-mail address:
3 Abbreviations used in this paper: GALT, gut-associated lymphoid tissue; LN, lymph node; Tg, transgenic; Tg+, OVA specific as identified by KJ1-26 mAb binding; CFSE, 5,6-carboxy-succinimidyl-fluoresceine-ester; OVA-TCR, DO11.10 OVA-transgenic mice; FSC, forward light scatter; Cyt c, cytochrome c; PP, Peyer’s patch.
Received for publication September 28, 1998. Accepted for publication March 1, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A. C. Nathwani, A. M. Davidoff, H. Hanawa, Y. Hu, F. A. Hoffer, A. Nikanorov, C. Slaughter, C. Y. C. Ng, J. Zhou, J. N. Lozier, et al. Sustained high-level expression of human factor IX (hFIX) after liver-targeted delivery of recombinant adeno-associated virus encoding the hFIX gene in rhesus macaques Blood, August 13, 2002; 100(5): 1662 - 1669. [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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C. Baron, I. McMorrow, D. H Sachs, and C. LeGuern Persistence of Dominant T Cell Clones in Accepted Solid Organ Transplants J. Immunol., October 15, 2001; 167(8): 4154 - 4160. [Abstract] [Full Text] [PDF]
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 A. George-Chandy, K. Eriksson, M. Lebens, I. Nordstrom, E. Schon, and J. Holmgren Cholera Toxin B Subunit as a Carrier Molecule Promotes Antigen Presentation and Increases CD40 and CD86 Expression on Antigen-Presenting Cells Infect. Immun., September 1, 2001; 69(9): 5716 - 5725. [Abstract] [Full Text] [PDF]
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A. L. Meyer, J. Benson, F. Song, N. Javed, I. E. Gienapp, J. Goverman, T. A. Brabb, L. Hood, and C. C. Whitacre Rapid Depletion of Peripheral Antigen-Specific T Cells in TCR-Transgenic Mice After Oral Administration of Myelin Basic Protein J. Immunol., May 1, 2001; 166(9): 5773 - 5781. [Abstract] [Full Text] [PDF]
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 L. M. Godsel, K. Wang, B. A. Schodin, J. S. Leon, S. D. Miller, and D. M. Engman Prevention of Autoimmune Myocarditis Through the Induction of Antigen-Specific Peripheral Immune Tolerance Circulation, March 27, 2001; 103(12): 1709 - 1714. [Abstract] [Full Text] [PDF]
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H. N. Shi, H. Y. Liu, and C. Nagler-Anderson Enteric Infection Acts as an Adjuvant for the Response to a Model Food Antigen J. Immunol., December 1, 2000; 165(11): 6174 - 6182. [Abstract] [Full Text] [PDF]
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 A. Hayday and J. L. Viney The Ins and Outs of Body Surface Immunology Science, October 6, 2000; 290(5489): 97 - 100. [Abstract] [Full Text]
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 K. M. SMITH, A. D. EATON, L. M. FINLAYSON, and P. GARSIDE Oral Tolerance Am. J. Respir. Crit. Care Med., October 1, 2000; 162(4): S175 - 178. [Abstract] [Full Text] [PDF]
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E. Blanas, G. M. Davey, F. R. Carbone, and W. R. Heath A Bone Marrow-Derived APC in the Gut-Associated Lymphoid Tissue Captures Oral Antigens and Presents Them to Both CD4+ and CD8+ T Cells J. Immunol., March 15, 2000; 164(6): 2890 - 2896. [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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C. Desvignes, N. Etchart, J. Kehren, I. Akiba, J.-F. Nicolas, and D. Kaiserlian Oral Administration of Hapten Inhibits In Vivo Induction of Specific Cytotoxic CD8+ T Cells Mediating Tissue Inflammation: A Role for Regulatory CD4+ T Cells J. Immunol., March 1, 2000; 164(5): 2515 - 2522. [Abstract] [Full Text] [PDF]
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) from
recipient mice were measured 0–6 h after feeding. CD69 expression was
detected on OVA-TCR+ lymphocytes in both peripheral and
mesenteric LNs 3–6 h after feeding OVA. Analyses from individual mice
are paired vertically.
