Direct Evidence That Functionally Impaired CD4
+ T Cells Persist In Vivo Following Induction of
Peripheral Tolerance
This information is current as
of June 16, 2017.
Kathryn A. Pape, Rebecca Merica, Anna Mondino, Alexander
Khoruts and Marc K. Jenkins
J Immunol 1998; 160:4719-4729; ;
http://www.jimmunol.org/content/160/10/4719
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References
Direct Evidence That Functionally Impaired CD41 T Cells
Persist In Vivo Following Induction of Peripheral Tolerance1
Kathryn A. Pape, Rebecca Merica, Anna Mondino, Alexander Khoruts, and Marc K. Jenkins2
A
lthough developing T cells specific for self peptide/
MHC molecules presented by thymic APCs are deleted
in the thymus (1, 2), this cannot be the mechanism of
immunologic tolerance for all self Ags. For example, intrathymic
clonal deletion could not eliminate CD41 T cells specific for self
peptide/MHC molecules derived from novel self proteins that appear in the blood after mature T cells have seeded the periphery
(e.g., puberty- or pregnancy-associated hormones). These Ags
would be accessed easily by naive self-reactive T cells because the
peripheral blood and lymphoid tissues contain many class II MHCexpressing, bone marrow-derived APCs that are capable of taking
up, processing, and presenting Ags to circulating T cells. Therefore, it is reasonable to assume that the immune system must be
able to silence the function of CD41 T cells in the periphery. This
has been well documented in studies in which soluble foreign Ags
are introduced into the bloodstream (3–7). However, the mechanisms by which the T cells are silenced under these circumstances
are not well understood.
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 –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
Department of Microbiology, Center for Immunology, University of Minnesota Medical School, Minneapolis, MN 55455
Received for publication November 19, 1997. Accepted for publication January
12, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from National Institutes of Health (AI27998,
AI35296, and AI39614 to M.K.J. and AI07313 to K.A.P.) and Howard Hughes Medical Institute (to A.K.).
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.
Copyright © 1998 by The American Association of Immunologists
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 TCRdependent proliferation that is acquired as a result of prior TCR
stimulation in the absence of APC-derived costimulatory signals or
proliferation (14 –16). Although functionally unresponsive T cells
have been identified in several in vivo systems (10, 12, 13, 17–25),
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
CD41 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 –32). It is also
possible that in vivo functional unresponsiveness is due to Agspecific suppression. It was reported recently that chronic activation of CD41 T cells in the presence of IL-10 generates IL-10-,
IL-5-, and TGF-b-secreting cells that suppress the proliferation of
CD41 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 CD41, TCR transgenic T cells that was accompanied by an
0022-1767/98/$02.00
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A small population of CD41 OVA-specific TCR transgenic T cells was tracked following the induction of peripheral tolerance by
soluble Ag to address whether functionally unresponsive, or anergic T cells, persist in vivo for extended periods of time. Although
injection of OVA peptide in the absence of adjuvant caused a transient expansion and deletion of the Ag-specific T cells, a
population that showed signs of prior activation persisted in the lymphoid tissues for several months. These surviving OVA-specific
T cells had long-lasting, but reversible defects in their ability to proliferate in lymph nodes and secrete IL-2 and TNF-a 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 CD41 T cells is generated in vivo after exposure
to soluble Ag. The Journal of Immunology, 1998, 160: 4719 – 4729.
4720
T CELL UNRESPONSIVENESS IN VIVO
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 CD41, KJ1-261 cells was measured by flow
cytometry.
Materials and Methods
Intracellular cytokine staining
Mice
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-a mAb (both from PharMingen) and washed once with the saponin buffer and twice with PBS. The PE channel fluorescence of 1000 to
2000 CD41, KJ1-261 cells or CD41, KJ1-262 cells was measured by flow
cytometry.
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 3 106 to 5 3 106 CD41,
KJ1-261 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-mm syringe filter, and 300 mg was injected in a volume of 0.25 ml into
the tail vein. For primary immunization, 30 or 300 mg of OVA peptide
323–339, or a mixture of 30 mg of OVA peptide 323–339 and 30 mg 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 mg 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-29-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, antiL-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
CD41, KJ1-261 or CD41, KJ1-262 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.
3
Abbreviations used in this paper: HA, hemagglutinin; BrDU, 5-bromo-29-deoxyuridine; PE, phycoerythrin.
In vitro cytokine assays
Lymph node cells (5 3 105) were cultured in triplicate wells with 2 3 105
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 mg/ml gentamicin sulfate, and 5 3
1025 M 2-ME. Aliquots (50 ml) of culture supernatant were removed from
each well at 24, 48, and 72 h after initiation of the culture.
In some experiments, KJ1-261 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-g, 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.
Results
A stable population of previously activated TCR transgenic T
cells remains after an injection of soluble Ag
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, CD41,
KJ1-261 DO11.10 T cells were readily detected by flow cytometry
in the lymph nodes 1 day (34, 40) or 13 days after transfer (Fig.
1A). 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
CD41, KJ1-261 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
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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.
The Journal of Immunology
4721
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 CD41, KJ1-262 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.
DO11.10 T cells from recipients injected previously with soluble
Ag accumulate poorly in vivo following a subsequent antigenic
challenge
The ability of soluble Ag to induce a hyporesponsive state in Agspecific 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).
The aforementioned experiments are complicated by the fact
that a fraction of the KJ1-261 cells present in the DO11.10 donor
mice expresses a second TCR containing an endogenous Va-chain
due to inefficient allelic exclusion (41). Some of these dual TCRexpressing 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 2B, 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
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FIGURE 1. Some DO11.10 T cells persist and
have an activated phenotype after OVA peptide injection. BALB/c recipients of DO11.10 T cells received nothing (A, B, C), an i.v. injection of 300 mg
of OVA peptide (D, E, F), or an s.c. injection of 300
mg of OVA peptide emulsified in CFA on the back
(G, H, I). Normal BALB/c mice that did not receive
DO11.10 T cells were used as negative controls (J).
Mice were sacrificed 12 (A, B, D, E, G, H) or 21 (C,
F, I) days after the OVA peptide injections, and
pooled brachial, axillary, and inguinal lymph node
cells were stained with anti-CD4 and KJ1-26 mAbs.
Some of the lymph node cells were stained with an
anti-CD45RB mAb, an anti-BrDU mAb, or an isotype control mAb along with the anti-CD4 and
KJ1-26 mAbs. In C, F, and I, the fluorescence intensity of CD41, KJ1-261 cells is shown.
4722
T CELL UNRESPONSIVENESS IN VIVO
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.
To conclude that the clonal expansion defect was correlated
with the persistence of Ag, it was necessary to monitor the in vivo
presence of the stimulatory OVA peptide/MHC molecules. A functional assay was devised because there are no reagents available
for physical detection of this complex. Normal BALB/c mice were
injected with OVA peptide. DO11.10 T cells were then transferred
into these mice at later times. If OVA peptide/I-Ad complexes
were present in a stimulatory form in the lymphoid tissues of the
recipient, then the freshly transferred DO11.10 T cells should recognize these complexes and acquire the light-scatter properties of
blasts. The sensitivity of this method is demonstrated by the finding that stimulatory OVA peptide/I-Ad complexes were detected in
the lymph nodes of mice that were injected 41 days earlier with
OVA peptide emulsified in IFA (Fig. 4A). 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.
Suboptimal accumulation of DO11.10 T cells from tolerized
mice in response to Ag plus adjuvant priming is due to
decreased proliferation
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
BrDU1, 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 BrDU1, 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.
DO11.10 T cells in tolerized mice have a cytokine production
defect
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,
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FIGURE 2. In vivo clonal expansion of DO11.10 T cells following a secondary antigenic challenge. BALB/c recipients of DO11.10 T cells (A and C)
or DO11.10 SCID T cells (B and C) were left uninjected or injected with OVA peptide, as described in Figure 1. Recipients were challenged with an s.c.
injection of 300 mg of OVA peptide emulsified in IFA 12 to 21 days after the initial injections. Brachial, axillary, and inguinal lymph nodes were pooled
at the time of challenge (baseline), or 3 or 5 days after challenge, and stained as described in Figure 1. In B, the fluorescence intensities of CD41, KJ1-261
DO11.10 SCID T cells present at the time of challenge are shown. The total number of KJ1-261 cells was determined by multiplying the percentage of
CD41, KJ1-261 cells by the total number of cells in the lymph nodes. The mean 6 SE of two to nine animals per group is shown in A, and the mean 6
SE of five to six animals per group is shown in C.
The Journal of Immunology
4723
since little or no IL-2 was detected if KJ1-261 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
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FIGURE 3. The defect in accumulation of
DO11.10 T cells in tolerized recipients is reversible. BALB/c recipients of DO11.10 T cells
were left uninjected, given one i.v. injection of
300 mg of OVA peptide on day 0, or weekly i.v.
injections of 300 mg of OVA peptide. Some of
the recipients were challenged with an s.c. injection in the back containing 300 mg of OVA
peptide emulsified in IFA 12 to 23 days after the
initial i.v. injections. The remaining recipients
were challenged in an identical fashion 49 to 75
days after the initial i.v. injections. Brachial, axillary, and inguinal lymph nodes were pooled at
the time of challenge (baseline) or 5 days after
challenge, and the percentage and total numbers
of CD41, KJ1-261 cells were determined, as described in Figures 1 and 2. In A, the percentage
of CD41, KJ1-261 cells before and after challenge on day 23 vs day 49 is shown on the dot
plots. CD41, KJ1-261 cells were detectable in
all recipients over the background staining observed in a BALB/c mouse. In B, the mean numbers of CD41, KJ1-261 cells 6 the 95% confidence limits for four to eight animals per group
are shown.
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.
This conclusion was supported by experiments in which intracellular lymphokine staining was used to directly measure IL-2
production in vivo (Fig. 7). 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 CD41, KJ1-262 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 adjuvantprimed recipients expressed intracellular IL-2 following an in vivo
4724
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-a. 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-a in response to in vivo
antigenic challenge (Fig. 7B).
Immune deviation and suppression do not account for the
hyporesponsive phenotype of DO11.10 T cells from tolerized
mice
It was possible that the loss of IL-2 and TNF-a 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 un-
treated or previously given an i.v. injection of OVA peptide, suggesting that a suppressive environment was not created by the
tolerizing Ag injection.
It was also possible that DO11.10 T cells from tolerized mice
were secreting inhibitory factors, such as IL-10 and TGF-b, which
have been shown to inhibit the proliferation of bystander T cells
(33, 44, 45). To determine whether this was occurring, the ability
of tolerized DO11.10 T cells to affect the in vivo behavior of a
second, bystander population of 6.5 TCR transgenic T cells, which
are specific for influenza HA peptide 111–119 bound to I-Ed class
II MHC molecules (36), was monitored in vivo. BALB/c mice
were injected with DO11.10 T cells. Recipient mice were then not
treated or were injected with OVA peptide i.v. Twelve days later,
the DO11.10 T cells in naive and tolerized mice were present at
similar numbers in the lymph nodes (Fig. 9, 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.
Discussion
Although previous studies on in vivo peripheral tolerance have
implicated a role for clonal anergy, it was not clear whether the
loss of T cell function observed in these studies was caused by an
inherent defect in the responsive T cells or was merely an apparent
defect truly related to cell death, immune deviation, or suppression. Much of the confusion in this area is related to the use of
systems in which it was not possible to unambiguously determine
whether the T cells bearing the appropriate TCRs are physically
present, or the use of TCR transgenic or superantigen systems that
contain abnormally high frequencies of Ag-specific T cells. We
were able to address these limitations by physically tracking the in
vivo fate of a small population of Ag-specific T cells that express
a fixed TCR of known specificity, in normal mice subjected to a
peripheral tolerance protocol. Furthermore, we relied almost exclusively on in vivo measures of T cell function to avoid in vitro
artifacts.
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
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FIGURE 4. Blastogenesis of DO11.10 T cells following transfer into
mice previously injected with OVA peptide. BALB/c mice were injected
i.v. with 300 mg of OVA peptide or s.c. in the back with 300 mg of OVA
peptide emulsified in IFA, 5, 10, or 41 days before adoptive transfer of
DO11.10 T cells. Brachial and axillary lymph node cells were prepared 3
days after transfer, and CD41, KJ1-261 cells were identified, as described
in Figure 1. Forward light scatter of CD41, KJ1-261 cells that were transferred into mice that received no prior Ag injections (open histograms) or
mice that were injected with OVA peptide in IFA (A) or alone (B) at the
indicated times (filled histograms) is shown. The histograms are representative of two independent experiments that yielded identical results.
T CELL UNRESPONSIVENESS IN VIVO
The Journal of Immunology
4725
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
FIGURE 6. DO11.10 T cells from tolerized mice
are defective in IL-2 production following in vitro
stimulation. BALB/c recipients of DO11.10 T cells
were left uninjected or were injected with OVA peptide, as described in Figure 1. Three (B and E),
twelve (A and D), twenty-one, or fifty-nine days (C
and F) later, brachial, axillary, inguinal, and cervical
lymph node cells were harvested and stained with
anti-CD4 and KJ1-26 mAbs. The remaining cells
were placed in culture with irradiated BALB/c
splenocytes, in the presence or absence of 5 mm of
OVA peptide. In some cases, DO11.10 T cells were
depleted with KJ1-26 mAb-coated magnetic beads
before culture. The concentration of IL-2 measured in
the supernatant from the Ag-stimulated wells is
shown in A, B, and C. The frequency of CD41, KJ1261 cells present in the lymph node cell suspensions
was multiplied by the number of total lymph node
cells/well to obtain the number of KJ1-261 cells
present at the time of culture initiation. The concentration of IL-2/well was then divided by the number
of CD41, KJ1-261 cells present (D–F). The mean 6
SD for five animals per group (A, B, D, and E) or two
animals per group (C and F) is shown.
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
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FIGURE 5. BrDU incorporation following antigenic challenge is impaired in DO11.10 T cells from tolerized recipients. BALB/c recipients of DO11.10
T cells were left uninjected (A and B), given one i.v. injection of 300 mg of OVA peptide (C and D), or two i.v. injections of 300 mg of OVA peptide,
1 wk apart (E and F). Two weeks after the last injection, recipients were challenged with 300 mg of OVA peptide emulsified in IFA, and 58 or 120 h later,
brachial, axillary, and inguinal lymph node cells were harvested and stained for detection of CD4 and the DO11.10 TCR, as described in Figure 1.
Representative histograms of anti-BrDU mAb and isotype control mAb staining in CD41, KJ1-261 cells are shown along with the percentage of CD41,
KJ1-261 cells that were BrDU1, the total number of cells that were CD41, KJ1-261, and the total number of CD41, KJ1-261 cells that were BrDU1
(mean 6 SD for two to three animals/group) at each time point.
4726
T CELL UNRESPONSIVENESS IN VIVO
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 CD41 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 BrDU1, 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 BrDU1 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 BrDU1
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 TCRexpressing memory cells from normal DO11.10 TCR transgenic
FIGURE 8. Intravenous injection of OVA peptide does not generate a
suppressive environment in BALB/c mice. BALB/c mice were left uninjected or were given an i.v. injection of 300 mg of OVA peptide. Mice in
both groups received DO11.10 T cells 11 days later. The following day, all
recipients were challenged with an s.c. injection of 300 mg of OVA peptide
emulsified in CFA in the back, and the percentage and total numbers of
CD41, KJ1-261 cells in the brachial, axillary, and inguinal lymph nodes,
at the indicated days, were determined as described in Figures 1 and 2. The
mean 6 SD for two to three animals per group is shown. Similar results
were obtained in a second experiment.
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FIGURE 7. DO11.10 T cells from tolerized mice are defective in intracellular IL-2 and TNF-a production following an in vivo antigenic challenge.
BALB/c recipients of DO11.10 T cells were left untreated, or were given two i.v. injections of 300 mg of OVA peptide 1 wk apart, or an s.c. injection in
the tail with 300 mg of OVA peptide emulsified in CFA. Two weeks after the last Ag injection, mice were challenged with an i.v. injection of 300 mg of
OVA peptide. At the time of challenge, or 1.5 or 6 h after challenge, cells from brachial, axillary, inguinal, and cervical lymph nodes, or spleen, were
immediately stained for detection of CD4, the DO11.10 TCR, and intracellular IL-2 or TNF-a. The amount of IL-2 staining in the CD41, KJ1-261 or CD41,
KJ1-262 population is expressed as a dot plot, with a box identifying the percentage of cells staining positive for IL-2 (A). The mean percentage of cells
staining positive for IL-2 or TNF-a 6 SD was determined for two to five animals per group (B).
The Journal of Immunology
4727
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-a, 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 –52).
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FIGURE 9. DO11.10 T cells in tolerized recipients do not mediate bystander suppression. BALB/c recipients of DO11.10 T cells were left uninjected
(DO11.10) or were given an i.v. injection of 300 mg of OVA peptide (DO11.10 tol). Twelve days later, some of the mice from each group received 6.5
T cells. The next day, mice were challenged with 30 mg of OVA peptide plus 30 mg of HA peptide emulsified in CFA. Brachial, axillary, and cervical lymph
node cells were assessed for the percentage (A and B) and total numbers (C) of CD41, KJ1-261 cells or CD41, 6.51 cells, as described in Figures 1 and
2, on the day of challenge or 5 days after challenge. The mean 6 SD for three animals per group is shown (n.a., not applicable). Similar results were obtained
in a second experiment.
4728
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 longlived, 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 –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 Agdependent 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 CD28dependent 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 – 65) that promote T cell survival
and acquisition of the capacity to produce lymphokines such as
IL-4 and IFN-g (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-g. 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.
Acknowledgments
We thank Dr. Daniel Mueller for critically reviewing the manuscript, and
Jennifer White for expert technical assistance.
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