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Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD8+ T cells are a component of the cellular
immune response critical for the clearance of intracellular pathogens
and certain tumors. Throughout the development of T cells there are
multiple chances for a cell to undergo apoptosis. One of the first
opportunities occurs in the thymus, where a developing cell can undergo
death by neglect if it lacks a TCR with sufficient affinity for MHC
class I complexed with the appropriate self ligand or death through
negative selection if the TCR/MHC interaction is too strong (1, 2). Throughout the double-positive
(CD8+CD4+) stages these
thymocytes are extremely sensitive to agents such as dexamethasone and
irradiation, which induce apoptosis through production of reactive
oxygen species and loss of mitochondrial function (3, 4).
When single-positive CD8 T cells migrate to the peripheral lymphoid
organs, they acquire an increased resistance to apoptosis compared with
thymocytes. These cells express moderate levels of the
anti-apoptotic protein Bcl-2 (5) and continue to
require MHC interaction for survival (6, 7). If a naive T
cell encounters its cognate peptide in the appropriate MHC context
along with costimulation it will become activated and begin to
proliferate. These cells also acquire effector functions such as
cytokine production and cytotoxicity. Once Ag is cleared, these cells
continue through a brief, Ag-independent replication phase where their
numbers continue to increase (8, 9, 10). At the completion of
this phase, Bcl-2 levels decrease substantially, and effector cells
begin to undergo a period of apoptosis in vivo. This period of
apoptosis lasts for 
2–3 wk, during which 
90% of the Ag-specific
CD8+ T cells undergo apoptosis (11).
The surviving memory cells can persist for extended periods and provide
long term protective immunity (12). Additionally, these
memory CD8+ T cells no longer require MHC class I
interactions for their survival in vivo (6).
Previously we have documented that Ag-specific memory CD8+ T cells contain increased levels of Bcl-2 compared with either naive or effector cells (5). This raised the question of whether memory cells are more resistant to apoptosis than naive cells. Previous studies have examined whether primed CD4 cells were more resistant to Fas-induced death in vitro. These authors found that memory cells underwent less death in vitro (13, 14).
We examined apoptosis of Ag-specific CD8+ memory T cells after whole body irradiation and during the contraction of an immune response following acute infection with lymphocytic choriomeningitis virus (LCMV)4 or vaccinia virus (VV) expressing the LCMV CD8+ T cell epitope gp33–41. We found that after whole body irradiation memory cells survived preferentially compared with naive cells. Rechallenge of immune mice generated a vigorous secondary response, and the amount of contraction in a secondary response was only 2- to 5-fold compared with a 10- to 15-fold decrease observed in a primary response. To equalize numbers of Ag-specific cells we adoptively transferred P14 transgenic CD8 naive or memory T cells (which express a TCR specific for gp33–41) into naive recipients and challenged with virus. After exposure to Ag we found not only that memory cells underwent faster expansion than naive cells during the effector phase, but that memory CD8 T cells underwent less death during the contraction phase.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to 8-wk-old C57BL/6, B6.PL-Thy1a/Cy, and BALB/c mice were purchased from National Cancer Institute (Frederick, MD). P14 TCR transgenic (H-2b and Thy1.2+) mice have been described previously (15). Thy1.1+ P14 transgenic mice were generated by crossing P14 mice onto a B6.PL-Thy1a/Cy (H-2b and Thy1.1+) background.
Viruses
For LCMV infection, mice were infected i.p. with 2 x 105 PFU of the Armstrong strain or i.v. with 2 x 106 PFU of the clone 13 strain. LCMV was prepared and quantitated as described previously (16). For vaccinia infections mice were infected i.p. with 5 x 106 PFU of VV-gp33, a recombinant VV that expresses the Db-restricted LCMV CD8 T cell epitope gp33–41. This virus and its preparation have been described previously (17).
Preparation of MHC class I tetramers
The construction and purification of LdNP118–126, Dbgp33–41, DbNP3936–404, and Dbgp276–286 have been described previously (11).
Surface and intracellular staining
All Abs were purchased from BD PharMingen (San Diego, CA).
Surface staining was performed as described previously
(11). For Bcl-2 staining, cells were first surface stained
with anti-CD8
and Dbgp33–41, and then
intracellular staining was performed with anti-Bcl-2 as described
previously (5). Bromodeoxyuridine (BrdU) staining was
performed using the BrdU Kit from BD PharMingen according to the
manufacturer’s instructions. For analysis of direct ex vivo apoptosis,
splenocytes were isolated, surface stained as described above, briefly
incubated with annexin V and 7-amino-actinomycin D (7-AAD) at
room temperature in the dark, and removed immediately. Samples were
obtained on a FACSCalibur instrument and analyzed using CellQuest
software (BD Biosciences, San Diego, CA).
Irradiation
LCMV immune mice were irradiated in a Gammacell irradiator (Atomic Energy of Canada, Ottawa, Canada) to receive 200, 400, or 600 rad whole body gamma irradiation.
Cell isolation
Lymphocytes were isolated from the spleen, lymph nodes, bone
marrow, and PBL as described previously (5, 11, 18). For
isolation of non-lymphoid tissues, mice were euthanized, the abdomen
was opened, the hepatic vein was cut, and 5 ml ice-cold PBS was
injected directly into the hepatic artery to perfuse the liver. The
gall bladder was removed, and the entire liver was excised. The liver
tissue was homogenized using a wire screen and was incubated in 0.25
mg/ml collagenase B (Roche, Mannheim, Germany) and 1 U/ml DNase (Sigma,
St. Louis, MO) at 37°C for 45 min. Digested liver was centrifuged,
and the pellet was resuspended in 5–10 ml 44% Percoll (Sigma). This
solution was underlaid with 56% Percoll and spun at 2000 rpm for 20
min at 20°C. The intrahepatic lymphocyte populations were harvested
from the interface, and RBC were lysed using 0.83% ammonium chloride,
washed, and counted. Lung lymphocytes were isolated in a similar
manner. At the peak of the response on day 7, 1.50–2 x
107 lymphocytes were isolated from the liver, of
which 80% were CD8+ T cells; from lung
5–10 x 106 cells were isolated, of which
80% were CD8+ T cells. By day 32 postinfection
we could isolate 2 x 106 cells from the
liver (25–44% CD8+ T cells) and 4–7 x
105 cells from the lungs (25%
CD8+ T cells).
Generation of memory CD8+ T cells
The P14 Tg memory cells used in experiments with VV-gp33 (Fig. 3
) were generated as follows. Naive P14 TCR transgenic cells
(104) were injected i.v. into naive C57BL/6
hosts. Four hours later these mice were infected with 2 x
105 PFU LCMV-Armstrong. Under these conditions we
have shown that 98% of the gp33-specific CD8+ T
cells are derived from transgenic precursors (19). After
60 days these mice were sacrificed, and the number of transgenic memory
cells was determined by staining with -CD8 and
Dbgp33–41. Splenocytes were transferred i.v.
from these LCMV-immune mice into naive C57BL/6 mice to ensure that the
recipient mice received 104 P14 memory
CD8+ T cells. These animals are referred to as
memory transfer. Naive transfer was performed by injecting
104 naive P14 transgenic cells i.v. into naive
C57BL/6 mice. These groups of mice were then infected with 5 x
106 PFU VV-gp33 i.p. and analyzed at the
indicated time points.
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9) were generated as follows. Naive P14 TCR transgenic cells
(104) were injected i.v. into naive C57BL/6
hosts. Four hours later, these mice were infected with 5 x
106 VV-gp33 i.p. After 60 days these mice were
sacrificed, and the number of transgenic memory cells was determined by
staining with -CD8 and Dbgp33–41.
Splenocytes were transferred i.v. from these VV-gp33-immune mice into
naive C57BL/6 mice to ensure that the recipient mice received
104 P14 memory CD8+ T
cells. These animals are referred to as memory transfer. Naive transfer
was performed by injecting 104 naive P14
transgenic cells i.v. into naive C57BL/6 mice. These groups of mice
were then infected with 2 x 105 PFU
LCMV-Armstrong i.p. and used at the indicated time points.
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Isolation and transfer of naive and memory P14 cells into the same animal
Naive cells were isolated from naive P14
Thy1.2+ mice and enumerated by staining with
-CD8 and Dbgp33–41. Enough splenocytes
were transferred i.v. to give the desired numbers of naive
gp33-specific CD8+ T cells. To generate
Thy1.1+ P14 memory cells,
104 naive Thy1.1+ P14
CD8+ T cells were transferred i.v. into naive C57
BL/6 hosts and infected with 2 x 105 PFU
LCMV-Armstrong i.p After 90 days these animals were sacrificed, and
memory cells were isolated by FACS sorting with CD8 and Thy1.1 Abs.
The isolated population was 98% pure. These cells were mixed with
naive P14 Thy1.2 splenocytes and injected i.v. into naive C57BL/6
recipients. These mice were infected with 2 x
105 PFU LCMV-Armstrong i.p. and analyzed at the
indicated time points.
Characterization of naive and memory P14 Tg CD8+ T cells
The naive P14 cells were
CD44low, CD122low, and
LFA-1low and did not produce IFN-
in a 4- to
6-h assay after stimulation with Ag. Memory P14 cells were
CD44high, CD122high,
LFA-1high, CD43low/int
(effector cells are CD43high),
CD25low, and CD69low and
rapidly (2–4 h) produced IFN- after stimulation with peptide.
Neither naive nor memory P14 cells exhibited direct ex vivo
cytotoxicity against virus-infected targets.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether memory cells were more resistant to apoptosis
than naive cells we subjected LCMV-immune mice to whole body
irradiation and examined the survival of naive and memory cells in the
same animal. After 18 h apoptosis was complete, and we analyzed
survival at this time point to minimize the effects of homeostatic
proliferation of the few remaining cells. Fig. 1A shows that adult BALB/c
mice contained 4 x 106 naive
(LFA-1low) CD8+ T cells and
1.5 x 106 memory
(LFA-1high) cells in the spleen. After whole body
irradiation both cell populations decreased, but naive cells declined
to 105 cells (40-fold drop), while
activated/memory cells only decreased to 3 x
105 (5-fold drop; Fig. 1B). In a
dose-dependent manner memory CD8+ T cells
became enriched in the spleen as they increased from 20% of the
total CD8 population in untreated mice to 76% after 600 rad.
LCMV-immune mice contained 10% splenic CD8+ T
cells specific for the LCMV CTL epitope NP118–126. After irradiation
(Fig. 1C), the total number of Ag-specific memory cells
decreased from 6 x 105 to 1.6 x
105 (4- to 5-fold drop; Fig. 1D), but
these cells also became enriched as they increased in a dose-responsive
manner from 15% at 200 rad to 46% of the total CD8 population after
600 rad. Similar trends were observed in the lymph nodes, bone marrow,
and PBL (data not shown).
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We next examined the expansion and contraction of
LCMV-specific CD8+ T cells during a secondary
infection. In this experiment and all subsequent ones in this paper we
will use the following nomenclature to describe the various
cell populations. Effector cells generated from naive cells will be
referred to as primary effectors and the surviving cells from the pool
of these primary effector cells as memory cells (naive
primary
effectorprimary memory). Ag-specific CD8+ T
cells generated upon secondary infection will be referred to as
secondary effector and secondary memory cells (primary
memorysecondary effectorssecondary memory), and those generated
after a tertiary infection will be referred to as tertiary effectors
and tertiary memory cells (secondary memorysecondary
effectorstertiary memory).
Groups of naive and LCMV-immune BALB/c mice were challenged with virus,
and the expansion and contraction of the CD8+ T cell
responses during the primary and secondary infections was monitored
with MHC class I tetramers. As shown in Fig. 2 (A and B)
contraction of the primary response was much greater (15-fold) than
that of the secondary response (2- to 3-fold). At the peak of the
primary response (day 8) there were 107
NP118-specific CD8+ T cells, and 90% of these
primary effector cells died by day 30 to give a memory pool of
7 x 105 cells (15-fold drop). In
striking contrast the secondary effectors only decreased from
107 at the peak (day 5) to 4 x
106 on day 36 (2.5-fold drop). Since the
LCMV-specific CD8+ T cell response in BALB/c mice
is heavily directed toward a single immunodominant epitope
(NP118–126), we also examined secondary responses in C57BL/6 mice that
elaborate sizable responses to three epitopes, gp33–41, NP396–404,
and gp276–286. Fig. 2C shows that for all three CD8
epitopes there was an 10- to 15-fold reduction during the primary
response and only a 3-fold reduction during the secondary
response.
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Upon secondary infection we observed significantly less death of effector cells than after primary infection. However, there is a vast difference in the precursor frequency of Ag-specific CD8+ T cells between naive and immune mice. Naive mice have been estimated to contain 100–500 precursors for any given epitope (19, 20). In LCMV-immune mice there are between 105–106 memory cells for the epitopes we analyzed. To reach 107 effector cells in immune mice requires a much fewer number of divisions compared with the primary response. It is possible that the differences in the amount of death we observed may simply be due to differences in the number of divisions that the T cells undergo during the expansion phase. To better control for this we used an adoptive transfer system where we transferred similar numbers of naive or memory transgenic cells specific for the LCMV Db-restricted epitope gp33–41 into naive recipients. Thus, in these experiments the starting point is the same for memory and naive CD8+ T cells. These mice were then challenged with either LCMV or VV expressing the gp33–41 epitope.
Fig. 3
(A and B)
shows analysis of primary and secondary CD8 responses in the spleen
after infection with recombinant VV expressing gp33. Early in the
response, on day 5 postinfection, secondary effector cells had expanded
to almost 10-fold greater numbers than primary effector cells. At the
peak of the response, on day 8, secondary effector cells were present
in greater numbers, and this difference was maintained as the response
contracted. When the decrease in cell numbers in both populations was
compared, we observed a 5-fold contraction of the primary effectors
compared with a 2.5-fold contraction of the secondary effectors (Fig. 3C).
In addition to examining how naive and memory cells respond in the
context of a recombinant vaccine (i.e., VV expressing gp33 epitope) we
also analyzed the responses following infection with the native virus.
To address this we transferred naive and memory Tg cells into naive
C57BL/6 hosts and then challenged them with LCMV. Five days
postinfection there were 2 x 106 secondary
effector and 4 x 105 primary effector cells
in the spleen (Fig. 4, A and
B). The number of secondary effectors reached a plateau on
day 6, edged slightly higher on day 7, and began to contract by day 8.
This contrasted with the primary effector pool, which plateaued on day
7, remained constant on day 8, and then began to contract. After viral
clearance the new memory pool was established, and the contraction of
the secondary response was significantly less than the primary
response. Comparison of the death phase revealed that transferred naive
cells underwent 18-fold reduction from the peak on day 8, while
transferred memory cells underwent 6-fold reduction from the peak on
day 7. In addition to examining cell numbers in the spleen we examined
other lymphoid and non-lymphoid tissues. Fig. 5 shows that lymphoid tissues such as the
bone marrow (A) and lymph nodes (B) contain
similar numbers of primary and secondary effector cells on days 6–8
postinfection, but by the establishment of memory there were more
secondary memory cells. Examination of cell numbers in two non-lymphoid
tissues the lung (Fig. 5C) and liver (Fig. 5D)
revealed similar trends.
In both the LCMV and vaccinia systems we observed differences in the
contraction of the immune response after stimulation of naive and
memory CD8+ T cells. One potential explanation
for this phenomenon is that the cytokine milieu during the secondary
infection is inherently different, and all cells, even primary
effectors, would undergo less contraction in this environment. Although
the majority of the LCMV response in the adoptive transfer model is
directed against donor gp33–41-specific cells, there is an easily
measurable host NP396–404 response. In both naive and memory cell
transfers the endogenous NP396-specific T cell response will represent
primary effector cells and would rule out nonautonomous factors
controlling cell fate decisions. When the NP396–404 response was
examined (Fig. 6) after transfer of
either naive or memory gp33-specific cells, a similar peak of
NP396+-specific T cells was observed on day 8,
and similar numbers were also observed after completion of the death
phase on day 32. Thus, the contraction of the primary effectors is
similar in both groups of mice (i.e., transgenic naive or memory cell
recipients).
Although we observed similar contraction of NP396-specific
CD8+ T cells in both primary and secondary
infections, these cells are of a different specificity than gp33. To
address the fate of naive and memory cells of the same specificity, we
transferred naive and memory P14 cells into the same mouse. These two
populations (i.e., naive and memory P14 cells) can be distinguished
from each other by staining with anti-Thy1.1 Abs, as the memory
cells were generated from Thy1.1+ P14 mice. Naive
and memory cells were mixed at various ratios (naive cells/memory
cells), transferred into naive recipients, and then infected with LCMV.
A longitudinal analysis of PBL was performed by retroorbitally bleeding
the recipient mice on days 5, 7, 15, and 33 postinfection. P14 cells
were identified by staining with -CD8 and
Dbgp33–41. Tetramer-positive cells were further
characterized to determine whether they were Thy1.1 positive (derived
from transferred memory P14 cells). The results of these experiments
are shown in Fig. 7. The data from the
control groups (naive or memory cell transfer only) are shown in Fig. 7A, and the experimental groups (both naive and memory cells
transferred into the same mouse) are shown in Fig. 7B. The
striking finding is that regardless of the ratio of naive/memory P14
cells on day 0, memory cells always contracted less than naive cells
following viral infection. Thus, as a result of this decreased
downsizing of the secondary effectors, Thy1.1 P14 cells became the
predominant Ag-specific cells in all groups of mice. This is summarized
in Fig. 7B, and raw data from a representative mouse are
shown in Fig. 7C. These results showing decreased
contraction of secondary effectors compared with primary effectors of
the same antigenic specificity (P14 Tg cells) and in the same animal
and prove unequivocally that this important difference in the response
of naive and memory CD8 T cells is due to intrinsic differences in the
(i.e., cell autonomous) and is not the result of environmental
effects.
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A). This shows that the
decreased downsizing of secondary effectors cannot be explained by
increased proliferation of these cells during the death phase. In fact,
if anything their proliferation was slightly less than that of primary
effectors during the contraction phase. In addition to measuring
proliferation we determined ex vivo apoptosis of primary and secondary
effector cells on days 7 and 15 postinfection (Fig. 8B).
High levels of annexin V binding are indicative of cells in the early
stages of apoptosis, while cells that bind both annexin V and 7-AAD are
in the later stages of apoptosis or are dead. Directly ex vivo many
more primary effector cells (56%) on day 7 were in the early stages of
apoptosis compared with secondary effector cells (28%). By day 15,
36% of primary effector cells were in the early stages of apoptosis
compared with 16% of secondary effector cells. Taken together, these
results (Fig. 8) show that decreased downsizing of secondary effectors
is most likely due to decreased apoptosis.
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A shows a longitudinal
analysis of the PBL of a representative animal as it passes through a
primary, secondary, or tertiary response. All animals started with
equal numbers of P14 TCR transgenic cells. Similar to secondary
effector cells, tertiary effector cells proliferated to a greater
extent than primary effector cells 5 days postinfection. These cells
also peaked at a level similar to that of the primary effector cells on
day 8 postinfection. By the time the memory phase was established, both
secondary and tertiary infections had generated greater numbers of
cells than the primary infection (Fig. 9C). Because of the
critical role of Bcl-2 in controlling cell death of CD8 cells we
examined its expression in primary, secondary, and tertiary effector
cells (Fig. 9). Fig. 9D shows that after T cell
activation (day 8 postinfection) Bcl-2 levels decreased in all three
populations compared with naive cells, but secondary and tertiary
effectors contained slightly higher levels of Bcl-2 than primary
effectors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Radiation-induced apoptosis is a critical technique used to treat certain malignancies. Sensitivity to radiation-induced death was traditionally assessed in clonogenic survival assays in vitro where the number of colony-forming cells was compared against varying doses of radiation. After irradiation it has been observed that cells with the greatest proliferative capacity, such as lymphocytes, are among the most sensitive to apoptosis. Teleologically this makes sense because of the risk of expanding a population of cells that harbor DNA damage that could eventually lead to malignancy. Why, then, do memory cells undergo less apoptosis than naive cells? Studies have shown that memory cells undergo a slow homeostatic proliferation, while naive cells undergo little or no proliferation (6). In theory, memory cells should be more sensitive to irradiation-induced death, as they cycle more and should have more of a chance for DNA damage-sensing mechanisms, such as the tumor suppressor p53, to kill the cells. Although multiple mechanisms may account for the radioresistance, this apparent conundrum could be explained by the increased expression of Bcl-2, as previous studies by Strasser (21) and colleagues have shown that lymphocytes that overexpress Bcl-2 are more resistant to irradiation-induced death in vitro.
Irradiation of Ag-specific CD8+ memory T cells
represents one type of apoptotic stimuli these cells may respond to;
the death that occurs after expansion induced by exposure to Ag during
reinfection or vaccine boosting represents another. Rechallenge of
LCMV-immune mice results in a vigorous amanestic response. The amount
of contraction of the secondary response was 2- to 5-fold compared
with the 10- to 15-fold contraction observed after primary
infection. One potential explanation for the difference observed in
contraction between the two infections is that the lower number of
divisions in secondary infection compared with primary infection causes
less death. We used an adoptive transfer system to illustrate that
differential death after exposure to Ag is an innate property of memory
compared with naive cells and is not solely due to differences in
proliferation. In the adoptive transfer experiment, equal cell numbers
of naive and memory cells were transferred. We found that memory cells
underwent greater expansion than naive cells. These results confirm and
extend a similar finding by Veiga-Fernandes et al. (22),
who demonstrated that memory cells specific for the HY Ag proliferated
faster than naive cells upon exposure to male splenocytes. We extend
these findings to demonstrate that memory cells also expand to greater
numbers more quickly in viral infections. At the peak of the response
the memory cells were either greater (VV) or almost equal (LCMV)
compared with the naive cell transfers. Previous studies by Zimmerman
et al. (23) have documented similar total expansion of
naive and memory cells at the peak of the LCMV response. When the
immune response had finished contracting, the cells generated from
memory cells had always contracted less. Most of these cells were
present not only in several lymphoid tissues (spleen, lymph nodes, and
bone marrow), but also in non-lymphoid tissues such as lung and liver
(Fig. 5). In addition to observing differential expansion and
contraction of naive and memory P14 cells when they were transferred
into separate mice, we observed similar trends when these cells were
transferred into the same mouse. This finding is important because it
allows us to rule out nonautonomous factors such as the cytokine milieu
as the main reason for the differential contraction of naive and memory
cells and shows that decreased downsizing of secondary effectors is due
to intrinsic differences between naive and memory cells.
Survival of secondary P14 Tg effectors was better than that of primary P14 effectors after challenge with either LCMV or VV-gp33. However, the overall contraction was greater after LCMV infection compared with VV-gp33 infection. One possible reason for this difference could be the duration of antigenic stimulation. There is likely to be more Ag after LCMV infection than after VV-gp33 infection, and a longer period of stimulation may result in greater downsizing of the response. Additional experiments will need to be performed to precisely define the reasons that underlie the differences we have observed between LCMV and VV-gp33 infections. These studies are beyond the scope of this work. However, in terms of the present study it is worth emphasizing again that regardless of the virus infection, the contraction of primary effectors was always greater than that of secondary effectors.
What is the mechanistic basis for the difference in the numbers of Ag-specific memory cells observed following primary and secondary infections? From our BrdU labeling experiments we demonstrate that the difference cannot be ascribed to excess proliferation during the contraction phase following viral clearance. Our data point to differences in apoptosis causing differences in cell numbers. This is also supported by our observation that secondary effector cells contained lower levels of annexin V binding directly ex vivo compared with primary effector cells on both days 7 and 15. Although we cannot formally exclude in vitro manipulation influencing cell death, it is important to note that secondary effector cells still contained fewer apoptotic cells than primary effectors. Apoptosis is a complex, multifactorial process that is controlled by many genes. In recent years considerable research has implicated mitochondria as a critical cellular component of apoptosis (24, 25). When cells are subjected to apoptotic stresses, mitochondria undergo changes in membrane permeability. These changes result in the release into the cytoplasm of proteins such as cytochrome c (26, 27), apoptosis-inducing factor (28), and second mitochondria-derived activator of caspase/Diablo (29). Once they enter the cytoplasm, these proteins activate and maintain caspase cascades and cause DNA degradation. Bcl-2 prevents apoptosis by preventing the release of these proteins into the cytosol (30). In our study we show that at the peak of the response secondary and tertiary effector cells contained slightly increased levels of Bcl-2 compared with primary effector cells. While increased Bcl-2 may partially explain increased survival, two lines of evidence suggest that other mechanisms may be involved as well. First, the Bcl-2 family is composed of many genes, some of which induce apoptosis. Increased survival could also be due to decreased levels of pro-apoptotic proteins such as Bad, Bax, Bim, or Bid (31). The second line of evidence is based on observations regarding the levels of reactive oxygen species and how these affects T cell apoptosis. Previous studies by Hildemann et al. (32) showed that treatment of superantigen-activated T cells with Mn(III) tetrakis(5,10,15,20-benzoic acid) porphyrin, a compound that catalyzes the destruction of reactive oxygen species, decreases T cell death in vitro. Early in LCMV responses (day 5), primary effector cells contain increased levels of superoxide compared with naive cells (J. Grayson and R. Ahmed, unpublished observations). The production of superoxide may cause intracellular damage culminating in apoptosis. It will be critical to determine whether secondary effector cells contain decreased levels of reactive oxygen species compared with primary effector cells.
In addition to documenting increased resistance to apoptosis of memory cells after irradiation and decreased downsizing of secondary effectors, we found that this property does not become altered following an additional (third) exposure to Ag. This finding has important implications for vaccination protocols. Many vaccines rely on initial priming followed by multiple boosts. It is unclear how much time is required after boosting for effector cells to reacquire the memory cell’s resistance to apoptosis and capacity for rapid proliferation. It is also critical to determine whether increased exposure to Ag selects for a subpopulation of memory cells that becomes more resistant to apoptosis with each boost. Since we only exposed memory cells to Ag twice, we do not know how this property will change with repeated boosting. Using the systems described in this paper, these key questions can be addressed in future studies. Our findings may also be useful in optimizing structured treatment interruption of HIV-infected individuals. This treatment results in increasing HIV load by cessation of anti-retroviral therapy for a brief period. In some patients (33) and in non-human primates (34, 35) this is followed by increases in the number of Ag-specific cells, which decline again after therapy is reinstituted. Although HIV infection is a complex process, with expansion of Ag-specific CD8 T cells being influenced by multiple factors, such as viral load, CD4 numbers, and the time of initial HIV exposure, structured treatment interruption is essentially a regimen involving a cyclical increase and decrease in Ag. General principles derived from studies of memory cells and their response to Ag may be applicable and provide guidelines for optimizing structured treatment interruption or therapeutic vaccination regimens.
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Footnotes |
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2 Current address: Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, NC 27104.
3 Address correspondence and reprint requests to Dr. Rafi Ahmed, Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, 1510 Clifton Road, Room G211 Rollins Research Building, Atlanta, GA 30322. E-mail address: ra{at}microbio.emory.edu
4 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; BrdU, bromodeoxyuridine; NP, nucleoprotein; VV, vaccinia virus; 7-AAD, 7-amino-actinomycin D.
Received for publication May 17, 2002. Accepted for publication August 7, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cell receptor diversity. Science 286:958.This article has been cited by other articles:
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 B. Li, M. VanRoey, F. Triebel, and K. Jooss Lymphocyte Activation Gene-3 Fusion Protein Increases the Potency of a Granulocyte Macrophage Colony-Stimulating Factor-Secreting Tumor Cell Immunotherapy Clin. Cancer Res., June 1, 2008; 14(11): 3545 - 3554. [Abstract] [Full Text] [PDF]
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 S.-J. Ha, S. N. Mueller, E. J. Wherry, D. L. Barber, R. D. Aubert, A. H. Sharpe, G. J. Freeman, and R. Ahmed Enhancing therapeutic vaccination by blocking PD-1-mediated inhibitory signals during chronic infection J. Exp. Med., March 17, 2008; 205(3): 543 - 555. [Abstract] [Full Text] [PDF]
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M. Shamim, S. G. Nanjappa, A. Singh, E. H. Plisch, S. E. LeBlanc, J. Walent, J. Svaren, C. Seroogy, and M. Suresh Cbl-b Regulates Antigen-Induced TCR Down-Regulation and IFN-{gamma} Production by Effector CD8 T Cells without Affecting Functional Avidity J. Immunol., December 1, 2007; 179(11): 7233 - 7243. [Abstract] [Full Text] [PDF]
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Z. Liu, H. Dai, N. Wan, T. Wang, S. Bertera, M. Trucco, and Z. Dai Suppression of Memory CD8 T Cell Generation and Function by Tryptophan Catabolism J. Immunol., April 1, 2007; 178(7): 4260 - 4266. [Abstract] [Full Text] [PDF]
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L. Krishnan, K. Gurnani, C. J. Dicaire, H. van Faassen, A. Zafer, C. J. Kirschning, S. Sad, and G. D. Sprott Rapid Clonal Expansion and Prolonged Maintenance of Memory CD8+ T Cells of the Effector (CD44highCD62Llow) and Central (CD44highCD62Lhigh) Phenotype by an Archaeosome Adjuvant Independent of TLR2 J. Immunol., February 15, 2007; 178(4): 2396 - 2406. [Abstract] [Full Text] [PDF]
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 J. J. Obar, S. Fuse, E. K. Leung, S. C. Bellfy, and E. J. Usherwood Gammaherpesvirus persistence alters key CD8 T-cell memory characteristics and enhances antiviral protection. J. Virol., September 1, 2006; 80(17): 8303 - 8315. [Abstract] [Full Text] [PDF]
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, Percentage of gp33-specific cells derived
from naive P14 cells; •, percentage of Ag-specific cells derived from
memory P14 cells. Note that in all groups of mice the contraction of
secondary effectors is less than that of primary effectors, such that
regardless of the ratio of naïve/memory cells transferred,
there were always more always more Ag-specific CD8+ T cells
derived from memory P14 cells on day 33 postinfection. An example of
the raw data from the 2.5:1 (naive/memory) cell ratio is shown in
C. In the dot plot on the left of the
figure the percentage of CD8+ T cells that are
Dbgp33–41+ is indicated in the left
corner of each plot. The dot plot in the right
column is gated on tetramer-positive cells and shows the
percentage of Ag-specific cells derived from naive (Thy1.2) or memory
(Thy1.1) P14 cells. Note that Thy1.1 gp33+ cells became the
predominant population by day 15 after infection.
