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Immunologie Cellulaire, Laboratoire de Biologie Moléculaire et Cellulaire, Ecole Normale Supérieure de Lyon Centre National de la Recherche Scientifique, Lyon, France; and
Laboratoire de Physiologie Animale, Université Libre de Bruxelles, Brussels, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and CD8
. Together these data indicate that
repeated exposure to soluble antigenic peptide in vivo can induce a
state of functional tolerance characterized by defective TCR signaling,
impaired proliferation, and increased sensitivity to cell
death. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The incidence of autoimmune diseases and the development of organ transplants have highlighted the need for protocols aimed at inducing a state of peripheral T cell tolerance. This can be achieved following in vivo treatment with Abs directed against TCR/CD3 complex components (23, 24, 25, 26), Abs to CD4 or CD8 coreceptor molecules (27), or superantigens (28, 29, 30, 31). However, these protocols lead to partial or total immunosupression. The induction of tolerance to a given Ag has therefore been investigated as a means of blocking specific responses without affecting the entire peripheral T cell pool. Peripheral tolerance to foreign antigenic peptides recognized by CD4 T cells can be established after exposure to soluble Ags administered orally (15, 16) or systemically (32, 33, 34, 35). While oral administration of Ag leads to TGF-ß-mediated immunosuppression (15, 16), systemic exposure to Ag is thought to load APCs without promoting their maturation and expression of costimulatory molecules. Deletion often accounts for the lack of specific T cell responses after treatment with polyclonal activators or antigenic peptides in vivo (30, 33), but survival of CD4 T cells that have become unresponsive to Ag in vivo has also been described (35).
Studies performed on CD4 T lymphocytes in vitro have indicated that induction of unresponsiveness or anergy leads to the inability of these cells to produce IL-2 and/or proliferate upon restimulation (36, 37, 38). Anergy induction also leads to alterations in the early signal transduction events that follow Ag receptor engagement. These include altered regulation of the inositol phosphates and calcium metabolism (39, 40), constitutive Fyn kinase activity (41), a partial block in TCR/CD3-induced tyrosine phosphorylation (39, 40), and diminished activation of the signaling pathways located downstream of ras (42, 43).
For CD8 T cells, multiple systemic exposures or high doses of Ag induce tolerance in vivo (12, 44, 45, 46). However, the mechanisms responsible for tolerance of specific CD8 T cells remain poorly understood. Deletion of Ag-specific T cells (12, 20) and down-regulation of TCR surface levels (14) have both been observed in TCR transgenic systems after tolerance induction with exogenous Ags. There is also evidence indicating that a subset of the CD8 T cells is rendered anergic after tolerance induction. These cells can survive in the absence of Ag and recover responsiveness over time (47).
We have previously shown that a single injection of soluble antigenic peptide induces a population of long-lived CD8 T cells that is hyper-reactive to Ag in vitro (48). In the present study we have used the F5 TCR transgenic system (49) to characterize the CD8 T cells that survive a tolerance induction protocol consisting of multiple exposures to soluble antigenic peptide. Our data show that 7 days after the last of three peptide injections administered at 4-day intervals, surviving CD8 T cells proliferate poorly in vitro and in vivo and have lost the ability to differentiate into cytotoxic effectors after in vitro stimulation in the absence of exogenously added cytokines. In addition, the tolerant F5 CD8 T cells are highly sensitive to cell death in vitro. Analysis of early signal transduction pathways revealed that these cells also had constitutively elevated cytoplasmic calcium levels and impaired early signal transduction in response to co-cross-linking of CD3 with CD8.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/10, F5, RAG-1-/-F53 TCR transgenic and RAG-1-/- mice were gifts from D. Kioussis (49). All animals were bred in the institute’s animal facility. Immunizations were performed by injecting 50 nmol of the A/NT/60/68 influenza virus nucleoprotein peptide Ala-Ser-Asn-Glu-Asn-Met-Asp-Ala-Met (NP366–374; Neosystems Laboratoire, Strasbourg, France) in PBS into the peritoneal cavity. Control mice either were not treated or were injected with PBS alone.
Cell culture and proliferation assays
Spleen and lymph node T cells were cultured in RPMI 1629 (Life Technologies, Gaithersburg, MD) supplemented with 2 mM L-glutamine (Life Technologies), 10 µg/ml gentamicin (Life Technologies), 6% FCS (Boehringer Mannheim, Mannheim, Germany), and 5 x 10-5 M ß-ME. F5 transgenic T cells (5 x 104 cells/well) were activated in 96-well plates with graded doses of peptide and 2 x 105 irradiated (3000 rad) C57BL/10 spleen cells and in the presence or the absence of 5% cell culture supernatant containing IL-2. The final IL-2 content ranged from 100–150 U/ml. On day 2, cells were pulsed for 16 h with 0.5 µCi of [3H]thymidine/well (2.0 Ci/mmol; Amersham, Aylesbury, U.K.) and were harvested on day 3.
Cytotoxicity assays
F5 TCR transgenic CD8 T cells (1.5 x 105 cells/well) were activated for 3 days in 24-well plates with 10 nM NP peptide and 2 x 106 irradiated C57BL/10 spleen cells in the presence or the absence of 5% cell culture supernatant containing IL-2 (100–150 U/ml). After 3 days in culture, live T cells were isolated by Ficoll-Hypaque (Lympholyte M, Cedarlane Laboratories, Hornby, Canada) gradient centrifugation. Target P815 cells expressing a transfected H-2Db molecule (P815-Db) were pulsed with 1 mM NP peptide for 3 h and further incubated with 51Cr for 1 h at 37°C. F5 transgenic T cells and targets were plated at E:T cell ratios ranging from 30/1 to 0.1/1 in round-bottom 96-well plates and incubated at 37°C in 5% CO2 for 4 h. Results are expressed as the percent lysis as a function of the E:T cell ratio. The percent lysis was calculated using the following equation: % lysis = 100 x test 51Cr released - spontaneous 51Cr released/maximum 51Cr released - spontaneous 51Cr released, where spontaneous release was determined using 51Cr-labeled targets in the absence of effectors and maximum release after treatment of 51Cr-labeled targets with 2% Triton X-100.
Fluorescence staining and flow cytometry analysis
Spleen cell suspensions were applied to Ficoll-Hypaque (Lympholyte M, Cedarlane Laboratories) gradient centrifugation. Cells were then washed twice with medium and resuspended in PBS containing 2% horse serum and 0.1% NaN3, and 106 cells were incubated with Ab for 45 min at 4°C. After two more washes with PBS/horse serum/NaN3, cells were incubated in the presence of second layer reagents (avidin-phycoerythrin or avidin-Tricolor, Caltag, Burlingame, CA) for 30 min at 4°C. Cells were then washed twice and analyzed on a FACScan (Becton Dickinson, Mountain View, CA). The Abs YTS169.4-PE and YTS169.4-FITC (anti CD8, Caltag Laboratories, Burlingame, CA), RR3-15-biotin (anti-Vß11), 145–2C11-FITC (anti-CD3), and 145-2C11-biotin were prepared in the laboratory.
Labeling of cells with CFSE
The method used for labeling cells with CFSE is a modification of the protocol described by Weston and Parish (50). Briefly, T cells were resuspended at a concentration of 1–5 x 107/ml in fully supplemented growth medium before the addition of CFSE at a final concentration of 10 mM. Cells were then incubated for 10 min at 37°C, and the reaction was stopped by adding 5 vol of ice-cold medium. After two washes, CFSE-labeled cells were resuspended in growth medium for in vitro culture or in PBS for i.p. injection into C57BL/10 recipients. To achieve higher levels of CFSE fluorescence, RAG-1-/-F5 and F1(RAG-1-/-F5 x RAG-1-/-) T cells were labeled with CFSE for 13 min instead of 10 min for some in vivo assays.
Analysis of intracellular calcium mobilization
This method has been described in detail previously (51). Total
lymph node cells were loaded at a final concentration of
107/ml in fully supplemented RPMI 1629 containing the
acetomethylester form of indo-1 at a concentration of 2 mM and 0.2%
F127 (Molecular Probes, Junction City, OR). Loading was conducted at
37°C for 40 min in the dark. Cells were then washed, resuspended in
fully supplemented medium, and stained at 4°C with PE-coupled Abs
directed against CD4 (YTS 191.1, Caltag) and B220 (Sigma, St. Louis,
MO). Analysis was performed on a Becton Dickinson FACS-Star Plus. The
UV excitation (351–364 nm) was provided by an argon laser; blue
emission was detected at 480–520 nm, and violet emission was detected
at 383–407 nm. Cells that were negative for PE staining were >95%
CD8+. For activation, samples of 2 x 106
LN cells were incubated for 5 min at 37°C with homemade biotinylated
mAbs to CD3 (mAb 145 2C11) and CD8 (YTS169.4).
Avidin was added 2 min after the beginning of the assay. Results are expressed as the mean indo ratio over time in the CD4-B220- population.
Tyrosine phosphorylation assays
CD8 lymph node T cells were purified with magnetic beads using a
negative selection strategy. Briefly, total lymph node cells were
incubated with anti-CD4 (GK1.5) and anti-B220 (RA3 6B2)
hybridoma culture supernatants for 45 min at 4°C. Cells were then
washed three times and incubated for 30 min at 4°C with
magnetic beads coupled to goat anti-rat IgG (H & L)
(Biomag, Perseptive Diagnostics, U.K.) at a ratio of 15
beads/cell. As determined by flow cytometric analysis, CD8 T
lymphocytes represented 90–95% of the cells that remained after
magnetic depletion. Samples of 1 x 107 CD8 T cells
were incubated at room temperature with biotinylated mAbs to CD3
(mAb 145 2C11) and CD8 (YTS169.4) for 5 min. After a 3-min
incubation with avidin at 37°C, cells were rapidly pelleted and lysed
for 15 min in 250 µl of ice-cold 1% Nonidet P-40, 0.25%
deoxycholate, 150 mM NaCl, 50 mM Tris (pH 7.5), 0.5% aprotinin, and 1
mM PMSF buffer containing 2 mM sodium vanadate. Lysates were cleared at
14,000 rpm for 10 min. Proteins were then separated by electrophoresis
on 4–20% SDS-acrylamide gradient gels (Novex, San Diego, CA) and
electroblotted onto polyvinylidene difluoride membranes (Bio-Rad,
Hercules, CA). Immunoblots were saturated for 1 h with 6% BSA in
Tris-buffered saline and incubated for 18 h with 0.75 µg/ml of a
rabbit anti-phosphotyrosine antiserum (Zymed, San Francisco, CA) in
6% BSA in TBS. Detection was performed using pig anti-rabbit
peroxidase Abs (Dako, Copenhagen, Denmark) followed by enhanced
chemiluminescence.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Multiple systemic exposures to a soluble viral antigenic peptide
has been shown to induce tolerance of CD8 peripheral T cells (44, 45, 46, 52). We first sought to determine how this tolerance induction protocol
affected the peripheral CD8 T cell pool in F5 TCR transgenic mice. F5
mice were given three injections of the cognate influenza virus
NP366–374 peptide at 4-day intervals. Seven days after the
last peptide injection, the number of F5 CD8 T cells that could be
recovered in the lymph nodes represented at least 30% of that in
control animals. A subtle decrease in the expression levels of the
transgenic TCR Vß11 chain, CD3, and CD8 could be detected on
the surface of CD8 T cells stimulated in vivo (Fig. 1
A). A survey of surface
molecules known to be up-regulated after in vivo T cell stimulation
revealed that CD44 and Ly-6C were the only markers that were
up-regulated on the surface of CD8 T cells stimulated 7 days before
analysis with either single or multiple peptide injections (data not
shown). When cells stimulated multiple times in vivo were cultured in
vitro in the presence of irradiated syngenic spleen cells and graded
doses of Ag, they proliferated poorly compared with control populations
(Fig. 1B) or CD8 T cells primed by a single peptide
injection (data not shown). Similar results were obtained when
proliferation was measured at different times after in vitro
stimulation (data not shown). Proliferative responses were partially
restored if tolerant F5 CD8 T cells were stimulated in the presence of
exogenously added IL-2 (Fig. 1B). These results indicated
that multiple peptide injections in vivo rendered F5 CD8 T cells
hyporesponsive to peptide stimulation in vitro.
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show that tolerant T cells failed to become efficient CTL. However, in
the presence of IL-2, tolerant T cells were able to differentiate into
cytotoxic effector cells. Based on the hyporesponsive phenotype
described above, F5 CD8 T cells stimulated by multiple peptide
injections in vivo will be called tolerant.
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A number of mechanisms could account for the poor peptide-specific
proliferative responses of tolerant T cells observed in vitro. These
include a lower frequency of peptide-responsive cells, an increased
rate of cell death, and slow or lack of proliferation in response to Ag
in vitro. To follow the fate of tolerant CD8 T cells in vitro, we have
used the fluorescent dye CFSE, which allows tracking of cell divisions
using flow cytometry. Divisions could be detected as early as 40 h
of in vitro stimulation in both control and tolerant CD8 T cell
populations and at peptide doses ranging from 0.15–5 nM (data not
shown). After 3 days in culture in the presence of 5 and 1.5 nM
peptide, several divisions could be detected in both control and
tolerant F5 CD8 T cell populations (Fig. 3A). However, the number of
cell divisions accomplished by activated tolerant T cell populations
after 3 days in culture was lower than that observed in control F5 CD8
T cells. Moreover, at this time point, the number of viable tolerant
CD8 T cells was significantly lower than that in control samples. At
low Ag doses (0.5 and 0.15 nM) and after 3 days in culture, these
numbers were consistently 10–20 times lower in tolerant T cell
populations than in control samples. By comparison, F5 CD8 T cells
stimulated by a single peptide injection 7 days before analysis
performed the same number of divisions as naive cells (Fig. 3B). Together, these data show that when tolerant F5 CD8 T
cells were cultured in vitro they performed fewer divisions in response
to peptide and were more sensitive to cell death compared with naive
populations.
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To determine whether hyporesponsiveness of tolerant CD8 T cells
was also detectable in vivo, control and tolerant CD8 T lymphocytes
were labeled with the fluorescent dye CFSE and injected into C57BL/10
recipients. Three days later, recipient mice were injected with
antigenic peptide, and CD8 T cell divisions were monitored 72 h
later. In these assays CD8 T cells from both F5 and
RAG-1-/-F5 mice were used. As shown in the
following section, multiple peptide injections into
RAG-1-/-F5 mice also induced a state of in
vitro hyporesponsiveness (see Fig. 5). The data presented in Fig. 4 show that while the overall majority of
naive RAG-1-/-F5 CD8 T cells recovered from the
lymph nodes of recipient mice were capable of dividing in response to
antigenic challenge (Fig. 4A), only a minor fraction of the
tolerant T cells appeared to have done so (Fig. 4B). As a
further indication of the diminished proliferation of tolerant CD8 T
cells, we found that there was no increase in the percentage of
CFSE-labeled lymph node CD8 T cells following peptide injection. In
contrast, control CFSE-labeled CD8 T cell populations increased three-
to sevenfold under the same experimental conditions (Fig. 4, A and B, and data not shown). This was not
dependent on the RAG-1-/- background, since similar
observations were made when tolerant F5 CD8 T cells were injected into
C57BL/10 mice (data not shown).
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Role of cell surface transgenic TCR expression levels in tolerant CD8 T cell response
The data presented above indicate that multiple injections of an
agonist peptide in vivo lead to hyporesponsiveness in vitro and in
vivo. Expression of a second TCR -chain has been described in mouse
T cells (53, 54). Since a clonotypic Ab is not available in the F5
system, we could not rule out the possibility that the F5 CD8 T cells
that survived this tolerance induction protocol were, in fact, cells
that did not fully respond to the antigenic peptide. It was thus
conceivable that multiple peptide injections selected cells that
expressed lower levels of the transgenic F5 TCR -chain. F5 mice
deficient for the RAG-1 enzyme were therefore used to determine whether
such cells contributed to the unresponsiveness to peptide challenge in
vitro. In these mice, only the transgenic TCR is expressed on the T
cell surface. RAG-1-/-F5 CD8 T cells that
survived multiple peptide stimulations in vivo expressed lower levels
of Vß11 on the cell surface (Fig. 5
A). This down-regulation of
Vß11 expression was more pronounced than that seen in the F5 CD8 T
cells and corresponded to a twofold decrease (see Fig. 1). As observed
in F5 peripheral CD8 T cells, a small but reproducible decrease in
CD8 surface expression was also detected in
RAG-1-/-F5 T cells stimulated by multiple
peptide injections in vivo (Fig. 5A). As seen using F5 CD8 T
cells, RAG-1-/-F5-tolerant CD8 T cells
proliferated poorly in response to graded doses of peptide in vitro
(Fig. 5B) and recovered some proliferative capacity when
cultured in the presence of IL-2. These results indicated that CD8 T
cells expressing only the F5 transgenic TCR can also be rendered
hyporesponsive to peptide restimulation in vitro. In addition, they
also show that other mature lymphocyte populations had no impact on
tolerance induction of F5 CD8 T cells.
To evaluate the contribution of the small TCR down-regulation to the
tolerant phenotype, RAG-1-/-F5 mice were
crossed with RAG-1-/- mice. The F1 progeny
carry half as many copies of the TCR and -ß transgenes. This
translates into cell surface TCR Vß11 levels half those seen in
RAG-1-/-F5 CD8 T cells and are also comparable
to those seen in tolerant RAG-1-/-F5 CD8 T
cells (Fig. 5A). However, despite lower cell surface levels
of TCR, F1 (RAG-1-/-F5 x
RAG-1-/-) CD8 T cells responses to peptide were similar
to those of control RAG-1-/-F5 CD8 T cells in
vitro as measured using thymidine incorporation (Fig. 5B).
Similar observations were made when comparing
RAG-1-/-F5 and F1
(RAG-1-/-F5 x RAG-1-/-)
CD8 T cell responses to peptide using CFSE-labeled cells transfered
into C57BL/10 recipients (Fig. 5C).
These results argue that lower levels of cell surface transgenic TCR cannot alone account for impaired proliferative responses in the CD8 T cell population that has been stimulated by multiple peptide injections in vivo.
Multiple exposures to antigenic peptide in vivo lead to a partial block of TCR signal transduction
T cell activation using partial agonist peptides (55),
superantigens (56, 57), and anti-CD3 Abs (26, 39) in vitro or in
vivo induces a state of T cell unresponsiveness that has been linked to
alterations in TCR signal transduction. They include increased baseline
activity of Fyn (42), inhibition of Ag receptor-mediated early tyrosine
phosphorylation events (26, 40, 41), and diminished intracellular
calcium mobilization (40, 41). As multiple injections of peptide in
vivo lead to inhibition of F5 CD8 T cell proliferation in vitro, we
investigated whether hyporesponsiveness correlated with early TCR
signaling defects. CD8+ T cells from F5 mice injected three
times with antigenic peptide were tested for their ability to respond
to co-cross-linking of CD3 with CD8. In these experiments, F5 CD8 T
cells were used because down-regulation of CD3 was less pronounced
than that seen in the RAG-/- background. In tolerant T
cells, basal levels of intracellular free calcium were higher than
those in control populations (Fig. 6A). In addition,
intracellular free calcium responses (Fig. 6B) and tyrosine
phosphorylation of several cellular substrates (Fig. 7) were both significantly reduced in
tolerant CD8 T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The hyporesponsive phenotype described in this study does not appear to be linked to activation-induced T cell short term desensitization. Indeed, the proliferative responses of CD8 T cells activated in vivo by a single peptide injection 7 days before analysis were identical with those of naive populations. This is in agreement with the observation that a single exposure to peptide administered 10 days before exposure to virus may protect mice from infection with lymphocytic choriomeningitis virus (44). It is also consistent with induction of a memory-like F5 CD8 T cell population after a single exposure to antigenic peptide in vivo (48). In addition, we have found that 7 days after a single peptide injection, calcium mobilization triggered by co-cross-linking of CD3 and CD8 was comparable to that seen in naive populations (data not shown).
A number of mechanisms have been proposed to account for hyporesponsiveness of tolerant peripheral T cells. One of these argues that T cell hyporesponsiveness after tolerance induction results from selection of a pre-existing population that cannot respond to peptide. Our results strongly argue against such a model by providing evidence indicating that hyporesponsiveness affected a population of cells that had responded to the first encounter with peptide. Indeed, using naive CFSE-labeled RAG-1-/-F5 CD8 T cells transferred into C57BL/10 recipients, we have shown that the overall majority of RAG-1-/-F5 CD8 T cells had proliferated 3 days after peptide injection. These data are in agreement with our previous report showing that the bulk of F5 CD8 T cells incorporate bromodeoxyuridine in response to a single peptide injection (48) and make it unlikely that surviving CD8 T cells are derived from a RAG-1-/-F5 CD8 T cells subset that did not respond to Ag.
Another mechanism that could account for hyporesponsiveness in the
surviving Ag-specific T cell population involves a decrease in the
number of cell surface TCR molecules capable of recognizing the
peptide. In the F5 system, this could be the consequence of a second
TCR -chain rearrangement that has been reported in humans (58) as
well as in normal and TCR transgenic mice (53, 54). One consequence of
dual TCR -chain rearrangement is the expression of two Ag receptors.
As a result, the number of cell surface TCRß receptors capable of
recognizing a given Ag may be decreased. Since the ability to transduce
activation signals correlates with TCR surface levels (59) in T cell
clones, responses to limiting doses of Ag may be diminished in T cells
expressing two Ag receptors. In vivo evidence for such a mechanism
comes from studies in transgenic systems in which skin grafts are not
rejected by CD8 T cells expressing lower levels of the Ag receptor (14, 60). In the F5 model, cells expressing a second TCR would be selected
after multiple stimulations in vivo and would become the bulk of the
peripheral CD8 T cell population that survived tolerance induction. The
fact that RAG-1-/-F5 CD8 T cells that cannot
express endogenous TCR -chain could be rendered tolerant argues
against such a mechanism.
Following multiple peptide injections, CD8 T cells from
RAG-1-/-F5 mice show some down-regulation of
the TCR. In some systems, this is thought to be responsible for the
lack of responsiveness of tolerant T cell populations to Ag (14, 60).
However, using T cells that express cell surface levels of TCR Vß11
comparable to those detected on the surface of tolerant
RAG-1-/-F5 CD8 T lymphocytes (see Fig. 5), we
found that this had no influence on the proliferative responses of
naive cells to peptide both in vitro and in vivo. These results show
that mechanisms other than TCR down-regulation are responsible for the
tolerant phenotype of F5 CD8 T cells. Indeed, modifications of TCR
signaling have been demonstrated in anergic CD4 T cells in vitro (39, 40).
In support of such a model, we found that F5 CD8-tolerant T cells displayed fundamental alterations in early TCR signaling pathways. We first detected high levels of intracellular Ca2+, an observation that has been reported in anergic CD4 T cell clones (41). We further demonstrated that tolerant CD8 T cells were partially defective for intracellular signals such as cytoplasmic free calcium increases and tyrosine phosphorylation triggered by TCR/CD3 co-cross-linking with CD8. These results are consistent with previous reports showing that T cells that survive in vivo stimulation with superantigens display altered Ag receptor signaling (56, 57). They also suggest that alterations in intracellular calcium metabolism and TCR signaling contribute to block tolerant CD8 T cell responses in vitro and in vivo. Further analysis will be required to determine whether tolerant CD8 T cells share with anergic CD4 T cell clones increased Fyn activity (41), high Rap-1-GTP content (61), and impaired TCR-mediated activation of ERK and JNK (42, 43).
Using CFSE-labeled cells adoptively transferred into C57BL/10 recipients, we have shown that while the overall majority of naive F5 CD8 T cells did divide in response to peptide, a large fraction of the tolerant T cell population did not proliferate in the recipient’s lymph nodes. These results clearly indicate that 10 days after the last exposure to peptide and 3 days after transfer into an environment free of Ag, a significant proportion of the F5 TCR transgenic CD8 T cells remained unresponsive to peptide in vivo. To determine whether cytokines produced by naive F5 CD8 T cells could drive tolerant T cells to divide in vivo, we also injected tolerant CFSE-labeled F5 CD8 T cells into F5 recipients. In these mice, the large majority of peripheral CD8 T cells are activated and divide in response to the same dose of peptide (39). However, we found no evidence suggesting that tolerance could be reversed under these experimental conditions (data not shown).
Some tolerant CFSE-labeled CD8 T cells were capable of dividing in response to peptide stimulation in C57BL/10 recipients. These cells did not correspond to a subset of CD8 T cells that has escaped tolerance induction in the donor. Indeed, the division profiles that can be detected 3 days after peptide injection in the recipient mice were strikingly different when comparing naive and tolerant F5 CD8 T cells. Activation of naive cells leads to a normal distribution of division peaks, with a maximum at four divisions. By contrast, division peak distribution was not normal when tolerant T cells were analyzed, since the peak corresponding to one division contained the largest number of cells. This observation could mean that tolerant CD8 T cells that divided also migrated rapidly out of the lymph nodes. However, based on our in vitro results showing that CFSE-labeled tolerant T cells were highly sensitive to cell death, it seems more likely that those F5 tolerant T cells that responded to peptide stimulation in vivo died.
By comparing the percentages of control and tolerant CFSE-labeled F5
CD8 T cells detected in the lymph nodes of C57BL/10 recipients
challenged with PBS, we also found that they were consistently lower in
mice injected with tolerant F5 CD8 T cells (data not shown). One
possibility is that the homing of naive and that of tolerant CD8 T
cells are different. However, there is also evidence suggesting that
tolerant T cells may have a shorter life span in vivo. First, T cells
rendered anergic in vivo gradually disappear when parked in a recipient
that does not express the Ag (62). Second, recent reports have shown
that survival of naive TCRß T cells in the periphery is dependent
on an environment expressing the restricting MHC (63, 64, 65). These
findings show that a survival signal relayed by TCR is required for
maintenance of mature T cells in peripheral lymphoid organs. This is
compatible with a model proposed by Raff and co-workers (66) in which
mammalian cells constitutively express proteins involved in triggering
apoptosis, such that survival of these cells would require signals to
inhibit this default death program. Since tolerant F5 T cells are
partially defective for TCR and coreceptor signaling in our system, it
is conceivable that the amount of signal transduced by the Ag receptor
complex and/or CD8 upon interaction with H-2 Db is not
sufficient to ensure survival of these cells in vitro and in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Patrice M. Dubois, Centre National de la Recherche Scientifique-Ecole Normale Supérieure de Lyon, UMR49, 46 allée d’Italie, 69364 Lyon Cedex 07, France. E-mail address:
3 Abbreviations used in this paper: RAG-1, recombination-activating gene-1; PE, phycoerythrin; CFSE, 5-carboxyfluorescein diacetate succinimidyl ester.
Received for publication March 5, 1998. Accepted for publication July 13, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chains on the surface of normal murine T cells. Eur. J. Immunol. 25:1617.[Medline]
chains on the surface of T cells in T cell receptor transgenic mice. J. Exp. Med. 178:1807. chains: dual receptor T cells. Science 262:422.This article has been cited by other articles:
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M. Pihlgren, M. Friedli, C. Tougne, A.-F. Rochat, P.-H. Lambert, and C.-A. Siegrist Reduced Ability of Neonatal and Early-Life Bone Marrow Stromal Cells to Support Plasmablast Survival J. Immunol., January 1, 2006; 176(1): 165 - 172. [Abstract] [Full Text] [PDF]
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W. Ise, K. Nakamura, N. Shimizu, H. Goto, K. Fujimoto, S. Kaminogawa, and S. Hachimura Orally Tolerized T Cells Can Form Conjugates with APCs but Are Defective in Immunological Synapse Formation J. Immunol., July 15, 2005; 175(2): 829 - 838. [Abstract] [Full Text] [PDF]
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W. L. Redmond, B. C. Marincek, and L. A. Sherman Distinct Requirements for Deletion versus Anergy during CD8 T Cell Peripheral Tolerance In Vivo J. Immunol., February 15, 2005; 174(4): 2046 - 2053. [Abstract] [Full Text] [PDF]
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 K. A. Mitchell and B. P. Lawrence Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Renders Influenza Virus-Specific CD8+ T Cells Hyporesponsive to Antigen Toxicol. Sci., July 1, 2003; 74(1): 74 - 84. [Abstract] [Full Text] [PDF]
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K. Asai, S. Hachimura, M. Kimura, T. Toraya, M. Yamashita, T. Nakayama, and S. Kaminogawa T Cell Hyporesponsiveness Induced by Oral Administration of Ovalbumin Is Associated with Impaired NFAT Nuclear Translocation and p27kip1 Degradation J. Immunol., November 1, 2002; 169(9): 4723 - 4731. [Abstract] [Full Text] [PDF]
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C. Ohlen, M. Kalos, L. E. Cheng, A. C. Shur, D. J. Hong, B. D. Carson, N. C.T. Kokot, C. G. Lerner, B. D. Sather, E. S. Huseby, et al. CD8+ T Cell Tolerance to a Tumor-associated Antigen Is Maintained at the Level of Expansion Rather than Effector Function J. Exp. Med., June 3, 2002; 195(11): 1407 - 1418. [Abstract] [Full Text] [PDF]
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 K. Steinbrink, E. Graulich, S. Kubsch, J. Knop, and A. H. Enk CD4+ and CD8+ anergic T cells induced by interleukin-10-treated human dendritic cells display antigen-specific suppressor activity Blood, April 1, 2002; 99(7): 2468 - 2476. [Abstract] [Full Text] [PDF]
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T. Walzer, C. Arpin, L. Beloeil, and J. Marvel Differential In Vivo Persistence of Two Subsets of Memory Phenotype CD8 T Cells Defined by CD44 and CD122 Expression Levels J. Immunol., March 15, 2002; 168(6): 2704 - 2711. [Abstract] [Full Text] [PDF]
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C. Arpin, G. Angelov, T. Walzer, M. Tomkowiak, and J. Marvel Hyperproliferative Response of a Monoclonal Memory CD8 T Cell Population Is Characterized by an Increased Frequency of Clonogenic Precursors J. Immunol., March 1, 2002; 168(5): 2147 - 2153. [Abstract] [Full Text] [PDF]
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K. A. Frauwirth, M.-L. Alegre, and C. B. Thompson CTLA-4 Is Not Required for Induction of CD8+ T Cell Anergy In Vivo J. Immunol., November 1, 2001; 167(9): 4936 - 4941. [Abstract] [Full Text] [PDF]
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R. Maile, B. Wang, W. Schooler, A. Meyer, E. J. Collins, and J. A. Frelinger Antigen-Specific Modulation of an Immune Response by In Vivo Administration of Soluble MHC Class I Tetramers J. Immunol., October 1, 2001; 167(7): 3708 - 3714. [Abstract] [Full Text] [PDF]
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N. Bercovici, A. Heurtier, C. Vizler, N. Pardigon, C. Cambouris, P. Desreumaux, and R. Liblau Systemic Administration of Agonist Peptide Blocks the Progression of Spontaneous CD8-Mediated Autoimmune Diabetes in Transgenic Mice Without Bystander Damage J. Immunol., July 1, 2000; 165(1): 202 - 210. [Abstract] [Full Text] [PDF]
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K. A. Frauwirth, M.-L. Alegre, and C. B. Thompson Induction of T Cell Anergy in the Absence of CTLA-4/B7 Interaction J. Immunol., March 15, 2000; 164(6): 2987 - 2993. [Abstract] [Full Text] [PDF]
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C. T. Nugent, D. J. Morgan, J. A. Biggs, A. Ko, I. M. Pilip, E. G. Pamer, and L. A. Sherman Characterization of CD8+ T Lymphocytes That Persist After Peripheral Tolerance to a Self Antigen Expressed in the Pancreas J. Immunol., January 1, 2000; 164(1): 191 - 200. [Abstract] [Full Text] [PDF]
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C. S. Schmidt and M. F. Mescher Adjuvant Effect of IL-12: Conversion of Peptide Antigen Administration from Tolerizing to Immunizing for CD8+ T Cells In Vivo J. Immunol., September 1, 1999; 163(5): 2561 - 2567. [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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