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Laboratory of Cellular and Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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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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The outcome of priming vs tolerance can be influenced by LPS
administration or anti-CD40 Ab, stimuli that are known to activate
APC and produce proinflammatory cytokines such as IL-6 and TNF-
(7, 13, 14). This suggests that the costimulatory
environment present during initial Ag exposure might be critical for
determining the eventual fate of the immune response. However, LPS can
be given up to 2 days after introduction of the (super)Ag,
demonstrating that the initial T cell encounter with an APC is not the
sole fate-determining event. These observations suggest that naive T
cells initially respond positively to the Ag, but then readjust their
response potential several days later in an adaptive process that
remains poorly understood (15). This re-evaluation step by
the cell could be critical for the decision to die, become a memory
cell, persist as an effector cell, or down-regulate responsiveness.
Parameters such as the remaining Ag concentration, the persistence of
costimulation (B7s, ICAM), the presence of CTLA-4 or programmed
death-1, the levels and types of cytokines being produced
(IL-10, TGF-
, IFN-
, IL-4), the presence of regulatory T cells
(Tr1, CD25+), and the density of responding T
cells could all be important variables in this decision-making
process.
To explore some of these variables in a simplified model system, we
studied the response of naive peripheral CD4+ T
cells to persistent Ag in vivo. A few such models already exist, but
they use heterogeneous populations of T cells and/or have not been
extensively examined during the adaptive phase (16, 17, 18).
To create a better model, we transferred recombination-activating gene
(Rag)42-/-
TCR transgenic (Tg) CD4+ T cells specific for
pigeon cytochrome c (PCC)/I-Ek
(19, 20) into a second Tg mouse expressing the Ag under
the control of an MHC class I promoter and an Ig enhancer
(21). This second Tg had been previously crossed by us to
a CD3
-/- mouse (22) to create a
host with no T cells. This allowed us to follow the fate of the
transferred, monospecific, naive CD4+ T cell
population in the absence of competition by other T cells.
Interestingly, this model yielded an enormous population of
hyporesponsive T cells whose intrinsic adaptive tolerance could be
induced to different degrees, even though Ag presentation was kept
constant at a very low level. These observations, plus the
reversibility of the state, suggest that CD4+ T
cells can tune their threshold of responsiveness in the face of
persistent Ag.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B10.A/SgSnAi (B10.A) mice and various knockout and Tg lines
crossed onto that background were bred at the National Institute of
Allergy and Infectious Diseases contract facility at Taconic Farms
(Germantown, NY), an American Association for the Accreditation of
Laboratory Animal Care-accredited specific pathogen-free barrier
facility. The derivation of the B10.A TCR-Cyt 5C.C7
Rag2-/- mouse was described previously
(20). This strain carries the V11/V3
CD4+ T cell Tg receptor specific for the PCC
peptide 81–104 bound to I-Ek. This mouse will be
referred to as the TCR-Tg. A second Tg mouse (called either RO or
membrane-associated PCC (mPCC)) was the generous gift of S.
Hedrick and S. Oehen (University of California, San Diego, CA)
(21). This mouse expresses a membrane-targeted form of PCC
under the control of an MHC class I promoter and an Ig enhancer. We
received the mice on a B6 background at N5 and crossed them twice to
B10.A, selecting for the MHCa haplotype. We then
crossed them with a CD3-/- mouse
(22), which we had also placed on a B10.A background with
two backcrosses and an intercross. The final B10.A mPCC Tg
CD3-/- mouse generated
will be called mPCC-CD3-/-. All mice used
from this strain were heterozygous for the mPCC transgene. As a no Ag
control in our experiments, the CD3-/- mouse
on a B10.A background was used (abbreviated as
CD3-/-).
Transfer and retransfer of Tg cells
Naive T cells (3 x 106) isolated
from the lymph nodes (LNs) (cervical, axillary, brachial, inguinal, and
mesenteric) of TCR-Tg mice (
95% CD4+ cells)
were injected i.v. into mPCC-CD3-/- or
CD3-/- mice. Before injection, the cells
were labeled with 10 µM CFSE (Molecular Probes, Eugene, OR), as
previously described (23). In one set of experiments, we
also injected naive CFSE-labeled TCR-Tg cells i.v. into
mPCC-CD3-/- mice that had previously
received unlabeled naive TCR-Tg cells 38 days before (full (D38)
hosts).
For the retransfer experiments, mPCC-CD3-/-
mice were sacrificed 38 days after the injection of naive TCR-Tg cells.
CD4+ T cells were purified from LNs (>90%
purity) by magnetic bead depletion of B cells, as well as macrophages
and dendritic cells, using a two-step procedure. The first depletion
was with sheep anti-mouse Ig-coated Dynabeads M-450 (Dynal, Lake
Success, NY), and the second was with a mixture of rat mAbs
(anti-Mac1, anti-B220, and anti-I-Ek;
BD PharMingen, Mountain View, CA), followed by sheep anti-rat
IgG-coated Dynabeads M-450. The TCR-Tg cells were then stained with
CFSE and 3 x 106 injected i.v. into
CD3-/- mice, into
mPCC-CD3-/- mice, or back into full (D38)
hosts. Spleen and LNs were removed from these mice and studied at
different time points after the retransfer. In one experiment, the
second hosts were injected i.p. at day 35 with 10 µg staphylococcal
enterotoxin A (Toxin Technologies, Sarasota, FL) in PBS, and the
cellular expansion was monitored after 2, 4, and 8 days.
Flow cytometry
Cell suspensions were stained with PE-labeled Abs: anti-CD4
(Caltag, San Diego, CA), anti-CD69, anti-CD25, and biotinylated
anti-V3 Abs revealed by streptavidin Tricolor (BD PharMingen).
Immunofluorescence analysis was performed on a FACScan cytometer (BD
Biosciences, Mountain View, CA), and data files were analyzed using
CellQuest software (BD Biosciences).
T cell proliferation assay and cytokine ELISA
Five thousand cloned A.E7 T cells or 10,000 purified TCR-Tg
CD4+ cells were cultured with graded
concentrations of PCC peptide (aa 81–104, synthesized and HPLC
purified by the National Institute of Allergy and Infectious Diseases
core facility (National Institutes of Health, Rockville, MD)) and
5 x 105 irradiated (3000 rad) spleen cells
as APC from CD3-/- mice or
mPCC-CD3-/- mice. In some experiments,
normal B10.A spleen cells were anti-Thy-1 depleted, irradiated, and
used as APC. In one experiment, the TCR-Tg CD4+
cells were preactivated by stimulating them with PCC and B10.A splenic
APC for 48 h, expanding them in 10 U/ml IL-2, and using them after
5 days of rest. The culture medium was RPMI 1640 (Biofluids, Rockville,
MD) supplemented with 10% FCS (Biofluids), 4 mM glutamine, 100 U/ml
penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME. The cultures
were set up in flat-bottom, 96-well plates (Costar 3596, Corning, NY).
After 48 h of culture, 100 µl of supernatant was removed for
cytokine assays, and the concentrations of all the cytokines were
determined by sandwich ELISA (Endogen, Woburn, MA, or R&D Systems,
Minneapolis, MN), according to the manufacturer’s instructions. The
wells were then pulsed with 1 µCi [3H]TdR
(6.7 Ci/mmol; ICN Biomedical, Costa Mesa, CA) and harvested 16 h
later. The incorporated [3H]TdR was measured by
scintillation counting in a Betaplate 1205 detector (Wallac,
Gaithersburg, MD). Dose-response curve data were fit with the nonlinear
regression software program of Prism GraphPad, and the
EC50 were calculated. To determine the ability of
the cells to re-express CD25 and CD69, they were cultured for 48 h
under the same conditions, collected, and then stained for flow
cytometry analysis, as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- APC in vivo
CD4+ TCR-Tg T cells were collected from the
LNs of naive mice and labeled in vitro with CFSE, and 3 x
106 cells were transferred i.v. into
mPCC-CD3-/- mice. One day later, 10–15% of
the cells could be recovered from spleen and peripheral LNs (Fig. 1
, left graph). These cells
were all activated, as assessed by size enlargement measured using flow
cytometry (data not shown) and CD25 and CD69 expression (see below).
The cells began to proliferate between days 1 and 2 in vivo, and 80%
of them had divided two or three times (Fig. 1, right graph,
mPCC-CD3-/-). A similar doubling time (7–12
h per cycle) was maintained between days 2 and 3. After day 3, the
expansion phase abated, reaching a maximum of 97-fold on day 4 (Fig. 1, left graph). However, after day 4 the T cell recovery began
to decline, suggesting either the onset of apoptosis, as has been
described in other model systems (24), or migration to
other tissues. The cell loss in this second phase of the response was
50%, and was completed by day 10–14. This was followed by a small
increase in cell number over the next 6 wk. In this third phase, which
can start as early as day 7 to 10, the cells have greatly curtailed
their in vivo responsiveness to the Ag. We will refer to this stage as
the adaptive phase.
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-/- host not
expressing the Ag, they failed to expand or divide during the first
week (Fig. 1
, left panel for cell number and right
panel labeled CD3-/- for CFSE
staining), even though in this particular experiment there were
slightly more cells recovered at day 1 than from the
mPCC-CD3-/- host. However, starting at day
7, one division was observed in a small subset (20%) of the cells. A
detectable increase in cell yield was seen by day 14, and by day 22,
75% of the cells had divided at least once and one-third of them more
than once. By 2 months, the net expansion was 4-fold. This response
is similar to the homeostatic T cell proliferation observed by others
in T cell-deficient hosts, which varies in magnitude and time of onset
depending on the particular Tg TCR being studied (25). In
our experiments, this phenomenon appears to be completely supplanted by
the expansion and deletion phases of the T cell response to
cognate Ag.
We also looked at the expression of early activation markers on the
CD4+ T cells isolated at different times after
transfer into the mPCC-CD3-/- hosts (Fig. 2). At 24 h, all the cells expressed
high levels of CD69, and 85% of them had up-regulated CD25. After this
initial full activation, both CD69 and CD25 levels were rapidly
down-regulated, and near the end of the expansion phase (day 3) and
during the deletion phase (day 8), these markers were no longer
detected at the cell surface. However, in the adaptive phase (from day
14 onward), 30–40% of the CD4+ T cells
re-expressed CD69 (only day 38 is shown in Fig. 2), although the level
of expression never achieved that observed on day 1. None of the cells
re-expressed CD25 (Fig. 2), but most were
CD44high (data not shown).
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The adapted T cells, isolated 38 days after transfer, were
restimulated in vitro for 2 days with different amounts of PCC peptide
and examined in this individual cell assay for their ability to
up-regulate CD69 and CD25 (Fig. 3).
Stimulation with 3 nM exogenous peptide did not induce detectable
increases of CD69 or CD25 on the adapted T cells, whereas 50–60% of
the naive T cells responded to the same concentration. In fact, the low
levels of CD69 on the freshly isolated adapted T cells disappeared in
culture. The adapted T cells did show a substantial CD69 and CD25
response (40%) at 100 nM, but even at very high concentrations of Ag
(1 µM) only 70% of them re-expressed CD25, and only 50%
significantly up-regulated CD69. The response patterns seen at these
high doses for the adapted cells resemble those seen for the naive T
cells at the low Ag doses (3–10 nM), suggesting a 100- to 300-fold
shift in the Ag dose-response curves.
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). One day after
transfer, the maximum proliferative response was 6-fold greater than
that observed with naive T cells, although the
EC50 values were similar (9 nM for naive vs 7 nM
for day 1) (Fig. 4A). This result suggests that activation
in vivo is a very effective means of preparing many
CD4+ T cells to subsequently enter cell cycle in
vitro without changing the Ag threshold for activation. However, after
7 days in vivo the T cells became hyporesponsive. The
EC50 was shifted to 50 nM, and the maximum
response returned to the level of naive cells. This 5-fold shift is
maintained out at 36 days (EC50 = 42 nM) (Fig. 4B). In contrast, if the T cells were transferred into a
CD3-/- host not expressing Ag, the cells
became hyperresponsive (EC50 = 1 nM) by day 36.
This suggests that the homeostatic response leads to a memory-like
state, whereas the response to persistent Ag leads by 7 days to an
anergic-like state.
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). On day 7, IL-2 production by adapted
T cells in response to 1 µM peptide was 15% of that observed for
naive T cells (Fig. 5A), and this hyporesponsiveness was
maintained out to 36 days (Fig. 5, A and C).
Naive T cells did not produce very much IL-4, IL-10, and IFN-
following stimulation in vitro for 48 h with 1 µM peptide.
However, after 24 h in the mPCC-CD3-/-
host, an enormous induction event occurred in the T cells, allowing
them to produce large amounts of these cytokines. IFN- production
was the greatest (Fig. 5A), rivaling that produced by T cell
clones such as A.E7. IL-4 and IL-10 production were also substantial
(Fig. 5B). However, 7 days after transfer, all the cytokine
production potentials were greatly reduced, and this reduction was
maintained out to day 36. By this time, IFN- production was only 5%
of that at day 1. IL-4 production was reduced to 5%, IL-3 production
to 3%, and IL-10 production to 2% (Fig. 5, A and
B). IL-3 production was also decreased relative to that of
naive T cells (9.5%). Because the IFN- response was initially so
large, the population remaining at day 36 gives the impression that the
T cells have differentiated predominantly toward a Th1 phenotype.
However, with regard to the adaptation process, it is clear that the
degree of down-regulation is comparable for all the cytokines.
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-/-
host not expressing the Ag, no difference from naive cells was noted
out to day 7 (data not shown). However, by 14 days the ability to
produce significant amounts of IFN- was detected, and by 36 days the
levels were comparable with those seen in the T cells adapted to the
PCC Ag (Fig. 5D). At this time point, though, a
dose-response curve shows that in fact the two populations are quite
different. Although the adapted cells required >10 nM peptide to
elicit a detectable IFN- response, the homeostatically expanded
population was still giving a strong response at concentrations as low
as 3 nM. For IL-2 production (Fig. 5C), these cells were
also slightly hyperresponsive in comparison with naive cells
(EC50 = 130 nM vs 330 nM). Other cytokine
production was either undetectable (IL-10) or only slightly more than
that of the naive cells (IL-3 and IL-4) (data not shown). Thus, again,
it appears that the homeostatic response leads to a hyperresponsive
state, whereas the persistent PCC Ag stimulation leads to a
hyporesponsive state. Adaptation in vivo is not a change in Ag presentation
The initial immune response during the first week after transfer
could potentially have led to destruction of the APCs in the
mPCC-CD3-/- host. If this process continued
into the adaptive phase, then it might have resulted in a new steady
state level of processed Ag, which was inadequate to strongly stimulate
the T cells. Two approaches were used to examine this possibility.
First, spleen cells were taken from either a naive
mPCC-CD3-/- mouse or a host containing day
40 or day 56 adapted T cells and used to stimulate either the T cell
clone A.E7 or preactivated CD4+ TCR-Tg T cells.
As shown in Fig. 6A, the
magnitude of the T cell proliferative response in the absence of any
added Ag was comparable for the two types of APC and similar to that
obtained when 0.3 nM peptide was added to normal B10.A spleen cells.
Furthermore, addition of exogenous PCC augmented the response of both
mPCC APCs with similar dose-response curves, suggesting that the
endogenous levels of processed Ag are the same in both mice. The
transition point for augmentation of the endogenous response was
carefully measured in Fig. 6B and found to occur for both
APC between 0.08 and 0.16 nM, emphasizing the relatively low amount of
PCC that is functionally expressed in vivo.
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-/- mouse that had been
reconstituted 38 days previously with Tg T cells and now contained
20–40 x 106 adapted
CD4+ cells. As shown in Fig. 7, the CFSE-labeled second cohort
expanded at the same rate and to the same magnitude as if they had been
injected into an empty mPCC-CD3-/- host (see
Fig. 1 for comparison). This experiment shows that the Ag
presentation in vivo has remained the same. Furthermore, there appeared
to be no competition or suppression from the adapted T cells in the
tolerant host. This rules out a role for active immunoregulation as a
mechanism to explain the proliferative unresponsiveness.
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11, anti-V3, or anti-CD4. Little or
no reduction of each of these molecules was detected (0–25%).
Furthermore, occasional slight reductions were also seen on T cells
that had come out of the state (see below), and thus there was no
correlation with the functionality of the cells (data not shown). Reversal of adaptation on retransfer of the T cells to a host not expressing the Ag
To determine whether the tolerant state was reversible, and thus
truly adaptable, CD4+ T cells were purified from
LNs of day 38 adapted mPCC-CD3-/- mice, and
3 x 106 of them (>90% purity) were
injected into a CD3-/- mouse not expressing
the Ag. For the first 7 days, these cells did not divide at all (Fig. 8A and CFSE data not shown).
Between days 7 and 36, a slow expansion occurred, culminating in a 6-
to 8-fold increase in cell number, which was maintained out to day 56
(Fig. 8B). This pattern appeared similar to that of naive T
cells in a CD3-/- host (Fig. 1).
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A). Within 1 day after
transfer, the proliferative dose-response curve had returned to the
normal pattern seen for naive T cells. Interestingly, by 7 days, the
cells had become hyperresponsive (EC50 = 2 nM vs
10 nM for naive cells). This increased sensitivity remained out to 56
days (EC50 = 3 nM). The ability to produce
cytokines in vitro was also assessed after retransfer (Fig. 9B). Both IL-2 and IFN- production increased with time.
For IFN-, the maximum response increased 5.7-fold by day 56 in
experiment 1 (Fig. 9B) or 3.6-fold by day 38 in experiment 2
(Fig. 9C). For IL-2, the maximum response increased
significantly by day 7, but did not reach the level of production of
naive T cells until day 56 (Fig. 9B). The IL-2
EC50 returned to that of the naive cells by day
38 (Fig. 9C). For IFN-, the EC50
decreased only slightly (2-fold) by day 38, compared with the initial
value at the time of retransfer (Fig. 9C). These results
suggest that the hyporesponsive state can reverse when the cells are
transferred into an environment that lacks the Ag. This reversal is
partially accomplished in the first week, suggesting that the cell
divisions that occur beyond day 7 (Fig. 8B) are not
essential to initiate this change in responsiveness.
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The in vivo response to Ag of day 38 adapted T cells was also
examined by injecting them back into day 38 adapted mice or into naive
mPCC-CD3-/- mice, both expressing the Ag. In
the former host, with resident tolerant cells, no proliferative
expansion of the new cohort was seen out to 7 days (data not shown).
Surprisingly, in the naive mPCC-CD3-/- empty
host, the cells began to expand after day 1, and the proliferation
lasted for several days (Fig. 8A). By 7 days, the new
population had expanded 13-fold. This level was maintained out to day
56 (Fig. 8B). In comparison with naive TCR-Tg
CD4+ cells (Fig. 1), the T cells adapted for 38
days were clearly hyporesponsive: the rate at which they doubled was
only once every 40 h vs 7–12 h for naive cells. Furthermore, the
maximum expansion was 4-fold less. Thus, the T cells stopped
dividing before the population had completed filling up the host.
Finally, the fact that this expansion did not occur on injection back
into a day 38 host, which already contained adapted T cells, suggests
that it is influenced by some parameter(s), such as cell density, in
addition to the presence of the Ag.
A functional analysis of the cells retransferred into the empty
mPCC-CD3-/- host is shown in Fig. 9. The in
vitro proliferative dose-response curve initially reverted to that of a
naive population (Fig. 9A, day 1), but by day 7 the cells
had again become hyporesponsive (EC50 = 24 nM).
Interestingly, this decreased sensitivity appeared to become even
greater after the cells stopped proliferating
(EC50 = 83 nM at day 56), and in fact surpassed
the hyporesponsiveness originally displayed by the transferred cells at
the time of retransfer (EC50 = 30 nM). The
acquisition of a deeper tolerant state was also seen for cytokine
production. The levels of both IL-2 and IFN- produced in response to
1 µM peptide in vitro initially increased 1 day after transfer into
the second mPCC-CD3-/- host, but then
declined slowly thereafter (Fig. 9B), reaching a 7- to
10-fold lower level of production by days 22–36 as compared with at
the time of retransfer (injected cells D0). In the second experiment,
shown in Fig. 9C, one can also observe from the
dose-response curves that the remaining responding cells became more
hyporesponsive with time. For IL-2 production, the curve shifted to
higher Ag concentrations by 5- to 10-fold between days 7 and 38. For
IFN-, a similar shift was seen between the initial dose-response
curve at the time of retransfer and that observed at day 38. Production
of IL-4 and IL-3 also diminished after retransfer (data not shown).
These experiments demonstrate that the level of adaptive tolerance can
be greatly influenced by environmental parameters other than just the
expressed levels of Ag and costimulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and demonstrated that at least 50–70% of the
cells were being affected. This adaptation to chronic Ag exposure
appeared to be in the T cell population itself and not due to changes
in the Ag presentation environment of the host, because a fresh cohort
of naive T cells injected into an adapted host expanded normally. This
experiment also ruled out active suppression as a mechanism. The
hyporesponsive state was not caused by TCR down-regulation and
persisted in vivo for months. We have also observed this state if the T
cells were transferred into an irradiated mPCC host containing normal T
cells, although the expansion phase in this case is not as great (data
not shown). The hyporesponsive state was reversible if the T cells were
transferred to a second CD3-/- host not
expressing the Ag. At least part of this reversal took place in the
absence of proliferation, suggesting that the original mechanism for
the limitation in cytokine production was not solely the loss of a
subset of cells by a deletional or migrational process. Finally, in one
preliminary experiment, stimulation of these second hosts at day 21
with 10 µg of the superantigen staphylococcal enterotoxin A
demonstrated that the adapted cells were proliferatively tolerant in
vivo. Expansion of the CD4+ T cell population was
4.5-fold less in the Ag-bearing mPCC-CD3-/-
host than in a CD3-/- host lacking the Ag.
The latter expansion (18-fold) was equivalent to that seen for a
control naive T cell population transferred into a
CD3-/- host under the same conditions
(16-fold).
One of the most surprising findings in our experiments was the
enhancement of the hyporesponsive state on retransfer of the cells into
a second, T cell-deficient, Ag-bearing host. The cells initially
appeared to partially recover from the tolerant state, both in terms of
proliferation and cytokine production, but then they gradually lost
sensitivity and eventually achieved a deeper level of unresponsiveness.
This more profoundly depressed state was not solely due to cell
deletion or redistribution in the second host, because the recovered
cells themselves clearly became more desensitized at later time points
(Fig. 9, A and C). The possibility that T cells
can reach different levels of tolerance has been suggested before by
Ferber et al. (30). However, in their model this
phenomenon was seen when the cells were exposed to different
concentrations of Ag in a serial manner. In our model, the Ag
presentation is the same on re-exposure. Thus, some other variable(s)
must have allowed the retransferred cells to readjust their activation
threshold.
Several possible mechanisms come to mind. The first is a change in cell
density. Only 3 million T cells, of 20–40 million in the first host,
were injected into the second host. As we showed in vitro a number of
years ago (31) and has been more recently demonstrated in
vivo for thymic selection (32) and peripheral expansion
(33, 34), T cells of similar specificity can compete with
one another for activation by a limited number of peptide/MHC
complexes. Thus, the lower density of T cells created on retransfer of
a small number of them into a second empty host would effectively allow
each cell to experience a higher concentration of Ag. This might be
adequate to activate the hyporesponsive T cells. This idea is supported
by the control experiment in which we transferred the T cells back into
a day 38 host filled with tolerant cells. Under these conditions, the
CFSE-labeled donor cells did not proliferate. Note that this T cell
competition argument did not seem to apply to the naive T cells when
they were injected into a day 38 tolerant host (Fig. 7). We think this
is because the naive T cells are more responsive than the tolerant
cells (compare Fig. 1 with Fig. 8A), and thus more effective
competitors for peptide/MHC complexes. Alternatively, they go to
different niches for their stimulation. In contrast, the expansion of
naive T cells can be affected by the presence of memory T cells
(26).
But even if the initial response on retransfer is a consequence of a
change in cell density, it is not obvious why this would lead to a
deeper state of tolerance, as opposed to a re-equilibration at the
original level, once the T cell numbers expanded again. One possible
explanation is that the expansion in the second host never reaches the
same population size achieved in the first host, because of the
impaired responsiveness of the retransferred T cells. In our
experiments, the difference in population sizes achieved was 4-fold.
In this scenario, the final differences in cell densities would lead to
a difference in effective Ag concentrations, which in turn would lead
to a difference in adjusted tolerance threshold levels. An alternative
possibility is that only the proliferative response is activated by the
change in cell density, but that this response itself brings about the
reprogramming. Thus, similar to the idea that the chromatin is opened
up at cytokine loci by appropriate signaling during proliferative
expansion (28, 29), a tolerogenic environment might only
effectively signal the closing down of a cytokine locus during a
proliferative cycle.
Another possible explanation for the threshold changes on retransfer is
that the costimulatory environment of the tolerant host has actually
been modified, but that this change only influences the adapted T cells
and not naive T cells. A host that has undergone an immune response
might contain many nonprofessional APC that bear up-regulated
costimulatory molecules in its tissues, such as B7-1 and B7h, induced
in response to inflammatory cytokines such as TNF- (35, 36). In a scenario in which negative regulation (e.g., CTLA-4)
is dominant in the adaptive phase of the immune response, the tolerant
T cells would be kept in check in the first host by CTLA-4/B7-1
interactions (37), whereas a naive cohort might be
unaffected (Fig. 7). However, transfer of the tolerant cells to a new
host would abruptly change the environment to one in which B7-1
expression is low. This would break the negative regulation cycle, and
allow the cells to proliferate until up-regulation of B7-1 and CTLA-4
expression could again establish negative regulation in the second
host. However, the final difference in activation thresholds would
still require some additional changes to take place during the
proliferative expansion, e.g., induction of more B7-1 in the
environment of the second host or a decrease in signaling potential in
the adapted T cell, as discussed above. Furthermore, arguing against
this model is a preliminary experiment in which we injected
anti-CTLA-4 mAb into tolerant mice for 5 days and did not enhance
proliferation in vivo or cytokine production in vitro (L. Chiodetti, N.
Singh, and R. Schwartz, unpublished observations).
What is the basis for the tolerance process? The absence of evidence for immunoregulation in the in vivo mixing experiment, coupled with the hyporesponsiveness of the T cells in vitro when they are stimulated with fresh APC and optimal Ag doses, suggests that the tolerance process is intrinsic to the T cells themselves. The shifts in the CD25, CD69, and proliferative dose-response curves in vitro all suggest a desensitization process. This is supported by the shift in cytokine production curves seen in the retransfer experiments, although a loss of cytokine-producing cells also contributes to the tolerance. Although this is the first clear description of an in vivo desensitization tolerance mechanism for CD4+ T cells, it appears to be comparable in some ways to the phenomenon of B cell clonal anergy described by Goodnow and colleagues (38). Their model also involves two Tg mice in which a monoclonal B cell population becomes hyporesponsive after transfer into an Ag-bearing host or when crossed to the Ag-bearing mouse. Biochemically, the B cells appeared to down-regulate their IgM receptors by 90% and to show a block in early tyrosine phosphorylation events. The cells persisted in this state for a long time provided that there were no other normal B cells around, and the process was reversible on transfer to a host that did not express the Ag. In our adaptive T cell model, receptor down-modulation does not appear to be a major component of the tolerance process. Rather, preliminary data suggest a 2-fold inhibition in the ability to activate (with plate-bound anti-TCR) the early response kinases of purified CD4+ T cells from the tolerant mice. This supports the idea of an adaptive process in which the T cell intrinsically alters its threshold for responding to signals through the TCR. One mechanism by which the T cells could enter such an altered state is through the expression of CTLA-4 (39, 40). This molecule is up-regulated following CD4+ T cell activation, and inhibits TCR signaling by bringing the phosphatase SHP-2 into the receptor activation complex (41). Another is through the negative feedback recruitment of SHP-1 to the TCR activation complex following TCR signaling (42).
No matter what the nature of the intrinsic tolerant state turns out to be, the ability of this mechanism to achieve variable levels of unresponsiveness in the presence of a constant amount of Ag presentation is still a new and surprising finding. This adaptive property emphasizes the ability of the responding T cell to set different thresholds for activation depending on multiple cues from the environment. Constant environments seem to be tolerated, whereas changing environments elicit a reaction. A similar notion of individual T cells adjusting their response potential has been proposed to explain positive selection to agonist peptides in the thymus (43). To us, this type of adaptive tolerance model is most consistent with the theoretical thinking of Grossman and Paul (44), in which multiple environmental cues are integrated to determine the response threshold for T cell reactivation following Ag exposure.
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Acknowledgments |
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Footnotes |
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2 
Current address: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322.
3 Address correspondence and reprint requests to Dr. Ronald H. Schwartz, National Institutes of Health, Building 4, Room111, MSC-0420, Bethesda, MD 20892-0420. E-mail address: rs34r{at}nih.gov
4 Abbreviations used in this paper: Rag, recombination-activating gene; LN, lymph node; mPCC, membrane-associated PCC; PCC, pigeon cytochrome c; Tg, transgenic.
Received for publication April 3, 2001. Accepted for publication June 5, 2001.
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). The spleen and LNs were removed at various times after transfer
and stained with anti-V
)
or 7 (
) days after transfer into mPCC-CD3
) and IFN-
). C and D, Cytokine dose-response
curves for IL-2 (
