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,
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Trudeau Institute, Saranac Lake, NY 12983;
Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105; and
Department of Pathology, University of Tennessee, Memphis, TN 38163
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to protective effects, primed CD4+ T cells have also been implicated in detrimental effects during viral infections. Presensitization with the major surface glycoprotein (G protein) of respiratory syncytial virus (RSV)3 led to severe pulmonary eosinophilia upon challenge with RSV (12, 13). This enhanced disease has been found to be associated with a strongly Th2-biased pulmonary CD4+ response triggered by the G protein of RSV (14, 15, 16). Similar phenomena have also been observed in influenza virus model (17). Although adoptive transfer of the virus-specific Th1 clones conferred complete protection against viral challenge, transfer of virus-specific Th2 clones resulted in exacerbated lung pathology upon challenge with the virus (17). This indicates that the quality or nature of CD4+ T cell populations primed may have a significant impact on the nature of the subsequent responses to infection.
Current influenza vaccines are composed of Formalin-inactivated trivalent influenza viruses and are designed to induce protective humoral immunity (18). This vaccination regimen also induces potent CD4+ T cell memory (19, 20). However, little is known about the impact of these primed CD4+ T cells on the immune response to heterologous viruses that are serologically distinct, but that share CD4+ T cell epitopes. In the present study, we addressed the issue of CD4+ T cell-mediated antiviral immunity in immunologically intact mice, using Sendai virus as a model system. Our results demonstrate that CD4+ T cell priming had a significant influence on the profile of T and B cell-mediated responses to acute Sendai virus infection and also influenced the establishment of long-term CD8+ T cell memory. Most important, we show in this work that CD4+ T cell priming leads to accelerated viral clearance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed under specific pathogen-free conditions before infection with Sendai virus at 6–10 wk of age.
Priming of mice with peptide Ags
Sendai virus hemagglutinin-neuraminidase (HN) peptides (HN421–436, HN559–574, HN163–178), nucleoprotein (NP) peptide (NP324–332), and matrix protein (M) peptide (M149–164) have been described previously (21, 22). The peptides were synthesized and purified with reverse-phase HPLC at St. Jude Children’s Research Hospital. Mice were primed with 50 µg of either the HN421–436 or HN559–574 synthetic peptides emulsified in 100 µl of CFA by s.c. injection into the base of the tail. Control mice received an equal amount of CFA prepared with PBS. Seven to ten days later, the animals were boosted with 50 µg of the corresponding peptides in IFA or PBS in IFA as a negative control.
Virus infections and assays
The Enders strain of Sendai virus (23) was grown, titrated, and stored, as described previously (7). Mice that had been primed with HN peptides 10–30 days previously were infected intranasally (i.n.) under anesthesia with 500 egg infectious doses (EID50) of Sendai virus. All infected mice were held under biosafety level 3 conditions. Virus titers of lung tissues were determined from lung homogenates by endpoint titration in embryonated hen eggs (logEID50), as described previously (7).
CD4+ T cell proliferation assay
Mice were killed 10–30 days after immunization with Sendai virus HN peptides, and single-cell suspensions were prepared from the inguinal and paraaortic lymph nodes. CD4+ T cells were enriched from pools of two to three spleens by negative selection with Dynabeads (Dynal, Oslo, Norway), as described previously (3). The enriched cell population usually comprised 82–87% CD4+ T cells, as determined by flow cytometry. Enriched CD4+ T cells (3–5 x 105) were then cultured with T cell-depleted, X-irradiated (3000 rad) splenocytes from syngeneic naive mice (2.5 x 105) in the presence of HN peptides (10 µg/ml) for 4 days in 200 µl of complete tumor medium (CTM) at 37°C with 10% CO2. CTM is prepared by supplementing MEM (Life Technologies, Rockville, MD) with essential and nonessential amino acids, sodium pyruvate, L-glutamine, sodium bicarbonate, gentomycin, penicillin G, streptomycin sulfate, and 2-ME, as described previously (24). [3H]thymidine (1 µCi/well; DuPont, Boston, MA) was added for the final 18 h of culture. Cells were harvested using an automatic cell harvester (Tomtec, Vallac, Gaithersburg, MD), and the incorporated radioactivity was determined by an automatic beta scintillation counter system (LKB, Vallac). The results are expressed as cpm ± SD of triplicate cultures.
Determination of virus-specific serum Ab titers by ELISA
Sendai virus-specific serum Ab titers were determined by ELISA, as described previously (25). Briefly, serial dilutions of test serum samples were incubated on plates coated with purified Sendai virus Ag preparation. Alkaline phosphatase-labeled goat anti-mouse secondary Abs (Southern Biotechnology Associates, Birmingham, AL) were added, followed by development of the color with the peroxidase substrate (p-nitrophenyl phosphate; Sigma, St. Louis, MO). In all assays, a pooled normal mouse serum collected from naive C57BL/6 mice was used to determine the endpoint titer of test serum samples. The endpoint was defined as the final dilution of a test sample whose OD value is greater than the mean plus 3 SD of data obtained with normal mouse serum.
Differential WBC counting of the bronchoalveolar lavage (BAL)
Cells from the BAL were pooled from three to four infected mice, washed once with CTM, and resuspended in 80 µl of CTM. The concentrations and percentages of pulmonary inflammatory cells, including total WBC, neutrophils, eosinophils, basophils, monocytes, macrophages, and lymphocytes, were counted and calculated by an automatic cell counter (Hemavet 3500; CDC Technologies, Oxford, CT). The criteria used for the differential cell count by the instrument are based on the cell size, number, and morphology of different types of murine WBC. The results are expressed as absolute numbers of each inflammatory cell type per lung.
Flow cytometry
Staining with Sendai virus MHC class I-peptide tetramers was
done as described previously (26). Briefly, B cells were
first depleted from spleen samples by panning on anti-mouse
IgG-coated flasks. Nonspecific staining was blocked in purified
anti-mouse CD16/CD32 (FcR
III/II receptor; PharMingen, San Diego,
CA). The cells were then stained with Sendai virus
NP324–332/Kb-PE or
NP324–332/Db-PE tetramer,
followed by staining with tricolor-conjugated anti-CD8 (Caltag,
Burlingame, CA) and FITC-conjugated anti-CD44 or anti-CD62L
(PharMingen). Two-color staining of BAL cells utilized FITC-conjugated
anti-CD4 and PE-conjugated anti-CD8 mAbs (PharMingen). Stained
samples were acquired on a Becton Dickinson FACScan flow cytometer, and
the data were analyzed using CellQuest software (Becton Dickinson
Immunocytometry System, San Jose, CA). For BAL samples, the results are
expressed as the absolute cell numbers of CD4+,
CD8+, or
NP324–332/Kb-tetramer+
cells in each lung, which were calculated based on the percentage of
tetramer+/CD4+ or
CD8+ cells among total lymphocytes, and the
absolute numbers of lymphocytes were determined from the total WBC
count of each lung. For spleen and MLN samples, the results are
expressed as the percentage of
NP324–332/Kb-tetramer+
cells among total CD8+ T cells.
Intracellular IFN- staining following peptide
stimulation
Cells from the BAL were pooled from 10 to 14 primed and infected
mice of each group and absorbed onto plastic dishes to remove
macrophages. The nonadherent cell populations were then cultured for
6 h in the presence or absence of 1 µg/ml of Sendai virus
NP324–332 peptide or 10 µg/ml HN peptides in
250 µl of CTM containing 10 µg/ml brefeldin A (BFA). After culture,
intracellular IFN- staining was performed, as described previously
(27). Briefly, the responder cells were washed twice with
PBS/BFA, blocked in mAb to FcRIII/II receptor, and stained with rat
anti-mouse CD8+ or
anti-CD4+ FITC conjugates (PharMingen). They
were then washed in PBS/BFA, fixed in 1% formaldehyde, and permeated
in PBS/0.5% Saponin (Sigma). The cells were stained with rat
anti-mouse IFN- PE or rat IgG1 PE isotype control. FACS analysis
was done as described above. The results are expressed as the
percentage of CD4+ IFN-+
or CD8+ IFN-+
double-positive populations among total CD4+ or
CD8+ cells after subtraction of background
obtained with an irrelevant Sendai virus peptide,
M149–164.
51Cr release assay
The cytotoxic activity of cells from the BAL of primed and infected mice was determined as described previously (28). Briefly, target cells (L-Kb and L-Db transfectants) were labeled with 51Cr (Na51CrO4; Amersham Life Science, Arlington Heights, IL) and then either pulsed with Sendai virus NP324–332 peptide or infected with Sendai virus. Unpulsed target cells were used as negative controls. Various numbers of BAL cells were incubated with 2 x 103 targets for 5 h. The percentage of specific 51Cr release was calculated by the formula: percent specific release = (experimental - spontaneous)/(maximal - spontaneous). Spontaneous release was typically <10% of the total release induced by 0.5% Triton X-100.
Single cell ELISPOT assay for Sendai virus-specific Ab-forming cell (AFC)
The ELISPOT assay for virus-specific AFC was done as described previously (29, 30) using nitrocellulose Multiscreen HA filtration plates (Millipore, Bedford, MA) coated with purified Sendai virion Ags. Virus-specific AFC-producing IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA were enumerated in single cell suspensions prepared from cervical lymph node (CLN) and MLN, and the results are expressed as mean ± SD.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have shown that the CD4+ T
cell response to Sendai virus infection in C57BL/6 mice is directed
against dominant
(HN421–436/Ab) and
subdominant (HN559–574/Ab)
epitopes derived from the viral HN protein (22). The goal
of the current study was to investigate the effect of priming
CD4+ T cells specific for these epitopes on
various aspects of the immune response to a subsequent virus infection.
Thus, we primed C57BL/6 mice in the base of the tail with either
HN421–436 or HN559–574
peptides in CFA and boosted them 7–10 days later with the same
peptides in IFA. To confirm priming of the mice, splenic
CD4+ T cells were restimulated in vitro with
either the HN421–436 or
HN559–574 peptides, or a
Kb-restricted Sendai virus nucleoprotein peptide
(NP324–332) as a negative control. As shown in
Fig. 1
, mice primed with the
HN421–436 peptide proliferated strongly in
response to the HN421–436 peptide in vitro, but
not to HN559–574 and
NP324–332 peptides. In contrast,
CD4+ T cells from mice primed with the
HN559–574 peptide proliferated exclusively to
the HN559–574 peptide, and not the other
peptides tested. CD4+ T cells taken from control
mice that had been primed with PBS/CFA responded to none of the
peptides tested. These results confirmed that vaccination with either
the HN421–436 or
HN559–575 peptides in CFA primed
CD4+ T cells specific for the corresponding
priming peptide.
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, a positive control serum obtained from
Sendai virus-infected mice contained a high titer of virus-specific
Abs, whereas none of three serum samples from CFA-,
HN421–436-, or
HN559–574-primed mice contained any
detectable Abs specific for Sendai virus. This demonstrates that
vaccination with either HN421–436 or
HN559–574 peptides did not induce virus-specific
Ab responses.
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To investigate the impact of CD4+ T cell
priming on the subsequent pulmonary responses to Sendai virus, we first
characterized a normal inflammatory process in the BAL of unvacccinated
mice after a primary infection with Sendai virus. Inflammatory
infiltrates consisted of a variety of inflammatory cells, including
neutrophils, eosinophils, basophils, monocytes, and lymphocytes (Fig. 2). Neutrophils were the predominant
inflammatory cell type during the first 7 days after infection. They
peaked on day 7 postinfection, accounting for 67% of the inflammatory
infiltrates, and dropped rapidly to background levels at about day 14
postinfection. Significant numbers of lymphocytes were first detected
on day 7, peaked on day 10 postinfection (65%), and decreased quickly
to background level within the next 3–5 days.
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Accelerated recruitment of HN-specific CD4+ T cells to the lung
We next asked whether CD4+ T cells primed by
this vaccination protocol were able to traffic to the site of an acute
infection and participate in the subsequent immune responses. Thus, we
compared the kinetics of CD4+ T cell recruitment
in HN421–436-immune and unprimed mice that had
been i.n. infected with Sendai virus. As shown in Fig. 3, the recruitment of
CD4+ T cells was significantly accelerated in the
BAL of HN421–436-immune mice. The accumulation
of CD4+ cells into the BAL peaked at day 7
postinfection (2.3 x 105/lung), and
remained relatively high for the rest of the 6-day period of
observation, whereas significant numbers of CD4+
T cells (1 x 105/lung) in the BAL of
unprimed animals were only detected after 7 days and peaked on day 10
postinfection. Interestingly, the peak number of
CD4+ T cells in unprimed mice was nearly twice as
high as that of HN421–436-primed mice (
4
x 105/lung). Priming with the subdominant
HN559–574 peptide had no effect on total
CD4+ T cell recruitment to the lung.
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staining to determine the
frequency of
HN421–436/Ab-specific
IFN-+ T cells in the BAL on days 7 and 10
postinfection. These time points were selected because maximal numbers
of CD4+ T cells into the lung were observed at
these times in HN421–436-primed and control mice
(Fig. 3
). As shown in Fig. 4, more than
14% of the CD4+ cells present in the BAL of
HN421–436-immune mice were specific for the
dominant HN421–436/Ab
epitope on day 7 after infection, compared with only about 4% of the
CD4+ cells in the BAL of control mice.
CD4+ T cells specific for the subdominant
HN559–574/Ab epitope were
only a minimal component of the inflammatory infiltrates of lungs in
all the three groups at this time point. Similar results were obtained
at day 10. Priming with the subdominant
HN559–574 peptide did not lead to an enhanced
recruitment of
HN559–574-/Ab-specific
CD4+ T cells in the lung.
CD4+ T cells producing IL-4, IL-5, and IL-10 were
not detectable in intracellular cytokine-staining assays (data not
shown). Taken together, the data demonstrate that the priming of
CD4+ T cells specific for the
HN421–436/Ab epitope
peptide resulted in an accelerated CD4+ T cell
response to a primary Sendai virus infection.
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Previous studies have established that
NP324–332/Kb-specific
CD8+ effector T cells dominate the inflammatory
infiltrates of the lung at about day 10 after Sendai virus infection
and play a major role in the clearance of the virus from respiratory
tract (7). Therefore, we investigated whether
CD4+ T cell priming to the
HN421–436 peptide influenced the
subsequent CD8+ T cell response to Sendai virus
infection. The kinetics of
NP324–332/Kb-specific
CD8+ T cell recruitment into the lung after
infection with Sendai virus was determined using tetramer reagents that
detect CD8+ T cells specific for either the
dominant (NP324–332/Kb) or
subdominant (NP324–332/Db)
class I-restricted epitopes (26, 32). Consistent with
previous observations (7), maximal
CD8+ T cell recruitment was observed in control
mice on day 10 postinfection (Fig. 5). T
cells specific for the dominant
NP324–332/Kb epitope were
recruited to the lung with similar kinetics and represented 50% of
the total CD8+ T cells in the lung at day 10
postinfection. In contrast, the kinetics of CD8+
T cell recruitment to the lungs of
HN421–436-primed mice was significantly
different. Peak numbers of CD8+ T cells were
detected at day 7 and fell rapidly by day 10. Interestingly, the
numbers of T cells specific for the
NP324–332/Kb epitope
stayed relatively low through day 14 postinfection.
CD8+ T cells specific for the subdominant
NP324–332/Db epitope were
not detectable in the pulmonary infiltrates of all three groups of mice
tested during the entire 14-day period of observation, consistent with
previous studies (26). The accumulation of total and
virus-specific CD8+ T cells to the respiratory
tract of the subdominant HN559–574-primed and
virus-infected mice was similar to that seen in control mice with
regard to their magnitude and kinetics.
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, strong
NP324–332/Kb-specific CTL
activity could be detected in the BAL of both
HN559–574-primed and adjuvant alone-primed mice
on day 10 after infection. However, as predicted by the
NP324–332/Kb tetramer
staining (Fig. 5),
NP324–332/Kb-specific CTL
activity was significantly reduced in the BAL of mice primed previously
with HN421–436 peptide. Consistent with
NP324–332/Db tetramer
staining (Fig. 5), no CTL activity against the subdominant
NP324–332/Db epitope
was detected in all the three groups of mice tested (Fig. 6).
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Significantly decreased frequencies of virus-specific long-term memory CD8+ T cells in peripheral lymphoid organs
Recent studies have shown that high frequencies of long-term
CD8+ T cell memory to both the dominant
NP324–332/Kb and the
subdominant NP324–332/Db
epitope could be detected in the spleen after a primary infection
(26). Given that CD4+ T cell priming
with the HN421–436 peptide resulted in a
dramatic decrease in the numbers of
NP324–332/Kb-specific CTL
effectors in pneumonic lung, we next asked whether this influenced the
establishment of memory CD8+ T cells. Thus, we
analyzed the frequencies and phenotypes of virus-specific memory
CD8+ T cells by staining spleen cells with the
NP324–332/Kb tetramer at
different time points after infection with Sendai virus. As shown in
Table II, high frequencies of
CD8+
NP324–336/Kb+-specific T
cells were detected in both infected control mice and
HN421–436-primed mice (ranging from 4.0–4.9)
during acute infection (day 12 postinfection). All of the
CD8+
NP324–332/Kb+-specific T
cells expressed a
CD44high/CD62Llow phenotype
at this time (data not shown). This indicates that priming with the
HN421–436 peptide does not affect the numbers of
NP324–332/Kb-specific T
cells in the peripheral lymphoid tissues during the acute infection.
However, analysis of T cell frequencies at day 31 after infection, when
immunological memory status is considered to have been established,
revealed a significant impact of HN421–436
peptide vaccination on the frequencies of Ag-specific memory
CD8+ T cells. Whereas the frequency of
CD8+
NP324–336/Kb-specific T
cells in spleen of unprimed animals was 2.4%, frequencies of
CD8+
NP324–336/Kb-specific T
cells were only 0.4% in spleen of HN421–436
peptide-primed memory mice. Similar results were obtained when analyzed
on day 60 after infection (data not shown). These results demonstrate
that vaccination based on priming CD4+ T cells
not only has a significant influence on the effector phase of
virus-specific CD8+ CTL responses, but also on
the absolute numbers of CD8+ memory T cells that
are established.
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The data to date demonstrate that priming with the
HN421–436 peptide resulted in significant
effects on the immune responses to Sendai virus infection.
Virus-specific CD4+ responses become dominant and
cytotoxic CD8+ T cell responses were reduced. To
investigate the biological consequence of these changes on virus
clearance, we assessed virus titers in the lung at various times after
infection. As shown in Fig. 7A, high titers of Sendai
virus were detected in the lung homogenates of CFA alone-primed control
mice on days 3 and 5. From day 7 on, virus titers dropped rapidly and
no virus was detected in all six animals tested on day 10. In contrast,
virus clearance was significantly increased in
HN421–436-primed animals. As shown in Fig. 7B, virus was completely eliminated from the lung tissues of
three mice on day 8, a 2-day advantage over control mice.
Interestingly, slightly accelerated viral clearance was also observed
following priming with the subdominant HN559–574
peptide. As shown in Fig. 7C, while none of the three
control mice cleared virus on days 8 and 9, virus was only detected in
two of three mice on day 8, and only one of three mice on day 9 from
the lung homogenates.
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Ab-mediated mechanisms play a key role in controlling secondary Sendai virus infections. In addition, it has been shown that Ab is able to control primary Sendai virus infections when CD8+ T cell responses are absent, albeit with slower kinetics (6, 7, 8). In light of the classic observation that preimmunization with carrier leads to accelerated and enhanced Ab response to hapten (34), we hypothesized that CD4+ T cell priming would result in accelerated and enhanced B cell responses to Sendai virus. To test this, we first used a single cell ELISPOT assay to determine the induction of virus-specific Ab-forming cells (AFC) in the MLN and CLN. These sites were chosen as they have been shown to contribute to the generation of virus-specific Abs in respiratory secretions and serum during acute Sendai virus infection (25).
The overall antiviral AFC response in the MLN was both accelerated and
enhanced in magnitude by previous immunization with
HN421–436 peptide. As shown in Fig. 8, the frequencies of AFC-producing
virus-specific IgM, IgG1, IgG2a, IgG2b, and IgG3 isotypes in the MLN of
HN421–436 peptide-vaccinated mice were higher
than those of control mice and could be detected 1 day earlier.
Immunization with the subdominant HN559–574
peptide was much less effective in promoting the antiviral AFC response
in the MLN. Only slightly increased numbers of AFC-producing IgG1 and
IgA were present in the CLN.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanism through which CD4+ T cell epitope vaccination resulted in significantly enhanced viral clearance is unclear. It was not due to an Ab response to the immunizing peptide since Sendai virus-specific Abs were not detected in the primed animals before infection. It is also unlikely that enhanced viral clearance was mediated by Ag-specific CD8+ CTL since these cells were significantly reduced in number. Similarly, the CD4+ T cells that were recruited rapidly to the lung were not themselves cytotoxic. One possibility is that the primed CD4+ T cells mediated an accelerated and enhanced antiviral Ab response, leading to an accelerated viral clearance. Indeed, the virus-specific AFC numbers were both accelerated and increased in magnitude in MLN in HN421–436 peptide-primed mice following Sendai virus infection. However, slightly enhanced Sendai virus-specific serum Abs were detected on day 8 after viral challenge in both HN421–436- and HN559–574-primed mice. The fact that only HN421–436-primed, but not HN 559–574-primed, mice cleared virus from the lung at this time point suggests that this level of Ab is not the primary factor in mediating accelerated viral clearance. Additional experiments are underway in our lab to formally rule out this possibility by testing the role of Ab in this system using B cell-deficient mice. Together, our data are consistent with the idea that primed CD4+ T cells are able to promote accelerated clearance of a Sendai virus infection in an Ab-independent manner.
Although the mechanism of CD4+ T cell-mediated
control of Sendai virus infection is unknown, the most likely candidate
is through the production of antiviral cytokines. For example, both Th1
(IFN-, IL-2, and TGF-ß) and Th2 cytokines (IL-4, IL-5, and IL-10)
have been identified during acute viral infections induced by
respiratory tract viruses, including Sendai virus (35, 36). It was found that adoptive transfer of large numbers of
activated Th1 clones specific for influenza A virus, but not Th2
clones, was able to confer protection against lethal viral challenge in
vivo (17). In the present study, we show that following
priming with the dominant Sendai virus HN epitope, the frequency of
IFN--producing CD4+ T cells was increased in
the pneumonic lung, while CD4+ T cells producing
IL-4, IL-5, and IL-10 were not detectable in intracellular
cytokine-staining assays (data not shown). This indicates that
CD4+ T cell priming to the
HN421–436 epitope using CFA as an adjuvant does
not change the normal cytokine pattern of the
CD4+ T cell response in the lung after Sendai
virus infection. Rather, it strengthens a virus-specific Th1 response.
It is possible that early or enhanced IFN- secretion could
facilitate viral clearance, as has been demonstrated in other systems
(37, 38). Other inflammatory cytokines, including
IFN-
ß, might also contribute to an accelerated viral clearance in
our system. In this regard, it should be noted that pulmonary
recruitment of monocytes/macrophages, the major cell source of
IFN-ß, was obviously accelerated and enhanced in
HN421–436-primed animal. Taken together, the
observed acceleration of viral clearance may reflect a combined effect
of different antiviral cytokines in pulmonary infiltrates.
The requirement of CD4+ T cell help for generation of CD8+ T cell responses is controversial. Numerous experiments have shown that depletion of CD4+ T cell populations had little effect on the generation of CD8+ cytotoxic effectors against influenza A virus, Sendai virus, or lymphocytic choriomeningitis virus (7, 39, 40, 41, 42). On the other hand, many recent studies have documented the critical involvement of CD4+ T cell help in the generation and maintenance of effective CD8+ responses against some chronic viral infections. For example, it was found that virus-specific CD8+ T cell responses were completely lost in CD4+ T cell-deficient mice chronically infected with MHV-68 or lymphocytic choriomeningitis virus (43, 44). In this study, our data suggest a new aspect of CD4+/CD8+ interactions in the Sendai virus model. Virus-specific CD4+ T cells induced by peptide resulted in decreased virus-specific CD8+ effector function, and decreased frequency of CD8+ long-term memory. The reasons for this are unclear. One possibility is that HN421–436-primed animals were able to clear virus more efficiently, resulting in a lower Ag load and reduced CD8+ T cell differentiation. Since memory T cells are derived, at least in part, from effector T cells (45), this would result in low frequency of CD8+ memory cells. This possibility is supported by a recent observation in an in vitro model system that differentiation of cytotoxic effectors was Ag dose dependent (46). However, an alternative possibility is that biased CD4+ expansion may negatively regulate the primary activation of CD8+ T cells, or the recruitment of activated T cells to the lung, possibly through the secretion of specific cytokines.
The negative effect of primed CD4+ T cells on the generation of virus-specific CD8+ memory may have significant implications for current vaccination strategies against respiratory virus infection. It has been shown that the CD8+ T cell arm is the major mediator of such a broad protection among serologically distinct influenza viruses (47). For example, infection of mice with an influenza A virus of one subtype led to induction of partial protection to infection with a virus of a different subtype (47, 48, 49). The results presented in this work imply that although primed, cross-reactive CD4+ T cells can enhance protection against challenge with a homologous virus, they could potentially block the development of cross-reactive CD8+ T cell memory and thereby reduce protection against some virus strains. These results argue that a vaccine approach that targets multiple arms of the immune system to develop broad immunity would be preferable.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David L. Woodland, Trudeau Institute, P.O. Box 59, Saranac Lake, NY 12983. E-mail address:
3 Abbreviations used in this paper: RSV, respiratory syncytial virus; AFC, Ab-forming cell; BAL, bronchoalveolar lavage; BFA, brefeldin A; CLN, cervical lymph node; CTM, complete tumor medium; EID50, egg infectious dose; ELISPOT, enzyme-linked immunospot; HN, hemagglutinin-neuraminidase; i.n., intranasal; MLN, mediastinal lymph node; NP, neucleoprotein; WBC, white blood cell.
Received for publication November 11, 1999. Accepted for publication January 6, 2000.
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D. Stober, I. Jomantaite, R. Schirmbeck, and J. Reimann NKT Cells Provide Help for Dendritic Cell-Dependent Priming of MHC Class I-Restricted CD8+ T Cells In Vivo J. Immunol., March 1, 2003; 170(5): 2540 - 2548. [Abstract] [Full Text] [PDF]
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G. M. Winslow, A. D. Roberts, M. A. Blackman, and D. L. Woodland Persistence and Turnover of Antigen-Specific CD4 T Cells During Chronic Tuberculosis Infection in the Mouse J. Immunol., February 15, 2003; 170(4): 2046 - 2052. [Abstract] [Full Text] [PDF]
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L. S. Cauley, T. Cookenham, T. B. Miller, P. S. Adams, K. M. Vignali, D. A. A. Vignali, and D. L. Woodland Cutting Edge: Virus-Specific CD4+ Memory T Cells in Nonlymphoid Tissues Express a Highly Activated Phenotype J. Immunol., December 15, 2002; 169(12): 6655 - 6658. [Abstract] [Full Text] [PDF]
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M. C. Wolkers, M. Toebes, M. Okabe, J. B. A. G. Haanen, and T. N. M. Schumacher Optimizing the Efficacy of Epitope-Directed DNA Vaccination J. Immunol., May 15, 2002; 168(10): 4998 - 5004. [Abstract] [Full Text] [PDF]
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M. M. McNeal, J. L. VanCott, A. H. C. Choi, M. Basu, J. A. Flint, S. C. Stone, J. D. Clements, and R. L. Ward CD4 T Cells Are the Only Lymphocytes Needed To Protect Mice against Rotavirus Shedding after Intranasal Immunization with a Chimeric VP6 Protein and the Adjuvant LT(R192G) J. Virol., January 15, 2002; 76(2): 560 - 568. [Abstract] [Full Text] [PDF]
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W. Zhong, A. D. Roberts, and D. L. Woodland Antibody-Independent Antiviral Function of Memory CD4+ T Cells In Vivo Requires Regulatory Signals from CD8+ Effector T Cells J. Immunol., August 1, 2001; 167(3): 1379 - 1386. [Abstract] [Full Text] [PDF]
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 R. J. Hogan, W. Zhong, E. J. Usherwood, T. Cookenham, A. D. Roberts, and D. L. Woodland Protection from Respiratory Virus Infections Can Be Mediated by Antigen-Specific Cd4+ T Cells That Persist in the Lungs J. Exp. Med., April 16, 2001; 193(8): 981 - 986. [Abstract] [Full Text] [PDF]
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R. J. Hogan, E. J. Usherwood, W. Zhong, A. D. Roberts, R. W. Dutton, A. G. Harmsen, and D. L. Woodland Activated Antigen-Specific CD8+ T Cells Persist in the Lungs Following Recovery from Respiratory Virus Infections J. Immunol., February 1, 2001; 166(3): 1813 - 1822. [Abstract] [Full Text] [PDF]
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 E. O’Neill, S. L. Krauss, J. M. Riberdy, R. G. Webster, and D. L. Woodland Heterologous protection against lethal A/HongKong/156/97 (H5N1) influenza virus infection in C57BL/6 mice J. Gen. Virol., November 1, 2000; 81(11): 2689 - 2696. [Abstract] [Full Text]
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), HN421–436-immune
mice (
), and HN559–574-immune mice (
) were
determined using an automatic cell counter. The data are representative
of two independent experiments.
) and subdominant (
) CD8+ epitopes of
Sendai virus NP molecule were determined by
NP324–332/Kb or
NP324–332/Db tetramer staining.
) 20 days before
were inoculated i.n. with 500 EID50 of Sendai virus. CLN
and MLN were sampled from five individual mice of each group at
indicated time points after infection. An ELISPOT assay was used to
determine the number of cells producing IgM, IgG1, IgG2a, IgG2b, IgG3,
and IgA specific for Sendai virus. The results are expressed as
mean ± SD of five individual mice in each group.