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B Cells Promote Resistance to Heterosubtypic Strains of
Influenza via Multiple Mechanisms
1
Javier Rangel-Moreno, Damian M. Carragher, Ravi S. Misra, Kim Kusser, Louise Hartson,
Amy Moquin, Frances E. Lund, and Troy D. Randall
2
Immunity to heterosubtypic strains of influenza is thought to be mediated primarily by memory T cells, which recognize
epitopes in conserved proteins. However, the involvement of B cells in this process is controversial. We show in this study
that influenza-specific memory T cells are insufficient to protect mice against a lethal challenge with a virulent strain of
influenza in the absence of B cells. B cells contribute to protection in multiple ways. First, although non-neutralizing Abs by
themselves do not provide any protection to challenge infection, they do reduce weight loss, lower viral titers, and promote
recovery of mice challenged with a virulent heterosubtypic virus in the presence of memory T cells. Non-neutralizing Abs
also facilitate the expansion of responding memory CD8 T cells. Furthermore, in cooperation with memory T cells, naive B
cells also promote recovery from infection with a virulent heterosubtypic virus by generating new neutralizing Abs. These
data demonstrate that B cells use multiple mechanisms to promote resistance to heterosubtypic strains of influenza and
suggest that vaccines that elicit both memory T cells and Abs to conserved epitopes of influenza may be an effective defense
against a wide range of influenza serotypes. The Journal of Immunology, 2008, 180: 454 – 463.
P
revious infection with influenza elicits immunity to sub-
sequent challenge infection with a normally lethal dose
of virulent virus. Immunity to the same serotype of vi-
rus (homotypic immunity) is primarily mediated by Abs to hem-
agglutinin (HA)
3
and neuraminidase (NA) that neutralize and
completely prevent infection by the challenge virus (1). In con-
trast, immunity to serotypically distinct strains of virus (het-
erosubtypic immunity) is thought to be mediated primarily by T
cells that recognize epitopes in conserved influenza proteins,
such as nucleoprotein (NP) (2, 3). Heterosubtypic immune re-
sponses cannot prevent infection, but do lower viral titers, ac-
celerate viral clearance, and reduce morbidity and mortality.
Thus, although vaccines that elicit heterosubtypic immunity
will not prevent yearly influenza epidemics, they should reduce
the morbidity and mortality associated with pandemic viruses
that express new HA and NA surface proteins.
Heterosubtypic immunity is easily demonstrated in laboratory
animals, including mice (4 – 6), pigs (7, 8), and cotton rats (9),
using pairs of viruses that express different subtypes of HA and
NA surface proteins. However, heterosubtypic immunity to influ-
enza is more difficult to demonstrate in humans, and there is some
controversy whether it exists at all (3). For example, children with
pre-existing immunity to H1N1 do not demonstrate any resistance
to infection with a live attenuated H3N2 influenza vaccine or ex-
hibit reduced viral shedding (10). In contrast, epidemiological data
suggests that there is cross-protection between strains of influenza
that are circulating at the same time (11, 12). Similarly, data from
the Cleveland Family Study show that adults recently recovered
from H1N1 infection were much less susceptible to infection with
H2N2 virus than children in the same households who were not
previously exposed to H1N1 (13). Although the interpretation
of these data is still being debated, multiple lines of evidence
demonstrate that cross-reactive T cells and even Abs are im-
portant for resistance to heterosubtypic strains of influenza (14 –
17). In part, the difference between humans and laboratory an-
imals in the effectiveness of heterosubtypic immunity may be
due to the short-lived nature of heterosubtypic effector mecha-
nisms (15) and to the fact that experiments in animals are per-
formed on timescales of months, whereas studies in humans test
resistance over several seasons. Thus, by gaining an under-
standing of the mechanisms by which heterosubtypic immunity
works, we should be able to develop vaccines that boost this
type of immunity in humans.
Although the mechanisms of heterosubtypic immunity have
been studied by numerous groups, a consensus view has not
emerged. Depletion studies showed that CD4 and CD8 T cells
play important roles in heterosubtypic immunity but that this
type of immunity is relatively short lived (15), possibly due to
the loss of effector memory cells in the lung airways (18). In
fact, most groups studying heterosubtypic immunity report CTL
responses that cross-react with both the priming and challenge
viruses (14, 19 –22). Moreover, immunization with conserved
proteins, like NP or matrix-2 (M2), leads to demonstrable het-
erosubtypic immunity (17, 21, 23, 24), as does immunization
with peptide Ags that elicit influenza-specific memory CD8 T
cells (22, 25–27). However, CD8 T cells appear to be dispens-
able for heterosubtypic immunity in some studies (16, 28), pos-
sibly because CD4, CD8, NK T, and
␥␦
T cells play partially
Trudeau Institute, Saranac Lake, NY 12983
Received for publication July 23, 2007. Accepted for publication October
26, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Trudeau Institute and National Institutes of Health
Grants AI072689, AI61511, and HL69409 (to T.D.R.), and AI68056 and AI50844 (to
F.E.L.). R.S.M. was supported by National Institutes of Health Training Grant
AI49823.
2
Address correspondence and reprint requests to Dr. Troy Randall, Trudeau In
-
stitute, 154 Algonquin Avenue, Saranac Lake, NY 12983. E-mail address: trandall@
trudeauinstitute.org
3
Abbreviations used in this paper: HA, hemagglutinin; NA, neuraminidase; NP, nu
-
cleoprotein; M, matrix;
MT, B6.129S2-Igh-6
tm1Cgn
/J; PA, acidic polymerase; PB,
basic polymerase; M2e, external domain of M2; EIU, egg infectious units.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
www.jimmunol.org
redundant roles in heterosubtypic immunity, and elimination of
any one of these populations does not substantially impair the
response as a whole (4).
Despite the well-established idea that heterosubtypic immu-
nity to influenza is mediated by cross-reactive T cells, isolated
reports suggest that B cells are also important for resistance to
heterosubtypic strains of influenza and may be more important
than CD8 T cells (16, 28). However, it is not clear how B cells
might mediate their protective effects. B cells could facilitate
heterosubtypic immunity by generating non-neutralizing Abs
that cross-react with conserved epitopes on the surface of the
challenge virus, despite extensive differences between the se-
quences of various HA and NA subtypes (29, 30). Although
these Abs would not be neutralizing, they might bind and help
eliminate free virions and infected cells. Alternatively, B cells
could generate high titers of Abs to highly conserved internal
proteins. Although these Abs would not bind to free virions or
infected cells, they might facilitate viral clearance through un-
known mechanisms. A final B cell-dependent mechanism of
heterosubtypic immunity is that memory T cells can help naive
B cells to differentiate and produce new Abs that help clear the
challenge infection (31). In this manuscript, we show that B
cells use a combination of these mechanisms to promote het-
erosubtypic immunity to influenza.
Materials and Methods
Mice
C57BL/6J and B6.129S2-Igh-6
tm1Cgn
/J (
MT) mice were obtained from
The Jackson Laboratory. All mice were on the C57BL/6J background and
were maintained in the animal facility at Trudeau Institute. Survival studies
were performed as time to morbidity (greater than 30% weight loss, poor
response to external stimuli, or inability to eat or drink) rather than time to
death. All procedures involving live animals were approved by the Trudeau
Institute Institutional Animal Care and Use Committees and were per-
formed in accordance with guidelines set by the National Research
Council.
Influenza infection and quantification
Primary infections were with 300 egg infectious units (EIUs) of influenza
A/X31 administered intranasally in 100
l. Secondary infections were with
1000 EIU influenza A/PR8/34 administered intranasally in 100
l. Viral
titers in the lungs of infected mice were quantified in embryonated eggs.
Briefly, lungs were homogenized in 2 ml of PBS and 500
l of this stock
was used to make 10-fold serial dilutions. One hundred
l of each dilution
was inoculated into chicken eggs. Allantoic fluid was harvested from in-
oculated eggs 4 days later, and infected eggs were scored by hemaggluti-
nation of chicken RBC. The viral-endpoint titer was defined as the highest
dilution in which two or more eggs (of three) positively scored in the
hemagglutination assay. Alternatively, virus was quantified using a viral
foci assay. Briefly, Madin Darby Canine Kidney cells were grown in 96-
well, flat-bottom plates until just confluent and then washed with HBSS.
Homogenized tissue samples were diluted in Zero Serum Media (Diagnos-
tic Hybrids) supplemented with 4
g/ml trypsin and applied to washed
Madin Darby Canine Kidney cells. Plates were centrifuged for 1.5 h at
800 ⫻ g, washed, and cultured overnight in Zero Serum Media/trypsin at
33°C. The next day, the medium was removed, and the cells were fixed
with 80% acetone and allowed to dry. The wells were rehydrated with PBS,
containing 2% FBS and 0.01% NaN
3
, and probed with mouse anti-
influenza A (Chemicon International). The primary Ab was detected with
biotinylated goat anti-mouse IgG (Chemicon International) followed by
alkaline phosphatase-conjugated streptavidin (DakoCytomation). Viral
foci were developed by incubating for 30 min with 5-bromo-4-chloro-3-
indolyl phosphate and Nitro Blue Tetrazolium tablets (Sigma Fast BCIP/
NBT from Sigma-Aldrich) dissolved in H
2
O. The resulting spots were
counted under a dissecting microscope.
Flow cytometry
Mice were sacrificed at the indicated times after infection, and tissues were
removed and mechanically disrupted by passage through a wire mesh. Live
leukocytes were obtained by density-gradient centrifugation using Lym-
pholyte-Poly (Cedarlane Laboratories) as a cushion. Cells were incubated
in 3% FBS in PBS containing 10
g/ml 2.4G2 to block Fc receptor bind-
ing, followed by the addition of fluorochrome-conjugated Abs or MHC
class I tetramers. All fluorochrome-conjugated Abs were obtained from BD
Biosciences. The MHC class I (H-2D
b
) tetramers containing NP
366 –374
or
acidic polymerase (PA)
224 –233
peptides used to identify influenza-specific
T cells were generated by the Trudeau Institute Molecular Biology Core
Facility. Flow cytometry was performed on a dual laser FACSCalibur (BD
Biosciences) available through the Flow Cytometry Core Facility at the
Trudeau Institute.
T cell enumeration and statistics
Influenza-specific CD8 T cells were enumerated in the spleens of infected
mice by first counting total live lymphocytes in the spleen using a hemo-
cytometer and multiplying that number by the frequency of propidium
iodide
⫺
CD8
⫹
tetramer
⫹
cells observed using flow cytometry. Flow cytom
-
etry data were first gated on live lymphocytes and then gated on CD8
⫹
cells. Tetramer
⫹
cells within this population are shown in Figs. 2–5. Sta
-
tistical differences between the numbers of influenza-specific CD8 T cell in
each group were calculated using an unpaired t test in the Prism graphics
and analysis program.
B cell purification
Single-cell suspensions from naive C57BL/6 mice were incubated on ice
with 2.4G2 at 10
g/ml for 10 min and then with 25
l anti-B220 MACS
beads per spleen equivalent for an additional 15 min on ice. After washing,
the cells were applied to a MACS CS column. Bound cells were collected,
washed, and injected into recipient mice. The purity of the B cell prepa-
rations was consistently above 95%.
Protein expression and purification
A cDNA encoding three tandem copies of the first 23 amino acids of
influenza M2 protein linked to the full-length influenza NP, and a C-ter-
minal 6Xhis tag was synthesized by GeneArt and subcloned into
pTricHis2C (Invitrogen Life Technologies). The resulting fusion protein
was expressed in Top10F’ Escherichia coli (Invitrogen Life Technologies).
Briefly, bacteria were grown to log phase and protein expression, induced
by adding isopropyl-

-D-thiogalactopyranoside to a final concentration of
1 mM. The cells were pelleted and resuspended in 25 ml of 50 mM
NaH
2
PO
4
, 300 mM NaCl, 10 mM Imidazole (pH 8.0) with 1 mg/ml ly
-
sozyme, 1
g/ml Pepstatin A, 5
g/ml Aprotinin, 1 mM PMSF, and 5
g/ml Leupeptin. Lysates were rocked at 4°C for 30 min and then soni-
cated on ice. DNase I (Invitrogen Life Technologies) was added to a final
concentration of 5
g/ml, and the lysates were rocked an additional hour
at 4°C. Lysates were clarified by centrifugation at 10,000 ⫻ g for 30 min.
The recombinant M2eNP-fusion protein was purified using the ProBond
Purification system from Invitrogen Life Technologies. Briefly, recombi-
nant proteins were dialyzed against PBS and sterile filtered before use.
Mice were immunized and boosted 10 days later with 20
g recombinant
protein in combination with 30
g LPS and 50
g anti-CD40 (10C8) as
adjuvants. Serum was obtained 28 days after initial immunization.
Purification of viral proteins
Viral proteins were purified using a protocol modified from Johansson et al.
(32). Virus was pelleted from infected allantoic fluid by centrifugation at
100,000 g for 1 h. Pelleted virus was resuspended in PBS and layered on
top of a 60–30% sucrose gradient. The gradient was centrifuged at 25,000
rpm in a SW50.1 rotor for 100 min. The band-containing virus was col-
lected, and virus was pelleted at 100,000 g for 4 h. The pellet was solu-
bilized in 15% n-octyl

-D glucopyranoside in 50 mM NaAcetate, 2 mM
CaCl
2
, and 0.2 mM EDTA (pH 7.0) and dialyzed against 50 mM NaAcetate,
2 mM CaCl
2
, and 0.2 mM EDTA (pH 7.0). The resulting protein was quan
-
tified and used to coat plates for ELISA.
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 30 min at 37°C, the precip-
itate was pelleted in a microcentrifuge, and the serum was removed. In-
fluenza-specific ELISAs were performed by coating plates with purified
viral proteins at 1
g/ml or with 2
g/ml M2
1–23
peptide (New England
Peptide). 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 Ab was detected with HRP-conjugated goat anti-mouse IgM or goat
anti-mouse IgG (Southern Biotechnology Associates).
455The Journal of Immunology
Results
B cells are required for optimal resistance to heterotypic
strains of influenza
It is commonly believed that memory T cells that recognize
epitopes in conserved internal proteins of influenza are responsible
for resistance to heterosubtypic strains of influenza (5, 6, 14).
However, the role of B cells in resistance to heterosubtypic strains
of influenza is unclear. To test the role of B cells in heterosubtypic
immunity to influenza, we used a pair of influenza viruses, A/X-31
(X31) and A/PR8/34 (PR8), that express different HA and NA
subtypes. Since X31 expresses the H3N2 subtypes of the HA and
NA coat proteins and PR8 expresses the H1N1 subtypes of HA and
NA, Abs generated to X31 do not neutralize PR8 (33). However,
the internal proteins of both viruses are the same and contain
epitopes that stimulate immunodominant T cell responses to both
viruses (33, 34). These cross-reactive T cells are thought to me-
diate the effects of heterosubtypic immunity (6, 14).
To test whether B cells were required for successful resistance
to challenge with a heterosubtypic strain of influenza, we infected
C57BL/6 and
MT mice with X31, allowed them to recover for 4
wk, and then challenged the immune mice as well as naive controls
with a high dose (5000 EIU) of PR8 (Fig. 1A). We found that
although X31-immune C57BL/6 mice all survived challenge in-
fection, the X31-immune
MT mice all succumbed to infection by
day 10 (Fig. 1B). In fact, the X31-immune
MT mice were no
more resistant to challenge with PR8 than naive
MT mice (Fig.
1B). We also observed that X31-immune C57BL/6 mice lost al-
most no weight after the challenge infection, whereas naive
C57BL/6, naive
MT, and X31-immune
MT mice all rapidly
lost weight over the first 10 days after infection, and only a few
naive C57BL/6 mice recovered (Fig. 1C). Consistent with the se-
verity of their illness, naive C57BL/6, naive
MT, and X31-im-
mune
MT mice had high viral titers in their lungs on day 6 after
challenge, whereas most of the X31-immune C57BL/6 mice had
very low titers of virus in their lungs at this point (Fig. 1D). To-
gether these data demonstrate that B cells are essential for reduced
morbidity and increased survival after challenge with a high dose
of a heterosubtypic strain of influenza.
To test whether B cells were also required for resistance to a low
dose of a heterosubtypic strain of influenza, we performed the
same experiment described above but challenged with a low dose of
virus (1000 EIU) of PR8. In this case, we found that only the naive
MT mice succumbed to infection (Fig. 2A). Moreover, the X31-
immune C57BL/6 mice lost very little weight, whereas the X31-
immune
MT and naive C57BL/6 mice lost weight through day 10
and then recovered (Fig. 2B). The viral titers in the lungs corre-
lated with the severity of weight loss, with high viral titers in naive
C57BL/6 and naive
MT mice and slightly lower titers in X31-
immune
MT mice (Fig. 2C). In contrast, X31-immune C57BL/6
mice had mostly cleared the virus from their lungs by day 6 after
infection (Fig. 2C). As expected, memory CD8 T cells responding
to influenza NP and PA were present in the spleens of X31-im-
mune mice on day 6 after infection but were not present at detect-
able frequencies in the spleens of naive mice at this time (Fig. 2D).
Interestingly, the number of influenza-specific CD8 T cells was
higher in X31-immune C57BL/6 mice than in X31-immune
MT
mice (Fig. 2E). However, it is difficult to compare these groups, as
the spleens in C57BL/6 mice are larger and have a higher total
cellularity than those in
MT mice (35). Together, these data dem-
onstrate that although B cell deficient memory mice can control
challenge infection with a low dose of heterosubtypic influenza
virus, morbidity is significantly higher, viral clearance is dramat-
ically impaired, and, despite the presence of memory CD8 T cells,
the numbers of memory CD8 cells are reduced.
Naive B cells cooperate with memory T cells to reduce
morbidity and accelerate recovery upon heterosubtypic
challenge infection
One somewhat trivial explanation for the importance of B cells in
resistance to heterosubtypic strains of influenza is that naive B
cells could be required to generate new strain-specific Abs that
eventually help to neutralize the challenge virus. In the absence of
these B cells and the Abs that they produce, the memory T cells
would be unable to clear the infection on their own. To test this
possibility, we infected C57BL/6 and
MT mice with X31 and
allowed them to recover for 4 wk. We then adoptively transferred
5 ⫻ 10
7
naive splenic B cells to groups of naive and X31-immune
C57BL/6 and
MT mice. A control group of naive
MT mice did
not receive B cells. All groups were then challenged with a low
dose of PR8 (Fig. 3A). As expected, the naive
MT mice that did
not receive B cells succumbed within 12–13 days after challenge
(Fig. 3B). However, all groups that received B cells survived (Fig.
3B). The X31-immune C57BL/6 mice that received B cells before
challenge did not exhibit weight loss, whereas the X31-immune
MT mice that received B cells lost some weight through day 7
and then recovered (Fig. 3C). In contrast, the naive C57BL/6 and
MT mice that received B cells exhibited substantial and equiv-
alent weight loss through day 10 before recovery (Fig. 3C).
FIGURE 1. Failure of heterosubtypic protection in B cell deficient mice. A, C57BL/6 and
MT mice were infected, or not, with 300 EIU of X31, allowed
to recover for 4 wk, and then challenged with 5000 EIU of PR8. B, Survival was monitored for 3 wk after challenge infection. C, Weight loss was measured
for 3 wk following challenge infection. D, Viral titers were measured in the lungs 6 days after challenge infection. There were 4–5 mice per group in each
panel. This experiment was performed twice with similar results.
456 B CELLS AND RESISTANCE TO HETEROSUBTYPIC INFLUENZA
The viral titers reflected the morbidity in each group, with high
viral titers in all naive mice, slightly reduced titers in X31-immune
MT, and very low titers in X31-immune C57BL/6 mice on day
6 after challenge (Fig. 3D). As expected, memory CD8 T cells
were observed in the spleens of X31-immune C57BL/6 and
MT
mice, but not in naive mice at day 6 after infection (Fig. 3E).
Again, the numbers of memory CD8 T cells were higher in
C57BL/6 mice than in
MT mice (Fig. 3F). We also found that
while X31-immune B6 mice had very high levels of X31-specific
IgG in their serum on day 6 after challenge infection (Fig. 3G), and
they had very low titers of PR8-specific IgG (Fig. 3H). In contrast,
neither X31-specific nor PR8-specific IgG were detected in any
other group on day 6 after infection (Fig. 3, G and H). However,
all groups had detectable titers of PR8-specific IgG by day 21 after
infection (Fig. 3H). Interestingly, the previously naive B6 mice
made very high levels of PR8-specific IgG, while X31-immune B6
mice made very low levels of PR8-specific IgG, possibly due to the
limited duration of Ag exposure in this group (Fig. 3D). Surpris-
ingly, the titers of PR8-specific IgG on day 21 in X31-immune
MT mice were similar to those in previously naive
MT mice
(both of which received B cells before challenge infection) (Fig.
3H), suggesting that memory CD4 cells in X31-immune
MT
mice do not accelerate the differentiation of naive B cells. These
results demonstrate that providing naive B cells to X31-immune
MT mice helps to minimize weight loss and accelerate recovery.
However, the presence of naive B cells in X31-immune
MT mice
is not sufficient to reduce viral titers or accelerate T cell recall
responses. Thus, whereas the presence of naive B cells helps
recovery from infection, presumably by generating new Abs,
additional B cell-dependent mechanisms must be involved in
the reduction of viral titers.
Non-neutralizing Abs facilitate viral clearance and promote
memory CD8 T cell expansion after heterosubtypic challenge
infection
To test whether Abs elicited by X31 infection provided some pro-
tection to challenge with PR8, we adoptively transferred nonim-
mune serum or X31-immune serum to either naive or X31-immune
MT mice and then challenged all groups with a low dose of PR8
(Fig. 4A). We found that naive mice all succumbed to infection,
regardless of whether they received naive or immune serum (Fig.
4B). In contrast, most X31-immune
MT mice given naive serum
and all
MT mice given X31-immune serum survived (Fig. 4B).
Despite the ability of both groups of X31-immune
MT mice to
recover from challenge infection, the group that received X31-
immune serum lost much less weight and recovered much more
quickly than those that received naive serum (Fig. 4C). Moreover,
X31-immune
MT mice that received X31-immune serum had
very reduced viral titers in their lungs compared with all other
groups on day 6 after challenge (Fig. 4D). As expected, we ob-
served memory CD8 T cells in X31-immune
MT mice on day 6
after challenge, but not in naive
MT mice (Fig. 4F). However,
the numbers of responding memory CD8 T cells were higher in
MT mice that received X31-immune serum than in
MT mice
that received naive serum. These data demonstrate that Abs gen-
erated to X31 (H3N2) contribute substantially to resistance to PR8
FIGURE 2. Influenza-specific memory T cells are not sufficient to provide efficient protection against heterosubtypic infection. C57BL/6 and
MT mice
were infected, or not, with 300 EIU of X31, allowed to recover for 4 wk, and then challenged with 1000 EIU of PR8. A, Survival was monitored for 3 wk
after challenge infection. B, Weight loss was measured for 3 wk following challenge infection. C, Viral titers were measured in the lungs 6 days after
challenge infection. D, NP- and PA-specific CD8 T cells were detected in the spleen by flow cytometry on day 6 after challenge infection. The numbers
in the panels indicate the frequency of Ag-specific cells in the CD8 population (mean ⫾ SD). The plots are gated on live CD8 T cells. E, The numbers
of splenic NP-specific CD8 T cells were calculated on day 6 after challenge infection. There were five mice per group in each panel. This experiment was
performed three times with similar results.
457The Journal of Immunology
(H1N1), despite the difference in the HA and NA subtypes of these
viruses. Importantly, however, Abs to heterosubtypic strains of
influenza only confer resistance in combination with memory T
cells and do not confer any resistance on their own.
We next wanted to determine whether the combined transfer of
naive B cells and X31-immune serum to X31-immune
MT mice
could restore their resistance to the level seen in X31-immune
C57BL/6 mice. Therefore, we compared the responses of naive
and X31-immune C57BL/6 mice (Fig. 5A, top row), naive and
X31-immune
MT mice (Fig. 5A, second row), naive and X31-
immune
MT mice that received 5 ⫻ 10
7
naive splenic B cells
(Fig. 5A, third row), and naive and X31-immune
MT mice that
received 5 ⫻ 10
7
naive splenic B cells as well as X31-immune
serum (Fig. 5A, bottom row). We found that while naive
MT
FIGURE 3. Naive B cells provide partial protection against heterosubtypic challenge. A, C57BL/6 and
MT mice were infected, or not, with 300 EIU
of X31 and allowed to recover for 4 wk. Some groups received 5 ⫻ 10
7
purified splenic B cells 1 day before challenge with 1000 EIU of PR8. B, Survival
was monitored for 3 wk after challenge infection. C, Weight loss was measured for 3 wk following challenge infection. D, Viral titers were measured in
the lungs 6 days after challenge infection. E, NP- and PA-specific CD8 T cells were detected in the spleen by flow cytometry on day 6 after challenge
infection. The numbers in the panels indicate the frequency of Ag-specific cells in the CD8 population (mean ⫾ SD). The plots are gated on live CD8 T
cells. F, The numbers of splenic NP-specific CD8 T cells were calculated on day 6 after challenge infection. There were five mice per group in each panel.
This experiment was performed three times with similar results. G and H, The serum titers of X31-specific (G) and PR8-specific (H) IgG were determined
by ELISA in all groups 6 days after challenge infection and 21 days after challenge infection.
458 B CELLS AND RESISTANCE TO HETEROSUBTYPIC INFLUENZA
mice succumbed to infection within 12 days, all naive C57BL/6
mice survived and most naive
MT mice that received B cells
survived, regardless of whether they received X31-immune se-
rum or not (Fig. 5B, left panel). However all groups of naive
mice exhibited similar signs of acute illness and weight loss
(Fig. 5C, left panel). In contrast, most X31-immune
MT mice
survived challenge infection without the addition of B cells or
X31-immune serum and all
MT mice that received B cells or X31-
immune serum survived (Fig. 5B, right panel). Moreover, all X31-
immune mice exhibited less weight loss than their naive counter-
parts (Fig. 5C, right panel). Although the transfer of naive B cells
and X31-immune serum clearly reduced weight loss and acceler-
ated recovery of X31-immune
MT mice, they still lost slightly
more weight than X31-immune C57BL/6 mice and recovered
more slowly (Fig. 5C, right panel).
A similar trend was observed when we examined viral titers in
the lungs on day 6 after infection. All groups of naive mice had
high viral titers (Fig. 5D, open symbols). In contrast, X31-immune
MT mice had slightly lower viral titers than the naive groups.
The transfer of naive B cells lowered viral titers further, and the
transfer of B cells as well as X31-immune serum lowered viral
titers even more, nearly to the levels seen in X31-immune
C57BL/6 mice (Fig. 5C, right panel). As expected, all groups of
X31-immune mice had memory CD8 T cells, whereas the groups
of naive mice did not (Fig. 5E). Interestingly, the addition of B
cells and X31-immune serum boosted the numbers of NP-specific
CD8 T cells to nearly the levels seen in X31-immune C57BL/6
mice. Together, these data demonstrate that, in combination with
memory T cells, naive B cells and X31-immune serum cooperate
to promote resistance to challenge infection with PR8.
Heterotypic Abs react with internal rather than coat proteins of
influenza
The above results demonstrate that Abs against X31 provide pro-
tection against challenge infection with PR8, despite the differ-
ences in HA and NA subtypes. Since Abs against the HA and NA
of one influenza subtype are usually minimally cross-reactive with
the HA and NA proteins of other influenza subtypes (32), these
data suggest that alternative B cell epitopes are important for het-
erosubtypic immunity. To directly demonstrate that Abs elicited by
X31 cross-reacted poorly with surface Ags from PR8, we coated
ELISA plates with viral proteins from X31 or PR8 and tested the
binding of Abs from the serum of mice previously infected with
X31 (Fig. 6A) or mice previously infected with PR8 (Fig. 6B). As
expected, Abs from X31-immune serum strongly reacted with
X31-coated wells and did not react with PR8-coated wells (Fig.
6A). Conversely, Abs from PR8-immune serum strongly reacted
with PR8-coated wells and did not react with X31-coated wells
FIGURE 4. X31-immune serum provides significant protection against heterosubtypic challenge in X31-immune
MT mice. A,
MT mice were
infected, or not, with 300 EIU of X31 and allowed to recover for 4 wk. Mice then received 400
l of serum from either normal donors or from
X31-immune donors 1 day before challenge with 1000 EIU of PR8. B, Survival was monitored for 3 wk after challenge infection. C, Weight loss
was measured for 3 wk following challenge infection. D, Viral titers were measured in the lungs 6 days after challenge infection. E, NP- and
PA-specific CD8 T cells were detected in the spleen by flow cytometry on day 6 after challenge infection. The numbers in the panels indicate the
frequency of Ag-specific cells in the CD8 population (mean ⫾ SD). The plots are gated on live CD8 T cells. F, The numbers of splenic NP-specific
CD8 T cells were calculated on day 6 after challenge infection. There were five mice per group in each panel. This experiment was performed three
times with similar results.
459The Journal of Immunology
(Fig. 6B). These data demonstrate that there is minimal cross-re-
activity of Abs elicited by viruses of one subtype with proteins
from heterosubtypic viruses. However, these data do not exclude
the possibility that a minor component of the polyclonal anti-sera
recognizes proteins that are conserved between viruses. To test this
possibility, we first coated plates with the external domain of M2
(M2e, a synthetic 23 amino acid peptide), which has the identical
sequence in both X31 and PR8 viruses (36), and tested the binding
of Abs from naive mice, X31 immune mice, or mice that had been
previously immunized with a recombinant fusion protein contain-
ing the M2e domain linked to the entire NP (M2e-NP) (Fig. 6C).
We found that sera from naive mice or from mice previously in-
fected with X31 did not react with M2e, even though M2e-specific
Abs were easily detected the sera of mice previously immunized
with recombinant M2e-NP (Fig. 6C). Thus, anti-M2e Abs present
in the serum of X31-immune mice are not likely to explain the
protective effects of the X31-immune serum when transferred to
X31 immune
MT mice.
Because X31 infection did not elicit detectable levels of Abs
against the surface proteins of PR8, we next tested whether X31
infection elicited Abs to internal proteins like NP that are con-
served in both viruses. Therefore, we next coated plates with re-
combinant NP and tested the binding of Abs from naive mice, X31
immune mice, or M2e-NP-immune mice (Fig. 6D). Although Abs
in naive sera did not react with recombinant NP, Abs in both X31-
immune sera and M2e-NP-immune sera reacted strongly with re-
combinant NP (Fig. 6D). These data demonstrate that although
Abs in X31-immune serum do not react with the HA and NA
proteins of PR8 or with the conserved external domain of M2, they
do react with at least one conserved internal protein that is highly
conserved between PR8 and X31. Together, these data suggest that
Abs to conserved internal proteins of influenza viruses promote
FIGURE 5. Naive B cells, immune serum, and memory T cells act together to protect against heterosubtypic challenge. A, C57BL/6 and
MT mice were
infected, or not, with 300 EIU of X31 and allowed to recover for 4 wk. Mice then received 5 ⫻ 10
7
splenic B cells alone or with 400
l of serum from
X31-immune donors 1 day before challenge with 1000 EIU of PR8. B, Survival was monitored for 3 wk after challenge infection. C, Weight loss was
measured for 3 wk following challenge infection. D, Viral titers were measured in the lungs 6 days after challenge infection. E, NP-specific CD8 T cells
were detected in the spleen by flow cytometry on day 6 after challenge infection. The numbers in the panels indicate the frequency of NP-specific cells
in the CD8 population (mean ⫾ SD). The plots are gated on live CD8 T cells. F, The numbers of splenic NP-specific CD8 T cells were calculated on day
6 after challenge infection. There were five mice per group in each panel.
460 B CELLS AND RESISTANCE TO HETEROSUBTYPIC INFLUENZA
resistance to challenge infection with heterosubtypic strains of
influenza.
Discussion
Memory T cells responding to epitopes in conserved internal pro-
teins of influenza are most often cited as the mechanism through
which heterosubtypic immunity is achieved (3). However, our data
clearly demonstrate that B cells and Abs are also important com-
ponents of heterosubtypic immunity. B cells are essential for sur-
vival after challenge infection with high doses of heterosubtypic
viruses and reduce weight loss and morbidity after challenge with
low doses of heterosubtypic viruses. This is consistent with other
reports showing that B cells are required for heterosubtypic
protection (16, 28) but contrasts with the conclusion that T cells
are the primary mediators of heterosubtypic immunity (15). In
part, the different conclusions of these studies may reflect which
parameters are measured. Survival is ultimately important but is
not a very nuanced measure of protection. In fact, we find that
X31-immune
MT mice can survive challenge with low doses
of PR8, but they lose significantly more weight and have much
higher viral titers than X31-immune C57BL/6 mice. In addition,
the virulence of the challenge virus is important. For example,
even naive
MT mice can clear the relatively avirulent X31
virus, but they rarely survive infection with the virulent PR8
virus (37). Thus, the importance of B cells in resistance to het-
erosubtypic infections is magnified by the dose and virulence of
the challenge virus.
Our studies also show that B cells promote immunity to hetero-
subtypic strains of influenza via multiple mechanisms. First, naive
B cells play an important role in the recovery of immune mice
from heterosubtypic challenge. Although this fits with the idea that
memory CD4 cells rapidly respond to challenge infection and pro-
mote a more robust primary B cell response to the challenge virus
(31), we found that the presence of memory T cells in
MT mice
made no difference in the magnitude of the B cell response to the
challenge virus. Nevertheless, during the recall response in
MT
mice, transferred naive B cells do generate PR8-specific neutral-
izing Ab, which prevents further infection by neutralizing virus,
targeting viral particles, and infected cells for clearance by phago-
cytic cells. As a result, naive B cells do not significantly accelerate
viral clearance over that seen in a normal primary response, but
they are essential for the resolution of infection.
Although B cells could also be facilitating the CD4 T cell re-
sponse by acting as APCs, we found that naive B cells from class
II deficient donors promoted recovery as effectively as normal B
cells (not shown). Given that B cells make a potent T cell inde-
pendent Ab response to influenza (37), we conclude that the
primary function of naive B cells upon heterosubtypic challenge
infection is to produce Ab rather than help CD4 T cells. Never-
theless, we have observed reductions in influenza-specific CD4
responses in
MT mice, consistent with published data (38). Thus,
impaired function of memory CD4 cells probably also contributes
to the immune defects in
MT mice that are responding to het-
erosubtypic influenza infections.
A second mechanism by which B cells promote resistance to
heterosubtypic challenge is via the production of Abs to the prim-
ing virus that cross-react with the challenge virus. In the presence
of cross-reactive memory T cells, these Abs reduce weight loss,
promote recovery, and, importantly, accelerate viral clearance
from the lungs. The presence of these Abs also promotes more
rapid expansion of cross-reactive memory T cells upon challenge
infection. These protective Abs are not neutralizing and, in our
studies, have no effect on challenge infection in the absence of
memory T cells. Moreover, the protective Abs elicited by X31
infection are not cross-reactive with the surface proteins of PR8
and do not bind to the external domain of M2. Our inability to
detect M2e-specific Abs in X31-immune serum may be due to
inefficient detection by our peptide based ELISA but may also
reflect the relatively poor Ab response to M2e during natural in-
fection (39). Regardless, the protective serum contains high titers
of Abs to conserved internal proteins, like NP, consistent with
previous reports (16).
Given that transferred X31-immune serum contains almost en-
tirely IgG and almost no detectable IgA (37), we conclude that the
majority of the protective effect conferred by transferred serum is
mediated by IgG. Nevertheless, the production of local IgA that
cross-reacts with both X31 and PR8 in the respiratory tract of
normal C57BL/6 mice will almost certainly play an important role
in protection against infection with heterosubtypic strains of influ-
enza (40). In fact, one of the reasons the transfer of immune serum
to
MT mice does not provide protection equivalent to that in
immune C57BL/6 mice may be that the local IgA component of
immunity is still absent in these recipients. Although the produc-
tion of IgA has been observed in
MT mice (41), we have not
detected significant levels of influenza-specific IgA in either the
bronchial lavage fluid or the serum of either naive or X31-immune
MT mice. However, we cannot discount the possibility that some
IgA is produced in these animals after X31 infection and that it
plays a role in the minimal level of protection observed in X31-
immune
MT mice. Despite this possibility, however, the influ-
enza-specific IgG in the transferred X31-immune serum clearly
plays an important role in resistance to heterosubtypic challenge.
Although transferred immune serum clearly confers additional
resistance to X31-immune mMT mice responding to heterosub-
typic strains of influenza, it is not clear how this effect is mediated.
It is plausible that IgG Abs in immune serum bind to NP and other
proteins released by infected cells and that immune complexes
containing these proteins are processed by APCs, which accelerate
and expand the memory CD8 T cell response. This hypothesis fits
with our data showing that serum Abs elicited by X31 do not
provide any protection against PR8 in the absence of memory T
cells. Moreover, we found that the transfer of immune serum to
immune
MT mice significantly boosted the CD8 T cell recall
response compared with that in
MT mice that received control
serum. Although we only examined splenic CD8 T cell responses
FIGURE 6. Abs against X31 cross-react with internal proteins of PR8. A
and B, Abs in serum from X31-immune mice (A) or PR8-immune mice (B)
were allowed to bind ELISA plates that were coated with proteins from dis-
rupted X31 or PR8 viruses. Abs in naive serum, X31-immune serum, or serum
from mice immunized with recombinant M2e-NP fusion protein were allowed
to bind plates coated with purified M2e peptide (C) or purified recombinant NP
protein (D). All plates were developed with anti-mouse IgG.
461The Journal of Immunology
in these experiments, it will be interesting to test in future exper-
iments whether immune serum also boosts CD8 recall responses in
the lung, as these T cells are likely to directly impact viral clear-
ance and facilitate recovery. As discussed above however, Abs,
particularly local IgA, may also work independently of any effects
on the T cell response.
Our studies clearly show that non-neutralizing influenza-specific
Abs in serum can provide significant protection to heterosubtypic
virus challenge, provided heterosubtypic T cells are present. In
contrast, other studies find that some non-neutralizing Abs provide
significant protection when transferred to naive recipients that do
not have influenza-specific memory T cells (30). One possible ex-
planation for this difference is that suboptimal amounts of Ab were
transferred in our experiments. However, we could easily detect
high titers of influenza-specific IgG in
MT recipient mice up to
15 days after infection that were comparable to the titers of influ-
enza-specific IgG seen in C57BL/6 mice 15 days after a primary
infection (not shown). Thus, it seems unlikely that Ab levels were
too low to act on their own. An alternative explanation for the
difference between our results and earlier studies may be found in
the fine specificity of the Abs elicited against particular viruses.
For example, Abs elicited by H3 significantly cross-react with H5
(28), and Abs elicited to H1 or H3 via natural infection of children
cross-react with H8 (42), whereas Abs to X31 (H3N2) do not
significantly cross-react with PR8 (H1N1) (Fig. 6). Interestingly, it
is also reported that anti-HA Abs do not confer any demonstrable
protection against challenge infections with heterosubtypic stains
of virus (10). Similarly, the transfer of anti-H1N1 or anti-H3N2
antiserum to naive mice did not protect against the opposite strain
of virus (43). Thus, it seems that the fine specificity of non-neu-
tralizing Abs for epitopes in HA and NA that are conserved be-
tween subtypes is probably important for determining whether
these Abs can facilitate protection by themselves or whether they
will work in conjunction with already primed cellular immune re-
sponses. Thus, it will be important to identify these conserved B
cell epitopes and define which ones elicit the most effective pro-
tection against heterosubtypic infections.
The results presented here have important implications for
influenza vaccine design. Given the numerous examples of
memory T cells that recognize conserved internal proteins, like
NP, PA, M2, and basic polymerase (PB)1 (14, 44) and given our
data showing that Abs to one or more of these conserved pro-
teins can facilitate T cell recall responses to these proteins, it
makes sense to design vaccines that elicit memory T cells and
high titers of Abs to these proteins. However, most influenza
vaccines are composed of fixed whole virus, split virus, or pu-
rified HA and NA subunits (45). These vaccines primarily elicit
Ab responses to the HA and NA subunits and, to a lesser extent,
Abs to abundant internal proteins, like NP. However, Abs are
not efficiently elicited to proteins present in low copy numbers
in virions, like PB1, PB2, PA, and M2, which are only ex-
pressed at 30 –60 molecules per virion. Even the neuraminidase
is expressed at only 100 molecules per virion compared with
500 for HA, 1000 for NA, and 3000 for M1. Thus, despite the
clear efficacy of using vaccination to elicit neutralizing Ab to
specific serotypes and subtypes of HA and NA, a complemen-
tary vaccination strategy is to also vaccinate against conserved,
low-abundance internal proteins in a way that maximizes both
T and B cell responses and promotes heterosubtypic immunity
to multiple strains of influenza.
Disclosures
The authors have no financial conflict of interest.
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463The Journal of Immunology
