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Journal of Virology, May 2001, p. 4435-4438, Vol. 75, No. 9
Department of Immunology, St. Jude
Children's Research Hospital, Memphis, Tennessee 38105
Received 21 November 2000/Accepted 29 January 2001
The cycling characteristics of CD8+ T cells specific
for two lytic-phase epitopes of murine gammaherpesvirus 68 ( Respiratory challenge with
murine gammaherpesvirus 68 ( In the present analysis, we used these CD4+
T-cell-deficient I-Ab CD8+ T cells were obtained from the spleen and
from the infected lungs (1) by bronchoalveolar lavage
(BAL). The experiments focus on the two most prominent The bromodeoxyuridine (BrdU)-positive (BrdU+)
CD8+ population showed maximal prevalence in both
the spleen and BAL populations recovered from the
I-Ab+/+ and
I-Ab
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.9.4435-4438.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Virus-Specific and Bystander CD8+
T-Cell Proliferation in the Acute and Persistent Phases of a
Gammaherpesvirus Infection
and
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ABSTRACT
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Abstract
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HV68)
have been analyzed for mice with high or low levels of virus
persistence. The extent of cell division is generally reflective of the
antigen load and suggests that
HV68 may be regularly reactivating
from latency for some months after the resolution of the acute phase of
the infectious process. Although
HV68 infection is also associated with massive proliferation of lymphocytes that are not obviously specific for the virus, the level of "bystander-induced" cycling in
a population of influenza virus-specific CD8+ T cells was
generally fourfold lower than the extent of cell division seen
for the antigen-driven,
HV68-specific response. The overall
conclusion is that turnover rates substantially in excess of 5 to 10%
over 6 days for CD8+ "memory" T-cell populations are
likely to be reflective of continued antigenic exposure.
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TEXT
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Abstract
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HV68) induces massive turnover in the
CD8+ T-cell compartment (13, 24).
This pattern of sustained lymphocyte proliferation persists long after
both
HV68 replicative infection is controlled in epithelial sites
and peak numbers of latently infected B lymphocytes are detected in the
spleen (4). These cycling CD8+ T
cells comprise at least two distinct populations. The first consists of
multiple sets of
HV68-specific CD8+ T cells
that are, in C57BL/6J (B6) mice, responding to at least six different
viral peptides derived from proteins expressed during the lytic phase
of the infectious process (18). The second is an unusual
CD8+ T-cell population that expresses a V
4
T-cell receptor chain and expands greatly in numbers after the end of
the initial, lytic phase of the infectious process (24).
These CD8+ V
4+ T cells
show some evidence of oligoclonality (14), although they
do not seem to be recognizing viral peptides or major
histocompatibility complex class I or II glycoproteins
(6). Furthermore, the dramatic increase in the size of the
CD8+ V
4+ set is totally
dependent on concurrent, CD40 ligand-mediated CD4+ T-cell help (3, 9). This effect
is not seen in major histocompatibility complex class
II
/
(I-Ab
/
) mice.
/
mice (12) and conventional B6
(I-Ab+/+) congenic mice to
characterize the proliferation of virus-specific CD8+ T cells under conditions of differential,
continuing
HV68 challenge. The
I-Ab+/+ group effectively controls
lytic
HV68 infection within 12 days of the initial intranasal (i.n.)
exposure, while I-Ab
/
mice show
persistent evidence of
HV68 replication in the respiratory tract and
succumb to a late-onset wasting disease that develops after 80 to 100 days (2, 4). Latently infected B cells and macrophages can
be demonstrated in both groups over the very long term by an
infectious-center assay and by limiting-dilution analysis.
HV68
epitopes, H2Dbp56 (Dbp56)
and H2Kbp79 (Kbp79). The
HV68 p56 peptide (AGPHNDMEI) is derived from a single-stranded DNA
binding protein, while the p79 peptide (TSINFKVI) is derived from the
large ribonucleotide reductase (18). The possible
contribution of "bystander" activation (8, 16, 22, 25,
26) to the total pool of cycling CD8+ T
cells was also analyzed for a "memory" population specific for the
prominent influenza virus
DbNP366 epitope
(10). Influenza-immune
I-Ab+/+ mice were first infected
intraperitoneally with H1N1 influenza A virus and then boosted at least
6 weeks prior to
HV68 challenge by respiratory infection with an
H3N2 virus that shares the same nucleoprotein gene (10,
15).
/
mice at day 20 after the
initial exposure to
HV68 (Fig. 1A to D). The percentages of BrdU+ T cells in the
splenic CD8+
Dbp56+ and
CD8+
Kbp79+ sets were also
remarkably concordant for the I-Ab+/+
and I-Ab
/
mice until day 60 after
the i.n.
HV68 challenge (Fig. 1B and C), with the values ranging
from >80% on day 20 to about 30% on day 60. The same was true for
the BAL populations until day 40, although there was evidence of more
cycling in the I-Ab
/
mice by day
50 (Fig. 1E and F). This result presumably reflects the persistent,
low-level production of lytic virus in the
I-Ab
/
mouse respiratory tract
(4).

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FIG. 1.
Kinetic analysis of BrdU incorporation in the
CD8+ Kbp79+ and CD8+
Dbp56+ sets during the first 60 days after
HV68 infection (13, 19, 23).
I-Ab+/+ and
I-Ab
/
mice were infected i.n.
with 2 × 103 PFU of
HV68 and given 0.8 mg of
BrdU/ml in their drinking water for 6 days prior to sampling. The
results for pooled spleen (A, B, and C) and BAL (D, E, and F)
populations from four or five mice at each time point show the
percentages of BrdU+ cells for all gated CD8+
lymphocytes (A and D) and for the virus-specific
Dbp56+(B and E) and
Kbp79+ (C and F) sets. The lymphocytes were
stained with anti-CD8-tricolor and phycoerythrin-labeled
Kbp79 or Dbp56 tetramers, fixed, and stained
with anti-BrdU-fluorescein isothiocyanate (13, 19, 23).
The cells were then analyzed on a FACScan flow cytometer using
CellQuest software (Becton Dickinson, Mountain View, Calif.). A minimum
of 2,000 CD8+ tetramer-positive events were analyzed for
each sample.
Analysis at later time points (days 70 to 90) showed a continuing
profile of greater cycling for both the total and the virus-specific CD8+ T-cell populations in the BAL populations
from the I-Ab
/
mice (Fig.
2D to F). This difference between the
I-Ab+/+ and
I-Ab
/
groups was also apparent
for the virus-specific CD8+ T cells recovered on
day 90 from the spleen (Fig. 2B and C), although the effect was not
evident in the total CD8+ T-cell population (Fig.
2A). A further pulse-chase analysis (12) also
indicated that both the CD8+
Dbp56+ (Fig.
3A and B) and the
CD8+
Kbp79+ (Fig. 3C and D) sets
were proliferating at a higher rate in the I-Ab
/
mice between day 50 and day
70 after infection.
|
|
Some evidence of bystander activation for the influenza virus
CD8+
DbNP366+
set was found in both the spleen and BAL populations recovered during
the first 18 days after i.n.
HV68 challenge (Table
1). Although we refer to this effect
throughout as bystander activation, a description that implies T-cell
receptor-independent stimulation by cytokines (8, 19,
26), it is also formally possible that there is some
unidentified cross-reactivity between a
HV68-encoded epitope and
DbNP366 (17).
The percentages of BrdU+
CD8+
DbNP366+ T
cells in the spleen were significantly increased (P < 0.05) over background values (day 0) at 12 and 16 days after i.n.
exposure to
HV68, although the prevalence of cycling
CD8+
DbNP366+ T
cells was three- to fourfold lower than that for the
CD8+
Dbp56+ and
CD8+
Kbp79+ sets (Table 1). The
results are very comparable to those found previously during the acute
phase of infection with influenza B virus (5) and suggest
that
HV68 has no particular propensity to drive the proliferation of
"irrelevant" CD8+ memory T cells.
|
In general, this analysis of cycling for the CD8+
Dbp56+ and
CD8+
Kbp79+ T-cell populations
in I-Ab+/+ and
I-Ab
/
mice indicates that the
extent of cell division above background levels is correlated with the
antigen load. Although every CD8+
DbNP366+ T
cell divides multiple times during the acute, antigen-driven phase of
the host response to the readily eliminated influenza A viruses
(11), the background turnover rates for influenza virus-specific CD8+
DbNP366+
memory T cells in the spleen average about 5% (5, 10)
(Table 1, day 0). Particularly in the secondary H3N2
H1N1 response
used to prime the mice analyzed for bystander activation in Table 1, the extent of BrdU incorporation drops to the level characteristic of
long-term memory as soon as the virus is eliminated from the lungs
(11).
Much higher levels of cycling (20 to 40%) (Fig. 1 and 2) were detected
for the CD8+
Dbp56+ and
CD8+
Kbp79+ T-cell populations
recovered from
HV68-infected
I-Ab+/+ mice for at least 2 months
after evidence of virus replication (4) could no longer be
found in the respiratory tract (day 12). This result suggests that,
although infectious virus cannot be recovered directly from the spleen
by a conventional plaque assay, there may be a continuing process of
reactivation from latency in the in vivo situation. Virus reactivation
could in turn lead to rapid CD8+ T-cell-mediated
elimination of the now productively infected B cells and macrophages
and, as a consequence, a progressive reduction in the extent of
HV68
latency. By 80 to 90 days after the initial
HV68 challenge, the
numbers of cycling CD8+
Dbp56+ and
CD8+
Kbp79+ T cells in
I-Ab+/+ mice had fallen (Fig. 2) to
levels more characteristic of those associated with "resting"
memory for other viruses. The counts continued to be high in the
I-Ab
/
congenic mice, where
evidence of continuing lytic infection was found in the lung epithelium
(4).
The results of these experiments thus support the idea that any evidence of CD8+ T-cell proliferation that is substantially above a " homeostatic background" (7, 21, 23) of 5 to 10% over 6 days is reflective of continued exposure to antigen (20). Evidence of bystander activation can be found at the acute phase of the host response to an unrelated pathogen, but any bystander proliferation is at a much lower level than that associated with the antigen-driven effect.
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ACKNOWLEDGMENTS |
|---|
We thank Gabriela Byers for technical assistance and Vicki Henderson for help with the manuscript.
This work was supported by U.S. Public Health Service grants AI29579, AI38359, and CA21765 and by the American Lebanese Syrian Associated Charities. G.T.B. is a C. J. Martin fellow of the Australian National Health and Medical Research Council.
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FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Immunology, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105. Phone: (901) 495-3470. Fax: (901) 495-3107. E-mail: peter.doherty{at}stjude.org.
Present address: Division of Immunology, The Walter and Eliza Hall
Institute of Medical Research, Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia.
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