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Department of Microbiology, Center for Immunology, University of Minnesota Medical School, Minneapolis, MN 55455
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in vivo
following an antigenic challenge. These defects were not associated
with the production of Th2-type cytokines or the capacity to suppress
the clonal expansion of a bystander population of T cells present in
the same lymph nodes. Therefore, our results provide direct evidence
that a long-lived population of functionally impaired Ag-specific
CD4+ T cells is generated in vivo after exposure to soluble
Ag. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peripheral deletion in response to soluble Ag is one mechanism by which the immune system could eliminate self-reactive T cells that escape thymic deletion. Experimental models in which superantigens are injected into normal mice (8, 9), or the relevant peptide Ags are injected into TCR transgenic mice (10, 11, 12, 13), have provided evidence for peripheral deletion. However, in both of these model systems, a population of T cells survives. It is possible that this survival is artifactual because the abnormally high number of specific T cells present in these situations makes complete deletion impossible. Alternatively, deletion may be only an intermediate step in peripheral tolerance, and additional mechanisms may be required to fully silence self-reactive peripheral T cells.
Another potential mechanism for silencing T cells is functional inactivation, often referred to as anergy. Based largely on in vitro experiments with Th1 clones, anergy is defined as a defect in TCR-dependent proliferation that is acquired as a result of prior TCR stimulation in the absence of APC-derived costimulatory signals or proliferation (14, 15, 16). Although functionally unresponsive T cells have been identified in several in vivo systems (10, 12, 13, 17, 18, 19, 20, 21, 22, 23, 24, 25), the failure of a common induction pathway or molecular defect to emerge from these studies has raised doubts about the relationship between the in vivo models and the in vitro paradigm based on Th1 clones (26). Doubts about the relevance of the Th1 clone model have been reinforced by some studies in which naive T cells, the probable targets of peripheral tolerance in vivo, did not become unresponsive when stimulated through the TCR in the absence of costimulatory signals (27, 28). These issues have led some to argue that functional inactivation is not an important peripheral tolerance mechanism, or is only a brief stage that tolerized T cells pass through before deletion (29).
Recent studies have implicated immune deviation as an alternative explanation for the apparent functional unresponsiveness of CD4+ T cells in vivo. For example, it has been reported that what was thought to be anergy based on loss of Th1 lymphokine production is really skewing of self-reactive T cells, such that they respond to Ag only by producing a limited set of Th2 cytokines that are not capable of supporting T cell growth (30, 31, 32). It is also possible that in vivo functional unresponsiveness is due to Ag-specific suppression. It was reported recently that chronic activation of CD4+ T cells in the presence of IL-10 generates IL-10-, IL-5-, and TGF-ß-secreting cells that suppress the proliferation of CD4+ T cells in vitro and prevent colitis in SCID mice (33).
We have attempted to address these mechanistic issues in detail by creating a situation in which a peptide/MHC-specific T cell population, large enough to be detected by flow cytometry following staining with an anti-clonotypic mAb, but small enough to behave in a physiologic manner when confronted with Ag in vivo, is monitored following the induction of peripheral tolerance. We demonstrated in a previous study that soluble Ag induces a transient accumulation and loss of adoptively transferred OVA-specific CD4+, TCR transgenic T cells that was accompanied by an apparent unresponsiveness in the surviving T cells (34). In this study, we found that a population of Ag-specific T cells, initially impaired in their ability to proliferate and produce lymphokines in vivo, survived for months after the initial Ag injection to the point in which they recovered from their unresponsive state. The unresponsive state was not associated with immune deviation or suppression. Therefore, tolerance to soluble Ag is maintained in part because the Ag-specific T cells that persist in the periphery possess an inherent activation defect during the time that the Ag is present.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DO11.10 BALB/c mice were produced by crossing the original DO11.10 TCR transgenic mice (35) for >10 generations to normal BALB/c mice. Offspring expressing the transgenic TCR were identified by flow-cytometric analysis of peripheral blood cells, as previously described (34). DO11.10 BALB/c mice were intercrossed, and offspring homozygous for the DO11.10 transgenes were identified by their ability to exclusively produce DO11.10 TCR-expressing offspring when crossed with BALB/c mice. DO11.10 BALB/c SCID mice were produced by crossing DO11.10 BALB/c mice for two generations with BALB/c SCID mice, and selecting offspring that contained DO11.10 T cells, but lacked B cells in the peripheral blood. The 6.5 TCR transgenic mice (36) were obtained from Dr. Hyam Levitsky (The Johns Hopkins University, Baltimore, MD). All TCR transgenic mice were housed in a pathogen-free facility according to National Institute of Health guidelines. Normal BALB/c mice, purchased from the National Cancer Institute (Frederick, MD) and housed in a conventional facility, were used as recipients of donor TCR transgenic cells.
Injections and treatment of mice
Lymph node and spleen cells (containing 2.5 x 106 to 5 x 106 CD4+, KJ1-26+ cells) from TCR transgenic donors were prepared for adoptive transfer, as previously described (34), and injected into the tail veins of recipient mice in a volume of 0.3 ml of PBS. For the systemic Ag injections, OVA peptide 323–339 was dissolved in PBS and passed though a 0.22-µm syringe filter, and 300 µg was injected in a volume of 0.25 ml into the tail vein. For primary immunization, 30 or 300 µg of OVA peptide 323–339, or a mixture of 30 µg of OVA peptide 323–339 and 30 µg of hemagglutinin (HA)3 peptide 111–119, was emulsified in CFA, and injected s.c. in a 0.1-ml vol into the tail or distributed between three sites on the back. For secondary challenge, 30 or 300 µg of OVA peptide was emulsified in IFA and injected s.c. in a 0.1-ml vol distributed between three sites on the back. 5-bromo-2'-deoxyuridine (BrDU; Sigma, St. Louis, MO) was administered daily in the drinking water (0.8 mg/ml).
Detection of TCR transgenic T cells
Lymph node cells were first incubated in staining buffer (HBSS plus 2% FCS and 0.2% sodium azide) on ice with culture supernatant containing anti-FcR mAb (2.4G2; American Type Culture Collection, Rockville, MD) for 10 min to block FcR. Phycoerythrin (PE)-labeled anti-CD4 (PharMingen, San Diego, CA) and biotinylated KJ1-26 (37) or biotinylated 6.5 (36) mAbs were added and incubated for an additional 20 min. After a wash in staining buffer, the cells were incubated with FITC-labeled streptavidin (Caltag, South San Francisco, CA) for 20 min, washed, and analyzed on a Becton Dickinson (Mountain View, CA) FACScan flow cytometer. Twenty thousand events that had the light-scatter properties of lymphocytes were collected.
Detection of cell surface activation markers
Lymph node cells were incubated on ice with 2.4G2 culture supernatant for 10 min, followed by Cy-Chrome-labeled anti-CD4 mAb (PharMingen), biotinylated KJ1-26 mAb, FITC-labeled anti-CD45RB mAb, anti-L-selectin mAb, or anti-LFA-1 mAb (all from PharMingen) for 20 min. The cells were washed and incubated on ice for 20 min with PE-labeled streptavidin (Caltag). The FITC channel fluorescence of 1000 to 2000 CD4+, KJ1-26+ or CD4+, KJ1-26- cells was measured.
Detection of BrDU incorporation
Lymph node cells were stained with Cy-Chrome-labeled anti-CD4 mAb, biotinylated KJ1-26 mAb, and PE-labeled streptavidin, as described above. The cells were fixed for 1 h in PBS containing 1% paraformaldehyde and 0.01% Tween-20, stored overnight in PBS, and stained for BrDU incorporation, according to a published protocol (38). Briefly, the cells were incubated at 37°C in 1 ml DNase buffer (0.15 mM NaCl, 4.2 mM MgCl2, 4.2 mM CaCl2, 50 Kunitz/ml DNase I (DN-25; Sigma), pH 5) for 20 min, washed, and suspended in PBS containing 2% mouse serum and 0.5% Tween-20. Each sample was then divided and incubated at room temperature with FITC-labeled anti-BrDU mAb or IgG1 control mAb (both from Becton Dickinson) for 30 min. After several washes, the FITC channel fluorescence of 1000 to 2000 CD4+, KJ1-26+ cells was measured by flow cytometry.
Intracellular cytokine staining
Lymph node cells were stained with Cy-Chrome-labeled
anti-CD4 mAb, biotinylated KJ1-26 mAb, and FITC-labeled
streptavidin, as described above. The cells were fixed for 20 min at
room temperature in PBS containing 2% formaldehyde, and permeabilized
with two washes in staining buffer containing 0.5% saponin (Sigma).
The permeabilized cells were incubated at room temperature for 30 min
with PE-labeled anti-IL-2 mAb or anti-TNF- mAb (both from
PharMingen) and washed once with the saponin buffer and twice with PBS.
The PE channel fluorescence of 1000 to 2000 CD4+,
KJ1-26+ cells or CD4+, KJ1-26-
cells was measured by flow cytometry.
In vitro cytokine assays
Lymph node cells (5 x 105) were cultured in triplicate wells with 2 x 105 irradiated (3000 rad) BALB/c splenocytes, with or without OVA peptide 323–339 in 0.2 ml of Eagle’s Hanks’ amino acids medium (Biofluids, Rockville, MD) supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, 20 µg/ml gentamicin sulfate, and 5 x 10-5 M 2-ME. Aliquots (50 µl) of culture supernatant were removed from each well at 24, 48, and 72 h after initiation of the culture.
In some experiments, KJ1-26+ cells were removed from cell suspensions by magnetic bead depletion. Streptavidin-coated magnetic beads (Promega, Madison, WI) were incubated with KJ1-26 mAb for 15 min on ice, washed, and then incubated with the lymph node cell suspensions, at a ratio of 10 beads/lymph node cell, for 30 min at 4°C. The beads and attached cells were then removed, and the lymph node suspensions were counted and placed in culture, as described above.
IL-2, IFN-
, IL-5, and IL-4 mAb pairs were purchased from PharMingen,
and the ELISAs performed on the culture supernatants, according to the
manufacturer’s directions, with known amounts of recombinant murine
cytokine (all from PharMingen) used to generate standard curves for
comparison.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To physically monitor Ag-specific T cells in vivo, T cells from
DO11.10 TCR transgenic mice (35), which express transgenes encoding a
TCR specific for chicken OVA peptide 323–339 bound to
I-Ad class II MHC molecules (39), were transferred
into BALB/c recipient mice and tracked with mAbs specific for CD4 and
the clonotypic TCR (KJ1-26) (37). As previously described,
CD4+, KJ1-26+ DO11.10 T cells were readily
detected by flow cytometry in the lymph nodes 1 day (34, 40) or 13 days
after transfer (Fig. 1
A). Intravenous
injection of OVA peptide was used to induce tolerance (34), and
injection of OVA peptide emulsified in CFA was used as a control for T
cell priming. Following both types of Ag injections, the DO11.10 T
cells expanded in the lymph nodes for several days (34). Most of the
DO11.10 T cells in the tolerized recipients then disappeared such that
by day 12, a population equal in size to the starting population
remained (Fig. 1D). These T cells had equivalent
levels of CD4, but expressed about twofold lower levels of TCR (Fig. 1D) than their counterparts in untreated recipients
(Fig. 1A). As previously reported (34), a
substantially larger population of DO11.10 T cells remained on day 12
in the OVA peptide plus CFA-primed recipients (Fig. 1G). All of the CD4+, KJ1-26+
cells detected came from the DO11.10 donors because these cells were
not detected in untransferred mice, either before (Fig. 1J) or after immunization with OVA peptide (34). The
populations of DO11.10 T cells that survived after day 12 in the OVA
peptide-injected groups had responded to Ag in the past because
they expressed low levels of CD45RB (Fig. 1, E and
H) and, in BrDU-treated recipients, stained positive
for BrDU (Fig. 1, F and I). This activated
phenotype was not observed if the DO11.10 T cells were from recipients
that did not receive Ag (Fig. 1, B and C),
nor was it observed in the CD4+, KJ1-26- cells
of the recipients (Fig. 1, B, E, and
H). Thus, a detectable, activated population of
DO11.10 T cells persisted after a tolerizing injection of OVA peptide,
indicating that deletion of all Ag-specific T cells is not the only
tolerance mechanism operating in this system.
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The ability of soluble Ag to induce a hyporesponsive state
in Ag-specific T cells was confirmed by measuring the capacity of
DO11.10 T cells to accumulate in the lymph nodes following a secondary
challenge with OVA peptide. Recipients that received no injection
(naive), an i.v. injection of OVA peptide (tolerized), or an s.c.
injection of OVA peptide emulsified in CFA (adjuvant primed) were given
an s.c. injection of OVA peptide in IFA 12 to 21 days after the initial
Ag injections. DO11.10 T cells accumulated dramatically in the draining
lymph nodes of previously naive recipients, achieving a peak level on
day 5 that was 30- to 60-fold greater than the starting level (Fig. 2A). In contrast, the
DO11.10 T cells in tolerized recipients achieved a peak response on day
5 that was, at best, 10-fold greater than the level present on the day
of challenge (Fig. 2A). The response in
adjuvant-primed recipients was more difficult to interpret because the
number of DO11.10 T cells present in the lymph nodes was at an elevated
level at the time of challenge. In this case, the challenge injection
caused DO11.10 T cells to accumulate maximally in the draining lymph
nodes on day 3, an earlier time than in the naive or tolerized
cases, but, like DO11.10 T cells from tolerized recipients,
the cells only expanded 10-fold over the already elevated
starting level (Fig. 2A).
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-chain due to
inefficient allelic exclusion (41). Some of these dual TCR-expressing
cells have a memory/activated phenotype (41), probably due to
activation by environmental Ags. The coexistence of DO11.10 T cells
with naive and memory phenotypes and more than one TCR is problematic
for several reasons. First, it is difficult to exclude the possibility
that a tolerizing injection of Ag deleted all of the initially naive
DO11.10 T cells and the surviving population consisted of the double
TCR-expressing cells. Second, it is possible that tolerance induction
depended on interactions between the naive and memory DO11.10 T cells.
To eliminate these possibilities, T cells from DO11.10 BALB/c SCID
mice, which exclusively express the DO11.10 TCR and a naive phenotype
(R. Merica, unpublished observation), were used for adoptive transfer.
As shown in Figure 2
B, a population of DO11.10 SCID T cells
was clearly present in recipients injected 12 days earlier with soluble
Ag, and these cells expressed less surface TCR than naive cells. Thus,
it is unlikely that dual TCR-expressing DO11.10 T cells account for the
surviving T cell population following tolerance induction. Furthermore,
like normal DO11.10 T cells, DO11.10 SCID T cells in tolerized
recipients expanded poorly in the draining lymph nodes following
secondary challenge with OVA peptide in IFA (Fig. 2C), demonstrating that initially naive DO11.10 T
cells can be tolerized in the absence of memory DO11.10 T cells. The in vivo accumulation defect of tolerized DO11.10 T cells is reversible
In several other cases, peripheral T cell tolerance was reversed
in the absence of the relevant Ag (17, 23, 42). To address this issue
in our system, we examined the clonal expansion of DO11.10 T cells from
tolerized recipients in response to Ag plus adjuvant challenge at
various times after the initial i.v. injection of OVA peptide. As shown
in Figure 3, A and
B, DO11.10 T cells expanded poorly in response to a
secondary challenge 12 to 23 days after the tolerizing injection of OVA
peptide, as compared with DO11.10 T cells from naive animals. In
contrast, DO11.10 T cells from naive or tolerized recipients expanded
similarly (Fig. 3, A and B) in response to
Ag challenge 49 to 75 days after the tolerizing injection. However, the
DO11.10 T cells remained in a hyporesponsive state in vivo if OVA
peptide was injected once per week until challenge at day 49 (Fig. 3B). Thus, the persisting DO11.10 T cells recovered
from the in vivo clonal expansion defect unless there was continuous
administration of the Ag.
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A). As shown
in Figure 4B, the DO11.10 T cells became blasts when
transferred into mice, 3 or 5, but not 10 days after the mice received
OVA peptide i.v. at the dose used in the tolerance experiments.
Therefore, based on this assay, stimulatory OVA
peptide/I-Ad complexes disappeared from the animals
sometime between 5 and 10 days after i.v. injection of OVA peptide.
Thus, disappearance of stimulatory OVA peptide/I-Ad
complexes preceded reversal of the in vivo clonal expansion defect in
the DO11.10 T cells, consistent with the idea that Ag persistence was
required for maintenance of the hyporesponsive state.
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The failure of DO11.10 T cells from tolerized recipients to
accumulate efficiently in lymphoid tissues following a second antigenic
challenge could be related to decreased proliferation, as would be
expected if they were functionally impaired, or to death of
proliferating cells. To differentiate between these two possibilities,
naive recipients, or recipients that were tolerized with one or two
i.v. injections of OVA peptide were challenged 2 wk after the second
i.v. injection with OVA peptide in CFA, and given BrDU until the time
of sacrifice. If tolerized DO11.10 T cells proliferated in vivo at the
same rate as naive DO11.10 T cells, but died at a higher rate, then the
surviving cells from the two groups would be expected to have
incorporated equal amounts of BrDU following Ag challenge. This was not
the case, because 58 h after challenge, 73% of the DO11.10 T
cells in previously naive recipients incorporated BrDU (Fig. 5A), whereas only 32.5
and 13.6% of the DO11.10 cells in recipients that were tolerized with
one or two i.v. injections of OVA peptide, respectively, incorporated
BrDU (Fig. 5, C and E). Therefore, the
poor in vivo accumulation of DO11.10 T cells in the lymph nodes of
tolerized mice correlated with reduced DNA synthesis. An increase in
the frequency and total number of BrDU+, DO11.10 T cells
was observed in both tolerized and naive recipients 5 days after
challenge (Fig. 5, B, D, and
F), the time of peak DO11.10 T cell accumulation
(Figs. 1, 2, and 5). However, the total number of BrDU+,
DO11.10 T cells present in tolerized mice was three- to sixfold lower
than in naive recipients, consistent with a model in which the
tolerized T cells do not proliferate as efficiently as their naive
counterparts.
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If the surviving DO11.10 T cells in tolerized recipients were
functionally impaired, then the reduced proliferation of DO11.10 T
cells in tolerized recipients following secondary challenge would be
expected to be associated with reduced T cell growth factor production.
Lymph node cells were taken from naive, tolerized, or adjuvant-primed
recipients, 12 days after Ag injection, and assayed for IL-2 production
following in vitro culture with OVA peptide (Fig. 6A) to address this
issue. IL-2 production was mainly dependent on the presence of the
transferred DO11.10 T cells, since little or no IL-2 was detected if
KJ1-26+ T cells were depleted from the lymph node cell
suspensions before culture (Fig. 6A). This finding
allowed for estimation of the amount of IL-2 produced per DO11.10 T
cell present at the time of culture initiation (Fig. 6D). In the lymph node cultures from naive
recipients, a significant amount of IL-2 was detected after the first
24 h, and the level consistently increased with each additional
day of culture. Cultures from adjuvant-primed recipients contained the
highest level of IL-2 after the first day, and the level of IL-2 did
not increase thereafter, potentially due to consumption. Cultures from
tolerized recipients differed, in that they did not contain detectable
IL-2 after the first day, and contained 10- to 20-fold lower levels of
IL-2 over the next 2 days than did cultures from naive recipients. In
contrast, when lymph node cells were placed in culture 3 days after a
tolerizing injection of OVA peptide (Fig. 6, B and
E), the pattern of IL-2 production was identical to
that observed in cultures from adjuvant-primed recipients. Finally,
when lymph node cells were placed in culture 59 days after a tolerizing
injection of OVA peptide, the pattern of IL-2 production in cultures
from tolerized recipients was identical to that observed in cultures
from naive recipients (Fig. 6, C and F).
Little or no IL-2 was detected from any of the groups when the lymph
node cells were cultured without Ag (data not shown). Together, these
results show that Ag-specific T cells acquire an IL-2 production defect
about 1 wk after i.v. injection of Ag, which is lost 8 wk later.
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). An i.v. injection of OVA
peptide was used to facilitate synchronous Ag presentation during
challenge. When naive recipients were injected with OVA peptide, about
15 and 30% of the DO11.10 T cells in the lymph nodes and spleen,
respectively, contained intracellular IL-2 6 h later (Fig. 7A, lower left panel, and Fig. 7B). If CD4+, KJ1-26- cells
were examined (Fig. 7A, right panels, and Fig. 7B), or if the recipients were not challenged with Ag
(Fig. 7A, upper left panel), less than
0.5% of the cells contained intracellular IL-2, demonstrating that
IL-2 production could only be detected in activated DO11.10 T cells.
Similar to DO11.10 T cells in naive recipients, 10 to 30% of the
DO11.10 T cells in the lymph nodes and spleen from adjuvant-primed
recipients expressed intracellular IL-2 following an in vivo challenge
with OVA peptide (Fig. 7B). However, less than 4% of
the DO11.10 T cells from the lymph nodes or spleen of tolerized
recipients expressed intracellular IL-2 following challenge (Fig. 7B). A similar defect was found for the production of
TNF-. Less than 4% of the DO11.10 T cells from tolerized
recipients, but about 12% of the DO11.10 T cells from naive or
adjuvant-primed recipients expressed intracellular TNF- in response
to in vivo antigenic challenge (Fig. 7B).
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It was possible that the loss of IL-2 and TNF- production in
DO11.10 T cells from tolerized recipients was related to immune
deviation, a situation in which the T cells acquire the ability to
produce potentially suppressive Th2 cytokines (31, 32, 43). This was
unlikely, since neither IL-4 nor IL-5 could be detected in the culture
supernatants of OVA peptide-stimulated lymph node cells from tolerized
mice (data not shown). In addition, IL-4 was not detected by
intracellular staining of DO11.10 T cells following an in vivo Ag
challenge of tolerized recipients (data not shown). However, it was
formally possible that i.v. injection of Ag induced the generation of
other regulatory cells or factors that suppressed the function of the
DO11.10 T cells upon in vivo challenge. To address this possibility,
DO11.10 T cells were transferred into untreated BALB/c mice or BALB/c
mice that were given an i.v. injection of OVA peptide 11 days before,
and then challenged the next day with OVA peptide in CFA. As shown in
Figure 8, the DO11.10 T cells expanded
equally well in mice that were untreated or previously given an i.v.
injection of OVA peptide, suggesting that a suppressive environment was
not created by the tolerizing Ag injection.
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, A and
C). Some of these recipients then were injected with
6.5 T cells, and the next day challenged with a mixture of the OVA and
HA peptides in CFA. Tolerance was induced in the recipients of a
previous OVA peptide i.v. injection, because DO11.10 T cells in these
mice expanded only 10-fold, whereas DO11.10 T cells in naive recipients
expanded 30- to 40-fold in the draining lymph nodes following the
challenge injection (Fig. 9, B and C).
However, the bystander population of 6.5 T cells expanded 30-fold in
the absence or presence of naive or tolerized DO11.10 T cells (Fig. 9, B and C), suggesting that stimulation of
tolerized DO11.10 T cells did not suppress the clonal expansion of a
bystander population of 6.5 T cells present in the same lymph node.
Identical results were obtained in an experiment in which the
recipients were challenged with purified dendritic cells that were
pulsed with a mixture of OVA and HA peptides, ensuring that the same
APCs would be presenting Ag to both DO11.10 and 6.5 T cells (data not
shown). The inability of tolerized DO11.10 T cells to suppress the
proliferation of bystander 6.5 T cells was not explained by the fact
that these T cells were incapable of being tolerized, because a prior
i.v. injection of HA peptide into 6.5 T cell recipients resulted in
poor in vivo clonal expansion (Fig. 10A) and poor in
vitro IL-2 production (Fig. 10B) from 6.5 T cells
upon secondary antigenic challenge. It was also notable that the
presence of an expanding population of 6.5 T cells, which presumably
would be producing IL-2, did not improve the clonal expansion of the
tolerized DO11.10 T cells (Fig. 9, B and
C), suggesting that a T cell growth factor response
defect may be involved in the unresponsive state.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although most of the DO11.10 T cells that expanded following injection of adjuvant-free soluble Ag were rapidly depleted from the lymph nodes, probably due to apoptosis (9, 10, 46), a stable population of DO11.10 T cells that displayed functional defects survived for at least 3 wk. In fact, a sizable population of DO11.10 T cells survived for several months after the Ag injection until they recovered their functional capability. However, the recovered DO11.10 T cells did not become activated at the late time points unless challenged with Ag probably because the relevant peptide/MHC molecules derived from the tolerizing Ag injection were no longer present. This finding highlights the fact that recovery of functional unresponsiveness by self Ag-reactive T cells would not necessarily lead to autoimmunity as long as the relevant self Ag is no longer expressed in the periphery.
An alternative explanation for why an unresponsive population of Ag-specific T cells would survive deletion is that the survivors possessed a preexisting activation defect, for example, due to low levels of adhesion molecules or a TCR mutation. This possibility was ruled out, however, by our finding that the majority of unresponsive DO11.10 T cells that persisted after i.v. Ag injection showed signs of prior activation.
The adoptive transfer system studied in this investigation provides direct evidence that not all Ag-specific CD4+ T cells are deleted during the induction of peripheral tolerance by soluble Ag. These findings argue against the idea that functionally impaired T cells are short-lived cells destined to die. A given T cell clone probably has the potential to react to multiple peptides presented in the context of a particular class II MHC molecule (47). Therefore, deleting a clone based on self-reactivity to one peptide would eliminate the ability of the immune system to use that clone to recognize other antigenic peptides. For example, if a single T cell clone was able to recognize both a peptide from human chorionic gonadotrophin, a hormone present only during pregnancy, and a peptide from influenza virus, deleting this T cell clone during pregnancy would partially compromise the ability of the immune system to respond to influenza virus. Thus, it would be advantageous if the immune system could simply silence a self-reactive T cell clone for the time that self Ag is expressed in the periphery.
DO11.10 T cells in tolerized animals did not accumulate efficiently in the lymph nodes following challenge with Ag. The finding that few BrDU+, DO11.10 T cells were detected 58 h after challenge suggests that a lack of proliferation, and not death of proliferating cells, is involved. However, the fact that the small number of DO11.10 T cells that accumulated in the lymph nodes 5 days after challenge were mostly BrDU+ suggests that tolerized DO11.10 T cells can proliferate to some extent in response to Ag. Because BrDU staining cannot detect the difference between one and multiple rounds of DNA synthesis, the presence of BrDU+ DO11.10 T cells following antigenic challenge of tolerized mice is consistent with the possibility that all of the DO11.10 T cells became hyporesponsive to some degree and underwent fewer rounds of DNA synthesis than naive cells. An alternative explanation would be that there was an outgrowth of a small subpopulation of DO11.10 T cells that escaped tolerance induction, and thus were able to proliferate normally in response to Ag. The double TCR-expressing memory cells from normal DO11.10 TCR transgenic mice (41) are unlikely to account for a population that escaped tolerance induction, since residual proliferation following Ag challenge of tolerized recipients was also noted when DO11.10 SCID T cells were used for adoptive transfer. However, these studies have not eliminated the possibility that a small number of the transferred transgenic T cells do not encounter an OVA peptide/MHC-bearing APC during the period of tolerance induction, and thus remain in a responsive state.
The reduced capacity of DO11.10 T cells from tolerized mice to
proliferate in response to antigenic stimulation was associated with
impaired production of IL-2 and TNF-, suggesting that an intrinsic
defect in T cell growth factor production was responsible as in the
case of Th1 clones (16). The DO11.10 T cells in tolerized mice
expressed about twofold lower levels of TCR than DO11.10 T cells from
naive mice. In addition, in some experiments the level of TCR
expression increased as the cells recovered their ability to respond to
Ag (K. A. P., unpublished observation). It is therefore
possible that the reduction in TCR expression was responsible for the
functional defects. However, it is difficult to believe that this
reduction is functionally significant given the low number of
surface TCRs that need to be engaged for signaling to occur (48, 49). Indeed, our finding that in vivo persistence of Ag was required to
maintain unresponsiveness suggests that the TCRs on tolerized DO11.10 T
cells must be capable of transducing some signals. It is thus more
likely that the tolerized DO11.10 T cells have a selective
TCR-signaling defect, such that the signals required to maintain the
unresponsive state are preserved, but those required for transcription
of lymphokine genes are not. A precedent for this situation has
recently been reported for unresponsive T cell clones, in which
TCR-mediated calcium mobilization occurs, but activation of certain
kinases does not (50, 51, 52).
It should be noted that the DO11.10 T cells did not manifest a lymphokine production defect at early times after administration of the tolerizing Ag. The time lag required for the defect to become apparent could be explained by delayed production of a repressor protein, as postulated for T cell clones (53). Alternatively, the population of DO11.10 T cells present at the peak of clonal expansion could be comprised of a predominant population of effector cells, which are not unresponsive, and a minor subpopulation, which is already unresponsive. If the effector cells are short-lived, as proposed by Swain et al. (54), and the unresponsive cells are long-lived, then the unresponsive phenotype would only become apparent as the effector cells died.
It was possible that the in vivo proliferation defect of the DO11.10 T cells in tolerized mice was associated with immune deviation or suppression. However, DO11.10 T cells from tolerized mice did not produce detectable levels of the Th2 cytokines, IL-4 and IL-5. Furthermore, naive DO11.10 T cells that were transferred into tolerized mice, which would be expected to contain the putative suppressive environment, responded normally following immunization. Finally, DO11.10 T cells from tolerized mice did not suppress the clonal expansion of activated bystander T cells within the same lymph nodes. Thus, there is no evidence that suppressive mechanisms contribute to the defects in IL-2 production and in vivo clonal expansion of the tolerized D011.10 T cells. In addition, the finding that the clonal expansion defect of unresponsive DO11.10 T cells was not corrected by T cell growth factors provided by bystander 6.5 T cells responding in the same lymph node suggests that this form of tolerance would not be easily broken by concomitant responses to irrelevant Ags.
Results from several sources are now consistent with the idea that the induction of peripheral T cell tolerance is dependent on an initial phase of T cell activation (8, 9, 10, 11, 34, 55). We have shown that DO11.10 T cells produce IL-2 early after injection of adjuvant-free soluble Ag and proliferate vigorously in the lymphoid tissues for several days (34, 56). Abbas and coworkers recently reported that blockade of the CD28-B7 pathway prevents this Ag-dependent clonal expansion and results in phenotypically naive T cells that are fully responsive to antigenic stimulation at later times (55). Furthermore, they showed that blockage of CTLA-4 converted an Ag injection that normally induces tolerance into one that induces priming. Together these results suggest that CD28-dependent activation and CTLA-4 engagement are actually required for in vivo peripheral tolerance to occur. This was not predicted by the in vitro model, in which a lack of CD28 signaling improves the induction of unresponsiveness in Th1 clones (57, 58). Thus, although the unresponsive T cells that survive in tolerized mice in vivo have some of the same lymphokine production defects as anergic Th1 clones, these T cells may enter this state via different mechanisms.
The DO11.10 T cells that survive in mice tolerized with Ag alone, or
primed with Ag plus adjuvant both show signs of prior activation, and
yet only the T cells from primed mice are rapid and potent lymphokine
producers. The simplest explanation for the functional differences is
that Ag presentation in the presence of adjuvant-induced inflammation
is required for naive T cells to become functional memory cells.
Inflammation enhances APC expression of B7 molecules, which might give
CD28 a competitive advantage over the negative regulator CTLA-4 (59),
allowing for enhanced T cell clonal expansion instead of tolerance.
Inflammation also induces cytokines (60, 61, 62, 63, 64, 65) that promote T cell
survival and acquisition of the capacity to produce lymphokines such as
IL-4 and IFN- (66, 67). In the absence of inflammation, Ag would be
presented by APC expressing low levels of B7, a situation in which only
transient clonal expansion occurs and negative regulation by CTLA-4 may
generate T cells that have lost the ability to produce IL-2 and have
not gained the capacity to respond to growth factors or produce IL-4 or
IFN-. The necessity for inflammation to produce the appropriate
environment for effective T cell responses would be a viable way to
limit the function of T cells specific for neo-self Ags that appear
late in life and facilitate the function of T cells specific for
microbial Ags (29, 68), which would always enter the system with an
adjuvant.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Marc K. Jenkins, University of Minnesota Medical School, Department of Microbiology and Center for Immunology, Box 334 UMHC, 420 Delaware Street S.E., Minneapolis, MN 55455.
3 Abbreviations used in this paper: HA, hemagglutinin; BrDU, 5-bromo-2'-deoxyuridine; PE, phycoerythrin.
Received for publication November 19, 1997. Accepted for publication January 12, 1998.
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R. Wang, Y. Wang-Zhu, C. R. Gabaglia, K. Kimachi, and H. M. Grey The Stimulation of Low-Affinity, Nontolerized Clones by Heteroclitic Antigen Analogues Causes the Breaking of Tolerance Established to an Immunodominant T Cell Epitope J. Exp. Med., October 4, 1999; 190(7): 983 - 994. [Abstract] [Full Text] [PDF]
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D. C. Tsitoura, R. H. DeKruyff, J. R. Lamb, and D. T. Umetsu Intranasal Exposure to Protein Antigen Induces Immunological Tolerance Mediated by Functionally Disabled CD4+ T Cells J. Immunol., September 1, 1999; 163(5): 2592 - 2600. [Abstract] [Full Text] [PDF]
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 C. A. Chambers, M. S. Kuhns, and J. P. Allison Cytotoxic T lymphocyte antigen-4 (CTLA-4) regulates primary and secondary peptide-specific CD4+ T cell responses PNAS, July 20, 1999; 96(15): 8603 - 8608. [Abstract] [Full Text] [PDF]
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R. Blackstock, K. L. Buchanan, Adekunle M. , Adesina, and J. W. Murphy Differential Regulation of Immune Responses by Highly and Weakly Virulent Cryptococcus neoformans Isolates Infect. Immun., July 1, 1999; 67(7): 3601 - 3609. [Abstract] [Full Text] [PDF]
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C. A. Blish, S. R. Dillon, A. G. Farr, and P. J. Fink Anergic CD8+ T Cells Can Persist and Function In Vivo J. Immunol., July 1, 1999; 163(1): 155 - 164. [Abstract] [Full Text] [PDF]
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J. Sun, B. Dirden-Kramer, K. Ito, P. B. Ernst, and N. Van Houten Antigen-Specific T Cell Activation and Proliferation During Oral Tolerance Induction J. Immunol., May 15, 1999; 162(10): 5868 - 5875. [Abstract] [Full Text] [PDF]
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J. M. Benson, S. S. Stuckman, K. L. Cox, R. M. Wardrop, I. E. Gienapp, A. H. Cross, J. L. Trotter, and C. C. Whitacre Oral Administration of Myelin Basic Protein Is Superior to Myelin in Suppressing Established Relapsing Experimental Autoimmune Encephalomyelitis J. Immunol., May 15, 1999; 162(10): 6247 - 6254. [Abstract] [Full Text] [PDF]
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H. Gudmundsdottir, A. D. Wells, and L. A. Turka Dynamics and Requirements of T Cell Clonal Expansion In Vivo at the Single-Cell Level: Effector Function Is Linked to Proliferative Capacity J. Immunol., May 1, 1999; 162(9): 5212 - 5223. [Abstract] [Full Text] [PDF]
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C. A. London, V. L. Perez, and A. K. Abbas Functional Characteristics and Survival Requirements of Memory CD4+ T Lymphocytes In Vivo J. Immunol., January 15, 1999; 162(2): 766 - 773. [Abstract] [Full Text] [PDF]
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A. J. Zajac, J. N. Blattman, K. Murali-Krishna, D. J.D. Sourdive, M. Suresh, J. D. Altman, and R. Ahmed Viral Immune Evasion Due to Persistence of Activated T Cells Without Effector Function J. Exp. Med., December 21, 1998; 188(12): 2205 - 2213. [Abstract] [Full Text] [PDF]
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