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Laboratory of Molecular Immunology, Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115
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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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(8, 9, 10, 11) has been shown to alter the effects of
CD4+CD25+ regulatory T
cells, thus suggesting that different mechanisms may be used by these
cells to induce suppression.
It was recognized early on that the nature of stimulation provided in
cocultures of CD4+ and
CD4+CD25+ cells determined
whether proliferative and cytokine responses would be suppressed. In
the mouse system, Thornton and Shevach (3) demonstrated
strong suppression in cocultures that were stimulated with soluble
CD3 while plate-bound (pb)CD3-activated cocultures did not induce
inhibition. Similarly, cocultures of human
CD4+CD25-
(CD4+) and
CD4+CD25+high cells
stimulated with a high concentration of pbCD3 did not inhibit
proliferation, while identical cultures stimulated with a lower,
suboptimal concentration of pbCD3 completely suppressed
proliferation and cytokine secretion (8). Similarly,
increasing the strength of signal with the addition of soluble CD28
to pbCD3 stimulation also abrogated suppression (3, 8).
Thus, stimuli that do not result in suppression can be thought of as
nonpermissive for suppression, and generally are signals that provide a
stronger signal through the TCR. Interestingly, these nonpermissive
stimuli, which result in strong T cell proliferation and cytokine
production, have traditionally been used to induce T cell clonal
expansion.
During ontogeny of the immune system, T cells expressing high-affinity TCRs for self Ags undergo clonal deletion in the thymus against a background of positive selection to MHC and self Ag (12, 13). Thus, the resultant T cell repertoire can be viewed as, in essence, an autoreactive population with low-affinity receptors for self Ags. In contrast, with inflammatory responses, the addition of costimulatory signals, including ligation of the CD28 receptor by B7.1 and B7.2, provides for strong clonal expansion of T cells in response to potentially lethal infections. This finding led us to postulate that CD4+CD25+high regulatory T cells selectively suppress target cells that received weak signals lacking costimulation while being unable to suppress responder T cells that had been induced by foreign microbial Ags to undergo clonal expansion via strong signals.
For CD4+CD25+high cells to
regulate immune responses in vivo, they must also overcome temporal and
spatial limitations of activation and target cell interaction. It has
been shown in both murine and human in vitro systems that contact is
required for suppression by
CD4+CD25+high regulatory
cells of the target responder CD4+ T cell
(3, 4, 8). Regulating responses in vivo is further
complicated by the fact that
CD4+CD25+high regulatory T
cells are found at a much lower frequency than responder
CD4+ T cells, representing 
1.5–3% of total
CD4+ T cells in peripheral blood
(8). CD4+CD25+
cells express CCR4 and CCR8 chemokine receptors that lead to selective
migration to dendritic cells at sites of inflammation and immune
activation in secondary lymphoid tissues (14). Presumably,
it is at these sites that the timing of activation and contact between
the responder and regulatory T cells, in addition to TCR signal
strength, determines whether the outcome will be suppression or
activation. Thus, it is important to determine whether
CD4+ responding T cells remain sensitive to
suppression for a certain period of time after TCR activation. If so,
the regulatory T cell would have a "window of opportunity" during
which it may suppress unwanted responses, such as those against
self Ags.
We have determined whether varying the strength of signal to either the
regulatory or responder T cell influences the kinetics and degree of in
vitro suppression. Varied strengths of signals that induced different
degrees of T cell activation were simulated by cross-linking the TCR by
the differential physical presentation of CD3 Abs in the presence or
absence of CD28 coligation. We also analyzed the suppression induced
under different signal strengths for sensitivity to blockade of GITR
and TGF-. The capacity to inhibit TCR signals generated via
engagement by MHC presentation of specific Ag was addressed by use of a
random copolymer in cocultures of CD4+ and
CD4+CD25+high cells. The
results demonstrate that highly activated CD4+ T
cells, as would occur during inflammatory responses, were absolutely
resistant to suppression by regulatory
CD4+CD25+high T cells,
while weakly activated CD4+ T cells, as would
occur during physiologic engagement of TCRs to self Ags in the absence
of costimulation, were exquisitely sensitive to suppression. Thus, this
in vitro model, depicting the importance of TCR signal strength on both
responder and regulatory populations, may reflect the in vivo
boundaries of suppression by
CD4+CD25+high regulatory T
cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells were cultured in RPMI 1640 media supplemented with 2 nM
L-glutamine, 5 mM HEPES, and 100 U/µg/ml
penicillin/streptomycin (all from BioWhittaker, Walkersville, MD), 0.5
mM sodium pyruvate, 0.05 mM nonessential amino acids (both from Life
Technologies, Rockville, MD), and 5% human AB serum (Gemini
Bio-Products, Woodland, CA) in 96-well U-bottom plates (CoStar,
Corning, NY). The CD3 (clone UCHT1 for pb assays and clone Hit3a for
soluble conditions) and CD28 (clone 28.2, at 5 µg/ml) Abs were
purchased from BD PharMingen (San Diego, CA). For soluble stimulation,
the CD3 (Hit3a) and CD28 (28.3) were used at a final
concentration of 2.5 µg/ml. The CD3 beads (catalog no. 111.14,
with SPV-T3b CD3 monoclonal) were purchased from Dynal Biotech (Lake
Success, NY) and used at a ratio of six beads to one responder T cell
per well. For pbCD3 stimulation, 50 µl of the CD3 Ab diluted in
PBS (Life Technologies) at a concentration of 2.5 µg/ml was added to
each culture well, incubated at 37°C for 4 h, and then washed
twice with PBS. For the Ab blocking studies, the reagents were
purchased from R&D Systems (Minneapolis, MN) and used at the final
concentration of 10 µg/ml: mouse IgG1 (MAB002), chicken IgY
(AB-101-C), hTGF- (IgG1 mAb, MAB240), hTGF- (polyclonal
chicken Ig,AF-101-NA), and hGITR (IgG1 mAb, MAB689).
Cell isolation
Human blood mononuclear cells were isolated from freshly drawn
human blood by Ficoll-Hypaque (Amersham Pharmacia Biotech, Piscataway,
NJ) gradient centrifugation. The
CD4+CD25-
(CD4+, or responder T cells) and the
CD4+CD25+high (regulatory T
cells) were isolated from 1–2 x 108 PBMCs
by sorting with a FACSVantage SE (BD Biosciences, Franklin Lakes, NJ).
These cells were incubated with 250 µl each CD4-CyChrome (no.
555348, IgG1; BD PharMingen) and CD25-PE (no. IM0479, IgG2a;
Immunotech, Brea, CA). Monocytic cells were selected against by
removing any cells that stained with the CD14-FITC (LPS receptor,
no. 30544X, IgG2a), CD32-FITC (Fc
RII, no. 30934X, IgG2b), and
CD116-FITC (GM-CSFR, no. 18774B, IgM), all purchased from BD
PharMingen. The sort gates were restricted to the population of
lymphocytes by means of their forward and side scatter properties,
excluding large, activated T cells. T cell-depleted accessory cells
(TdACs) were isolated by negative selection of PBMCs by incubation with
CD2-coated beads purchased from Dynal Biotech (catalog no. 111.01)
followed by irradiation at 3300 rad.
Cell stimulation assays
In all assays using CD3 stimulation, the
CD4+CD25- cells were
plated at 2.5 x 103/well, while the
CD4+CD25+high cells were
plated at 1.25 x 103/well. Thus upon
coculture, the cells were combined at a 2:1 ratio. All wells received
1 x 104 TdACs. To determine proliferation,
half of the culture supernatant (100 µl) was removed from each well
before 1 µCi of [3H]thymidine (NEN, Boston,
MA) was added on day 5 for the final 16 h of culture before
harvesting. For the experiments addressing the time course of coculture
initiation, the separately stimulated cultures of
CD4+CD25-/TdAC,
CD4+CD25+high/TdAC, and
"TdAC only" were set up in 100 µl total volume, spun gently to
initiate the stimulation, and then were resuspended and combined at 15,
38, or 60 h after initiation of stimulation. The time was counted
from the initial activation of the cultures, and the assays were all
similarly pulsed with [3H]thymidine on day 5,
as described above. In the separate stimulation and time course
coculture experiments shown in Figs. 2
and 3, the positive control
cocultures consisted of responder cell cultures that had been combined
with TdAC cultures that had received the identical stimulation as its
corresponding regulatory cell cultures. To interpret the data, the
results of the positive control cocultures (addition of TdAC-only
cells) are compared with the results of the cocultures that had
received the identically activated regulatory cells. For the
experiments using glatiramer acetate (GA; Copaxone; kindly supplied by
Teva Marion Partners, Kansas City, MO), the cultures were
activated at the designated concentration and the number of cells was
increased 4-fold, although the 2:1 ratio was maintained. These
Ag-specific assays were pulsed with
[3H]thymidine on day 7 after stimulation. All
assays exhibited <10% SEM and were repeated in a minimum of three
independent experiments using blood from different donors.
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The supernatants that were removed before addition of
[3H]thymidine and were diluted and analyzed on
Immulon 4 ELISA plates (Dynex Technologies, Chantilly, VA) using the Ab
pairs: IFN- (M-700A and M-701-B Biotin; Endogen, Woburn, MA), IL-10
(nos. 18551D and 18562D-Biotinylated; BD PharMingen), and IL-13 (554570
and Biotinylated no. 555054; BD PharMingen) developed with an
avidin-peroxidase conjugate (1/10,000 dilution) (A-7419; Sigma-Aldrich,
St. Louis, MO) and tetramethylbenzidine peroxide substrate (no.
50-76-06; Kirkegaard & Perry Laboratories, Gaithersburg, MD). Instead
of IL-4, IL-13 was assayed as a prototypical Th2 cytokine due to
limitations in the detection of IL-4 in culture supernatants of human T
cells likely due to its consumption.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As a result of the strength of the generated TCR signals,
different methods of
CD4+CD25- T cell
stimulation cause markedly different levels of proliferation. As shown
in Fig. 1, a high concentration of
pbCD3 is a very strong stimulus that when used in the coculture of
CD4+CD25- responder cells
and CD4+CD25+high cells did
not result in inhibition of proliferation. With pbCD3 stimulation,
the coculture exhibited 109% of the proliferation induced by the
culture of responder cells alone. The addition of soluble CD28
costimulation to the pbCD3 further enhanced the proliferative
response and again did not result in suppression of the coculture. In
contrast, stimulation with either of the weaker stimuli, bead-bound
CD3 or soluble CD3 and CD28, gave lower levels of responder
cell proliferation that were significantly reduced by 80 and 73%,
respectively, upon coculture with
CD4+CD25+high cells.
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The previous results suggest that strong stimulation either inactivates the suppressive function of the CD4+CD25+high regulatory cells or induces the CD4+CD25- responder T cells to become resistant to suppression. To address this question, responder and regulatory T cells were separately stimulated with the different stimuli and combined at the indicated times after intiation of activation. As all T cell cultures were stimulated in the presence of irradiated TdACs, cultures of "TdACs only" were also stimulated under the same conditions and similarly combined with the various activated cultures of responding T cells to serve as controls. Thus, the proliferation that resulted from the addition of the activated TdAC-only cultures provided the baseline for comparison of regulatory cell function apparent in the cocultures receiving the identically activated regulatory TdAC cultures. The kinetic experiments were performed three times with cells isolated from different donors, and produced virtually identical results.
CD4+CD25+high regulatory
cells or TdACs that had been given no stimulation or were preactivated
with CD3 beads (weakest signal), soluble CD3/CD28 (weak
signal), or pbCD3 (strong signal) were added to cultures of CD3
bead-stimulated (weakest stimulus) responder CD4+
T cells at 15, 38, or 60 h after activation (Fig. 2
A). Irrespective of the
activation signal provided to regulatory
CD4+CD25+high T cells,
there was strong inhibition of proliferation of the CD3
bead-activated responder CD4+ T cells when
cultures were initiated within 15 or 38 h of activation. As the
coculture of CD4+CD25+high
regulatory cells that were not preactivated also resulted in strong
inhibition of responder cell proliferation at 15 and 38 h, it is
possible that the regulatory cells were activated as a consequence of
residual CD3 stimuli. Similarly, the addition of regulatory cells
stimulated with either of the two weaker stimuli of CD3 beads or
soluble CD3/CD28 resulted in significant inhibition of responder
CD4+ T cells in cocultures initiated up to
38 h after activation. Interestingly, the addition of regulatory
cells that had been preactivated with the strong nonpermissive stimulus
(pbCD3) was able to suppress proliferation by 90%, but only in
cocultures established by 15 but not 38 h after activation.
Because the same CD4+ T cells were suppressed at
38 h when combined with regulatory cells preactivated with weaker
stimuli, the CD4+CD25+high
regulatory cells rapidly lost their ability to inhibit proliferation
after receiving strong activation signals.
In the next series of experiments, responder T cells were activated
with soluble CD3/CD28, a slightly stronger stimulus than CD3
beads (Fig. 2B). The addition of
CD4+CD25+high regulatory T
cells activated by the different stimuli resulted in suppression of
responder CD4+ T cell proliferation in all cases,
but only if the cocultures were initiated within 15 h after
activation. The sensitivity of these CD4+
responder cells preactivated with soluble CD3/CD28 to inhibition
is much different from that exhibited by the CD4+
T cells stimulated with CD3 beads, indicating different kinetics of
suppression. In this study, suppression occurred when coculture was
initiated within 15 h, indicating that responder
CD4+ T cells became resistant to regulation more
quickly when they were stimulated with a stronger TCR stimulus.
In the next series of experiments, the responder T cells were activated
with pbCD3, a strong nonpermissive stimulus (Fig. 2C). In
general, the regulatory
CD4+CD25+high T cells did
not suppress the highly activated CD4+ responder
cells, although these regulatory cells were capable of suppressing
CD4+ cells stimulated with weaker stimuli. As an
exception, the addition of
CD4+CD25+high cells that
had been preactivated with soluble CD3/CD28 did suppress the
pbCD3-stimulated responder T cells by 50% if coculture was
initiated 15 h after activation. Thus, stimulation with soluble
CD3/CD28 produces regulatory cells with the highest levels of
suppressive activity.
The stronger the stimulation signal given to the responder cells, the less sensitive the cells are to inhibition of cytokine secretion
The supernatants isolated from these timed cocultures were tested
for the presence of IFN- (Th1), IL-13 (Th2), and IL-10 as shown in
Fig. 3. IL-10 was only reproducibly
detected in supernatants from cocultures established with responder
cells stimulated with pbCD3. Furthermore, we could not detect IL-10
from cultures of regulatory cells alone unless IL-2 was provided in
addition to strong TCR stimulation (data not shown). In general, the
secretion of IL-13 and IFN- in these cocultures was similar to their
corresponding proliferation profiles. The strongly stimulated responder
cells exhibited the least inhibition of cytokine secretion, while the
more weakly activated responder cells exhibited greater inhibition upon
coculture with the differentially preactivated regulatory cells.
Cocultures established with pbCD3-stimulated responder T cells
demonstrated inhibition of cytokine production in the absence of
coincident inhibition of proliferation. The cocultures established with
strongly preactivated responder cells (pbCD3, Fig. 3C)
and regulatory cells (pbCD3) exhibited no reduction in
proliferation, yet showed modest inhibition of IL-13 and IFN-
production. In contrast, cocultures of the same strongly activated
responder cells with the soluble CD3/CD28 activated
CD4+CD25+high cells
exhibited marked inhibition of cytokine production if they were
established by 15 h, similar to their proliferation profile.
Cocultures established with the weakest preactivated regulatory cells
(CD3 beads) exhibited a small but consistent reduction in IL-13,
while there was no suppression of IFN- secretion. Lastly, there was
no inhibition of either cytokine by cocultures with
CD4+CD25+high cells that
had not been preactivated. Thus, in cocultures with strongly activated
responder T cells, the stronger the preactivation stimulus provided to
the CD4+CD25+high cells,
the more capable they were of inhibiting the secretion of cytokines for
longer periods of time. In contrast, all the cocultures that
wereestablished with the more weakly activated responder T
cells (CD3 beads in Fig. 3A, soluble CD3/CD28 in
Fig. 3B) exhibited significant reduction in the secretion of
both IL-13 and IFN- regardless of the preactivation status of the
cocultured regulatory cells. These data suggest that there is an
interval of time after activation during which
CD4+CD25- T cells are
sensitive to regulation of their cytokine production. Furthermore, the
length of this interval directly depends upon the strength of the
signals that the different T cells encountered.
There is no association between IL-10 secretion and inhibition of responder CD4+ T cell responses
As IL-10 was only detected in the cocultures established with
pbCD3-stimulated responder cells, Fig. 3D depicts only
the IL-10 produced by this stimulation condition. The addition of
regulatory cells was not required for production of IL-10, as it was
found in cocultures receiving the TdAC-only cultures, indicating that
the responder CD4+CD25-
cells are secreting the IL-10. Cocultures of CD4+
T cells receiving
CD4+CD25+high and TdAcs
alone that had received either no preactivation or CD3 bead
stimulation produced the most IL-10. The addition of all
CD4+CD25+ regulatory cells,
except those stimulated with CD3 beads, to pbCD3-stimulated
CD4+ responder cells resulted in either no change
or increases in IL-10 production. In contrast, CD3 bead-stimulated
CD4+CD25+high cells
consistently suppressed IL-10 secretion. Thus, it would appear that
although both CD3 bead and soluble CD3/CD28 preactivated
regulatory cells can suppress the production of IL-13 and IFN-, only
the CD3 bead preactivated regulatory cells inhibit the production of
IL-10. Thus, subtle differences exist between regulatory cells
depending upon the nature of the activation stimuli.
Depending upon the stimulation conditions, the regulation of
CD4+ cell proliferation by
CD4+CD25+high cells exhibited modest
sensitivity to the neutralization of GITR, while blocking TGF- had
no effect
Cocultures of responder and regulatory cells stimulated with
soluble CD3/CD28 demonstrated no change in the extent of the
inhibition, regardless of the presence of mouse IgG1, TGF-, or
GITR (Fig. 4B). In
contrast, stimulation of cocultures with CD3-beads produced a slight
but consistent diminution from 91% suppression with control Ig to 78%
suppression in the presence of GITR (Fig. 4A). In
contrast, the addition of either the monoclonal TGF- or
polyclonal chicken TGF- did not affect the inhibition. Thus,
although blocking GITR resulted in a markedly increased level of
responder cell proliferation to both types of stimuli, it only slightly
reduced the inhibition in those cocultures that had received CD3
bead stimulation.
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Having demonstrated that regulation in part depends on the
strength of TCR signals delivered by different physical presentation of
Abs to CD3 and/or CD28, we next attempted to determine whether the
strength of the signal in an Ag-specific system would also alter the
level of suppression mediated by
CD4+CD25+high T cells. The
ability to address this question is a function of T cell precursor
frequency for the Ag to be examined. For example, previous studies from
our laboratory have demonstrated that human CD4+
T cell responses to myelin basic protein are found in
1/106 CD4+ T cells.
Analysis of such responses and their subsequent inhibition would be
very difficult in light of the limiting numbers of
CD4+CD25+high cells that
can routinely be isolated. We found that the frequency of T cells
reactive to GA, which is a collection of random polymers of glutamate,
lysine, alanine, and tyrosine, is relatively high (up to 1/5000
CD4+ T cells) (15). Furthermore, we
and others have shown that an in vitro primary response against
GA can be generated from cells isolated from normal healthy
controls without immunization. To ascertain whether
CD4+CD25+high cells could
inhibit Ag-specific responses, the fewest
CD4+CD25- cells that could
be seeded per well and give 100% response was determined to be 1
x 104. To assess the ability of
CD4+CD25+high cells to
suppress responses to GA as a model Ag, cocultures were established at
a 1:2 (regulatory:responder) ratio at two different concentrations of
GA to mimic high and low signal strength conditions (16).
CD4+CD25+high cells
inversely suppressed the proliferative response to GA in a
dose-dependent fashion (Fig. 5). That is,
with higher doses of GA, there was less suppression by regulatory
CD4+CD25+high cells. There
was no suppression of the no-Ag cultures since they were at background
levels even without the addition of regulatory cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The suppression mediated by nonpreactivated regulatory cells was less complete than that mediated by weakly preactivated regulatory cells. Unstimulated CD4+CD25+high regulatory cells isolated ex vivo were observed to suppress the proliferation and cytokine production by weakly activated CD4+ responder T cells. However, the nonpreactivated regulatory cells were less able than those that were given some means of preactivation to affect the cytokine secretion by strongly activated responder T cells. Thus, while it is possible that CD4+CD25+high regulatory cells are capable of directly suppressing ex vivo, it is most likely that unstimulated CD4+CD25+high regulatory cells were activated by residual anti-CD3 stimuli at the time the cocultures were established. It has recently been shown that human CD4+CD25+ T cells require activation to regulate CD4+ cells. Specifically, human CD4+CD25+ regulatory cells that were activated and fixed still inhibited responder CD4+ cells, while resting and fixed CD4+CD25+ regulatory cells were not functionally competent to suppress (18, 19). Together, these data suggest a change on the surface of activated CD4+CD25+ that is important for their regulatory function. Our data suggest that the expression or change in expression of this molecule on the cell surface is affected by the type of TCR signal delivered to the regulatory cell, as differentially stimulated regulatory cells demonstrate altered capacity to inhibit proliferation or cytokine production.
It was important to examine whether changing the strength of signal
delivered by a peptide MHC complex through the TCR also influenced
regulatory/responder T cell function. We took advantage of the random
copolymer Ag GA to evaluate the capacity of
CD4+CD25+high regulatory T
cells to mediate Ag-specific suppression. GA was chosen because a
relatively high frequency of HLA-DR-restricted
CD4+ T cells respond to the peptide complex and
higher in vitro concentrations lead to higher T cell activation states
(15, 16). Increasing the Ag concentration is equivalent to
increasing the strength of the signal delivered by CD3 stimulation.
We found that responder CD4+ cells were less
responsive to suppression via
CD4+CD25+high regulatory
cells with increasing concentrations of GA. Thus, although similar to
our results with increasing CD3 stimulation, the results with
increasing Ag-specific activation may reflect the more physiologic
MHC/Ag engagement of the TCR as it occurs in vivo.
Our data highlight how subtle changes in T cell activation protocols
result in marked differences in T cell function. Different physical
methods of Ab presentation, the use of different CD3 Abs, the use of
serum or serum-free media, and the presence or absence of accessory
cells all have different effects on T cell activation. Thus, a strong
stimulus that has been defined as optimal for T cell growth will likely
become a weaker suboptimal stimulus if the T cells are cultured in the
absence of serum and feeders. This assumption is supported by recent
data using a serum and accessory cell-free system where a 100-fold more
responder cells were required to achieve similar levels of
proliferation (19). A number of groups demonstrate
suppression under conditions of pbCD3/soluble CD28 stimulation,
while others do not. This fact is likely related to the in vitro
conditions regarding the presence of serum and APCs (3, 8)
(18, 19, 20). In the studies presented in this report,
conditions such as serum and APCs were held constant so that TCR
engagement could be studied in its least artificial in vitro state.
These data suggest that different strengths or modes of stimulation may
alter the mechanism by which
CD4+CD25+high cells
regulate immune responses. Different modes of action are suggested by
the finding that CD3 bead-stimulated regulatory cells inhibited
IL-10 production, while regulatory cells stimulated with the stronger
stimuli, which suppressed cytokine production and/or proliferation of
various target T cells, did not suppress IL-10 secretion.
Differentially stimulated regulatory cells also exhibit differences in
their abilities to inhibit strongly stimulated responder cell
proliferation (soluble CD3/CD28 regulatory cells) or to function
through a manner that is sensitive to inhibition by blockade of GITR
(CD3 bead stimulation). Thus, it is likely that
CD4+CD25+high cells use
multiple methods to regulate differentially triggered target T
cells.
The ability of
CD4+CD25+high regulatory
cells to secrete the immunosuppressive cytokines TGF- or IL-10 and
their involvement in regulation are unclear (3, 5, 6, 10, 21). We have not been able to detect IL-10 secretion from
cultures of CD4+CD25+high
cells alone under conditions in which they can suppress
CD4+ responder cells. This discrepancy with other
reports may be due to our use of a highly pure population of regulatory
cells that are isolated by cell sorting instead of magnetic bead
isolation, and results in a CD25+ population that
is >98% CD45RO+, which has been shown to
distinguish cells with suppressive activity (5). Nakamura
et al. (10) suggest that the cell contact in these
cocultures is necessary for the responding T cells to interact with
membrane-bound TGF- on the cell surface of
CD4+CD25+ cells. Yet,
completely functional and suppressive
CD4+CD25+ cells have been
isolated from spleens of IL-10-/- mice and from
thymocytes of neonatal TGF--/- mice
(3, 22). Recent reports suggest a model of "infectious
tolerance" in which the
CD4+CD25- T cells that
have interacted with
CD4+CD25+ cells become
anergic and tolerogenic themselves via a mechanism that involves
autologous production of TGF- and IL-10 (18, 19).
However, our data indicate that while interaction with
CD4+CD25+ cells may lead to
CD4+ cell anergy, it may not always lead to the
generation of tolerogenic CD4+ cells that can
suppress other cells through the secretion of immunosuppressive
cytokines. Because IL-10 was secreted in cocultures that show no
inhibition themselves, the secretion of IL-10 appears to result from
the type of stimulation and not from the presence of functionally
suppressed or anergized target T cells. This conclusion is emphasized
by the inability to identify IL-10 secretion in the suppressive
cocultures established with CD3 bead- or soluble
CD3/CD28-stimulated responder cells. Thus, not all suppressive
interactions result in the production of tolerogenic
CD4+CD25- cells
suppressing through IL-10. We are currently investigating whether the
CD4+CD25- cells cocultured
under weakly stimulatory conditions with the
CD4+CD25+high cells may in
fact be tolerogenic, but through a different mechanism.
In summary, we generated an in vitro model in humans that allowed
differential activation of regulatory
CD4+CD25+high and responder
CD4+ T cells. Responder
CD4+ T cells become refractory to suppression by
regulatory CD4+CD25+high
cells with strong engagement of the TCR by either prolonged CD3
stimulation or high concentrations of Ag/HLA-DR complexes. Our data are
consistent with a model where
CD4+CD25+high regulatory T
cells suppress the physiologic activation of autoreactive T cells
associated with low signal strength while T cells activated during
inflammatory responses associated with high signal strength are
refractory to this mechanism of suppression.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Clare Baecher-Allan, Laboratory of Molecular Immunology, Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: callan{at}rics.bwh.harvard.edu
3 Abbreviations used in this paper: GITR, glucocorticoid-induced TNFR; GA, glatiramer acetate; pb, plate-bound; TdAC, T cell-depleted accessory cell.
Received for publication August 18, 2002. Accepted for publication October 2, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155:1151.[Abstract]
. J. Exp. Med. 194:629.1 production and responsiveness. J. Exp. Med. 196:237.This article has been cited by other articles:
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  M. Bonelli, A. Savitskaya, K. von Dalwigk, C. W. Steiner, D. Aletaha, J. S. Smolen, and C. Scheinecker Quantitative and qualitative deficiencies of regulatory T cells in patients with systemic lupus erythematosus (SLE) Int. Immunol., July 1, 2008; 20(7): 861 - 868. [Abstract] [Full Text] [PDF]
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N. Oberle, N. Eberhardt, C. S. Falk, P. H. Krammer, and E. Suri-Payer Rapid Suppression of Cytokine Transcription in Human CD4+CD25 T Cells by CD4+Foxp3+ Regulatory T Cells: Independence of IL-2 Consumption, TGF-beta, and Various Inhibitors of TCR Signaling J. Immunol., September 15, 2007; 179(6): 3578 - 3587. [Abstract] [Full Text] [PDF]
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X. Wu, K. Roelofs-Haarhuis, J. Zhang, M. Nowak, L. Layland, E. Jermann, and E. Gleichmann Dose dependence of oral tolerance to nickel Int. Immunol., August 13, 2007; (2007) dxm066v1. [Abstract] [Full Text] [PDF]
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Q. Xu, J. Lee, E. Jankowska-Gan, J. Schultz, D. A. Roennburg, L. D. Haynes, S. Kusaka, H. W. Sollinger, S. J. Knechtle, A. M. VanBuskirk, et al. Human CD4+CD25low Adaptive T Regulatory Cells Suppress Delayed-Type Hypersensitivity during Transplant Tolerance J. Immunol., March 15, 2007; 178(6): 3983 - 3995. [Abstract] [Full Text] [PDF]
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X. Valencia, C. Yarboro, G. Illei, and P. E. Lipsky Deficient CD4+CD25high T Regulatory Cell Function in Patients with Active Systemic Lupus Erythematosus J. Immunol., February 15, 2007; 178(4): 2579 - 2588. [Abstract] [Full Text] [PDF]
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A. Joetham, K. Takada, C. Taube, N. Miyahara, S. Matsubara, T. Koya, Y.-H. Rha, A. Dakhama, and E. W. Gelfand Naturally Occurring Lung CD4+CD25+ T Cell Regulation of Airway Allergic Responses Depends on IL-10 Induction of TGF-beta J. Immunol., February 1, 2007; 178(3): 1433 - 1442. [Abstract] [Full Text] [PDF]
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K. Hirahara, L. Liu, R. A. Clark, K.-i. Yamanaka, R. C. Fuhlbrigge, and T. S. Kupper The Majority of Human Peripheral Blood CD4+CD25highFoxp3+ Regulatory T Cells Bear Functional Skin-Homing Receptors J. Immunol., October 1, 2006; 177(7): 4488 - 4494. [Abstract] [Full Text] [PDF]
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F. Billiard, E. Litvinova, D. Saadoun, F. Djelti, D. Klatzmann, J. L. Cohen, G. Marodon, and B. L. Salomon Regulatory and Effector T Cell Activation Levels Are Prime Determinants of In Vivo Immune Regulation J. Immunol., August 15, 2006; 177(4): 2167 - 2174. [Abstract] [Full Text] [PDF]
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K. Wing, Z. Fehervari, and S. Sakaguchi Emerging possibilities in the development and function of regulatory T cells Int. Immunol., July 1, 2006; 18(7): 991 - 1000. [Abstract] [Full Text] [PDF]
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) preactivated by the designated stimuli
to cultures of previously stimulated CD4+CD25-
cells. The CD4+CD25- cells were preactivated
by different stimuli: A, 
(CD4+CD25+high), 
(CD4+CD25-), and 
(Cocultures) represent
the levels of thymidine incorporation measured at day 5 from triplicate
cultures (mean ± SEM). This is representative of three
separate experiments performed with cells from different donors.
)
against two doses of GA (40 and 100 µg/ml) were assayed after 7 days
in vitro. [3H]Thymidine was added to the cultures at day
7 and harvested after 16–18 h. The assays were performed in
quadruplicate and triplicate, the symbols represent the means of the
total proliferative response, and the errors represent SEM. This has
been repeated >3 times with cells isolated from different healthy
donors; all repeats demonstrate suppression of GA responses that
decreases at high Ag dose.