By
From the * Beirne B. Carter Center for Immunology Research, the Department of Medicine, the § Department of Microbiology, and the
Department of Pathology, University of Virginia Health Sciences
Center, Charlottesville, Virginia 22908
In the adaptive immune response to most viruses, both the cellular and humoral arms of the immune system play complementary roles in eliminating virus and virus-infected cells and in promoting recovery. To evaluate the relative contribution of CD4+ and CD8+ effector T lymphocytes in virus clearance and recovery, we have examined the host response to lethal type A influenza virus infection in B lymphocyte-deficient mice with a targeted disruption in the immunoglobulin mu heavy chain. Our results indicate that naive B cell-deficient mice have a 50- 100-fold greater susceptibility to lethal type A influenza virus infection than do wild type mice. However, after priming with sublethal doses of influenza, immune B cell-deficient animals show an enhanced resistance to lethal virus infection. This finding indicates that an antibody-independent immune-mediated antiviral mechanism accounts for the increased resistance to lethal virus challenge. To assess the contribution of influenza-specific CD4+ and CD8+ effector T cells in this process, defined clonal populations of influenza-specific CD4+ and CD8+ effector T cells were adoptively transferred into lethally infected B cell-deficient mice. Cloned CD8+ effectors efficiently promoted recovery from lethal infection, whereas cloned CD4+ T cells conferred only partial protection. These results suggest that memory T lymphocytes can act independently of a humoral immune response in order to confer resistance to influenza infection in immune individuals. The potential implications of these results for vaccination against human influenza infection are discussed.
For many viral infections, it has been suggested that the
cell-mediated immune response plays a dominant role
in recovery from disease, whereas the humoral, or B cell,
response is important for protection against free virus and
in preventing reinfection upon secondary exposure to an
identical or cross-reactive virus (1, 2). In the case of influenza virus pneumonia, the disease can be cured either by a
CD8+ T cell response alone (3, 4), or, in the absence of
CD8+ T cells, by a CD4+ T cell and concomitant B cell
response (5, 6). In addition, studies examining the T cell-B
cell interactions occurring in response to influenza virus infection have found that (a) adoptively transferred CD4+ T
cells can provide help for the production of neutralizing
anti-influenza virus antibody (7); (b) this antibody response
leads to virus clearance from the lungs of infected T cell-
deficient (nude) mice (7); and (c) transfer of neutralizing anti-hemagglutinin (HA) antibody alone into infected SCID mice
(deficient in T and B cells) leads to influenza virus clearance
from the lungs of these immunodeficient mice (2, 8).
Since both activated CD4+ and, to a lesser extent, activated CD8+ T lymphocytes produce cytokines that can
augment B lymphocyte differentiation and the production
of neutralizing antibodies, the precise role of T cells in orchestrating recovery from experimental influenza infection
is not well defined. To assess the contribution of T lymphocyte effector activity, we have examined the response of mice with a targeted mutation in the membrane exon of
the immunoglobulin µ chain gene (B cell-deficient or µ knockout [KO] mice) to lethal primary and challenge type
A influenza virus infection and the effectiveness of transferred CD4+ and CD8+ T cells in virus clearance. We find
that while B cell-deficient mice are more susceptible to lethal influenza virus infection than are wild-type mice, earlier vaccination of µKO mice leads to enhanced resistance
to lethal influenza challenge. Also, CD8+ T cells are more
efficient than CD4+ T cells in promoting virus clearance
and recovery in the absence of B lymphocytes. Our results
indicate that the presence of influenza-specific memory
T lymphocytes in an immune individual results in increased resistance to subsequent infection in the absence of neutralizing antibody.
Animals.
Pathogen-free male and female C57Bl/6 (H-2b)
mice were purchased from Taconic Farms, Inc. (Germantown, NY)
and used at 6-12 wk of age. Breeding pairs of B cell-deficient
(H-2b) mice with a targeted mutation in the membrane exon of
the immunoglobulin µ chain gene, or µKO mice, provided by
K. Rajewsky (University of Cologne, Germany) (9), were bred
and maintained in a colony at the University of Virginia, and
were used at 6-12 wk of age.
Viruses.
Influenza virus strain A/JAPAN/57 (A/JAPAN/
305/57 [H2N2]) was grown in the allantoic cavity of 10-d-old
embryonated hens' eggs and stored as infectious allantoic fluid as
previously described (10). Determination of virus titer, expressed
as hemagglutinating units, was done as previously described (10).
Influenza virus strain A/JAPAN/57 used for intranasal inoculation and adoptive transfer procedures was mouse adapted by serial
passage in mouse lung and is lethal at concentrations as low as one
hemagglutinating units (equivalent to 104 EID50, or egg infectious
dose, units). For procedures requiring a sublethal challenge of influenza, an egg-adapted, antigenically identical strain of A/JAPAN/
57 was used for intranasal inoculation. This preparation is not lethal for the mice used in these studies at concentrations equivalent to 108 EID50 units.
Intranasal Influenza Virus Inoculation.
Intranasal inoculation of
mice and determination of LD50 values was performed as previously described (11). To evaluate a dose response to intranasal virus, groups of 5-10 age-matched animals received serial 10-fold
dilutions of allantoic fluid in cold PBS ranging from 10 Bulk and Clonal T Lymphocyte Lines.
Spleen cells from µKO
and C57Bl/6 mice which had received a sublethal intranasal dose
of A/JAPAN/57 12 d before were harvested and processed as
previously described (11). Subsequently, these bulk cultures were
stimulated in vitro with influenza A/JAPAN/57 virus-infected,
gamma-irradiated (2,000 rad) C57Bl/6 spleen cells every 7-10 d
in complete medium (IMDM [GIBCO BRL, Gaithersburg, MD]),
10% HIFCS, 1% glutamine, 5 × 10 Assays for Cell-mediated Cytotoxicity.
The 51Cr-release cytotoxicity assay and lysis calculations were carried out as previously described in detail (10). Effector/target ratios ranged from 5:1 to
50:1 depending on assay. Spontaneous release was <15%.
Adoptive Transfer Procedure.
Adoptive transfer of day 6 viable
cloned cells was performed as previously described (11, 12). 8-12-wk-old C57Bl/6 and B cell-deficient mice were intranasally inoculated with 10 LD50 influenza A/JAPAN/57 virus, and within
30 min 107 clone cells in 0.5 ml Iscove's medium were injected
intravenously. Control mice were injected intravenously with 0.5 ml Iscove's medium alone. Mice were watched daily for 21 d for
morbidity and/or mortality.
To assess the impact of the presence or absence of B cells
on susceptibility to infection with influenza virus, cohorts
of age-matched C57Bl/6 and µKO mice were inoculated
intranasally with varying doses of infectious mouse-adapted
A/JAPAN/57 influenza virus (10
It is well established that the host immune response to
primary influenza virus infection results both in a cell-mediated immune response with the induction of CD8+
CTLs and activated CD4+ T cells, and in a humoral response
with the production of neutralizing antibody (13). Therefore, one likely explanation for increased susceptibility of
µKO mice to influenza infection is the inability of µKO mice
to mount a neutralizing antibody response. Indeed, µKO
mice are unable to mount a serum influenza-specific antibody response after influenza infection (data not shown).
To determine if recovery from primary intranasal influenza infection in µKO mice correlated with the presence
of cellular immune effectors, e.g., CTLs, at the site of infection, groups of µKO and age-matched C57Bl/6 mice were
sublethally infected with the mouse-adapted A/JAPAN/57
virus. On day 12 after infection, the mice were killed, lungs
were excised, and mononuclear cells infiltrating the lungs
were collected and tested for in vitro cytolytic activity. As
Fig. 2 a shows, mononuclear cells from the lungs of infected µKO mice and from conventional mice exhibited a
comparable degree of specific cytolytic activity on virus-infected target cells in vitro. Similar results were obtained
when T cells were obtained at d 12 from lungs of wild-type
and B cell-deficient mice recovering from nonlethal intranasal infection with an attenuated A/JAPAN/57 virus
preparation (data not shown).
The finding of virus-specific cytolytic activity in the lungs
of µKO mice recovering from sublethal primary infection
raised the possibility that primary infection would also result
in the development of virus-specific memory CD8+ (and
presumably CD4+) T lymphocytes capable of responding
to challenge infection with virus. To further examine this
hypothesis, two experimental approaches were employed.
First, immune splenocytes from µKO and C57Bl/6 mice
were taken 28 d after priming by sublethal infection with attenuated virus. These splenocytes were restimulated once
in vitro and subsequently tested for CTL activity. As shown
in Fig. 2 b, the in vitro secondary CTL response to A/JAPAN/57-infected stimulators between vaccinated µKO and
C57Bl/6 mice was comparable. Second, groups of µKO and
wild-type mice were first vaccinated by intranasal infection
with a live attenuated (egg-adapted) A/JAPAN/57 virus
preparation. This attenuated virus stock has an LD50 for µKO mice ~1,000-fold higher than the challenge mouse-adapted stock and is uniformly nonlethal for conventional
mice. It clears from the lungs of both conventional and B
cell-deficient mice by day 14 after infection (data not
shown). 28 d after vaccination, when CTL activity was no
longer detectable in the lungs of infected animals, the vaccinated mice were challenged by intranasal infection with
the aggressive mouse-adapted A/JAPAN/57 virus.
Fig. 3 shows the results of this priming/challenge study.
As expected, conventional mice, which have neutralizing
antiviral antibody both in the circulation and locally in the
respiratory tract, were uniformly resistant to lethal infection
with mouse-adapted virus at virus doses up to 106 EID50
units (i.e., the equivalent of an innoculum of 103 LD50
doses for a naive conventional mouse). By contrast, challenge infection of B lymphocyte-deficient µKO mice resulted in death (Fig. 3). However, the immune µKO mice
demonstrated a 100-fold greater resistance to challenge infection with mouse-adapted virus than did naive µKO mice
(LD50 values of 101.7 EID50 units and 104 EID50 units for
mouse-adapted virus in naive and vaccinated µKO mice,
respectively). These results suggest that the enhanced resistance to lethal virus challenge observed in the primed B
cell-deficient mice was due to the activation of virus-specific memory T lymphocytes in response to challenge infection.
In view of the evidence for both antibody independent
clearance of virus during primary infection of µKO mice
and enhanced resistance of these mice to lethal infection after priming, it was of interest to assess the contribution of
virus-specific CD4+ and CD8+ effector T lymphocytes to
recovery from infection in B cell-deficient mice. To examine this, we adoptively transferred clonal populations of
A/JAPAN/57 virus-specific CD4+ and CD8+ T lymphocytes into lethally infected conventional and µKO mice. The clones used for these studies were H-2b haplotype-restricted CD4+ T cells (i.e., 4D7) and CD8+ T cells (i.e.,
11E4 and B1.11), which have been previously characterized by us and have been shown to promote recovery
when adoptively transferred into lethally infected C57Bl/6
mice (11). Fig. 4 shows the survival data for one adoptive
transfer (representative of five independent experiments)
carried out with these influenza-specific CD4+ and CD8+
T lymphocyte clonal effectors. As a control for each experiment, the same number of influenza-specific CD4+ or CD8+
T cell clones were adoptively transferred into lethally challenged C57Bl/6 mice, and all three clones consistently promoted 100% survival as previously published by this laboratory (11). The results of the adoptive transfers into C57Bl/6
mice are not shown as they are identical to those in our
previous publication (11), but results of the viral challenge
without concomitant clone transfer are shown in Fig. 4. In
addition to promoting recovery in C57Bl/6 mice, both
CD8+ T cell clones efficiently promoted recovery in lethally infected µKO mice (Fig. 4). On the other hand, the
adoptively transferred clone of CD4+ T cells conferred partial protection in infected µKO mice with only 20% of the
clone recipients surviving lethal infection (Fig. 4). In four
other independent transfer experiments using these clonal
populations of CD4+ and CD8+ T cells, the overall survival for lethally infected µKO recipients of the virus-specific CD4+ T cell clone ranged from 20 to 60%, whereas
the CD8+ T cell clones reproducibly promoted recovery of
100% of infected µKO recipients.
This difference in the efficiency of virus clearance by
CD4+ and CD8+ immune effectors was not a unique property of these three cloned T cell populations. In companion
experiments, bulk cultures of A/JAPAN/57 immune splenocytes produced outcomes similar to their clonal counterparts upon adoptive transfer into lethally infected conventional and B cell-deficient recipients. Fractionated bulk populations of activated CD4+ T cells promoted recovery
in 100% of conventional recipients and in only 40%-60%
of µKO recipients (data not shown). Like the virus-specific
CD8+ clones (Fig. 4), bulk populations of CD8+ CTL effectors were uniformly protective after adoptive transfer into either conventional or B cell-deficient recipients.
In this report we have examined recovery from lethal
pulmonary influenza infection in µKO mice. We found that
B lymphocyte-deficient mice show greater susceptibility to
lethal primary influenza infection than do conventional
mice with an intact B cell compartment. However, after
vaccination with attenuated virus, the µKO mice demonstrate enhanced resistance to secondary challenge infection
with virulent virus. This finding is consistent with the presence of virus-specific memory T lymphocytes, which can respond to and promote recovery from lethal challenge infection, in the vaccinated animals. Finally, we have observed
that virus-specific CD4+ and CD8+ effector T lymphocytes differ in their capacity to clear virus and to promote
recovery in infected B cell-deficient recipients.
The essential role of neutralizing antiviral antibody in
protection against reinfection has been well established for
influenza and most other viruses (2, 14, 16). The finding
that B lymphocyte-deficient mice demonstrate increased
sensitivity to lethal primary infection with type A influenza
suggests an important role for antiviral antibody in recovery
from primary influenza virus infection. Palladino et al. have
previously provided compelling evidence for a critical role
of neutralizing antibody in virus clearance during primary
influenza infection in the mouse (2). The findings reported
here support this concept. However, although no gross abnormalities in immune responsiveness have been reported to date in µKO mice (9, 17, 18), more subtle alterations in
the host response to influenza in animals lacking mature
B220+ B cells (e.g., delayed T cell response kinetics) may
contribute to their increased susceptibility to lethal influenza infection. It is noteworthy that Topham et al. recently
reported efficient clearing of influenza HKX-31 influenza
strain in µKO mice after sublethal infection (19). The findings reported here are in agreement with those results; however, our results further suggest that although virus clearance can be achieved in the absence of mature B cells, a
humoral immune response to the virus also makes an important contribution to the control of virus replication and
recovery from primary infection with a virulent virus strain.
A characteristic feature of influenza infection in the human is the susceptibility of immune individuals (primed by
earlier vaccination or natural infection) to infection with
serologically distinct variant virus strains of the same type A
influenza subtype as that which arises in the human population through antigenic drift (20). Since these circulating viruses also share conserved antigenic epitopes recognizable by
human and murine CD4+ and particularly CD8+ T lymphocytes (13, 21) the contribution in immune individuals of influenza-specific memory T lymphocytes directed to these
conserved antigenic epitopes in resistance to and recovery
from subsequent infection with drift variants has generally
been considered minimal (1). The results reported here suggest otherwise. Earlier priming of B lymphocyte-deficient
µKO mice with a live attenuated virus resulted in a >100-fold increase in the resistance of vaccinated mice to lethal
challenge infection. This finding implies that, like preexisting neutralizing anti-influenza antibody, memory T lymphocytes present in influenza-immune individuals can act to modify the course and severity of subsequent influenza
infections. In a recent report on the outcome of lymphocytic choriomeningitis virus (LCMV) infection in B lymphocyte-deficient mice, the presence of LCMV-specific
memory T cells induced by earlier vaccination likewise resulted in enhanced resistance to challenge infection (22).
Our finding that the secondary CTL response elicited in
primed B cell-deficient mice is comparable to the CTL response in immune animals with an intact B lymphocyte
compartment is in keeping with other recent reports in viral and nonviral models that demonstrated normal development and maintenance of CD8+ T cell memory in mice
lacking mature B lymphocytes (22, 23). However, as noted
above, T lymphocytes which differentiate in the absence of
B cells may develop compensatory mechanisms not normally
expressed by T lymphocytes developing and responding in
the presence of an intact B cell compartment. To assess the relative contribution of CD4+ and CD8+ T cells to virus
clearance and recovery in µKO mice, we chose to adoptively transfer defined clonal and bulk populations of virus-specific effector CD4+ and CD8+ T cells derived from B
cell-positive donors into lethally infected µKO recipients.
The results of this adoptive transfer analysis were clear
cut. Virus-specific CD8+ T lymphocyte effectors were
equally effective at protecting from lethal infection and at
promoting recovery in both conventional and B lymphocyte-deficient recipients. On the other hand CD4+ T lymphocyte effectors were as effective as CD8+ T lymphocytes
in protecting mice with an intact B cell compartment, but
promoted recovery in only a fraction of lethally infected µKO recipients. This result is in keeping with earlier observations of Scherle and co-workers that influenza-specific
CD4+ T lymphocytes function in vivo to clear influenza
infection primarily by collaborating with B lymphocytes in
the production of antiviral antibody (7, 24).
However, it should be emphasized that we reproducibly
observed virus clearance and recovery in 20-60% of lethally
infected B lymphocyte-deficient mice after transfer of activated virus-specific CD4+ T cells. Thus, one or more antibody-independent effector mechanisms appear to be used
by CD4+ effector T cells to confer resistance to lethal challenge in some µKO recipients. Since the CD4+ T cell clone
used in this study, 4D7, has been previously shown to express virus-specific MHC class II-restricted cytolytic activity in vitro (11) direct cytolysis of influenza-infected respiratory epithelial cells would be the probable mechanism to
account for the antiviral effect of transferred CD4+ T cell
effectors in µKO recipients. The recent studies of Tripp et
al. on virus clearance by CD4+ T cells in influenza-infected
MHC class II-deficient mice provide compelling evidence
against direct cytolysis as a critical in vivo effector mechanism of virus-specific effector CD4+ T lymphocytes (25).
A more likely explanation for the antiviral effect of transferred CD4+ T cells in µKO mice is that upon contact
with infected MHC class II-positive cells the CD4+ effectors release proinflammatory cytokines that could accelerate the generation of specific CD8+ T cell effectors in the infected recipient and/or amplify nonadaptive innate immune effectors mechanism with antiviral activity.
The ultimate goal of vaccination against viruses is to prevent the establishment of infection and this can be most effectively achieved by the presence of preexisting neutralizing antibody at the initial site of infection. In the case of
type A influenza virus and other viruses that can undergo
extensive antigenic variation, vaccination to prevent infection is difficult to achieve. Therefore, it may be more realistic to also consider vaccination strategies that lessen the severity of influenza infection. The results of the murine
influenza model reported here strongly suggest that, in an
immune individual, primed populations of influenza-specific memory T lymphocytes can act to modify the course and
severity of subsequent influenza infection. These findings
further imply that to be most effective a vaccine against human influenza should not only elicit a neutralizing antibody
response but should also efficiently prime virus-specific memory CD8+ and CD4+ T lymphocytes.
2 to 10
6
and animals were watched daily for morbidity and/or mortality. Each group consisted of both male and female mice as no differences have been observed when using age-matched mice.
5 M 2-ME, and antibiotics)
with 15 U/ml human recombinant IL-2 (huRIL-2; Biosource
International, Inc., Camarillo, CA). The procedures developed to
establish and maintain the T cell clones used in these studies have
been described in detail elsewhere (11).
3-10
7 dilutions of a stock
preparation with an infectious titer of 107 EID50/ml in embryonated eggs). As shown in Fig. 1, µKO mice were ~100-fold more susceptible to lethal virus infection than
were wild-type C57Bl/6 mice. The LD50 of this mouse-adapted A/JAPAN/57 influenza virus was 103.8 EID50 units
in wild-type C57Bl/6 mice and 101.7 EID50 units in µKO
mice, respectively. The titer of mouse-adapted virus used
for these studies was ~107 EID50 units, which corresponds
to an LD50 of 103 EID50 units in C57Bl/6 mice and an
LD50 of 101-102 EID50 units in µKO mice.
Fig. 1.
B cell-deficient mice are more susceptible to influenza viral
challenge. µKO (a) and C57Bl/6 (b) mice were intranasally inoculated with varying doses (, 10
3 dilution;
, 10
4 dilution;
, 10
5 dilution;
, 10
6 dilution; and
, 10
7 dilution) of mouse adapted influenza virus
and watched for 21 d for morbidity and mortality.
[View Larger Version of this Image (22K GIF file)]
Fig. 2.
µKO mice can initiate and maintain an influenza-specific
CTL response after challenge with influenza virus. (a) Lungs were removed from µKO (shaded bars) and C57BL/6 (open bars) mice on day 12 after intranasal viral challenge with a sublethal dose of A/JAPAN/57 (attenuated strain). Cell suspensions from the lungs were obtained by processing through a sieve, and were Ficoll purified and plated for a final effector/targer (E/T) ratio of 50:1. Assay time was 6 h and spontaneous
release for all targets was <20%. Results of two separate experiments with
two mice per experimental group are shown. (b) Influenza specific bulks
from four individual µKO (shaded bars) and from C57BL/6 (open bars)
mice were tested for their ability to lyse uninfected and A/JAPAN/57 infected class II negative (EL4) and class I and II positive (LB15.13) target cells in a standard chromium release assay. Assay time = 6 h; E/T = 10:1.
Spontaneous release for all targets was <15%. Experiment is representative of three separate experiments.
[View Larger Version of this Image (43K GIF file)]
Fig. 3.
B cell-deficient mice are more susceptible to rechallenge
with influenza. Groups of 7-12 age-matched µKO (open symbols) and C57Bl/6 (closed symbols) mice were intranasally infected with attenuated
A/JAPAN/57. 28 d later the animals were rechallenged intranasally with
the dilutions of mouse adapted A/JAPAN/57: , 10
1 dilution;
, 10
2
dilution;
, 10
3 dilution;
, 10
4 dilution. Animals were followed for
21 d for mortality. Data is representative of two experiments.
[View Larger Version of this Image (16K GIF file)]
Fig. 4.
CD8+, but not CD4+, T cell clones promote full recovery in
B cell-deficient mice. B cell-deficient (µKO) mice were infected intranasally with a 10 LD50 dose of mouse-adapted A/JAPAN/57 virus followed
by an intravenous injection of 107 cells. The cells transferred included
4D7 (, a CD4+ clone), 11E4 (
, a CD8+ clone), and B1.11 (
, a
CD8+ clone). Mice receiving intranasal influenza, but no cell transfer, are
denoted as
. C57Bl/6 mice receiving intranasal influenza without cell
transfer are also shown (
). Each group represents 5-7 animals. Adoptive
transfer of these clones and media control into lethally challenged C57Bl/
6 mice was done simultaneously as a control.
[View Larger Version of this Image (14K GIF file)]
Address correspondence to Dr. Thomas J. Braciale, University of Virginia Health Sciences Center, Beirne B. Carter Center for Immunology Research, HSC-MR4-4012, Charlottesville, VA 22908. Phone: 804-924-1219; FAX: 804-924-1221; E-mail: tjb2r{at}virginia.edu Dr. Mary Beth Graham's present address is University of Illinois at Chicago, Department of Medicine (M/C 733, Rm. 1158 MBRB), 900 S. Ashland Ave., Chicago, IL 60607. E-mail: mbgraham{at}uic.edu
Received for publication 11 August 1997 and in revised form 14 October 1997.
This work was supported by Physician Scientist Award K11-AI01086 (M.B. Graham) from the National Institute of Allergy and Infectious Diseases and National Institutes of Health grants HL-33391, AI-15608, and AI-28317 (T.J. Braciale).
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