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in HSV-1-Specific CD8+ T Cells Identifies Distinct Responding Subpopulations During the Primary Response to Infection1
Department of Microbiology and Immunology, Louisiana State University Health Sciences Center School of Medicine, Shreveport, LA 71130
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-staining assay. It revealed that 
6%
of the CD8+ T cells were specific for the gB epitope.
Phenotypic analysis of the IFN--producing gB-specific
CD8+ T cells generated in the PLN during the course of the
acute infection expressed the CD44high CD25+
phenotype on days 3–5 postinfection. Surprisingly, IFN--producing
CD8+ T cells expressed the CD44high
CD25- phenotype on days 5–8 postinfection, in contrast to
expectations for a CD8+ effector T cell. IFN--producing
CD25- CD8+ T cells were detected in the PLN on
day 21 postinfection, long after infectious virus had been cleared.
Throughout the response, the spleen was found to be the major reservoir
of gB-specific CD8+ T cells, even during the peak of the
response. In contrast to the gB-specific CD8+ T cell
population within the PLN, the entire gB-specific CD8+ T
cell population within the spleen was CD25-. Collectively,
these results suggest the generation of subpopulations of
virus-specific CD8+ T cells, distinguished by the
expression of CD25, during the acute phase of the primary response to a
localized viral infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain), CD11a, CD11b, and
CD49d and down-regulating the lymph node
(LN)3 homing receptor,
CD62L (2, 3, 4, 5, 6). Whether the expression of these surface
markers precedes the attainment of effector function and whether their
levels are maintained on the CD8+ T cell
effectors is not fully understood.
The specific immune response elicited to acute HSV-1 infection of the
hind footpad in the C57BL/6 (B6) mouse model involves the accumulation
of CD8+ T cells in the popliteal LN (PLN)
draining the site of infection (7). This increase in the
CD8+ T cell numbers appears as two waves; an
early peak on day 5 postinfection (p.i.), corresponding to the onset of
viral clearance, and a second peak on day 8 p.i., when infectious
HSV-1 is no longer detectable in the skin (7). In B6 mice,
the HSV-1-specific CD8+ T cell response is
predominantly restricted to H-2Kb
(8) and is almost exclusively directed against an epitope
(498SSIEFARL505) derived
from the envelope glycoprotein, glycoprotein B (gB)
(9, 10, 11, 12, 13). Recently, the relationship between the
CD8+ T cell phenotype and the frequency of the
CTL precursor population, at the peak of the response, was assessed by
limiting dilution analysis (LDA) subsequent to FACS (7).
These studies revealed that the HSV-1-specific effectors were found
exclusively within the CD44high
CD25+ CD8+ T cell
population. However, only 0.3–0.4% of the CD8+
T cell population within the PLN possessed gB-specific cytolytic
activity as estimated by LDA. There are two, not mutually exclusive,
explanations for this low frequency of HSV-1-specific cytolytic
CD8+ T cells. First, a great proportion of the
activated CD8+ T cells may not be specific for
HSV-1 but may have been nonspecifically activated, either by bystander
activation (14) or by cross-reactive recognition
(15, 16). Second, the LDA may have underestimated the
frequency of HSV-1-specific CTL precursors, hence, the true frequency
may be considerably higher than 0.3–0.4% (17). The
intracellular IFN--staining assay has recently been demonstrated to
be 10- to 15-fold more sensitive in the detection of lymphocytic
choriomeningitis virus (LCMV)-specific CD8+ T
cells than the conventional LDA (17, 18). Therefore, it
was of interest to employ this assay to obtain a better picture of the
size of the Ag-specific CD8+ T cell population
generated in response to a localized virus infection, which by the LDA
was shown to give a much weaker stimulus to the immune system than
LCMV. Also, by using a more sensitive means of detecting the
HSV-1-specific CD8+ T cells, a more extensive
kinetic analysis of the surface-marker expression on the Ag-specific
CD8+ T cell population generated in response to a
localized virus infection could be performed.
The intracellular IFN--staining assay was, according to previous
studies (17, 18), 10- to 15-fold more sensitive in the
detection of Ag-specific CD8+ T cells than the
conventional LDA. The phenotypic analysis of surface-marker expression
on the gB-specific IFN--producing CD8+ T cells
revealed, in agreement with previous studies of cytolytic function
(7, 19), that the CD8+ T cell
effectors expressed high levels of the activation marker CD44.
Surprisingly, although previous studies showed that both precursor and
effector HSV-1-specific CTL were found exclusively within the
CD44high CD25+
subpopulation of CD8+ T cells in the PLN at the
peak of the response (day 5 p.i.) (7, 19), the
intracellular IFN--staining assay detected IFN--producing
CD44high CD25-
CD8+ T cells at this time point. By day 8
p.i., the CD25- effector subpopulation
constituted the sole population of gB-specific
CD8+ T cells in the PLN. Speculation that this
effector subpopulation might represent an early memory cell population
lead us to perform a phenotypic characterization of the gB-specific
CD8+ T cell population within the spleen,
reservoir of the T cell memory population following viral clearance.
These studies showed that a subpopulation of gB-specific
CD8+ T cells harboring a classical memory
phenotype (CD44high CD25-
CD62Llow) could be detected within the spleen
even during the acute phase of the primary response. By day 21
p.i. the gB-specific CD8+ T cell population
within the PLN had been vastly reduced (0.5% of the total
CD8+ T cells), but, as expected, a population of
Ag-specific CD8+ T cells remained within the
spleen, representing about 3.7% of the total
CD8+ T cell population. This suggests the
presence of two separate populations of effectors during the acute
phase of the primary response, of which one may persist as a memory
CD8+ T cell population.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male B6 (H-2b) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) at 4–5 wk of age. Animals were used between 6 and 12 wk of age.
Virus
HSV-1 strain Patton, originally obtained from Dr. R. Tenser (Pennsylvania State University College of Medicine, Hershey, PA), was plaque-purified four times on Vero cell monolayers and established as a stock by infection of Vero cells at a multiplicity of infection of 0.01. Virus present in culture supernatant and virus released from infected cells by a freeze/thaw cycle and a 1-min round of sonication were pooled, titrated on Vero cell monolayers, and stored at -80°C before use.
HSV-1 immunization of B6 mice
Mice were anesthetized by i.p. injection of 60 mg/kg of sodium
pentobarbital (Butler, Columbus, OH). Mice were then given 5 x
106 pfu HSV-1 in 50 µl of TBS in each hind
footpad using a modification of the previously described multiple
puncture injection method (20). Briefly, 50 µl of virus
was delivered s.c. into the hind footpad, which was then repeatedly
punctured with a 27-gauge needle for 15 s. At intervals following
infection, lymphocytes within the PLN were isolated for phenotypic
analysis.
Abs and reagents for surface staining
The following panel of mAb and reagents were used for phenotypic
analysis of lymphocytes in the PLN: APC anti-CD8 (clone 53-6.7;
Caltag Laboratories, Burlingame, CA), biotin anti-CD25 (clone 7D4;
PharMingen, San Diego, CA), biotin anti-CD44 (clone IM7;
PharMingen), PE anti-CD25 (clone PC61; PharMingen), biotin
anti-CD62L (clone Mel14; PharMingen), and streptavidin-peridinin
chlorophyll protein (Becton Dickinson, San Jose, CA).
Intracellular IFN- staining
LN or splenic lymphocytes were cultured for 5 h in 96-well
U-bottom microtiter plates (Costar, Cambridge, MA) at a concentration
of 1 x 106 cells/well in 0.2 ml complete
medium with 1 µl/ml brefeldin A (GolgiPlug; PharMingen). Spleen cells
pulsed with vesicular stomatitis virus (VSV) nucleoprotein
(52RGYGYQGL59) or HSV-1
glycoprotein B
(498SSIEFARL505)
peptides were used as stimulators (12). The stimulators
were prepared by incubating 1 x 106 spleen
cells in 500 µl IMDM, 10% FCS containing 10-2
M VSV or HSV-1 peptides (Louisiana State University Health Sciences
Center Core Laboratories, New Orleans, LA) at a final concentration of
50 µM for 3 h at 37°C. A total of 3 x
104 stimulators were added per well containing
1 x 106 LN cells. As a positive control,
spleen cells were stimulated nonspecifically by addition of PMA (0.005
µg/ml) and ionomycin (0.5 µg/ml). After 5 h of culture, the
cells were spun down and surface-stained in PBS, supplemented with 2%
FCS and 0.5% sodium azide, with the above-listed panel of Abs. After
washing the unbound Ab, cells were subjected to intracellular cytokine
stain using the Cytofix/Cytoperm kit (PharMingen) according to
manufacturer’s instructions. For intracellular IFN- staining, the
FITC-conjugated monoclonal rat anti-mouse IFN- Ab (clone XMG1.2;
PharMingen) or its isotype control Ab (rat IgG1) was used. Flow
cytometric analysis was performed on a FACScaliber instrument (Becton
Dickinson, San Jose, CA) by the Core Facility for Flow Cytometry at
Louisiana State University Health Sciences Center at Shreveport. The
data were analyzed using CellQuest software (Becton Dickinson).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing CD8+ T cells are specific to
the HSV-1 gB peptide
To perform a phenotypic analysis of the effector
CD8+ T cells accumulating within the draining PLN
during acute HSV-1 infection, intracellular IFN- staining of HSV-1
gB-stimulated CD8+ T cells was employed. Fig. 1
A shows that about 6% of the
total CD8+ T cells within the PLN of day
5-infected mice were IFN--positive following 5 h stimulation
with the gB peptide. In comparison, about 15% of the CD8 T cells in
the spleen were capable of producing IFN- when stimulated
nonspecifically (Fig. 1B). Verification of the specificity
of the CD8+ T cell response was conducted in part
by using stimulators pulsed with a nonspecific peptide derived from the
VSV glycoprotein. As shown in Fig. 1C, IFN--positive
CD8+ T cells were not detected following 5 h
stimulation with the nonspecific epitope. Also, LN cells from
uninfected mice, as expected, failed to give rise to any
CD8+ effectors following gB stimulation (Fig. 1D), suggesting that the IFN--producing
CD8+ T cells had been activated by the HSV-1
infection, specifically.
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mAb was verified by
including a FITC isotype control mAb in the intracellular
IFN--staining assay. Fig. 1
D shows that only a very small
proportion of FITC-positive cells from HSV-1-infected mice were
detected following gB stimulation using the isotype control mAb. In the
following results, the number of FITC-positive cells detected by the
isotype control mAb, for each combination of surface-staining reagents,
was subtracted from the signal from the FITC anti-mouse IFN- mAb
when calculating the number of IFN--producing
CD8+ T cells.
Kinetics of IFN--producing CD8+ T cells in the PLN
Previous studies revealed that the frequency of activated
(CD44high CD25+)
CD8+ T cells, generated in response to acute
HSV-1 infection, reaches a peak on day 5 p.i. followed by a
decline to normal levels by days 7–9 p.i. (7). This peak
of activated CD8+ T cells was found to correlate
with cytolytic function when assessed by both bulk culture and LDA
51Cr release assays (7, 19, 21).
Using the intracellular IFN--staining assay, the peak of activated
(CD25+) CD8+ T cells was
also found to correlate with effector function when assessed by the
ability to produce IFN-. The number of IFN--producing gB-specific
CD8+ T cells rose from about 2.5 x
104 per LN on day 4 p.i. to about 8.0
x 104 on day 5, followed by a gradual decline to
about 3.5 x 104 on day 8 p.i. (Fig. 2).
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+ CD8+
T cells
To establish the phenotype of the IFN--producing
CD8+ T cells, surface staining of activation
markers was performed in conjunction with the intracellular cytokine
staining. HSV-1-specific CD8+ CTL precursors
express high levels of CD44 (CD44high) (7, 19), known to be up-regulated on activated T lymphocytes
(4, 6, 22) and long-term memory T lymphocytes
(23, 24, 25, 26). It was anticipated that
CD8+ T cell effectors, when assessed by the
ability to secrete IFN-, would therefore express the
CD44high phenotype. The results shown in Fig. 3 show that this was indeed the case. All
of the IFN--producing CD8+ T cells were
CD44high. Thus, regardless of whether
HSV-1-specific CD8+ T cell effector function is
assessed by cytolytic potential or by IFN- production,
CD8+ T cell effectors express high levels of the
activation marker CD44.
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-chain, CD25,
at the peak of the response (day 5 p.i.) (19).
Employing the more sensitive intracellular IFN--staining assay, it
was observed that IFN- production also closely correlated with
expression of CD25 to day 5 p.i. (Fig. 4). Beyond day 5 p.i., the frequency
of IFN--producing CD25+
CD8+ T cells diminished, returning to
preinfection levels by day 8 p.i. During this period, a population
of CD25- IFN-+
CD8+ T cells could be detected. The
CD25- IFN-+
CD8+ T cells were present as early as day 5
p.i., became more prevalent than the CD25+
IFN-+ CD8+ T cells from
day 6 p.i., and constituted the sole population of
IFN--producing CD8+ T cells by day 8 p.i.
Therefore, staining for intracellular IFN- revealed an
HSV-1-specific CD8+ T cell subpopulation not
previously observed using other methods of detection.
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-production by HSV-1-specific memory phenotype
CD8+ T cells
Because infectious virus is cleared from the footpads by day
8 p. i., one would expect the vast majority of gB-specific
CD8+ T cells within the PLN to have undergone
activation-induced cell death by day 21 p.i. However, a memory
population of gB-specific CD8+ T cells should be
present in circulation and principally reside within the spleen. To
verify this, spleen and PLN cells were harvested on day 21 p.i.
and subjected to the intracellular IFN- staining assay. As expected,
very few IFN--producing CD8+ T cells could be
detected within the PLN at this time point (Fig. 5B). The small population that
could be detected expressed a classical memory phenotype:
CD44high CD25-
CD62Llow. About 4.2% of the total
CD8+ T cell population within the spleen was gB
specific and, likewise, harbored a surface-marker phenotype
characteristic of a memory cell population (Fig. 5B),
suggesting the establishment of a virus-specific
CD8+ T cell memory population within the
spleen.
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-staining assay. Indeed,
about 3.7% of the total CD8+ T cell population
within the spleen was gB specific on day 5 p.i. (Fig. 5A). When the frequencies of IFN--positive cells within
the spleen and PLN were converted to actual cell numbers, the spleen
was found to be the main reservoir of gB-specific
CD8+ T cells during both the acute and early
memory phases of the primary response (Fig. 5C). As observed
previously, the majority of the gB-specific CD8+
T cells in the PLN expressed CD25, characteristic of recently activated
effector CD8+ T cells, whereas the splenic
gB-specific CD8+ T cells were
CD25- (Fig. 5, A and
B). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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staining,
coincided precisely with the frequency of Ag-specific CD8+
T cells estimated by class I MHC tetramer staining during primary and
secondary responses to influenza virus (27). Hence, the
model that most of the CD8+ T cell expansion
during viral infections was not Ag specific, but represented bystander
activation and/or cross-reactive stimulation of nonspecific cells, had
to be revised. However, the LCMV model is characterized by an unusually
large expansion of the CD8+ T cell population
compared with other viral infections, due principally to viral
replication within the spleen. Compared with the LCMV model, HSV-1
infection appears to provide a weaker stimulus to
CD8+ T cells and does not result in as massive an
expansion of the CD8+ T cell population.
Previous studies have shown that only 0.3–0.4% of the
CD8+ T cell population within the PLN, at the
peak of the response (day 5 p.i.), is gB specific following acute
cutaneous infection of B6 mice when estimated for cytolytic function
under LDA culture conditions (7). In contrast, employment
of the intracellular IFN- staining assay in the present studies
revealed that the CD8+ T cells recognizing the
single immunodominant HSV-1-specific gB epitope represented about 6%
of the total CD8+ T cell population within the
PLN. Hence, as observed in the LCMV model (17, 18), the
intracellular IFN--staining assay was about 10- to 15-fold more
sensitive in its ability to detect virus-specific
CD8+ T cells than the LDA approach. Furthermore,
this frequency is similar to one reported in a recent study analyzing
the contribution of the gB epitope to the overall cytotoxic T cell
response to HSV-1 infection of B6 mice (9). Staining with
gB-containing MHC class I tetramer complexes has revealed a slightly
higher frequency of HSV-1-specific CD8 T cells in the spleen and PLN
(manuscript in preparation). This difference may be caused by the
presence of effector-function-negative gB-specific CD8+ T
cells that were recently identified during chronic LCMV infection of
mice (28).
The LN draining the site of a localized viral infection has
traditionally been considered the primary site for priming and
expansion of the Ag-specific CD8+ T cell
population during the acute phase of the primary response. Following
viral clearance, the spleen then becomes the major reservoir of
Ag-specific CD8+ T cells, harboring the memory
population for the lifetime of the animal. In accordance, attempts in
our laboratory to isolate cytolytic lymphocytes from the spleen at the
peak of the primary response (day 5 p.i.) have failed (our
unpublished observation). However, a recent study has shown that the
spleen, not the draining LN, can be the major reservoir of the
Ag-specific CD8+ T cell population even during
the acute phase of the primary response to a localized virus infection
(29). Our studies confirm this finding. During the peak of
the primary response (day 5 p.i.), the spleen contained roughly
3 x 105 gB-specific
CD8+ T cells, whereas only about 6 x
104 gB-specific CD8+ T
cells could be detected within the PLN by the intracellular
IFN--staining assay. It is of interest to note that that the
gB-specific CD8+ T cells within the spleen, both
during the acute phase of the response and 2 wk following viral
clearance, harbored a surface-marker phenotype characteristic of memory
cells: CD44high CD25-
CD62Llow. In contrast, the majority of the
gB-specific CD8+ T cells within the PLN expressed
CD25 at the peak of the response. However, by day 8 p.i. the
entire gB-specific CD8+ T cell population within
the PLN had attained a memory cell phenotype and retained this
phenotype following viral clearance of the footpads, at least until day
21 p.i. As expected, the vast majority of the virus-specific
CD8+ T cells within the PLN disappeared by day
21 p.i. and constituted <0.5% of the total
CD8+ T cell population at this site. By day
45 p.i., gB-specific CD8+ T cells were
virtually absent from the PLN, whereas this cell population constituted
about 2.3% of the CD8+ T cells in the spleen
(data not shown). The presence of CD25-
gB-specific CD8+ T cells, expressing IFN- in
response to Ag stimulation, within the PLN at the peak of the response
was surprising because previous studies using a cell sorting and LDA
approach had shown that the cytolytic effectors expressed CD25. Also, a
very recent study, employing the intracellular IFN--staining assay,
indicated that all of the gB-specific, IFN--expressing
CD8+ T cells were found to coexpress CD25
(9). The less-sensitive LDA, in comparison to the
intracellular IFN--staining assay, is likely responsible for the
failure to detect the small population of CD25-
effectors using the cell sorting and LDA approach. Concerning the
latter study, differences in the in vitro-stimulation conditions may
explain why the IFN--producing gB-specific
CD8+ T cells all coexpressed CD25. In this study,
the restimulation cultures contained IL-2 that may have selected for
gB-specific CD8+ T cells expressing
CD25.
Several possible, not mutually exclusive, scenarios may explain the presence of CD25- gB-specific CD8+ T cells within the PLN and spleen during the acute phase of the primary response. First, a continuous down-regulation of the CD25 marker on the CD25+ subpopulation within the PLN, following a period of effector function, and the subsequent migration of these cells to the spleen could give rise to CD25- gB-specific CD8+ T cells at both sites. Due to the high numbers of gB-specific CD8+ T cells in the spleen on day 5 p.i. this would demand either extensive migration of the CD25- cells from the PLN to the spleen or extensive expansion of these cells once they have reached the spleen. The latter possibility is supported by a recent study that revealed that a large portion of the virus-specific CD8+ T cells in the spleen during a localized infection were actively dividing (29). Alternatively, the CD25- gB-specific CD8+ T cells within the spleen may constitute a separate population of CD25- gB-specific CD8+T cells. In this instance, because the spleen is not a site of HSV-1 replication, the virus-specific CD8+ T cell population could have become activated and expanded following interaction with dendritic cells, carrying the gB peptide, that had migrated from the HSV-1-infected footpads. Also in this scenario, one would expect the splenic gB-specific CD8+ T cells to be actively dividing. Finally, the CD25- and CD25+ gB-specific CD8+ T cells detected within the PLN during the acute phase of the primary response may constitute two separate populations of effectors, of which only one persists as a memory T cell population following migration to the spleen. The two latter scenarios would both lend support to a recent study suggesting the development of memory T cells, as a separate cell population, during the primary response (30). Future studies will aim at distinguishing between these possibilities.
In summary, by employment of the intracellular IFN--staining assay,
6% of the CD8+ T cells within the PLN were found
to be gB-specific at the peak of the primary response to acute
cutaneous HSV-1 infection. Surprisingly, the spleen was the major
reservoir of gB-specific CD8+ T cells even during
the acute phase of the response. Furthermore, whereas the majority of
the effectors within the PLN expressed CD25 at the peak of the
response, CD25 expression was not detected on the splenic
virus-specific CD8+ T cells. This may suggest the
presence of separate subpopulations of gB-specific
CD8+ T cells during the acute phase of the
primary response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stephen R. Jennings, Department of Microbiology and Immunology, Louisiana State University Health Sciences Center School of Medicine, 1501 Kings Highway, P.O. Box 33932, Shreveport, LA 71130.
3 Abbreviations used in this paper: LN, lymph node; FP, footpad; PLN, popliteal LN; LDA, limiting dilution analysis; p.i., postinfection; B6, C57BL/6; gB, glycoprotein B; LCMV, lymphocytic choriomeningitis virus; VSV, vesicular stomatitis virus.
Received for publication December 9, 1999. Accepted for publication May 31, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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