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Unité du Développement des Lymphocytes, Centre National de la Recherche Scientifique, Unité de Recherche Associée 1961, Institut Pasteur, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Useful surface markers for the discrimination between functional
subsets of CD4 T cells are CD25 and CD45RB. CD25 is a component of the
IL-2R and is transiently expressed on CD4 T cells after activation
(11), and CD25+ T cells make up
approximately 10% of the peripheral CD4 T cell pool. Transfer of
CD25-depleted splenic cells into nude mice of susceptible strains leads
to the development of organ-specific autoimmune diseases, which can be
prevented by the cotransfer of purified CD25+ CD4
T cells (2, 3). Moreover, the lack of
CD25+ cells allows for efficient clearance of
tumors, demonstrating the active suppression of anti-self immune
responses by regulatory T cells (12).
CD25+ CD4 T cells were found to produce high
levels of TGF-
and IL-10 compared with
CD25- CD4 T cells (3). Recently, it
was shown that the protective effect of CD25+ CD4
T cells is dependent on CTLA-4 (13).
CD45RB is another marker of activation used for the discrimination of different CD4 T cell subsets. This surface molecule is up-regulated during thymic development (14), and its expression on naive CD4 T cells decreases upon activation (15). According to this marker, roughly one-third of CD4 T cells are activated in unmanipulated mice.
In an experimental system of IBD several groups showed that after
transfer into immunodeficient recipients, naive
CD45RBhigh CD4 T cells cause a wasting disease
characterized by intestinal inflammation (7, 8). High
levels of IFN-
and TNF-
are found in both the spleen and the
intestine of the recipients (8, 16, 17, 18), and injected T
cells can expand at least 200-fold under specific pathogen-free (SPF)
conditions (10). In contrast, naturally activated
CD45RBlow CD4 T cells usually do not induce
disease, expand less, and protect the recipients from naive T
cell-induced IBD (7, 8, 10, 16, 17). This protective
effect is dependent on TGF- and IL-10 (17, 19, 20, 21, 22). The
CD45RBlow CD4 T cell pool contains most of the
CD25+ CD4 T cells; the latter contribute
one-third to the pool of CD45RBlow CD4 T cells.
In a recent study Read et al. showed that the protection from IBD in
this experimental system is enriched in the CD25+
T cell population within the CD45RBlow CD4 T cell
pool and is also CTLA-4 dependent (23).
The general mechanisms regulating the expansion and survival of peripheral CD4 T cells are to date not well understood (for review, see Ref. 24). The peripheral T cell pool is divided into a naive and an activated/memory compartment, which are apparently independently regulated (25, 26). The size of the activated/memory CD4 T cell pool is controlled by regulatory T cells within this pool (10). The administration of recombinant murine IL-10 protected recipients reconstituted with CD45RBhigh CD4 T cells from disease and decreased the number of recovered splenic CD4 T cells (17), suggesting a role for IL-10 in the regulation of the expansion of peripheral CD4 T cells.
Here, we investigated the survival, the dynamics of expansion, and the homeostatic equilibrium of different peripheral CD4 T cell subpopulations from normal donors upon transfer into T and B cell-deficient mice, their regulatory properties, and the role of IL-10 in the expansion process. Our results show that although both CD25+ and CD25- CD45RBlow CD4 T cell pools contain regulatory T cells, only the CD25+ population can efficiently regulate the size of the activated/memory CD4 T cell compartment via a mechanism involving the production of IL-10. Furthermore, our results show that control of CD4 T cell peripheral expansion and disease prevention are largely independent processes. Finally, the data demonstrate that although the CD25+ CD4 T cell population reaches a homeostatic equilibrium at low cell numbers, a fraction of these cells has a high potential of expansion upon transfer into alymphoid recipients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6-Ly5.2 mice were obtained from Janvier (Le Genest-St-Isle, France). C57BL/6-Ly5.1, C57BL/6 recombination-activating gene-2-deficient (RAG-2°) and C57.BA (Thy1.1) mice were purchased from CDTA (Orleans, France). All animals were kept under SPF conditions in the animal facilities of the Institut Pasteur (Paris, France). C57BL/6-IL-10-deficient (IL-10°) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were bred under SPF conditions in our animal facilities. Donors and recipients were sex-matched and were used at 6–12 wk of age. IL-10° donors were used at 5–8 wk of age.
Antibodies
The following mAbs were used: anti-Ly5.1-FITC or -PE (clone A.20); anti-CD4-biotin, -FITC, -PE, -TriColor, or -allophycocyanin (L3T4); anti-CD8-FITC or -PE (CT-CD8a); anti-CD45RB-PE (23G2); anti-CD25-FITC (7D4) or -PE (PC61); anti-CD38-FITC (90); anti-CD69-PE (H1.2F3); anti-CD44-PE (IM7.8.1); and anti-Thy1.1-PE (Ox-7). All Abs were purchased from PharMingen (San Diego, CA) or Caltag (Burlingame, CA).
T cell preparations
Before sorting, splenic single-cell suspensions were first enriched for CD4+ or CD25+ cells by positive selection on midiMACS columns (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer’s instructions. In brief, cells were first incubated with biotinylated anti-CD4 Abs for 20 min on ice in PBS supplemented with 0.5% FCS, then incubated in the same buffer with streptavidin-microbeads for 15 min. The magnetically labeled positive fraction was retained on a midiMACS column. Alternatively, for the enrichment of CD25+ cells, FITC-labeled anti-CD25 Abs and anti-FITC-microbeads were used. In all cases, after enrichment the cells were labeled with anti-CD45RB-PE, anti-CD4-Tricolor, and anti-CD25-FITC Abs for 20 min on ice and then sorted on a FACStarPlus (Becton Dickinson, Mountain View, CA). For CD45RB, the brightest 40–50% and the dimest 20–30% of CD4+ cells were sorted as high and low, respectively. CD25+ and CD25- CD4 T cells were sorted out of the CD45RBlow population. The purity of the sorted populations was routinely >96%.
Labeling with CFSE
Labeling of cells with CFSE was performed as previously described (27). In brief, after washing FACS-sorted CD4 T cells twice in PBS, the cells were resuspended at 1–2 x 107/ml PBS and incubated 10 min at room temperature with CFSE at a final concentration of 6 µM. This solution was incubated with an equal volume of FCS for an additional 2 min before washing twice with PBS.
Cell transfers
RAG-2° mice were injected iv. or i.p. with 3 x 105 CD4 T cells from FACS-sorted subpopulations. When cells were coinjected (at a ratio of 1:1), Ly5.1+ and Ly5.2+ donor cells were used. For CFSE-labeled cells, 0.5–1.5 x 106 CD4 T cells from Ly5.1+ origin were injected i.v. Here, coinjections were made with Ly5.1+ and Thy1.1+ donors at a ratio of 1:1.
Preparation of intestinal cells
Whole intestines were first flushed extensively to eliminate the lumen content, then were longitudinally opened and cut into 1- to 2-cm pieces. These were incubated twice in OptiMEM medium (Life Technologies, Gaithersburg, MD) containing 5% FCS and 450 U of collagenase (Sigma, St. Louis, MO) for 20 min at 37°C. After filtering through gauze, lymphoid cells were isolated on a 40% Percoll gradient. The cells were then washed and stained for fluorocytometric analysis.
Flow cytometric analysis
Single-cell suspensions from spleen; axillary, inguinal, and mesenteric lymph nodes; or intestine were incubated for 20 min at 4°C in microtiter plates with 50 µl of the appropriate Ab preparations in PBS supplemented with 3% FCS and 0.01% azide. When possible, one million cells were stained. Alternatively, the whole organ cell suspension was used. The Ab concentrations used were tested for optimal stainings of splenic control samples before experimental use. Dead cells were excluded from the analysis by propidium iodide. Blood samples were first stained with appropriate Abs before lysing erythrocytes with FACS Lysing Solution (Becton Dickinson). Flow cytometric analysis was performed on a FACScan (Becton Dickinson) using CellQuest software (Becton Dickinson).
Statistical analysis
Unless otherwise indicated, analysis was performed using the unpaired t test. In cases where the variances between compared groups were significantly different, the unpaired t test was modified with Welch’s correction. The data were considered significantly different at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Splenic CD4 T cells from normal unmanipulated mice were sorted
into four subpopulations according to the expression of CD45RB and CD25
markers: 1) CD45RBhigh cells, hereafter denoted
RBhigh; 2) CD45RBlow
cells (of which about one-third is CD25+),
denoted RBlow; 3) CD45RBlow
cells, which were depleted of CD25+ cells,
denoted 25- RBlow; and 4)
CD45RBlow cells expressing the CD25 marker,
hereafter referred to as 25+
RBlow CD4 T cells (Fig. 1
A).
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Mice injected with (naive) RBhigh T cells
invariably developed signs of wasting. Only one of eight animals
survived for 12 wk after transfer (Fig. 1
B), and all
recipients lost weight (Fig. 1C), developed diarrhea, and
had a markedly enlarged colon upon analysis. Noninjected control
RAG-2° mice kept under the same conditions never developed signs of
wasting or diarrhea (data not shown). In the group of animals that
received the total pool of RBlow cells, one of
seven mice became sick (Fig. 1C) and was sacrificed 8 wk
after transfer (Fig. 1B). Half of the recipients of the
25- RBlow pool remained
healthy throughout the experimental period of 3.5 mo (Fig. 1C). The other half suffered from wasting, but the disease
progressed more slowly compared with that in recipients of
RBhigh T cells, and only two of them lost >20%
of their initial body weight within 12 wk after transfer (Fig. 1B). The only experimental group in which all recipients
invariably gained weight and did not develop signs of wasting was the
one injected with 25+ RBlow
T cells (Fig. 1C). Thus, the incidence of a wasting disease
in alymphoid recipients after transfer of different CD4 T cell
populations appears to correlate with the frequency of
CD25+ cells in the transferred population.
Peripheral expansion of CD4 T cell subsets: CD25+ CD45RBlow CD4 T cells reach homeostatic equilibrium at low cell numbers
To assess the accumulation and the respective homeostatic
equilibrium of the injected T cell populations, at the time of
sacrifice the number of CD4+ cells was scored in
the spleen; axillary, inguinal, and mesenteric lymph nodes; as well as
blood and intestine of all the recipients described in the previous
section. In this series of transfers, on the average, 2.9 x
106 CD4 T cells were recovered from mice injected
with RBhigh T cells (Fig. 1D). In
animals injected with the total pool of naturally activated/memory
RBlow T cells, we could only score half the
number of cells found in the previous group (on the average, 1.4
x 106; p < 0.03), as shown
previously (10). Interestingly, the number of cells (on
the average, 2.9 x 106) obtained from mice
reconstituted with 25-
RBlow T cells was similar to that scored in
recipients of RBhigh T cells regardless of the
state of health of the recipients (Fig. 1D) and the
time point of sacrifice. Finally, recipients of
25+ RBlow T cells only
yielded approximately the number of cells injected (on the average,
3.9 x 105), that is, 7-fold less compared
with the 25- RBlow
(p < 0.0001) or the
RBhigh (p < 0.001)
population and about 4-fold less compared with recipients of
unfractionated RBlow T cells
(p < 0.01; Fig. 1D).
The organ distribution of the CD4+ cells in all
groups of mice is shown in Table I. The
majority of T cells were found in the spleen, accounting for roughly
half of the recovered CD4 T cells. In the intestine, with the exception
of recipients of the 25+
RBlow fraction, similar numbers of CD4 T cells
were recovered in both healthy and sick animals in all other groups
(Table I). However, it cannot be formally excluded that the observed
differences in cell numbers between different CD4 T cell subsets are
due not to different expansions of these cells, but to a differential
migration pattern predominantly into other organs, such as liver, lung,
or bone marrow, which have not been investigated here.
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Both CD25+ and CD25- CD45RBlow CD4 T cells contain regulatory cells capable of preventing a wasting disease induced by naive CD4 T cells
In the experiments described above (see Fig. 1), half of the
recipients of the 25-
RBlow T cell population became sick, whereas the
other half remained healthy for at least 3 mo. This differential
outcome could be the result of differences in the frequency of
potentially pathogenic or, alternatively, of regulatory T cells in the
individual inoculums of this CD4 T cell subset.
To directly assess the presence of regulatory activity in the two
CD45RBlow subpopulations, RAG-2° recipients
were coinjected with 3 x 105
RBhigh T cells and 3 x
105 CD4 T cells of either the
25+ or 25-
RBlow T cell subset (Fig. 2A). The majority of the
animals injected with these mixtures were protected from disease (Fig. 2B), and a similar fraction of animals in both groups
developed a wasting disease with similar kinetics (Fig. 2C).
In conclusion, the CD25-
CD45RBlow T cell subpopulation contained
sufficient regulatory activity to prevent CD4 T cell-induced wasting in
40% of the recipients.
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The cell recovery from recipients of the total
RBlow T cell pool was significantly different
from that of either 25- or
25+ subfractions (see Fig. 1). This could be
interpreted to indicate that the restricted expansion of total
RBlow T cells after transfer into RAG-2°
hosts was due to control of the accumulation of
25- RBlow T cells exerted
by 25+ RBlow T cells. To
investigate whether the CD25+ T cell pool is
indeed responsible for control of the size of the activated/memory CD4
T cell compartment, we analyzed the coinjected recipients (see Fig. 2)
for the level of T cell reconstitution either 12–14 wk after transfer
or when the recipients dropped to <80% of their initial weight. The
identification of the origin of each donor population was based on the
expression of the Ly5.1 and Ly5.2 markers.
The total CD4 T cell recovery from mice injected with mixed
25- RBlow and
RBhigh T cells was >4-fold higher compared with
that in animals that received mixed 25+
RBlow and RBhigh cells
(p < 0.04, by unpaired t test with
Welch’s correction; Fig. 2D). In the group cotransferred
with 25- RBlow and
RBhigh T cells both populations expanded to
similar numbers as those scored in animals injected with either
population alone (Figs. 2E and 1D), with a
similar distribution of each subset in all organs (Table II). Again, no significant difference was
observed between healthy and sick animals (Fig. 2E).
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E). However, approximately 4-fold less T cells of
originally RBhigh T cells were scored in these
animals compared with recipients of RBhigh T
cells injected alone (p < 0.004) or coinjected
with 25- RBlow T cells
(p < 0.05, by unpaired t test with
Welch’s correction). T cell numbers were mostly reduced in spleen,
blood, and intestine (Table II). Interestingly, the relative frequency
of originally 25+ RBlow to
RBhigh T cells in the intestine was 1:10 to 1:20,
since 25+ RBlow T cells
were again barely detected in the intestine (Table II). This ratio was
independent of the health state of the mice, and the presence of
25+ RBlow T cells did not
inhibit the activation of the original RBhigh T
cells, as assessed by the low density of the CD45RB marker (Fig. 3).
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CD25+ CD45RBlow CD4 T cells can expand in vivo
CD25+ T cells do not expand upon stimulation with anti-CD3 Abs in vitro (28, 29), which was interpreted to reflect an anergic state of these cells (28). The reconstitution of RAG-2° mice with 25+ RBlow cells yielded approximately the number of cells injected (see above). However, because in transfer experiments only a minority of the injected cells survives in the host 24–48 h after transfer (10), this suggested that the CD25+ T cell population could nevertheless expand to a certain extent in the recipients.
To more accurately address this issue, CD4 T cells from normal
Ly5.1+ donors were sorted into three
subpopulations according to the expression of CD25 and CD45RB and were
labeled with CFSE. Then, 0.5–1.5 x 106
cells of each subset were separately injected into congenic
Ly5.2+ RAG-2° hosts. Thirty-six to 48 h
after transfer, donor-derived CD4 T cells were analyzed in the
peripheral lymphoid organs, the blood, and the gut. While no donor
cells were recovered from the intestine, the bulk of the populations
were found in the spleen. Naive RBhigh T cells
survived much better than naturally activated
25- or 25+
RBlow T cells (Fig. 4C). On the average 2.6% of
the RBhigh T cell population could be recovered
at this early time point, whereas only 1.1 and 0.3% of injected
25- and 25+
RBlow T cells, respectively, were recovered. At
this time point, very few of the injected cells had divided, as
assessed by CFSE staining (data not shown).
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C, 4- and 6.8-fold the number of
injected cells were recovered from recipients of
25- RBlow and of
RBhigh T cells, respectively. In contrast,
recipients of 25+ RBlow T
cells contained 20% of the number of injected cells. However, taking
into account the differential survival of the injected populations in
the three groups of recipients, the increase in cell numbers over the
surviving populations on day 2 was 60-fold for the
25+ RBlow T cell population
(corresponding to five rounds of division of the surviving T cells),
compared with 360- and 260-fold for the 25-
RBlow and the RBhigh T cell
populations (equivalent to seven or eight rounds of division),
respectively (Fig. 4C). In conclusion, these results show that a fraction of the 25+ RBlow cells has an expansion capacity not very different from that of the 25- RBlow or RBhigh T cells. The lower reconstitution capacity of CD25+ cells 3.5 mo after transfer probably reflects a difference in the homeostatic regulation of steady state numbers in these populations.
CD25+ CD45RBlow CD4 T cells regulate the size of the CD45RBhigh CD4 T cell compartment early after transfer
In these series of experiments CFSE-labeled
25+ RBlow and
RBhigh T cells were also coinjected into RAG-2°
recipients, and the mice were analyzed 2, 6, and 12 days later. On day
2 after transfer no significant cell division was detected (Fig. 4A). On day 12 after transfer the
25+ RBlow population had
expanded as much as the same population injected alone (Fig. 4A). In contrast, the RBhigh
population in the coinjected animals showed a higher frequency (22%)
of cells that were CFSE positive after 12 days compared with the ones
isolated from hosts receiving RBhigh T cells
alone (3%; Fig. 4A). Moreover, at this time point the
absolute number of RBhigh T cells recovered from
mice injected with this population alone was 10-fold higher than that
in the mice coinjected with 25+
RBlow T cells. Interestingly, 6 days after
transfer 60–80% of the 25+ CD4 T cells found in
the spleen of the hosts had lost the CFSE staining, indicating that
they had already gone through at least six or seven rounds of division.
In contrast, at this time point >90% of the
RBhigh T cells did not divide more than once
(Fig. 4B). The same result was observed when the two
populations were independently injected (data not shown).
These experiments show that a fraction of 25+ RBlow T cells have a quite remarkable potential of expansion, and that by day 6 they were already engaged in cell division. In addition, they show that the homeostatic activity of 25+ RBlow on RBhigh T cells operates shortly after transfer.
The expression of the CD25 molecule on CD25+ CD45RBlow CD4 T lymphocytes in vivo is not stable and is influenced by the presence of other CD4 T cells
The frequency of CD25+ T cells is constant throughout the adult life of normal mice (30), which led to the hypothesis that these cells constitutively express this molecule (29, 31). Since T cells express high levels of CD25 upon activation (11), it was also suggested that CD25+ T cells are continuously activated in vivo (32). Here we analyzed whether the reconstitution of the CD25 compartment is dependent on the origin of the injected cells.
At sacrifice, the peripheral lymphoid organs of RAG-2° animals
injected with different CD4 T cell subsets, as shown in Figs. 1 and 2,
were analyzed for the expression of CD25 on the recovered CD4 T cells
as well. Interestingly, the frequency of lymph node T cells that
stained positively for CD25 was similar in the recipients of
25+ and 25-
RBlow cells (Fig. 5). In the spleen, the frequency of
CD25+ cells was 13.9% (SEM = 3.0;
n = 8) within the group that received
25- RBlow cells compared
with 19.4% (±1.1; n = 4) in the recipients of
25+ RBlow cells. The values
observed were independent of the state of health of the mice. In
contrast, transfers of naive RBhigh cells
reconstituted the CD25 compartment to a lower extent (in the spleen
5.6% (±2.0); n = 5).
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, for expression of the CD25 marker.
Originally RBhigh T cells showed similar
frequencies of CD25-expressing cells in mesenteric lymph nodes
regardless of the presence of other T cells in the injected recipients
(5.8% (±1.2; n = 7) in the presence of
25- RBlow cells and 8.5%
(±3.0; n = 6) in the presence of
25+ RBlow T cells). The
frequency of CD25-expressing cells in the originally
25- RBlow population was
modestly increased by the presence of RBhigh
cells in mesenteric lymph nodes, but was within the same range as in
animals injected with 25-
RBlow cells alone (25-
RBlow alone, 18.9% (±2.0; n =
8); 25- RBlow in
coinjections, 22.5% (±3.2; n = 7); Fig. 5). In the
presence of RBhigh cells, the donor
CD25+ population consisted mostly of
CD25+ cells in the mesenteric lymph nodes (70.3%
(±3.6; n = 6) compared with 21.9% (±4.1;
n = 4) in recipients of CD25+
cells alone). These findings were similar in spleen and pooled axillary
and inguinal lymph nodes. We conclude that for the majority of transferred CD25+ CD4 T cells, the expression of the CD25 molecule requires the presence of other CD4 T cells. In addition, the 25- RBlow population can generate higher frequencies of CD25+ cells than RBhigh T cells upon transfer.
CD25+ CD45RBlow CD4 T cells from IL-10° mice cannot efficiently regulate peripheral CD4 T cell numbers
Administration of rIL-10 leads to decreased numbers of splenic T cells recovered from mice injected with RBhigh cells (17), and IL-10° mice develop, apart from IBD, splenomegaly (33). Given these data, we hypothesized that IL-10 plays a role not only in the protection from disease, but also in regulation of the expansion of CD4 T cells. Earlier studies have shown that IL-10° mice at 6 wk of age contain normal numbers of thymocytes and splenic T cells (19). To confirm and extend these findings, we analyzed the thymus, spleen, lymph nodes, blood, and intestine of 6-wk-old IL-10° and wild-type (wt) mice for CD4 and CD8 T cells. Indeed, IL-10° T cells were indistinguishable from wt mice with regard to numbers and expression of CD45RB, CD25, CD38, CD69, and CD44, including the presence of CD25+ CD4+CD8- thymocytes (data not shown), suggesting that the development of both CD4 and CD8 T cells is not strongly affected by the lack of IL-10.
To address the question of whether IL-10 is necessary for efficient
control of the size of the activated T cell pool, we performed the same
transfer experiments described above with donor cells from healthy
IL-10° mice bred onto the C57BL/6 background. Transfer of 3 x
105 sorted RBhigh or
25- RBlow cells from
IL-10° donors induced wasting in all RAG-2° recipients, suggesting
that the regulatory T cells in the 25-
RBlow population are IL-10 dependent. The wasting
in mice injected with IL-10° 25-
RBlow cells developed somewhat faster than that
in hosts of IL-10° RBhigh T cells (Fig. 6A). Both populations expanded
to a similar level (Figs. 6C and 1D), with a
comparable organ distribution of the recovered T cells (Table III) as the corresponding populations
from wt animals.
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B). From these mice, on the
average, 1.1 x 106 CD4 T cells could be
recovered, representing 3-fold more than from animals that received wt
25+ T cells (p < 0.02;
Figs. 6C and 1D). This was largely due to
increased cell numbers in spleen and blood of the recipients (Table III), and IL-10° 25+
RBlow cells were found in the intestine only in
very low numbers. The difference in cell recovery was not due to an
increased survival of IL-10°CD4 T cells, since similar numbers were
obtained 2 days after transfer into RAG-2° recipients compared with
the corresponding wt population (data not shown). Furthermore, the
frequency of CD25-expressing cells among the recovered IL-10°CD4 T
cells was for all populations indistinguishable from that of the
corresponding wt CD4 T cells (data not shown).
Consistent with previous results (22), when 3 x
105 IL-10° 25+
RBlow cells were injected together with 3 x
105 wt RBhigh cells, the
IL-10°CD25+ T cells could not prevent wasting
in the RAG-2° hosts (Fig. 6, A and B). The
numbers of cells recovered from mice coinjected with IL-10°
25+ RBlow T cells and wt
RBhigh cells were not significantly different
from those recovered from recipients of either population alone (Figs. 6C and 1D). Interestingly, the expression of CD25
as well as the organ distribution of the coinjected populations were
very similar to those in the recipients of wt T cells (data not shown
and Table III).
As shown above, regulation of the peripheral expansion of
RBhigh by 25+
RBlow cells was already effective by 12 days
after transfer. We ascertained the lack of regulatory activity of
IL-10° T cells by coinjecting normal CD25-
cells with IL-10°CD25+ T cells at different
ratios into RAG-2° recipients. As shown in Fig. 7, IL-10°, but not wt
CD25+, cells showed a complete absence of
regulatory effect on the expansion of CD25-depleted CD4 T cells. Even
at a ratio of six IL-10°CD25+ T cells to one wt
CD25- cells, no signs of inhibitory activity
were detected.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We conclude that control of CD4 T cell peripheral expansion and disease prevention are largely independent processes. Furthermore, we show that the mechanism underlying the regulation of the size of the peripheral T cell compartment is IL-10 dependent. Our results also provide the first description of the population dynamics of CD25+ T cells upon in vivo transfer. They establish that although CD25+ T cells reach a homeostatic equilibrium at low cell numbers, a fraction of these cells has a high potential of expansion.
CD25+ CD4 T cells were described to contain regulatory CD4 T cells in several experimental systems, including the one used here (2, 3, 12, 23, 30, 34). However, there is increasing evidence that regulatory CD4 T cells exist in the CD25- compartment as well (35, 36). Our data showing the capacity of the CD25- CD45RBlow CD4 T cells to protect from wasting disease are well in line with a recent report (23) in which a similar reduction in the incidence of CD4 T cell-induced IBD was observed at a comparable CD25-/RBhigh T cell ratio.
This observation raises the question of the relationship between
CD25+ and CD25- regulatory
CD4 T cells. At this point we cannot exclude that the two subsets
differ in their development, function, and/or specificity, although
this is not very likely. First, the expression of CD25 on different CD4
T cell subsets is not stable after transfer into alymphoid hosts, and
in the case of CD25+ T cells it is dependent on
the presence of other CD4 T cells. It is thus possible that in normal
animals CD25 expression is dynamic and, therefore, the marker is not
identifying the entire pool of regulatory T cells. Secondly, both
CD25+ and CD25- regulatory
T cells depend on IL-10 for efficient disease protection. Furthermore,
CD25- regulatory T cells do not compensate for a
block of the function of CD25+ regulatory T cells
when the protective activity of total CD45RBlow T
cells is inhibited by anti-TGF- or anti-CTLA-4 Ab treatment
(20, 23).
The dissociation between protection of disease and systemic (and local) regulation of CD4 T cell numbers observed in our studies indicates that both processes are to a large extent independently regulated. This may be the result of a quantitative difference in the number of regulatory CD4 T cells required to control both processes. Disease protection may require lower numbers of regulatory T cells and/or rely on the presence of appropriate TCR specificities in the pool of regulatory T cells. Thus, it can be effective even in the absence of efficient growth control. The lack of appropriate specificities would also explain why the CD25+ CD4 population did not confer protection from wasting in some cases, while showing a quite efficient growth inhibitory activity on other CD4 T cells in the sick recipients. Similar observations were described in other systems: tolerance can be ensured when T cells expand (37), and differentiation can take place in the absence of overt proliferation of T cells (38, 39).
Taken together, it is possible that regulatory CD25- T cells are descendants of thymic regulatory CD25+ T cells (34, 35) and represent an alternative state of the same functional pool of peripheral regulatory T cells. CD25+ CD4 T cells might be enriched for regulatory T cells simply because they are activated (effector state), but regulatory T cells might become CD25- T cells in the absence of the appropriate stimuli (memory state).
In contrast to our previous report (10), we could now recover sizable numbers of CD4 T cells from the intestines of healthy recipients. Moreover, similar numbers of intestinal T cells were observed between healthy and sick recipients within the same experimental group. However, this appears to represent a different organ distribution of the cells rather than a higher level of T cell expansion, because the differences in total cell numbers from recipients of CD45RBlow T cells compared with recipients of CD45RBhigh T cells was in this study very similar to what we reported previously. This argues for a systemic regulation of peripheral CD4 T cell numbers and not for a compartmentalized control in individual organs. The increased frequency of intestinal CD4 T cells reported here could perhaps reflect a subclinical state of inflammation in these overall healthy mice due to an unbalanced ratio of regulatory to target CD4 T cells. Nevertheless, a >3-fold reduction in the number of intestinal T cells belonging to the transferred CD45RBhigh CD4 T cell population was observed in coinjections with the CD25+ CD45RBlow T cells. This is consistent with our previous report, namely that regulatory CD4 T cells inhibit the accumulation of CD4 T cells in the intestine. The observation that similar T cell numbers are scored in the intestines of sick and healthy animals reinforces the conclusion that T cell expansion and incidence of disease are not directly linked.
Our studies also assessed the proliferative potential and the homeostatic equilibrium of peripheral CD25+ CD4 T cells. The idea has been that regulatory T cells have a limited capacity of expansion, perhaps as a result of their own growth inhibitory activity. This is in line with the inability of these cells to proliferate in vitro upon stimulation unless exogenous IL-2 is added. Here we provide evidence that a fraction of CD25+ CD45RBlow cells is capable of considerable in vivo proliferation despite the fact that the population reaches a homeostatic equilibrium at low cell numbers. The present data do not provide information on the rate of apoptosis occurring after each round of division, but extensive apoptosis during the expansion process will only increase the number of cell divisions required to account for the observed cell numbers.
The reasons why the homeostatic equilibrium of CD25+ CD45RBlow T cells is reached at low cell numbers are nevertheless unclear at this point. It could be that these cells are driven and/or regulated by different growth factors or have limited functional niches compared with the other CD4 T cell populations.
Asseman et al. (22) demonstrated that the IBD protective function of regulatory CD4 T cells is IL-10 dependent. The lack of efficient growth inhibitory activity of CD25+ T cells from IL-10° mice reveals a role for this IL in peripheral T cell homeostasis. In the results presented here CD25+ T cells from IL-10° mice showed many characteristics of wt CD25+ CD4 T cells, and most recipients of IL-10°CD25+ T cells remained healthy, although this subset contained potentially aggressive T cells. This suggests that the CD25+ pool of IL-10°CD4 T cells, although not homogeneous, is highly enriched for cells of the regulatory lineage, which, in the absence of IL-10, have a higher potential of expansion.
Other groups reported a linkage between the susceptibility to autoimmune diseases and the balance between IL-12 and IL-10 as well as a role for IL-12 in CD4 T cell expansion (40, 41). It is thus possible to envisage that IL-10 produced by regulatory T cells leads to down-regulation of IL-12 production by APC, resulting in decreased levels of IL-2 and, in turn, restricted CD4 T cell expansion. Our data show that regulatory CD25+ T cells prevent extensive T cell expansion and do not seem to interfere significantly with the activation of naive T cells in the recipient.
The onset of IBD and splenomegaly in IL-10° mice occurs relatively
late in life compared with other situations in which deregulation of
peripheral T cell homeostasis is already apparent 3–4 wk after birth.
This strongly suggests that factors other than IL-10 are involved in
the regulation of peripheral T cell numbers. Indeed, spontaneous
autoimmune disease and disruption of T cell homeostasis were recently
described in mice transgenic for a T cell-targeted dominant negative
TGF- receptor (42, 43). It is worth pointing out,
however, that whatever the cellular interactions or mechanisms that
delay the development of disease in IL-10° mice, they are disrupted
in the transfer experiments presented here. Thus, further studies are
needed to dissect the dependence of T cell homeostasis from cytokines
produced by regulatory T cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Oliver Annacker, Unité du Développement des Lymphocytes, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France.
3 Current address: Laboratorio de Imuno-Regulaçao e Microbiologia, Centro de Pesquisas Gonçalo Moniz-FIOCRUZ, R. Waldemar Falcco 121 Brotas, Salvador, Bahia, Brasil CEP 40295-001.
4 Abbreviations used in this paper: IBD, inflammatory bowel disease; SPF, specific pathogen-free; RAG-2°, recombination activating gene-2 deficient; IL-10°, IL-10 deficient; wt, wild type.
Received for publication October 23, 2000. Accepted for publication December 15, 2000.
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M. P. Eggena, B. Barugahare, N. Jones, M. Okello, S. Mutalya, C. Kityo, P. Mugyenyi, and H. Cao Depletion of Regulatory T Cells in HIV Infection Is Associated with Immune Activation J. Immunol., April 1, 2005; 174(7): 4407 - 4414. [Abstract] [Full Text] [PDF]
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S. Zelenay, T. Lopes-Carvalho, I. Caramalho, M. F. Moraes-Fontes, M. Rebelo, and J. Demengeot Foxp3+ CD25- CD4 T cells constitute a reservoir of committed regulatory cells that regain CD25 expression upon homeostatic expansion PNAS, March 15, 2005; 102(11): 4091 - 4096. [Abstract] [Full Text] [PDF]
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R. Setoguchi, S. Hori, T. Takahashi, and S. Sakaguchi Homeostatic maintenance of natural Foxp3+ CD25+ CD4+ regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization J. Exp. Med., March 7, 2005; 201(5): 723 - 735. [Abstract] [Full Text] [PDF]
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T. Barthlott, H. Moncrieffe, M. Veldhoen, C. J. Atkins, J. Christensen, A. O'Garra, and B. Stockinger CD25+CD4+ T cells compete with naive CD4+ T cells for IL-2 and exploit it for the induction of IL-10 production Int. Immunol., March 1, 2005; 17(3): 279 - 288. [Abstract] [Full Text] [PDF]
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C. M. Freeman, B.-C. Chiu, V. R. Stolberg, J. Hu, K. Zeibecoglou, N. W. Lukacs, S. A. Lira, S. L. Kunkel, and S. W. Chensue CCR8 Is Expressed by Antigen-Elicited, IL-10-Producing CD4+CD25+ T Cells, Which Regulate Th2-Mediated Granuloma Formation in Mice J. Immunol., February 15, 2005; 174(4): 1962 - 1970. [Abstract] [Full Text] [PDF]
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A. M. Hanash and R. B. Levy Donor CD4+CD25+ T cells promote engraftment and tolerance following MHC-mismatched hematopoietic cell transplantation Blood, February 15, 2005; 105(4): 1828 - 1836. [Abstract] [Full Text] [PDF]
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 C Veltkamp, R B Sartor, T Giese, F Autschbach, I Kaden, R Veltkamp, D Rost, B Kallinowski, and W Stremmel Regulatory CD4+CD25+ cells reverse imbalances in the T cell pool of bone marrow transplanted TG{varepsilon}26 mice leading to the prevention of colitis Gut, February 1, 2005; 54(2): 207 - 214. [Abstract] [Full Text] [PDF]
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C. J. Workman and D. A. A. Vignali Negative Regulation of T Cell Homeostasis by Lymphocyte Activation Gene-3 (CD223) J. Immunol., January 15, 2005; 174(2): 688 - 695. [Abstract] [Full Text] [PDF]
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M. A. Curotto de Lafaille, A. C. Lino, N. Kutchukhidze, and J. J. Lafaille CD25- T Cells Generate CD25+Foxp3+ Regulatory T Cells by Peripheral Expansion J. Immunol., December 15, 2004; 173(12): 7259 - 7268. [Abstract] [Full Text] [PDF]
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O. R. Millington, A. McI. Mowat, and P. Garside Induction of Bystander Suppression by Feeding Antigen Occurs despite Normal Clonal Expansion of the Bystander T Cell Population J. Immunol., November 15, 2004; 173(10): 6059 - 6064. [Abstract] [Full Text] [PDF]
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E. Xystrakis, A. S. Dejean, I. Bernard, P. Druet, R. Liblau, D. Gonzalez-Dunia, and A. Saoudi Identification of a novel natural regulatory CD8 T-cell subset and analysis of its mechanism of regulation Blood, November 15, 2004; 104(10): 3294 - 3301. [Abstract] [Full Text] [PDF]
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H. C. Dujardin, O. Burlen-Defranoux, L. Boucontet, P. Vieira, A. Cumano, and A. Bandeira Regulatory potential and control of Foxp3 expression in newborn CD4+ T cells PNAS, October 5, 2004; 101(40): 14473 - 14478. [Abstract] [Full Text] [PDF]
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J. Machen, J. Harnaha, R. Lakomy, A. Styche, M. Trucco, and N. Giannoukakis Antisense Oligonucleotides Down-Regulating Costimulation Confer Diabetes-Preventive Properties to Nonobese Diabetic Mouse Dendritic Cells J. Immunol., October 1, 2004; 173(7): 4331 - 4341. [Abstract] [Full Text] [PDF]
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S. Makita, T. Kanai, S. Oshima, K. Uraushihara, T. Totsuka, T. Sawada, T. Nakamura, K. Koganei, T. Fukushima, and M. Watanabe CD4+CD25bright T Cells in Human Intestinal Lamina Propria as Regulatory Cells J. Immunol., September 1, 2004; 173(5): 3119 - 3130. [Abstract] [Full Text] [PDF]
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U. Wagner, M. Pierer, M. Wahle, F. Moritz, S. Kaltenhauser, and H. Hantzschel Ex Vivo Homeostatic Proliferation of CD4+ T Cells in Rheumatoid Arthritis Is Dysregulated and Driven by Membrane-Anchored TNF{alpha} J. Immunol., August 15, 2004; 173(4): 2825 - 2833. [Abstract] [Full Text] [PDF]
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E. Nishimura, T. Sakihama, R. Setoguchi, K. Tanaka, and S. Sakaguchi Induction of antigen-specific immunologic tolerance by in vivo and in vitro antigen-specific expansion of naturally arising Foxp3+CD25+CD4+ regulatory T cells Int. Immunol., August 1, 2004; 16(8): 1189 - 1201. [Abstract] [Full Text] [PDF]
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P. Hoffmann, R. Eder, L. A. Kunz-Schughart, R. Andreesen, and M. Edinger Large-scale in vitro expansion of polyclonal human CD4+CD25high regulatory T cells Blood, August 1, 2004; 104(3): 895 - 903. [Abstract] [Full Text] [PDF]
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K. Inagaki-Ohara, T. Chinen, G. Matsuzaki, A. Sasaki, Y. Sakamoto, K. Hiromatsu, F. Nakamura-Uchiyama, Y. Nawa, and A. Yoshimura Mucosal T Cells Bearing TCR{gamma}{delta} Play a Protective Role in Intestinal Inflammation J. Immunol., July 15, 2004; 173(2): 1390 - 1398. [Abstract] [Full Text] [PDF]
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L. Graca, A. Le Moine, C.-Y. Lin, P. J. Fairchild, S. P. Cobbold, and H. Waldmann Donor-specific transplantation tolerance: The paradoxical behavior of CD4+CD25+ T cells PNAS, July 6, 2004; 101(27): 10122 - 10126. [Abstract] [Full Text] [PDF]
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M. K. Lee IV, D. J. Moore, B. P. Jarrett, M. M. Lian, S. Deng, X. Huang, J. W. Markmann, M. Chiaccio, C. F. Barker, A. J. Caton, et al. Promotion of Allograft Survival by CD4+CD25+ Regulatory T Cells: Evidence for In Vivo Inhibition of Effector Cell Proliferation J. Immunol., June 1, 2004; 172(11): 6539 - 6544. [Abstract] [Full Text] [PDF]
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O. Joffre, N. Gorsse, P. Romagnoli, D. Hudrisier, and J. P. M. van Meerwijk Induction of antigen-specific tolerance to bone marrow allografts with CD4+CD25+ T lymphocytes Blood, June 1, 2004; 103(11): 4216 - 4221. [Abstract] [Full Text] [PDF]
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P. L. Vieira, J. R. Christensen, S. Minaee, E. J. O'Neill, F. J. Barrat, A. Boonstra, T. Barthlott, B. Stockinger, D. C. Wraith, and A. O'Garra IL-10-Secreting Regulatory T Cells Do Not Express Foxp3 but Have Comparable Regulatory Function to Naturally Occurring CD4+CD25+ Regulatory T Cells J. Immunol., May 15, 2004; 172(10): 5986 - 5993. [Abstract] [Full Text] [PDF]
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S. P. Cobbold, R. Castejon, E. Adams, D. Zelenika, L. Graca, S. Humm, and H. Waldmann Induction of foxP3+ Regulatory T Cells in the Periphery of T Cell Receptor Transgenic Mice Tolerized to Transplants J. Immunol., May 15, 2004; 172(10): 6003 - 6010. [Abstract] [Full Text] [PDF]
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I. M. de Kleer, L. R. Wedderburn, L. S. Taams, A. Patel, H. Varsani, M. Klein, W. de Jager, G. Pugayung, F. Giannoni, G. Rijkers, et al. CD4+CD25bright Regulatory T Cells Actively Regulate Inflammation in the Joints of Patients with the Remitting Form of Juvenile Idiopathic Arthritis J. Immunol., May 15, 2004; 172(10): 6435 - 6443. [Abstract] [Full Text] [PDF]
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T. W. Vahlenkamp, M. B. Tompkins, and W. A. F. Tompkins Feline Immunodeficiency Virus Infection Phenotypically and Functionally Activates Immunosuppressive CD4+CD25+ T Regulatory Cells J. Immunol., April 15, 2004; 172(8): 4752 - 4761. [Abstract] [Full Text] [PDF]
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L. A. Yi, S. Hajialiasgar, and E. Chuang Tyrosine-mediated inhibitory signals contribute to CTLA-4 function in vivo Int. Immunol., April 1, 2004; 16(4): 539 - 547. [Abstract] [Full Text] [PDF]
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B. Martin, A. Banz, B. Bienvenu, C. Cordier, N. Dautigny, C. Becourt, and B. Lucas Suppression of CD4+ T Lymphocyte Effector Functions by CD4+CD25+ Cells In Vivo J. Immunol., March 15, 2004; 172(6): 3391 - 3398. [Abstract] [Full Text] [PDF]
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M. Hesse, C. A. Piccirillo, Y. Belkaid, J. Prufer, M. Mentink-Kane, M. Leusink, A. W. Cheever, E. M. Shevach, and T. A. Wynn The Pathogenesis of Schistosomiasis Is Controlled by Cooperating IL-10-Producing Innate Effector and Regulatory T Cells J. Immunol., March 1, 2004; 172(5): 3157 - 3166. [Abstract] [Full Text] [PDF]
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J. Huehn, K. Siegmund, J. C.U. Lehmann, C. Siewert, U. Haubold, M. Feuerer, G. F. Debes, J. Lauber, O. Frey, G. K. Przybylski, et al. Developmental Stage, Phenotype, and Migration Distinguish Naive- and Effector/Memory-like CD4+ Regulatory T Cells J. Exp. Med., February 2, 2004; 199(3): 303 - 313. [Abstract] [Full Text] [PDF]
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X. Zhang, D. N. Koldzic, L. Izikson, J. Reddy, R. F. Nazareno, S. Sakaguchi, V. K. Kuchroo, and H. L. Weiner IL-10 is involved in the suppression of experimental autoimmune encephalomyelitis by CD25+CD4+ regulatory T cells Int. Immunol., February 1, 2004; 16(2): 249 - 256. [Abstract] [Full Text] [PDF]
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L. A. Stephens, A. N. Barclay, and D. Mason Phenotypic characterization of regulatory CD4+CD25+ T cells in rats Int. Immunol., February 1, 2004; 16(2): 365 - 375. [Abstract] [Full Text] [PDF]
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L. E. Marra, Z. X. Zhang, B. Joe, J. Campbell, G. A. Levy, J. Penninger, and L. Zhang IL-10 Induces Regulatory T Cell Apoptosis by Up-Regulation of the Membrane Form of TNF-{alpha} J. Immunol., January 15, 2004; 172(2): 1028 - 1035. [Abstract] [Full Text] [PDF]
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H. Jonuleit and E. Schmitt The Regulatory T Cell Family: Distinct Subsets and their Interrelations J. Immunol., December 15, 2003; 171(12): 6323 - 6327. [Full Text] [PDF]
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T. R. Malek The main function of IL-2 is to promote the development of T regulatory cells J. Leukoc. Biol., December 1, 2003; 74(6): 961 - 965. [Abstract] [Full Text]
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C. Cozzo, J. Larkin III, and A. J. Caton Cutting Edge: Self-Peptides Drive the Peripheral Expansion of CD4+CD25+ Regulatory T Cells J. Immunol., December 1, 2003; 171(11): 5678 - 5682. [Abstract] [Full Text] [PDF]
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A. Foussat, F. Cottrez, V. Brun, N. Fournier, J.-P. Breittmayer, and H. Groux A Comparative Study between T Regulatory Type 1 and CD4+CD25+ T Cells in the Control of Inflammation J. Immunol., November 15, 2003; 171(10): 5018 - 5026. [Abstract] [Full Text] [PDF]
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S. Fisson, G. Darrasse-Jeze, E. Litvinova, F. Septier, D. Klatzmann, R. Liblau, and B. L. Salomon Continuous Activation of Autoreactive CD4+ CD25+ Regulatory T Cells in the Steady State J. Exp. Med., September 2, 2003; 198(5): 737 - 746. [Abstract] [Full Text] [PDF]
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T. L. Denning, H. Qi, R. Konig, K. G. Scott, M. Naganuma, and P. B. Ernst CD4+ Th Cells Resembling Regulatory T Cells That Inhibit Chronic Colitis Differentiate in the Absence of Interactions Between CD4 and Class II MHC J. Immunol., September 1, 2003; 171(5): 2279 - 2286. [Abstract] [Full Text] [PDF]
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, wt CD25- CD4 T cells injected alone; 
,
CD25- CD4 T cells injected with the indicated
CD25+ CD4 T cell population. Differentiation of the origin
of the cells was achieved using Thy1.1 and/or Ly5.1 congenic
markers.