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-Deficient Mice Make Delayed, But Effective, T and B Cell Responses to Influenza1
Trudeau Institute, Saranac Lake, NY 12983
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- (LT-/-) mice are
thought to be unable to generate effective T and B cell responses. This
is attributed to the lack of lymph nodes and the disrupted splenic
architecture of these mice. However, despite these defects we found
that LT-/- mice could survive infection with a
virulent influenza A virus. LT-/- mice and normal
wild-type mice infected with influenza A generated similar numbers of
influenza-specific CD8 T cells that were able to produce IFN-
and
kill target cells presenting influenza peptides. Furthermore
influenza-infected LT-/- mice produced high titers of
influenza-specific IgM, IgG, and IgA. However, both CD8 and B cell
immune responses were delayed in LT-/- mice by 2–3
days. The delayed cellular and humoral immune response was sufficient
to mediate viral clearance in LT-/- mice that were
infected with relatively low doses of influenza virus. However, when
LT-/- mice were infected with larger doses of
influenza, they succumbed to infection before the immune response was
initiated. These results demonstrate that neither LT nor
constitutively organized lymphoid tissues, such as lymph nodes and
spleen, are absolutely required for the generation of effective
immunity against the respiratory virus influenza A. However, the
presence of LT and/or lymph nodes does accelerate the initiation of
immune responses, which leads to protection from larger doses of
virus. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to the cytotoxic activity of CD8 cells and the B cell
helper activity of CD4 cells, both CD4 and CD8 T cells secrete a
variety of cytokines that may interfere with viral replication and
stimulate local immune mechanisms (11, 12, 13). One such
cytokine, lymphotoxin-
(LT)3 is a member
of the TNF family of cytokines (14) and is active as
either a soluble homotrimer (15) or in combination with
LT
as a membrane-bound heterotrimer (16). Soluble LT
can signal through the TNF receptor type 1 (TNFR1) (p55) as well as
TNFR2 (p75), while membrane-bound LT signals through the LTR
(17). Like TNF-, LT can trigger apoptosis,
differentiation, proliferation, and the expression of a variety of
inflammatory cytokines and chemokines depending on the responding cell
type and the receptor that it binds (18). Although LT
is produced by a variety of lymphocytes (19), it is
produced at high levels by activated type 1 CD4 (Th1) and CD8 (Tc1) T
cells (20) and is thought to play an important role in the
effector functions of these cells.
Another important activity of LT is to induce the development of
organized lymphoid tissues (21).
LT-/- mice entirely lack lymph nodes and
Peyer’s patches and have a disrupted splenic architecture
(22). The pathways behind these defects have been studied
in detail, and it is now known that loss of LT (23),
LTR (24), or NF-
B-inducing kinase (a molecule in the
LTR signaling pathway) (25) leads to defects in
lymphoid organ development, the disruption of splenic organization, and
the loss of follicular dendritic cells (FDCs) and germinal centers.
Given the absence of lymph nodes and Peyer’s patches in
LT-/- mice and the lack of FDCs, organized B
cell follicles and germinal centers in the spleens of these mice, it is
not surprising that LT-/- mice are defective
in isotype switching (26) and affinity maturation
(27) and generally make poor Ab responses
(28). Furthermore, given that LT facilitates dendritic
cell (DC) maturation (29) and induces the expression of
chemokines that coordinate lymphocyte migration (30), it
is also not surprising that mice defective in the LT signaling pathway
make defective cellular immune responses. For example, mice that have a
loss of function mutation in NF-B-inducing kinase
(aly/aly mice) do not make detectable primary CTL responses
to vesicular stomatitis virus or lymphocytic choriomeningitis virus
(LCMV) and make a reduced CTL response to vaccinia virus (31, 32). Similarly, LT-/- mice have
severely impaired cellular immune responses to LCMV (33)
and cannot clear HSV (34). However,
LT-/- mice are not completely unable to
mount anti-viral responses, as LT-/-
mice can clear murine gammaherpes virus 68 (MHV-68) infection, albeit
with delayed kinetics (35). Likewise,
LT-TNF-/- mice can clear vaccinia virus and
LCMV after a significant delay (36). Together, these
results demonstrate that mice lacking an intact LT signaling pathway
have clear defects in cellular immunity to viral infections. However,
it remains unclear whether cellular immunity is inefficient in these
mice due to lack of CTL priming, an inability to expand Ag-specific T
cells, or alterations in the differentiation of Ag-specific T cells to
functional effector cells.
To determine the basis for the defective cellular immune responses
observed in LT-/- mice, we compared
LT-/- and normal wild type (WT) mice for
their ability to generate Ag-specific CD8 T cells to influenza and to
clear virus. We found that although LT-/-
mice were somewhat more susceptible to infection with high doses of
influenza virus, they were competent to generate influenza-specific CD8
T cells that produced IFN- and exhibited killing activity.
Furthermore, influenza-infected LT-/- mice
could generate B cells that produced isotype-switched,
influenza-specific Abs. However, unlike the immune response in WT mice,
the immune response to influenza in LT-/-
mice was delayed by several days. Thus, when
LT-/- mice were infected with larger doses
of virus, they succumbed to infection before specific adaptive immunity
could be generated. Together, these results suggest that neither the LT
signaling pathway nor the presence of organized lymphoid tissues is
absolutely required for T or B cell effector generation or function.
However, an intact LT signaling pathway and the presence of organized
lymphoid organs do facilitate the rapid induction of immune
responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (WT mice) and
C57BL/6.129Ltatm1Dch
(LT-/- mice) were obtained from The Jackson
Laboratory (Bar Harbor, ME). Both strains were bred and maintained in
the Animal Breeding Facility at Trudeau Institute. All procedures using
animals were approved by the Trudeau Institute institutional animal
care and use committee and were conducted according to the principles
outlined by the National Research Council.
Influenza infection and quantification
Mice were infected intranasally with 100 egg infectious units (EIU) of influenza A/PR8/34 in 100 µl unless otherwise indicated. Viral titers in the lungs of infected mice were quantified in embryonated eggs. Briefly, lungs were homogenized in 2 ml of PBS, and 500 µl of this stock was used to make 10-fold serial dilutions. One hundred microliters of each dilution was inoculated into each of three eggs. Allantoic fluid was harvested from inoculated eggs 4 days later, and infected eggs were scored by hemagglutination of chicken RBC. The viral end-point titer was defined as the highest dilution in which two or more eggs positively scored in the hemagglutination assay.
Flow cytometry
Mice were sacrificed at the indicated time points, and the spleens and lungs (without external bronchi and trachea) were removed and mechanically disrupted by passage through a wire mesh. Live leukocytes were obtained by density gradient centrifugation using Lympholyte-Poly as a cushion (Cedarlane, Hornby, Canada). Cells were incubated in 3% FCS in PBS containing 10 µg/ml 2.4G2 to block Fc receptor binding, followed by the addition of fluorochrome-conjugated Abs or MHC class 1 tetramers. All fluorochrome-conjugated Abs were obtained from BD PharMingen (San Diego, CA). The MHC class I tetramer H-2Db containing nucleoprotein (NP)366–374 peptide used to identify influenza-specific T cells was generated by the Trudeau Institute Molecular Biology Core Facility. Flow cytometry was performed on a dual laser FACSCalibur available through the Flow Cytometry Core Facility at Trudeau Institute.
Intracellular cytokine staining
Live leukocytes obtained from the spleens and lungs of infected
mice were cultured at 5 x 106/ml in 200
µl of complete RPMI containing 10 µg/ml brefeldin A and 10 U/ml
IL-2. Cells were stimulated with influenza
NP366–374 peptide or influenza
polymerase-2 (PA)224–233 peptide at 1 µg/ml or
with 5 ng/ml PMA and 650 ng/ml of A23187 for 5 h. Simulated cells
were then washed, blocked with 2.4G2, and probed with anti-CD8
before fixation in 4% paraformaldehyde in PBS. Fixed cells were washed
with 0.1% Triton X-100/3% FCS in PBS and probed with
fluorochrome-labeled anti-IFN- in the same buffer.
CTL assays
EL4 cells were cultured with no peptide, influenza NP366–374 peptide or influenza PA224–233 peptide at 1 µg/ml and were labeled overnight with 51Cr. After labeling, the EL4 targets were washed and cultured at 104 cells/well. Live leukocytes from the lungs of infected mice were added to the labeled EL4 cells at E:T cell ratios between 0.1:1 and 100:1 (in triplicate), and the cells were cultured for 5 h. Supernatants were then collected, and the released radioactivity was counted in a gamma counter. Spontaneous 51Cr release was determined from labeled EL4 cells in the absence of effectors, and maximum possible release was determined from EL4 cells cultured in 1% Triton X-100.
Serum collection and ELISAs
Blood was obtained from euthanized mice by severing the renal artery and pipetting into a 1.5-ml tube. After clotting for 2 h at 37°C, the precipitate was pelleted in a microcentrifuge, and the serum was removed. Influenza-specific ELISAs were performed by coating plates with purified virus at 1 µg/ml. Serum samples were diluted in 3-fold serial dilutions in PBS with 10 mg/ml BSA and 0.1% Tween 20 before incubation on coated plates. Bound Ig was detected with HRP-conjugated goat anti-mouse IgM, goat anti-mouse IgG, or goat anti-mouse IgA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- mice are more susceptible to influenza
infection
To determine whether the lack of lymph nodes and the disorganized
splenic structure in LT-/- mice conferred
increased susceptibility to influenza infection, we intranasally
inoculated groups of LT-/- and WT mice with
50, 240, 1200, or 6000 EIU of influenza A/PR8/34 (PR8) and monitored
the survival and morbidity (weight) of the infected mice over the next
30 days. As shown in Fig. 1
, both WT and
LT-/- mice succumbed to 6000 EIU of PR8 with
similar kinetics. However, there were small, but consistent,
differences in the survival of LT-/- and WT
mice at the intermediate doses of virus. For example, while all
LT-/- mice succumbed to infection with 1200
EIU, only three of five of the WT mice died at this dose of virus.
Furthermore, while two of five LT-/- mice
succumbed to 240 EIU, none of the WT mice died. All mice in both groups
survived 50 EIU of influenza. The survival of the
LT-/- mice infected with lower doses of
virus was not due to a nonproductive infection, as all mice in each
group exhibited signs of morbidity, including temporary weight loss
(Fig. 1), hunched posture, and ruffled fur. Together, these results
suggest that although the LD50 of PR8 for
LT-/- mice is 
5-fold lower than that for
WT mice, LT-/- mice do make an immune
response that is protective against lower doses of virus.
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-/- mice
To determine whether the increased susceptibility of
LT-/- mice to influenza was due to an
inability to mount a protective CD8 T cell response, we examined CD8 T
cell activation in the spleen and lungs of flu-infected WT and
LT-/- mice. Groups of WT and
LT-/- mice were infected with a dose of PR8
sufficient to induce infection, but not death, in either group of mice
(100 EIU). Five mice from each group were sacrificed at 0, 6, 9, 12,
15, and 21 days postinfection, and the cells from lung and spleen were
pooled. The accumulation of activated influenza
NP366–374 specific CD8 T cells was determined by
flow cytometry using an
H2-DbNP366–374 tetramer.
As shown in Fig. 2A,
influenza-specific CD8 T cells first appeared in the spleens of WT mice
on day 9, increased in frequency on day 12, and declined on days 15 and
21. The influenza-specific CD8 T cells appeared to have an activated
phenotype, as assessed by reduced expression of CD62L (Fig. 2). Unlike
what we observed in the spleens of flu-infected WT animals, activated
influenza-specific CD8 T cells did not appear in the spleens of
LT-/- mice until day 12. However, despite
the delayed appearance of these cells, the frequency of activated,
flu-specific CD8 T cells on days 12, 15, and 21 was increased in
LT-/- mice compared with that in WT mice.
When the total number of activated, Ag-specific cells in the spleen was
calculated, we found that a similar number of activated, flu-specific
CD8 T cells accumulated in the spleens of
LT-/- and WT mice, even though the
appearance of these cells was delayed by 3 days in
LT-/- mice (Fig. 2B).
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C). Again, the appearance of activated Ag-specific CD8 T
cells in the lungs of LT-/- mice was delayed
until day 12. However, unlike what we observed in the spleen, the
frequency of activated, influenza-specific CD8 T cells was lower in the
lungs of LT-/- mice than in the lungs of WT
mice at all time points (Fig. 2C). Despite the dramatic
reduction in the frequency of activated NP-specific CD8 T cells in the
lungs of LT-/- mice, however, the total
number of these cells was actually higher in the lungs
LT-/- mice than in WT mice at later time
points (Fig. 2D). This is due to the substantially higher
inflammatory response that occurs in the
LT-/- lung. Although we recovered similar
numbers of leukocytes from the lungs of uninfected WT and
LT-/- mice (an average of 1 x
106 cells/animal), the numbers of leukocytes
recovered from the lungs of influenza-infected WT mice peaked on day 9
(an average of 4 x 106 cells/animal), while
the numbers of leukocytes recovered from lungs of influenza-infected
LT-/- mice peaked between days 12 and 15 (an
average of 13 x 106 cells/animal).
Appearance of influenza-specific IFN--producing CD8 T cells is
delayed in LT-/- mice
To determine whether the tetramer-binding CD8 T cells observed in
LT-/- mice could make IFN- upon
restimulation, we isolated cells from the lungs and spleens of
influenza-infected WT and LT-/- mice at
various times after infection and cultured the cells for 5 h with
influenza NP366–374 peptide, influenza
PA224–233 peptide, or no peptide. The stimulated
cells were subsequently analyzed for IFN- production by
intracellular cytokine staining. IFN- was produced by CD8 T cells
from the lungs of both WT and LT-/- mice in
response to NP peptide (Fig. 3A), as well as PA peptide
(Fig. 3B), but not in the absence of peptide (Fig. 3C). While NP- and PA-specific IFN--producing CD8 T cells
appeared by day 9 in WT mice, their appearance was delayed in
LT-/- mice until day 12. Similar results
were observed in the spleen (not shown). When the total number of
Ag-specific IFN--producing CD8 T cells was determined, we found that
these cells were present at much lower levels in the lungs and spleens
of LT-/- mice compared with WT mice on day 9
postinfection. However, by days 15–21 the number of Ag-specific,
IFN--producing CD8 T cells in the lungs and spleens of
LT-/- mice exceeded that in WT mice (Fig. 3D).
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-/-
mice
To next determine whether the LT-/-
mice could generate effective CTLs, we isolated cells from the
bronchial alveolar lavage (BAL) and the lung tissue of
influenza-infected WT and LT-/- mice at
various times after infection and cultured the cells for 5 h with
51Cr-labeled EL4 target cells in the presence of
influenza NP366–374 peptide, influenza
PA224–233 peptide, or no peptide. As shown in
Fig. 4, robust CTL activity in response
to both NP and PA peptides was observed in WT cells on day 9. WT CTL
activity was reduced on day 12 (particularly to the PA peptide) and was
absent by day 15. No NP-specific CTL activity was observed in
LT-/- cells on day 9, but, by days 12–15,
NP-specific CTL activity could be observed in BAL and lung tissue
isolated from LT-/- mice. However, a higher
E:T cell ratio was needed to observe significant killing of the
targets. Furthermore, the CTL activity directed toward the PA peptide
in LT-/- mice was very weak and was observed
only on day 15. Together these data indicate that influenza-specific
CTLs can be generated in LT-/- mice.
However, the frequency of these cells is reduced due to the
increased lung inflammatory response in the infected
LT-/- mice.
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-/- mice, but are present in the lungs after
influenza infection
Although CTL activity is thought to be the principal form of
immunity responsible for resistance to influenza in a primary response
(9), the production of high affinity isotype switched Abs
is thought to provide the major source of protection against secondary
infection with homotypic virus (10). Since high affinity
isotype-switched B cells are generated in germinal centers
(37), we first examined whether the production of germinal
center B cells was altered in virus-infected
LT-/- mice. To determine the frequency of
germinal center B cells at various times after infection we isolated
cells from the lungs and spleens of influenza-infected WT and
LT-/- mice and analyzed the expression of
Fas and peanut agglutinin (PNA) on CD19+ B cells
by flow cytometry. As expected, we found that the frequency (Fig. 5A) and total number (Fig. 5B) of
FashighPNAhighCD19+
germinal center B cells increased rapidly in the spleens of
influenza-infected WT mice. Additionally, in agreement with numerous
reports demonstrating the inability of LT-/-
mice to make germinal centers in the spleen, we observed that the
frequency (Fig. 5A) and total number (Fig. 5B) of
germinal center B cells remained near background levels in the spleens
of influenza-infected LT-/- mice.
Surprisingly, however, we observed B cells with a germinal center
phenotype in the lungs of both WT and LT-/-
mice after influenza infection (Fig. 5C). The frequency of
germinal center B cells in the lung was slightly higher in
LT-/- mice than in WT mice, particularly on
days 12 and 15 postinfection (Fig. 5C), and the absolute
number of these cells was much higher in
LT-/- mice at these times (Fig. 5D). Although we did observe a transient population of
CD19+PNA+Fasint
cells on day 6 in both WT and LT-/- mice
(Fig. 5C, circled population), these cells did not have the
classic phenotype of germinal center cells (T. D. Randall,
unpublished observations); thus, we did not include them in our
calculation of the absolute number of germinal center B cells on day 6
(Fig. 5D).
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The appearance of B cells with a germinal center phenotype in the
lungs of influenza-infected LT-/- mice
suggested that the production of isotype-switched B cells and Abs may
also be intact in these mice. To test this hypothesis, cells were
isolated from the lungs of WT and LT-/- mice
21 days postinfection, and the frequency of
CD19+syndecan+ plasma cells
that expressed IgM, IgG1, IgG2a/b, IgG3, or IgA was determined. As
shown in Fig. 6A, similar
frequencies of IgM+, IgG1+,
IgG2a/b+, and IgA+ plasma
cells were found in WT lungs. In contrast, in the lungs of
LT-/- mice the frequencies of
IgM+ and IgA+ plasma cells
were increased relative to the frequencies of the IgG isotypes. These
differences were maintained when the absolute numbers of IgM- and
IgA-expressing plasma cells present in the lungs of WT and
LT-/- mice were calculated (Fig. 6B). Finally, the frequencies of
IgG1+-, IgG2a/b+-, and
IgG3+-expressing B cells that were not plasma
cells (isotype switched and syndecan-) were
higher in WT mice than in LT-/- mice.
However, the frequency of isotype-switched
IgA+syndecan- cells was
similar in WT and LT-/- mice (Fig. 6A).
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-/- mice were
differentiating into class-switched, Ab-secreting cells, this analysis
did not distinguish between Ag-specific and nonspecific B cells.
Therefore, to confirm that influenza-specific, isotype-switched Ab was
being produced, we performed ELISAs to determine the relative
concentration of influenza-specific Ab in the serum of animals infected
for various times. As shown in Fig. 6C, we found that the
concentration of influenza-specific IgM rapidly increased in WT mice,
peaked between days 9 and 12, and then gradually declined on days 15
and 21. Influenza-specific IgM appeared with slightly delayed kinetics
in the serum of LT-/- mice, but peaked
between days 12 and 21 at a level higher than that observed in WT mice.
Although influenza-specific IgG was barely detectable in WT mice on day
6, it rapidly climbed on days 9 and 12 and continued to increase on
days 15 and 21 (Fig. 6C). Influenza-specific IgG was not
detected until day 9 in LT-/- mice; however,
by day 15 the titer of flu-specific IgG climbed >1000-fold. Despite
the large burst of influenza-specific IgG produced by the
LT-/- mice, the titer of this Ab remained
10-fold less than that observed in WT mice. Finally, although the
number of IgA-producing plasma cells was increased in the lung tissue
of LT-/- mice compared with WT mice (Fig. 6B), the titers of influenza-specific IgA were similar in
the serum of these animals (Fig. 6C).
Viral clearance is delayed in LT-/- mice
The appearance of CTLs and influenza-specific Abs in
LT-/- mice coupled with the survival of
these mice at low doses of virus suggested that adaptive immune
mechanisms could clear virus in these mice. To test this directly, we
assayed viral burden in the lungs of influenza-infected WT and
LT-/- mice. As shown in Fig. 7, the viral burden in WT and
LT-/- was similarly high on day 6, a time
when there was little evidence of adaptive immune responses in either
strain of mice. While the viral burden rapidly declined on days 8 and
10 in WT mice, the viral burden remained high on these days in
LT-/- mice. Finally, however, virus was
cleared in both WT and LT-/- mice by days
15–20.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- mice
The data presented here clearly demonstrate that
LT-/- mice can generate Ag-specific CD8 T
cells in response to viral infection. Furthermore, the
influenza-specific T cells from LT-/- mice
expanded (Fig. 2) and differentiated into competent effector cells that
could make IFN- upon restimulation (Fig. 3), could kill
peptide-loaded target cells (Fig. 4), and could clear virus in vivo
(Fig. 7). In fact, the only major difference between the CD8 T cell
response of the LT-/- mice and WT mice was
that there was a 2- to 3-day delay in the appearance of Ag-specific CD8
T cells in LT-/- mice (
Figs. 2–4). These
results are in apparent contrast with other studies (31, 32, 33, 34, 36) in which mice deficient in the LT signaling pathway were
unable to make normal CTL T cell responses to viruses such as LCMV,
HSV, and vesicular stomatitis virus. However, the conclusions from some
of these earlier studies were based on data from early time points
after virus infection (6–8 days); thus, it is likely that CTL
responses were not observed in LT-/-
(33, 34), aly/aly (31, 32), or
TNF-/-LT-/-
(36) mice because CTLs had not yet developed at the time
points that were analyzed. We would have arrived at similar conclusions
had we only looked for influenza-specific CTL responses on days
9–10.
The delayed accumulation of virus-specific CD8 T cells is not the only
reason that reduced CTL activity was observed in previous studies using
LT signaling-deficient mice. Since CTL activity is a function of CTL
frequency, rather than total CTL number, CTL activity can be reduced
(as we observed in Fig. 4, day 12) even though the total number of
virus-specific cells may actually be similar between WT and
LT-/- mice (as we observed in Fig. 2, day
12). This difference between frequency and total number of Ag-specific
CD8 T cells is due to the more severe inflammatory response that occurs
in influenza-infected LT-/- mice. This
increased inflammatory response is most likely a result of prolonged
virus production in the LT-/- mice (see
Fig. 7).
Other studies also reported that IFN- production by virus-specific
CD8 T cells is reduced in mice defective in LT signaling (34, 35, 38). Again, this could be an issue of timing. We found that
IFN- production by virus-specific CD8 T cells was delayed in
LT-/- mice (Fig. 3), but this delay
corresponded with the delayed appearance of virus-specific CD8 T cells
(Fig. 2). Furthermore, although one study demonstrated that similar
numbers of Ag-specific CD8 T cells could be generated in WT and
LT-/- mice after infection or immunization,
those cells exhibited reduced killing activity and were unable to
produce IFN- (34). Unlike our studies, however, in
which we examined T cell function immediately ex vivo, that study
expanded T cells in vitro for 5 days before assay. Thus, it is unclear
whether the observed defects in the in vitro cultured
LT-/- CD8 T cells are representative of how
these cells behave in vivo.
Although we feel that timing is one critical difference between our
results and those of others, the type of infection or its location
within the body may also significantly impact how
LT-/- mice respond to the infection. For
example, it is reported that LT-/- mice make
poor CD8 T cells responses to LCMV (31, 32, 36, 38), a
systemic virus, and HSV (34), a neurotropic virus, while
we found that LT-/- mice could generate
CD8-mediated immunity to influenza, a respiratory virus. Interestingly,
our data are most consistent with the reported resistance of
LT-/- mice to another respiratory virus,
MHV-68 (35). In that study CTL activity was intact,
IFN- was produced (although at lower levels), and virus was cleared
with delayed kinetics from the lungs of MHV-68-infected
LT-/- mice. This raises the possibility that
lymphotoxin and/or lymph nodes may not be absolutely required to
generate immunity to respiratory viruses, but may be necessary to
generate CD8 T cell responses to viruses with other tropisms.
In previous studies we found that nasal-associated lymphoid tissue
(NALT) is present in LT-/- mice
(39), even though these mice are completely devoid of
lymph nodes and Peyer’s patches. These studies also showed that the
presence of NALT in LT-/- mice is not
sufficient to generate Ag-specific T or B cells or to clear influenza
virus from the lungs by day 10 (39). This is entirely
consistent with our current results, which show that the generation of
influenza-specific T cells (
Figs. 2–4), the production of
influenza-specific IgG (Fig. 6), and the clearance of virus (Fig. 7)
are all severely impaired before day 10 in
LT-/- mice. However, our current results
also demonstrate that immune responses are eventually generated in
LT-/- mice by day 12. These results suggest
that the spleen and/or NALT of LT-/- mice
contribute to immune responses even though the lymphoid areas of these
tissues are not organized in a normal fashion. However, we cannot
distinguish the relative importance of the spleen or NALT in the
generation of immunity to influenza in LT-/-
mice at this time.
Formation of germinal centers and isotype-switched Ab responses in
LT-/- mice
Initial experiments with LT-/- mice
immunized with experimental Ags such as SRBC or haptenated proteins
demonstrated that the formation of germinal centers and the production
of isotype-switched Abs to T-dependent Ags were severely impaired in
these mice (22, 27, 28, 40). The defective class-switched
Ab response was attributed primarily to the inability of
LT-/- mice to organize either primary or
secondary lymphoid follicles in the spleen, rather than to the lack of
lymph nodes, since the transfer of WT cells to
LT-/- mice enabled the mice to form germinal
centers and produce isotype-switched Abs (26, 40, 41).
Interestingly, there are a few reports that
LT-/- B cells are able to isotype-switch in
the apparent absence of germinal center formation, particularly in
response to viral infections (31, 35). In agreement with
these reports we found that Ab production in response to influenza
infection is not impaired per se, as the total numbers of Ab-secreting
cells in the lungs of LT-/- mice is actually
greater than that in WT mice (Fig. 6). However, while the production of
IgM and IgA was increased in LT-/- mice
(Fig. 6, B and C), the production of IgG was
decreased (Fig. 6C), suggesting that switching to some, but
not all, isotypes was impaired. This might be due to the loss of LT
signaling in B cells or to the lack of FDCs and an inability to form
germinal centers in the spleen.
As previously reported we found no evidence for germinal center
formation in the spleens of LT-/- mice (Fig. 5A), suggesting that isotype switching in response to virus
infection must have occurred independently of splenic germinal centers.
Interestingly, we observed B cells with a germinal center phenotype in
the lungs of LT-/- as well as WT mice (Fig. 5C). Although the lung is not a site in which germinal
centers are typically believed to form, there are reports that
organized lymphoid tissue and even germinal centers can be found in the
lung after immunization (42). Unlike bronchus-associated
lymphoid tissue, which is constitutively present in some species, these
organized lymphoid areas are not found in naive mice and must be
induced by infection or inflammation (43, 44, 45). Once
formed, however, they may serve as sites that can initiate primary
immune responses (46, 47). If germinal centers are formed
in the lungs of LT-/- mice after infection,
this would suggest that the mechanism controlling their formation in
the lung is LT independent and must be fundamentally different from the
LT-dependent mechanism that controls germinal center formation in
lymphoid tissues such as the spleen. These questions are currently
being addressed.
Why are immune responses to influenza delayed in
LT-/- mice?
Since LT-/- T cells are not thought to
have intrinsic defects in activation (22, 38), the
observed delay in CD8 T cell responses in
LT-/- mice is most likely due to a defect in
Ag presentation. This could be manifest at the level of DC activation,
DC migration from the lung to the spleen, or the ability of T cells to
find APCs. Any or all of these mechanisms could be operating in
LT-/- mice. For example, LT can induce DC
maturation and activation. Furthermore, the ability of T cells to
interact with DCs is impaired in LT-/- mice
at several levels. First, there are no lymph nodes in
LT-/- mice (22, 28), the
traditional site of T cell priming. Second, the expression of the
chemokines B lymphocyte chemoattractant, secondary lymphoid tissue
chemokine, and EBV-induced molecule-1 ligand chemokine is dependent on
LT (30); thus, the ability of lymphocytes and DCs to
home from peripheral tissues to secondary lymphoid organs is disrupted.
This is dramatically demonstrated by the paucity of DCs in the spleens
of LT-/- mice (29), even though
the number of DCs in the lungs of uninfected
LT-/- mice is normal (T. D. Randall,
unpublished observations) or elevated (29). Together,
these defects could easily explain the observed delay in T cell
activation observed in the LT-/-
mice.
In summary, we show that LT-/- mice are
capable of mediating all aspects of a primary cellular and humoral
anti-influenza response. Although the generation of
influenza-specific CD8 T cells was delayed, once formed, those cells
could kill Ag-loaded target cells, produce IFN-, and clear virus in
vivo. Furthermore, LT-/- mice produced
increased levels of influenza-specific IgM, normal levels of IgA, and
only slightly decreased levels of IgG, demonstrating that B cell
activity was largely intact. Together, these results suggest that the
presence of lymph nodes and an organized spleen are not absolutely
required for the generation of immunity, but that they facilitate the
rapid induction of immune responses.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Troy Randall, Trudeau Institute, P.O. Box 59, 100 Algonquin Avenue, Saranac Lake, NY 12983. E-mail address: trandall{at}trudeauinstitute.org
3 Abbreviations used in this paper: LT, lymphotoxin-; BAL, bronchial alveolar lavage; DC, dendritic cell; EIU, egg infectious unit; FDC, follicular dendritic cell; LCMV, lymphocytic choriomeningitis virus; MHV-68, murine gammaherpes virus 68; NALT, nasal-associated lymphoid tissue; NP, nucleoprotein; PA, influenza polymerase-2; PNA, peanut agglutinin; PR8, influenza A/PR8/34; TNFR1, TNFR type 1; WT, wild type.
Received for publication May 16, 2002. Accepted for publication September 3, 2002.
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)
and LT
) were intranasally infected with
6000, 1200, 240, and 50 EIU of influenza A/PR8/34 in 100 µl. Survival
and weight were monitored daily over 30 days following infection. The
weight shown is the average weight of all mice within a group at each
time point. The data shown are representative of three independent
experiments. The differences in survival between WT and
LT
) and LT
) in the lungs
and spleens of WT (
), LT
