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Departments of
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Oral Biology and
Microbiology, Immunobiology Vaccine Center, University of Alabama Medical Center, Birmingham, AL 35294;
Department of Clinical Bio-regulatory Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan; and
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Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2) and Th2-type
(e.g., IL-4, IL-5, IL-6, and IL-10) responses were maintained in
CD40L-/- mice orally immunized with OVA, whereas these
cytokine responses in CD40L+/+ mice were significantly
reduced. In addition, splenic CD4+ T cells from
CD40L-/- mice orally immunized with OVA provided B cell
help in Ag-specific Ab-forming cells when the cells were cultured with
naive B cells in the presence of Ag and CD40L-transfected cell lines.
In contrast, an identical culture condition containing splenic
CD4+ T cells from orally tolerized CD40L+/+
mice did not exhibit helper activity. Taken together, these findings
indicate that CD40L and CD40 interactions are essential for the
induction of systemic T cell unresponsiveness to orally administered
Ag. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-12 production 6, 7 . The administration of repeated low doses or a high dose of soluble Ag via the mucosal route (e.g., oral or nasal) can result in the induction of IgA Ab responses in the mucosal compartment, with an otherwise unresponsiveness in the systemic immune compartment 8, 9 . This phenomenon was originally termed oral tolerance 10 ; however, a more generalized term, mucosally induced tolerance, was recently introduced 9 because Ag-specific unresponsiveness can be induced by the nasal as well as oral route 11 . It has been shown that the T cell compartment of the CD4 phenotype played important roles in the induction of systemic unresponsiveness to mucosally administered Ag 12, 13 . For example, CD4+ T cells anergized by a high dose of oral Ag were implicated in the induction of mucosally induced tolerance 14 . In vitro treatment with anti-CD8 mAb during oral immunization with Ag did not influence reductions in Ag-specific Ab or T cell proliferative responses 15, 16 . Finally, Ag-specific tolerance was induced in ß2 microglobulin gene-depleted mice given OVA orally 17 .
Costimulatory molecules may be of importance in the induction of systemic unresponsiveness. For example, one recent study reported that selective blockade of B7-CTLA-4 interactions completely inhibited systemic tolerance induced by a high dose of Ag 18 . Further, blockade of CD40L-CD40 interactions by anti-CD40L Ab results in tolerance to the hapten 2,4-dinitrofluorobenzene-induced, Th1-type of contact hypersensitivity 19 . Furthermore, anergic Th1-type cells expressed reduced levels of CD40L expression 20 . Despite these recent studies, the precise roles for costimulatory signals in the induction and maintenance of mucosally induced tolerance remain undefined.
In the present study, we assessed the role of CD40L-CD40 interactions for the regulation of T cell unresponsiveness in response to orally administered Ag using CD40L-/- mice. Our results show that CD40L-CD40 interactions are required for the induction of systemic T cell unresponsiveness, which follows when Ag is given by the oral route.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation and initial characterization of CD40L-/- mice have been described previously 4, 5 . CD40L-/- mice used throughout this study were kindly obtained from Dr. Charles R. Maliszewski at Immunex Research and Development Corporation (Seattle, WA) and maintained as random hybrids on a C57BL/6 x 129/J background. CD40L-/- and CD40L+/+ mice were genotyped by a standard PCR method. All mice were maintained in flexible Trexler isolators and have remained pathogen-Ab negative. At 5–6 wk of age, the mice were removed from the colony isolator unit, housed in microisolator cages in horizontal laminar flow cabinets, and provided sterile food and water ad libitum. The mice were between 7 and 10 wk of age at the beginning of individual experiments.
Immunization
To induce systemic unresponsiveness to the chosen Ag OVA (Fraction V; Sigma, St. Louis, MO), a standard oral administration protocol was used in this study 9 . In some experiments, hen egg white lysozome (HEL; Sigma) was also used as an additional model Ag. Mice were given 25 mg of Ag dissolved in 0.25 ml of PBS by gastric intubation. Control mice received oral PBS only. Seven days later, mice were immunized via the s.c. route with 100 µg of OVA or HEL emulsified in 100 µl of CFA (Difco Laboratories, Detroit, MI). The mice were sacrificed 14 days after the s.c. immunization to examine Ag-specific T cell responses.
Measurement of delayed-type hypersensitivity (DTH) responses
A standard protocol for the measurement of DTH responses was employed in this study 9 . Briefly, 20 µg of OVA or HEL in 20 µl of PBS was injected into the left ear pinna, and the right ear pinna received PBS as a control. Ear swelling was measured 24 h later with a dial thickness gauge (Ozaki Manufacturing, Tokyo, Japan). The DTH responses were expressed as the increase of ear swelling after Ag injection following subtraction of swelling in the control site.
OVA-induced splenic CD4+ T cell proliferation
The spleen was removed aseptically and single-cell suspensions were prepared in RPMI 1640 (Cellgro Mediatech, Washington, DC) containing HEPES buffer (15 mM), L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml). CD4+ T cells were purified by the magnetic activated cell sorter system (Miltenyl Biotec, Sunnyvale, CA) as described previously 21 . Briefly, cells were incubated in a nylon wool column (Polysciences, Warrington, PA) to remove B cells and macrophages. Enriched T cell populations were then incubated with biotinylated anti-CD4 (GK 1.5) followed by streptavidin-conjugated microbeads and passed through the magnetized column. The purified T cell fractions were >95% CD4+ and were >99% viable. Cells were resuspended in complete medium (RPMI 1640 containing 10% heat-inactivated FBS). Purified CD4+ cells (4 x 106 cells/ml) were cultured with or without 1 mg/ml of OVA or HEL in the presence of T cell-depleted irradiated (3000 rads) splenic APCs from naive F2 (C57BL/6 x 129/J) mice in 96-well or 24-well tissue culture plates (Corning Glass Works, Corning, NY) for 4 days at 37°C in a moist atmosphere of 5% CO2 in air. To measure Ag-specific T cell proliferation, 0.5 µCi of tritiated [3H]thymidine (Amersham, Arlington Heights, IL) was added for the final 18 h of incubation. The cells were harvested, and the amount of [3H]thymidine incorporation was determined by scintillation counting. In some experiments, culture supernatants were harvested after 2 and 4 days of the incubation and were then subjected to cytokine-specific ELISA. For cytokine-specific mRNA analysis, CD4+ T cells were harvested after 2 days of incubation and were then subjected to cytokine-specific semiquantitative RT-PCR assays.
Semiquantitative analysis of cytokine-specific mRNA
For evaluation of cytokine-specific mRNA levels in OVA-stimulated CD4+ T cells, a semiquantitative RT-PCR was employed 22 . Total RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroform extraction procedure and 25 µg/ml of extracted RNA was subjected to reverse transcriptase reaction using Superscript II Reverse Transcriptase (Life Technologies, Gaithersburg, MD). The cDNA from 5 ng of RNA was used for each cytokine-specific PCR. For the amplification of cDNA, 35 cycles of reaction programmed as 1 min at 95°C and 1 min at 60°C was performed. The PCR product was quantitated by capillary electrophoresis with the laser-induced fluorescence detection system (CE-LIF, LIF-P/ACE; Beckman Instruments, Fullerton, CA) as described previously 22 . The fluorescence content of each cytokine-specific RT-PCR product was expressed as the peak area of relative fluorescence light units. The level of amplified cytokine cDNA was normalized to the corresponding ß-actin levels as 100%.
Cytokine-specific ELISA
Cytokine levels of culture supernatants were measured by an
ELISA as previously described 9 . In brief, the immunoplates (Nunc,
Naperville, IL) were coated with anti-IFN- (R4–6A2),
anti-IL-2 (JES6–1A12), anti-IL-4 (BVD4–1D11), anti-IL-5
(TRFK-5), anti-IL-6 (MP5–20F3), or anti-IL-10 (JES5–2A5) mAbs
(PharMingen, San Diego, CA). After blocking with 3% BSA in PBS,
samples and serial two-fold dilutions of standards were added to
duplicate wells and incubated overnight at 4°C. The wells were washed
and incubated with biotinylated anti-IFN- (XMG1.2),
anti-IL-2 (JES6–5H4), anti-IL-4 (BVD6–24G2), anti-IL-5
(TRFK-4), anti-IL-6 (MP5–32C11), or anti-IL-10 (JES5–16E5)
mAbs, respectively. After incubation, peroxidase-labeled
anti-biotin mAb (Vector Laboratories, Burlingame, CA) was added and
developed with 1.1 mM 2,2'-azino-bis (3-ethylbenz-thiazoline-6-sulfonic
acid) (Sigma) in 0.1 M citrate-phosphate buffer, pH 4.2, containing
0.01% H2O2. Standard curves were generated
using mouse rIFN-, rIL-2, rIL-4, rIL-5, rIL-6 (Genzyme, Cambridge,
MA), or rIL-10 (PharMingen).
Th cell assay for B cell responses
To assess Th cell activity in the CD4+ T cell subset
from CD40L-/- mice given OVA orally, a modified in vitro
B cell culture system was employed 23 . In some experiments, murine L
cells stably transfected with plasmid expressing CD40L were used as a
source for costimulatory signal provided via the ligand 24 . Purified
splenic CD4+ T cells (2.5 x 106 cells/ml)
from CD40L-/- mice orally immunized with OVA or PBS were
cocultured with or without CD40L-expressing L cells (irradiated at
10,000 rads; 1 x 105 cells/ml) in the presence of
naive B cells (2.5 x 106 cells/ml), feeder cells, and
OVA (1 mg/ml) for 5 days at 37°C with 5% CO2 in air.
Nonadherent cells were harvested and then assessed for IgM, IgG, and
IgA Ab-forming cell (AFC) responses with an OVA-specific enzyme-linked
immunospot (ELISPOT) assay. In brief, 96-well nitrocellulose plates
(Millititer HA; Millipore, Bedford, MA) were coated with OVA (1 mg/ml)
overnight at 4°C and blocked with PBS containing 3% BSA. After
addition of appropriate dilutions of cells in RPMI 1640 containing 10%
FCS, the plates were incubated for 4 h at 37°C with 5%
CO2 in air. OVA-specific IgM, IgG, and IgA AFC were
detected with peroxidase-labeled anti-mouse µ-, -, and
-chain-specific Abs (Southern Biotechnology Associates, Birmingham,
AL) and visualized by adding 3-amino-9-ethylcarbazole in 0.1 M sodium
acetate buffer (Moss, Pasadena, MA). Spots representing AFCs
were counted with the aid of a stereo microscope.
Statistics
The data are expressed as the mean ± SEM and compared using an unpaired Mann-Whitney U test. The results were analyzed using the Statview II statistical program (Abacus Concepts, Berkeley, CA) for Macintosh computers and were considered to be statistically significant if p values were <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the potential role of CD40L-CD40 interactions for
the development of mucosally induced tolerance, CD40L-/-
and CD40L+/+ mice were given 25 mg of OVA or PBS orally and
then examined for DTH responses by measuring OVA-induced ear swelling
(Fig. 1
). Both CD40L+/+ and
CD40L-/- mice given PBS showed significant ear swelling
(50–70 µm). As expected, the levels of OVA-specific DTH responses
were significantly reduced in CD40L+/+ mice given oral OVA
in comparison to those mice given oral PBS. Conversely, no reductions
of OVA-specific DTH responses were observed in CD40L-/-
mice given oral OVA. Similar results were obtained when HEL was used as
Ag (Fig. 1).
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). CD4+ T
cells isolated from spleen of CD40L+/+ mice orally
immunized with OVA or HEL showed significantly lower Ag-specific
proliferative responses than did those of CD40L+/+ mice
given oral PBS. In contrast, identical levels of Ag-specific T cell
proliferative responses were seen in CD40L-/- mice given
oral Ags or PBS. Th1- and Th2-type cytokine responses are not down-regulated in CD40L-/- mice by prior oral administration of a high dose of OVA
Because the levels of Ag-specific DTH and T cell proliferative
responses were identical between CD40L-/- mice given Ag
or PBS by the oral route, it was important to assess cytokine synthesis
by Ag-specific splenic CD4+ T cells isolated from orally
treated CD40L-/- or CD40L+/+ mice. Levels of
Th1-type (IFN- and IL-2) and Th2-type (IL-4, IL-5, IL-6, and IL-10)
cytokines were significantly reduced in culture supernatants of splenic
CD4+ T cells from CD40L+/+ mice given oral OVA
when compared with those of mice given oral PBS (Fig. 2). These findings are consistent with
the fact that decreased T cell proliferative responses were also seen
in CD40L+/+ mice orally immunized with OVA. In contrast,
similar levels of Th1- and Th2-type cytokine responses were noted in
CD40L-/- mice given oral OVA or PBS. Further, it is
important to point out that the levels of IL-2 synthesis, where low
production is an indicator of T cell anergy 25, 26 , was maintained in
both groups of CD40L-/- mice (Fig. 2; 7.7 ± 0.2 vs
8.5 ± 0.3 U/ml).
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). Taken
together, these findings indicate that T cell unresponsiveness, which
is normally induced by a high dose of oral Ag, was not established in
mice deficient in the CD40L-CD40 signaling pathway. CD4+ T cells from orally treated CD40L-/- mice provide B cell help in the presence of CD40L transfectants
To further confirm the presence of intact T cell functions
in CD40L-/- mice after oral administration of a high dose
of OVA, Th cell functions for B cell responses were evaluated. Splenic
CD4+ T cells isolated from CD40L-/- mice were
cocultured with CD40L transfectants in the presence of OVA, APCs, and
naive B cells for 5 days. OVA-specific AFCs were assessed by the
ELISPOT assay. When appropriate costimulatory signals through
CD40L-CD40 were substituted by CD40L transfectants, CD4+ T
cells from CD40L-/- mice given oral OVA were able to
provide efficient Th cell function for the induction of OVA-specific
IgM and IgG AFCs. In contrast, splenic CD4+ T cells from
CD40L+/+ mice given oral OVA only provided low levels of B
cell help with OVA-specific IgM and IgG AFC responses when compared
with CD40L+/+ mice given oral PBS
(p < 0.05, Table I). Lower numbers of OVA-specific IgM and
IgG AFC were detected in the cultures containing CD4+ T
cells from CD40L-/- mice without CD40L transfectants or
with control murine L cells that did not express CD40L. In addition,
very low numbers of Ag-specific IgM and IgG AFC were seen in wells that
did not contain CD4+ T cells (Table I). These data indicate
that not only Ag-induced DTH, T cell proliferative, and cytokine
responses, but also Ag-specific Th cell functions for B cell responses
were maintained in CD40L-deficient mice even after oral administration
of a high dose of Ag.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is now generally agreed that mucosally induced tolerance is established and maintained at the level of T cells 13, 14 . Two distinct forms of T cell unresponsiveness, e.g., active suppression and clonal deletion and/or anergy, which were induced by low and high doses of mucosally administered Ag, respectively, have been proposed as major mechanisms for the induction of mucosally induced tolerance 8, 13, 26 . Repeated administration of low doses of Ag induced active suppression that was mediated by the production of suppressive cytokines such as TGF-ß, IL-4, and IL-10 8 . In contrast, a high dose of Ag elicited clonal deletion 13 and/or anergy, which was characterized by the absence of T cell proliferation and decreased IL-2 synthesis as well as IL-2R expression 25, 26, 27 . Oral administration of a high dose of OVA induced diminished levels of OVA-specific proliferative and cytokine responses including IL-2 synthesis by splenic CD4+ T cells in CD40L+/+ mice, suggesting that systemic T cell unresponsiveness is mediated by clonal anergy. Further, the fact that feeding of OVA failed to reduce T cell proliferation and cytokine synthesis in CD40L-/- mice indicates that CD40L-CD40 interactions play a critical role in the induction of systemic T cell unresponsiveness.
It has been reported that T cell anergy could be induced when T cells were activated without adequate costimulatory signals normally provided by APCs 18, 28 . Interestingly, however, our findings showed that T cell unresponsiveness could not be induced in the absence of costimulatory signals resulting from CD40L-CD40 interactions. These results are consistent with other findings that CD4+ T cell proliferative responses were maintained in CD40L-/- mice after priming with myelin basic protein in CFA 29 . Thus, CD40L-CD40 interactions could be critical for induction of T cell unresponsiveness. In this regard, previous studies have demonstrated that CD40L and CD40 interactions are involved in the regulation of CD80/CD86 expression by APCs 30 . In addition, the interaction of CD80/CD86 and CTLA-4 provide negative signals for the inhibition of activated T cells 31, 32 . One possible mechanism is that the lack of a CD40L and CD40 interaction leads to the subsequent loss of negative signals generated by interaction of CD80/CD86 with CTLA-4 in CD40L-/- mice. To support this view, a recent study showed abrogation of high-dose mucosally induced tolerance in mice treated with CTLA-4-Ig fusion protein 18 . Our results do suggest that the interactions of CD40L and CD40 are necessary for the expression of CD80/CD86 molecules that affect T cell activation or unresponsiveness; however, the relationship between T cell activation and unresponsiveness as determined by CD28 or CTLA-4 engagement of CD80/CD86 molecules remains to be defined.
Our results revealed that the levels of IFN- synthesis from
OVA-stimulated splenic CD4+ T cells were significantly
lower in CD40L-/- mice than those of the same genetic
background. In this regard, it was shown that CD40L-CD40 interactions
are more critical for the generation of Th1- than Th2-type responses.
Thus, these costimulatory interactions are associated with IL-12
secretion by both macrophages and dendritic cells for the subsequent
promotion of IFN- production by Th1 cells 7, 33 . In fact, recent
studies also demonstrated that the inability of CD40L-/-
mice to control a parasite infection was due to impaired IL-12
production 6, 34 . In support of this, our separate study demonstrated
that the levels of Th1-type (e.g., IFN- and IL-2) but not Th2-type
(e.g., IL-4, IL-5, IL-6, and IL-10) cytokines were significantly lower
in anti-CD3 Ab-stimulated splenic CD4+ T cells isolated
from naive CD40L-/- mice than from control mice
(unpublished data). Additionally, recent studies have suggested that
IFN- plays a central role for induction of mucosally induced
tolerance 9 . In support of this notion, initial priming of Th1-type
responses (e.g., IFN- synthesis) could occur before mucosally
induced tolerance is established 35 . Moreover, repeated oral feeding
of high doses of OVA to OVA TCR-transgenic mice resulted in a
IFN--dominant immune response in Peyer’s patches (PP) 36 . Based
upon these reports, a potentially interesting experiment would be
treatment of CD40L-/- mice with exogenous IFN-, which
may restore systemic T cell unresponsiveness. However, it also remains
possible that this cytokine treatment may fail totally, because an
appropriate Th1 induction pathway for oral tolerance may not be
triggered due to the lack of organized PP, an important inductive site
for the mucosal immune system in CD40L-/- mice.
Alternatively, IFN- treatment may enhance MHC class II expression by
intestinal epithelial cells, which leads to active Ag presentation for
Ag-specific immune responses instead of tolerance induction. Indeed, it
has been shown that enhanced MHC class II expression by intestinal
epithelial cells does occur in IFN--treated mice with subsequent
abrogation of mucosally induced tolerance 37 . Despite this
likelihood, our present findings suggest that another explanation for
the lack of mucosally induced tolerance in the CD40L-/-
mice fed a high dose of Ag could be their impaired ability to induce
Th1-type responses.
The other possible explanation for the lack of T cell unresponsiveness to orally ingested Ag in CD40L-/- mice is that these mice do not form distinct geminal centers in PP 4 . In this regard, it has been shown that oral immunization induced Ag-specific immune responses in both systemic and mucosal compartments, and these immune responses were strongly associated with Ag uptake and processing pathways in PP 38 . Thus, it remains possible that induction of systemic unresponsiveness to orally administered Ag requires the presence of normal immunocompetent cells in PP. To support this view, it has been shown that oral tolerance to a high dose of Ag was due to clonal deletion of T cells in the PP 13 . Thus, it will be important to determine whether lack of T cell tolerance in CD40L-/- mice is due to the absence of actual CD40L-CD40 interactions or to a loss of germinal center formation that occurred subsequent to the disruption of the CD40L gene. To address this important issue, we are currently investigating mucosally induced tolerance in mice treated with a lymphotoxin ß receptor Ig fusion protein in utero because this treatment resulted in the absence of PP but an otherwise normal lymph node development 39 .
It has been shown that TGF-ß is a key cytokine for the induction of
low-dose oral tolerance 8 . Evidence from studies in OVA-transgenic
mice has revealed increased production of this cytokine in PP of mice
with high-dose oral tolerance 36, 40 . However, a recent study in
TGF-ß1-/- mice demonstrated that this cytokine was not
essential for the induction of low- or high-dose oral tolerance 41 .
To further address a potential role for TGF-ß in mucosally induced
tolerance, it will be interesting to assess the levels of TGF-ß
produced in CD40L-/- mice fed a high dose of Ag.
According to recent findings, which showed that reduced IFN- or
IL-12 synthesis resulted in the enhancement of TGF-ß production 36, 42 , it can be predicted that increased TGF-ß production would most
likely occur in CD40L-/- mice, because these mice
manifest an impairment of Th1-type responses. Studies along these lines
are currently under investigation in our laboratory.
In summary, the present study showed that the absence of CD40L-CD40 interactions prevented the development of systemic T cell unresponsiveness which is normally induced by a high oral dose of Ag. Although the precise mechanisms for the role of CD40L-CD40 signal pathways in the induction of mucosally induced tolerance needs to be further investigated, our study suggests that regulatory signals transduced by CD40L-CD40 play an important role in the induction and maintenance of mucosally induced tolerance.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kohtaro Fujihashi, Department of Oral Biology, Immunobiology Vaccine Center, University of Alabama Culture, Medical Center, BBRB 761, Birmingham, AL 35294-2170. E-mail address:
3 Abbreviations used in this paper: AFCs, Ab-forming cells; CD40L, CD40 ligand; DTH, delayed-type hypersensitivity; ELISPOT, enzyme-linked immunospot; HEL, hen egg white lysozome; PP, Peyer’s patch.
Received for publication June 1, 1998. Accepted for publication October 28, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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knockout mice. J. Immunol. 160:1687.ß TCR+ intestinal intraepithelial lymphocytes: Th1- and Th2-type cytokine production by CD4+CD8- and CD4+CD8+ T cells for helper activity. Intern. Immunol. 5:1473. treatment. J. Immunol. 144:4163.[Abstract]
/ß complex is required for the development of peripheral lymphoid organs. J. Exp. Med. 184:1999. have opposing effects, while TGF-ß positively regulates its own production. J. Immunol. 160:5719.This article has been cited by other articles:
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  D. W. Smith and C. Nagler-Anderson Preventing Intolerance: The Induction of Nonresponsiveness to Dietary and Microbial Antigens in the Intestinal Mucosa J. Immunol., April 1, 2005; 174(7): 3851 - 3857. [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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S. Nierkens, P. van Helden, M. Bol, R. Bleumink, P. van Kooten, S. Ramdien-Murli, L. Boon, and R. Pieters Selective Requirement for CD40-CD154 in Drug-Induced Type 1 Versus Type 2 Responses to Trinitrophenyl-Ovalbumin J. Immunol., April 15, 2002; 168(8): 3747 - 3754. [Abstract] [Full Text] [PDF]
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 K. Fujihashi, T. Dohi, P. D. Rennert, M. Yamamoto, T. Koga, H. Kiyono, and J. R. McGhee Peyer's patches are required for oral tolerance to proteins PNAS, March 13, 2001; 98(6): 3310 - 3315. [Abstract] [Full Text] [PDF]
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L. M. Howard and S. D. Miller Autoimmune Intervention by CD154 Blockade Prevents T Cell Retention and Effector Function in the Target Organ J. Immunol., February 1, 2001; 166(3): 1547 - 1553. [Abstract] [Full Text] [PDF]
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T. Koga, J. R. McGhee, H. Kato, R. Kato, H. Kiyono, and K. Fujihashi Evidence For Early Aging in the Mucosal Immune System J. Immunol., November 1, 2000; 165(9): 5352 - 5359. [Abstract] [Full Text] [PDF]
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L. Liu, V. K. Kuchroo, and H. L. Weiner B7.2 (CD86) But Not B7.1 (CD80) Costimulation Is Required for the Induction of Low Dose Oral Tolerance J. Immunol., August 15, 1999; 163(4): 2284 - 2290. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or Ag (OVA or HEL) (
) followed by s.c. immunization
with OVA or HEL in CFA. The details of the protocol are described in
Material and Methods. The results of T cell
proliferative responses were expressed as cpm of the OVA- or
HEL-stimulated mice minus cpm of the medium-only cultures. Baseline
values for splenic CD4+ T cell proliferation in medium only
were always below 300 cpm. No detectable DTH or CD4+ T cell
proliferative responses were seen in mice fed Ag or PBS without
systemic immunization. The results expressed are the mean ± SEM
of three separate experiments containing five mice per group. (*,
p < 0.05 vs PBS orally immunized group).