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*
Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark; and
Institute for Advanced Study, Princeton, NJ 08540
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-deficient (IFN--/-) mice inoculated with
intermediate doses of a slowly replicating strain of lymphocytic
choriomeningitis virus become chronically infected. In such mice a
hypercompensated CTL response is observed that partially controls virus
replication. Here we have investigated whether CD4+ Th
cells are required to establish and maintain this new equilibrium. The
absence of IFN- does not impair the generation of IL-2-producing
CD4+ cells, and depletion of these cells precipitates
severe CD8+ T cell-mediated immunopathology in
IFN--/- mice, indicating an important role of
CD4+ T cells in preventing this syndrome. Analysis of organ
virus levels revealed a further impairment of virus control in
IFN--/- mice following CD4+ cell
depletion. Initially the antiviral CTL response did not require
CD4+ cells, but with time an impaired reactivity toward
especially the glycoprotein 33–41 epitope was noted. Enumeration of
epitope-specific (glycoprotein 33–41 and nucleoprotein 396–404)
CD8+ T cells by use of tetramers gave similar results.
Finally, limiting dilution analysis of CTL precursors reveal an
impaired capacity to sustain this population in
CD4+-depleted mice, especially in mice also deficient in
IFN-. Thus, our findings disclose that T cell help is required to
sustain the expanded CTL precursor pool required in
IFN--/- mice. This interpretation is supported by
mathematical modeling that predicts an increased requirement for help
in IFN--/- hosts similar to what is found with fast
replicating virus strains in normal hosts. Thus, the functional
integrity of CD8+ effector T cells is one important factor
influencing the requirement for T cell help during viral
infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The kinetics of initial virus multiplication and subsequent virus distribution seem to play a critical role in determining the outcome of a noncytocidal virus infection. Parameters such as cell tropism and the replication rate of the virus as well as the dose of infection are important determinants in this context. Thus, rapidly replicating, invasive strains are more likely to exhaust the immune response and cause persistence than are slowly replicating ones, and the same is true for a high dose vs a low dose infection (1, 5, 6, 7, 8, 9).
With regard to the host, T cell responder status and thus initial T
cell precursor frequencies are important in influencing the outcome of
infection, as is the extent of the subsequent clonal expansion
(1, 3, 4, 9, 10, 11). The CD8+ T cell
population constitutes an important part of the antiviral immune
response. Virus-specific CD8+ CTL are crucial for
elimination of infected cells (12, 13), and in addition,
CD8+ cell-mediated production of antiviral
cytokines such as IFN- may further reduce viral replication
(14). In contrast, the role of CD4+
T cells is less clearly defined (15).
CD4+ T cells are important in regulating the
immune response, and besides providing help to B cells, they may exert
direct effector function by generating an abundance of cytokines
(16). Additionally, they are believed to be important for
the conditioning of APCs, making these capable of delivering
costimulatory signals to the Ag-specific CD8+
cells (17, 18, 19). Thus, CD4+ T cells
probably contribute to the antiviral CD8+ T cell
response in two ways: 1) CD4+ T cell/APC
interaction secures an optimally stimulating APC, and 2)
CD4+ cells provide most of the IL-2 involved in
driving the essential clonal expansion (20).
Infection of mice with lymphocytic choriomeningitis virus (LCMV) is a typical example of an infection with a noncytolytic virus, and this model highlights the crucial role for CD8+ T cells in resolution of infection (12, 13, 21, 22). However, studies concerning the contribution of CD4+ cells to resolution of this infection are ambiguous. By use of either CD4-depleted mice, CD4 knockouts, or MHC class II-deficient mice, it has been shown that CD4+ cells are dispensable for the initiation of the primary CTL response, but are pivotal for the long term maintenance of CD8+ effector cell activity and an expanded CTLp pool (21, 23, 24, 25, 26). Furthermore, under conditions of high dose infection or infection with rapidly replicating and disseminating LCMV strains, CD4+ cells are required to prevent rapid exhaustion of the CTL pool (27, 28). In contrast, CD4+ cells do not seem to be required for clearance of less invasive strains of LCMV, e.g., LCMV Armstrong (23, 28, 29).
As mentioned above, IFN- may be important in reducing viral
spreading and is thus likely to be an additional parameter important in
the virus-host balance. However, the role of IFN- in the outcome of
an LCMV infection has, like that of CD4+ cells,
been somewhat elusive. Whereas this cytokine in general is said to be
primarily important for resolution of infections with cytolytic viruses
(30), we have recently shown that absence of IFN-
during LCMV infection may have serious consequences for the outcome of
infection with this noncytolytic virus. Thus, while wild-type mice
control an infection with the vicerotropic and rapidly invasive LCMV
Traub strain, IFN--/- mice develop a severe
CD8+ T cell-mediated wasting disease and succumb
to the infection (31). In contrast, challenge with the
less invasive LCMV Armstrong strain does not lead to wasting disease in
IFN--/- mice. However, a chronic state of
infection is induced in the majority of these mice. Notably, virus
persistence is not the result of CTL exhaustion; instead, virus
coexists with augmented ex vivo CTL activity (32), and
analysis of cell cycle status and activation markers indicates that the
CD8+ T cell population is subject to permanent
stimulation and increased cellular turnover. Whether
CD4+ cells play a role in maintaining this
delicate new balance, e.g., by delivering help to the
CD8+ cell-mediated immune response in these mice,
is not known. Therefore, in the present study we examined the
importance of CD4+ cells for
CD8+ T cell-mediated virus control under these
conditions. Our study reveals that CD4+ cells are
crucial for maintaining the CD8+ T cell-mediated
control of LCMV Armstrong infection in
IFN--/- mice. Most striking, the clinical
effect of CD4+ T cell depletion is severe wasting
and death. Thus, our findings underscore that
CD4+ T cells are pivotal for the host to adapt in
a situation where the functional integrity of the generated effector T
cells is perturbed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (B6) wild-type mice were obtained either from
Bomholtgaard (Ry, Denmark) or The Jackson Laboratory (Bar Harbor, ME).
IFN--deficient (IFN--/-) mice
(C57BL/6-ifg<tm1>) were also derived from The Jackson Laboratory
either directly or as the progeny of breeder pairs. Seven- to 10-wk-old
mice were used in all experiments, and animals were always allowed to
acclimatize to the local environment for at least 1 wk before use. All
animals were housed under specific pathogen-free conditions as
validated by screening of sentinels. All animal experiments were
conducted according to national guidelines.
Virus
LCMV of the Armstrong strain (clone 53b) was used in all experiments. Mice to be infected received a dose of 4800 PFU in an i.v. injection of 0.3 ml.
Depletion of CD4+ T cells
The 
CD4 mAb GK1.5 was used. Mice to be depleted received a
dose of 0.1 ml of clarified ascetic fluid in 0.5 ml of PBS i.p. on days
-1, 0, 2, 5, 9, and 16 postinfection (p.i.). Flow cytometric analysis
consistently revealed <1% CD4+ T cells in such
treated mice.
Virus titration
Organ virus titers were assayed by intracerebral inoculation of 10-fold dilutions of a 10% organ suspension into young adult Swiss mice. Titration end points were calculated by the Kärber method and are expressed as the mean lethal dose (LD50).
Cell preparations
Spleens from mice were aseptically removed and transferred to HBSS. Single-cell suspensions were obtained by pressing the organs through a fine sterile steel mesh, and erythrocytes were lysed by 0.83% NH4Cl treatment. The cells were washed twice with HBSS, and cell concentration was adjusted in RPMI 1640 containing 10% FCS supplemented with 2-ME, L-glutamine, and penicillin-streptomycin solution.
Limiting dilution analysis
CTLp frequencies were determined as previously described (32). Briefly, 3-fold dilutions of responder cells were added in 100 µl of medium to round-bottom 96-well microtiter plates. Replicates (24 wells) were plated for each responding cell dilution and cocultured with 100 µl (3 x 105 cells) of 2500 rad gamma-irradiated, T cell-depleted syngeneic splenocytes pulsed with either glycoprotein 33–41 (gp33–41) or nucleoprotein 396–404 (np396–404; the two immunodominant MHC class I-restricted peptides of LCMV in H-2b mice) (33, 34, 35) or unpulsed splenocytes. The medium contained 10 U/ml of human rIL-2. Three identical sets of cultures were initiated with different stimulators and incubated for 7 days at 37°C in a humidified atmosphere. On day 4, 20 µl of medium with IL-2 (100 U/ml) was added to the cultures. The contents of individual wells were tested for cytotoxicity at the end of the culture period by incubating each well with 5000 51Cr-labeled, peptide-pulsed or unpulsed EL-4 cells (H-2b, MHC-I+II-) for 6 h. Wells were considered positive if the cytotoxic activity exceeded the average + 3 x SD of the spontaneous release of target cells incubated with medium alone. Minimal estimates of pCTL frequencies were obtained according to the Poisson distribution.
Cytotoxicity assays
Virus-specific CTL activity was assayed in a standard 51Cr release assay using EL-4 cells pulsed for 1 h at 37°C with LCMV gp33–41 or np396–404 peptide; unpulsed EL-4 cells served as a control target. The assay time was 5 h, and the percent specific release was calculated as described previously (36).
mAb for flow cytometry
The following mAbs were all purchased from PharMingen (San
Diego, CA) as rat anti-mouse Abs: FITC-conjugated anti-CD49d
(common -chain of lymphocyte Peyer’s patch adhesion molecule-1 and
very late Ag-4), PE- and Cy-Chrome-conjugated anti-CD8a,
biotinylated anti-CD44, FITC-conjugated anti-CD4 (for control
of cell depletion, RM4-4 not cross-reacting to GK1.5 was used),
PE-conjugated anti-IFN-, and PE-conjugated anti-IL-2.
MHC/peptide tetramers for flow cytometry
H-2Db/gp33–41 and H-2Db/np396–404 tetramers were obtained through the National Institute of Allergy and Infectious Disease tetramer facility and the National Institutes of Health AIDS Research and Reference Reagent Program.
Flow cytometric analysis
Cells (1 x 106) were stained with directly labeled mAb in staining buffer (10% rat serum, 1% BSA, and 0.1% NaN3 in PBS) for 20 min in the dark at 4°C and subsequently washed. If biotin-conjugated Ab was used, cells were additionally incubated with streptavidin-Tricolor or streptavidin-Cy-Chrome (Caltag, San Francisco, CA), washed, and fixed with 1% paraformaldehyde (37, 38, 39, 40). For visualization of LCMV-specific cytokine-producing CD4+ cells, splenocytes were incubated with MHC class II (Ab)-restricted gp66–81 peptide at 1 µg/ml for 5 h in the presence of IL-2 (50 U/ml) and monensin (3 µM) (41). After incubation cells were surface stained as described, washed, permeabilized, and stained with cytokine-specific mAbs. Finally, cells were washed and fixed as previously described. For tetramer staining, cells were incubated with the tetramers at pretitrated optimal concentrations for 1 h at room temperature, followed by surface labeling as described above.
Cells were analyzed using a FACSCalibur (Becton Dickinson, San Jose, CA), and at least 104 cells were gated using a combination of low angle and side scatter to exclude dead cells and debris. Data analysis was conducted using Cell-Quest software (Becton Dickinson).
Mathematical modeling
We describe a mathematical model that takes into account the
dynamics between LCMV replication and an LCMV-specific CTL response.
The model includes four variables: susceptible uninfected host cells
(x), infected cells (y), LCMC-specific CTLp
(w), and LCMV-specific CTL effectors (z). The
model is given by the following set of differential equations: 1)
x = 
- dx -
(
xy/(qz + 1)); 2) y =
(xy/(qz + 1)) - ay -
pyz; 3) w = csy -
cgyw - bw; and 4) z =
cgyw - hz. Susceptible host cells are
produced at a rate , die at a rate dx and
become infected by LCMV at a rate xy. Infected
cells die at a rate ay. CTLp are assumed to proliferate, but
do not have anti-viral activity. They proliferate at a rate
csy and decay at a rate bw. The parameter
c denotes the CTL responsiveness, determined by the efficacy
of recognition of Ag in conjunction with MHC class I. The parameter
s denotes the level of CD4+ T cell
help. Strong helper cell responses increase the rate of CTLp expansion.
When simulating LCMV dynamics in CD4+
cell-deficient hosts, we assume in accord with experimental data
(23, 25, 26) that the initial phase of CTL expansion is
not impaired by the absence of help. Instead, help becomes important
for CTL expansion after a defined time threshold (25, 26).
Note that the model assumes that CD4+ T cell help
promotes CTL expansion, but not differentiation. CTLe differentiate
from precursors at a rate cgyw and die at a rate
hz. CTLe are assumed not to proliferate at a significant
rate and have anti-viral activity. The model includes two modes of
CTL-mediated antiviral activity: 1) CTL lyse infected cells at a rate
pyz; and 2) CTL also secrete IFN-, which inhibits viral
replication at a rate qz.
LCMV can establish an infection if its basic reproductive ratio
(R0 = /da) is greater than unity. In
this case, the system can converge to one of two different equilibria.
If the CTL responsiveness and/or the level of
CD4+ cell help lies below a threshold, the CTL
response goes extinct, resulting in uncontrolled virus replication.
This is described by the following equilibrium expressions: 1)
x* = /; 2) y* = / -
d/; 3) w* = 0; and 4) z* = 0. A
sustained CTL response becomes established at significant levels if
csy* >> 0. Virus replication in
the presence of the CTL response is described by a third degree
polynomial. For simplicity the expressions are not written out
here.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- mice
Although the CD8+ T cell response in
IFN--/- mice infected with LCMV Armstrong
has previously been extensively characterized (29, 31, 32, 42), the CD4+ response has not been
analyzed before. To determine whether an unimpaired
CD4+ T cell response was generated in the absence
of IFN-, wild-type mice and IFN--/- mice
were infected with 4800 PFU of LCMV Armstrong, and on days 7 and
14 p.i. spleen cells were harvested and stimulated with an
immunodominant MHC class II-restricted LCMV-derived peptide
(gp66–81) for 5 h. Following in vitro
stimulation, IFN-- and IL-2-producing CD4+
cells were quantitated using a flow cytometry. As evident from the
analysis on both day 7 (Fig. 1
) and day
14 p.i. (percentage of CD4+ cells also
IL-2+: IFN--/- mice,
2.0 (1.3–2.1); wild-type mice, 1.6 (1.3–2.7); median (ranges) of four
mice per group), similar numbers of LCMV-specific IL-2 producing
CD4+ cells were generated in the absence of
IFN-. Therefore, the following experiments were aimed at evaluating
the functional importance, if any, of CD4+ cells
in LCMV-infected IFN--/- mice.
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-/-
mice
Infection of immune-competent mice with the slowly replicating
LCMV Armstrong strain induces a potent CD8+ T
cell mediated immune response which rapidly controls the infection in
all organs. Under these conditions depletion of
CD4+ cells does not affect the outcome of
infection (23, 26, 28, 29). In contrast,
IFN--/- mice infected with this virus
develop a chronic infection characterized by long-standing coexistence
of virus and significant ex vivo virus-specific CTL activity
(32). To investigate whether CD4+
cells play a role under these conditions, the course of LCMV Armstrong
infection was studied in CD4-depleted
IFN--/- mice. To start with, clinically
relevant parameters such as survival and weight loss were determined.
Notably, depletion of CD4+ cells resulted in a
severe wasting disease in IFN--/- mice that
terminated by the death of all treated animals within 3–4 wk p.i.
(Fig. 2, A and B).
In contrast, wasting was not observed in CD4-competent
IFN--/- mice except in a few cases in which
transient weight loss was noted. Nor did we observe wasting in
CD4-depleted wild-type mice and except for CD4-depleted
IFN--/- mice all mice survived the infection
(Fig. 2A). Depleting infected
IFN--/- mice of both
CD4+ and CD8+ cells
prevented both wasting disease and death (Fig. 2B),
demonstrating that CD8+ effector cells are
causing the symptoms observed in CD4-depleted infected
IFN--/- mice.
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-/- mice
CD4+ T cells has previously been found to be
redundant for the immune-mediated control of acute LCMV Armstrong
infection in wild-type mice (23, 26, 28, 29). To see
whether CD4+cells were important for virus
control in IFN--/- mice, organ virus titers
in CD4-depleted IFN--/- mice were assayed 7
and 14 days after infection (Fig. 3).
Seven days p.i., CD4-depleted IFN--/- mice
contained levels of virus in spleen, liver, and lungs similar to those
in infected CD4+ cell-competent
IFN--/- mice and infected wild-type mice.
However, on day 14 p.i. organ virus levels in CD4-depleted
IFN--/- mice were substantially elevated
compared with those in matched CD4-intact mice. Thus, depletion of
CD4+ cells further impaired the capacity of
IFN--/- mice to contain the infection.
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and
CD4+ cells
The coincidence of higher organ virus levels and
CD8+ T cell-mediated wasting in CD4-depleted,
infected, IFN--/- mice suggested that the
disease in these mice was the result of a partially impaired CTL
response leading to a chronic immunological conflict (4).
Therefore, direct analysis of ex vivo cytotoxicity was performed.
Previous results have shown that CTL activities in infected
IFN--/- mice and wild-type mice are of
similar magnitude the first 2 wk into the infection, but stay high in
mutants while gradually disappearing in wild-type mice as the infection
is controlled (32). The responsiveness of splenic effector
cells from infected IFN--/- mice depleted of
CD4+ cells was assayed and compared with that of
their CD4+ cell-competent counterparts. The
responses to two immunodominant MHC class I-restricted peptide epitopes
(gp33–41 and np396–404,
respectively) were determined on days 7 and 14 p.i. (Fig. 4). Although both groups of animals were
clearly able to raise a CTL response that killed target cells pulsed
with either peptide, analysis of per cell cytolytic activity on day
14 p.i. revealed that depletion of CD4+
cells significantly impaired the response to
gp33–41-pulsed target cells in
IFN--/- mice (Fig. 4C). Indeed,
in two independent experiments only two of seven CD4-depleted mice
exhibited gp33–41-specific killing within the
range of the response in CD4-intact animals. A trend toward a reduced
response was also noted for np396–404-pulsed
target cells. Notably, the total number of splenocytes in CD4-depleted
mice was about half that in undepleted mice on day 14 p.i.
Therefore, total cytolytic capacity per spleen was consistently lower
in the absence of CD4+ cells.
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-/- mice, nondepleted
IFN--/- mice, and wild-type mice regardless
of peptide epitope and concentration (data not shown). Thus, CTLs
generated in IFN--/- mice do not exhibit any
compensatory increase in average avidity.
Depletion of CD4+ cells affects the number of
virus-specific cells in IFN--/- mice
To directly enumerate virus-specific CD8+ T
cells, tetramer staining was performed in infected CD4-depleted and
CD4-competent, IFN--/- and wild-type mice.
Spleen cells harvested from mice infected 7 and 14 days previously were
allowed to bind to fluorescence-stained tetramer complexes containing
MHC class I (H-2Db) expressing either
gp33–41 or np396–404 and
were subsequently analyzed by flow cytometry (Fig. 5, A and C).
Generally, a trend toward higher numbers of virus-specific
CD8+ cells was observed in
IFN--/- mice at both time points. With
regard to the effects of CD4+ cell depletion,
marginal differences were detected when the mice were analyzed on day
7 p.i. However, when analyzed on day 14 p.i. (Fig. 5C), CD4 depletion was associated with lower numbers of both
gp33–41- and np396–404-specific cells in IFN-
-/- mice. In relative terms the difference was most
pronounced for gp33–41-specific cells
(
3-fold) consistent with the result of the CTL analysis. In
contrast, CD4 depletion had little if any effect on the number of
virus-specific cells in wild-type mice infected 14 days previously.
This pattern suggests that IFN--/- mice, due
to their genetic defect, compensate by generating more virus-specific
CD8+ cells during the acute infection, and that
the ability to maintain this compensation requires
CD4+ cells.
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-/- mice
in the absence of CD4+ cells
Finally, we investigated whether the absence of
CD4+ cells affected the ability to generate and
maintain an expanded CTLp population in infected
IFN--/- mice. Limited dilution assays were
performed with splenic cells from infected
IFN--/- and wild-type mice either depleted
of CD4+ cells or left untreated (Fig. 5
, B and D). Particularly on day 14 p.i. (in
nine of nine mice tested), but also in some (three of nine) animals
analyzed on day 7 p.i., CTLp frequencies for
gp33–41- and
np396–404-specific CTLs in CD4-depleted knockout mice
were below the ranges observed in their CD4+
cell-competent counterparts, a tendency that was not nearly as marked
when comparing CD4-depleted and CD4-competent wild-type mice.
Interestingly, the relative differences between CD4-depleted and
CD4-competent IFN--/- mice were more
pronounced at the CTLp level than at the level of tetramer-positive
cells. This is consistent with the interpretation that CTLp, as
detected by limiting dilution analysis, constitutes a minor subset of
less differentiated cells, giving rise to the
CD8+ effectors eventually detected by tetramer
(and CTL) analysis. Taken together, the above results suggest that
CD4+ cells are pivotal to maintain the more
extensive expansion of virus-specific CTLp required to control LCMV
Armstrong strain infection in IFN--/- mice,
and that elimination of CD4+ cells shifts the
balance toward exhaustive differentiation of virus-specific
CD8+ T cells.
Mathematical modeling of the consequences of CD4+ cell
deficiency in the presence or the absence of IFN-
In parallel to experimental testing, we also used a mathematical
model to investigate the effect of CD4 depletion in
IFN--/- mice. The present mathematical model
represent an extended version of the model recently used to
successfully describe the course of infection in
IFN--/- mice. The model was modified as
described in Materials and Methods to accommodate the
experimental observation that CTL activation and expansion in wild-type
mice initially is CD4 independent, but requires
CD4+ T cells for a sustained response. We also
assume that infection occurs with a relatively slowly replicating
strain. Simulations of the dynamics of CTL and virus load are shown in
Fig. 6. In wild-type mice a sustained CTL
memory response is generated that resolves the primary infection and
ensures long term immunological control and clearance.
IFN--/- mice are characterized by low level
persistent virus replication. However, the CTL response is still
sustained and controls the infection over the long term. The higher
virus load maintains a higher number of CTL in the memory phase
compared with the wild type, and the small bursts of virus drives the
CTL number up, which can keep viral replication in check. The
validity of this prediction has previously been documented
experimentally (32). CD4+ helper
cell deficiency also results in only a small loss of virus control.
However, the simulation suggests that over time the CTL response slowly
decays that can lead to reduced levels of virus control over the long
term. Consistent with this prediction, a slow decline in the number of
CTLp has previously been observed in
CD4+-deficient mice infected with LCMV Armstrong
strain (26). Hosts deficient in both
CD4+ cell help and IFN- production show a
rapid loss of virus control and a fast loss of the LCMV-specific CTL
response.
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, which shows the effect of
CD4+ helper cell deficiency in
IFN--/- hosts on the levels of CTL-induced
pathology (characterized by a low overall number of host cells). In the
presence of CD4+ cell help, the model suggests
that the host experiences transient immunopathology followed by
relatively efficient virus control. In contrast, in the absence of
help, severe and sustained immunopathology is predicted, which, in
practical terms, corresponds to wasting and death of the host.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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have previously
been found to contribute to resolution of infections with LCMV, and the
absence of either tends to shift the balance toward a more chronic
infection. The importance of IFN- critically depends on the tropism
and dose of infection. Thus, while infection of
IFN--/- mice with an intermediate dose of
the viscerotropic LCMV Traub strain is associated with severe
immunopathology and death (31), infection of these mice
with even higher doses of the less invasive LCMV Armstrong strain
develops into a chronic infection characterized by coexistence of virus
and hypercompensated CTL effector function (32). The
impact of CD4+ cells on the outcome of infection
also varies with tropism and dose of the virus strain. Thus following
infection with high doses of rapidly invasive LCMV strains,
CD4+ cells are required to prevent rapid
exhaustion of the CD8+ T cell response and
associated chronic infection (27, 28). Similarly,
CD4+ cells are important during long term control
of infection with lesser doses of rapidly invasive LCMV strains even if
the primary virus control may not require this subset (2).
In contrast, CD4+ cells play only a minor role
during acute infection as well as in the long term control of slowly
replicating LCMV strains (27, 28). However, if the role of
CD4+ cells depends upon the rate of virus spread,
it might be expected that these cells would also be needed for the
control of infections even with slowly replicating viruses in hosts
partially impaired in antiviral effector capacity. In line with this
intuitive deduction, the outcome of mathematical simulations suggests
that in IFN--/- mice,
CD4+ cells are required for a sustained CTL
response and permanent control of infection even with a slowly
replicating virus. Therefore, this study was undertaken to test
whether, as predicted, CD4+ T cells contribute to
the CD8+ cell-mediated virus control under the
conditions of the chronic infection previously observed in
IFN--/- mice infected with the slowly
replicating LCMV Armstrong strain.
The present findings disclose that CD4+ T cells
are pivotal for the control of an LCMV Armstrong strain infection in
IFN--/- mice. Thus, in the absence of
CD4+ cells, LCMV Armstrong-infected
IFN--/- mice develop severe
CD8+ cell-mediated wasting and die about 3 wk
after infection. Elimination of CD4+ cells is
also accompanied by an impaired ability to limit the virus spread in
these mice; thus, at 2 wk p.i. virus levels in spleen, liver, and
lungs are higher in IFN--/- mice
following CD4 depletion. This together with a partially impaired CTL
response readily explains the occurrence of fatal immunopathology
following CD4 depletion (4).
As to the mechanism(s) through which CD4+ cells
could influence the outcome of LCMV infection in
IFN--/- mice, several possibilities have to
be considered. First, CD4+ cells might be
antiviral effectors in their own right. However, there is no precedence
in the entire LCMV literature for such a scenario. Therefore, the
possibility that this should be the case in animals in which the
capacity to produce IFN- is abolished seems extremely unlikely.
Second, CD4+ cells could function through help to
B cells. Even though neutralizing Abs may contribute to the long term
control of infection with certain LCMV strains (2, 43, 44), Abs have never been found to play a role during the acute
phase of the infection. Furthermore, the evidence for generation of
neutralizing Abs in the context of LCMV Armstrong infection is entirely
negative. Therefore, it seems most appropriate to focus on the role of
CD4+ cell help in the generation and maintenance
of the antiviral CTL response.
Previous results have clearly established that
CD4+ T cells are not required for generation of
primary LCMV-specific CTL effectors (21, 23, 24, 25). The
present data confirm that this holds true in
IFN--/- mice. However, it has also
previously been documented that CD4+ cells are
required to maintain an expanded virus-specific CTLp pool (25, 26). This is normally of little practical consequence to
CD4-deficient mice infected with a slowly replicating LCMV strain such
as LCMV Armstrong, probably because the initial burst of CTL activity
suffices to drive the virus load almost to the level of extinction.
However, if the antiviral capacity of the generated effectors is
reduced, as in IFN--/- mice, virus
replication is not as efficiently curtailed, and a high level of CTL
activity needs to be sustained for a longer period. Under these
conditions the role of CD4+ cells in sustaining
an expanded CTLp pool becomes exposed even in mice infected with a
slowly replicating LCMV strain. What seems to be happening is that a
higher antigenic load gradually becomes established in
IFN--/- mice, and this leads to augmented
CTL differentiation and a higher number of CD8+
effector cells. Because the CTLp pool is less efficiently sustained in
the absence of CD4+ cells, the increased usage
slowly depletes this population in CD4-deficient mice, and a downward
spiral is initiated. Fewer CTLp gradually leads to fewer CTL effectors
further impairing the capacity to control the infection. The
consequence is a sustained Ag load, which, in turn, further drives the
available CTLp toward terminal differentiation. Eventually this may
result in complete depletion (exhaustion) of the CTLp pool
(8). However, if this process is not rapid, the host is
likely to succumb to the immunopathology that is induced in the futile
attempt to control the infection (4). The above
interpretation is supported by mathematical simulation. The chosen
model is based on relatively simple assumptions already experimentally
substantiated in the literature. Although the theoretical results do
not prove anything by themselves, the fact that there is consistency
between theoretical and experimental data provides important support
for the feasibility of the suggested scenario. In this context it may
be relevant to point out that this model not only provides predictions
that fit the present experimental observations, but it also
appropriately predicts a number of other outcomes already
experimentally documented.
In many respects our present results match those previously obtained by infection of CD4-deficient, but otherwise immunocompetent, mice with high doses of rapidly invasive LCMV strains (27, 28). However, there are two important differences. First, in the present study the failure to control the infection relates to an impaired host response, not to a factor intrinsic to the virus. Thus, we show here that CD4+ T cells are pivotal for the host to adapt in a situation where the functional capacity of the generated effector T cells is impaired. In other words, a qualitative (functional) defect within the effector subset substantially shifts the threshold for CD8+ cell exhaustion. Second, following infection with high doses of invasive viruses, exhaustion occurs so rapidly that most animals survive. Although this may be considered an advantage in certain respects, the rapid exhaustion also blurs the picture, while the more gradual depletion we observed allows a better separation of different phases. Thus initially a higher CD8+ T cell response is noted, and it is only with time that the depletion becomes obvious at all levels, first and most pronounced at the CTLp level and later also at the CD8+ effector level. Therefore, the present data confirm and extend the idea that CD4+ T cells are required to protect against exhaustive differentiation of the generated CTLp following an increased/chronic demand. Precisely how this effect is obtained is not certain, but production of IL-2 may be a critical factor (20).
The present study underscores the interdependence of CD4+ and CD8+ T cells in antiviral immunity. Thus, CD4+ cells are pivotal for CD8+ T cell-mediated control not only when high doses of fast replicating virus are introduced, but also following challenge with "normal" virus, provided that the functional capacity of the generated virus-specific effector T cells is impaired, i.e., a deficient CD4+ T cell response markedly reduces the capacity of the host to provide a flexible immune response. This finding is clearly of importance when trying to understand the complex cellular interactions that may take place during chronic viral infections in humans, e.g., hepatitis B and HIV infection. In this context it may be relevant to note that a good and sustained CD4+ T cell response is generally required for slow progression in AIDS patients (45), who are also noted for functional defects within their HIV-specific CD8+ T cells (46). Thus, declining CD4+ T cell numbers in individuals already impaired in effector cell capability may be what precipitates the complete immunological collapse in AIDS patients.
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2 Address correspondence and reprint requests to Dr. Allan Randrup Thomsen, Institute of Medical Microbiology and Immunology, The Panum Institute, 3C Blegdamsvej, Copenhagen, DK-2200 N, Denmark.
3 Abbreviations used in this paper: CTLp, CTL precursor; LCMV, lymphocytic choriomeningitis virus; IFN--/-, IFN--deficient; p.i., postinfection; np, nucleoprotein.
Received for publication November 6, 2000. Accepted for publication December 18, 2000.
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