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Center for Neurologic Diseases, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 following anti-CD3 stimulation.
CD25+CD4+ cells from fed mice suppressed the
proliferation of CD25-CD4+ T cells in vitro
more potently than CD25+CD4+ T cells isolated
from unfed mice, and this suppression was partially reversible by IL-10
soluble receptor or TGF- soluble receptor and high concentration of
anti-CTLA-4. With anti-CD3 stimulation,
CD25+CD4+ cells from unfed mice secreted
IFN-
, whereas CD25+CD4+ cells from fed mice
did not. Adoptive transfer of CD25+CD4+ T cells
from fed mice suppressed in vivo delayed-type hypersensitivity
responses in BALB/c mice. These results demonstrate an Ag-specific in
vivo method to activate CD25+CD4+ regulatory T
cells and suggest that they may be involved in oral
tolerance. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in
CD25+CD4+ T cell-mediated
suppression remains to be determined because
CD25+CD4+ T cells contain
mRNA for IL-4, IL-10, and TGF- (8).
Oral tolerance refers to systemic Ag hyporesponsiveness that occurs
after oral Ag administration and can occur by multiple mechanisms
(26, 27). One of the major mechanisms of oral tolerance is
the induction of regulatory CD4+ T cells that
mediate active suppression by producing immunomodulatory cytokines such
as IL-4, IL-10, and TGF-, with a unique role for TGF- (6, 28). Oral tolerance is effective in suppressing animal models of
autoimmune diseases, such as experimental autoimmune encephalomyelitis,
uveitis, arthritis, and diabetes, and is also being tested in human
autoimmune diseases (reviewed in Ref. 27). Transfer of CD4
T cells from myelin basic protein
(MBP)3-fed mice
prevents experimental autoimmune encephalomyelitis induced by
immunization with MBP in MBP transgenic (Tg) mice
(29).
Given that oral tolerance is a crucial immunologic process associated with the absorption and processing of proteins via the gut-associated lymphoid tissue and that CD25+CD4+ cells appear to be a biologically important class of regulatory cells, we asked whether oral Ag administration activated CD25+CD4+ regulatory T cells in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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OVA TCR Tg mice on the BALB/c background, clone DO11.10, which recognizes the 323–339 peptide fragment of OVA, were originally obtained from Dr. K. Murphy (30) and housed under specific pathogen-free conditions in the Animal Resource Facility of Harvard Medical School. Tg mice (6–8 wk old) were used throughout the studies. Tg mice were typed by staining peripheral blood leukocytes with FITC-conjugated anti-clonotype-spcific mAb KJ 1-26 (Caltag Laboratories, Burlingame, CA) and PE-conjugated anti-CD4 mAb (BD PharMingen, San Diego, CA). Female BALB/c mice (6–8 wk old) were purchased from The Jackson Laboratory (Bar Harbor, ME).
Immunization
DO11.10 or BALB/c mice were immunized s.c. at the flank with 100 µg of OVA (Sigma, St. Louis, MO) in 50 µl of PBS and 50 µl of CFA containing 200 µg of Mycobacterium tuberculosis (H37Ra; Difco, Detroit, MI), or i.v. injected with 25 mg of OVA once.
Induction of oral tolerance
OVA TCR Tg mice (6–8 wk old) were fed 20 mg/ml OVA or 20 mg/ml BSA (Sigma) in the drinking water for 5 days.
Abs and reagents
Abs used in cell staining were purchased from BD
PharMingen: biotinylated anti-CD25 (7D4), PE-conjugated anti-CD25
(PC61), PE- or FITC-conjugated anti-CD4 (L3T4), FITC-conjugated
anti-CD62 ligand (anti-CD62L) (MEL-14), FITC-conjugated anti-CD69
(H1.2F3), FITC-conjugated anti-CD44 (IM7), FITC-conjugated
anti-CD45RB (16A), purified anti-CD3
(145-2C11),
purified anti-CD28 (37.51), and purified anti-FcR.
Anti IL-10 and anti TGF-1 blocking Abs, recombinant mouse IL-10
soluble receptor (sR), recombinant human TGF- sRII/Fc chimera, human
IL-3 sR, and human TNFRI/Fc chimera were purchased from R&D
Systems (Minneapolis, MN).
Flow cytometric analysis
Three-color flow cytometry was performed as previously described (31). Briefly, 0.5–1 x 106 cells in 50 µl from spleens or lymph nodes, or purified T cells from spleens or lymph nodes, were incubated in staining buffer (PBS with 4% BSA and 0.1% sodium azide) for 5 min. The cells were then stained with biotinylated mAb in staining buffer for 30 min, washed twice, then incubated with a mixture of PE- and FITC-conjugated mAb (0.5 µg/sample), streptavidin-RED670 (1 µl/sample; Life Technologies, Rockville, MD), or FITC-conjugated streptavidin (BD PharMingen) for 30 min. The cells were washed twice and then fixed in PBS with 1% formaldehyde. The analysis was performed on a FACScan flow cytometer with CellQuest software (BD Biosciences, Mountain View, CA). All procedures were performed on ice until analysis.
For analysis of intracellular CTLA-4, sorted CD25+CD4+ or CD25-CD4+ T cells were fixed with Cytofix/Cytoperm solution (catalog no. 2075KK; BD PharMingen) according to the suggested protocol and then incubated with PE-conjugated anti-CD25 (1 µg/106 cells) on ice for 30 min in the dark. Cells were washed and analyzed immediately by flow cytometry.
Cell purification
Spleens and lymph nodes (axillary, inguinal, and mesenteric) were removed from OVA TCR Tg mice and prepared into single cell suspensions. CD4+ T cells were isolated using T cell subset columns (R&D Systems) according to the suggested protocol. To separate CD25+CD4+ T cells and CD25-CD4+ T cells, the enriched CD4+ T cells were incubated with biotin-conjugated anti-CD25 (10 µg/108 cells) in staining buffer on ice for 30 min, and washed twice. The cells were then incubated with FITC-conjugated streptavidin (10 µg/108 total cells) on ice for another 30 min. Finally, CD25+CD4+ T cells and CD25-CD4+ T cells were sorted by flow cytometry on a FACStar cell sorter (BD Biosciences). In adoptive transfer of experiments, purified CD4+ T cells were incubated with biotin-conjugated anti-CD25 (10 µg/108 cells) on ice for 30 min, followed by streptavidin MicroBeads (Miltenyi Biotec, Auburn, CA) for 15 min at 4°C. Magnetic separation was performed with a LS+ positive selection column according to the suggested protocol.
Preparation of APCs
Splenocytes from unfed OVA TCR Tg mice were incubated with anti-Thy 1.2 culture supernatants (HO-13.4) for 30 min on ice, followed by treatment with rabbit complement (Accurate Chemical and Scientific, Westbury, NY) for 45 min at 37°C. The procedure was repeated a total of two times for each experiment.
Proliferation assay
Proliferation assays were performed as previously described (32). Briefly, CD25-CD4+ T cells were cultured with 200 µg/ml OVA for 72 h in U-bottom 96-well plates at 5 x 104/well with the indicated numbers of CD25+CD4+ T cells and 1 x 105 cells/well of APC, in DMEM supplemented with 10% heat-inactivated FBS (BioWhittaker, Walkersville, MD), 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME (Sigma). Cultures were pulsed with 1 µCi of [3H]TdR per well (NEN, Boston, MA) 72 h later and harvested 16 h later. For splenocytes, cells were cultured in 96-well plates at 5 x 106 cells/ml in complete DMEM, pulsed with [3H]TdR 48 h later, and harvested 16 h later.
Cytokine ELISA
For cytokine assays, splenocytes were cultured at 1 x
106 cells/well in 200 µl of Ex Vivo 20 medium
(BioWhittaker) with various Ag concentrations. Supernatants were
collected after 24 h for IL-2, 48 h for IL-4, IL-10, and
IFN-, and 72 h for TGF-1. Purified
CD25+CD4+ and
CD25-CD4+ T cells were
cultured in 48-well plates (0.5 ml) at 2 x
106/ml in Ex Vivo 20 medium and stimulated with
10 µg/ml plate-bound anti-CD3 mAb alone or plus anti-CD28 mAb
(5 µg/ml). Supernatants were collected 48 h after culture.
Quantitative ELISA for IL-2, IL-4, IL-10, and IFN- were performed
using paired Abs and recombinant cytokines from BD PharMingen per the
manufacturer’s recommendations. TGF-1 was measured by TGF-1 Emax
ImmunoAssay System (Promega, Madison, WI). Briefly, the flat-bottom
96-well plates were coated overnight at 4°C with mouse
anti-TGF-1 mAbs in 100 µl of 0.1 N carbonate buffer, pH 8.2.
The plates were then washed three times with PBS containing 0.05%
Tween 20, and blocked with 1% BSA in PBS. After washing,
the plates were incubated with culture supernatants that were treated
with 2 µl of 1 N HCl at room temperature for 15 min followed by the
addition of 2 µl of 1 N NaOH overnight at 4°C. The plates were
washed again and incubated with rabbit anti-TGF-1 polyclonal
Ab for 2 h at room temperature, followed by anti-rabbit
IgG HRP for 1 h. Color was developed with one component
tetramethylbenzidine reagent (Kirkegaard and Perry
Laboratories, Gaithersburg, MD).
Adoptive transfer of CD25+CD4+ and CD25-CD4+ T cells and induction of delayed-type hypersensitivity (DTH) responses
CD25+CD4+ and CD25-CD4+ T cells from unfed or OVA-fed OVA TCR Tg mice were purified by MACS and immediately injected i.v. into 6- to 8-wk-old BALB/c mice at 1 x 106 cells/mouse. One day after transfer, the mice were immunized in the footpad with 100 µg of OVA in 0.1 ml of CFA containing 200 µg of M. tuberculosis. Thirteen days after immunization, mice received s.c. injections of 20 µl of OVA (1 mg/ml in PBS) in the left ear and 20 µl of PBS in the right ear. Ear thickness was measured 24 h later in a blinded fashion using a calipermeter (Mitutoyo, Osaka, Japan). Results are shown as the differences between the left and right ear thickness.
Statistical analysis
Differences in the percentages of T cells and ear thickness in mice were analyzed for significance using Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We asked whether oral administration of Ag was associated with
changes in CD25-CD4+ or
CD25+CD4+ T cells. To
address this question, 6- to 8-wk-old OVA TCR Tg mice were fed with 20
mg/ml OVA or BSA in the drinking water and sacrificed at day 6.
Lymphoid cells in inguinal lymph node (ILN), mesenteric lymph node
(MLN), Peyer’s patches (PP), and spleens from these mice were analyzed
by flow cytometry. We found a significant decrease in the percentage of
CD4+ T cells in OVA-fed mice in ILN, MLN, and
spleen, but not in the control mice fed BSA (Table I
). In terms of
CD25+ and CD25- cells, OVA
feeding resulted in significantly reduced numbers of
CD25-CD4+ T cells in
spleen, ILN, and MLN of OVA-fed mice. At the same time, the percentage
of CD25+CD4+ T cells
increased in OVA-fed mice compared with controls (Table I, Fig. 1A). PP from OVA-fed mice
showed no significant change in the percentage of
CD4+,
CD25-CD4+, or
CD25+CD4+ T cells at day 6,
as compared with control.
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B, there was a 3-fold increase in
CD25+CD4+ T cells within
the purified CD4+ T cells in OVA-fed mice as
compared with BSA-fed mice (13.3 vs 4.3%). We also found significantly
increased absolute numbers of
CD25+CD4+ T cells in the
spleens from OVA-fed mice as compared with those from unfed mice
(29.7 ± 8.9 x 105 vs 13.1 ±
4.4 x 105, p < 0.01).
Furthermore, this increase in the percentage of
CD25+CD4+ T cells persisted
in MLN and ILN from OVA-fed mice for as long as 4 wk postfeeding (Fig. 1C), at a time when the percentages of
CD25-CD4+ T cells returned
to their prefed values (62% in ILN and 59% in MLN). Similar
activation of CD25+CD4+ T
cells was observed when 1 mg of OVA323–339
peptide was used for feeding and in vitro stimulation (data not shown).
No significant changes were observed in OVA TCR Tg mice fed 20
mg/ml BSA.
To determine whether the changes we observed occurred on
clonotype-positive cells, we measured the percentage of
KJ1-26+ and
KJ1-26-CD25+ T cells
within the CD4 subset after feeding. Lymphoid cells from unfed (water),
BSA-fed, or OVA-fed mice were stained with anti-CD4, anti-CD25,
plus anti-clonotype TCR (KJ1-26). As shown in Table II, we found a significant increase in
the percentages of
KJ1-26+CD25+CD4+
T cells in ILN, MLN, and spleen from OVA-fed mice, whereas the
percentages of
KJ1-26-CD25+CD4+
T cells did not change significantly. Thus, oral administration of Ag
expanded clonotype-specific
CD25+CD4+ T cells.
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A,
CD25+CD4+ T cells increased
in the PP on days 1 and 2 of feeding after which they returned to
baseline levels. A large increase of
CD25+CD4+ T cells was
observed in the ILN beginning on day 3, at which time an increase in
the spleen and MLN was also seen. For
CD25-CD4+ T cells (shown
in Fig. 2B), a decrease was observed in all tissues
following feeding except for PP where cells increased on days 2 and 3,
after which they declined.
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Phenotypic characterization of CD25+CD4+ T cells purified from fed mice
To further characterize the
CD25+CD4+ T cells from
OVA-fed OVA TCR Tg mice, CD4+ T cells were
isolated on a T cell subset column and sorted on the basis of surface
CD25 expression. The resulting purity of the sorted
CD25+CD4+ T cells or
CD25-CD4+ T cells was
98%. We then tested the
CD25+CD4+ and
CD25-CD4+ populations for
expression of CD69, CD62L, and CD44 pre- and postfeeding. As shown in
Fig. 3, a higher proportion of
CD25+CD4+ T cells from fed
mice expressed CD69 as compared with the cells from unfed mice.
Approximately 60% of
CD25-CD4+ T cells
expressed CD69 after feeding. Both
CD25+CD4+ and
CD25-CD4+ T cells from fed
mice had down-regulation of CD62L and up-regulation of CD44 as compared
with unfed mice.
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To functionally characterize
CD25+CD4+ T cells from
OVA-fed mice, we measured proliferative responses in vitro and the
ability of CD25+CD4+ T
cells to act as regulatory cells. As shown in Fig. 4A, there were only minimal
proliferative responses in vitro when
CD25+CD4+ T cells were
stimulated with OVA plus APC, whereas
CD25-CD4+ T cells from fed
mice had prominent proliferative responses. Furthermore, although
CD25+CD4+ T cells from
OVA-fed mice showed minimal proliferative responses to OVA, they
completely suppressed the proliferation of
CD25-CD4+ T cells from fed
mice when both populations were cultured in a 1:1 ratio (Fig. 4A).
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B,
CD25+CD4+ T cells from
OVA-fed mice were more potent at suppressing the proliferation of
CD25-CD4+ T cells than
CD25+CD4+ T cells from
unfed mice. When 0.85 x 104
CD25+CD4+ T cells
from OVA-fed mice were added to 5 x
104
CD25-CD4+ cells, there was
a 75% decrease in proliferative responses, whereas with the same
number of CD25+CD4+ cells
from unfed mice, proliferative responses were decreased only by 12.5%.
CD25+CD4+ cells from fed
mice suppressed CD25-CD4+
cells from unfed mice (Fig. 4B) equally as well as they
suppressed CD25-CD4+ cells
from fed mice (Fig. 4A). CD25+CD4+ T cells from OVA-fed mice have regulatory properties in vivo
To assess the in vivo activity of
CD25+CD4+ regulatory T
cells generated by oral feeding, we tested whether
CD25+CD4+ T cells from
OVA-fed OVA TCR Tg mice could inhibit Ag-specific immune responses when
transferred into BALB/c mice. Purified
CD25+CD4+ or
CD25-CD4+ T cells (1
x 106) from OVA-fed mice were injected i.v. into
BALB/c mice. One day later, BALB/c mice were immunized in the footpad
with OVA/CFA. DTH responses and in vitro proliferative responses to OVA
were measured on day 14. As shown in Fig. 5A, DTH responses were
significantly suppressed in mice that received
CD25+CD4+ T cells from both
unfed and fed mice, but no significant effect was observed in those
that received CD25-CD4+ T
cells from unfed or OVA-fed mice. As shown in Fig. 5B, in
vitro proliferative responses of splenocytes to OVA were also
suppressed following transfer of CD25+CD4+ T
cells from both unfed and OVA-fed mice, whereas enhancement of
proliferation was observed when
CD25-CD4+ T cells from
both unfed and fed mice were transferred. Ag-specific proliferation in
the lymph node and secretion of IL-2 and IFN- in lymph node cells
and splenocytes were also suppressed in mice receiving
CD25+CD4+ cells from fed
mice. Furthermore,
CD25+CD4+ cells from
OVA-fed mice suppressed BSA responses in vitro by only 19% when added
to CD25-CD4+ cells from
BSA-immunized mice, but if OVA was added, in vitro suppression rose to
72% (Fig. 5C). This is consistent with reports that
suppression is triggered in an Ag-specific fashion, but then acts in an
Ag-nonspecific fashion (17).
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To further characterize the properties of
CD25+CD4+ T cells from fed
vs unfed mice, we analyzed the cytokine profiles of
CD25-CD4+ and
CD25+CD4+ T cells from
unfed and OVA-fed OVA TCR Tg mice. Cells were stimulated by plate-bound
anti-CD3 mAb. As shown in Fig. 6, CD25-CD4+ T cells from
either unfed or fed mice produce IL-2 and IFN-, but no IL-10 or
TGF-. CD25+CD4+ T cells
from unfed mice produce minimal amounts of IL-2 and similar amounts of
IFN- as CD25-CD4+ T
cells, whereas CD25+CD4+ T
cells from fed mice produce neither IL-2 nor IFN-.
CD25+CD4+ T cells from
unfed mice secrete IL-10 and small amounts of TGF-1 (80 pg/ml),
whereas the production of TGF-1 increases 3-fold (260 pg/ml) in
CD25+CD4+ T cells from
OVA-fed mice. CD25+CD4+ T
cells from both unfed and fed mice secrete similar amounts of IL-10.
There was no detectable IL-4 secretion by
CD25+CD4+ T cells from
either unfed or OVA-fed mice.
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by CD25+CD4+ T
cells from either unfed or fed mice (data not shown). These results
suggest that inhibition of B7/CD28 signaling or enhancement of
B7/CTLA-4 signaling might represent an important step in the generation
and maintenance of the anergic state of
CD25+CD4+ T cells.
The suppressive activity of CD25+CD4+ T
cells is partially mediated by IL-10 and TGF-1
Because CD25+CD4+ T
cells from both unfed and OVA-fed OVA TCR Tg mice secrete both IL-10
and TGF-1, we tested whether these cytokines play a role in
mediating the suppressor function of
CD25+CD4+ T cells in vitro.
We first tested anti-TGF- and anti-IL-10 Ab and found no
effect (data shown in Fig. 7A). However, others have
reported that role for IL-10 in the function of regulatory cells that
inhibits colitis could be demonstrated only by using an anti-IL-10R
Ab (34). Thus, we tested the effect of TGF- sR and
IL-10 sR. As shown in Fig. 7B, the in vitro suppressive
activities of CD25+CD4+ T
cells were partially reversible by the addition of recombinant TGF-
sR or IL-10 sR to the culture medium at a concentration of 10 µg/ml,
demonstrating that
CD25+CD4+ T cells mediate
their suppressive effects, in part, via IL-10 or TGF-. No additional
reversal was observed at doses of 100 µg/ml. However, the mixture of
TGF- sR and IL-10 sR did not completely block the
CD25+CD4+ T cell-mediated
suppression. Thus, although the suppressive activity of these cells
could be shown to involve IL-10 and TGF-, other mechanisms and
factors appear to be involved.
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CTLA-4 is a CD28 homolog expressed on activated T cells and, upon
ligation with CD80 or CD86 on APCs, results in inhibition of T cell
activation. Recently, it has been shown that CTLA-4 is constitutively
expressed on CD25+CD4+ T
cells and involved in mediation of suppressive activity of
CD25+CD4+ T cells in vitro
and in vivo (21, 22). We then tested the effect of oral Ag
on the expression and function of CTLA-4 on
CD25+CD4+ T cells. As shown
in Fig. 8A, CTLA-4 was
expressed on only 0.2% of
CD25-CD4+ T cells from
unfed mice, which increased slightly to 2.6% following feeding.
In contrast, CTLA-4 was expressed on 12.5 ± 2.9%
CD25+CD4+ T cells from
unfed mice, which increased to 23.5 ± 1.6% in fed mice as
measured by intracellular staining. Inhibition of CTLA-4 signaling in
vitro by high concentrations of anti-CTLA-4 Ab (100 µg/ml)
completely abrogated the suppressive activity of
CD25+CD4+ T cells from
unfed mice, but only partially reversed suppression by
CD25+CD4+ T cells from fed
mice (data shown in Fig. 8B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 after anti-CD3 stimulation than
CD25+CD4+ T cells from
unfed mice and do not secrete IFN-, whereas unfed
CD25+CD4+ T cells do.
Others have reported that
CD25+CD4+ T cells require
activation in vitro to exert their suppressive function (10, 17). We found that mucosal administration of Ag might provide an
effective in vivo method of activating regulatory
CD25+CD4+ T cells for fully
immunosuppressive effects. We also demonstrate that TGF- and IL-10
appear to play a partial role in the suppressive properties of
CD25+CD4+ T cells in
vitro.
CD25+CD4+ regulatory T
cells were first described by Sakaguchi et al. (1) and
Taguchi and Nishizuka (2). Since then a great deal has
been learned about their function and suppressive properties both in
vitro and in vivo. Sakaguchi’s group (8, 10, 13, 19, 25)
has shown their importance in preventing the development of autoimmune
disease and their dual property as anergic and suppressive cells. They
have shown that removal of this population may induce tumor immunity
(14, 15) and that
CD25+CD4+ regulatory T
cells may require CTLA-4 but not CD28 as a costimulatory molecule for
functional activation (20). Shevach’s group has also
investigated this regulatory T cell population and found that
CD25+CD4+ T cells represent
a unique lineage of immunoregulatory cells (9) that can
suppress polyclonal T cell activation in vitro by inhibiting IL-2
production (11). They have also defined the specificity of
CD25+CD4+ cells in a
postthymectomy autoimmune gastritis model (16). In terms
of mechanism of action, they have reported that the suppressor effector
function of CD25+CD4+
immunoregulatory T cells requires activation via the TCR to become
suppressive, but once activated, the suppressor function is nonspecific
(17). Although it is clear that
CD25+CD4+ regulatory T
cells have potent in vivo and in vitro biologic effects, the precise
mechanism of action and induction in vivo remain to be defined. Read et
al. (21) report that CTLA-4 plays an important role in the
function of CD25+CD4+ cells
that control intestinal inflammation. Cederbom et al. (24)
have suggested that
CD25+CD4+ regulatory T
cells down-regulate costimulatory molecules on APCs, and Salomon et al.
(22) report that B7/CD28 costimulation is essential for
the homeostasis of
CD25+CD4+ immunoregulatory
T cells that control autoimmune diabetes. It has also been reported
that regulatory CD4+ cells that express
IL-2R
-chain (CD25) are resistant to clonal deletion
(12).
Our findings demonstrate that
CD25+CD4+ T cells with
potent immunoregulatory function are activated following oral Ag
administration. Their suppressive properties in vitro are partially
dependent on IL-10 and TGF-. Recently, Annacker et al.
(35) showed that naive
CD25+CD4+ T cells regulate
the expansion of peripheral CD4 T cells through the production of IL-10
because CD25+CD45
RBlowCD4+ cells from
IL-10-deficient mice do not protect from wasting disease induced by
naive CD45RBhighCD4+ T
cells. It has been reported that
CD25+CD4+ regulatory cells
produce IL-10 and are resistant to deletion in vivo (12).
Sakaguchi (8) has reported that there is increased
expression of IL-10 and TGF- message in
CD25+CD4+ T cells, and
Powrie reports that
CD45RBlowCD4+ T
cell-mediated suppression of colitis also involves IL-10
(34) and TGF- (36), although the latter
could only be shown in vivo. However, Shevach’s group
(17) did not identify any known cytokine-mediated
mechanisms in their studies even though they reported that the effector
mechanism is nonspecific. Our finding that the use of IL-10 sR and
TGF- sR was required to show a partial in vitro effect is consistent
with the experience of Powrie (34) in the colitis model.
Nonetheless, a complete understanding of the precise mechanism of
action of these cells remains to be determined by further in vivo
testing and cloning of the cells. The enhanced suppressive effect of
CD25+CD4+ T cells from fed
mice did not seem to be related solely to TGF- or IL-10 as there was
equivalent partial reversal by IL-10 sR or TGF- sR neutralization in
both populations. However,
CD25+CD4+ T cells from fed
mice have a higher proportion of CTLA-4-expressing cells, which could
explain the difference. It is likely that
CD25+CD4+ regulatory T
cells comprise different subpopulations depending on how they are
isolated and the function being tested.
Regarding oral tolerance, we have previously shown that orally
administered Ag induces TGF--secreting regulatory cells termed Th3
cells (37). An important question is the relationship of
Th3 cells to the CD25+CD4+
T cells reported here. Like orally induced
CD25+CD4+ T cells, Th3
cells are resistant to deletion (28). There is no
phenotype marker (e.g., CD25) for Th3 cells as there is for
CD25+CD4+ regulatory cells.
Of note, in our study we only observed an increase in
CD25+CD4+ T cells when a
high dose of Ag was placed in the drinking water or was gavaged to OVA
TCR Tg mice. We did not find a similar increase when naive mice were
fed and subsequently immunized even though TGF--secreting Th3 cells
can be induced by low-dose feeding followed by immunization in normal
mice. Furthermore, the suppressive effect of Th3 cells are easily
reversed in vitro with anti-TGF- Ab, something we did not
observe with induced
CD25+CD4+ cells even though
there was partial reversal with TGF- sR. Thus, it appears that
orally induced CD25+CD4+
regulatory cells are distinct from the Th3 cells we have described,
although they both can be induced in the gut following orally
administered Ag, and depend in different ways on TGF- for their
effector functions. These observations suggest that there are unique
properties in the gut that lead to the generation of both orally
induced CD25+CD4+
regulatory cells and Th3 type regulatory cells. This could be related
to the local milieu in the gut and costimulatory molecules such as
B7.2, because Th3 cells are preferentially generated by CD86
costimulation (38). Furthermore, the gut appears to
preferentially lead to expression of CTLA-4, and we have observed that
CTLA-4 may be involved in some forms of oral tolerance
(39). It has been shown recently that cell
contact-dependent suppression by CD4+CD25+
regulatory cells is mediated by cell surface-bound TGF-
(40). Although one can generate
CD25+CD4+ cells following
i.v. administration indicating cell activation, this phenotype does not
persist and these CD25+CD4+
cells do not have suppressive properties. Similarly, inducing CD25 in
vitro on CD25- cells does not induce a
suppressive phenotype (11). An important question
regarding expansion of
CD25+CD4+ T cells following
oral Ag is whether oral Ag induces
CD25+CD4+ regulatory cells
de novo or expands an already existing cross-reactive regulatory T cell
population. This question is currently being addressed in our
laboratory by oral Ag administration of OVA TCR Tg mice on the
RAG-/- background. In a recently published
paper, Thorstenson and Khoruts (41) transferred T cells
from RAG2-/- DO 11.10 mice to BALB/c mice and
reported the generation of anergic and potentially immunoregulatory
CD25+CD4+ T cells in vivo
after induction of peripheral tolerance with i.v. or oral Ag. In
preliminary experiments, we have found that directly feeding DO 11.10
mice on the RAG2-/- background led to increased
numbers of CD25+CD4+
cells, and spleen cells from fed animals produced less IL-2 and
had decreased proliferative responses as compared with unfed mice. Thus
it appears that oral Ag may be inducing
CD25+CD4+ regulatory cells
de novo, through more studies are required in this area.
Our findings identify an approach by which CD25+CD4+ regulatory T cells can be activated in vivo which provides an avenue for further understanding of both the biology of CD25+CD4+ T cells and mechanisms of oral tolerance.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Howard L. Weiner, Center for Neurologic Diseases, Harvard Medical School, Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, HIM 730, Boston, MA 02115. E-mail address: hweiner{at}rics.bwh.harvard.edu
3 Abbreviations used in this paper: MBP, myelin basic protein; Tg, transgenic; ILN, inguinal lymph node; MLN, mesenteric lymph node; PP, Peyer’s patches; CD62L, CD62 ligand; sR, soluble receptor; DTH, delayed-type hypersensitivity.
Received for publication May 18, 2001. Accepted for publication August 13, 2001.
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B. Dubois, L. Chapat, A. Goubier, M. Papiernik, J.-F. Nicolas, and D. Kaiserlian Innate CD4+CD25+ regulatory T cells are required for oral tolerance and inhibition of CD8+ T cells mediating skin inflammation Blood, November 1, 2003; 102(9): 3295 - 3301. [Abstract] [Full Text] [PDF]
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A. Kitani, I. Fuss, K. Nakamura, F. Kumaki, T. Usui, and W. Strober Transforming Growth Factor (TGF)-{beta}1-producing Regulatory T Cells Induce Smad-mediated Interleukin 10 Secretion That Facilitates Coordinated Immunoregulatory Activity and Amelioration of TGF-{beta}1-mediated Fibrosis J. Exp. Med., October 20, 2003; 198(8): 1179 - 1188. [Abstract] [Full Text] [PDF]
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D. A. Horwitz, S. G. Zheng, and J. D. Gray The role of the combination of IL-2 and TGF-{beta} or IL-10 in the generation and function of CD4+ CD25+ and CD8+regulatory T cell subsets J. Leukoc. Biol., October 1, 2003; 74(4): 471 - 478. [Abstract] [Full Text] [PDF]
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 T. Kaji, S. Hachimura, W. Ise, and S. Kaminogawa Proteome Analysis Reveals Caspase Activation in Hyporesponsive CD4 T Lymphocytes Induced in Vivo by the Oral Administration of Antigen J. Biol. Chem., July 18, 2003; 278(30): 27836 - 27843. [Abstract] [Full Text] [PDF]
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W. W. J. Unger, W. Jansen, D. A. W. Wolvers, A. G. S. van Halteren, G. Kraal, and J. N. Samsom Nasal tolerance induces antigen-specific CD4+CD25- regulatory T cells that can transfer their regulatory capacity to naive CD4+ T cells Int. Immunol., June 1, 2003; 15(6): 731 - 739. [Abstract] [Full Text] [PDF]
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C. Vasu, R.-N. E. Dogan, M. J. Holterman, and B. S. Prabhakar Selective Induction of Dendritic Cells Using Granulocyte Macrophage-Colony Stimulating Factor, But Not fms-Like Tyrosine Kinase Receptor 3-Ligand, Activates Thyroglobulin-Specific CD4+/CD25+ T Cells and Suppresses Experimental Autoimmune Thyroiditis J. Immunol., June 1, 2003; 170(11): 5511 - 5522. [Abstract] [Full Text] [PDF]
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M. Paas-Rozner, M. Sela, and E. Mozes A dual altered peptide ligand down-regulates myasthenogenic T cell responses by up-regulating CD25- and CTLA-4-expressing CD4+ T cells PNAS, May 27, 2003; 100(11): 6676 - 6681. [Abstract] [Full Text] [PDF]
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S. Grundstrom, L. Cederbom, A. Sundstedt, P. Scheipers, and F. Ivars Superantigen-Induced Regulatory T Cells Display Different Suppressive Functions in the Presence or Absence of Natural CD4+CD25+ Regulatory T Cells In Vivo J. Immunol., May 15, 2003; 170(10): 5008 - 5017. [Abstract] [Full Text] [PDF]
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C. Vasu, S. R. Gorla, B. S. Prabhakar, and M. J. Holterman Targeted engagement of CTLA-4 prevents autoimmune thyroiditis Int. Immunol., May 1, 2003; 15(5): 641 - 654. [Abstract] [Full Text] [PDF]
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A. Lundgren, E. Suri-Payer, K. Enarsson, A.-M. Svennerholm, and B. S. Lundin Helicobacterpylori-Specific CD4+ CD25high Regulatory T Cells Suppress Memory T-Cell Responses to H. pylori in Infected Individuals Infect. Immun., April 1, 2003; 71(4): 1755 - 1762. [Abstract] [Full Text]
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N. M. Tsuji, K. Mizumachi, and J.-i. Kurisaki Antigen-specific, CD4+CD25+ regulatory T cell clones induced in Peyer's patches Int. Immunol., April 1, 2003; 15(4): 525 - 534. [Abstract] [Full Text] [PDF]
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T. Oida, X. Zhang, M. Goto, S. Hachimura, M. Totsuka, S. Kaminogawa, and H. L. Weiner CD4+CD25- T Cells That Express Latency-Associated Peptide on the Surface Suppress CD4+CD45RBhigh-Induced Colitis by a TGF-{beta}-Dependent Mechanism J. Immunol., March 1, 2003; 170(5): 2516 - 2522. [Abstract] [Full Text] [PDF]
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C. Montagnoli, A. Bacci, S. Bozza, R. Gaziano, P. Mosci, A. H. Sharpe, and L. Romani B7/CD28-Dependent CD4+CD25+ Regulatory T Cells Are Essential Components of the Memory-Protective Immunity to Candida albicans J. Immunol., December 1, 2002; 169(11): 6298 - 6308. [Abstract] [Full Text] [PDF]
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J. Lehmann, J. Huehn, M. de la Rosa, F. Maszyna, U. Kretschmer, V. Krenn, M. Brunner, A. Scheffold, and A. Hamann Expression of the integrin alpha Ebeta 7 identifies unique subsets of CD25+ as well as CD25- regulatory T cells PNAS, October 1, 2002; 99(20): 13031 - 13036. [Abstract] [Full Text] [PDF]
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H. Y. Wu, F. J. Ward, and N. A. Staines Histone Peptide-Induced Nasal Tolerance: Suppression of Murine Lupus J. Immunol., July 15, 2002; 169(2): 1126 - 1134. [Abstract] [Full Text] [PDF]
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 P. M. Cobelens, A. Kavelaars, R. van der Zee, W. van Eden, and C. J. Heijnen Dynamics of mycobacterial HSP65-induced T-cell cytokine expression during oral tolerance induction in adjuvant arthritis Rheumatology, July 1, 2002; 41(7): 775 - 779. [Abstract] [Full Text] [PDF]
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ear thickness = thickness of left ear - thickness of
right ear. Data shown are the results of two independent experiments
with values for ear thickness from individual mice (6–8 mice/group).
*, p < 0.01 vs HBSS, CD25- fed, or
CD25- unfed. B, Spleen cells from BALB/c
mice receiving HBSS (triangles), CD25-CD4+ T
cells from fed mice (open circles), or unfed mice (open squares), or
CD25+CD4+ T cells from fed (filled circles) or
unfed (filled squares) were prepared on day 14 after immunization and
cultured at 5 x 105 cells/well in 10% DMEM with the
indicated concentration of OVA. The culture was pulsed 48 h later,
and cells were harvested 12 h later. Results are mean values of
two experiments. C, BALB/c mice were immunized with 100
µg of BSA in CFA. At day 14 after immunization,
CD25-CD4+ T cells from those mice were sorted
and then cocultured at 5 x 105/well with purified
CD25+CD4+ T cells (1 x
105/well) from OVA TCR Tg mice fed 20 mg/ml OVA in drinking
water for 5 days in the presence of APC and 200 µg/ml BSA with
or without 200 µg/ml OVA. The culture was pulsed at 72 h and
harvested 12 h later. The results are mean values (±SD) of two
experiments.
