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Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-
production by both primary and long-term memory T
cells was observed in 
60 min after peptide stimulation. Although the
on-rate kinetics of cytokine production were nearly identical,
activated T cells produced more IFN-, but less TNF-, than memory
T cells. Ag-specific cytokine synthesis was not a constitutive process
and terminated immediately following disruption of contact with
peptide-coated cells, demonstrating that continuous antigenic
stimulation was required by both T cell populations to maintain
steady-state cytokine production. Upon re-exposure to Ag, activated T
cells resumed cytokine production whereas only a subpopulation of
memory T cells reinitiated cytokine synthesis. Analysis of cytokine
profiles and levels of CD8, LFA-1, and CTLA-4 together revealed a
pattern of expression that clearly distinguished in vivo-activated T
cells from memory T cells. Surprisingly, CTLA-4 expression was highest
at the early stages of the immune response but fell to background
levels soon after viral clearance. This study is the first to show that
memory T cells have the same Ag-specific on/off regulation of cytokine
production as activated T cells and demonstrates that memory T cells
can be clearly discriminated from activated T cells directly ex vivo by
their cytokine profiles and the differential expression of three
well-characterized T cell markers. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Infection of adult mice with the natural murine pathogen lymphocytic
choriomeningitis virus
(LCMV),3 provides one
of the best-characterized model systems for studying antiviral T cell
responses and cytokine production (4, 5, 17, 19, 20, 21, 22, 23, 24, 25, 26). LCMV
infection is cleared within 8–10 days, predominantly by
CD8+ T cells (27, 28, 29, 30) in a
perforin-dependent manner (31, 32). The peak of the
effector CD8+ T cell response, as demonstrated by
the ability to lyse virus-infected target cells directly ex vivo,
occurs at 
8 days postinfection (p.i.). The effector phase of the
immune response declines sharply by 20–30 days p.i., but protection
against secondary challenge is maintained by a stable population of
memory CD8+ T cells (9). In the
absence of pre-existing antiviral Abs, these cells can protect against
a subsequent viral challenge (21). In BALB/c mice, >95%
of the CD8+ T cell response is mounted against a
single immunodominant epitope, LCMV NP118–126
(33), and studies comparing TCR Vß usage and CDR3 length
in CD8+ T cells indicate that memory T cells are
stochastically selected from this primary T cell pool
(34). The LCMV model system therefore provides the unique
opportunity to compare activated and memory T cell populations that
have the same Ag specificity and conserved CDR3 and Vß usage in a
nontransgenic mouse model.
In this study, we compared virus-specific cytokine production by activated T cells at the peak of the effector T cell response to that of long-term memory T cells. After peptide stimulation directly ex vivo, both activated and memory CD8+ T cells initiated cytokine production within 60 min, and the maximum number of responding antiviral T cells was attained within 4–6 h poststimulation. Remarkably, disruption of T cell contact with peptide-coated APCs resulted in the immediate termination of cytokine production in both populations, and re-exposure to specific peptide Ag led to reinitiation of cytokine production by the majority of activated T cells and a subpopulation of memory T cells. Thus, both primary and memory T cells were able to cycle cytokine production on, off, and on again in an exquisitely sensitive response to Ag contact. Virus-specific CD8+ T cells expressed high levels of LFA-1 (CD11a/CD18) at all time points examined, thus confirming its usefulness in distinguishing activated/memory T cells from naive T cells. Direct ex vivo expression of CTLA-4 (CD152) was limited to the first 4–8 days p.i. and declined to baseline levels by 15 days p.i., indicating that it is predominately found in virus-specific CD8+ T cells during the early in vivo stages of T cell activation. CD8 expression on antiviral T cells was high at early time points (4–6 days p.i.) but was substantially down-regulated by 8–15 days p.i. before returning to normal levels on the surviving memory T cells examined between 30 and 300 days p.i. These results demonstrate that although memory T cells are similar to activated T cells in terms of their on/off rates of Ag-specific cytokine production, memory T cells can be clearly distinguished from both activated and naive T cells by the differential expression of CD8, LFA-1, and CTLA-4. Together, these findings document functional and phenotypic changes that occur as the activated Ag-specific CD8+ T cell population matures into the memory T cell pool.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LCMV immune mice were obtained by injecting 5- to 15-wk-old BALB/c mice i.p. with 2 x 105 PFU of LCMV-Armstrong (Arm-53b) and were used at the indicated time points. BALB/cByJ mice were purchased either from The Jackson Laboratory (Bar Harbor, ME) or from The Scripps Research Institute animal facility. The fibroblast cell line, BALB Clone 7, was grown in RPMI 1640 containing 10% FBS, L-glutamine, and antibiotics.
Peptide
HPLC-purified (>95% pure) LCMV NP118–126 peptide (PeptidoGenic, Livermore, CA) was dissolved in sterile PBS at 10 mg/ml and stored at -80°C until use.
Intracellular cytokine staining and flow cytometry
Spleen cells from LCMV-infected or naive mice were cultured at
37°C in 6% CO2 for the indicated amount of
time in the presence or absence of 10-7 M peptide (LCMV
NP118–126) in RPMI 1640 containing 10% FBS, 20
mM HEPES, L-glutamine, and antibiotics. Brefeldin A (Sigma,
St. Louis, MO) was added at a final concentration of 2 µg/ml. For
on-rate experiments, brefeldin A was included during the entire culture
period (0.5–6 h). Cells were immediately placed on ice, washed, and
stained for CD8 (PharMingen, San Diego, CA) in round-bottom 96-well
plates. The cells were washed and permeabilized using the
Cytofix/Cytoperm kit (PharMingen) according to the manufacturer’s
directions. Intracellular Ags were detected using PE-conjugated mAbs
specific for TNF- or CTLA-4 and FITC-conjugated anti-IFN- Abs
(PharMingen). Samples were resuspended in PBS containing 2%
formaldehyde and acquired on either a FACScan or FACSCalibur flow
cytometer (80,000–200,000 gated events acquired per sample) and
analyzed using Cellquest software (Becton Dickinson, San Jose,
CA).
Biotinylation and peptide loading of stimulator cells
BALB clone 7 cells were harvested by trypsinization and counted. After washing once with PBS to remove FBS and other soluble proteins, the cells were resuspended at 107/ml and biotinylated for 20 min on ice in PBS supplemented with 1 mM MgCl2 and 0.1 mM CaCl2 and containing 1.0 mg/ml freshly added NHS-biotin (Calbiochem, La Jolla, CA). Biotinylation was quenched by adding RPMI 1640 containing 10% FBS, and the cells were washed twice before being resuspended in RPMI 1640 containing 10% FBS and 10-7 M peptide (LCMV NP118–126). The cells (2 x 107/ml) were incubated at 37°C for 1 h with occasional mixing and washed extensively to remove unbound peptide. Biotinylation of targets had no effect on peptide presentation over a range of peptide doses (10-5–10-8 M).
Off-rate kinetics of cytokine production
Spleen cells (106/well) from LCMV-infected
mice were stimulated with 2 x 105
biotinylated fibroblasts coated with 10-7 M
peptide (LCMV NP118–126) for 5 h at 37°C
in 6% CO2. Streptavidin-coated magnetic beads
(Perceptive Biosystems, Framingham, MA) were added at a concentration
of 40 beads per biotinylated stimulator cell and mixed by pipetting.
After 20 min at 37°C, the cultures were resuspended by pipetting, and
biotinylated cells were removed by placing the 96-well plate over a
magnet (Perceptive Biosystems) for 2 min. This resulted in >95%
removal of biotinylated stimulator cells. (To ensure that a stimulatory
amount of peptide was not released from these cells, we cultured
peptide-coated cells at 37°C for up to 5 h, collected the
supernatant, and mixed this 1:1 with fresh spleen cells from
LCMV-infected mice (at day 8 p.i.). No IFN- production by
virus-specific CD8+ T cells was observed.) The
nonbiotinylated splenocytes were recovered by collecting 100 µl of
supernatant which was immediately added to wells containing 4 x
105 peptide-coated or uncoated fibroblast APCs
(prewarmed to 37°C) to observe the off rate of cytokine production.
Brefeldin A (Sigma) was added to a final concentration of 2 µg/ml for
the final 60 min of culture to facilitate intracellular cytokine
accumulation. Cycloheximide (Sigma) was added to a group of
peptide-stimulated cultures at a final concentration of 100 µg/ml.
One group of cultures was stimulated with soluble anti-CD3 (10
µg/ml) and anti-CD28 (2.5 µg/ml; PharMingen) and assayed under
the same conditions as the peptide-stimulated cultures described above.
At the indicated time points, cells were removed from the culture
plates, washed, and placed at 4°C overnight in PBS containing 10%
FBS and anti-CD8. The next day intracellular cytokine staining was
performed according to the manufacturer’s directions.
Statistics
Statistical differences between cytokine levels of activated (day 8) and memory T cells were determined by the Student t test using SigmaStat (SPSS, Chicago, IL) software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adult BALB/c mice were infected with LCMV and were used either at
8 days postinfection to study activated T cell responses at the peak of
the effector T cell response, or at 280–300 days p.i. to study memory
T cell responses. Fig. 1
shows FACS
analysis of IFN- production by these two T cell populations
following direct ex vivo stimulation with the immunodominant peptide
LCMV NP118–126. IFN- was undetectable in both
day 8 and memory CD8+ T cells immediately ex
vivo, suggesting that cytokines are not constitutively expressed by
either cell population. In contrast, IFN- production was readily
detectable in both T cell populations within 60 min of peptide
stimulation. The maximum number of responding T cells detected by
intracellular IFN- staining was attained within 6 h and was
similar to that previously identified using peptide-tetramer staining
(4). Cytokine production was not observed after peptide
stimulation of naive T cells, indicating that only primed
virus-specific T cells were activated to produce cytokines under these
in vitro conditions (data not shown and see Ref. 4). More
detailed analyses of cytokine production by day 8 and memory T cells
are shown in Fig. 2. As shown previously
(26), IFN- production by day 8 T cells began within 30
min poststimulation, and the maximum number of
IFN-+ CD8+ T cells was
observed by 6 h poststimulation (Fig. 2A). Likewise,
TNF- production was detectable within 30 min poststimulation and
peak numbers were attained within 6 h (Fig. 2B). Memory
T cells responded with nearly identical kinetics, and maximal
production of both cytokines was observed by 6 h poststimulation
(Fig. 2, A and B). To compare the on-rate
kinetics of day 8 and memory T cell cytokine production on the same
relative scale, the number of peptide-specific
CD8+ T cells producing IFN- or TNF- at each
time point was plotted as a percentage of the maximum response. As
shown in Fig. 2C, the kinetics of IFN- production was
strikingly similar in day 8 and memory T cell populations. In each
case, the time required to reach 50% of the maximal number of
cytokine-producing cells was between 90 and 120 min poststimulation.
Similar analysis of TNF- production (Fig. 2D) showed
again that both T cell populations reached 50% maximum cytokine
production within 2 h of stimulation directly ex vivo. These
results demonstrate that, upon encountering the appropriate peptide/MHC
complex, memory T cells express antiviral cytokines with kinetics that
are nearly superimposable with those of activated T cells analyzed
during the peak of the effector T cell response.
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and TNF- that were
produced after peptide stimulation directly ex vivo (Fig. 3). CD8+ T cells
from mice at 8 days p.i. and memory T cells from mice at 295 days
postinfection were stimulated in parallel for 6 h with peptide,
and the levels of IFN- and TNF- were compared by quantitating the
mean fluorescence intensity (MFI) of cytokine staining. As shown in
Fig. 3, day 8 T cells produced more IFN- than did memory T cells
(p = 0.02). In contrast, TNF- production was
much higher in memory T cells than in day 8 T cells
(p < 0.001). It is striking that, although
activated and memory CD8+ T cells have similar on
rates of cytokine synthesis (Fig. 2
), they plateau at significantly
different levels.
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The regulatory requirements that determine when cytokine
production is turned off is a largely unappreciated and unexplored
aspect of T cell-mediated immunity. Without proper control mechanisms
in place, unrestrained cytokine production might be harmful or even
lethal to the host. Our study (Figs. 1 and 2), as well as others
(4, 35, 36) have found that without peptide stimulation,
the vast majority of virus-specific CD8+ T cells
are not expressing cytokines when analyzed directly ex vivo, even at
the peak of the antiviral immune response. One explanation of these
findings is that T cells may rapidly terminate cytokine production
in the absence of continuous Ag contact. To ascertain the role of
peptide/MHC contact in the maintenance of sustained cytokine
production, we developed a novel technique to quickly and synchronously
separate responding T cells from peptide-coated APCs using magnetic
bead depletion. This allowed us to determine how rapidly both day 8 and
memory T cells were able to down-regulate cytokine synthesis upon
disruption of Ag contact. LCMV-specific T cells were stimulated for
5 h with biotinylated, peptide-coated fibroblasts as APCs to
generate a high level IFN- production (Fig. 4, t = 5). One group was
left unmanipulated and samples were harvested at the indicated time
points. The cognate interaction between the T cells and the APCs in two
other groups was disrupted by pipetting and the APCs were removed from
the cultures using streptavidin-magnetic beads. The recovered spleen
cells were then plated either into wells containing uncoated APCs, to
monitor the down-regulation of IFN- production, or into wells
containing peptide-coated APCs, to determine whether cytokine
production could be restored if the CD8+ T cells
again came into contact with their cognate Ag. As shown for day 8 cells
in Fig. 4A, if stimulated cultures were left unmanipulated,
then cytokine production by virus-specific T cells was maintained at a
steady-state level for the duration of the experiment. Addition of
cycloheximide, a protein synthesis inhibitor, resulted in a rapid
decline in the number of IFN--producing T cells. Disruption of T
cell contact with peptide-coated APCs resulted in an equally rapid
reduction (Fig. 4A, on/off), indicating that cytokine
production ceased immediately upon Ag disengagement. Down-regulation of
IFN- production was not a procedural artifact, since T cells
stimulated with soluble anti-CD3/anti-CD28 showed no
alterations in cytokine production after an identical regimen of in
vitro manipulations. In addition, if T cells were restimulated on
peptide-coated APCs (Fig. 4A, on/on), then the percentage of
IFN-+CD8+ T cells
rebounded to the initial numbers observed before APC removal. In
parallel experiments, memory T cells from mice at 280 days p.i. were
stimulated with peptide-coated APCs and then treated as described above
(Fig. 4B). Similar to day 8 T cells, memory T cell cultures
maintained nearly steady-state IFN- production while in contact with
peptide-coated APCs. After removal of the stimulatory cells, the number
of cells producing IFN- dropped by >90%, a decline almost
identical to that observed following the administration of
cycloheximide. In contrast to day 8 T cells, only about 30% of the
original IFN-+ memory T cells could be
recovered if they were restimulated on fresh peptide-coated targets.
The results of these experiments indicate that if Ag-specific T cells
are not in direct contact with a target cell presenting a stimulatory
level of peptide, then cytokine production is rapidly turned off until
the appropriate peptide/MHC complex is encountered.
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There are several activation/memory markers that distinguish
between naive and Ag-experienced T cells (9, 11, 12, 13, 14, 15).
However, functional and phenotypic markers that specifically
distinguish between activated T cells and memory T cells in normal
nontransgenic mice have not been described previously. In this study,
we evaluated patterns of cytokine production and CD8 expression levels
over the course of both primary and secondary LCMV infections to
determine whether either of these criteria corresponded with an
activated phenotype or a memory phenotype. At the indicated days p.i.,
IFN- and TNF- production by virus-specific
CD8+ T cells were assayed after 6 h of
peptide stimulation (Fig. 5). At 5–8
days p.i., the virus-specific T cell population was a mixture of both
IFN-+TNF-- cells and
IFN-+TNF-+ cells at a
ratio of 1:1–1:3. By 15 days p.i., the ratio of
IFN-+TNF-- to
IFN-+TNF-+ cells was
about 1:5. This trend continued until day 60, at which point the ratio
had shifted to at least 1:50, indicating that 98% of the responding
memory T cells produced both IFN- and TNF- simultaneously. This
maturational shift in cytokine profiles is not unique to BALB/c mice;
an identical shift in cytokine production was observed following acute
LCMV infection of C57BL/6 mice (data not shown). Likewise, recombinant
vaccinia virus infection resulted in a similar maturational shift in
cytokine profiles during the virus-specific CD8+
T cell response (data not shown). Therefore, with the aid of
intracellular cytokine staining directly ex vivo, we have identified a
previously unrecognized shift in cytokine profiles which appears to be
common to several virus infections and is not influenced by the strain
or MHC haplotype of the mice. To determine whether the
IFN-+TNF-+ memory
phenotype would persist if the T cells were re-exposed to Ag in vivo,
LCMV-immune mice were rechallenged with virus and assayed directly ex
vivo for cytokine production (Fig. 5). Before secondary challenge,
10% of CD8+ T cells were virus specific. By 3
days p.i., this number had increased to 15%, and by 5 days p.i.,
40% of CD8+ T cells were epitope specific
(Fig. 5). Moreover, the increase in total virus-specific
CD8+ T cells was even greater than indicated by
the above percentages due to cell proliferation; the total number of
virus-specific T cells in the spleen had doubled by 3 days p.i. and
increased by >10-fold by 5 days p.i. The ratio of
IFN-+TNF-- to
IFN-+TNF-+ T cells at
day 135 was 1:50 (Fig. 5, secondary, day 0) but, by 3 days
postsecondary infection,
IFN-+TNF-- cells
were more numerous, and the ratio had shifted to 1:10. By day 5
postsecondary infection, the ratio had shifted further to 1:3, but by
21 days the ratio began to revert to a memory phenotype and >80% of
virus-specific T cells produced both IFN- and TNF-. Therefore, at
the peak of the antiviral response (during primary or secondary
infection), there are two populations of virus-specific T cells,
IFN-+TNF-- and
IFN-+TNF-+, but after
resolving the infection, the memory T cell pool becomes double
positive.
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and 2, we noted
that CD8 expression by virus-specific day 8 T cells was significantly
lower than that seen in responding memory T cells analyzed at 295 days
p.i. (66% reduction, p < 0.001). We therefore
conducted a more extensive analysis of CD8 expression over the course
of both primary and secondary virus infections (Fig. 5). CD8 expression
on virus-specific T cells was visualized using two colors to depict
different levels of CD8; green events represent high CD8 expression
(within the range of CD8 levels on naive T cells assayed in parallel)
and red events represent down-regulated levels of CD8 expression. At
5–6 days p.i., CD8 levels were high (MFI, 1610 and 1160, respectively)
but by the peak of the primary T cell response at 8 days p.i., CD8
levels were sharply down-regulated on almost 90% of the virus-specific
T cells (MFI, 695). By 60 days p.i., cytokine production had matured to
an IFN-+TNF-+ memory
phenotype and the level of CD8 had returned to high levels (MFI, 1230).
Following reinfection with LCMV, CD8 levels on virus-specific T cells
were down-regulated by 50% at day 5 postsecondary infection. By 21
days postsecondary infection, CD8 levels had started to increase as the
T cell population reverted to the
IFN-+TNF-+ memory
phenotype. The low levels of CD8 observed on activated T cells at day
8 p.i. was not due to the in vitro stimulation/staining procedure
since CD8 levels are already low at this time point even in the absence
of peptide stimulation (Fig. 1 and see Refs. 4, 15, 37). This demonstrates that CD8 expression on virus-specific T
cells is transiently reduced during the course of an acute viral
infection, but returns to high levels during the memory phase of the
immune response. LFA-1 and CTLA-4 levels on activated and memory CD8+ T cells
Above we describe differences in cytokine production which provide
evidence indicating that activated CD8+ T cells
can be distinguished from memory cells. Although we also found that CD8
expression levels vary during the course of virus infection, this
parameter alone is insufficient to distinguish activated T cells from
memory T cells. To determine whether a combination of phenotypic
markers could be used to discriminate between these two
CD8+ T cell populations, we characterized LFA-1
and CTLA-4 expression in virus-specific
(IFN-+) CD8+ T cells at
each stage of the antiviral immune response (Fig. 6). LFA-1 was expressed at a high level
on >95% of antiviral CD8+ T cells at every time
point examined, confirming previous studies that describe this as an
excellent marker for distinguishing activated/memory T cells from naive
T cells (11). There were no substantial differences in
LFA-1 expression on virus-specific CD8+ T cells
examined between 5 and 60 days p.i. (Fig. 6 and Table I), indicating that this marker alone is
insufficient for segregating activated T cell populations from memory T
cell populations. In striking contrast, CTLA-4 expression was high in
activated virus-specific T cells at the early stages of the immune
response but was sharply down-regulated as T cells entered the memory
phase. At 4 days p.i., only 1–2% of splenic
CD8+ T cells were virus specific, but they
already expressed high levels of CTLA-4 (MFI, 880; data not shown). As
shown in Fig. 6, >90% of virus-specific CD8+ T
cells at 5 and 6 days p.i. expressed CTLA-4 at high levels (MFI,
570–590) compared with naive T cells (MFI, 30). At 8 days p.i., only
50% of LCMV-specific T cells expressed CTLA-4. However, >80% of
the blast-size CD8+IFN-+
cells at this time point expressed CTLA-4, albeit at intermediate
levels (data not shown). This suggests again that the most highly
activated cells continued to express CTLA-4. By 15 days p.i., CTLA-4
expression had declined further and was indistinguishable from naive T
cell expression levels. Similar kinetics of CTLA-4 expression was
observed in virus-specific CD4+ T cells in
C57BL/6 mice after LCMV infection (data not shown), indicating that
early high level expression of CTLA-4 is not limited to the
CD8+ T cell subset. A summary of CD8, LFA-1, and
CTLA-4 expression on Ag-specific T cells during the course of acute
viral infection is given in Table I. Together, these results show that
activated/memory T cells can be discriminated from naive T cells on the
basis of LFA-1 expression and demonstrate for the first time that CD8
and CTLA-4 expression levels can be used in combination to distinguish
activated T cells from memory T cells. In addition, these results
suggest that primary CD8+ T cells may be further
subdivided into two groups, early activated
(CD8highCTLA-4high) and
late activated
(CD8lowCTLA-4int/low), both
of which can be clearly distinguished from memory T cells
(CD8highCTLA-4low).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF- levels differed
significantly between these T cell populations, and the activated T
cell pool contained both
IFN-+TNF-- and
IFN-+TNF-+ cells,
whereas memory T cells almost exclusively expressed the
IFN-+TNF-+ phenotype.
Furthermore, analyses of CD8, LFA-1, and CTLA-4 levels in
virus-specific CD8+ T cells revealed a pattern of
expression that distinguished activated T cell populations from memory
T cell populations.
Cytolytic activity is a defining feature of activated
CD8+ T cells, and, since this activity is
typically lost by 30 days p.i. (9), it is often used to
distinguish effector T cells from memory T cells (18). An
exception to this rule is that some studies using TCR transgenic
CD8+ T cells find relatively strong cytolytic
activity for months in the apparent absence of antigenic stimulation
(38, 39). Although nontransgenic memory T cells may
demonstrate some low-level CTL activity (40), most memory
CD8+ T cells in immune hosts require
restimulation for 15 h or more before high-level cytolytic
activity is observed (41, 42). We show here that, in
contrast to lytic functions, there is little difference in the on-rate
kinetics of cytokine production by activated and memory T cell
populations (Figs. 1 and 2). We find that activated and memory T cells
initiate IFN- and TNF- production within 30–60 min of Ag
contact, and the rates at which virus-specific cells are recruited into
cytokine production are almost superimposable. The maximum number of
responding cells was attained by 6 h poststimulation, but the
resulting levels of cytokine production differed significantly;
activated cells produced more IFN-, whereas memory cells produced
more TNF-. This suggests that there may be a preset maximum
production level for each cytokine, based on the activation state of
the T cell upon Ag contact. Our results confirm and extend a recent
study which showed rapid onset of IFN- production but delayed
perforin-mediated lytic activity in transgenic T cells
(42). Why does a dichotomy exist between cytokine
production and cytolytic activity? Perhaps the lag period observed
before the acquisition of perforin-mediated lytic activity is
beneficial to the host. Direct lysis of a target cell is irreversible,
and, if this is the first response to Ag contact, then low-level
cross-reactivity or bystander activation may lead to the inappropriate
destruction of uninfected cells. However, by first secreting antiviral
cytokines, the activated memory T cell may impede viral growth while
preparing to mount a cytolytic response. The perforin lag period may be
assured by simple biochemical constraints; it takes up to 24–48 h for
perforin, granzymes, and other constituent granule proteins to be
synthesized and assembled (43).
We and others have found that, even at the peak of the antiviral immune
response, cytokine-producing CD8+ T cells are not
detectable ex vivo in the absence of peptide stimulation (4, 26, 44). The biological implication of this observation, that most
virus-specific CD8+ T cells may not be actively
producing cytokines in vivo, has not been previously appreciated. We
have shown that activated (day 8) T cells can rapidly terminate
cytokine production upon disruption of Ag contact (26),
but down-regulation of cytokine responses in memory
CD8+ T cells has not been previously described.
Remarkably, when peptide-coated APCs were removed, LCMV-specific
primary and memory T cells terminated IFN- production very rapidly
at a rate nearly identical to that observed in cultures treated with
cycloheximide (Fig. 4, A and B). Nevertheless,
IFN-+ cells remained detectable for 1–2 h
after removal of peptide or addition of cycloheximide, presumably
reflecting the kinetics of IFN- release, degradation, and so forth.
Therefore, if CD8+ T cells had been actively
producing cytokines in vivo at the time of cell harvest, then they
would have remained detectable, even in the absence of peptide
stimulation. Since no such cells were detected, we suggest that the
vast majority of virus-specific T cells are not actively producing
cytokines in vivo and instead rapidly initiate cytokine production in
response to Ag contact. What are the biological implications of such
rapid down-regulation of cytokine synthesis? It is critical that
cytokine secretion be tightly regulated, because these molecules can be
toxic and are responsible for many symptoms of disease
(45, 46, 47, 48, 49). Therefore, by limiting cytokine production to
periods of direct contact with infected targets, the release of
inflammatory and antiviral cytokines is confined specifically to sites
of infection.
One intriguing difference between day 8 and memory T cells was the
ability to resume cytokine production following restimulation with
peptide-coated APCs. Essentially all of the day 8 T cells that
responded to the primary stimulation were able to reinitiate cytokine
production after secondary stimulation with peptide-coated targets
(Fig. 4A). In contrast, only about 30% of memory T cells
were able to resume cytokine production after restimulation (Fig. 4B). It is possible that some T cells were lost when the
APCs were depleted, but this is unlikely because day 8 cells and memory
T cells were assayed in parallel; no loss of day 8 T cells was observed
even though these cells may be more likely to be removed from the
cultures when the APCs were depleted, as they have a slightly higher
avidity for peptide/MHC than do memory T cells (our unpublished
results). It is possible that memory T cells may have become anergic or
that the shut-down of cytokine production may have occurred to begin
cell division or the production of cytolytic proteins. These questions
may be best addressed by using peptide-tetramer staining to identify
Ag-specific T cells, independent of their effector functions. This
strategy has been used successfully to identify nonresponding
(IFN--) LCMV-specific
CD8+ T cells in chronically infected mice
(50) and to identify dysfunctional virus-specific T cells
circulating in the liver (35).
Long-term T cell memory was maintained by T lymphocytes that, upon
peptide stimulation, produced both IFN- and TNF- simultaneously
(Fig. 5). This was in contrast to day 8 CD8+ T
cells, which comprised both
IFN-+TNF-- and
IFN-+TNF-+
populations. The mechanisms that regulate these differing patterns of
cytokine production in the two CD8+ T cell
populations remain obscure and are under investigation. Direct ex vivo
analysis of cytokine expression patterns from 4 to 300 days p.i.
demonstrated that differentiation from an activated phenotype to a
memory phenotype was completed within 30–60 days after viral
challenge. Following reinfection of LCMV-immune mice with virus, memory
T cells and their progeny dominate the antiviral immune response
(9). Interestingly, our study shows that within 5 days
after secondary infection, virus-specific cytokine profiles changed
from a memory phenotype to an activated phenotype similar to that
observed during the primary response (Fig. 5). This demonstrates that
memory T cells (or their progeny) can alter their preset Ag-specific
cytokine expression patterns. The induction of
IFN-+TNF-- T cells
may protect the host against TNF--mediated toxicity during the early
stages of the secondary infection. Alternatively, the
IFN-+TNF--
subpopulation may represent T cells that are in the process of being
purged from the system and are simply losing the ability to produce
both cytokines as they undergo activation-induced cell death.
Experiments are under way to distinguish between these
possibilities.
The identification of surface markers that discriminate between naive T
cells and activated/memory T cells has been instrumental in the
characterization of T cell-mediated immune responses. A recent study
has used transgenic technology to mark memory T cell precursors
(51) but, until now, no markers had been identified which
specifically distinguished activated T cells from memory T cells in
normal mice. Our analysis of activation markers on virus-specific T
cells revealed that both day 8 and memory T cells were uniformly
LFA-1high, CD2high, and
VLA-4high (data not shown). We chose LFA-1 for
further analysis because it is a commonly used and well-characterized
marker of T cell activation/memory (11). At all time
points studied (day 4–300 p.i.), virus-specific T cells expressed high
levels of LFA-1. In contrast to constitutively high LFA-1 expression,
we found that CD8 expression levels varied considerably during the
course of infection; high CD8 levels were observed at 4–6 days p.i.
followed by greatly decreased CD8 levels at 8–15 days p.i. (Fig. 5).
By 60 days p.i., CD8 levels on the majority of virus-specific memory T
cells reverted to high levels (Fig. 5). Furthermore, we demonstrated
that CD8 expression was greatly reduced at 5–8 days following
secondary infection, indicating that a transient reduction in CD8
levels occurs regardless of whether the responding cells were derived
from a naive T cell population or from a memory T cell pool. The
observed down-regulation of CD8 expression is not limited to LCMV
infection; we found a similar degree of CD8 down-regulation following
recombinant vaccinia virus infection in which 50% of
NP118–126-specific CD8+ T
cells have reduced levels of CD8 expression at 8 days p.i. (data not
shown). Down-regulated CD8 expression also occurs on a substantial
population of splenic T cells at 8 days after infection with either
LCMV, influenza, vaccinia, or vesicular stomatitis virus (4, 5, 15, 37, 44), although these findings were not discussed. Recent
evidence indicates that, if mature CD8+ T cells
do not simultaneously engage their TCR and CD8 coreceptor, the cells
down-regulate CD8 expression and eventually die by apoptosis
(52). Thus, CD8 down-regulation may be a mechanism for
purging the immune system of excess CD8+ T cells
after resolving acute infection. This hypothesis corresponds
well with our data showing that CD8 down-regulation occurs
before progression into the memory phase, after which virus-specific T
cells expressing high levels of CD8 are maintained at nearly
steady-state levels (4, 9).
When first discovered, CTLA-4 was considered an activation Ag since it
shared homology with CD28, and anti-CTLA-4 Abs were found to act
synergistically with anti-CD28 Abs in promoting T cell activation
(53). However, more recent studies suggest that CTLA-4
expression has an inhibitory role in T cell activation
(54) and may abrogate cell cycle progression (55, 56). It is thought that CTLA-4 may exert its inhibitory effect
by interfering with TCR signal transduction (57) or by
binding to the B7 ligand, thus preventing CD28-mediated T cell
activation. Mice deficient in CTLA-4 succumb within 2–4 wk of birth to
a massive lymphoid expansion (58, 59), which appears to be
driven by endogenous Ag, since CTLA-4- cells
bearing transgenic TCRs do not expand to the same extent
(60). The role of CTLA-4 expression during acute viral
infection is unclear. Adoptive transfer/challenge experiments using TCR
transgenic CTLA-4-deficient CD8+ T cells have
shown that these cells respond normally to viral infection and do not
demonstrate unrestrained lymphoproliferation upon Ag stimulation, but
the interpretation of these results is complicated by the rejection of
the transgenic cells at 2 wk after transfer due to mismatched minor
histocompatibility loci (61). In our studies, we found
CTLA-4 expression to be highest during the early stages of the immune
response (days 4–6 p.i.), at which time the LCMV-specific
CD8+ T cells are rapidly expanding and have an
approximate division rate of 6–8 h (4). From 5 to 8 days
p.i., most of the infectious virus has been cleared and the doubling
time correspondingly decreases to 24–30 h. Although CTLA-4 levels were
declining by 8 days p.i. (Fig. 6), expression remained highest in the
blasting cells (data not shown), consistent with the hypothesis that
CTLA-4 is important during T cell expansion. We are now conducting
experiments to determine whether CTLA-4 expression is playing a
positive (62, 63) or negative (54) role in
virus-specific T cell expansion in vivo. In either case, our study
provides convincing evidence that CTLA-4 expression, in combination
with CD8 and LFA-1 levels, is a useful phenotypic marker for
distinguishing between activated T cells and memory T cells.
In this study, we analyzed virus-specific T cell responses directly ex vivo without extensive in vitro manipulation and in a nontransgenic model system. This allowed the function and phenotype of activated and memory T cells to be directly compared during both primary and secondary virus infections. In addition to demonstrating similar on/off regulation of cytokine production, these analyses show that CD8, LFA-1, and CTLA-4 expression levels can be used in combination to readily distinguish between naive, activated, and memory T cell populations.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mark K. Slifka, Department of Neuropharmacology, CVN-9, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address:
3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; NP, nucleoprotein; p.i., postinfection; MFI, mean fluorescence intensity.
Received for publication July 30, 1999. Accepted for publication October 12, 1999.
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G. T. Belz, W. Xie, and P. C. Doherty Diversity of Epitope and Cytokine Profiles for Primary and Secondary Influenza A Virus-Specific CD8+ T Cell Responses J. Immunol., April 1, 2001; 166(7): 4627 - 4633. [Abstract] [Full Text] [PDF]
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 V. P. Badovinac, A. R. Tvinnereim, and J. T. Harty Regulation of Antigen-Specific CD8+ T Cell Homeostasis by Perforin and Interferon-gamma Science, November 17, 2000; 290(5495): 1354 - 1357. [Abstract] [Full Text]
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V. P. Badovinac, G. A. Corbin, and J. T. Harty Cutting Edge: OFF Cycling of TNF Production by Antigen-Specific CD8+ T Cells Is Antigen Independent J. Immunol., November 15, 2000; 165(10): 5387 - 5391. [Abstract] [Full Text] [PDF]
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P. Shankar, M. Russo, B. Harnisch, M. Patterson, P. Skolnik, and J. Lieberman Impaired function of circulating HIV-specific CD8+ T cells in chronic human immunodeficiency virus infection Blood, November 1, 2000; 96(9): 3094 - 3101. [Abstract] [Full Text] [PDF]
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D. E. Hassett, M. K. Slifka, J. Zhang, and J. L. Whitton Direct Ex Vivo Kinetic and Phenotypic Analyses of CD8+ T-Cell Responses Induced by DNA Immunization J. Virol., September 15, 2000; 74(18): 8286 - 8291. [Abstract] [Full Text]
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) after
LCMV infection were stimulated with peptide for the indicated periods
of time and analyzed by flow cytometry. The percentage of
IFN-
