Download Persistence T Cell Function during Viral + CD8 Memory Generation

Memory Generation and Maintenance of
CD8 + T Cell Function during Viral
Persistence
This information is current as
of June 18, 2017.
Stephanie S. Cush, Kathleen M. Anderson, David H.
Ravneberg, Janet L. Weslow-Schmidt and Emilio Flaño
J Immunol 2007; 179:141-153; ;
doi: 10.4049/jimmunol.179.1.141
http://www.jimmunol.org/content/179/1/141
Subscription
Permissions
Email Alerts
This article cites 94 articles, 53 of which you can access for free at:
http://www.jimmunol.org/content/179/1/141.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2007 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
References
The Journal of Immunology
Memory Generation and Maintenance of CD8ⴙ T Cell
Function during Viral Persistence1
Stephanie S. Cush,* Kathleen M. Anderson,* David H. Ravneberg,* Janet L. Weslow-Schmidt,*
and Emilio Flaño2*†
I
mmunological memory is one of the hallmarks of the adaptive immune system and it can be functionally defined as the
stronger protective response of the host to secondary Ag
challenge (1, 2). It, thus, allows the immune system to respond
more vigorously to infectious pathogens that have been encountered previously. Upon primary activation by Ag, CD8⫹ T cells
follow a program of proliferation and differentiation into effectors
that control the infection (3). After this expansion phase, the majority of Ag-specific CD8⫹ T cells undergo programmed cell
death, leaving a population of memory CD8⫹ T cells (4). A rapid
and efficient recall response is partially due to the ubiquitous presence of increased frequencies of memory T cells in peripheral
tissues and secondary lymphoid organs (5). In addition, several
characteristics of memory T cell populations contribute to enhanced secondary responses including the ability to self-renew in
the absence of Ag, higher activation status and reduced costimulatory requirements, expression of lymphoid homing molecules
and homeostatic cytokine receptors, higher frequency than naive
precursors, rapid proliferation upon secondary stimulation, and the
ability to maintain the integrity of the T cell repertoire (1, 6, 7).
Nevertheless, some of these characteristics may be arbitrary (i.e.,
markers, Ref. 8) or not universal (i.e., long lived, Ref. 9). The
heterogeneity of memory T cells in phenotype, function, and
location has led to the idea that they can be divided in two major
*Center for Vaccines and Immunity, Columbus Children’s Research Institute, Columbus, OH 43205; and †College of Medicine, The Ohio State University, Columbus,
OH 43210
Received for publication January 9, 2007. Accepted for publication April 27, 2007.
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 in part by National Institutes of Health Grant AI-59603
and by Columbus Children’s Research Institute.
2
Address correspondence and reprint requests to Dr. Emilio Flaño, Center for Vaccines and Immunity, Columbus Children’s Research Institute, Columbus, OH 43205.
E-mail address: [email protected]
www.jimmunol.org
subsets: effector (CD62LlowCCR7low) and central-memory
(CD62LhighCCR7high) cells based on their expression of lymphoid homing receptors (10).
Memory CD8⫹ T cells during viral infections play a major role
in protection by rapid recognition and lysis of virus-infected cells.
The contribution of memory CD8⫹ T cells to recall responses is
well-defined during acute infections where Ag is cleared (11, 12).
There is, however, little and contradictory information regarding
memory generation during persistent infections. This is of special
relevance because persistent infectious diseases are a major health
concern worldwide (13, 14). The current paradigm, mostly derived
from studies with lymphocytic choriomeningitis virus (LCMV)3
and HIV, is that persistent infection leads to some degree of CD8⫹
T cell effector dysfunction or deletion and lack of T cell memory
formation (15–19). CD8⫹ T cell dysfunction during chronic infection has been raised as one reason for pathogen persistence (20).
This loss of function in the presence of persistent Ag follows a
progression in the sequence of cytotoxicity, IL-2, TNF-␣, and
IFN-␥ production, exhaustion, and deletion (20, 21). However,
these findings appear to be at odds with situations where viruses
persist and CD8⫹ T cell functions are not apparently compromised
(22–25), with reports of residual Ag presentation (26) or with a
possible role for Ag in the maintenance of immunological memory
(27). ␥-herpesviruses (␥HV) are characterized by the establishment of lifelong asymptomatic infection, the persistence in ⬎90%
of the human population, and the association with a large list of
life-threatening conditions (28, 29). CD8⫹ T cells are thought to be
the main effectors of long-term virus control (28, 30, 31). How do
CD8⫹ T cell populations maintain control of infection without
3
Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus;
␥HV68, murine ␥-herpesvirus 68; LDA-PCR, limiting dilution nested PCR; RT, room
temperature; d.p.i., days postinfection; m.p.i., months postinfection; MLN, mediastinal lymph node.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
During infection with viruses that establish latency, the immune system needs to maintain lifelong control of the infectious agent
in the presence of persistent Ag. By using a ␥-herpesvirus (␥HV) infection model, we demonstrate that a small number of
virus-specific central-memory CD8ⴙ T cells develop early during infection, and that virus-specific CD8ⴙ T cells maintain functional and protective capacities during chronic infection despite low-level Ag persistence. During the primary immune response,
we show generation of CD8ⴙ memory T cell precursors expressing lymphoid homing molecules (CCR7, L-selectin) and homeostatic cytokine receptors (IL-7␣, IL-2/IL-15␤). During long-term persistent infection, central-memory cells constitute 20 –50% of
the virus-specific CD8ⴙ T cell population and maintain the expression of L-selectin, CCR7, and IL-7R molecules. Functional
analyses demonstrate that during viral persistence: 1) CD8ⴙ T cells maintain TCR affinity for peptide/MHC complexes, 2) the
functional avidity of CD8ⴙ T cells measured as the capacity to produce IFN-␥ is preserved intact, and 3) virus-specific CD8ⴙ T
cells have in vivo killing capacity. Next, we demonstrate that at 8 mo post-virus inoculation, long-term CD8ⴙ T cells are capable
of mediating a protective recall response against the establishment of ␥HV68 splenic latency. These observations provide evidence
that functional CD8ⴙ memory T cells can be generated and maintained during low-load ␥HV68 persistence. The Journal of
Immunology, 2007, 179: 141–153.
142
T CELL MEMORY DURING VIRAL PERSISTENCE
pathogen recrudescence during persistent infections? Humans encounter different pathogens through life of which a considerable
number reach a persistent, parasitic, commensal, or a latency state
and the human system has to provide lifelong protection. Herpesvirus infections are a clear example: initial infection with CMV or
EBV may occur early in childhood, which demonstrates that the
immune system is capable of controlling infection for 70 years or
more. Although persistent infections induce a continual evolution
of the function and numbers of T cells that are specific for the
infecting pathogen, there seems to be a substantial difference between infections with impaired CD4⫹ T cell help and/or a high
antigenic load such as HIV or LCMV-clone 13 and infections with
intact CD4⫹ T cell help and low-load Ag persistence, such as
herpesvirus infections (32, 33).
To understand more clearly how CD8⫹ memory T cells are
generated, maintained, and function during low-load persistent viral infections, we studied their fate and function during the acute
and long-term response to a ␥HV infection. Using a murine model
of infection with ␥HV68, we show that Ag-specific CD8⫹ T cell
function is maintained during long-term, low-level viral and Ag
persistence.
Materials and Methods
Mice and viral infection
C57BL/6J and BALB/cJ mice were obtained from The Jackson Laboratory,
Harlan Farms, or were bred at Columbus Children’s Research Institute
(CCRI). ␥HV68, clone WUMS, was propagated and titered on monolayers
of NIH3T3 fibroblasts. Mice were housed in BL2 containment under pathogen-free conditions. The Institutional Animal Care and Use Committee at
the CCRI approved all the animal studies described here. Mice were anesthetized with 2,2,2,-tribromoethanol and intranasally inoculated with 1000
PFU ␥HV68 in 30 ␮l of HBSS.
Virus analysis
To determine the number of cells containing ␥HV68 genomes long-term
postinfection, a combination of limiting dilution analysis and nested PCR
(LDA-PCR) was used (34). Splenocytes were isolated, RBC were lysed,
and the number of cells per spleen was determined. Cells were serially
diluted with NIH3T3 cells in 96-well plates, lysed, and viral DNA was
amplified by PCR using primers specific for ␥HV68 Orf50. A 2-␮l aliquot
of the product was reamplified using nested primers. The final product was
analyzed by ethidium bromide staining of DNA after electrophoresis in 3%
agarose gel. This procedure was able to consistently detect a single copy of
target sequence. Twelve replicates were assessed for each cell dilution and
linear regression analysis was performed to determine the reciprocal frequency (95% degree of confidence) of cells positive for ␥HV68 DNA. At
least three independent experiments were used to determine the mean reciprocal frequency and SD of ␥HV68 DNA in splenocytes. Infectious center assays were performed as described (35).
Ag-presentation assays
Splenocytes were isolated and processed into single-cell suspensions using
collagenase D (5 mg/ml) treatment for 45 min. Cells were next incubated
with 2 mM PBS/EDTA for 10 min at room temperature to disrupt multicellular complexes. Cells were stained with an anti-CD11c Ab conjugated
to magnetic beads (Miltenyi Biotec) and separation columns were used to
purify cells into CD11c⫹ and CD11c⫺ fractions. A total of 3 ⫻ 104 APCs
from each fraction were plated with 1 ⫻ 105 ␥HV68-specific lacZ-inducible T cell hybridomas (49100.2 and 4943.4) (36) in 96-well plates in
triplicate and incubated overnight. ␤-galactosidase activity was assessed in
individual wells as described (36). Background stimulation was determined
by using APCs from naive spleens as stimulators.
Flow cytometry analysis
Splenocytes were processed into single-cell suspensions as described
above. Cells were stained with Fc-block (CD16/32) and then washed. The
cells were then stained with a combination of the following ␥HV68-specific MHC tetramers: ORF6487– 495/Db, ORF61524 –531/Kb, ORF65131–140/
Dd and M291–99/Kd, and Abs against CD62L (MEL-14), CD8 (53-6.7),
CD43 (1B11), CD122 (TM-b1), CD127 (A7R34), CD44 (IM7), and CD4
(GK1.5). All Abs were purchased from eBioscience with the exception of
CD43 (BD Pharmingen). MHC tetramers were generated as described (37)
or obtained from the National Institutes of Health Tetramer Core Facility.
The surface expression of CCR7 was analyzed using the recombinant ligand CCL19 Ig followed by anti-human Fc-biotin and streptavidin-PE.
This process can be slightly detrimental for the intensity of the tetramer
staining. Flow cytometry data were acquired on FACSCalibur or BD LSR
(BD Biosciences) and analyzed using FlowJo software. Gates were set
using negative controls, isotype controls, and following the staining pattern
of each marker on the bulk lymphocyte and CD8 population. For any given
activation marker and tetramer combination, all the analysis gates are identical in size and position.
T cell avidity assays
Tetramer dilution. Single-cell suspensions were obtained as indicated
above. Cells were plated in flasks coated with anti-mouse IgG plus IgM
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 1. Long-term infection is characterized
by low viral titers and low Ag persistence. A, Analysis
of ␥HV68 titers during long-term latency. The number
of latently infected cells (left y-axis) in the spleen of
C57BL/6 (f) and BALB/c (䡺) mice was determined using LDA-PCR assay at 5 m.p.i. The number of lytically
infected cells (ND, not detected, right y-axis) was determined using a modified plaque assay. Three individual mice were independently analyzed at each time
point; error bars represent SD. B, Analysis of ␥HV68
antigenic load in mice during long-term latency. Spleen
cells from mice 4 m.p.i. were used as stimulators in an
in vitro assay with two lacZ-inducible ␥HV68-specific
T cell hybridomas to determine the number of APCs
capable of presenting viral Ags on their cell surface.
Left panel, Hybridoma 4943 specific for peptide
ORF61/Kb; right panel, hybridoma 49100 specific for
peptide ORF6/Db. Dendritic cells were purified by antiCD11c Ab labeling and magnetic bead enrichment
(75% purity).
The Journal of Immunology
143
Abs (Jackson Immunochemicals) for 1 h to enrich for T cells. The nonadherent cells were incubated with Fc-block (CD16/32) and washed. A total
of 2 ⫻ 106 cells were plated per well in 96-well plates and stained with
ORF6487–495/Db at 2-fold dilutions for 1 h at room temperature (RT). Cells
were washed and subsequently incubated with anti-CD8 Ab, washed, and
then fixed with 1% paraformaldehyde. The results were expressed as the
percentage of tetramer/CD8 double-positive cells over the log concentration of tetramer. To compare groups within each experiment, the data were
normalized to the response at the highest concentration of tetramer. The
affinity, KD, is derived from the slope by KD ⫽ 1/slope.
Tetramer decay. Cells were processed, enriched by panning and Fcblocked as described above. After staining with ORF6487–495/Db and antiCD8 Ab for 1 h at RT, cells were washed twice and incubated in an excess
of anti-Db 28-14-8s purified Ab and incubated at 37°C to allow tetramer
dissociation. The anti-Db Ab was used to block rebinding of the tetramer to
the TCR. Decay was followed at intervals from 0 to 50 min at which times
the cells were fixed with 1% paraformaldehyde. The results were expressed
as the percentage of tetramer/CD8 double-positive cells over time. To compare groups within each experiment, the data were normalized to the percentage of maximum tetramer binding over time. The half-life was derived
from the slope by t1/2 ⫽ ln2/slope.
Intracellular cytokine staining. Single-cell suspensions were obtained as
indicated above. A total of 2 ⫻ 106 cells/sample were incubated in com-
plete medium in the presence of IL-2 (10 U/ml), brefeldin A (10 ␮g/ml),
and 10-fold dilutions of purified ORF6487–495 peptide for 5 h at 37°C. As
positive control, cells were stimulated with PMA/ionomycin. After Fcblocking, the cells were stained with Abs anti-CD44 and anti-CD8, fixed,
permeabilized, and stained with anti-IFN-␥ or isotype control Abs. The
data were expressed as the percentage of IFN-␥/CD8 double-positive cells
over the log peptide concentration after subtracting the background obtained
from the negative controls. The data were normalized to the response at the
highest concentration of peptide. The EC50 was determined as the peptide
concentration provoking a response halfway between baseline and maximum.
In vivo cytotoxicity assay
Single-cell suspensions obtained from naive spleens were pulsed with
ORF6487– 495 peptide or irrelevant influenza NP366 –374 peptide (1 ␮g/106
cells) for 2 h at 37°C. The NP-pulsed control cells were stained with 50 nM
CFSE and the relevant targets with 0.5 ␮M CFSE for 15 min at 37°C. The
cells were thoroughly washed, combined in a 1:1 ratio, and a total of
0.5–1 ⫻ 107 cells were injected i.v. into mice. The presence of CFSEpositive cells was analyzed in spleen cell suspensions from the recipient
mice 6 or 40 h later. Percent-specific killing was calculated according to the
formula percent-specific killing ⫽ (1 ⫺ (ratio of naive recipients/ratio of
infected recipients) ⫻ 100), where ratio ⫽ (number of CFSEhigh/number of
CFSElow).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 2. Memory CD8⫹ T cells predominate during long-term infection. Temporal kinetic analysis of the effector and memory CD8⫹ T cell
response to ␥HV68. A, Representative dot plots showing the CD62L/CD43 distribution of a population of virus-specific CD8 T cells (ORF61524 –531/Kb)
at different times after infection (14 days, 3 and 18 mo). Note the progressive increase in CD62L⫹CD43⫺ ␥HV68-specific CD8⫹ T cells. Cells have
been previously gated as tetramer⫹CD8⫹. B, Evolution of the different populations of Ag-specific CD8⫹ T cells during ␥HV68 infection. CD62L⫺
CD43⫹: f; CD62L⫺CD43⫺: Œ; CD62L⫹CD43⫺: F. Data are presented for ORF6487– 495/Db-specific CD8⫹ T cells in spleen and lung. Similar
kinetics were found for ORF61524 –531/Kb-specific CD8⫹ T cells. C, All the Ag-specific CD8⫹ T cell populations analyzed in spleen and MLN were
highly heterogeneous at 9 m.p.i. No significant differences were found between lytic-cycle epitopes (ORF61524 –531/Kb and ORF6487– 495/Db) and
latent-cycle epitopes (M291–99/Kd). All the analyses were performed in C57BL/6 and BALB/c mice. Three individual mice were analyzed at each
time point; error bars represent SD.
144
T CELL MEMORY DURING VIRAL PERSISTENCE
Adoptive cell transfers
Splenocytes from BALB/cJ mice at 8 mo post-␥HV68 inoculation were
processed into single-cell suspensions as described above and stained with
anti-CD4 and CD8 Abs. Cells were purified using a FACSVantage with
Diva option and 4 ⫻ 105 purified cells (purity 99%) were transferred i.v.
into naive BALB/cJ mice. The recipient and control mice were infected
with ␥HV68 24 h later. Viral latency was determined in the spleen on
day 16.
Statistical analysis
The data were fitted to linear and nonlinear regression lines. The form with
the highest R2 was used in further comparisons between groups. Early and
late groups were compared using two-way ANOVA with group and replicate as main effects while controlling for concentration or time. The extra
sum-of-squares F test was used to compare the models and reject or not the
simpler null hypothesis model. Statistical analyses were run several times
if there were outliers in the data. One analysis included all observations. A
second analysis was run excluding outliers that were outside a range of 3
SDs. There was one observation dropped from the tetramer dilution studies, none from the tetramer decay studies, and one from the IFN-␥ studies.
The statistical analysis was performed using GraphPad Prism and SigmaStat
software.
␥HV persistence: low antigenic load
␥HVs are characterized by the establishment of lifelong asymptomatic infection by, among other strategies, persisting in a latent
state in memory B cells. There is, however, a low level of viral
reactivation from latency that is thought to contribute to an active
process of infection of new cell reservoirs and to the release of
infectious viral particles (28, 34, 38). Thus, ␥HVs represent a good
model to study immune responses during conditions of low-level
Ag persistence. To study the low levels of viral latency during
␥HV68 persistent infection, we determined the viral load in the
spleen during long-term ␥HV68 infection of mice. We used a sensitive LDA-PCR assay for ␥HV68 orf50 to detect the number of
cells carrying viral DNA. The data show that there were consistently ⬍500 latently infected cells per spleen in C57BL/6 and
BALB/c mice at 5 months postinfection (m.p.i.) (Fig. 1A). In addition, we sought to determine the number of infectious viral particles at the same times postinfection using a plaque assay. The
data show that no infectious virus was detected (Fig. 1A), indicating that there was lack of lytic virus or that the number of lytic
viral particles at 5 m.p.i. is very low and thus, below the detection
level of this assay. Altogether, these data indicate that the viral
load during long-term ␥HV68 carrier state is very low and suggest
that the levels of persistent Ag in the host must also be low. To
determine the levels of persistent Ag during long-term infection,
we used two lacZ⫹-inducible T cell hybridomas specific for two
class-I-restricted lytic-phase epitopes from the ssDNA-binding
protein (ORF6487– 495/Db) and from the large RNase reductase
subunit (ORF61524 –531/Kb) of ␥HV68. As shown in Fig. 1B, hybridoma stimulation with bulk spleen cells did not detect the presence of viral Ags on 4 m.p.i. splenocytes. However, enrichment of
CD11c⫹ cell fraction using magnetic beads induced the stimulation of ␥HV68-specific hybridomas. No stimulatory capacity could
be detected in the CD11c⫺ population. Together, these data demonstrate the during persistent ␥HV infection, a small population of
APCs, likely dendritic cells, is capable of presenting lytic cycleassociated viral Ags on the cell surface in the context of MHC
molecules.
Generation of central-memory CD8⫹ T cells during virus
persistence
Previous studies have shown that there is a shift in the composition
of the memory T cell pool from an effector-memory to a centralmemory phenotype over time (11, 12, 39). However, during
FIGURE 3. Flow cytometry analysis of Ag-specific CD8⫹ T cells. A,
Representative staining showing tetramer vs CD8 dot plots for each one of
the tetramers used in this study. B, Distribution of CD122 and CD127
staining on spleen cells; CD8⫹ T cells and tetramer⫹/CD8⫹ T cells. M291–99/
Kd staining on day 28 is shown. Gates were set using negative controls,
isotype controls, and following the staining pattern of each marker on the
bulk lymphocyte and CD8⫹ population. For any given activation marker,
the analysis gates are identical in size and x-axis position. Numbers represent the percentage of positive cells in each quadrant/gate.
chronic LCMV infection, the presence of persistent Ag is thought
to prevent T cell memory generation (15). We sought to gain a
clearer understanding of the generation and heterogeneity of different subsets of memory CD8⫹ T cells in lymphoid organs and
the respiratory tract during low-level Ag persistence using ␥HV68
infection as a model. We performed a temporal kinetic analysis of
the evolution of ␥HV68-specific CD8⫹ T cells in two different
mouse strains: C57BL/6 and BALB/c. To track Ag-specific T
cells, we used tetramers against three different CD8⫹ T cell
epitopes (lytic cycle: ORF61524 –531/Kb and ORF6487– 495/Db, latent cycle: M291–99/Kd). We analyzed the level of cell surface expression of the lymphoid homing molecule L-selectin (CD62L)
and the activation-associated glycoform of CD43 (1B11) to distinguish subpopulations of effector and memory CD8⫹ T cells.
Up-regulation of L-selectin expression on Ag-experienced cells is
a common characteristic of memory T cells and their precursors (5,
12). The activation-associated glycoform of CD43 (1B11) has
been extensively used to distinguish memory from effector T cells
in mice (40, 41). Our data show that Ag-specific CD8⫹ T cells
with a central-memory phenotype (CD62LhighCD43low) could be
readily detected at 14 days postinfection (d.p.i.) in spleen and their
frequency increased over time during the course of persistent infection (Fig. 2, A and B). Central-memory CD8⫹ T cells constituted between 15 and 50% of the ␥HV68-specific CD8⫹ T cells
independent of the tissue or epitope analyzed during long-term
infection (9 m.p.i.) (Fig. 2C). The kinetic analysis also shows that
the effector CD8⫹ T cell population (CD62LlowCD43high) contracts from day 14 postinfection until day 90 (Fig. 2B). This process is accompanied by a progressive increase in the frequency of
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Results
The Journal of Immunology
145
effector-memory T cells (CD62LlowCD43low). This shift in the
composition of the T cells from effectors (CD43high) to memory
cells (CD43low) stabilizes 3 mo postinfection and the frequency of
total memory CD8⫹ T cells plateaus and remains remarkably stable until 18 mo postinfection, constituting 70 –90% of the CD8⫹ T
cells for any epitope analyzed. There is, however, a shift in the
composition of the CD8⫹ memory T cell pool over time that starts
3 mo postinfection with central-memory T cells (CD62Lhigh
CD43low) gradually increasing their frequency while the effectormemory subset (CD62LlowCD43low) contracts (Fig. 2B). Similar
kinetics were found for ORF6487– 495/Db-specific CD8⫹ T cells
(data not shown). Altogether, the data show that virus-specific
CD8⫹ T cells primarily had an effector-memory phenotype and
were heterogeneous long-term post-virus inoculation in both lymphoid organs and the lung. In addition, a population of virus-specific CD8⫹ T cells resembling a central-memory phenotype could
be detected at early stages postinfection and progressively increased its frequency over time.
Our previous data suggest the possibility that memory CD8⫹ T
cell precursors are being generated during the initial stages of the
adaptive immune response to a ␥HV infection. To investigate this
possibility, we analyzed the expression of two of the hallmarks of
bona fide memory T cells on virus-specific CD8⫹ T cells: homeostatic-cytokine receptors (IL-7 and IL-15) (15, 39) and lymphoid
homing molecules (L-selectin and CCR7) (5, 12). The strategy for
the gating of these populations is described in Fig. 3. The analysis
was performed 14 d.p.i., which marks the peak of ␥HV68 latency
and of viral Ag expression (36, 42). As shown in Fig. 4, A and B,
subsets of ␥HV68-specific CD8⫹ T cells expressing L-selectin,
CCR7, and receptors for IL-7 and IL-2/IL-15␤ on their cell surface
could be detected in bone marrow and spleen 2 wk post-virus
inoculation. These data indicate that phenotypically “normal”
memory CD8⫹ T cell precursors are being generated during the
peak of the T cell response to a persistent ␥HV infection. It should
be noted that the analysis of C57BL/6 mice at 14 d.p.i. shows two
populations of tetramer-positive cells, one which is tetramer low
and positive for L-selectin, IL-2/IL-15␤, IL-7␣, and CCR7, and
one which is tetramer high and negative for the markers tested.
Transient loss of tetramer binding and TCR/CD8 down-regulation
has been observed after T cell activation (43– 45) and could be
explained by the asymmetric T cell lymphocyte division after
Ag stimulation (46). To determine whether memory T cell precursors generated during primary infection had a central- or
effector-memory phenotype and their anatomical distribution,
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 4. CD8⫹ memory T cells are generated
during the early phase of infection. A and B, Memory
CD8⫹ T cell precursors are detected in bone marrow
(A) and spleen (B) of 14 d.p.i. ␥HV68-infected
C57BL/6 mice. The subset of Ag-specific cells precursors that express L-selectin, CD122, CD127, and
CCR7 (x-axis) is shown inside the gate against the
binding of ORF61524 –531/Kb and ORF6487– 495/Db (yaxis). The numbers indicate the percentage of cells inside the gate. The cells have been previously gated as
CD8⫹tetramer⫹. C and D, Central and effector-memory cells are present in lymphoid organs of infected
mice 14 days after virus inoculation. The number
(C) of CD8⫹ORF6487– 495/Db⫹ lymphocytes expressing CD62LhighCD127high (central-memory
cells), CD62LlowCD127high (effector-memory cells),
and CD62LlowCD127low (effectors) and their frequency (D) among the CD8⫹ORF6487– 495/Db⫹ lymphocyte population is shown in bone marrow, MLN,
and spleen. Data correspond to six to nine individual
mice per group. Error bars, SD.
146
T CELL MEMORY DURING VIRAL PERSISTENCE
we analyzed simultaneously the presence of L-selectin and
IL-7R on ORF6487– 495/Db-specific CD8⫹ T cells in different
lymphoid tissues (Fig. 4C). Ag-specific CD8⫹ T cells consisted
of cells with an effector phenotype (CD62LlowCD127low), centralmemory phenotype (CD62LhighCD127high), and effector-memory
phenotype (CD62LlowCD127high) in mediastinal lymph node (MLN),
spleen, and bone marrow. The analysis shows that memory populations constitute a significant fraction of the cells in the lymphoid tissues analyzed. Although most of the memory cells are found in the
spleen, Fig. 4D shows that their frequencies are higher in bone marrow and MLN than in spleen (CD62LhighCD127high: bone marrow
8.7%, MLN 10.7%, and spleen 3.3%; CD62LlowCD127high: bone
marrow 9.8%, MLN 12.1%, and spleen 7.3%). Similar numbers and
frequencies were obtained using CD43 and CD62L to divide the subpopulations of Ag-specific cells (data not shown). Taken together,
these results provide strong evidence that during primary ␥HV68 infection CD8⫹ memory T cell precursors with a central-memory and
effector-memory phenotype are being generated within lymphoid
tissues.
Memory CD8⫹ T cells express homeostatic cytokine receptors
and lymphoid homing molecules during virus persistence
Continuous Ag stimulation prevents the expression of homeostatic
cytokine receptors and lymphoid homing molecules on CD8⫹
memory T cells during LCMV persistence, which partially contributes to explain their dysfunction (15). Thus, we analyzed the
expression of L-selectin, CCR7, IL-2/IL-15␤ receptor, and IL-7R
on ␥HV68-specific CD8⫹ T cells during long-term infection. The
analysis of CD8⫹ T cells specific for two lytic cycle epitopes
(ORF61524 –531/Kb, ORF6487– 495/Db) during long-term infection
of C57BL/6 mice shows that virus-specific CD8⫹ T cells have
down-regulated the expression of IL-2/IL-15␤ receptor, with only
a small fraction of the tetramer-positive cells expressing CD122 on
their cell surface (1– 4%) (Fig. 5). However, the majority of the
virus-specific CD8⫹ T cells show surface expression of the IL-7R
(70 – 80%). In addition, a subpopulation of the Ag-specific CD8⫹
T cells express the lymphoid homing molecules L-selectin and
CCR7 on their cell surface (20 –30%). These data are in contrast
with results from other chronic infections (15) and indicate that
CD8⫹ T cells expressing molecular determinants of central-memory cells are maintained during persistent ␥HV68 infection. To
determine whether the down-regulation of the IL-2/IL-15␤ receptor is specific for lytic cycle Ags or it also occurs on CD8⫹ T cells
specific for latent cycle epitopes, we did a temporal kinetic analysis of ␥HV68-specific CD8 T cells in BALB/c mice comparing
the response of a lytic-epitope (ORF65131–140/Dd) and a latent
epitope (M291–99/Kd). As shown in Fig. 6, CD8⫹ T cells specific
for the latent epitope M291–99/Kd maintain the expression of the IL2/IL-15␤ receptor over time. Lytic epitope-specific CD8⫹ T cells,
however, progressively down-regulate the surface expression of the
IL-2/IL-15␤ receptor. Both populations of CD8⫹ T cells maintain the
expression of IL-7R and L-selectin. Together, these data indicate that
during long-term persistent ␥HV infection, memory CD8⫹ T cells
retain the expression of homeostatic cytokine receptors (IL-7) and
lymphoid homing molecules (L-selectin, CCR7) at their cell surface.
However, CD8⫹ T cells specific for lytic cycle Ags (ORF65131–140/
Dd, ORF61524 –531/Kb, and ORF6487– 495/Db) specifically down-regulate CD122 surface expression.
Ag-specific CD8⫹ T cells maintain TCR affinity during virus
persistence
High-avidity CD8⫹ T cells are thought to play a major role in
terminating viral infections (47, 48). The kinetics and affinity of
TCR-peptide/MHC interactions drive the selection of TCR clones
during the course of an immune response resulting in an overall
increased affinity for Ag (49). This mechanistic model suggests
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 5. Lytic epitope-specific
memory CD8⫹ T cells express L-selectin, CCR7, and IL-7␣ receptor but
down-regulate IL-2/IL-15␤ receptor
expression during long-term infection. Analysis of bone marrow and
spleen cells from mice at 9 m.p.i. with
␥HV68. Cells were previously gated
as CD8⫹tetramer⫹: ORF6487– 495/Db
is shown on A and ORF61524 –531/Kb
on B. The plots show tetramer staining (y-axis) vs memory markers
(CD62L, CD122, CD127, and CCR7
on x-axis). Note that the expression of
IL-2/IL-15␤ receptor is down-regulated while the rest of memory markers remain expressed at significant
levels. The numbers indicate the percentage of cells inside the gate. Representative plots from three independent experiments with similar results
are shown.
The Journal of Immunology
that continuous Ag stimulation influences the CD8⫹ T cell pool by
inducing changes in TCR usage or affinity that translate in the
selection of high-affinity immunodominant clones (50). Other
studies show that high-avidity CD8⫹ T cells are more susceptible
to apoptosis or clonal senescence under conditions of excessive Ag
load (51, 52) which may explain a decrease in immunodominance
and clonal dominance during memory to herpesvirus persistence
compared with primary responses (53). In addition, the down-regulation of the IL-2/IL-15B receptor expression on lytic Ag-specific
CD8⫹ T cells during persistent ␥HV68 infection may be related to
a decrease in clonal affinity. IL-15/IL-15R signaling mimics TCR
cross-linking (54), activates CD8⫹ T cells (55), and mediates avidity maturation of CD8⫹ T cells at the single cellular level by increasing TCR functional affinity (56). Thus, it is possible that in
the presence of low levels of persistent Ag, high-avidity memory
CD8⫹ T cells are being continuously stimulated by both Ag and
IL-15 and driven to clonal exhaustion.
High-avidity CD8⫹ T cells bind preferentially under conditions
of limited peptide/MHC complex availability (57, 58). To compare
the relative TCR affinity of ORF6487– 495/Db-specific CD8⫹ T cells
during early (14 –35 days) and long-term infection (at least
3 m.p.i.), we performed a dose-response titration using tetramer
dilution under conditions ranging from saturation to limiting availability (Fig. 7A). The data show that the normalized titration
curves for early and late time points postinfection were superimposable (Fig. 7B). The analysis of the data indicated the fit was the
same for linear and nonlinear approaches. Thus, in this situation,
the simpler linear model was chosen (R2-early: 0.9887, R2-late:
0.9674). The slopes, elevations, and y intercepts of both curves
were not significantly different and a common slope (9.2409) and
common y intercept for all the data (95.6591) could be determined.
There was no effect of the time postinfection on the affinity of
tetramer binding (KD-early: 0.1017, KD-late: 0.1157). These results indicate that there was no significant change in the average
TCR peptide/MHC affinity over time between primary infection
and ␥HV68 persistence.
The half-life of the TCR-peptide/MHC complex is related to the
strength of signaling (59, 60) and the rate of dissociation of peptide-MHC tetrameric reagents from the T cell surface is related to
the off rate of the TCR-peptide/MHC interaction (49). Thus, we used
tetramer decay analysis to compare the binding of ORF6487– 495/Db to
␥HV68-specific CD8⫹ T cells during early and long-term infection by measuring the dissociation rate using flow cytometry
(Fig. 7C). The normalized results show that there were no major
differences in the rate of tetramer dissociation time between
CD8⫹ T cells analyzed at early and late time points during
infection (Fig. 7D). The mathematical analysis indicated a better fit for the nonlinear one-phase exponential decay function
(R2-early: 0.9481, R2-late: 0.9510), so that the functional form
was used in comparing groups. The comparison of fit indicated
that the preferred model was one common curve fitting all data
sets independently of group and a common half-life value was
determined as 17.19 min. (K: 0.0403). Altogether, the tetramer
dilution and tetramer decay analyses demonstrate that Ag-specific CD8⫹ T cells exhibit similar TCR-peptide/MHC interaction affinities during primary and persistent ␥HV68 infection.
These data suggest that persistent Ag expression during ␥HV68
infection does not substantially modify the TCR affinity of
CD8⫹ T cells.
Ag-specific CD8⫹ T cells maintain functional avidity during
virus persistence
The functional avidity of CD8⫹ T cells is mediated by the interaction between TCR/CD8␤ and peptide/MHC complexes but also
by several other mechanisms such as the contribution of accessory
molecules in lipid raft recruitment of signaling proteins and regulatory pathways (61– 63). Persistent viral infections are thought to
lead to different degrees of CD8⫹ T cell dysfunction (15, 16, 33).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 6. Latent epitope-specific CD8⫹ T cells maintain the expression of lymphoid homing molecules and homeostatic cytokine receptors
during long-term infection. Contour plots show the temporal evolution of
memory CD8⫹ T cells in BALB/c mice for a latent epitope (M291–99/Kd,
A) and for a lytic epitope (ORF65131–140/Dd, B). Spleen cells were previously gated as CD8⫹tetramer⫹. Tetramer staining is plotted on the y-axis
and the specific memory marker on the x-axis (CD122, CD127, CD62L).
Note that memory CD8⫹ T cells specific for the latent epitope maintain
expression of the memory markers while cells specific for the lytic epitope
progressively lose surface expression of IL-2/IL-15␤ receptor by day 90
after infection. Representative plots from three independent experiments
with similar results are shown. C, Bar diagrams show the frequency of
IL-2/IL-15␤-expressing cells for the two epitopes described above. Error
bars, SD. ⴱ, p ⬍ 0.05.
147
148
T CELL MEMORY DURING VIRAL PERSISTENCE
This progressive decrease in T cell function during viral persistence has been partially attributed to triggering of inhibitory pathways (17, 64). To compare the functional capabilities of ␥HV68
primary and long-term CD8⫹ T cell responses, we sought to determine their effectiveness in IFN-␥ secretion or in cytotoxic killing. These two functions were chosen because they characterize
the early (cytotoxicity) and final (IFN-␥) stages of CD8⫹ T cell
functional impairment (20).
We measured the capacity of ␥HV68-specific CD8⫹ T cells to
secrete IFN-␥ in response to saturating and limiting availability of
peptide at early and late times postinfection. The data show intracellular IFN-␥ staining of splenic ORF6487– 495/Db-specific CD8⫹
T cells (Fig. 8A). The data points show that the normalized titration
curves for early and late time points postinfection overlapped (Fig.
8B). A better fit of IFN-␥ production data over peptide concentration was obtained with a nonlinear sigmoidal dose-response curve
analysis (R2-early: 0.8096, R2-late: 0.9082). The comparison of fits
indicated that the preferred model was one common curve fitting
all data sets independently of group and a common peptide concentration provoking a response halfway between baseline and
maximum was determined (EC50: 2.229 ⫻ 10⫺4). There were no
significant differences in IFN-␥-secreting capacity of each CD8⫹ T
cell population defined as the negative log of the peptide concentration that resulted in 50% maximal IFN-␥ production. These data
indicate that Ag persistence during ␥HV infection has no detri-
mental effect on the functional capacity of virus-specific CD8⫹ T
cells to produce IFN-␥ in response to antigenic stimulus.
We next compared the cytotoxic activity of ORF6487– 495/Dbspecific CD8⫹ T cells directly in vivo in mice 3 wk, 3 mo, and 8
mo postinfection. The elimination of ORF6487– 495-loaded targets
was assessed 6 and 40 h after transfer. Specific target elimination
occurred with faster and more efficient kinetics in mice at 3 wk
postinfection, which correlated with a higher frequency of
ORF6487– 495/Db-specific CD8⫹ T cells (Fig. 9). Mice at 3 and 8
mo postinfection had low specific killing rates 6 h after transfer
(5–14% specific killing) but were capable of killing specific targets
after a 40-h interval (65– 88% specific killing). In addition, no
major differences in specific killing were found between mice at 3
and 8 mo postinfection. It should be noted that 1) during long-term
infection, the frequency of splenic ORF6487– 495/Db-specific CD8⫹
T cells is 5- to 20-fold lower than during acute infection (Fig. 9)
and 2) at 3 and 8 m.p.i., the ␥HV68-specific CD8⫹ T cell pool
predominantly consists of effector-memory cells (as shown in Fig.
2). Altogether, these data indicate that CD8⫹ T cells during persistent ␥HV68 infection are capable of mounting a specific CTL
response in vivo although with slower kinetics that during acute
infection. The analysis of IFN-␥ production and cytotoxicity provides evidence of the functional capacity of virus-specific CD8⫹ T
cells during persistent infection.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 7. The TCR affinity of virus-specific CD8⫹ T cells does not change from primary infection to viral persistence. A and B, Measurement of TCR
affinity in CD8⫹ORF6487– 495/Db⫹ cells by peptide/MHC tetramer dilution analysis. Lymphocytes were obtained from spleens of mice infected 14 days
before (early group) or 3 mo before (late group), pooled, enriched by panning, and stained with serial 2-fold dilutions of ORF6487– 495/Db tetramer
conjugated to APC at concentrations ranging from 1/50 to 1/100,000. All the cells were subsequently stained with anti-CD8 Ab. Data are representative
from three independent experiments with similar results. A, Representative FACS plots of the CD8-ORF6487– 495/Db staining. B, Graphic representation of
the normalized frequency of tetramer binding and linear regression analysis for the experiment shown in A. To normalize the groups, the data were expressed
as the percentage of maximum tetramer binding. Inset, Raw data. C and D, Measurement of TCR affinity in CD8⫹ORF6487– 495/Db⫹ cells as determined
by peptide/MHC tetramer dissociation. Lymphocytes were sampled and processed as above, stained with ORF6487– 495/Db tetramer, washed, saturated with
anti-Db Ab, and incubated at RT to allow for tetramer dissociation. At different time points cells were sampled and stained with anti-CD8 Ab. Data are
representative from two independent experiments with similar results. C, Representative FACS plots of the CD8-ORF6487– 495/Db staining at different time
points during tetramer decay. D, Graphic representation of the normalized frequency of tetramer binding over time and nonlinear one-phase exponential
decay regression analysis of the experiment shown in C. To normalize the groups, the data were expressed as the percentage of maximum tetramer binding.
Inset, Raw data.
The Journal of Immunology
149
latency (Fig. 10). In addition, the data revealed that the addition of
CD4⫹ T cells did not improve the recall response of CD8⫹ T cells
as measured by protection. These data show that memory CD8⫹ T
cells from ␥HV68-infected mice can respond to a secondary virus
challenge by decreasing the number of latently infected cells in the
spleen during the establishment of latency phase.
Discussion
CD8⫹ T cells generated during long-term viral persistence can
mediate protection to ␥HV68 latency during a recall response
Memory T cells mediate faster and more effective responses to
secondary pathogen challenge than naive T cells (1, 2). To determine whether T lymphocytes from ␥HV68-immune mice were capable of preventing the establishment of viral latency, we tested
the capacity of CD8⫹ T cells from ␥HV68-infected mice to protect
during a recall response. CD8⫹ T cells were FACS purified from
BALB/c mice that were intranasally inoculated with 1000 PFU of
␥HV68 8 mo before. During the sorting procedure, B cells were
excluded by CD19 staining to avoid carrying latently infected B
cells in the purified populations. A total of 4 ⫻ 105 CD8⫹ T cells
or a 1:1 mixture of CD4⫹ and CD8⫹ T cells were i.v. inoculated
in 300 ␮l of PBS into naive BALB/c recipients. The recipient mice
were intranasally inoculated with ␥HV68 the following day and
viral splenic latency was analyzed at the peak of latently infected
B cell expansion on day 16. The results show that adoptive transfer
of CD8⫹ T cells from persistently infected mice reduces the number of ␥HV68-infected cells during the establishment of splenic
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 8. The functional avidity of virus-specific CD8⫹ T cells does
not change from primary infection to viral persistence as measured by their
IFN-␥ production capacity. Lymphocytes were obtained from spleens of
three to four mice infected 5 wk before (early group) or 3 mo before (late
group), pooled, enriched by panning, and stimulated with different concentrations of ORF6487– 495 peptide. All the cells were subsequently processed for IFN-␥ intracellular cytokine staining and surface stained with
anti-CD8 Ab. Data are representative from three independent experiments.
A, Representative FACS plots of the intracellular IFN-␥ staining at two
peptide concentrations for the early and late groups. B, Graphic representation of the normalized frequency of tetramer binding over time and nonlinear sigmoidal dose response regression analysis of the experiment
shown in A. To normalize the groups, the data were expressed as the percentage of maximum IFN-␥ staining at 1 ␮g/ml. Inset, Raw data minus
background (isotype-control intracellular staining).
Persistent infectious diseases are a major health concern (13, 14).
The general consensus is that chronic viral infections lead to some
degree of CD8⫹ T cell effector dysfunction or deletion and lack of
T cell memory formation (15–17). ␥HVs are very successful
pathogens that persistently infect ⬎90% of the human population
and initial infection may occur early in childhood (28). In this
report, we demonstrate that herpesvirus-specific central-memory
CD8⫹ T cells develop during infection and that herpesvirus-specific CD8⫹ T cells maintain functional and protective capacities
during long-term persistent infection.
Our results show that during persistent infection with ␥HV68,
there are consistently less than 500 latently infected cells per
spleen. Despite this very low level of viral latency, splenic dendritic cells but not B cells are capable of presenting viral lyticphase epitopes to ␥HV68-specific CD8⫹ T cell hybridomas. This
finding is unexpected as germinal center and memory B cells constitute the major viral reservoir during long-term latency (65, 66).
It suggests that dendritic cells cross-present lytic-phase viral Ags
during ␥HV persistence and supports the existence of continuous
viral reactivation during the latency phase of infection (38, 67, 68).
Reduced levels of latency and continuous presentation of lyticepitope Ags have relevant implications for the maintenance of
CD8⫹ T cell memory. Low-level latency together with low viral
gene expression (69) probably represents a very efficient immune
escape strategy and explains the low frequency of latent-phase
epitope-specific CD8⫹ T cells during ␥HV68 persistence (70).
Continuous presentation of lytic viral epitopes explains the delayed generation of central-memory CD8⫹ T cells and why effectors and effector-memory cells still constitute a large fraction of the
CD8⫹ T cell response during long-term latency (50 – 80%, Figs. 2,
4, and 5). It is possible that these lytic epitope-specific CD8⫹ T
cells partially represent secondary effectors or de novo generated
effectors as result of virus reactivation. In this sense, it has been
recently suggested that low-level TCR stimulation that occurs during HSV latency results in the maintenance of an activated but
functional CD8-memory pool (71) and that newly recruited naive
CD8⫹ T cells influence the heterogeneous CD8⫹ T cell response
during polyoma virus persistence (72).
Our data show that memory CD8⫹ T cell precursors could be
detected at early stages during primary infection and that effectormemory cells constitute the dominant CD8⫹ T cell subset once
primary infection has been resolved. The kinetic analysis of the
␥HV68-specific CD8⫹ T cell response shows that activated CD8⫹
T cells (CD62LlowCD43high) contract over a period of 3 mo postviral inoculation. This slow process can be in part explained for the
prolonged infectious mononucleosis-like syndrome in ␥HV68-infected mice (73) which results in exacerbated and prolonged CD8⫹
T cell activation and expansion (74, 75). In addition, ␥HV68 persistent replication and reactivation from latency (34, 76, 77) will
contribute to keep virus-specific CD8⫹ T cells stimulated. Although the frequency of memory CD8⫹ T cells remains stable
during long-term viral persistence, there is a shift in the ratio between effector-memory and central-memory subsets, with centralmemory cells progressively increasing their frequency over time.
Selective expression of IL-7R identifies CD8⫹ T cells that generate memory cells (78, 79). In addition to this marker, we have also
150
T CELL MEMORY DURING VIRAL PERSISTENCE
FIGURE 10. Long-term CD8⫹ T cells can a mediate protective recall
response to ␥HV latency. Spleen cells from BALB/c donor mice
8 m.p.i. were sorted by FACS. A total of 4 ⫻ 105 immune CD8⫹ or a
1:1 mixture of CD4⫹/CD8⫹ cells was adoptively transfer by i.v. injection into naive mice. Control mice received no cells. Mice were challenged with ␥HV68 24 h later. The number of latently infected spleen
cells was determined by an infectious center assay on day 16 after
challenge. Error bars represent SD.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 9. Long-term CD8⫹ T cells maintain cytotoxic capacity during persistent viral infection. Cytotoxicity was assessed by an in vivo CTL assays. Spleen
cells from naive mice were loaded with ␥HV68
ORF6487– 495 or with influenza NP peptides and stained
with different amounts of CFSE before injection into
naive control mice or into mice at different time points
after ␥HV68 inoculation. Killing was calculated 6 h (left
column) or 40 h (middle column) after transfer. Histograms show representative killing data from the analysis
of individual naive mice and from mice at 3 wk, 3 mo,
and 8 m.p.i. Bar diagrams show specific killing data
from four to five individual mice per time point. The
frequency of ORF6487– 495/Db⫹CD8⫹ T cells was determined by FACS staining of spleen cells and is shown on
the right column as representative dot plots and as a bar
diagram. Error bars, SD.
identified memory precursors using the expression of the IL-15R
and the lymphoid homing molecules L-selectin and CCR7 (15).
Our data correlate with results from acute and persistent infections
that demonstrate that memory cell precursors exist at the peak of
the CD8⫹ T cell response (39, 79, 80). The generation and stability
of CD8⫹ T cell memory or CD8⫹ T cell function during Ag persistence is poorly understood. It has been suggested that long-term
T cell memory might not develop in conditions of chronic Ag
stimulation (6). Infections with high load of persisting Ag result in
the suppression of IL-7 and IL-15 receptor expression and downregulation of lymphoid homing molecules on virus-specific CD8⫹
T cells, and this correlates with progressive T cell exhaustion (15,
20, 81). Our results show that IL-7R, L-selectin, and CCR7 expression are maintained on 20 –70% of Ag-specific CD8⫹ T cells
during viral persistence 9 m.p.i. This represents a small fraction of
the whole CD8⫹ population compared with that of acute respiratory infections, where most of the CD8⫹ T cell population is constituted by memory cells (82). It is also possible that small differences on the level of expression of phenotypic markers between
The Journal of Immunology
contribution of long-term T cell populations to the control of viral
latency. Although Ag persistence has been linked to poor CD8⫹ T
cell cytotoxic and recall responses (15), this was not the case during ␥HV68 infection. CD8⫹ T cells from persistently infected
mice were capable of mounting cytotoxic responses in vivo, albeit
with slower kinetics than during acute infection. These differences
can be explained by the reduced frequency of Ag-specific CD8⫹ T
cells and the contraction of effector cells after 3 m.p.i. In addition,
long-term CD8⫹ T cells were capable of eliciting protection
against the establishment of viral latency after adoptive transfer,
which supports the maintenance of CD8⫹ T cell function during
␥HV68 persistence. Our findings indicate that protective memory
can be generated during herpesvirus infections and suggest that the
model of CD8⫹ T cell linear differentiation that postulates centralmemory T cells as the end of a maturation process (12) also applies
with an extended time frame to infections with low levels of persistent Ag. These findings have important implications for our understanding of immune control and protection against pathogens or
Ags that persist in the host and for the design of vaccination
strategies.
Acknowledgments
We thank C. McAllister and the Morphology Core for assistance with
FACS sorting, J. Haynes and the Biostatistics Core for help with the statistical analysis, J. Cyster for CCL19 Ig, C. Walker for critical comments
to the manuscript, and M. Blackman for her continued support.
Disclosures
The authors have no financial conflict of interest.
References
1. Dutton, R. W., L. M. Bradley, and S. L. Swain. 1998. T cell memory. Annu. Rev.
Immunol. 16: 201–223.
2. Woodland, D. L. 2003. Cell-mediated immunity to respiratory virus infections.
Curr. Opin. Immunol. 15: 430 – 435.
3. Kaech, S. M., and R. Ahmed. 2001. Memory CD8⫹ T cell differentiation: initial
antigen encounter triggers a developmental program in naive cells. Nat. Immunol.
2: 415– 422.
4. Sprent, J., and D. F. Tough. 2001. T cell death and memory. Science 293:
245–248.
5. Lefrancois, L., and D. Masopust. 2002. T cell immunity in lymphoid and nonlymphoid tissues. Curr. Opin. Immunol. 14: 503–508.
6. Lanzavecchia, A., and F. Sallusto. 2005. Understanding the generation and function of memory T cell subsets. Curr. Opin. Immunol. 17: 326 –332.
7. Lefrancois, L. 2006. Development, trafficking, and function of memory T-cell
subsets. Immunol. Rev. 211: 93–103.
8. Badovinac, V. P., and J. T. Harty. 2006. Programming, demarcating, and manipulating CD8⫹ T-cell memory. Immunol. Rev. 211: 67– 80.
9. Robertson, J. M., M. MacLeod, V. S. Marsden, J. W. Kappler, and P. Marrack.
2006. Not all CD4⫹ memory T cells are long lived. Immunol. Rev. 211: 49 –57.
10. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two
subsets of memory T lymphocytes with distinct homing potentials and effector
functions. Nature 401: 708 –712.
11. Roberts, A. D., K. H. Ely, and D. L. Woodland. 2005. Differential contributions
of central and effector memory T cells to recall responses. J. Exp. Med. 202:
123–133.
12. Wherry, E. J., V. Teichgraber, T. C. Becker, D. Masopust, S. M. Kaech, R. Antia,
U. H. Von Andrian, and R. Ahmed. 2003. Lineage relationship and protective
immunity of memory CD8 T cell subsets. Nat. Immunol. 3: 225–234.
13. Morens, D. M., G. K. Folkers, and A. S. Fauci. 2004. The challenge of emerging
and re-emerging infectious diseases. Nature 430: 242–249.
14. Rappuoli, R. 2004. From Pasteur to genomics: progress and challenges in infectious diseases. Nat. Med. 10: 1177–1185.
15. Wherry, E. J., D. L. Barber, S. M. Kaech, J. N. Blattman, and R. Ahmed. 2004.
Antigen-independent memory CD8 T cells do not develop during chronic viral
infection. Proc. Natl. Acad. Sci. USA 101: 16004 –16009.
16. Zajac, A. J., J. N. Blattman, K. Murali-Krishna, D. J. Sourdive, M. Suresh,
J. D. Altman, and R. Ahmed. 1998. Viral immune evasion due to persistence of
activated T cells without effector function. J. Exp. Med. 188: 2205–2213.
17. Barber, D. L., E. J. Wherry, D. Masopust, B. Zhu, J. P. Allison, A. H. Sharpe,
G. J. Freeman, and R. Ahmed. 2006. Restoring function in exhausted CD8 T cells
during chronic viral infection. Nature 439: 682– 687.
18. Day, C. L., D. E. Kaufmann, P. Kiepiela, J. A. Brown, E. S. Moodley, S. Reddy,
E. W. Mackey, J. D. Miller, A. J. Leslie, C. DePierres, et al. 2006. PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease
progression. Nature 443: 350 –354.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
memory cells generated during acute infections or ␥HV68 persistence may be biologically relevant. Nevertheless, virus-specific
CD8⫹ T cells with characteristics of memory cells are maintained
during long-term persistent ␥HV68 infection, which suggests that
T cells may not need to fully rest from Ag exposure during conditions of low-level Ag persistence to differentiate into memory T
cells expressing lymphoid homing molecules and homeostatic cytokine receptors. It should be noted that differences in Ag load, T
cell precursor frequency, or TCR avidity can also influence the
expression of various phenotypic markers (such as CD62L and
IL-7R) and the capacity to generate a diversity of cytokines (such
as IFN-␥, TNF-␣, and IL-2), suggesting that the phenotype of
memory T cells is ultimately a consequence of molecular events
occurring during the Ag-driven phase of the response (83, 84).
IL-15 is implicated in CD8⫹ T cell proliferation, survival, and
avidity (56, 85, 86). Our data indicate that IL-2/IL-15␤ receptor
expression is down-regulated on lytic epitope-specific CD8⫹ T
cells during long-term ␥HV68 persistence, which contrasts with its
expression on latent epitope-specific CD8⫹ T cells. Interestingly,
EBV infectious mononucleosis induces a permanent deficit in IL15R expression on CD8⫹ T cells (87). It has been shown that
␥HV68-specific CD8⫹ T cell maintenance can be independent of
IL-15 (88). The functional implications of impaired IL-15 signaling during ␥HV infections are unclear and we could not find functional deficiencies at the level of IFN-␥ production and cytotoxic
capacity on lytic epitope-specific CD8⫹ T cells. However, functional and phenotypic differences between latent and lytic Ag-specific CD8⫹ T cells have been described elsewhere (89 –91).
The interaction between herpesviruses and the host presents several specific characteristics that are relevant to the analysis of the
CD8⫹ T cell response: 1) continuous presence of persistent Ag
may impact the evolution of the cognate response, 2) ␥HV persistence is characterized by low-level viral latency and antigenic load,
and T cells should compete for this limited resource, and 3) antigenic mutation is not likely to affect the T cell response. Our findings indicate that Ag persistence during ␥HV68 infection does not
modify TCR affinity and it has no detrimental effect on the functional capacity of virus-specific CD8⫹ T cells to produce IFN-␥ or
to kill in response to antigenic stimulus. These results are in concordance with data from murine CMV infection that suggest that
CD8⫹ T cell avidity does not change over time (92) and with
studies that indicate that the TCR memory repertoire to persistent
herpesvirus infections, although heterogeneous, can be remarkably
stable (50, 93). Altogether, these findings derived from herpesvirus
infections argue against the idea that the continuous presence of
Ag will eventually drive T cells into some degree of functional
exhaustion (16, 20, 21). These differences are likely to be explained by the low-level Ag persistence during herpesvirus infections. In this sense, during HSV latency in neurons, virus-specific
CD8⫹ T memory cells retain their capacity to produce IFN-␥ (94).
Although the TCR usage between T cells during primary and
memory ␥HV responses is similar, there is a shift in clonotypes
during memory formation (95). ␥HV68 persistence has also been
associated with enhanced effector functions when compared with
nonpersistently infected mice (96). These conflicting results may
reflect differences in the experimental systems and differences between human and murine studies. Regardless, these studies shed
light on the idea that CD8⫹ T cells during herpesvirus persistence
maintain their functional ability to control infection and suggest
that low-level Ag persistence may not intrinsically be a detrimental
factor for T cell function. It should also be noted that this process
might be mediated by the continuous recruitment of naive T cells
during viral persistence (72). Our analysis of CD8⫹ T cell protection using adoptive transfers allowed us to determine the specific
151
152
47. Alexander-Miller, M. A., G. R. Leggatt, and J. A. Berzofsky. 1996. Selective
expansion of high- or low-avidity cytotoxic T lymphocytes and efficacy for adoptive immunotherapy. Proc. Natl. Acad. Sci. USA 93: 4102– 4107.
48. Gallimore, A., T. Dumrese, H. Hengartner, R. M. Zinkernagel, and H. G.
Rammensee. 1998. Protective immunity does not correlate with the hierarchy of
virus-specific cytotoxic T cell responses to naturally processed peptides. J. Exp.
Med. 187: 1647–1657.
49. Savage, P. A., J. J. Boniface, and M. M. Davis. 1999. A kinetic basis for T cell
receptor repertoire selection during an immune response. Immunity 10: 485– 492.
50. Price, D. A., J. M. Brenchley, L. E. Ruff, M. R. Betts, B. J. Hill, M. Roederer,
R. A. Koup, S. A. Migueles, E. Gostick, L. Wooldridge, et al. 2005. Avidity for
antigen shapes clonal dominance in CD8⫹ T cell populations specific for persistent DNA viruses. J. Exp. Med. 202: 1349 –1361.
51. Alexander-Miller, M. A., G. R. Leggatt, A. Sarin, and J. A. Berzofsky. 1996.
Role of antigen, CD8, and cytotoxic T lymphocyte (CTL) avidity in high dose
antigen induction of apoptosis of effector CTL. J. Exp. Med. 184: 485– 492.
52. Anderton, S. M., C. G. Radu, P. A. Lowrey, E. S. Ward, and D. C. Wraith. 2001.
Negative selection during the peripheral immune response to antigen. J. Exp.
Med. 193: 1–11.
53. Davenport, M. P., C. Fazou, A. J. McMichael, and M. F. Callan. 2002. Clonal
selection, clonal senescence, and clonal succession: the evolution of the T cell
response to infection with a persistent virus. J. Immunol. 168: 3309 –3317.
54. Liu, K., M. Catalfamo, Y. Li, P. A. Henkart, and N. P. Weng. 2002. IL-15 mimics
T cell receptor crosslinking in the induction of cellular proliferation, gene expression, and cytotoxicity in CD8⫹ memory T cells. Proc. Natl. Acad. Sci. USA
99: 6192– 6197.
55. Weng, N. P., K. Liu, M. Catalfamo, Y. Li, and P. A. Henkart. 2002. IL-15 is a
growth factor and an activator of CD8 memory T cells. Ann. NY Acad. Sci. 975:
46 –56.
56. Oh, S., L. P. Perera, D. S. Burke, T. A. Waldmann, and J. A. Berzofsky. 2004.
IL-15/IL-15R␣-mediated avidity maturation of memory CD8⫹ T cells. Proc.
Natl. Acad. Sci. USA 101: 15154 –15159.
57. Yee, C., P. A. Savage, P. P. Lee, M. M. Davis, and P. D. Greenberg. 1999.
Isolation of high avidity melanoma-reactive CTL from heterogeneous populations using peptide-MHC tetramers. J. Immunol. 162: 2227–2234.
58. Daniels, M. A., and S. C. Jameson. 2000. Critical role for CD8 in T cell receptor
binding and activation by peptide/major histocompatibility complex multimers.
J. Exp. Med. 191: 335–346.
59. Alam, S. M., P. J. Travers, J. L. Wung, W. Nasholds, S. Redpath, S. C. Jameson,
and N. R. Gascoigne. 1996. T-cell-receptor affinity and thymocyte positive selection. Nature 381: 616 – 620.
60. Lyons, D. S., S. A. Lieberman, J. Hampl, J. J. Boniface, Y. Chien, L. J. Berg, and
M. M. Davis. 1996. A TCR binds to antagonist ligands with lower affinities and
faster dissociation rates than to agonists. Immunity 5: 53– 61.
61. Konig, R. 2002. Interactions between MHC molecules and co-receptors of the
TCR. Curr. Opin. Immunol. 14: 75– 83.
62. Davis, S. J., and P. A. van der Merwe. 2006. The kinetic-segregation model: TCR
triggering and beyond. Nat. Immunol. 7: 803– 809.
63. Greenwald, R. J., G. J. Freeman, and A. H. Sharpe. 2005. The B7 family revisited. Annu. Rev. Immunol. 23: 515–548.
64. Moser, J. M., J. Gibbs, P. E. Jensen, and A. E. Lukacher. 2002. CD94-NKG2A
receptors regulate antiviral CD8⫹ T cell responses. Nat. Immunol. 3: 189 –195.
65. Flano, E., I. J. Kim, D. L. Woodland, and M. A. Blackman. 2002. ␥-herpesvirus
latency is preferentially maintained in splenic germinal center and memory B
cells. J. Exp. Med. 196: 1363–1372.
66. Kim, I. J., E. Flano, D. L. Woodland, F. E. Lund, T. D. Randall, and
M. A. Blackman. 2003. Maintenance of long term ␥-herpesvirus B cell latency is
dependent on CD40-mediated development of memory B cells. J. Immunol. 171:
886 – 892.
67. Laichalk, L. L., and D. A. Thorley-Lawson. 2005. Terminal differentiation into
plasma cells initiates the replicative cycle of Epstein-Barr virus in vivo. J. Virol.
79: 1296 –1307.
68. Kim, I. J., C. E. Burkum, T. Cookenham, P. L. Schwartzberg, D. L. Woodland,
and M. A. Blackman. 2007. Perturbation of B cell activation in SLAM-associated
protein-deficient mice is associated with changes in gammaherpesvirus latency
reservoirs. J. Immunol. 178: 1692–1701.
69. Virgin, H. W. t., R. M. Presti, X. Y. Li, C. Liu, and S. H. Speck. 1999. Three
distinct regions of the murine gammaherpesvirus 68 genome are transcriptionally
active in latently infected mice. J. Virol. 73: 2321–2332.
70. Usherwood, E. J., D. J. Roy, K. Ward, S. L. Surman, B. M. Dutia,
M. A. Blackman, J. P. Stewart, and D. L. Woodland. 2000. Control of gammaherpesvirus latency by latent antigen-specific CD8⫹ T cells. J. Exp. Med. 192:
943–952.
71. Sheridan, B. S., K. M. Khanna, G. M. Frank, and R. L. Hendricks. 2006. Latent
virus influences the generation and maintenance of CD8⫹ T cell memory. J. Immunol. 177: 8356 – 8364.
72. Vezys, V., D. Masopust, C. C. Kemball, D. L. Barber, L. A. O’Mara,
C. P. Larsen, T. C. Pearson, R. Ahmed, and A. E. Lukacher. 2006. Continuous
recruitment of naive T cells contributes to heterogeneity of antiviral CD8 T cells
during persistent infection. J. Exp. Med. 203: 2263–2269.
73. Blackman, M. A., E. Flano, E. Usherwood, and D. L. Woodland. 2000. Murine
␥-herpesvirus-68: a mouse model for infectious mononucleosis? Mol. Med. Today 6: 488 – 490.
74. Tripp, R. A., A. M. Hamilton-Easton, R. D. Cardin, P. Nguyen, F. G. Behm,
D. L. Woodland, P. C. Doherty, and M. A. Blackman. 1997. Pathogenesis of an
infectious mononucleosis-like disease induced by a murine ␥-herpesvirus: role
for a viral superantigen? J. Exp. Med. 185: 1641–1650.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
19. Trautmann, L., L. Janbazian, N. Chomont, E. A. Said, S. Gimmig, B. Bessette,
M. R. Boulassel, E. Delwart, H. Sepulveda, R. S. Balderas, et al. 2006. Upregulation of PD-1 expression on HIV-specific CD8⫹ T cells leads to reversible
immune dysfunction. Nat. Med. 12: 1198 –1202.
20. Wherry, E. J., J. N. Blattman, K. Murali-Krishna, R. van der Most, and
R. Ahmed. 2003. Viral persistence alters CD8 T-cell immunodominance and
tissue distribution and results in distinct stages of functional impairment. J. Virol.
77: 4911– 4927.
21. Fuller, M. J., and A. J. Zajac. 2003. Ablation of CD8 and CD4 T cell responses
by high viral loads. J. Immunol. 170: 477– 486.
22. Hislop, A. D., N. E. Annels, N. H. Gudgeon, A. M. Leese, and A. B. Rickinson.
2002. Epitope-specific evolution of human CD8⫹ T cell responses from primary
to persistent phases of Epstein-Barr virus infection. J. Exp. Med. 195: 893–905.
23. Hislop, A. D., M. Kuo, A. B. Drake-Lee, A. N. Akbar, W. Bergler, N.
Hammerschmitt, N. Khan, U. Palendira, A. M. Leese, J. M. Timms, et al. 2005.
Tonsillar homing of Epstein-Barr virus-specific CD8⫹ T cells and the virus-host
balance. J. Clin. Invest. 115: 2546 –2555.
24. Rickinson, A. B., M. F. Callan, and N. E. Annels. 2000. T-cell memory: lessons
from Epstein-Barr virus infection in man. Philos. Trans. R. Soc. Lond. B. Biol.
Sci. 355: 391– 400.
25. Kemball, C. C., E. D. Lee, V. Vezys, T. C. Pearson, C. P. Larsen, and
A. E. Lukacher. 2005. Late priming and variability of epitope-specific CD8⫹ T
cell responses during a persistent virus infection. J. Immunol. 174: 7950 –7960.
26. Zammit, D. J., D. L. Turner, K. D. Klonowski, L. Lefrancois, and L. S. Cauley.
2006. Residual antigen presentation after influenza virus infection affects CD8 T
cell activation and migration. Immunity 24: 439 – 449.
27. Gray, D. 2002. A role for antigen in the maintenance of immunological memory.
Nat. Rev. Immunol. 2: 60 – 65.
28. Rickinson, A. B., and E. Kieff. 1996. Epstein-Barr Virus. In Fields Virology.
D. M. K. B. N. Fields and P. M. Howley, eds. Lippincott-Raven Publishers,
Philadelphia, pp. 2397–2446.
29. Moore, P. S., and Y. Chang. 2003. Kaposi’s sarcoma-associated herpesvirus immunoevasion and tumorigenesis: two sides of the same coin? Annu. Rev. Microbiol. 57: 609 – 639.
30. Munz, C. 2005. Immune response and evasion in the host-EBV interaction. In
Epstein-Barr Virus. E. R. Robertson, ed. Caister Academic Press, Norfolk, pp.
197–231.
31. Doherty, P. C., J. P. Christensen, G. T. Belz, P. G. Stevenson, and M. Y. Sangster.
2001. Dissecting the host response to a ␥-herpesvirus. Philos. Trans. R. Soc.
Lond. B. Biol. Sci. 356: 581–593.
32. Appay, V., D. F. Nixon, S. M. Donahoe, G. M. Gillespie, T. Dong, A. King,
G. S. Ogg, H. M. Spiegel, C. Conlon, C. A. Spina, et al. 2000. HIV-specific
CD8⫹ T cells produce antiviral cytokines but are impaired in cytolytic function.
J. Exp. Med. 192: 63–75.
33. Klenerman, P., and A. Hill. 2005. T cells and viral persistence: lessons from
diverse infections. Nat. Immunol. 6: 873– 879.
34. Flano, E., I. J. Kim, J. Moore, D. L. Woodland, and M. A. Blackman. 2003.
Differential ␥-herpesvirus distribution in distinct anatomical locations and cell
subsets during persistent infection in mice. J. Immunol. 170: 3828 –3834.
35. Cardin, R. D., J. W. Brooks, S. R. Sarawar, and P. C. Doherty. 1996. Progressive
loss of CD8⫹ T cell-mediated control of a ␥-herpesvirus in the absence of CD4⫹
T cells. J. Exp. Med. 184: 863– 871.
36. Liu, L., E. Flano, E. J. Usherwood, S. Surman, M. A. Blackman, and D. L.
Woodland. 1999. Lytic cycle T cell epitopes are expressed in two distinct phases
during MHV-68 infection. J. Immunol. 163: 868 – 874.
37. Altman, J. D., P. H. Moss, P. R. Goulder, D. H. Barouch, M. G. McHeyzerWilliams, J. I. Bell, A. J. McMichael, and M. M. Davis. 1996. Phenotypic analysis of antigen-specific T lymphocytes. Science 274: 94 –96.
38. Grundhoff, A., and D. Ganem. 2004. Inefficient establishment of KSHV latency
suggests an additional role for continued lytic replication in Kaposi sarcoma
pathogenesis. J. Clin. Invest. 113: 124 –136.
39. Kaech, S. M., S. Hemby, E. Kersh, and R. Ahmed. 2002. Molecular and functional profiling of memory CD8 T cell differentiation. Cell 111: 837– 851.
40. Hogan, R. J., E. J. Usherwood, W. Zhong, A. A. Roberts, R. W. Dutton,
A. G. Harmsen, and D. L. Woodland. 2001. Activated antigen-specific CD8⫹ T
cells persist in the lungs following recovery from respiratory virus infections.
J. Immunol. 166: 1813–1822.
41. Onami, T. M., L. E. Harrington, M. A. Williams, M. Galvan, C. P. Larsen,
T. C. Pearson, N. Manjunath, L. G. Baum, B. D. Pearce, and R. Ahmed. 2002.
Dynamic regulation of T cell immunity by CD43. J. Immunol. 168: 6022– 6031.
42. Flano, E., D. L. Woodland, M. A. Blackman, and P. C. Doherty. 2001. Analysis
of virus-specific CD4⫹ T cells during long-term gammaherpesvirus infection.
J. Virol. 75: 7744 –7748.
43. Daniels, M. A., L. Devine, J. D. Miller, J. M. Moser, A. E. Lukacher, J. D.
Altman, P. Kavathas, K. A. Hogquist, and S. C. Jameson. 2001. CD8 binding to
MHC class I molecules is influenced by T cell maturation and glycosylation.
Immunity 15: 1051–1061.
44. Kambayashi, T., E. Assarsson, B. J. Chambers, and H. G. Ljunggren. 2001. IL-2
down-regulates the expression of TCR and TCR-associated surface molecules on
CD8⫹ T cells. Eur. J. Immunol. 31: 3248 –3254.
45. Drake, D. R., 3rd, R. M. Ream, C. W. Lawrence, and T. J. Braciale. 2005.
Transient loss of MHC class I tetramer binding after CD8⫹ T cell activation
reflects altered T cell effector function. J. Immunol. 175: 1507–1515.
46. Chang, J. T., V. R. Palanivel, I. Kinjyo, F. Schambach, A. M. Intlekofer,
A. Banerjee, S. A. Longworth, K. E. Vinup, P. Mrass, J. Oliaro, et al. 2007.
Asymmetric T lymphocyte division in the initiation of adaptive immune responses. Science 315: 1687–1691.
T CELL MEMORY DURING VIRAL PERSISTENCE
The Journal of Immunology
86. Pulle, G., M. Vidric, and T. H. Watts. 2006. IL-15-dependent induction of 4-1BB
promotes antigen-independent CD8 memory T cell survival. J. Immunol. 176:
2739 –2748.
87. Sauce, D., M. Larsen, S. J. Curnow, A. M. Leese, P. A. Moss, A. D. Hislop,
M. Salmon, and A. B. Rickinson. 2006. EBV-associated mononucleosis leads to
long-term global deficit in T-cell responsiveness to IL-15. Blood 108: 11–18.
88. Obar, J. J., S. G. Crist, E. K. Leung, and E. J. Usherwood. 2004. IL-15-independent proliferative renewal of memory CD8⫹ T cells in latent gammaherpesvirus
infection. J. Immunol. 173: 2705–2714.
89. Catalina, M. D., J. L. Sullivan, R. M. Brody, and K. Luzuriaga. 2002. Phenotypic
and functional heterogeneity of EBV epitope-specific CD8⫹ T cells. J. Immunol.
168: 4184 – 4191.
90. Hislop, A. D., N. H. Gudgeon, M. F. Callan, C. Fazou, H. Hasegawa, M. Salmon,
and A. B. Rickinson. 2001. EBV-specific CD8⫹ T cell memory: relationships
between epitope specificity, cell phenotype, and immediate effector function.
J. Immunol. 167: 2019 –2029.
91. Obar, J. J., S. G. Crist, D. C. Gondek, and E. J. Usherwood. 2004. Different
functional capacities of latent and lytic antigen-specific CD8 T cells in murine
gammaherpesvirus infection. J. Immunol. 172: 1213–1219.
92. Munks, M. W., K. S. Cho, A. K. Pinto, S. Sierro, P. Klenerman, and A. B. Hill.
2006. Four distinct patterns of memory CD8 T cell responses to chronic murine
cytomegalovirus infection. J. Immunol. 177: 450 – 458.
93. Levitsky, V., P. O. de Campos-Lima, T. Frisan, and M. G. Masucci. 1998. The
clonal composition of a peptide-specific oligoclonal CTL repertoire selected in
response to persistent EBV infection is stable over time. J. Immunol. 161:
594 – 601.
94. Khanna, K. M., R. H. Bonneau, P. R. Kinchington, and R. L. Hendricks. 2003.
Herpes simplex virus-specific memory CD8⫹ T cells are selectively activated and
retained in latently infected sensory ganglia. Immunity 18: 593– 603.
95. Callan, M. F., C. Fazou, H. Yang, T. Rostron, K. Poon, C. Hatton, and
A. J. McMichael. 2000. CD8⫹ T-cell selection, function, and death in the primary
immune response in vivo. J. Clin. Invest. 106: 1251–1261.
96. Obar, J. J., S. Fuse, E. K. Leung, S. C. Bellfy, and E. J. Usherwood. 2006.
Gammaherpesvirus persistence alters key CD8 T-cell memory characteristics and
enhances antiviral protection. J. Virol. 80: 8303– 8315.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
75. Flano, E., C. L. Hardy, I. J. Kim, C. Frankling, M. A. Coppola, P. Nguyen,
D. L. Woodland, and M. A. Blackman. 2004. T cell reactivity during infectious
mononucleosis and persistent gammaherpesvirus infection in mice. J. Immunol.
172: 3078 –3085.
76. Gangappa, S., S. B. Kapadia, S. H. Speck, and H. W. t. Virgin. 2002. Antibody
to a lytic cycle viral protein decreases gammaherpesvirus latency in B-cell-deficient mice. J. Virol. 76: 11460 –11468.
77. Kim, I.-J., E. Flano, D. L. Woodland, and M. A. Blackman. 2002. Antibodymediated control of persistent ␥-herpesvirus infection. J. Immunol. 168:
3958 –3964.
78. Holmes, S., M. He, T. Xu, and P. P. Lee. 2005. Memory T cells have gene
expression patterns intermediate between naive and effector. Proc. Natl. Acad.
Sci. USA 102: 5519 –5523.
79. Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh, and
R. Ahmed. 2003. Selective expression of the interleukin 7 receptor identifies
effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 4:
1191–1198.
80. Wong, P., and E. G. Pamer. 2003. CD8 T cell responses to infectious pathogens.
Annu. Rev. Immunol. 21: 29 –70.
81. Chen, G., P. Shankar, C. Lange, H. Valdez, P. R. Skolnik, L. Wu, N. Manjunath,
and J. Lieberman. 2001. CD8 T cells specific for human immunodeficiency virus,
Epstein-Barr virus, and cytomegalovirus lack molecules for homing to lymphoid
sites of infection. Blood 98: 156 –164.
82. Hikono, H., J. E. Kohlmeier, K. H. Ely, I. Scott, A. D. Roberts, M. A. Blackman,
and D. L. Woodland. 2006. T-cell memory and recall responses to respiratory
virus infections. Immunol. Rev. 211: 119 –132.
83. Kedzierska, K., N. L. La Gruta, S. J. Turner, and P. C. Doherty. 2006. Establishment and recall of CD8⫹ T-cell memory in a model of localized transient
infection. Immunol. Rev. 211: 133–145.
84. La Gruta, N. L., S. J. Turner, and P. C. Doherty. 2004. Hierarchies in cytokine
expression profiles for acute and resolving influenza virus-specific CD8⫹ T cell
responses: correlation of cytokine profile and TCR avidity. J. Immunol. 172:
5553–5560.
85. Melchionda, F., T. J. Fry, M. J. Milliron, M. A. McKirdy, Y. Tagaya, and
C. L. Mackall. 2005. Adjuvant IL-7 or IL-15 overcomes immunodominance and
improves survival of the CD8⫹ memory cell pool. J. Clin. Invest. 115:
1177–1187.
153