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The Wistar Institute, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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5 and 20 days from lung and
nose, respectively. The low mortality of Th cell-depleted BALB/c mice
suggested that B cells contributed to recovery in a Th-independent
manner. This was verified by showing that transfer of 8–10 million T
cell-depleted naive spleen cells into µMT(-CD4) mice 1 day before
infection reduced mortality to 0%. The mechanism by which B cells
improved recovery was investigated. We found no evidence that they
operated by improving the lung-associated Tc response. Treatment of
infected µMT(-CD4) mice with normal mouse serum spiked with
hemagglutinin-specific IgM did not reduce mortality. Taken together,
the data show that 1) the Tc response is capable of resolving
autonomously (in conjunction with innate defenses) influenza virus
infections, although with substantial delay compared with intact mice,
and 2) B cells can contribute to recovery by a Th-independent
mechanism. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mouse has been a valuable and extensively used model to study the
mechanisms that protect against or promote recovery from this
infection. Evidence indicates that components of both innate
(10, 11, 12) and adaptive (13, 14) immune systems
contribute to the control of the infection and that activities provided
by CD4+ helper (Th) and B cells (15, 16) or CD8+ cytotoxic T (Tc) cells
(17, 18, 19) can independently resolve it, although the latter
are generally believed to be more effective. The recovery process
mediated by Th and B cells appears to depend largely on the generation
of a Th-dependent antiviral Ab response, as neither Th
(19, 20, 21) nor B cells (16) are capable of
resolving the infection on their own, while infection in SCID mice can
be cured by treatment with Abs specific for the viral hemagglutinin
(HA) molecule (13, 22). The high therapeutic efficacy of
these Abs appears to be due to their ability to concomitantly suppress
yield of progeny virus from infected cells and prevent released progeny
virus to spread the infection to new host cells (13, 23).
The Tc cell-mediated recovery process has been shown to rely mainly on
the perforin/granzyme- and Fas-mediated killing of infected host cells
(14, 24), while secretion of cytokines such as IFN-
,
which may inhibit virus spread by inducing cellular resistance to
infection, does not appear to play a significant role (25, 26), at least in the intact mouse (27), but may
become important if the Tc activity is being tested at its therapeutic
threshold (28). The above implies that effector Tc (eTc)
are capable of killing infected epithelial cells before the release of
progeny virus. This is surprising in the case of an acute infection in
which the eTc has available only a short window of time (between
expression of viral peptides by MHC class I and release of virions) to
perform this task (29, 30). The massive recruitment of
virus-specific eTc into the cellular exudate of the infected airways at
the time of virus clearance would be consistent with such a scenario
(14). However, since evidence for the autonomy of
Tc-mediated clearance was obtained in the study of influenza viruses of
low pathogenicity, such as X31 (17, 18) and B/AA
(19), we wondered whether eTc-mediated control mechanisms
would be similarly effective also against a more pathogenic, and
perhaps more rapidly replicating, influenza virus strain like PR8.
There is evidence from other virus systems that rapidly replicating
viruses such as vesicular stomatitis virus and Semliki Forest virus or
more virulent variants of lymphocytic choriomeningitis virus are not
effectively controlled by Tc (30, 31, 32, 33, 34). In addition, two of
the influenza virus studies (17, 18) had been done in mice
that contained B cells, although no Th cells. Therefore, the conclusion
that Tc resolved the infection autonomously in these mice assumed that
the B cells made no significant contribution to recovery without help
from Th cells.
In this study, we used B cell-deficient mice (µMT) of BALB/c background, which were additionally depleted of Th cells by chronic treatment with anti-CD4 Ab GK1.5, to test the ability of the Tc response to autonomously resolve the highly pathogenic PR8 and the less pathogenic X31 virus infections. The study confirmed that the Tc response has the basic capability to autonomously (in conjunction with innate defense) resolve these infections, but with substantial delay compared with immunologically intact mice, which resulted in high mortality in infection with the pathogenic PR8 strain. The study further showed that B cells contributed to the recovery process by a Th-independent mechanism of still undefined nature.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice were purchased from Harlan Sprague Dawley (Indianapolis, IN) and used for experiments at 2–3 mo of age. The µMT deletion in C57BL/6 x 129 (35) was backcrossed for six generations to BALB/c and then maintained by brother-sister breeding as homozygous µMT(-/-) line. Offspring was used at 2–4 mo of age. All mice were maintained in microisolators under specific pathogen-free conditions.
Media and solutions
ISC-CM consists of Iscove’s Dulbecco’s medium (Life Technologies, Gaithersburg, MD), supplemented with 0.05 mM 2-ME, 0.005 mg/ml transferrin (Sigma, St. Louis, MO), 2 mM glutamine (JRH Biosciences, Lenexa, KS), and 0.05 mg/ml gentamicin (Mediatech, Herndon, VA). ISC-CM was further supplemented, as indicated, with FCS (HyClone Laboratories, Logan, UT), BSA (Sigma), normal mouse serum (Harlan Sprague-Dawley), 1 mg/ml geneticin (Sigma), or 0.1 mg/ml 5-bromo-2'-deoxyuridine (Sigma).
Cell lines and hybridomas
P1.HTR (36), a highly transfectable variant of the DBA mastocytoma cell line P815 (H-2d, Ia negative), was maintained in ISC-CM 5% FCS + 5-bromo-2'-deoxyuridine. P1.HTR transfected with B7.1 (HTR-7.1) was kindly provided by Dr. F. Gajewski (37) and maintained in ISC-CM + geneticin. A20 is a B cell line (H-2d, Ia positive) and was maintained in ISC-CM 2% FCS. Madin-Darby canine kidney (MDCK) cells were maintained in ISC-CM 5% FCS. The rat anti-mouse CD4 hybridoma GK1.5 (ATCC TIB 207) was grown in ISC-CM 5% FCS and Ab purified by adsorption/elution from protein A-Sepharose.
Virus and infectivity titration
The influenza type A virus strain A/PR/8/34 Mt.S (H1N1) (PR8)
was originally obtained from Mt. Sinai Hospital, New York, NY.
Infectious stock was grown in the allantoic cavity of embryonated
hen’s eggs. Influenza virus B/Lee/40 (Lee) was also grown in
embryonated hen’s eggs. Titers of infectious virus were determined in
MDCK microcultures, as described (16, 38), and usually
expressed as TCID50 (50% tissue culture
infectious dose). One TCID50 of PR8 is equivalent
to 4 EID50 (50% egg infectious dose) and to
0.25 MID50 (50% mouse infectious dose).
Measurement of antiviral Ab concentration by ELISA
The ELISA was performed essentially as described
(39). In brief, the solid-phase immunoadsorbent was
prepared by adsorbing purified virus (25 µl containing 25 HAU of
virus, 175 ng viral protein) into wells of round-bottom polyvinyl
plastic plates. Before assay, the wells were washed and blocked by
incubation with PBS 1% BSA. Test samples and purified anti-HA Ab
standards of IgG or IgM isotypes were diluted in PBS 1% BSA and
incubated (25 µl/well, quadruplicates) for 90 min with the viral
immunoadsorbent. The plates were then washed and bound Ab detected by
successive incubations with biotinylated C
- or
CH-specific mAbs, streptavidin-AP (Sigma), and
pNPP (Sigma), with washes between each incubation step. The color
intensity (A405–750) was determined
(Emax; Molecular Devices, Sunnyvale,
CA); the OD of test samples (normal and experimental mouse sera) was
compared with the one seen with purified Ab standard and expressed as
µg Ab/ml, using the SOFTmax software (Molecular Devices, Sunnyvale,
CA). The control sera included a pool and several individual samples
from roughly age-matched naive BALB/c mice. Sera from experimental mice
were obtained at termination of experiments and tested
individually.
CD4 T cell depletion and infection of mice
Three days before infection, mice were injected i.p. with 200 µg of purified Ab GK1.5 in PBS. The same treatment was repeated 1 and 7 days after infection and thereafter at 7-day intervals until termination of experiments. For infection, mice were anesthetized by i.p. injection of 0.2 ml ketamine (10 mg/ml PBS)/xylazine (2 mg/ml) and allowed to aspirate into the lower RT a droplet (30–50 µl) of virus that had been applied to the nares.
Isolation of lung-associated cells
Mice were anesthetized by i.p. injection of ketamine/xylazin and
exsanguinated by heart puncture. For bronchoalveolar lavage (BAL), the
trachea was exposed, the thorax opened, and a 20-gauge needle inserted
into the trachea right below the larynx. A sample of 0.5 ml of PBS was
then injected into the RT and slowly withdrawn and collected; this
procedure was repeated with three 0.5-ml samples of PBS. For virus
titration, lung lobes were removed, quickly frozen, and stored frozen
until disruption of the tissue for determination of lung-associated
infectious virus titer, as described (16, 38). For
isolation of lung leukocytes, lung lobes, free of macroscopically
visible lymph nodes, were sliced into small tissue fragments by means
of a tissue chopper (Brinkmann, Westbury, NY). The chopped up tissue
was then incubated for 60 min at 37°C in ISC-CM (5 ml/lung)
supplemented with 50 U/ml of collagenase (Worthington Biochemical,
Freehold, NJ) and 70 U/ml DNase I (Boehringer Mannheim, Indianapolis,
IN). The digest was then pipetted several times through a narrow
pipette orifice, the large cell fragments let to settle, and the
supernatant transferred into a 15-ml centrifuge tube, one tube per
lung. The pipetting was repeated with a second batch of ISC-CM, without
enzymes. The pooled cell suspension was then mixed with 1/2 vol of
100% Percoll (Pharmacia, Piscataway, NJ) and underlayed with 1.5–2
ml of 70% Percoll. The tube was centrifuged for 10 min at 700 x
g at room temperature. The cells at the 33:70% interface
were harvested, washed once in ISC-CM 5% FCS, and used for
51Cr release assay and FCM analysis.
51Cr release assay
The assay was performed as described (20) and used infected P1.HTR cells as targets.
Cytokine secretion
Lung leukocytes (106/ml) were incubated
with PR8- or Lee-infected HTR-7.1 cells (106/ml)
in ISC-CM 5% FCS for 24 h at 37°C. The concentration of
IFN-, IL-4, IL-5, and TNF-
in the culture medium was determined
by capture ELISA using commercially available Ab pairs (PharMingen, San
Diego, CA), essentially as recommended by the manufacturer.
Depletion of T and B cells in vitro
Two different protocols were used to deplete cell subsets from spleen cell suspensions in vitro. Procedure A: The spleen cell suspension (2–3 x 107/ml ISC-CM, 5% FCS) was incubated with frequent resuspension for 30 min at 4°C with Dynabeads (Dynal S.A., Oslo, Norway) displaying anti-CD8 Ab 53-6.72 or anti-CD4 Ab GK1.5. The beads were prepared by coupling the respective Abs to tosyl-activated beads according to the manufacturer’s protocol. The bead:cell ratio was 7 x 106 anti-CD8 beads and 14 x 106 anti-CD4 beads:107 spleen cells. Free cells were separated from those with attached beads in a magnetic field. Procedure B: Spleen cells (107/ml ISC-CM, 5% FCS, 0.5% normal mouse serum) were incubated for 30 min at 4°C with anti-B220 ZA3-3A1/6.1 (ATCC TIB146) and/or 53-6.72 and GK1.5. Each Ab was used at 1 µg/106 cells. The cells were then washed three times with cold ISC-CM, 5% FCS, resuspended in that medium at 2–3 x 107 cell/ml, and incubated for 30 min at 4°C with Dynabeads displaying the rat Ig-specific Ab Rt2-8. Free cells were then separated from those with attached beads in a magnetic field.
FCM analyses
FITC-labeled 53-6.72 and PE-labeled GK1.5 and RA3-6B2 were purchased from PharMingen. Rt2-8 (rat Ig specific) was labeled with the FluoroTag FITC conjugation kit (Sigma), according to the manufacturer’s protocol. Culture fluid from the CD45-specific hybridoma M5/114.15.2 (ATCC TIB 120) was used for identification of leukocytes by indirect staining.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B cell-deficient µMT mice (on BALB/c background) were depleted
of Th cells by treatment with the CD4-specific Ab GK1. The Ab treatment
(200 µg purified GK1.5 per i.p. injection) was started 3 days before
infection, and repeated on day 1 and day 7 after infection and at 7-day
intervals thereafter until termination of experiments. It resulted in
extensive depletion of Th cells, as generally fewer than 2% of cells
in mediastinal lymph nodes (MedLN) and spleen scored positive for CD4
and/or residual cell-bound rat Ig when tested by FCM (data not shown).
As shown in Fig. 1
A, infection
of µMT(-CD4) mice with a small dose (50
TCID50, 10 MID50) of PR8
virus resulted in a severe disease with progressive loss of body weight
and usually lethal outcome. Overall, 80% of µMT(-CD4) mice died
after PR8 infection (Table I). The high
mortality was not due to an excessive virus challenge dose, as
immunologically intact BALB/c mice recovered readily from the same
virus challenge without much morbidity (weight loss) and no mortality
(Fig. 1B and Table I). In addition, it could not be
attributed to the anti-CD4 Ab treatment per se or the lack of Th
cells, as similarly Th-depleted BALB/c(-CD4) mice had no difficulties
in controlling the infection (Fig. 1C).
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A). By
contrast, immunologically intact BALB/c mice, which had been infected
with partially to 100% lethal doses of PR8, usually died in the second
week of infection (Fig. 2B). Importantly, virus that was
immunologically indistinguishable from PR8 could consistently be
isolated from the RT of µMT(-CD4) mice that died at late stages of
infection (data not shown). These findings indicated that the high
mortality resulted from the inability of these mice to resolve the PR8
infection.
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The large difference in mortality between µMT(-CD4) and
BALB/c(-CD4) mice was surprising, as B cells are not known to
contribute to the control of this infection in the absence of Th cells
(14, 16). Two possible explanations came to mind: 1) The
institute-bred µMT mice carried a subclinical disease that enhanced
the severity of the viral infection. 2) B cells, present in
BALB/c(-CD4) but not µMT(-CD4), contributed to virus control,
possibly because of help provided by residual Th cells. To distinguish
between these possibilities, we tested whether transfer of B cells into
µMT(-CD4) mice improved their ability to control the infection. In
the first experiment, µMT(-CD4) mice were injected i.v. with
107 T cell-depleted spleen cells (6 x
106 B cells) from naive BALB/c mice 1 day before
virus challenge. As shown in Table II,
all B cell-injected µMT(-CD4) mice were able to control the
infection, while 80% of the PBS-injected control µMT(-CD4) mice
died. In the second experiment, µMT(-CD4) mice were injected with T
cell-depleted or T and B cell-depleted spleen cells, and recovery was
again dependent on the transfer of B cells. These findings indicated
that the high mortality of µMT(-CD4) mice was not the result of an
underlying disease and pointed to an inherent difficulty of the host’s
defense system to control the PR8 virus infection in the absence of
both B and Th cells.
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, most sera from Th cell-depleted mice
contained only minimally higher amounts of virus-reactive Ab than
normal BALB/c mouse serum (mean of 3 µg/ml). With the exception of
one B cell-reconstituted µMT(-CD4) mouse, this Ab consisted entirely
of IgM, as no difference between these sera and normal mouse serum was
seen if the assay was developed with an IgG-specific reagent (data not
shown). The mean virus-reactive Ab concentration in BALB/c(-CD4) mice
was 7.6 (±4) µg/ml, i.e., 3.5 µg/ml above the mean of normal
mouse serum. To test whether natural serum Ab and the minimal
presumably Th-independent Ab response seen in BALB/c(-CD4) mice was
responsible for their recovery, infected µMT(-CD4) mice were treated
repetitively with serum Ig from naive BALB/c mice (3 mg/injection) and
HA-specific IgM (10 µg/injection). The latter dosage was chosen to
result in an HA-specific serum Ab concentration roughly equivalent to
the one seen in BALB/c(-CD4) mice (Fig. 3) (23). This
treatment failed to improve recovery in two independent experiments
(Table II
, Expt. 3) and made us consider the possibility that the B
cells contributed to the recovery through a mechanism other than Ab
secretion.
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We next considered the possibility that B cells were involved in
the induction of the virus-specific Tc response. This was assessed by
testing leukocytes isolated from the lungs of infected mice directly ex
vivo (without restimulation) for cytotoxic activity against PR8- and
Lee-infected target cells in vitro. Lee is an influenza type B virus
that is immunologically non-cross-reactive with PR8 and thus measures
the contribution of nonspecific cytotoxicity in these assays. In the
experiments shown in Fig. 4, one lung
lobe from each individual mouse was used for determination of the virus
titer and the other, after pooling within each group, for isolation of
leukocytes and determination of lung-associated cytotoxic activity.
These experiments showed that µMT(-CD4) mice generated eTc responses
that were comparable with those mounted by BALB/c(-CD4) and intact
BALB/c mice during the early phase (day 7, day 10) of the infection,
both in terms of cytotoxic activity per lung-derived leukocyte (Fig. 4A) and cellularity (Fig. 4A, insets). At day 14
and day 20, the eTc response in µMT(-CD4) mice even exceeded those
seen in the other groups of mice, although all or part of this
increased cytotoxicity may have been due to the higher nonspecific
cytotoxic activity exhibited by the cells from µMT(-CD4) mice at
later time points. However, in spite of the strong eTc response in the
lung, none of the µMT(-CD4) mice had managed to clear the infection
by day 14 (Fig. 4B), and two mice tested at day 20 still
contained high titers of virus in their lungs (
), while one had
apparently been successful in clearing it. The latter mouse (
)
looked healthy, and was for that reason tested separately also in the
cytotoxicity assay. In marked contrast, all BALB/c and BALB/c(-CD4)
mice had cleared the infection by day 10 and day 14, respectively.
Thus, the difficulty in resolving the PR8 virus infection could not be
attributed to an obvious deficit in the size or activity of the
lung-associated virus-specific eTc response of µMT(-CD4)
mice.
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(Fig. 5A) and no
detectable IL-4 and IL-5 (data not shown) and, thus, comprised mainly
Tc1. Next, we tested whether Tc were effectively recruited into the
airway lumen by measuring the cytotoxic activity of cells recovered by
BAL. Similar numbers of leukocytes were recovered by BAL from both
groups of mice (data not shown), and the BAL cells exhibited comparable
cytotoxic activity (Fig. 5B). Finally, we considered the
possibility that a Tc activity, which may be important for virus
clearance in vivo, but was not revealed by the above assays in vitro,
was defective in Tc populations generated in the absence of B cells.
For instance, low TCR avidity has been reported to result in stronger
reduction of Tc activity in vivo than in vitro (40, 41).
Using inhibition of cytotoxic activity by anti-CD8 Ab as correlate
of TCR avidity (40), no difference between eTc populations
was evident (Fig. 5, C and D). In addition, we
compared lung-derived leukocytes for clearance activity in vivo by
adoptive transfer (i.v., 5 x 105 CD8 T
cells/mouse) into PR8-infected SCID mice. These experiments (not shown)
were inconclusive because cells from neither group of donor mice showed
significant clearance activity in vivo, in spite of exhibiting high
cytotoxic activity in vitro.
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Virus isolated from µMT(-CD4) mice is recognized by eTc generated in these mice
The coexistence of high virus titers and seemingly strong eTc
populations in the lungs of µMT(-CD4) mice over many days of
infection raised the question as to whether the virus that grew in
these mice could indeed be recognized by the eTc cells present in their
lungs. To test this, virus isolated from lungs of two µMT(-CD4)
mice, one 14 and the other 36 days after infection, was expanded by
passage in embryonated hen’s eggs and then used to infect target cells
for 51Cr release assay. As shown in Fig. 6, lung-derived eTc from µMT(-CD4)
mice killed these target cells as effectively as eTc from intact BALB/c
mice. Thus, the failure of µMT(-CD4) mice to control the infection
could not be explained by emergence of escape mutants in these
mice.
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PR8 is a relatively pathogenic virus (LD50
300 MID50 in immunologically intact mice; Fig. 2), and we wondered whether the severe morbidity induced by this virus
may be responsible for the reduced clearance activity of Tc. X31 virus
is a reassortant between PR8 and Aichi/68(H3N2). It has a similar mouse
infectivity (1 MID50 5–10
TCID50) and produces similar peak virus titers in
the lung as PR8, but is much less pathogenic. Therefore, a larger
infection dose could be used, which resulted in infection of both nasal
and lower RT (epithelium of nose is less susceptible to infection than
epithelium of lower RT). Upon infection with 104
TCID50 of X31, µMT(-CD4) mice (Fig. 7A, open symbols) showed only
minimally greater weight loss than intact BALB/c mice (Fig. 7A, closed symbols), and all µMT(-CD4) mice survived the
infection. Nevertheless, virus clearance was delayed compared with
intact BALB/c mice. In the case of the lung, all intact mice had
cleared the virus by day 10, while only 60% of the µMT(-CD4) mice
had cleared it by day 15, and two of six mice still contained residual
virus by day 21. Overall, X31 was cleared from the lung more
effectively than PR8, as all PR8-infected µMT(-CD4) mice tested
between day 14 (Fig. 3) and 16 (not shown) still contained high virus
titers in their lungs (log10: 5.5 ± 1.1,
n = 9). Interestingly, virus clearance from the nose
was much more delayed than clearance from the lung, and 50% of the
µMT(-CD4) mice still showed high virus titers at this site 28 days
after infection, and one of three mice tested positive for virus at day
48. Judging from the body weight curve (Fig. 7A), the
protracted nasal infection had not much impact on the general health
status of these mice. These findings indicate that the Tc response has
the basic capability to resolve autonomously an influenza virus
infection if the infection per se does not result in excessive
deterioration of the general health status, but clearance remains
delayed compared with intact mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Infection
with the less pathogenic X31 virus strain could be resolved in
µMT(-CD4) mice without mortality, but virus clearance from the lung
and nasal epithelium was delayed by 5 and 20 days, respectively,
compared with intact BALB/c mice. The difficulty of µMT(-CD4) mice
in controlling influenza virus infections was not due to a poorer
health status of the institute-bred µMT mouse line compared with
commercially obtained BALB/c mice, because injection of the
µMT(-CD4) mice with 8–10 million T cell-depleted spleen cells from
naive mice before PR8 virus challenge reduced mortality to 0% (Table II). Furthermore, it could not be attributed to the anti-CD4 Ab
treatment per se, which was applied to deplete Th cells in vivo,
because the same Ab treatment did not aggravate the disease in
BALB/c(-CD4) mice (Fig. 1). Most importantly, it could not be
attributed to a measurable defect of the virus-specific lung-associated
eTc response of µMT(-CD4) mice (42), as the latter did
not differ in any of several parameters tested from the responses
generated by immunologically intact or Th cell-depleted BALB/c(-CD4)
mice, including total lung-associated cellularity, recruitment of Tc
into the bronchoalveolar compartment, their avidity (as assessed by
inhibition with anti-CD8 Ab), cytotoxic activity, and secretion of
IFN- in vitro. The apparently normal eTc response of µMT(-CD4)
mice is in agreement also with studies by other investigators showing
that Th cell deficiency did not diminish the size of the initial Tc
responses to influenza (43) and Sendai virus
(44). Although Th cell deficiency diminished the
generation of virus-specific memory Tc (43 , and our
unpublished observation), this could not explain the delayed virus
clearance or high mortality because BALB/c(-CD4) mice recovered
normally. Thus, a virus-specific Tc response, comparable in size and
activity with the one generated in immunologically intact mice, cleared
the infections with significant delay compared with intact mice. This
shows unequivocally that B and/or Th cells make an important
contribution to the clearance of these viruses in the immunologically
intact mouse.
We are aware of only a single previous study, conducted by Epstein et
al. (19), in which the efficacy of the primary Tc response
in clearance of a viral infection was studied in the absence of both Th
and B cells. The study made use of B cell-deficient
(JH- and C-deleted, termed doubly inactivated,
DI) mice of C57BL/6 background that were depleted of Th cells by
treatment with the same Ab as used in this study and tested for ability
to resolve a pulmonary infection with an influenza virus strain of type
B. The study revealed no substantial difference in clearance of B/AA in
Th-depleted DI mice compared with immunologically intact mice, in that
virus was cleared from the lungs of intact mice between day 7 and day
11 and from the lungs of Th-depleted DI mice between day 7 and day 14.
Clearance from the nasal epithelium, which showed the most pronounced
delay in our study (Fig. 7B), was not monitored. The use of
mice of different genetic background and of different virus strains and
the lack of data regarding virus clearance from the nose make a
comparison of the two studies difficult.
An interesting finding was the greatly different efficacy of
Tc-mediated clearance of X31 from epithelia of upper and lower RT.
Thus, while 40% of µMT(-CD4) mice had managed to reduce the
infection in the lung to undetectable level by day 15, all of these
mice still contained high virus titers in the nose, and it took another
10 to 15 days until the nasal infection was reduced to undetectable
level in 50% of the mice (Fig. 7B). Whether the nasal
infections were indeed completely cleared in these mice or only
suppressed to undetectable level, similar to findings made with
lymphocytic choriomeningitis virus-infected B cell-deficient mice
(45, 46), remains to be determined. Possible reasons for
the delayed Tc-mediated clearance could be a less effective recruitment
of Tc into the cellular exudate of the nose and/or a lower
susceptibility of nasal epithelial cells to Tc-mediated control
mechanisms (47). The greatly delayed virus clearance
indicates that B and/or Th cells play a dominant role in virus
clearance from this site in intact mice, probably through production of
Th-dependent secretory IgA (48, 49).
Most unexpected was the finding that naive B cells made a significant
contribution to virus control in Th cell-depleted mice. Thus, injection
of 8–10 million T cell-depleted spleen cells from naive BALB/c mice
into µMT(-CD4) mice 1 day before infection with PR8 reduced
mortality from 80% to 0% (Table II). This provided a straightforward
explanation also for the finding that B cell-containing BALB/c(-CD4)
mice had no difficulty in resolving the PR8 infection (Fig. 1 and Table I). The mechanism through which naive B cells contributed to the
recovery process remains unclear. 1) The possibility that B cells
functioned as important stimulators for the virus-specific Tc response
is not supported by the finding that µMT(-CD4) and BALB/c(-CD4)
mice mounted lung-associated Tc responses of comparable strength (Fig. 4). In addition, evidence from other experimental systems has indicated
that B cells tend to suppress rather than promote Tc responses
(50, 51). 2) A role of B cells in the recruitment of Tc to
the site of infection, e.g., through secretion of proinflammatory or
Tc-attracting chemokines (52, 53), is not supported by the
finding that Tc activity was of similar strength in BAL fluid obtained
from µMT(-CD4) and BALB/c(-CD4) mice (Fig. 5). 3) B cells may
contribute to the recovery process through Th-independent Ab
production, as reported for other virus infections (54, 55). Although we cannot exclude this possibility, several
observations appear to argue against it. First, the amount of
Th-independent Ab produced in BALB/c (-CD4) mice in the course of
infection was small and barely (by 3–4 µg/ml) exceeded the level of
the background or natural Ab detected by our assay in normal mouse
serum (Fig. 3). In addition, given that the BALB/c(-CD4) mice
contained initially roughly 10 times more B cells
(108) than µMT(-CD4) mice after transfer of
107 T cell-depleted spleen cells, an even lower
concentration of Ab would be expected in the latter mice. Second,
repetitive treatment of infected µMT(-CD4) mice with normal mouse
serum Ig (3 mg/injection, as substitute for natural Ab) and HA-specific
Ab of IgM isotype (10 µg/injection, as substitute for Th-independent
Ab) did not improve virus control (Table II). Third, IgM, the main
isotype of the T-independent response, has previously been shown to
have very low therapeutic activity in virus-infected SCID mice even
when administered repetitively and in high dosage (13).
Although these indirect observations do not support a role for the
Th-independent Ab production in this system, they cannot not exclude it
either. Ultimately, the process by which B cells operate here can
probably only be resolved unequivocally by testing the activity of B
cell populations that cannot produce virus-specific Ab (e.g.,
expressing a transgenic BCR of known specificity) or cytokines, or do
not display certain surface components.
In conclusion, the results of this study show that the Tc response is
capable of resolving autonomously (in conjunction with innate defenses)
influenza type A virus infections, but virus clearance is delayed
compared with immunologically intact mice, particularly from nasal
epithelium and to lesser extent from the lower RT. This shows that the
highly effective clearance seen in immunologically intact mice cannot
be attributed solely to the Tc response and that non-Tc-mediated
activities make relevant contributions as well. As shown in this study,
these include, among others, a Th-independent B cell activity. Although
this B cell activity is not capable of controlling the infection on its
own (14, 16), it clearly makes a significant contribution
in the presence of a Tc response. This is similar to findings made with
Th cells that are incapable of controlling the infection on their own
(19, 20, 21) but, as shown by the lower mortality of
nondepleted µMT mice (Table I), improve resistance in the presence of
a Tc response. The high effectiveness of the intact host defense
appears to be due to many additive and synergistic interactions between
distinct defense mechanisms.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Walter Gerhard, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address:
3 Abbreviations used in this paper: RT, respiratory tract; BAL, bronchoalveolar lavage; DI, doubly inactivated; Tc, CD8+ T cell; eTc, effector Tc; FCM, flow cytometry; HA, hemagglutinin; MDCK, Madin-Darby canine kidney; MID50, 50% mouse infectious dose; TCID50, 50% tissue culture infectious dose.
Received for publication August 25, 1999. Accepted for publication December 17, 1999.
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gene. J. Exp. Med. 178:1725.-dependent clearance of influenza A virus and protection from consolidating pneumonitis in nitric oxide synthase 2-deficient mice. J. Exp. Med. 188:1541. to clear an acute viral infection. Virology 229:349.[Medline]
(MIP-1a) and MIP-1ß chemokine production in human B cells. J. Immunol. 162:4455.This article has been cited by other articles:
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|
|
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|
|
|
|
|
|
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|
|
|
|
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|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
 J. D. CRAPO, A. G. HARMSEN, M. P. SHERMAN, and R. A. MUSSON Pulmonary Immunobiology and Inflammation in Pulmonary Diseases Am. J. Respir. Crit. Care Med., November 1, 2000; 162(5): 1983 - 1986. [Full Text]
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), B cell-reconstituted µMT(-CD4) (
), BALB/c(-CD4) (
), and intact BALB/c (
). The E:T ratio is
based on total cell count. B, One lung lobe from each
individual mouse included in this experiment was tested for virus
titer. The threshold of virus detection is indicated by the broken
line. The data are from two separate experiments, the first testing the
indicated number of mice 10 and 14 days, and the second 7 and 20 days
after infection. Data similar to the ones shown here for day 14 and 20
were obtained with mice tested in different experiments at days 15 and
16 after infection.
, 
) or with
virus isolated from the lung of µMT(-CD4) mice 14 days (
) or
35 days (
) after infection with PR8. The E:T ratio is based on
total cell count. The lung cells contained 64% (µMT(-CD4)) and 27%
(BALB/c) CD8+ T cells. Similar results were obtained with
lung cells isolated from other mice 16 and 20 days after
infection.