By
From the Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892
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Abstract |
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Peripheral tolerance may be maintained by a population of regulatory/suppressor T cells that
prevent the activation of autoreactive T cells recognizing tissue-specific antigens. We have previously shown that CD4+CD25+ T cells represent a unique population of suppressor T cells
that can prevent both the initiation of organ-specific autoimmune disease after day 3 thymectomy and the effector function of cloned autoantigen-specific CD4+ T cells. To analyze the
mechanism of action of these cells, we established an in vitro model system that mimics the
function of these cells in vivo. Purified CD4+CD25+ cells failed to proliferate after stimulation
with interleukin (IL)-2 alone or stimulation through the T cell receptor (TCR). When cocultured with CD4+CD25 cells, the CD4+CD25+ cells markedly suppressed proliferation by
specifically inhibiting the production of IL-2. The inhibition was not cytokine mediated, was
dependent on cell contact between the regulatory cells and the responders, and required activation of the suppressors via the TCR. Inhibition could be overcome by the addition to the cultures of IL-2 or anti-CD28, suggesting that the CD4+CD25+ cells may function by blocking
the delivery of a costimulatory signal. Induction of CD25 expression on CD25
T cells in vitro
or in vivo did not result in the generation of suppressor activity. Collectively, these data support the concept that the CD4+CD25+ T cells in normal mice may represent a distinct lineage
of "professional" suppressor cells.
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Introduction |
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It is widely accepted that the development of autoimmune disease involves a breakdown in the mechanisms that control self- versus nonself-discrimination. The primary mechanism that leads to tolerance to self-antigens is thymic deletion of autoreactive T cells. However, some autoreactive T cells may escape thymic deletion or recognize antigens expressed only extrathymically. T cell anergy (1) and T cell ignorance/indifference (2) have been proposed as the primary mechanisms used to control these potentially harmful populations. However, anergic or ignorant T cell populations have the potential to be activated when their target self-antigens are released into the lymphoid system during the course of an infection or when they are activated by cross-reactive epitopes present on infectious agents (3). Thus, these "passive" mechanisms for self-tolerance may not be sufficient to completely control potentially pathogenic T cells. Over the past 10 years, evidence has accumulated for an "active" mechanism of immune suppression in which a distinct subset of cells suppresses the activation of autoreactive T cells that have escaped the passive mechanisms of tolerance (4).
A number of organ-specific autoimmune diseases can be
induced in rodent strains that do not normally develop autoimmunity by procedures that interfere with normal T
cell maturation or by rendering the animals partially T cell
deficient (5). In general, a defined subset of T cells from
syngeneic healthy donors can prevent the development of
autoimmunity on transfer to lymphopenic recipients, indicating that the normal immune system contains immunoregulatory T cells that can prevent the activation of autoreactive T cells (6). For example, Powrie et al. have shown
that colitis can be induced in immunodeficient SCID mice
by transfer of the CD45RBhigh subset of CD4+ T cells from
normal mice, but not by the CD45RBlow population (7).
The CD45RBlow population, when transferred together
with the CD45RBhigh population, completely inhibited development of the disease. Evidence for the existence of
regulatory T cells has also been obtained in both the BioBreeding rat and nonobese diabetic mouse strains that
spontaneously develop autoimmune diabetes (8, 9). Immunoregulatory T cells are also likely to be responsible for the
relative resistance of mice that express a transgenic TCR
specific for a peptide from myelin basic protein to the development of experimental allergic encephalomyelitis,
whereas mice that express the same TCR on a recombination activating gene (RAG)-deficient (/
) background are
highly susceptible (10).
Although many of these studies have demonstrated that
the immunoregulatory T cells are present in the subpopulation of CD4+ cells that express activation/memory markers, a more detailed phenotypic characterization of the suppressor population is lacking. Studies using two different
model systems have suggested that a potent CD4+ immunoregulatory T cell population that can be defined by expression of the IL-2R chain (CD25) is responsible for the
prevention of certain autoimmune diseases. In the first
model system (11, 12), genetically susceptible mice that
were thymectomized on day 3 of life (d3Tx)1 developed
organ-specific autoimmune disease involving one or more
organs. The disease process was mediated by CD4+ T cells;
however, CD4+ T cells from normal adult mice could inhibit the development of disease in the d3Tx animals if
they were transferred by day 14 of life. Furthermore, the
inhibitory activity was completely contained in the minor
(10%) subset of CD4+ T cells that coexpressed CD25 (13,
14). In the second model, when CD4+CD25+ T cells were
depleted from CD4+ T cells isolated from peripheral lymphoid tissues of normal adult mice and the remaining
CD4+CD25
cells injected into nu/nu mice recipients, the
recipients developed a high incidence of organ-specific autoimmune disease (13, 15). Again, cotransfer of populations
enriched in CD4+CD25+ prevented the induction of disease by the CD4+CD25
population. We have also recently demonstrated that CD4+CD25+ T cells can inhibit
the capacity of a cloned line of autoantigen-specific effector
cells to transfer disease to nu/nu recipients (16). Thus, the
CD4+CD25+ population can inhibit both the induction
and effector function of autoreactive T cells.
Although the existence of immunoregulatory T cell
populations has been amply documented, the activity of
these suppressor populations has been measured in vivo in
model systems that require weeks to months of assessment
of disease activity. Therefore, it has proven difficult to determine their mechanism of action, antigen specificity, or
cellular targets. In this report, we demonstrate that the population of CD4+CD25+ T cells present in the peripheral
lymphoid system of normal mice is a potent inhibitor of
polyclonal T cell activation in vitro. Suppression is mediated by a cytokine-independent, cell contact-dependent mechanism that requires activation of the suppressor cell
via the TCR. The CD4+CD25+ cells inhibit the induction
of IL-2 production in the responder CD4+CD25 population, and suppression can be overcome by the addition of exogenous IL-2 or enhancement of endogenous IL-2 production.
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Materials and Methods |
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Mice.
Female BALB/c and C57BL/6 mice were obtained from the National Cancer Institute (Frederick, MD). C57BL/10 mice were obtained from Taconic Farms (Germantown, NY). DO.11.10-TCR transgenic SCID mice were bred in our own facilities (14). IL-4Media, Reagents, and Antibodies.
All cells were grown in RPMI 1640 (Biofluids, Rockville, MD) supplemented with 10% heat-inactivated FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), 2 mM L-glutamine, 10 mM Hepes, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate (all from Biofluids), and 50 µM 2-ME (Sigma Chemical Co., St. Louis, MO). PMA, ionomycin, and Con A were purchased from Sigma Chemical Co. Biotin-conjugated anti-CD25 (7D4), FITC-conjugated streptavidin, PE-conjugated anti-CD45RB (16A), PE-conjugated anti-CD62L (Mel-14), PE-conjugated anti-CD69, PE-conjugated anti-CD5, anti-CD28, anti-CD40 (HM40-3), anti-B7-2 (GL1), anti-CTLA-4, anti-IL-2 (S4B6), anti-IL-4 (11B11), anti-IL-10 (JES5-2A5), or (SXC-1 and SXC-2) and anti-IFN-Cell Purification.
LNs (axillary, inguinal, superficial cervical, mandibular, and mesenteric) were harvested from 8-10-wk-old female mice. They were mashed through a wire mesh into HBSS/5% FCS to prepare single cell suspensions. LN preparations were then enriched for T cells on T cell columns (R&D Systems). To purify CD4+CD25+ cells, the enriched T cells were incubated with biotin-conjugated anti-CD25 (15 µg/108 cells) in PBS/2% FCS for 15 min at 4°C, washed, incubated with FITC-conjugated streptavidin (15 µg/108 cells) in PBS/2% FCS for 15 min at 4°C, and washed. The cells were then incubated with anti- FITC microbeads (Miltenyi Biotec, Inc., Auburn, CA) for 15 min at 4°C and magnetic separation was performed with a VS+ positive selection column according to the suggested protocol (Miltenyi). The retained cells were eluted from the column as purified CD4+CD25+ cells. The flow-through was then passed over CD4+ subset columns (R&D Systems) to obtain CD4+CD25Proliferation Assays.
CD4+CD25Reverse Transcriptase PCR Reactions.
CD4+CD25Cytokine ELISA and Northern Blot Analysis.
Cultures for Northern blot analyses and ELISAs were carried out in 24-well plates (0.8 ml) with 5 × 105 CD4+CD25 ![]() |
Results |
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The CD4+CD25+ population typically represented
5-8% of the total LN population or 10-15% of CD4+ cells
(Fig. 1 A, left). This population could be isolated easily to
levels of 90-95% purity (Fig. 1 A, right) with the magnetic anti-FITC microbead procedure (see Materials and Methods), and the labeled cells could then be analyzed directly
on the FACS® or placed into culture. When compared
with CD4+CD25 T cells, the CD4+CD25+ cells were
similar in their pattern of expression of CD5, had a slightly
higher proportion of CD62Llow cells, and had a higher proportion of CD69+ cells (Fig. 1 B). They had an unusual
pattern of expression of CD45RB, and were composed
primarily of cells that expressed intermediate and low levels. Thus, although modestly enriched in cells that express
activation/memory cell markers, the CD4+CD25+ population contained a significant proportion of cells with a naive/resting phenotype. All of the CD4+CD25+ cells expressed TCR-
/
at a level similar to that of the CD4+
CD25
population; the percentage of cells in both populations that expressed a given V
was also identical (data not
shown).
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Although their constitutive expression of CD25 raised
the possibility that the CD4+CD25+ population might be
hyperresponsive to stimulation with IL-2 and/or via the
TCR, they were completely unresponsive to stimulation with
high concentrations of IL-2, soluble anti-CD3, plate-bound anti-CD3, and Con A in the presence of T cell-depleted
spleen cells as AC (Fig. 1 C). Moreover, they were also unresponsive to stimulation with anti-CD3 and optimal concentrations of anti-CD28. However, the addition of IL-2
did restore responsiveness to soluble anti-CD3 to levels
similar to those observed with the CD4+CD25 population.
In addition, the CD4+CD25+ responded normally when
stimulated with the TCR-independent stimuli, phorbol ester and calcium ionophore or phorbol ester and IL-2.
When the CD4+CD25+ population was
cocultured with CD4+CD25 cells, marked suppression of
the response to stimulation with soluble, but not plate-bound, anti-CD3 was observed (Fig. 2 A). Moreover, responses to Con A in the presence of AC were similarly inhibited and the suppression was not restricted by the MHC
as CD4+CD25+ T cells from BALB/c mice suppressed the
response of CD4+CD25
T cells from C57BL/6 mice (data
not shown). In multiple experiments of this type, significant suppression (>70%) of the response to soluble anti-CD3 was observed at a final ratio of suppressors/responders of 1:4 and complete suppression was seen at ratios of 1:2.
Suppression was not overcome by increasing the concentration of soluble anti-CD3 (data not shown). Most importantly, suppression required activation of the CD4+CD25+
population, as the CD25+ cells were unable to suppress the
antigen-specific responses of CD4+CD25
T cells from
mice that expressed an antiovalbumin transgenic TCR, but
could readily suppress the response of the same cells to anti-CD3 (Fig. 2 B).
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It seemed likely that
one mechanism by which the CD4+CD25+ T cells could
mediate suppression was by the secretion of suppressor cytokines. Supernatants derived from anti-CD3-stimulated
CD4+CD25+ contained low levels of IL-10, but contained
no detectable levels of IL-2, IL-4, or IFN- (data not shown).
When the CD4+CD25+ cells were examined for cytokine
gene expression by reverse transcriptase (RT)-PCR, IL-2
mRNA was not detected, but it was readily detectable when CD4+CD25
cells were stimulated with anti-CD3
(Fig. 3). This result is consistent with the failure of the
CD4+CD25+ T cells to proliferate when stimulated with
anti-CD3. Activated CD4+CD25+ cells did not express
significant amounts of IL-4 mRNA, but the levels of IL-10
mRNA were increased in both unstimulated and anti-CD3-stimulated CD4+CD25+ populations when compared with CD4+CD25
cells. FasL message was lower in
resting CD4+CD25+ cells and was not induced by stimulation, whereas TNF-
was upregulated comparably in both
CD25+ and CD25
populations. These results are in contrast to the results of Asano et al. (13), in which unstimulated CD4+CD25+ cells expressed IL-2, IL-4, and TGF-
,
as well as IL-10.
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The IL-10 ELISA and RT-PCR data raised the possibility that IL-10 might mediate the suppressive activity of the
CD4+CD25+ cells. However, when CD4+CD25+ were
separated from CD4+CD25 cells by culture in a TranswellTM, suppression was not observed (Fig. 4 A). Furthermore, the addition of neutralizing antibodies to IL-10 as
well as other known suppressive cytokines alone or in combination also failed to abrogate suppression (Fig. 4 B). As it
remained possible that an unknown suppressor cytokine was operative in this model, supernatants were collected from
stimulated CD4+CD25+ cells or stimulated CD4+CD25
cells coincubated with CD4+CD25+ cells and tested for
their capacity to suppress the responses of freshly explanted
CD4+CD25
cells (Fig. 4 C); however, no suppression
was seen. Lastly, to definitively rule out the involvement of
IL-4 or IL-10 in this system, CD4+CD25+ T cells were
purified from mice genetically deficient in these cytokines.
CD4+CD25+ cells from both of the knockout mice were
comparable to controls in their ability to suppress (Fig. 5).
Collectively, these studies demonstrate that CD4+CD25+
cells do not mediate their suppressive effects by secretion of a soluble suppressor factor.
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As an initial approach to an analysis of the
cell surface molecules that might be involved in mediating
cell contact-dependent suppression in this in vitro model,
we added various antibodies to cell surface antigens that
could be targets of the CD4+CD25+ cells. We focused our
efforts on the two major pathways involved in the interaction between CD4+ cells and AC, namely CD28-CTLA-4/B71/2 and the CD40/CD40L pathways. As previously
reported (20), addition of either anti-CTLA-4 or anti-CD40 had modest enhancing effects on the responses of the CD4+CD25 cells, but had no effects on the suppression of the responses by the CD4+CD25+ cells (Fig. 6).
Anti-B7-2 completely inhibited stimulation of the CD4+
CD25
population, as B7-2 is the major costimulatory molecule expressed by resting T cell-depleted spleen cells that
were used as AC. Most importantly, the addition of anti-CD28 or exogenous IL-2 not only markedly enhanced the
responses of the CD4+CD25
cells, but almost completely
abrogated the suppressive effects due to CD4+CD25+ cells.
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The neutralization of suppression by the addition of IL-2
or anti-CD28 to the cocultures of CD4+CD25+ and
CD4+CD25 populations, as well as the ability of exogenous IL-2 to overcome the nonresponsiveness of the
CD4+CD25+ cells when cultured alone (Fig. 1), raised the
possibility that the CD25+ population was exerting its inhibitory effects by binding IL-2 and functioning as an "IL-2
sink". We initially examined IL-2 production in anti-CD3-stimulated cultures of CD4+CD25
, CD4+CD25+,
and the mixture of the two populations by ELISA. IL-2
was readily detectable in the supernatants of CD4+CD25
cells cultured alone, undetectable in the supernatants of CD4+
CD25
cells, and markedly reduced in the supernatants of
the coculture (Fig. 7 A). This result is consistent with the
possibility that the CD4+CD25+ population was blocking
the production of IL-2 by the CD4+CD25+ cells, but it
did not rule out the possibility that the CD4+CD25+ cells
inhibited proliferation by sequestering IL-2. Therefore, we
directly examined the induction of IL-2 mRNA by Northern blot analysis. IL-2 mRNA was readily detected when
the CD4+CD25
cells were cultured alone, but was undetectable when the CD4+CD25
and CD4+CD25+ were
cocultured (Fig. 7 B). Although the level of
-actin mRNA was slightly lower in the cocultures, longer exposure of this blot did not reveal IL-2 mRNA. In other experiments, a
faint band for IL-2 mRNA could sometimes be seen. In
any case, these results strongly support the view that the
CD4+CD25+ population exerts its inhibitory effects by blocking the induction of IL-2 production by the CD4+CD25
cells at the level of RNA transcription.
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We and others have shown
that the CD4+CD25+ population is solely responsible for
inhibition of the induction of autoimmunity after 3dTx
(13, 14). In addition, we have also demonstrated that the
induction of CD25 expression on CD25 T cells derived
from mice that express a transgenic TCR on a SCID background was not sufficient to suppress the induction of autoimmune disease after 3dTx in vivo. This result raised the possibility that the CD4+CD25+ cells present in normal
mice represent a unique cell population. To extend these
studies to our in vitro model, we purified CD4+CD25
from normal mice, cultured them for 24 h in the presence
of Con A to induce CD25 expression, and then mixed them
with freshly isolated CD4+CD25
cells. Although >80%
of the Con A-stimulated CD25
cells expressed CD25
(data not shown), no suppression of the proliferative responses of the CD4+CD25
cells was observed (Fig. 8 A).
This result demonstrates that the mere expression of CD25
is insufficient to mediate suppression.
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It has been postulated that the induction of autoimmunity after 3dTx is secondary to a deficiency of the CD4+
CD25+ population that develops later during ontogeny (13).
Paradoxically, a greater percentage (25-30 versus 10%) of
CD4+ T cells in 3dTx mice express CD25 compared with
normal mice (18). To rule out the possibility that the in
vivo induction of CD25 would render cells suppressive,
CD4+CD25+ cells were purified from 3dTx mice between
4 and 6 mo of age and added to cultures of CD4+CD25
cells from normal BALB/c mice. No suppression was observed in these cocultures (Fig. 8 B). These results again
support the view that the CD4+CD25+ cells present in
normal mice are a unique suppressor population and that
the CD4+CD25+ in the 3dTx mice are autoimmune effector cells.
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Discussion |
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The role of immunoregulatory or suppressor T cells has
been well documented in the immunologic literature over
the past 25 years. However, a great deal of controversy remains as to their lineage, antigen specificity, and mechanism of action. The concept of antigen-specific suppressor
factors that acted in a complex cascade has not been validated by biochemical and molecular studies (21). Many of
the older in vivo studies on the activity of suppressor cells
have been reinterpreted as being secondary to alterations in
the Th1-Th2 balance that was induced by different modes
of antigen delivery. Most recent data on the importance of
suppressor cells has been derived from their involvement in
mediating transplantation tolerance and in preventing the
induction of autoimmune disease. The induction of autoimmune disease after 3dTx has been considered for many
years to be secondary to a deficiency of T suppressor cells
that normally develop after day 3 of life. The suppressor cells in this model belong to the minor subpopulation of
CD4+ T cells that coexpress CD25 and are capable of suppressing the induction of autoimmunity after 3dTX, the
induction of autoimmunity induced by transfer of CD4+
CD25 T cells to nu/nu recipients, and the capacity of activated autoantigen-specific T cell clones to induce disease in
nu/nu recipients.
The goal of our study was to develop an in vitro model
system in which the function of these potent CD4+CD25+
T cells could be analyzed. We found that the CD4+CD25+
cells were themselves completely nonresponsive to stimulation by TCR-derived signals in the presence or absence of
costimulation. More importantly, they could adoptively
suppress the responses of CD4+CD25 cells in coculture
studies. Although suppression in many model systems in
vivo and in vitro is mediated by the secretion of one of the
suppressor cytokines (IL-4, IL-10, or TGF-
), the mechanism of suppression by CD4+CD25+ cells appeared to be
mediated by a contact-dependent mechanism. No suppression was seen when the suppressors were separated from the responders by a semipermeable membrane, and supernatants from stimulated CD4+CD25+ cells could not transfer suppression. Neutralizing antisuppressor cytokine mAbs
alone or in combination also failed to abrogate suppression.
Finally, to rule out the involvement of the newly described
(22) T regulatory 1 population that exerts bystander suppression by the secretion of IL-10, we demonstrated that
CD4+CD25+ cells from both IL-4
/
and IL-10
/
donors
were as effective as T cells from wild-type mice in mediating suppression in vitro. We have also shown that these
same CD4+CD25+ cells from cytokine-deficient mice are
capable of mediating suppression of autoimmune disease in
vivo when they are transferred to d3Tx mice (McHugh,
R., A.M. Thornton, and E.M. Shevach, unpublished observations). Although we were unable to neutralize suppression in vitro with anti-TGF-
, it still remains possible
that TGF-
may play some role in the action of these cells
in vivo, as TGF-
/
mice develop spontaneous inflammatory disease of a variety of organs and have an increase in
Th1 inflammatory cytokines (23). The potential relationship of the CD4+CD25+ population to the CD45RBlow
cells whose ability to protect animals from inflammatory
bowel disease could be reversed by anti-TGF-
(24) and
with TGF-
-producing suppressor populations (the so-called Th3 cells) induced by oral delivery of antigen (25,
26) remains to be determined.
The requirement for cell contact to observe suppression
raised the possibility that the CD4+CD25+ population
might be mediating suppression by actually killing the responder T cell population by the Fas/FasL pathway. However, no evidence for cell death was observed in the cocultures and identical numbers of viable cells were recovered
in the presence or absence of the CD4+CD25+ cells after
24 h of culture (data not shown). Furthermore, we could
not detect mRNA by RT-PCR for the FasL after stimulation of the CD4+CD25+ cells with anti-CD3, whereas it
was readily induced in the CD4+CD25 population. Another potentially trivial mechanism by which the CD25+
population might be mediating inhibition was by passively
absorbing IL-2 produced by the responder cells. We definitively ruled out this possibility by demonstrating that the
suppressor cells almost completely inhibited IL-2 gene
transcription and hence IL-2 production in the responder
T cell population. Inhibition of IL-2 gene transcription has
also been observed with suppressor T cell populations isolated from animals that have been recently subjected to total lymphoid irradiation (TLI; reference 27). No information is available on the phenotype of the suppressor cells in
that model, but spleen cells from TLI-treated animals contain increased numbers of CD4
CD8
CD3low cells. Although the binding of IL-2 by the CD4+CD25+ cells appeared to play no role in suppression, it is still possible that
the expression of CD25 may be related to the functional
capacity of these cells to inhibit the development of autoimmune effector cells as IL-2
/
, IL-2R
-chain (CD122)
/
,
and CD25
/
mice develop multiple manifestations of inflammatory autoimmune diseases (28). These studies
suggest that IL-2 itself may either directly or indirectly play
an important role in the development and/or function of
this unique population of CD25+ suppressor cells.
There are a number of unique aspects of suppression in
this in vitro model which may offer some insight into the
physiological function of the CD4+CD25+ cells in vivo.
First, suppression in vitro required that the suppressor population be exposed and presumably activated via the TCR
since the antigen-specific response of naive T cells from TCR transgenic mice was not suppressed by the CD4+
CD25+ cells, whereas the responses of the same cell mixture to anti-CD3 stimulation were completely suppressed.
Second, the responses to soluble anti-CD3 in the presence
of normal T cell-depleted spleen cells were easily suppressed, whereas the responses to plate-bound anti-CD3
were unaffected. This result may be secondary to a qualitatively distinct activating signal by plate-bound mAb, but is
also consistent with the possibility that the target of the
suppressor population is actually the AC rather than the responding T cell. Lastly, suppression could be overcome in
the coculture studies by the addition of IL-2 or by enhancing endogenous IL-2 production by the addition of anti-CD28 to the cultures. It thus appears that the induction of
the IL-2R on the responder population is not blocked by
the addition of the suppressor population, and we have
confirmed this by FACS® analysis (data not shown). Again,
this observation is consistent with the possibility that the
AC is the target of the suppressor cell; however, as of yet
we have not been able to demonstrate that the suppressor
cells interfered with the delivery of either positive or negative costimulatory signals. The addition of anti-CTLA-4 did not reverse suppression (Fig. 6), and CD4+CD25+ cells
from either CD28/
or CD40L
/
mice were fully capable of inhibiting anti-CD3 stimulation in vitro (data not
shown).
It should also be noted that the nonresponsiveness of the
purified CD4+CD25+ to stimulation with anti-CD3 could
be overcome by the addition of exogenous IL-2. It is possible that the anergic state of the CD4+CD25+ population
was broken by the addition of IL-2. Alternatively, as the
CD4+CD25+ population is heterogeneous in expression of
the membrane markers we have studied (Fig. 1 B), the response seen in the presence of IL-2 may be secondary to
CD25+ conventional memory T cells that "contaminate"
the suppressor cell population. Surprisingly, the addition of
anti-CD28 had no effect on the proliferative responses of
the CD4+CD25+ population, while abrogating the suppression in the cocultures of the CD25+ and CD25 cells.
In the latter case, the anti-CD28 may directly stimulate the
CD25
cells to produce IL-2, whereas in the former the
vast excess of suppressors may still be capable of inhibiting
anti-CD28-induced IL-2 production by the few conventional memory cells present in the CD25+ pool.
Sakaguchi et al. (32) have proposed that any CD25+ cell,
rather than a distinct, functional CD4+CD25+ subset, may
mediate suppression. We have previously shown that the
induction of CD25 on a homogeneous population of
CD25 T cells derived from the TCR-transgenic SCID
mouse was insufficient to prevent the induction of post-3dTX autoimmunity in vivo. Similarly, suppression of proliferation was not seen when CD25+ cells, generated by in
vitro mitogen stimulation of CD25
cells, were added to
fresh CD25
populations. Not surprisingly, CD25+ T cells
derived from 3dTx animals were also unable to mediate suppression. In both of these latter situations, enhancement
of proliferation was usually observed. We have been unable
to detect any differences in the level of expression of the
/
-TCR between CD25+ and CD25
cells; the pattern
of V
usage was also identical in both populations. One caveat in the interpretation of these results is the potential heterogeneity of the CD25+ population. Studies are now in
progress to determine whether the suppressive activity of the
CD25+ cells can be localized to a smaller subpopulation
(e.g., CD69+, CD62Llow, CD45RBlow or int). It is possible
that restrictive TCR usage might be observed if suppressor
activity can be localized to a more defined subpopulation of
CD25+ cells. Our data support the concept that the
CD4+CD25+ may represent a distinct lineage of professional suppressor cells.
One of the major drawbacks of this in vitro model is that as yet we have been unable to separate the requirements for activation of the suppressor populations from those of the responder populations and this has hindered our further analysis of the antigen specificity (if any) of the suppressor cells and their cellular targets. Several studies (33, 34) have presented evidence that the CD4+CD25+ population in the 3dTx model recognizes the same set of autoantigens as the autoantigen-specific effector pool, but these findings have not been confirmed (35). No data are available as to the antigen-specificity of the regulatory cells in the models where lymphopenic animals are reconstituted with CD45RBlow cells although it has been proposed that the T cells that mediate dominant tolerance recognize the autoantigen or peptides derived from it (36) and that both the effector and suppressor repertoires are generated in the thymus. No studies have suggested that receptor-based immunoregulation (37, 38) is operative in these model systems. Although our in vitro studies on polyclonal activation do not address the issue of the antigen-specificity of suppressor cells, they are most consistent with a model in which the suppressor and effector populations compete at the AC surface for antigen and/or costimulatory signals. To a certain extent, our results resemble the observations of Lombardi et al. (39), who have shown that one mechanism by which anergic antigen-specific T cell clones inhibit proliferative responses of normal T cell clones in cocultures is by competition for antigen or costimulatory signals generated at the surface of the AC. Cobbold et al. (40) have also proposed that the suppressor populations induced by nondepleting anti-CD4 mAbs in a model of infectious transplantation tolerance are alloantigen-specific cells that are incapable of differentiating into effectors, but function by inhibiting the delivery of antigen/costimulatory signals to the effector population. One possibility is that the CD4+CD25+ cells are themselves specific for ubiquitously expressed autoantigens. Such cells would have escaped negative selection in the thymus because they would have downregulated their TCR signaling properties and their capacity to differentiate into effector cells. However, they could compete for costimulatory signals with low affinity autoreactive precursor cells on the surface of the same AC. The in vitro model system described in this report should facilitate identification of the antigen specificity and the mechanism of suppression of these potent regulatory cells.
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Footnotes |
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Address correspondence to Ethan M. Shevach, LI, NIAID, NIH, Bldg. 10, Rm. 11N311, 10 Center Dr., MSC 1892, Bethesda, MD 20892-1892. Phone: 301-496-6449; Fax: 301-496-0222; E-mail: ems1{at}box-e.nih.gov
Received for publication 19 March 1998 and in revised form 29 April 1998.
Abbreviations used in this paper AC, accessory cell; d3Tx, thymectomized on day 3 of life; FasL, Fas ligand; RT, reverse transcriptase.
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References |
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