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Department of Pathology, University of Massachusetts Medical Center, Worcester, MA 01655
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-secreting cells in the
spleen during an acute LCMV infection. CD8+ T cells made up
the majority of the IFN--producing cells, but analysis of the cell
culture supernatants by ELISA showed that the CD4+ T cells
produced more IFN- on a per cell basis. Using limiting dilution
assays, we examined the CD4+ T cell precursor (Thp)
frequency in C57BL/6 mice infected with LCMV. The virus-specific Thp
frequency increased from <1/100,000 in uninfected mice to a peak of
1/600 in purified splenic CD4+ T cell populations by 10
days postinfection with LCMV. After the peak of the response, the Thp
frequency decreased only about twofold per CD4+ T cell to
1/1200 and remained stable into long term memory. In contrast to the
highly activated CD4+ T cells recovered during the acute
LCMV infection, the memory CD4+ T cells were maintained at
a lower activation state as judged by cell size and ability to secrete
IFN-. Thus, like the CD8+ T cell frequencies, the
CD4+ T cell frequencies remain elevated after the acute
infection subsides and stay elevated throughout long term immunity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although usually not as vigorously stimulated as CD8+ T cells, CD4+ T cells also become activated and respond to viral infections (4). In some but not all systems, their response is required for the induction of CD8+ CTL, and even in systems such as LCMV, where their response is not required for CTL induction (5, 6, 7), CD4+ T cells may be required for the long term maintenance of CD8+ T cell memory (8). Surprisingly little quantitative information, however, is known about the acute CD4+ T cell responses to experimental viral infections and about the frequencies and stability of virus-specific CD4+ T cells in the memory state. How memory CD4+ T cells may contribute to the stability of memory CD8+ T cells is also unclear. To begin to address these issues, we have undertaken a quantitative analysis of the mouse CD4+ T cell response to LCMV, a virus for which the CD8+ T cell response has been very well analyzed. We show here that, like the CD8+ T cell frequencies, the CD4+ T cell frequencies remain elevated after the acute infection subsides and stay elevated throughout long term immunity, albeit in a less active state.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (H-2b) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and used at 2 to 14 mo of age for most experiments. The LCMV Armstrong strain was propagated in BHK21 baby hamster kidney cells (9). Mice were inoculated i.p. with 7 x 104 plaque-forming units of LCMV diluted 1:100 in PBS in 0.1 ml vol/mouse. This dilution in PBS was done to prevent CD4+ T cell activity against cell debris or FCS Ags present in the virus stock.
Cell preparations and lymphocyte phenotyping
Mice were killed by cervical dislocation, and spleens were removed aseptically. Splenic leukocytes were obtained by preparing single-cell suspensions from spleens and treating them with 0.84% NH4Cl to lyse Es, as described previously (10). Cell culture medium used was RPMI medium (Sigma, St. Louis, MO) containing 10% FCS (Sigma), 100 U/ml penicillin G, 100 mg/ml streptomycin sulfate, 2 mM L-glutamine, 5 x 10-5 M 2-ME, 0.1 mM sodium pyruvate (Life Technologies, Grand Island, NY), 0.1 mM nonessential amino acids (Life Technologies), and 10 mM HEPES. Splenic leukocytes from individual animals were analyzed in two-color mode on a FACScan cytometer (Becton Dickinson, Mountain View, CA). The mAb used for staining were phycoerythrin (PE) conjugated (H129.19 anti-CD4 and PK136 anti-NK1.1) or FITC conjugated (53-6.7 anti-CD8, IM7 anti-CD44, RA3-6B2 anti-CD45R/B220, MEL-14 anti-CD62L, and H1.2F3 anti-CD69). All mAb were purchased from PharMingen (San Diego, CA). Approximately 1 x 106 cells were stained, and 5,000 to 10,000 events were acquired from each preparation. The data were analyzed using either PC-Lysis or Cell Quest software (Becton Dickinson).
ELISPOT assays
ELISPOT assays for cytokine-secreting cells were performed based
on slight modifications of established protocols (11, 12). Briefly,
96-well nitrocellulose-based microtiter plates (Millititer HA;
Millipore, Bedford, MA) were coated overnight at 4°C with 50
µl/well of anti-cytokine mAb diluted in PBS. After the plates
were washed with PBS, all wells were blocked with 200 µl of RPMI 1640
containing 10% FCS for 2 h at 37°C. After a washing with PBS,
the lymphocyte populations (either splenic leukocytes or FACS-separated
T cell populations) were then added to the wells (1 x
105 to 1.3 x 104 or from 1 x
105 to 7.8 x 102 cells/well for
responders with 3 x 104 LCMV-infected irradiated
(2000 rads) peritoneal exudate cells (PEC) as stimulators) in RPMI
1640, 10% FCS (200 µl/well total volume) and incubated for 20 h
at 37°C. After the wells were washed in PBS-Tween, biotinylated
anti-cytokine mAb were added, diluted in PBS containing 10% FCS
(100 µl/well), and incubated overnight at 4°C. Plates were washed
in PBS-Tween, and 100 µl of an anti-biotin mAb conjugated with
peroxidase (1/250 dilution in PBS, 10% FCS; Jackson Immunoresearch
Laboratories, West Grove, PA) per well were added, followed by another
overnight incubation at 4°C. Spots representing individual
cytokine-secreting cells were visualized by developing with the
substrate 3-amino-9-ethylcarbazole and counted using an Olympus SZ-STS
Stereozoom microscope (Lake Success, NY). All assays were performed in
triplicate. Mean numbers of cytokine-secreting cells were calculated
from the triplicate assays. The results shown are means ± SD for
two to three separate experiments. The following pairs of mAb were used
in the ELISPOT assays: anti-IL-4, BVD4-1D11, and
biotinylated BVD6-24G2; anti-IFN-, R4-6A2, and biotinylated
XMG1.2. The mAb were used at a concentration of 2 µg/ml, with the
exception of R4-6A2, which was used at 10 µg/ml. All of the mAb were
obtained from PharMingen.
Restimulation in vitro and cytokine ELISA
Unsorted splenic leukocytes or FACS-separated T cells were
resuspended at a final density of 1 x 106 cells
per ml in RPMI 1640 supplemented as above. Responders (100 µl) were
added to 3 x 104 LCMV-infected irradiated (2000 rads)
stimulator PEC in RPMI 1640 (200 µl/well total volume) and incubated
at 37°C. Supernatants were harvested at 24, 48, 72, and 96 h and
stored at -80°C before being assayed for cytokines by ELISA.
Dynatech Immulon 4 plates (Dynatech, Chantilly, VA) were coated by
incubation overnight at 4°C with anti-cytokine mAb diluted in PBS
(50 µl per well). The plates were then washed with PBS-0.05% Tween
20 after each of the following steps. Plates were blocked with PBS-10%
FCS (200 µl/well) for 2 h at room temperature. Samples were
added at 100 µl/well, and a standard curve was constructed for each
plate by using eight twofold dilutions of recombinant cytokine, and the
plates were again incubated overnight at 4°C before the addition of
biotinylated anti-cytokine mAb at 100 µl/well. After a 1-h
incubation at room temperature, 100 µl of avidin-peroxidase (1/400
dilution in PBS, 10% FCS; obtained from Sigma) per well were added,
and the plates were incubated at room temperature for 30 min before
detection with 100 µl of the peroxidase substrate,
3,3',5,5'-tetramethylbenzidine dihydrochloride (Sigma) dissolved in
0.05 M phosphate-citrate buffer, pH 5.0. The reaction was stopped with
25 µl of 2 N H2SO4. Plates were read at 450
nm using a THERMOMAX plate reader and analyzed using
SoftMax 2.3 (both from Molecular Devices, Menlo Park, CA). The pairs of
anti-cytokine mAb listed above for the ELISPOT assays were used in
the ELISA, all at a concentration of 2 µg/ml. Recombinant murine
IFN- and IL-4 used as standards were obtained from PharMingen.
Cell sorting and T cell precursor frequency analysis
CD4 limiting dilution assay (LDA) were performed based on
modifications of established protocols (13, 14, 15). Splenic leukocytes
from a pool of three to four mice were stained with PE-conjugated
anti-CD4 and FITC-conjugated anti-CD8. They were then sorted in
two-color mode on a FACStarPlus flow cytometer from
Becton Dickinson. Sorted CD4+ and CD8+ cells
were consistently >94% pure. Cultures were set up in 96-well U-bottom
plates (Falcon, Lincoln Park, NJ) in a final volume of 200 µl in RPMI
medium. Dilutions of sorted CD4+ T cells in 100 µl
were plated as replicates of 16 or 24 microcultures with 3 x
104 uninfected (mock-infected with BHK21 cell supernatant)
or LCMV-infected irradiated (2000 rads) PEC added in an additional 100
µl. The cultures were incubated at 37°C in 5% CO2. At
various times after the initiation of the cultures, the plates were
centrifuged at 1000 rpm for 5 min, 50-µl aliquots of the
supernatants were transferred to new plates, and IL-2 activity was
measured using the CTLL-2 biologic assay. Briefly, the CTLL-2 cells
were grown in 25-cm2 flasks in RPMI 1640 medium
supplemented with 1 U/ml of recombinant IL-2 (Cellular Products,
Buffalo, NY). The CTLL-2 cells were washed three times with RPMI 1640,
and 5 x 103 CTLL-2 cells in 50 µl were added to 50
µl of the cell culture supernatants in 96-well plates and incubated
for 18 h at 37°C in 5% CO2. Wells were then pulsed
with 1 µCi of [3H]TdR in 50 µl of RPMI for 6 h.
The plates were harvested onto glass fiber filters, and
[3H]TdR incorporation was assayed in a Wallac Betaplate
scintillation counter (Wallac Gaithersburg, MD). In two experiments,
IL-4 activity was determined by the proliferation of the IL-4-dependent
cell line, CT.4S (provided by Dr. William Paul, National Institutes of
Health, Bethesda, MD). Briefly, 50-µl aliquots of the supernatants
were incubated for 24 h with 5 x 103 CT.4S cells
in 50 µl and then pulsed overnight with 1 µCi of
[3H]TdR. Microcultures giving values in the CTLL-2 or
CT.4S assay of >3 SDs above the mean values obtained for APC alone
were scored as positive. The frequencies were corrected for the purity
of the sorted CD4+ populations. Frequencies were calculated
using 
2 analysis according to the method of Taswell (16)
on a computer program provided by Dr. Richard Miller (University of
Michigan, Ann Arbor, MI). The frequencies reported against uninfected
APC are estimated, given that the curves generated from the responder
dilution series often yielded linear regression lines that never
crossed the 37% negative well threshold. Thus, these frequencies may
reflect slight overestimates of the background response to uninfected
APC. However, the response against uninfected APC was always
significantly lower than that against virus-infected APC in all of the
LCMV-infected mice.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Figure 1
shows the percentage of
CD4+ and CD8+ T cells in the spleen of
C57BL/6 mice acutely infected with LCMV. Also shown in Figure 1 is the
twofold increase in the total leukocyte number in the spleen that
occurs during the acute LCMV infection. The increase in the percentage
of CD8+ T cells combined with the increase in the total
number of cells results in a massive 5- to 20-fold expansion in the
number of CD8+ T cells in the spleen during LCMV infection
(10). In contrast, there is only a modest increase in the total number
of CD4+ T cells in the spleen, because the percentage of
CD4+ T cells has decreased at the peak in the leukocyte
number (Fig. 1). Although the total CD4+ T cell number
remains stable, further analyses revealed evidence of CD4+
T cell activation as shown by blastogenesis and activation Ag
expression. The mean percentages of blast-sized CD4+ T
cells from eight individual mice per group were as follows: day 0, 8;
day 3, 9; day 5, 16; day 7, 25; day 9, 21; day 11, 19; day 15, 11.
Figure 2 shows the distribution of
CD4+ T cells following an acute infection with LCMV with
regard to the Ags CD44 (Pgp-1), a marker the cell surface expression of
which increases on activated T cells, and CD62L (MEL-14), a molecule
with a cell surface expression that decreases on activated T cells
(17). Table I shows that there is an
increase in the mean fluorescence intensity (MFI) of CD44 on gated
CD4+ T cells, and as can be seen in Figure 2, this increase
in the MFI is mainly due to an increase in the relative proportion of
CD4+ cells expressing a high level of CD44 rather than all
of the CD4+ T cells staining more highly with CD44.
Likewise, Table I shows that there is a decrease in the MFI of CD62L on
gated CD4+ T cells, and as can be seen in Figure 2, this
decrease in the MFI seems to be due to an increase in the relative
proportion of CD4+ cells expressing a low level of CD62L.
We also performed a more detailed time course of the cell surface
expression of CD44 and CD62L at days 0, 3, 5, 7, 9, 11, and 15
postinfection (p.i.). The peak in cell surface expression of the
activation marker CD44 was at day 11 p.i. and correlated with the
lowest cell surface expression of CD62L (data not shown).
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). In
contrast to CD44 and CD62L discussed above, CD4+ T cell
surface expression of the very early activation Ag CD69 peaked much
earlier, at 3 days p.i., and declined thereafter (data not shown).
CD45RB cell surface expression has been shown to increase on effector
CD4+ T cells (17). In agreement with this, we observed an
increase in the cell surface expression of CD45RB on CD4+ T
cells at day 10 p.i. with LCMV. Interestingly, while CD45RB
expression increases on activated cells and then decreases on memory
CD4+ T cells, a sialated form of this protein (CZ-1),
previously shown to be bright on activated cells, remains high on
memory CD4+ T cells, and this is reflected in the higher
expression of CZ-1 vs CD45RB on CD4+ immune cell
populations (day 60, Table I) (18). Thus, LCMV infection induces an
activated phenotype on CD4+ T cells that peaks by day
11 p.i. Cytokine secretion during the acute LCMV infection
The above experiments show that CD4+ T cells
expressing an activated cell surface phenotype are induced following an
acute LCMV infection. Since one of the primary effector functions of
CD4+ T cells is the secretion of various cytokines, we
utilized the ELISPOT assay to assess IFN- and IL-4 production at the
single-cell level in C57BL/6 mice acutely infected with LCMV.
Uninfected mice (day 0) had no detectable IFN-- or IL-4-secreting
cells (hereafter referred to as IFN-+ and
IL-4+ cells) in the spleen. Following an infection with
LCMV, the frequency of IFN-+ cells per spleen leukocyte
started to increase by day 5 p.i. and reached its peak by day
9 p.i. The mean numbers of IFN--secreting cells per
105 splenic leukocytes from eight individual mice per group
were as follows: day 0, 0; day 3, 0; day 5, 6; day 7, 177; day 9, 346;
day 11, 178; day 15, 124. On a per spleen basis, the peak in the total
number of IFN-+ cells was even more pronounced at days 7
to 11 p.i., because the number of cells in the spleen had nearly
doubled (Fig. 1). By day 15 p.i., the frequency of
IFN-+ cells had started to decline. The decline in the
frequency of IFN-+ cells after days 7 to 11 p.i.
coincided with the clearance of the virus and the decline of the
CD8+ CTL response. In contrast to IFN-, few
IL-4+ cells could be detected in the spleen, agreeing with
previous work suggesting that LCMV induces primarily a Th1
response (19).
CD4+ and CD8+ T cells from the spleens of mice
that had been acutely infected with LCMV were purified by cell sorting
to determine whether virus-specific CD4+ T cells
contributed to the secretion of IFN- in the ELISPOT assays described
above. Table II, which presents the
results from two to three separate experiments per time point, shows
that CD8+ T cells made up the majority of IFN--secreting
cells during the acute LCMV infection. However, there were detectable
frequencies of LCMV-specific CD4+ T cells secreting
IFN-. Furthermore, we noticed in these assays that, while on average
11-fold more CD8+ than CD4+ T cells secreted
IFN-, the CD4+ "spots" in the assays were
consistently larger. This suggested that the CD4+ T
cells may be making more IFN- on a per cell basis than the
CD8+ T cells. Previous work with in vitro systems has shown
that CD4+ T cells can secrete higher levels of cytokines
than similarly stimulated CD8+ T cells (20). To examine
whether this were the case during LCMV infection, we FACS-purified
CD4+ and CD8+ T cells at various times p.i.,
restimulated them in vitro with virus, and performed ELISA assays on
the culture supernatants. Table II shows that CD4+ T cells
secreted as much IFN- protein into the cell culture supernatant as
CD8+ T cells, even though there were 11-fold more
CD8+ T cells secreting IFN- than CD4+ T
cells, as detected with ELISPOT assays. This indicates that the
CD4+ T cells make more IFN- on a per cell basis than
do the CD8+ T cells.
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in the 20-h assay was
greatly reduced compared with cells isolated during the acute LCMV
infection. We were unable to detect any CD4+ T cells
secreting IFN- from the memory mice. This reduced frequency likely
reflects a decrease in the activation status of these cells rather than
a reduction in the frequency of cells capable of secreting IFN-, as
discussed below. The relatively short assay time (20 h) of the ELISPOT
assay is probably not long enough to fully stimulate the memory T cells
as opposed to the already activated cells taken from the acute LCMV
infection. Quantitation of the CD4+ T cell precursor during the acute LCMV infection and on into memory
To quantitate the virus-specific CD4+ T cell
response during LCMV infection, an assay for LCMV-specific Thp was
adapted from the method used by recent studies in the influenza and
Sendai virus systems (14, 15, 21). Figure 3 shows a typical regression line
obtained from C57BL/6 mice during the acute infection with LCMV (day 7)
and into memory (day 60). We performed kinetic studies (Fig. 3) to
confirm that we were measuring the peak of the IL-2 production in
the LDA cultures. These kinetic studies revealed that high frequencies
of Thp during the acute LCMV infection and into memory were found after
at least 48 h following the initiation of the culture. These
results and interpretations agree with the initial studies performed
using Sendai virus (14).
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). The
LCMV-specific Thp frequency rose from <1/100,000 in a naive animal
(day 0) to 1/600 by day 10 p.i. with LCMV. This peak in the Thp
frequency by day 10 p.i. corresponds with that of the activation
markers CD44 and CD62L discussed above. Interestingly, the Thp
frequency per CD4+ T cell dropped only twofold from the
peak of the response (days 9–11) on into long term memory. This
relatively small reduction in the Thp frequency mirrors the twofold
drop in frequency that we have previously reported for the
CD8+ T cell response during LCMV infection (1). In fact, on
a per spleen basis, the decline in the total number of CD4+
memory T cells is even less than the decline in the total number of
CD8+ memory T cells, because the percentage of
CD4+ T cells increases between the peak of the acute
infection and memory, whereas the percentage of CD8+ T
cells decreases (see Fig. 1).
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, treatment with anti-CD4 mAb blocked all of the virus-specific
IL-2 production, returning the precursor frequency to a level
comparable to that obtained against uninfected APC. Second, because
there was the possibility that the CTLL-2 cells used to monitor IL-2
production in these LDA could be detecting IL-4, we added anti-IL-4
mAb to the assays. Table IV shows that there was no effect (within the
95% confidence limits) in three of the four experiments when
anti-IL-4 mAb was added to the LDA cultures. In one experiment,
using LCMV-immune animals, the presence of the anti-IL-4 mAb only
slightly (within the 95% confidence limits) reduced the resulting
precursor frequency. In two additional experiments, we used the very
IL-4-sensitive CT.4S cells line in the LDA. No IL-4 could be detected
using this indicator cell line at day 15 or 240 p.i. in the LDA.
LCMV infection is thought to stimulate primarily a Th1 cytokine profile
in the responding T cell populations (19). The anti-IL-4 blocking
experiments and use of the CT.4S cells support this, suggesting that
the LCMV-specific CD4+ T cells are primarily of a Th1
phenotype.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in short-term assays, the Thp frequencies 1 year after
infection are only twofold lower than their peak at day 10 p.i.
Virus-specific CD8+ CTL memory has also been shown to be
long-lived (2, 22), and we recently reported that the frequency of
LCMV-specific precursor CTL per CD8+ T cell drops only
twofold from its peak, as the acute LCMV infection converts into a
memory state (1). This 2-fold drop per CD8+ T cell occurs
during the more global 5- to 10-fold drop in the total CD8+
T cell number per spleen, as the immune response silences and the
CD8/CD4 ratio converts from 2:1 to 1:2, as shown in Figure 1. A similar
observation has been made in an LCMV-specific CD8+ TCR
transgenic model, in which the frequency of adoptively transferred
transgenic T cells reached 70% of the CD8+ T cells during
the peak of the acute infection and remained at 27% in the memory
state (23). Thus, in the wake of a substantial reduction in the number
of CD8+ T cells, the proportion of virus-specific cells
within this compartment is, like our present observation with
CD4+ T cells, only moderately modulated, in a manner
consistent with a 1:1 dilution with naive cells. These results indicate
that after an acute viral infection resolves, both the CD4+
and CD8+ T cell pools remain heavily biased with
virus-specific memory T cells.
Little quantitative information has been generated on the stability of
CD4+ T cells in viral infections. Some quantitation of
CD4+ T cell precursors has been performed in the Sendai and
influenza virus systems (14, 15, 21). In each of these systems, the
observed peak in Thp frequency is 1/100, higher than that we have
observed here for LCMV-specific Thp. However, the Thp frequency in the
memory state in each of these systems is near 1/1400, very close to the
frequency of 1/1200 we have shown here for LCMV-specific Thp (Table III). In addition, Topham et al. (15) show that the influenza-specific
Thp frequency remains relatively stable in the spleen for 6 mo but
drops off during this time in both the mediastinal and cervical lymph
nodes. Thus, in both the Sendai and influenza virus systems, there
seems to be a larger, 10-fold drop in the Thp frequency from the peak
of the response into long term memory due to a higher peak in the Thp
frequency (4, 15). We have not seen such a drop in the LCMV system for
either CD4+ or CD8+ T cells (Table III and 1 . Whether this reflects differences in the immune response to these
viruses or differences in the assay systems is unclear. T cells in the
resting memory state may have different activation requirements than
those during the acute infection, and subtle differences in the LDA
might affect that outcome. Earlier work on LCMV has suggested more
extreme drops on CTP precursor (CTLp) frequencies between the acute
infection and the memory state (2, 8, 24), but analysis of our own data
and of recent data from other laboratories indicates that there is very
little drop when the assays are optimized and the data are expressed as
CTLp per CD8+ T cell (1, 2, 23).
Factors contributing to the maintenance and preservation of memory T
cells are poorly understood. Recent work has indicated that the
presence of class II and class I MHC molecules is required for the
respective survival of CD4+ and CD8+ T cells in
vivo (25, 26). This argues that the TCR on memory cells need to be
triggered for their survival, but this is likely to be a lower affinity
event than that which initially activated the cells, because memory
cells can persist in vivo in the absence of the Ag to which they were
initially generated (2, 22). The potent CD8+ CTL response
during the LCMV infection does not require CD4+ T cells for
its induction (5, 6, 7), but in the absence of CD4+ T cells
LCMV-specific CD8+ CTL memory rapidly wanes (8). This
suggests that the CD8+ T cells receive growth or survival
factors from the CD4+ T cells to perpetuate in this
environment devoid of the strong Ag stimulus that elicited the
CD8+ T cell response in the first place. At least some
memory CD8+ T cells appear to be in a higher activation
state than memory CD4+ T cells. At any given time, a higher
percentage of CD8+ T cells is undergoing blastogenesis than
CD4+ T cells (27), and Table II shows that in 20-h ELISPOT
assays, some IFN- production can be detected in the CD8+
T cell population but not in the CD4+ T cell population. We
have recently reported that enriched blast-sized CD8+ T
cells >1 year after the infection has cleared can still mediate direct
cytotoxic activity against sensitive targets (28). We are left with the
irony that the memory CD8+ T cells are more active
than the memory CD4+ T cells and yet require the
CD4+ T cells to maintain their activity. Whether autologous
stimulation is sufficient for the maintenance of the more dormant
CD4+ T cell population is not known.
In this study, we utilized a panel of cell surface markers to define
activated CD4+ T cells and showed that by day 10 p.i.
with LCMV, there was an increase in the cell surface expression of
activation markers such as CD44, CD49d, LFA-1, and CZ-1 on gated
CD4+ T cells (Fig. 2 and Table I). While there was little
increase in the absolute numbers of CD4+ cells in the
spleen expressing these activation markers, there was an increase in
the relative proportion of CD4+ cells expressing these
activation markers (Fig. 2). This observation agrees well with a recent
study using the Traub strain of LCMV in BALB/c mice (29). Our previous
work has shown that many of these CD4+ T cells are
blast-sized at day 6 p.i., in agreement with an activated state
(18). Here we report that 25% of the CD4+ T cells in the
spleen were blast-sized at day 7 p.i. These data contrast with a
report by Christensen et al. using the Traub strain of LCMV in BALB/c
mice, in which they show that only 2% of CD4+ T cells
exhibit increased DNA content during the peak of the infection (30).
The discrepancy between these findings and ours may be due to the
differences in the strains of mice or viruses that were used in these
studies. Even though 25% of the CD4+ T cells were
blast-sized, there was little or no increase in the total number of
CD4+ T cells in the spleen during an acute LCMV infection
(Fig. 1). This observation highlights a difference between the LCMV
virus system and other protein-Ag systems in which peptide-specific
CD4+ T cells have been shown to expand in number (31, 32).
Interestingly, the peak of expression of the very early activation Ag
CD69 occurred at 3 days p.i., well before the peak of the other markers
discussed above (Table I). This expression pattern parallels the type I
IFN response in these mice (33). To our knowledge, type I IFN has not
been previously reported to up-regulate CD69 expression on
CD4+ T cells, but it has been shown to be induced by
IFN-
on NK cells (34).
The frequency of IFN--secreting cells in the spleen peaked by day
9 p.i. in general agreement with Gessner et al. (Table II) (35, 36). Our frequency of IFN- producing splenocytes at day 7 p.i.
was 10-fold higher than that reported by Cousens et al. (37) using the
same strain of mice and virus used here. The reason for this
discrepancy may be due to us adding LCMV-infected PEC to our ELISPOT
assays to enhance the virus-specific stimulation during these assays.
We found consistently more CD8+ T cells secreting IFN-
than CD4+ T cells during the acute LCMV infection, as
detected at the single-cell level utilizing the ELISPOT assay. This
ratio was not surprising, given that there were 10-fold more CTLp
than Thp at these time points during the acute LCMV infection (1).
Interestingly, in four independent experiments, the CD4+
and CD8+ T cells secreted comparable amounts of IFN-
protein into the supernatant, even though there were many more
IFN--secreting CD8+ T cells than CD4+ T
cells as detected by ELISPOT assays (Table II). This observed
difference may be explained by fact that LCMV infection induces
CD8+ CTL with high cytotoxic activities that may be capable
of destroying virus-infected APC and thus reducing the amount of
IFN- that is produced by the CD8+ CTL (38). However,
this seems unlikely given that the number of LCMV-infected APC used in
these assays is in great excess over the frequency of responding
CD8+ T cells. Of interest is that recent studies have found
that CTL clones with high cytotoxic activity produced low amounts of
cytokines (39).
Examination of the CD4+ Thp frequency during an acute LCMV
infection and on into the memory state revealed that within 10 days
p.i. with LCMV, the Thp frequency rose from <1/100,000 to 1/600.
The Thp frequency remained elevated in immune animals, dropping only
twofold to 1/1200, and remained very stable into long term memory.
While the CD4+ T cell response to LCMV resembles that of
the CD8+ T cells, there are significantly fewer
virus-specific CD4+ than CD8+ T cells. It is
not clear whether the difference in precursor frequencies between the
CD4+ and CD8+ T cells reflects real differences
or is due to the inherent limitations of the CD4+ Thp LDA.
The efficiency of these assays is understood not to be 100%, and
studies have provided indirect evidence that most of the activated and
proliferating CD8+ T cells during the acute LCMV infection
are LCMV specific (40), even though only 3 to 4% can be detected by
LDA. It is also possible that some virus-specific Thp secrete cytokines
other than IL-2 (such as IFN- or TNF-) and would not be detected
by the CTLL-2 assay used here.
In the present study, we have shown that there is little change in the frequency of virus-specific CD4+ T cells from the peak of the response into long term memory. Thus, the host T cell repertoire remains biased with a high frequency of LCMV-specific memory CD4+ T cells after clearance of the acute virus infection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Raymond M. Welsh, Department of Pathology, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, MA 01655. E-mail address:
3 Abbreviations used in this paper: CTLp, cytotoxic T lymphocyte precursor; Thp, T cell precursor; LCMV, lymphocytic choriomeningitis virus; PE, phycoerythrin; PEC, peritoneal exudate cells; MFI, mean fluorescence intensity; p.i., postinfection; LDA, limiting dilution assay.
Received for publication November 19, 1997. Accepted for publication March 4, 1998.
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References |
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