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and IL-2 Shape a Population of Regulatory Cells That Controls CD4+ T Cell Numbers1
* Lymphocyte Population Biology, Unité de Recherche Associée Centre National de la Recherche Scientifique 1961, Institut Pasteur, and
Institut National de la Santé et de la Recherche Médicale Unité 345, Centre Hospitalo-Universitaire Necker, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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- and
IL-2-deficient mice is due to the lack of a population of regulatory
cells essential for CD4 T cell homeostasis. In chimeras reconstituted
with bone marrow cells from IL-2R-deficient donors,
restitution of a population of CD25+CD4+ T
cells prevents the chaotic accumulation of lymphoid cells, and rescues
the mice from autoimmune disease and death. The reintroduction of
IL-2-producing cells in IL-2-deficient chimeras establishes a
population of CD25+CD4+ T cells, and restores
the peripheral lymphoid compartments to normal. The
CD25+CD4+ T cells regulated selectively the
number of naive CD4+ T cells transferred into T
cell-deficient hosts. The CD25+CD4+/naive CD4 T
cell ratio and the sequence of cell transfer determines the homeostatic
plateau of CD4+ T cells. Overall, our findings demonstrate
that IL-2R is an absolute requirement for the development of the
regulatory CD25+CD4+ T cells that control
peripheral CD4 T cell homeostasis, while IL-2 is required for
establishing a sizeable population of these cells in the peripheral
pools. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mutant IL-2R-/- mice represent a paradigm
for perturbed lymphocyte homeostasis (10). They develop
massive enlargement of peripheral lymphoid organs associated with T and
B cell expansion and autoimmune disease (10), indicating
that IL-2R is essential for the control of the size of the
peripheral lymphoid compartment. It was generally believed that the
defect in IL-2R-/- mice was cell autonomous
and that IL-2R regulated the balance between clonal expansion and
cell death following lymphocyte activation (10, 11). Thus,
in the absence of the negative signals mediated by IL-2R, T cells
would undergo uncontrolled expansion (10). However, it was
recently shown that when placed in a normal environment, TCR transgenic
(Tg)3
IL-2R-/- cells exhibited normal clonal
contraction after Ag-induced expansion (12), suggesting
that activation-induced cell death (AICD) is kept and that IL-2R
signals could also control bystander T cell activation
(12). Alternatively, IL-2R could be required for the
development and/or the function of a subpopulation of T cells capable
of regulating peripheral T cell homeostasis. Different lines of
evidence seem to support this latter alternative. First, recent
findings showed that wild-type T cells could control the
expansion of IL-2R
-deficient T cells in mixed bone marrow
(BM) chimeras, a property attributed to a population of
cytotoxic CD8 T cells (13). Secondly, previous studies
have also shown that Ag-induced expansion of TCR Tg IL-2-deficient T
cells could be controlled by CD25+ T cells
(14). Finally, results indicate that regulatory
CD45RBlowCD25+CD4+
T cells limit naive CD4 T cell expansion and suggest that they may play
a role in the control of peripheral T cell numbers
(15).
We decided to investigate if populations of regulatory
CD25+CD4+ T cells
(16) could prevent the chaotic lymphocyte accumulation in
IL-2R-/- mice and control the expansion of
peripheral naive CD4 T cells. We found that
CD25+CD4+ T cells could
indeed control peripheral T cell accumulation and composition in mouse
chimeras reconstituted with BM cells from
IL-2R-/- mice and rescued these mice from
death. Similarly, recombination-activating gene
(Rag)2-/- chimeras reconstituted with a
mixture of BM cells from IL-2R and IL-2-deficient donors remained
healthy, and the number and distribution of CD25+
and CD25-CD4 T cells was as in normal mice. To
relate these effects to homeostatic control of the CD4 peripheral T
cell pool, we examined the selectivity and quantitative requirements
ruling the expansion of naive
CD45RBhighCD25-CD4+
and
CD45RBlowCD25+CD4+
T cells transferred into CD3
-/- T
cell-deficient host mice (17). We found that the two cell
populations show different homeostatic plateaus and that
CD25+CD4+ T cells can
selectively inhibit the peripheral expansion of the naive CD4 T cells,
but not CD8 T cells in a dose-dependent manner.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6.Ly5.2 mice from Iffa-Credo (L’Arbresle, France),
B6.CD3-/- (17),
B6.IL-2R-/- (10),
B6.TCR-/- (18), and
C57BL/6.Ly5.1 mice from the Centre de Development des Techniques
Avancées-Centre National de la Recherche Scientifique
(Orléans, France), B6.IL-10-/-
(19) from The Jackson Laboratory (Bar Harbor, ME) and
B6.IL-2-/- (20) from our breeding
facilities or from Dr. A. Schimpl (Institute for Virology and
Immunobiology, University of Wurzburg, Germany) were matched for age
(6–12 wk) and sex.
Cell sorting and cell transfers
Lymph node (LN) cells from the Ly5.2 and Ly5.1 donor mice
were first enriched for CD4+ T cells by negative
selection using a Dynal MPC6 MACS (Dynal Biotech, Great Neck,
NY). Briefly, cells were incubated with a mixture of rat Abs
directed to mouse B220 (RA3-6B2), Mac1 (CD11b), and CD8 (53–6.7),
all from BD PharMingen (San Diego, CA), followed by sheep anti-rat
Ig-coated Dynabeads (Dynal Biotech). After removing the positive
fraction, >90% of the remaining population was
CD4+. These cells labeled with the appropriate
combinations of anti-CD4 (L3T4/RM4-5), anti-CD45RB, and
anti-CD25 (784), Abs were sorted on a
FACStarPlus (BD Biosciences, Mountain View, CA).
The purity of the sorted
CD45RBhighCD25-CD4+
and
CD45RBlowCD25+CD4+
populations varied from 96–99.9%.
Intact nonirradiated B6.CD3-/-
(17) hosts were injected i.v. with the purified CD4 T cell
populations alone or mixed at different cell ratios. By using mice
differing by Ly5 allotypes, we were able to discriminate the cells
originating from the different donor mice. Host mice were sacrificed at
different time intervals after cell transfer. Spleen, inguinal, and
mesenteric LN cell suspensions were prepared and the number and
phenotype of the cells from each donor population evaluated. The total
peripheral T cells showed in the results represent the number of cells
recovered in the host’s spleen added to twice the number of cells
recovered from the host’s inguinal and mesenteric LNs.
Labeling with CFSE
Cells were labeled with CFSE as described (21). Briefly, sorted CD4+ T cells (107/ml) were incubated for 10 min at 37°C with CFSE (10 µM).
BM chimeras
Host 8-wk-old Rag2-/- B6 mice were lethally irradiated (900 rad) with a 137Ce source and received i.v. 2–4 x 106 T cell-depleted BM cells from different donor mice, mixed at different ratios. T cell depletion (<0.1%) was done in a Dynal MPC6 MACS after incubating the BM cells with anti-CD4, anti-CD8, and anti-CD3 biotinylated Abs followed by streptavidin-coated Dynabeads. By using donor and host mice that differ according to Ly5 allotype markers, we were able to discriminate between the T cells originating from the different donors.
Flow cytometry analysis
The following mAbs were used: anti-CD3 (145-2C11),
anti-CD4 (L3T4/RM4-5), anti-CD69 (H1.2F3), anti-CD25 (784),
anti-CD45RB, anti-CD24/HSA (M1/69), and anti-TCR (H57)
from BD PharMingen, and anti-CD44 (IM781) and anti-CD62L
(MEL14) from Caltag Laboratories (San Francisco, CA). Cell surface
four-color staining was preformed with the appropriate
combinations of FITC, PE, tricolor, PerCP, biotin, and APC-coupled Abs.
Biotin-coupled Abs were secondary labeled with APC-, tricolor-, (Caltag
Laboratories) or PerCP-coupled (BD Biosciences) streptavidin. Dead
cells were excluded during analysis according to their light-scattering
characteristics. All acquisitions and data analysis were performed with
a FACSCalibur (BD Biosciences) interfaced to the Macintosh CellQuest
software.
Statistical analysis
Sample means were compared using the unpaired Student’s t test. In case the variances of the two samples were considerably different, the data were log-transformed to see if the variances become more similar. If so, the unpaired t test was applied to the log-transformed data. Otherwise, Sattertwhaite’s approximation was applied. Sample means were considered significantly different at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- mice and prevent death of chimeras
reconstituted with BM cells from IL-2R-/- mice
Mutant IL-2R-/- mice develop massive
lymphocyte accumulation and autoimmune disease (10). It
was proposed that in the absence of negative signals mediated through
IL-2R, T cells would undergo uncontrolled expansion suggesting that
the IL-2R-/- defect was cell autonomous
(10). We asked if a population of normal T cells could
control the chaotic accumulation of T cells in
IL-2R-/- mice. We examined whether mature
CD25+CD4+ T cells could
alter the number and state of activation of CD4 T cells in
B6.Rag2-/- chimeras reconstituted with 100% BM
cells from B6.IL-2R-/- donors. Using these
BM chimeras rather than intact B6.IL-2R-/-
mice allowed us to increase the number of mice studied. We found that
all chimeras injected exclusively with BM cells from
B6.IL-2R-/- donors died within 40–50 days
(Fig. 1
A), with overt signs of
runting, anemia, and in some mice, lymphoid hyperplasia (up to 300
x 106 lymphocytes). In contrast, the BM chimeras
that received 105
CD25+CD4+ T cells from
normal B6.Ly5.1 donors 2 wk after BM reconstitution were all alive 6 mo
later (Fig. 1A and data not shown). The effects of the
CD25+CD4+ T cells were
time-dependent since only 50% of the chimeras survived if we delayed
their transfer to 4 wk after BM injection (Fig. 1A). These
differences suggest that either control of T cell numbers is more
efficient earlier when the number of peripheral
IL-2R-/- T cells is lower, or it may require
the continuous presence of "regulatory" T cells at the onset of T
cell production. The surviving chimeras remained healthy, the
hematocrit levels were normal (40–45%), and the total number of T
cells was similar (47.1 ± 7.7 and 81 ± 9.7 x
106 for the two groups of BM chimeras injected
with CD25+CD4+ T cells,
respectively) to control mice (40–60 x 106
CD4 T cells). The composition of the peripheral LN T cells was as in
normal mice comprising 5–10% of
CD45RBlowCD25+CD4+
T cells, all of B6.Ly5.1 origin, and 50–60% of nonactivated
CD45RBhighCD25-CD4+
T cells all from IL-2R-/- origin (Fig. 1B). This is in contrast to donor
IL-2R-/- mice, where most (>80%) T cells
have an activated phenotype (Ref. 10 ; data not shown). We
should point out that the transfer of up to 2 x
105
CD25-CD4+ T cells did not
rescue the CD25-/- BM chimeras (data not
shown).
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-/- T cells, lethally irradiated
lymphopenic B6.Rag2-/- mice were reconstituted
with a mixture of T cell-depleted BM cells. In brief, 50% of the
injected BM cells were from B6.Ly5.2IL-2R-/-
donors and the remaining 50% from
B6.Ly5.2.TCR-/- (unable to generate T cells)
and normal B6.Ly5.1 donors mixed at different ratios. By keeping the
fraction of cells from IL-2R-/- donors in
the injected BM cohort at 50%, we fixed the rate of production of
IL-2R-/- T cells in all chimeras studied.
Thus, the resulting chimeras should all have the same number of
peripheral IL-2R-/- T cells
(1). By mixing BM cells from B6 normal and
B6.TCR-/- donors, we reduced the number of
competent precursors available for thymus colonization and
regeneration, as the normal competent precursor cells are diluted among
incompetent precursors from the TCR-/-
mutant donors (1). With this strategy, we could evaluate
the role of different numbers of normal T cells in the control of a
fixed number of IL-2R-/- T cells. We found
that all the chimeras that could only generate
IL-2R-/- T cells, i.e., which received 50%
BM cells from B6.IL-2R-/- donors and 50% of
BM cells from B6.TCR-/- donors, died 40–50
days after reconstitution (Fig. 2A). The presence of 5% cells
from normal donors in the thymus of the chimeras was sufficient to
rescue 80% of the mice (Fig. 2A). The presence of 10 or
50% of normal cells rescued 100% of the chimeras. In the 50/50
B6.Ly5.2IL-2R-/-/B6.Ly5.1 chimeras,
the representation of the two types of donor cells remained unchanged
both in the thymus and in the peripheral T cell pools (data not shown).
In the surviving BM chimeras containing a fraction of cells from normal
donors, the total number of peripheral T cells and the relative
distribution of the CD25+ and
CD25- CD4 T cell subsets were as in normal mice
(Fig. 2B). Thus, a developing population of normal CD4 T
cells controlled the accumulation and state of activation of the
IL-2R-/- T cells. These results show that
the lethal accumulation of peripheral T cells in
IL-2R-/- mice is not cell autonomous, but
due to the lack of a population of CD4+ T cells
essential for peripheral T cell homeostasis. We demonstrated that
expression of the IL-2R chain is required for the generation of this
population of regulatory CD4 T cells.
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-/- BM chimeras
With age, IL-2-/- mice develop fatal
inflammatory bowel disease (IBD) and lymphocyte proliferation
(20). The peripheral T cell compartments of these mice
show overt signs of T cell activation and lack a well-defined
population of CD25+, which never exceeds 1–2%
of the CD4+ T cells (14, 22). We
investigated whether the
CD25+CD4+ T cells from
IL-2-/- mice could rescue the defects of CD4 T
cell homeostasis observed in IL-2R-/- mice.
We reconstituted lethally irradiated lymphopenic
B6.Rag2-/- mice with a 50/50 mixture of T
cell-depleted BM cells from B6.IL-2R-/- and
B6.IL-2-/- donors. Control mice received BM
cells from either B6.IL-2R-/- or
B6.IL-2-/- mice equally mixed at 50/50 with BM
cells from B6.TCR-/- donors. The presence of
50% of BM cells from IL-2-/- donors rescued
the totality of the IL-2R-/- BM chimeras
(Fig. 3A) and restored the CD4
T cell populations to normal. In the peripheral T cell pools of these
chimeras, the number and the distribution of
CD25+ and CD25- CD4 T
cells was as in normal mice (Fig. 3B). Thus, in presence of
IL-2, the hemopoietic precursors from IL-2-deficient donor mice
generated a stable population of mature peripheral
CD25+CD4+ regulatory T
cells able to control the homeostasis of the CD4 T cell
compartment. Upon secondary transfer, this population of
IL-2-/-CD25+CD4+
cells was able to rescue B6.IL-2R-/-
chimeras (data not shown). Chimeras injected with BM cells from
B6.IL-2-/- donors alone show a normal number of
CD25+ and CD25- CD4 T
cells (data not shown). This finding indicates that in
Rag2-/- hosts, resident non-T cells can provide
a source of endogenous IL-2 (23) sufficient to compensate
for the lack of its production by the IL-2-/-
hemopoietic cells. Overall, these results demonstrate that IL-2 is
required for the establishment of a stable population of
CD25+CD4+ regulatory T
cells in the peripheral pools. In absence of this, population control
of CD4 T cell numbers is lost and the mice develop lymphoid hyperplasia
and autoimmune diseases. Overall, these findings indicate that
populations of CD25+ and
CD25-CD4 T cells may have different homeostatic
properties and that they may regulate each other. We decided to
investigate this possibility using a cell transfer strategy.
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Peripheral T cells, when transferred into T cell-deficient hosts,
are capable of considerable expansion (5, 6, 7), but their
number is controlled at a homeostatic plateau. To investigate the
homeostasis of peripheral CD4+ T cell
subpopulations, different numbers of purified
CD4+ cells, that is,
CD45RBhighCD25-,
CD45RBlowCD25-, and
CD45RBlowCD25+ cells
were i.v. transferred into syngeneic CD3-/-
T cell-deficient hosts. In hosts receiving as few as 5 x
103 and as many as 105
cells,
CD45RBhighCD25-CD4+
T cells (from now on referred to as naive CD4) expanded to reach stable
equilibrium at 
1–2 x 107 cells, 10–12
wk after transfer (Fig. 4). In mice
injected with the same number of
CD45RBlowCD25+CD4+
T cells (from now on referred to as
CD25+CD4+), these cells
also expanded but reached equilibrium at 10-fold lower values, i.e., at
1–2 x 106 cells/hosts (Fig. 4). Transfer
of increasing numbers (>105) of cells did not
modify the final cell recovery (data not shown). These results indicate
that naive CD4+ and
CD25+CD4+ T cells are both
able to expand and accumulate at the periphery, but their final number
is regulated at different homeostatic plateau levels. It should be
noted that at later times after transfer, mice injected with naive
CD4+ T cells developed a wasting autoimmune
disease and eventually died (>15 wk), while hosts of
CD25+CD4+ T cells remained
healthy (data not shown). Total nonseparated LN
CD4+ T cells containing 10%
CD25+ cells expanded to a plateau of 1–2
x 107 cells, but fail to develop signs of
wasting disease (data not shown). Activated
CD45RBlowCD25-CD4+
T cells expanded to similar plateaus as naive
CD4+ T cells (data not shown).
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Cellular competition and the presence or absence of resident T
cell populations can alter the peripheral fate of newly arriving thymus
emigrants (8, 9, 24). To truly establish the homeostasis
of the transferred peripheral T cells, we asked if the presence of a
resident T cell population could interfere with the expansion of a
second newly injected cell population or whether the injection of a new
population could modify the fate of a resident population. We
"parked" 5 x 104 Ly5.1 naive
CD4+ T cells in different hosts. Seven weeks
later, each host received the same number of a second population of
Ly5.2 naive CD4+ or
CD25+CD4+ T cells.
Age-matched control mice received either the first or the second
population alone. We sacrificed the mice at 7 (before the second
injection) or 14 wk after the first injection. After transfer, naive T
cells acquired a CD45RBlow activated/memory
phenotype, but only a few (1–2%) became CD25low
(data not shown). In mice injected sequentially with two populations of
naive CD4+ T cells, the expansion of both
populations was limited through competition and they shared the
peripheral compartment of the host (Fig. 5A). The total T cell recovery
was the same as in mice injected with either population alone
(2 x 107). The transfer of 5 x
104
CD45RBlowCD25+CD4+
cells into mice injected 7 wk before with
CD45RBhighCD25-CD4+
cells suppressed significantly (p < 0.001)
further expansion of the established 5 x
106
CD45RBhighCD25-CD4+-derived
T cell population (Fig. 5B). The total T cell recovery
diminishes accordingly. The number of cells recovered from the second
population of
CD45RBlowCD25+CD4+
T cells did not change. These results show that a limited number
(5 x 104) of newly transferred
CD45RBlowCD25+CD4+
T can suppress the expansion of an abundant (5 x
106) population of resident
CD4+ T cells.
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C). In the
mice hosting the first population of
CD25+CD4+ T cells, newly
transferred naive CD4+ T cells expanded and
induced a 3- to 4-fold increase (p < 0.01) in
the number of resident CD4 T cells from
CD25+CD4+ origin (Fig. 5D). These results show that newly injected naive
CD4+ T cells helped the growth of the progeny of
CD25+CD4+ T cells
(p < 0.01). In contrast, the resident
CD25+CD4+ T cell progeny do
not significantly (p = 0.5) inhibit the growth
of newly transferred naive CD4+ T cells. In hosts
that received CD25+CD4+
cells, only 30% of the recovered T cells remained
CD25+ (data not shown). This could
represent true phenotypic changes or the expansion of a few contaminant
CD4 T cells in the injected CD25+ population. We
investigated the suppressive capacities of the resident cells that
express or not CD25. For this purpose,
CD25+CD4+ T cells were
parked for 7 wk in host mice. At the end of this time period,
CD25+CD4+ T cells and
CD45RBlowCD25-CD4+
T cells derived from the parked population were injected alone or
coinjected with naive CD4+ T cells from different
Ly5 donors into secondary CD3-/- hosts.
Although the resorted CD25+ cells retained the
capacity to suppress the growth of naive CD4+ T
cells (p < 0.001), the suppressive capacity of
the resorted CD25- cells was absent or reduced
(p = 0.21; Fig. 6). These results suggest that the
suppressor effects correlate with the surface expression of
CD25.
|
We showed that
CD25+CD4+ T cells could
control chaotic accumulation of IL-2R-/- T
cells. We decided to investigate whether they could also control the
homeostatic plateau of naive CD4 T cells transferred to T
cell-deficient hosts. We quantified both the numbers of cells involved
and the selectivity of the interactions. We transferred
104 purified naive Ly5.1
CD4+ T cells or CD8 T cells alone or coinjected
with variable numbers of Ly5.2
CD25+CD4+ T cells, ranging
from 3 x 103–105,
into CD3-/- hosts (Fig. 7, A and B). We
found that the CD25+CD4+ T
cells limited the accumulation of naive CD4+ T
cells (15) and that their suppressive effects were
dose-dependent (Fig. 7A). Increasing numbers of
CD25+CD4+ T cells
progressively suppressed the expansion of the cotransferred naive
CD4+ T cells, and at a 10:1 cell ratio, we
recovered 10-fold less T cells from naive CD4+
origin (p < 0.005). Total T cell recovery
diminished according to the level of suppression, that is, overgrowth
of the coinjected CD25+CD4+
T cells did not compensate for the lack of expansion of the naive T
cells (p-NS) (Fig. 7B). We also
found that CD25+CD4+ T
cells did not affect the growth of coinjected total LN CD8 T cells
(p = 0.4; Fig. 7C), indicating that
their inhibitory effects are lineage-specific. By varying either the
number of T cells injected, or the ratio
CD25+/CD25- T cells, we
found that the number of T cells from naive
CD25-CD4 origin recovered was not dependent on
the number of cells transferred, but determined by the
CD25+/CD25- ratio present
in the inoculum (Fig. 7D). These results raised the
possibility that the
CD25+CD4+ T cells might
have blocked division of the naive CD4+ T cells.
To test this possibility, we compared the fate of CFSE-labeled naive T
cells transferred alone (Fig. 7E, top
panel) or in the presence of an excess of
CD25+ cells (Fig. 7E, bottom
panel). Three days after transfer (Fig. 7E), the
patterns of dilution of the CFSE labeling were similar in both groups
of mice, and we recovered an identical number of cells in the two
groups of host mice (data not shown). At day 10, the majority of the
transferred cells were CFSE-, indicating that
these cells underwent several rounds of division. However, the fraction
of CFSE- cells was higher, and we recovered
27-fold more CD4 T cells in the mice injected with naive T cells in
absence of CD25+ cells (Fig. 7E). The
differences in total cell recovery could be due to either an increase
in cell survival or to an increase in the rate of cell division of the
CD4+CD25- naive T cells
when transferred alone. Thus, the present results do not allow
discriminating between these two possibilities or if the increase of
the number of cells corresponds to an increased fraction of cells that
enter cell cycle or to a reduced cell cycle time. Studies on the
annexin V labeling of the transferred populations were not conclusive
(data not shown). On the whole, these results indicate that the
suppressive effects are not obtained through complete block of
proliferation, but do not allow us to distinguish whether they affect
the rate of cell expansion or the survival (accumulation) of the newly
generated T cells. In contrast, we found that activated
CD45RBlowCD25-CD4+
T cells did not control expansion of naive CD4 T cells (data not
shown).
|
-/- CD4 T cells
Finally, we studied whether the same forms of interaction also
applied to populations of CD4 T cells from
IL-2R-/- and IL-2-/-
mice. We found that
CD25+CD4+ T cells from
normal donors inhibited the expansion of the CD4+
T cells from IL-2R-/- mice transferred into
CD3-/- hosts (p <
0.05; Fig. 8A). Similarly,
naive CD4+ T cells from
IL-2-/- origin expanded and were suppressed
(p < 0.01) while
CD25+CD4+ T cells from
IL-2-/- mice slightly suppressed the expansion
of naive CD4+ T cells from normal donors as well
as CD25+CD4+ T cells from
normal donors at a 1:1 cell ratio (Fig. 8B). Altogether,
these findings suggest that the control exerted by the
CD25+CD4+ T cells on the
accumulation of peripheral CD4 T cells in the
IL-2R-/- is due to their ability to regulate
peripheral CD4 T cell homeostasis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-2R-/- mutant mice are reported as a
paradigm for perturbed lymphocyte homeostasis (10). The
lack of the IL-2R was believed to impair AICD in vivo
(10), to modify the balance between clonal expansion and
cell death, resulting in the deregulation of both the size and content
of the peripheral lymphoid compartments. The primary uncontrolled T
cell activation lead subsequently to secondary policlonal B cell
activation and autoantibody production. However, recent findings have
shown that when placed in a normal environment, TCR Tg
CD25-/- T cells exhibited a significant
reduction in Ag-induced expansion due to normal AICD (12).
This observation was interpreted as indicating that the regulatory role
of IL-2R signals was mediated through the control of bystander T
cell activation (12). We now demonstrate that the chaotic
lymphocyte accumulation developed in adult
IL-2R-/- mice is not cell autonomous: it is
due to the lack of a T cell population essential for the homeostasis of
peripheral T cell numbers. Two main lines of evidence support this
conclusion. First, the presence of a limited number of
CD25+CD4+ T cells rescues
mouse chimeras reconstituted with BM from
IL-2R-/- mice from chaotic lymphocyte
accumulation, polyclonal B and T cell activation, and death, and
restores the peripheral lymphoid compartment to normal. Second, we show
that CD25+CD4+ T cells
inhibit peripheral expansion of CD4 T cells from
IL-2R-/- mice transferred into T
cell-deficient adoptive hosts. Moreover, by using IL-2-deficient mice,
which also develop lymphoid hyperplasia and autoimmunity late in life
(20), we show that IL-2 is required for the establishment
of a stable and sizeable population of peripheral
CD25+CD4+ regulatory cells.
Thus, IL2-R/IL-2 signals are not involved in control of bystander
activation, but are instead required for the generation and peripheral
expansion/survival of a population of regulatory CD4 T cells essential
for peripheral CD4 T cell homeostasis.
We further dissected and quantified the type of cell interactions
involved in peripheral homeostasis by following the fate of separated
populations of CD4 T cells transferred into immune-deficient hosts
(7). We found that after transfer into
CD3-/- T cell-deficient hosts, purified
naive CD4+ T cells expanded to reach a stable
plateau at 1–2 x 107 cells,
independently of the number of injected cells. When a second population
of naive CD4+ cells was transferred into the same
hosts, the growth of each population was limited and the total T cell
recovery was the same as in mice injected with only one population. We
did not observe an overt advantage of either the tenant or the newcomer
cells. These findings confirmed that cellular rivalry could alter the
fate of T cells at the periphery (8, 9, 24), and attested
that the expansion of the transferred naive CD4+
T cells is under homeostatic control (7). We found that
the accumulation of the
CD25+CD4+ T cells in T
cell-deficient hosts is limited by a homeostatic plateau which
singularly operates at values 10-fold lower than for total
CD4+ or naive CD4+ T cells,
i.e., at 1–2 x 106 cells/host. We
confirmed by the cotransfer of these two T cell populations that the
presence of CD25+CD4+ T
cells limited the expansion of the naive CD4+ T
cells (15). Total T cell recovery diminished accordingly
to the levels of suppression thus, excluding the presence of
competition between the two populations.
We expanded these observations and showed that the inhibitory effects
were dose-dependent and lineage-specific, as they did not affect naive
CD8+ T cell expansion in vivo. However, lineage
specificity seems dependent on the experimental conditions, as
CD25+CD4+ T cells were
shown to control memory but not naive CD8+ T
cells (33), and to suppress both
CD4+ and CD8+ T cell
activation in vitro (34, 35). Suppression of naive
CD4+ T cell growth was obvious when the number of
CD25+CD4+ T cells exceeded
the number of the naive CD4+ T cells by a factor
of 10, less noticeable and variable when the two populations were
present at similar numbers. Thus, the physiological relevance of the
CD25+CD4+ T cells could be
arguable since in normal mice their number rarely exceeds 10% of the
total CD4+ T cell pool. However, upon sequential
cell transfers, we found that the injection of a limited number (5
x 104) of
CD25+CD4+ T cells arrests
the growth of an expanding population of 5 x
106 resident naive
CD4+-derived T cells. More importantly, a limited
number of CD25+CD4+ T cells
rescues mice reconstituted with BM cells from
IL-2R-/- donors, controls the chaotic T cell
accumulation, and reestablishes a peripheral T cell pool with a normal
subpopulation composition. Thus, the suppressive effects mediated by
the CD25+CD4+ T cells have
physiological relevance since they were also obtained at physiological
ratios of CD25+ to CD25-.
The changes in effectiveness observed between the different
experimental protocols and schedules of injection may be due to the
different fraction/number of activated cells to be suppressed.
Immediately following transfer, most naive CD4 T cells are activated,
while later, near steady state equilibrium, only a fraction
proliferates (7). Alternatively, they could be due to the
different capacity of the regulatory cells to suppress homeostatic
proliferation, occurring in the cell transfer experiments, and natural
T cell proliferation and reconstitution occurring in the BM chimeras.
Finally, the homing of the
CD25+CD4+ T cells could
also differ between the different experimental protocols used. In
experiments where the
CD25+CD4+ T cells were
transferred at the same time as the naive CD4 T cells, differential
homing abilities of the two populations could explain the apparently
too high CD25+:CD25- ratio
needed in the cotransfer experiments. The homing of the
CD25+CD4+ T cells could be
favored by the presence of activated T cells in the periphery of the
host mice as it could happen in the sequential transfer
experiments.
How do the CD25+CD4+ T
cells regulate homeostasis of naive CD4+ T cells?
To investigate the possible effects of
CD25+CD4+ T cells in
blocking the proliferation of CD25- cells, we
have transferred CFSE-labeled
CD25-CD4+ T cells alone or
with an excess of CD25+CD4 regulatory T cells
(Fig. 7E). The dilution of CFSE labeling observed in
both situations is similar, but the accumulation of cells observed in
the CFSE-negative fraction of cells accounts for the differences in the
number of cells recovered. Thus, homeostatic proliferation could be
said to occur in both situations, but its extent could be limited only
when CD25+CD4+ regulatory T
cells are present. The possibility that CD25+CD4
regulatory T cells are capable of inhibiting the extent of homeostatic
proliferation suggests the interesting possibility that the observed
regulation of self-reactive responses is just a side effect of a
broader function of these cells in the control of peripheral T cell
numbers. If these cells control the magnitude of expansion of all naive
CD25-CD4+ T cells, this
may also include expansion of self-reactive clones present within that
population. However, the opposite can also be true, and the control of
self-reactive responses may result in the control of total cell numbers
recovered. In this study, we show that the presence of T regulatory
(Treg) cells prevents the activation of CD4 T cells from
CD25-/- origin, including self-reactive clones,
and allows the establishment of a normal size naive peripheral T cell
compartment; sequential cell transfer, the
CD25+CD4+ regulatory T
cells suppress the expansion of activated T cells engaged in
homeostatic proliferation, reducing the number of cells recovered.
It has been shown that IL-10 mediates the regulatory functions of the
CD25+CD4+ T cells
(28, 36, 37, 38, 39), but in vitro studies have excluded the role
of IL-10 in CD25+ T cell-mediated suppression
(40). We examined the role of IL-10 in the suppression of
T cell proliferation in vivo; in contrast to a previous report
(15), we found that
CD25+CD4+ T cells from
IL-10-/- mice inhibit the expansion of naive T
cells as effectively as CD25+ cells form
wild-type mice (data not shown). This indicates that the effects of the
CD25+CD4+ T cells on T cell
expansion are IL-10-independent. However, we confirmed that
CD25+ cells from IL-10-/-
mice failed to protect against the wasting disease induced by the naive
CD4+ T cells (data not shown). TGF has also
been implicated in IBD protection, and recent claims suggest that it
may play a role in T cell homeostasis (41, 42, 43). However,
we found that both naive and activated CD4 T cells expressed similar
levels of mRNAs for the three subforms of TGF (data not shown).
Moreover, CD25+ T cells from TGF-deficient
mice are referred to be suppressors (44). We also found
that CD25+CD4+ cells from
TNF--/- and LT-/-
mice inhibit expansion of naive T cells (data not shown), excluding
their role in this process. By using lpr and gld
mutant mice, we excluded a possible role of Fas/Fas ligand
interactions in these processes (data not shown). The possible
involvement of CTLA-4 in T cell homeostasis is also unlikely, as it has
also been shown that
CD25+CD4+ T cells from
CTLA-4-deficient mice exhibit suppressor activity
(45). Recent results implicating the
glucocorticoid-induced TNFR (TNFRS18) in the regulatory activity of the
CD25+ T cells (46, 47) are not
conclusive (44). Other possibilities are under
investigation.
The suppressive capacity of the CD25+CD4+ T cells, while maximal upon injection, was virtually lost when these cells were parked for 2 mo in the hosts. It is possible that regulation of existing and newly transferred cells differs, and/or that "parked" cells may evolve functionally. Upon secondary transfer, we showed that the parked CD25+CD4+ T cell retained their suppressive abilities. Our in vivo observations contrast with recent in vitro data showing that T cells having lost CD25 expression suppress expansion of naive CD4 T cells (48). This apparent discrepancy may simply reflect differences in the in vitro and in vivo behavior of the Treg cells. Other reports have shown that the CD25+ cells progeny of in vivo activated naive CD4+ T cells were not able to confer effective protection of disease (49) or to control in vitro T cell proliferation (40). Moreover, CD25-CD4+ thymocytes, when transferred to immune-deficient hosts, cannot generate a Treg cell population and induce autoimmune disease (50). In addition, the autoimmune manifestations that occur in neonatal thymectomized mice (day 3) correlate with the absence of CD25+CD4+ T cells, and the reintroduction of CD25+ cells generated in 3-day-old Tx mice was unable to prevent disease (40), which could be avoided by CD25+CD4+ T cells from normal donors (28). These observations indicate that the regulatory functions may be a property of a specific cell subpopulation, but that inside this subpopulation these functions correlate with the expression of the CD25 marker and may require continuous T cell stimulation (27, 32). The inhibitory effects may require direct T-T cell interactions (40) or act via a third party presenting cell. We found that the initial CD25+:CD25- ratio strictly determined the final number of CD4 T cells, suggesting a direct relationship between the two populations. Inhibition does not seem to require Ag specificity or mutual cognate recognition by the interacting cells, since in vitro the two populations do not need to recognize the same ligand (51). However, maintenance of the Ag-specific regulatory cells seems to require the continuous presence of the Ag (32).
To the question of whether the regulatory cells may represent a
separate CD4+ T cell lineage (52),
the answer is yes. The IL-2R-/- mutant mice
lack these cells. The transfer of a limited number of
CD25+CD4+ T cells in mouse
chimeras reconstituted with BM cells from
IL-2R-/- mice prevents lethal lymphocyte
accumulation. However, delayed transfer of the
CD25+CD4+ cells was less
effective in protection, stressing the importance of regulatory/naive
cell ratios at the time of the initial peripheral seeding. The presence
of as few as 5% of cells from a normal donor developing in the thymus
of the chimeras suffices to reestablish full control of the number and
state of activation of the peripheral CD4
IL-2R-/- T cells. It has been shown that
CD25+CD4+ T cells are
generated in the thymus (38, 50, 53). Our findings support
this claim. We also found that transferred
CD25+CD4+ T cells can
persist for prolonged periods in absence of the thymus, as observed in
the IL-2R-/- BM chimeras. Thus, these cells
can be either long-lived or capable of self renewal at the periphery.
Interestingly, in the different mixed
IL-2R-/-/normal BM chimeras, the number of
peripheral CD25+CD4+ T
cells was the same independently of the fraction of normal cells
present in the BM inoculum. IL-2-deficient mice lack a significant
number of CD25+CD4+ cells
at the periphery (38) and develop lymphoid hyperplasia and
IBD (20). Recent studies suggested that disturbed
peripheral homeostasis in IL-2-deficient mice resulted from either an
IL-2-dependent AICD defect and/or the lack of
CD25+CD4+ regulatory cells
(14). We found that
IL-2-/--derived cells rescue the
IL-2R-/- BM chimeras from death, and that in
these chimeras, the number and the distribution of
CD25+ and CD25-CD4 T cells
in the peripheral T cell compartments was as in normal mice. Thus, in
the presence of IL-2, the BM precursors from IL-2-deficient donor mice
generated a well-defined population of
CD25+CD4+ regulatory T
cells capable to control the number of CD4 T cells in the peripheral
compartments. These findings suggest that IL-2 is required for the
peripheral survival and maintenance of the subset of the
CD25+ regulatory cells produced by the thymus.
Production of IL-2 by proliferating CD4 T cells may also contribute to
the survival of the regulatory cells. Thus, we may envisage a feedback
loop in which expanding naive CD4 T cells contribute to their own
regulation. The role of IL-2/IL-2R interactions in T cell homeostasis
is further supported by results showing that
IL-2R-/- T cells in mice reconstituted with
a mixture of IL-2R-/- T and
IL-2R+ BM cells did not expand or develop into
an abnormally activated stage (13). However, in this last
study, the cells responsible for the homeostatic control were believed
to be CD8 T cells (13) and not the
CD25+CD4+ T cells that we
now identified as capable of controlling IL-2R-deficient T cells. It
is possible that other cell populations contribute to regulate
peripheral T cell pools.
In summary, we demonstrate the role of T cell interactions in the
control of the size of the peripheral CD4+ T cell
pool. We show that homeostasis of peripheral CD4+
T cells follows subpopulation structure,
CD25+CD4+ T cells limiting
the accumulation of dividing naive CD4+ T cells.
We found that IL-2R-deficient mice lack a subpopulation of
regulatory cells essential for CD4 T cell homeostasis. Adoptive
replacement of the
CD25+CD4+ T cell population
prevents the chaotic accumulation of lymphoid cells in the peripheral
compartments of IL-2R-deficient mice. It also prevents the
subsequent polyclonal T and B cell activation, autoimmune hemolytic
anemia, and IBD observed in IL-2R-/- mice
(10). We also show that IL-2-deficient mice also lack a
sizeable population of
CD25+CD4+ T cells that
expands in presence of IL-2 to control autoimmunity and lymphoid
hyperplasia in IL-2R chimeras. To conclude, we show that the
mechanism by which IL-2R and IL-2 play an essential role on T cell
homeostasis is by shaping a population of
CD25+CD4+ regulatory T
cells that control peripheral CD4 T cell numbers. We demonstrate that
IL-2R is an absolute requirement for the generation of the
regulatory cells. These cells generate in the thymus in the absence of
IL-2, but require IL-2 to establish a stable functional population in
the peripheral compartments.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Antonio A. Freitas, Lymphocyte Population Biology Unit, Unité de Recherche Associée Centre National de la Recherche Scientifique 1961, Institut Pasteur, 25 Rue du Dr. Roux, 75015 Paris, France. E-mail address: afreitas{at}pasteur.fr
3 Abbreviations used in this paper: Tg, transgenic; AICD, activation-induced cell death; BM, bone marrow; Rag, recombination-activating gene; LN, lymph node; Treg, T regulatory; IBD, inflammatory bowel disease.
Received for publication April 29, 2002. Accepted for publication August 19, 2002.
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S. Lepage and R. Lapointe Melanosomal Targeting Sequences from gp100 Are Essential for MHC Class II-Restricted Endogenous Epitope Presentation and Mobilization to Endosomal Compartments Cancer Res., February 15, 2006; 66(4): 2423 - 2432. [Abstract] [Full Text] [PDF]
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 S. Koonpaew, S. Shen, L. Flowers, and W. Zhang LAT-mediated signaling in CD4+CD25+ regulatory T cell development J. Exp. Med., January 23, 2006; 203(1): 119 - 129. [Abstract] [Full Text] [PDF]
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 J. Harnaha, J. Machen, M. Wright, R. Lakomy, A. Styche, M. Trucco, S. Makaroun, and N. Giannoukakis Interleukin-7 Is a Survival Factor for CD4+ CD25+ T-Cells and Is Expressed by Diabetes-Suppressive Dendritic Cells Diabetes, January 1, 2006; 55(1): 158 - 170. [Abstract] [Full Text] [PDF]
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M. Mesel-Lemoine, M. Cherai, S. Le Gouvello, M. Guillot, V. Leclercq, D. Klatzmann, V. Thomas-Vaslin, and F. M. Lemoine Initial depletion of regulatory T cells: the missing solution to preserve the immune functions of T lymphocytes designed for cell therapy Blood, January 1, 2006; 107(1): 381 - 388. [Abstract] [Full Text] [PDF]
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 T. Muthukumar, D. Dadhania, R. Ding, C. Snopkowski, R. Naqvi, J. B. Lee, C. Hartono, B. Li, V. K. Sharma, S. V. Seshan, et al. Messenger RNA for FOXP3 in the urine of renal-allograft recipients. N. Engl. J. Med., December 1, 2005; 353(22): 2342 - 2351. [Abstract] [Full Text] [PDF]
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B. Knoechel, J. Lohr, E. Kahn, J. A. Bluestone, and A. K. Abbas Sequential development of interleukin 2-dependent effector and regulatory T cells in response to endogenous systemic antigen J. Exp. Med., November 21, 2005; 202(10): 1375 - 1386. [Abstract] [Full Text] [PDF]
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L. Li, W. R. Godfrey, S. B. Porter, Y. Ge, C. H. June, B. R. Blazar, and V. A. Boussiotis CD4+CD25+ regulatory T-cell lines from human cord blood have functional and molecular properties of T-cell anergy Blood, November 1, 2005; 106(9): 3068 - 3073. [Abstract] [Full Text] [PDF]
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F. Ghiringhelli, P. E. Puig, S. Roux, A. Parcellier, E. Schmitt, E. Solary, G. Kroemer, F. Martin, B. Chauffert, and L. Zitvogel Tumor cells convert immature myeloid dendritic cells into TGF-{beta}-secreting cells inducing CD4+CD25+ regulatory T cell proliferation J. Exp. Med., October 3, 2005; 202(7): 919 - 929. [Abstract] [Full Text] [PDF]
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 Z. Fehervari and S. Sakaguchi CD4+ regulatory cells as a potential immunotherapy Phil Trans R Soc B, September 29, 2005; 360(1461): 1647 - 1661. [Abstract] [Full Text] [PDF]
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N. Bosco, F. Agenes, and R. Ceredig Effects of Increasing IL-7 Availability on Lymphocytes during and after Lymphopenia-Induced Proliferation J. Immunol., July 1, 2005; 175(1): 162 - 170. [Abstract] [Full Text] [PDF]
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A. Gruber and T. Brocker MHC Class I-Positive Dendritic Cells (DC) Control CD8 T Cell Homeostasis In Vivo: T Cell Lymphopenia as a Prerequisite for DC-Mediated Homeostatic Proliferation of Naive CD8 T Cells J. Immunol., July 1, 2005; 175(1): 201 - 206. [Abstract] [Full Text] [PDF]
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G. Darrasse-Jeze, G. Marodon, B. L. Salomon, M. Catala, and D. Klatzmann Ontogeny of CD4+CD25+ regulatory/suppressor T cells in human fetuses Blood, June 15, 2005; 105(12): 4715 - 4721. [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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A. L. Bayer, A. Yu, D. Adeegbe, and T. R. Malek Essential role for interleukin-2 for CD4+CD25+ T regulatory cell development during the neonatal period J. Exp. Med., March 7, 2005; 201(5): 769 - 777. [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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P. A. Antony, C. A. Piccirillo, A. Akpinarli, S. E. Finkelstein, P. J. Speiss, D. R. Surman, D. C. Palmer, C.-C. Chan, C. A. Klebanoff, W. W. Overwijk, et al. CD8+ T Cell Immunity Against a Tumor/Self-Antigen Is Augmented by CD4+ T Helper Cells and Hindered by Naturally Occurring T Regulatory Cells J. Immunol., March 1, 2005; 174(5): 2591 - 2601. [Abstract] [Full Text] [PDF]
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V. Sanchez-Guajardo, J. A. M. Borghans, M.-E. Marquez, S. Garcia, and A. A. Freitas Different Competitive Capacities of Stat4- and Stat6-Deficient CD4+ T Cells during Lymphophenia-Driven Proliferation J. Immunol., February 1, 2005; 174(3): 1178 - 1187. [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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P. O. Anderson, A. Sundstedt, Z. Yazici, S. Minaee, R. Woolf, K. Nicolson, N. Whitley, L. Li, S. Li, D. C. Wraith, et al. IL-2 Overcomes the Unresponsiveness but Fails to Reverse the Regulatory Function of Antigen-Induced T Regulatory Cells J. Immunol., January 1, 2005; 174(1): 310 - 319. [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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R. M. Teague, R. M. Tempero, S. Thomas, K. Murali-Krishna, and B. H. Nelson Proliferation and Differentiation of CD8+ T Cells in the Absence of IL-2/15 Receptor {beta}-Chain Expression or STAT5 Activation J. Immunol., September 1, 2004; 173(5): 3131 - 3139. [Abstract] [Full Text] [PDF]
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 D. Jones, S. Ibrahim, K. Patel, R. Luthra, M. Duvic, and L. J. Medeiros Degree of CD25 Expression in T-Cell Lymphoma Is Dependent on Tissue Site: Implications for Targeted Therapy Clin. Cancer Res., August 15, 2004; 10(16): 5587 - 5594. [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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S. J. Bensinger, P. T. Walsh, J. Zhang, M. Carroll, R. Parsons, J. C. Rathmell, C. B. Thompson, M. A. Burchill, M. A. Farrar, and L. A. Turka Distinct IL-2 Receptor Signaling Pattern in CD4+CD25+ Regulatory T Cells J. Immunol., May 1, 2004; 172(9): 5287 - 5296. [Abstract] [Full Text] [PDF]
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B. H. Nelson IL-2, Regulatory T Cells, and Tolerance J. Immunol., April 1, 2004; 172(7): 3983 - 3988. [Abstract] [Full Text] [PDF]
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M. Schmidt-Supprian, J. Tian, E. P. Grant, M. Pasparakis, R. Maehr, H. Ovaa, H. L. Ploegh, A. J. Coyle, and K. Rajewsky Differential dependence of CD4+CD25+ regulatory and natural killer-like T cells on signals leading to NF-{kappa}B activation PNAS, March 30, 2004; 101(13): 4566 - 4571. [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. Fujimoto, H. Tsutsui, O. Xinshou, M. Tokumoto, D. Watanabe, Y. Shima, T. Yoshimoto, H. Hirakata, I. Kawase, K. Nakanishi, et al. Inadequate induction of suppressor of cytokine signaling-1 causes systemic autoimmune diseases Int. Immunol., February 1, 2004; 16(2): 303 - 314. [Abstract] [Full Text] [PDF]
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 S. Xiao, S.-s. J. Sung, S. M. Fu, and S.-T. Ju Combining Fas Mutation with Interleukin-2 Deficiency Prevents Colitis and Lupus: IMPLICATING INTERLEUKIN-2 FOR AUTO-REACTIVE T CELL EXPANSION AND Fas LIGAND FOR COLON EPITHELIAL CELL DEATH J. Biol. Chem., December 26, 2003; 278(52): 52730 - 52738. [Abstract] [Full Text] [PDF]
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J. Ermann and C. G. Fathman Costimulatory signals controlling regulatory T cells PNAS, December 23, 2003; 100(26): 15292 - 15293. [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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M. A. Burchill, C. A. Goetz, M. Prlic, J. J. O'Neil, I. R. Harmon, S. J. Bensinger, L. A. Turka, P. Brennan, S. C. Jameson, and M. A. Farrar Distinct Effects of STAT5 Activation on CD4+ and CD8+ T Cell Homeostasis: Development of CD4+CD25+ Regulatory T Cells versus CD8+ Memory T Cells J. Immunol., December 1, 2003; 171(11): 5853 - 5864. [Abstract] [Full Text] [PDF]
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Q. Tang, K. J. Henriksen, E. K. Boden, A. J. Tooley, J. Ye, S. K. Subudhi, X. X. Zheng, T. B. Strom, and J. A. Bluestone Cutting Edge: CD28 Controls Peripheral Homeostasis of CD4+CD25+ Regulatory T Cells J. Immunol., October 1, 2003; 171(7): 3348 - 3352. [Abstract] [Full Text] [PDF]
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A. Antov, L. Yang, M. Vig, D. Baltimore, and L. Van Parijs Essential Role for STAT5 Signaling in CD25+CD4+ Regulatory T Cell Homeostasis and the Maintenance of Self-Tolerance J. Immunol., October 1, 2003; 171(7): 3435 - 3441. [Abstract] [Full Text] [PDF]
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H. von Boehmer Dynamics of Suppressor T Cells: In Vivo Veritas J. Exp. Med., September 15, 2003; 198(6): 845 - 849. [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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C. Asseman, S. Read, and F. Powrie Colitogenic Th1 Cells Are Present in the Antigen-Experienced T Cell Pool in Normal Mice: Control by CD4+ Regulatory T Cells and IL-10 J. Immunol., July 15, 2003; 171(2): 971 - 978. [Abstract] [Full Text] [PDF]
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T. Barthlott, G. Kassiotis, and B. Stockinger T Cell Regulation as a Side Effect of Homeostasis and Competition J. Exp. Med., February 17, 2003; 197(4): 451 - 460. [Abstract] [Full Text] [PDF]
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) or 4 wk (
) later.
B, Phenotypic characterization of the peripheral LN CD4
T cells in a chimera reconstituted with BM cells from
B6.Ly5.2IL-2R
) or the second (
